Download FONGAnti-inflammatoryRole2010

The Anti-inflammatory Role for IkappaB Kinase
(IKK) Beta Through Inhibition of ‘Classical’
Macrophage Activation
Carol Ho Yan Fong
A thesis submitted for the degree of Doctor of Philosophy
Queen Mary, University of London
March 2010
Centre of Cancer and Inflammation
Institute of Cancer and the CR-UK Clinical Centre at Bart & The
London
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Abstract
Recent research has revealed a role of NF-B in the resolution of inflammation.
Using Cre-lox mediated gene targeting, IKK was selectively deleted in
macrophages (IKKβ∆Mye). From in vitro studies, LPS stimulated IKKMye
macrophages increased STAT1 phosphorylation, iNOS, MHC II and IL-12
production, suggesting negative cross talk between NF-B and STAT1 signalling
pathways.
Since IKK is required for TNF gene expression and TNF
signalling, I investigated the hypothesis that TNF inhibits ‘classical’
macrophage activation through IKK activation. Macrophages from p55-/- and
mice treated with anti-TNF antibody show increased STAT1 activation and IL12 expression after LPS and IFN stimulation. BMDM infected with adenovirus
expressing IKKβ dominant negative rescued the inhibitory effect of TNFα on IL12p40 production, indicating TNFα inhibits IL-12p40 via IKKβ activation.
Macrophages are antigen presenting cells while IL-12 and MHC II are critical
factors for TH1 cell development. I thus investigate the inhibitory effects of
IKKβ∆Mye macrophages in TH1 responses. FACS analysis showed higher MHC
II, costimulatory molecules expression on IKKβ∆Mye macrophages after LPS
stimulation. In a DTH model, recall assay has shown increased antigen-specific
IFN production from IKKMye splenocytes compared to IKKβF/F splenocytes.
Furthermore, IFN production was greatly enhanced by CD4+ OTII T cells cocultured with IKKMye macrophages. Further analysis of CD4+ OTII T cells
with qRT-PCR showed increased TH1 genes including IRF1, IFN, IL-12R1
and IL-12R2 and reduced TH2 marker IL-4. In addition to the enhanced
antigen-specific T cell responses, IKKMye macrophages also increased anti2
tumour immunity. Injection of H-Y positive MB49 tumour cells into IKKF/F and
IKKMye female mice has shown tumour rejection, but no tumours were rejected
after CD8+ T cells depletion, suggesting tumour rejection is associated with
enhanced CTL activity. Taken together, these studies demonstrated the negative
regulatory roles of IKK in macrophage activation and their impact to the innate
and adaptive immunity.
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List of Contents
ABSTRACT ................................................................................................................. 2
LIST OF CONTENTS ................................................................................................ 4
LIST OF TABLES .................................................................................................... 11
LIST OF LEGENDS ................................................................................................. 12
ACKNOWLEDGEMENTS ...................................................................................... 16
STATEMENT OF ORIGIN ..................................................................................... 18
ABBREVIATIONS ................................................................................................... 19
1. INTRODUCTION ................................................................................................. 23
1.1 MACROPHAGES AND THEIR ROLES IN INNATE AND ADAPTIVE IMMUNITY ........... 23
1.2 MACROPHAGE HETEROGENEITY ......................................................................... 23
1.3 PATHOGEN RECOGNITION ................................................................................... 27
1.4 NON SIGNALLING PATTERN RECOGNITION RECEPTORS ....................................... 27
1.5 PHAGOCYTOSIS .................................................................................................. 29
1.6 TOLL LIKE RECEPTORS SIGNALLING .................................................................... 29
1.7 TNF SIGNALLING PATHWAY ............................................................................ 36
1.8 NF-КB SIGNALLING PATHWAY ........................................................................... 37
1.9 IFN SIGNALLING PATHWAY ................................................................................ 39
1.10 RESOLUTION OF INFLAMMATION ...................................................................... 41
1.11 NEGATIVE REGULATION OF SIGNALLING .......................................................... 41
1.12 APOPTOSIS ....................................................................................................... 42
1.13 NF-ĸB AND THE RESOLUTION OF INFLAMMATION............................................. 43
1.14 NF-ĸB AND APOPTOSIS ..................................................................................... 45
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1.15 NF-B AND MACROPHAGE ACTIVATION ........................................................... 47
1.16 MACROPHAGES BRIDGE INNATE AND ADAPTIVE IMMUNITY ............................. 48
1.17 THE EFFECTOR FUNCTIONS OF T CELLS............................................................. 49
1.18 T CELL POLARISATION ...................................................................................... 50
1.19 NF-B, INFLAMMATION AND CARCINOGENESIS ............................................... 53
1.20 AIM OF THE THESIS ........................................................................................... 56
2. METHODS AND MATERIALS ......................................................................... 57
2.1 MICE .................................................................................................................. 57
2.2 CELL LINES AND PRIMARY CELLS ....................................................................... 57
2.2.1 Cell lines ............................................................................................. 57
2.2.2 Macrophage Colony-Stimulating Factor derived bone marrow derived
macrophages ................................................................................................ 58
2.2.3 Thioglycolate elicited macrophages ................................................... 59
2.3 DELAYED-TYPE HYPERSENSITIVITY .................................................................... 60
2.3.1 Immunisation ....................................................................................... 60
2.3.2 CD4+ T cell isolation .......................................................................... 61
2.3.3 CFSE labelling .................................................................................... 62
2.3.4 Allogenic Mixed lymphocyte reaction ................................................. 62
2.3.5 Characterisation of OTII specific CD4+ T cells ................................. 63
2.3.6 Characterisation of CD4+T cells isolated from mBSA immunised mice
...................................................................................................................... 63
2.3.7 mBSA recall assay ............................................................................... 63
2.4 MB49 TUMOUR MODEL ...................................................................................... 64
2.4.1 Preparation of MB49 tumour cells for injection ................................. 64
2.4.2 Tumour digestion ................................................................................ 64
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2.4.3 H-Y recall assay .................................................................................. 65
2.5 RNA .................................................................................................................. 66
2.5.1 RNA extraction with QIAamp RNA blood mini kit............................ 66
2.5.2 RNA extraction with Tri-reagent......................................................... 67
2.5.3 DNase treatment of RNA ..................................................................... 67
2.5.4 Quantitative real-time polymerase chain reaction ............................. 68
2.5.5 Ribonuclease protection assay ............................................................ 71
2.5.6 Polyacryamide gel preparation .......................................................... 73
2.6 GENOTYPING FOR IKKΒF/F AND IKKΒΔMYE MICE ................................................ 74
2.6.1 Polymerase chain reaction .................................................................. 74
2.7 ADENOVIRUS PRODUCTION................................................................................. 76
2.7.1 Initial adenoviral amplification .......................................................... 76
2.7.2 Cell factory -10TM preparation and infection...................................... 76
2.7.3 Cesium Chloride purification.............................................................. 78
2.7.4 Determination of particle counts ........................................................ 79
2.7.5 The tissue culture infectious dose 50 Assay ........................................ 80
2.7.6 Infecting bone marrow derived macrophages with adenovirus .......... 81
2.8 IMMUNOBLOTTING ............................................................................................. 82
2.8.1 Preparation and quantification of whole cell protein lysates ............. 82
2.8.2 Sodium dodecyl suphate-polyacrylamide gel electrophoresis ............ 84
2.9 ELISA ................................................................................................................ 86
2.10 FLUORESCENCE ACTIVATED CELL SORTING ANALYSIS ..................................... 88
2.10.1 Cell surface staining ......................................................................... 88
2.10.2 Intracellular staining ........................................................................ 88
2.11 STATISTICAL ANALYSIS .................................................................................... 90
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2.12 FULL COMPANY NAME AND LOCATION ............................................................. 91
3. RESULTS .............................................................................................................. 92
3.1 NEGATIVE REGULATORY ROLE OF IKK IN MACROPHAGE ACTIVATION ............. 92
3.1.1 Genotyping of IKKF/F and IKKMye mice ........................................ 93
3.1.2 IKK inhibits STAT1 activation .......................................................... 96
3.1.3 IKKβ suppresses expression of iNOS and IRF1 mRNA ...................... 99
3.1.4 IKK inhibits IL-12 production ........................................................ 102
3.1.5 TNFα inhibits STAT1 activation in macrophages in vivo ................. 103
3.1.6 TNF inhibits STAT1 activation in macrophages in vitro ................ 107
3.1.7 TNF inhibits STAT1 through p55 ................................................... 112
3.1.8 Inhibition of IKK activity in vitro with adenovirus expressing IKK
dominant negative inhibitor ....................................................................... 116
3.1.9 Ad-IKKdn infected BMDM increased STAT1 phosphorylation and
iNOS expression ......................................................................................... 117
3.1.10 TNFα inhibits IL-12p40 production through activation of IKKβ ... 120
3.1.11 Summary.......................................................................................... 122
3.1.12 Discussion ....................................................................................... 123
3.1.12.1 Mechanisms of IKK inhibition of classical macrophage
activation ................................................................................................ 123
3.1.12.2 TNF and inhibition of classical macrophage activation ........ 124
3.1.12.3 Experimental limitations .......................................................... 126
3.2 INHIBITORY ROLES OF IKK IN ADAPTIVE IMMUNITY ...................................... 130
3.2.1 IKKMye macrophages express higher levels of CD80, CD86, MHCII
and low CD124/IL-4 receptor  ................................................................ 131
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3.2.2 Bacteria elicited peritoneal macrophages from IKKMye mice express
high levels of CD80, CD86, MHC II and low IL-4R ............................... 134
3.2.3 Mix lymphocyte reaction ................................................................... 136
3.2.3.1 CD4+ T cell isolation .................................................................. 136
3.2.3.2 Optimisation of MLR ................................................................. 138
3.2.3.3 Capacity of different macrophages in promoting T cell
proliferation ............................................................................................ 140
3.2.3.4 IKKMye macrophages increase allogenic T cell proliferation . 142
3.2.4 Delayed type hypersensitivity ............................................................ 143
3.2.4.1 Immunisation ............................................................................. 143
3.2.4.2 Dose response assay for mBSA induced T cell proliferation .... 145
3.2.4.3 IKKβ∆Mye splenocytes increase mBSA specific IFNγ production
ex-vivo .................................................................................................... 146
3.2.4.4 Priming mBSA specific T cell responses in IKK∆Mye mice ..... 148
3.2.4.5 Secondary mBSA specific response elicited by IKKMye
macrophages in vitro .............................................................................. 150
3.2.4.6 IKKMye macrophages increase antigen specific IFN production
by OTII T cells ....................................................................................... 152
3.2.4.7 IKKMye macrophages increase TH1 specific gene expression in
OTII cells ............................................................................................... 154
3.2.5 Inhibitory roles of TNF on macrophage APC activity ................... 158
3.2.5.1 TNF inhibits MHC II and CD80 expression on BMDM ......... 158
3.2.5.2 TNF inhibits allogenic T cell proliferation .............................. 160
3.2.5.3 TNFα treatment inhibits BMDM induced IFN production by
OTII cells ............................................................................................... 161
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3.2.5.4 TNFα inhibits TH1 specific gene expression.............................. 163
3.2.6 Summary............................................................................................ 165
3.2.7 Discussion ......................................................................................... 167
3.2.7.1 NF-B and APC function of macrophages ................................ 167
3.2.7.2 IKK activation in macrophages and generation of antigenspecific T cell responses......................................................................... 170
3.2.7.3 The role of TNF in TH1 responses ........................................... 174
3.3 IKKMYE MACROPHAGES INCREASE ANTI-TUMOUR IMMUNITY ....................... 176
3.3.1 Targeting IKK in macrophages inhibits MB49 tumour growth in
female mice................................................................................................. 177
3.3.2 Targeting IKK in macrophages does not inhibit MB49 tumour
growth in male mice ................................................................................... 178
3.3.3 IKKMye splenocytes increase H-Y specific IFN production ex-vivo
.................................................................................................................... 180
3.3.4 Targeting IKK in macrophages inhibits the immunosuppressive
tumour microenvironment .......................................................................... 182
3.3.5 MB49 tumour rejection in IKKMye female mice is CD8 dependent
.................................................................................................................... 186
3.3.6 MB49 tumour rejection in male mice after Dxr treatment is CD8 T cell
dependent ................................................................................................... 188
3.3.7 Summary............................................................................................ 190
3.3.8 Discussion ......................................................................................... 192
3.3.8.1 IKK deletion in macrophages enhanced H-Y antigen specific
anti-tumour immunity ............................................................................ 192
3.3.8.2 Mechanisms for tumour rejection in IKKβ∆Mye mice ................. 194
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3.3.8.3 IKK deletion in macrophages response to chemotherapy........ 195
3.3.8.4 IKK maintains tumour-induced tolerance ................................ 195
4. GENERAL DISCUSSION ................................................................................. 197
4.1 IKK/NF-B INHIBITS CLASSICAL MACROPHAGE ACTIVATION........................ 197
4.2 NF-B-STAT SIGNALLING AND REGULATION OF MACROPHAGE ACTIVATION . 198
4.2.1 Cytokine production .......................................................................... 200
4.2.2 iNOS-Arginase balance..................................................................... 200
4.2.3 APC activity ...................................................................................... 201
4.3 MOLECULAR MECHANISMS OF MACROPHAGE REGULATION BY IKK .............. 202
4.4 IL-4 SIGNALLING AND STAT6 ......................................................................... 204
4.4.1 STAT1 and STAT6 ............................................................................. 204
4.4.2 STAT6 and NF-B ............................................................................. 206
4.4.3 IKK/NF-B and IL4R expression ................................................. 206
4.5 INHIBITION OF CLASSICAL MACROPHAGE ACTIVATION BY TNF ..................... 208
5. FUTURE WORK ................................................................................................ 212
5.1 REGULATION OF SOCS BY IKK ..................................................................... 212
5.2 ANTIGEN PROCESSING AND PRESENTATION ...................................................... 213
5.3 T CELL PRIMING IN VIVO ................................................................................... 213
5.5 STAT6 SIGNALLING ......................................................................................... 214
5.6 TUMOUR-INDUCED TOLERANCE........................................................................ 215
6. REFERENCES .................................................................................................... 216
7. PUBLICATION……………………………………………………………235
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List of Tables
TABLE 1. ROI, RNI ...................................................................................................... 29
TABLE 2. EXOGENOUS AND ENDOGENOUS TLR LIGANDS ............................................ 32
TABLE 3 STIMULUS USED IN EXPERIMENTS .................................................................. 59
TABLE 4 PRIMER SEQUENCES FOR QRT-PCR ............................................................... 70
TABLE 5 PCR PRIMER SEQUENCES ............................................................................... 75
TABLE 6 MASTER MIX OF PCR REAGENTS ................................................................... 75
TABLE 7 DETERMINATION OF PARTICLE COUNTS ......................................................... 79
TABLE 8 TCID 50 ASSAY............................................................................................. 80
TABLE 9 SUPPLEMENT PER ML OF WHOLE CELL LYSIS BUFFER ..................................... 83
TABLE 10 ANTIBODIES FOR WESTERN BLOTTING ........................................................ 85
TABLE 11 ANTIBODIES FOR ELISA .............................................................................. 87
TABLE 12 ANTIBODIES USED FOR FACS ANALYSIS...................................................... 89
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List of Legends
FIGURE 1.6 1 LOCATION OF TLRS ................................................................................ 30
FIGURE 1.6 2 TNF/IL-1/TLR SIGNALLING ................................................................... 35
FIGURE 3.1.1 1 SELECTIVE DELETION OF IKK IN MACROPHAGES USING CRE-LOX
RECOMBINASE ......................................................................................................
94
FIGURE 3.1.1 2 PCR ANALYSIS OF IKBKB AND CRE TRANSGENIC MICE ........................ 95
FIGURE 3.1.1 3 LEVEL OF IKK EXPRESSION IN IKKΒF/F AND IKKΒ∆MYE
PERITONEAL MACROPHAGES
................................................................................ 95
FIGURE 3.1.2 1 IKK INHIBITS STAT1 ACTIVATION AND INOS EXPRESSION .............. 98
FIGURE 3.1.3 1 IKK INHIBIT INOS IN THE MRNA LEVEL ......................................... 100
FIGURE 3.1.3 2 IKKΒ INHIBITS MHC II EXPRESSION.................................................. 101
FIGURE 3.1.4 1 IKK INHIBITS IL-12 PRODUCTION .................................................... 102
FIGURE 3.1.5 1 BLOCKING TNF IN VIVO INCREASED STAT1 PHOSPHORYLATION
AND INOS EXPRESSION ......................................................................................
104
FIGURE 3.1.5 2 TNF SELECTIVELY INHIBIT IL-12 PRODUCTION ............................... 106
FIGURE 3.1.6 1 INHIBITION OF STAT1 PHOSPHORYLATION IN BMDM PRETREATED WITH TNF ........................................................................................
108
FIGURE 3.1.6 2 REDUCTION OF IL-12 PRODUCTION BY TNF PRE-TREATED MCSFBMDM .............................................................................................................. 109
FIGURE 3.1.6 3 REDUCED MRNA LEVEL OF IL-12P40 AND IP10 BY TNF PRETREATED MCSF-BMDM ...................................................................................
111
FIGURE 3.1.7 1 THE INHIBITORY EFFECT OF TNF ON STAT1 WAS MEDIATED BY
P55 .....................................................................................................................
113
FIGURE 3.1.7 2 INCREASED IL-12 PRODUCTION FROM P55-/- PERITONEAL
MACROPHAGES ...................................................................................................
12
115
FIGURE 3.1.8 1 EFFICIENCY OF ADENOVIRUS INFECTION ............................................ 116
FIGURE 3.1.9 1 AD-IKKDN INFECTED BMDM INCREASED STAT1
PHOSPHORYLATION ............................................................................................
118
FIGURE 3.1.9 2 IKKDN INFECTED BMDM INCREASED IL-12P40 PRODUCTION ......... 119
FIGURE 3.1.10 1 IKKΒ INHIBITION RESCUED TNFΑ MEDIATED SUPPRESSION OF IL12P40 PRODUCTION ............................................................................................ 120
FIGURE 3.1.10 2 TNF AND IKK INHIBITS IL-10 PRODUCTION ............................... 121
FIGURE 3.2.1 1 IKKMYE MACROPHAGES EXPRESS HIGHER LEVEL OF CD80, CD86
AND MHCII .......................................................................................................
132
FIGURE 3.2.1 2 TG ELICITED PERITONEAL IKKMYE MACROPHAGES EXPRESS
REDUCED IL-4R ...............................................................................................
133
FIGURE 3.2.2 1 BACTERIA ELICITED PERITONEAL MACROPHAGES FROM IKKMYE
MICE EXPRESS HIGH COSTIMULATORY MOLECULES, MHC II AND LOW IL-4R .
135
FIGURE 3.2.3.1 1 ISOLATION OF CD4+ T CELLS WITH MAGNETIC BEAD SEPARATION . 137
FIGURE 3.2.3.2 1 OPTIMIZATION OF THE RATIO OF T CELLS AND MACROPHAGES ....... 139
FIGURE 3.2.3.3 1 ACTIVITY OF DIFFERENT MACROPHAGES IN ALLOGENIC MLR ........ 141
FIGURE 3.2.3.4 1 IKKMYE MACROPHAGES INDUCED AN ENHANCED ALLOGENIC T
CELL PROLIFERATION ......................................................................................... 142
FIGURE 3.2.4.1 1 SPLENOCYTES FROM MBSA IMMUNISED MICE ELICITED ANTIGEN
DEPENDENT CYTOKINE PRODUCTION EX-VIVO ..................................................... 144
FIGURE 3.2.4.2 1 OPTIMISATION OF THE DOSE OF MBSA FOR T CELL PROLIFERATION ASSAY .... 145
FIGURE 3.2.4.3 1 IKKΒ∆MYE SPLENOCYTES INCREASED MBSA SPECIFIC IFNΓ
PRODUCTION EX-VIVO .........................................................................................
146
FIGURE 3.2.4.3 2 IKKΒ∆MYE SPLENOCYTES DECREASED MBSA SPECIFIC IL-10
PRODUCTION EX-VIVO .........................................................................................
13
147
FIGURE 3.2.4.4 1 IKKMYE INCREASES TH1 CELLS DEVELOPMENT IN VIVO ................ 149
FIGURE 3.2.4.5 1 IKK∆MYE MACROPHAGES INCREASE MBSA SPECIFIC IFN
PRODUCTION IN VITRO .........................................................................................
151
FIGURE 3.2.4.6 1 IKK∆MYE MACROPHAGES ENHANCE OVA323-339 SPECIFIC T CELL
ACTIVATION IN VITRO
......................................................................................... 153
FIGURE 3.2.4.7 1 IKK∆MYE MACROPHAGES INCREASE EXPRESSION OF TH1
SELECTIVE GENES IN OTII CELLS
....................................................................... 157
FIGURE 3.2.5.1 1 INHIBITION OF CD80 AND MHC II BY TNFΑ IN BMDM ................ 159
FIGURE 3.2.5.2 1 TNFΑ TREATMENT OF BMDM REDUCED ALLOGENIC T CELL
PROLIFERATION ..................................................................................................
160
FIGURE 3.2.5.3 1 TNFΑ TREATMENT INHIBITS BMDM INDUCED IFN PRODUCTION
BY OTII CELLS ...................................................................................................
162
FIGURE 3.2.5.4 1 TNF TREATMENT OF BMDM INHIBITS CHARACTERISTIC TH1
GENE EXPRESSION IN OTII CELLS .......................................................................
164
FIGURE 3.3.1 1 TARGETING IKK IN MACROPHAGES PROMOTES REJECTION OF
MB49 TUMOURS IN FEMALE MICE ...................................................................... 177
FIGURE 3.3.2 1 TARGETING IKK IN MACROPHAGES DOES NOT AFFECT MB49
TUMOUR GROWTH IN MALE MICE ........................................................................
179
FIGURE 3.3.3 1 H-Y RESTIMULATION ASSAY WITH SPLENOCYTES FROM MB49
TUMOUR-BEARING FEMALE MICE
....................................................................... 181
FIGURE 3.3.4 1 GENE EXPRESSION IN MB49 TUMOURS FROM IKKF/F AND
IKKMYE FEMALE MICE .................................................................................... 183
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FIGURE 3.3.4 2 CD3+ TUMOUR INFILTRATING LYMPHOCYTES FROM IKKΒ∆MYE MICE
EXPRESSED HIGHER INTRACELLULAR IFNΓ ........................................................
184
FIGURE 3.3.4 3 IL-4R AND MHC II EXPRESSION ON TAM FROM MB49 TUMOUR ... 186
FIGURE 3.3.5 1 TUMOUR REJECTION IN IKKMYE FEMALE MICE WAS MEDIATED BY
CD8+ T CELLS .................................................................................................... 187
FIGURE 3.3.6 1 TARGETING IKK IN TAM INCREASED RESPONSE TO
CHEMOTHERAPY .................................................................................................
189
FIGURE 4.4.1 1 COMPETITION BETWEEN STAT1 AND STAT6 FOR SBE ................... 205
FIGURE 4.4.1 2 IKK ACTIVATION PROMOTES IL-4R EXPRESSION ......................... 208
FIGURE 4.5 1 POSSIBLE MECHANISMS OF THE INHIBITORY EFFECT OF IKK/TNF
ON STAT1 SIGNALLING .................................................................................... 211
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Acknowledgements
The first person I would like to thank is my supervisor, Toby Lawrence, for
giving me the opportunity to do a PhD in his lab. For all his support and
guidance throughout the four years. Thank for all his valuable comments that
have continuously inspired me and improve my ‘scientific creativity’, a special
thank to Toby for being a tough, high standard supervisor who has leaded me to
achieve what I have achieved. Also, Toby has introduced me to the exciting
scientific world and strongly built up my interest in science, this has leaded me to
see myself as a scientist as my long-term career. Finally, I would like to thank
him for his advises and effort on reading my thesis.
The second person I would like to thank is Magali Bebien for teaching me
experimental techniques, for the friendship and for sharing all the happiness and
sadness. Thank for her support and encouragement throughout my study. Magali
has always made my working days more enjoyable and keeping me good
company.
The third person I would like to thank is Andy Chan. I would like to thank him
for his support in the past 8 years. Thank for keeping me positive and provided
me a happy, comfortable environment to rest after all the long working days.
Thank for being patience and understanding in my moody days. Thank for being
with me throughout the difficult time and encourage me all the way through.
16
Finally, a huge thank to my parent, brother, aunties and uncles for all their
support. Thank you to my parent to send me abroad and received education in
UK. This has given me the opportunity to see the other side of the world and
develop a strong, independent Carol. This has equipped me with all I need to do
what I want in my life.
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Statement of Origin
All work described in this thesis was performed by myself.
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Abbreviations
A
AAD
ad
AEBSF
AICD
AP
APC
Arg-1
BMDM
BSA
C/EBP
CaCl
CF
CFA
CIITA
Cn
CO2
ConA
CPE
cpm
CR
CsCl
CFSE
CTL
DAMP
DCs
DC-SIGN
DD
DEPC
DMEM
DNA
dNTP
DTH
DTT
Dxr
EDTA
EGF
EGTA
Ampere
7-amino-actinomycin D
Adenovirus
4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride
Activation Induced Cell Death
Activating Protein
Antigen Presenting Cell
Arginase 1
Bone Marrow Derived Macrophages
Bovine Serum Albumin
CCAAT/Enhancer Binding Protein
Calcium Chloride
Cell Factory
Complete Freund Adjuvant
Class II Transactivator
Cryptococcus neoformans
Carbon Dioxide
Concanavalin A
Cytopathic Effect
Counts Per Minute
Complement Receptor
Cesium Chloride
Carboxyfluorescein Succinimidyl Ester
Cytotoxic T Lymphocytes
Damage Associated Molecular patterns
Dendritic Cells
Dendritic Cell-Specific ICAM-3 Grabbing non-Integrin
Death Domain
diethylpyrocarbonate
Dulbecco's Modified Eagle Medium
Deoxyribonucleic Acid
Deoxyribo Nucleotide Triphosphate
Delayed Type Hypersensitivity
Dithiothreitol
Doxorubicin
Ethylenediaminetetraacetic Acid
Epidermal Growth Factor
Ethylene Glycol Tetraacetic Acid
ELISA
ES
FACS
FADD
FBS
FGF
xg
Enzyme Linked Immunosorbent Assay
Embryonic Stem
Fluorescence Activating Cell Sorting
Fas Associated Protein with Death Domain
Fetal Bovine Serum
Fibroblast Growth Factor
gravity
19
g
G
GAS
GBS
GFP
h
HCl
gram
Gauge
Gamma interferon Activation Site
Group B Streptococcus
Green Fluorescence Protein
Hour
Hydrochloric Acid
HDACs
HLA
HMGB1
HSP
i.p
ICSBP
IFA
IFN
Ig
IKK
IL
IL-4R
In
iNOS
IRAK
IRF
ISGF
IB
JAK
JNK
KCl
KIR
LPS
LT
Lys
Lys
M-MLV
M.O.I
M1
M2
MAL
MAP
MB
MBP
mBSA
MCSF
MDSC
MEF
MFI
MgCl
MHC
min
Histone Acetyltransferases and Deacetylases
Human Leukocyte Antigen
High Mobility Group Box 1
Heat Shock Proteins
Intraperitoneal
IFN Consensus Binding Protein
Incomplete Freund Adjuvant
Interferon
Immunoglobulin
IB kinase
Interleukin
IL-4 Receptor Alpha
Inches
Inducible Nitric Oxide Synthase
Interleukin Receptor Associated Kinase
IFN Responsive Factor
IFN Stimulated Gene Factor
Inhibitor of B
Janus Activated Kinase
c-Jun NH2 terminal Kinase
Potassium Chloride
Kinase Inhibitory Region
Lipopolysaccharide
Lymphotoxin
Lysine
Lysosome
Moloney Murine Leukaemia Virus
Multiplicity Of Infection
Type I Classically Activated Macrophage
Type II Alternative Activated Macrophage
MyD88-Adaptor Like
Mitogen Activated Protein
Murine Bladder
Mannose Binding Protein
Methylated Bovine Serum Albumin
Murine Colony Stimulating Factor
Myeloid Derived Suppressor Cell
Mouse Embryonic Fibroblast
Mean Fluorescence Intensity
Magnesium Chloride
Major Histocompatibility complex
Minute
20
MKK
ml
mLDL
MLR
mM
MR
mRNA

MyD88
NaCl
NaHPO4
NaVO4
neo
NF-B
ng
NIK
NK
NLS
NO
o
C
OVA
PAMP
PBS
PCR
PDKI
PGE2
PGN
PIPIB
PKC
PMA
PMSF
PNPP
Mitogen Activated Protein Kinase Kinase
Millilitre
Modified Lipoprotein
Mixed Lymphocyte Reaction
Millimolar
Mannose Receptor
Messenger RNA
Micro
Myeloid Differentiation Primary Response Gene 88
Sodium Chloride
Sodium phosphate
Sodium Orthovanadate
Neomycin
Nuclear Factor Kappa B
Nanogram
NF-B Inducing Kinase
Natural Killer
Nuclear Localization Sequence
Nitric Oxide
Celsius
Ovalbumin
Pathogen Associated Molecular Pattern
Phosphate Buffered Saline
Polymerase Chain Reaction
3-Phosphoinositide-Dependent Kinase 1
Prostaglandin E2
Peptidoglycan
Plasma Membrane Intrinsic Protein IB
Protein Kinase C
Phorbol Myristate Acetate
Phenylmethanesulphonylfluoride
p-Nitrophenyl Phosphat
PRR
PS
PTB
PTP
PVDF
qRT-PCR
RANKL
RHD
RIP
RNA
RNI
ROI
RPA
rpm
RPMI
RSV
s.c
Pattern Recognition Receptor
Phosphatidyserine
Phosphotyrosine Binding
Protein Tyrosine Phosphatases
Polyvinylidene difluoride
Quantitative Real Time Polymerase Chain Reaction
Receptor Activator of NF-B Ligand
Rel-Homology Domain
Receptor Interacting Protein
Ribonucleic Acid
Reactive Nitrogen Intermediate
Reactive Oxygen Intermediate
Ribonuclease protection assay
Rotation Per Minute
Roswell Park Memorial Institute
Respiratory Syncytial Virus
Subcutaneous
21
SARM
SBE
SDS
SDS-PAGE
sec
Ser
SH2
SHP1
siRNA
SLE
SOCS
SP
STAT
TAB
TACE
TAK
TAM
TANK
TBE
TBS
TC-PTP
TCID
TCR
tg
TGF
TH
TICAM
TIL
TIR
TIRAP
TLR
TNF
TNFR
TPCK
TRADD
TRAF
TRAM
Treg
TRIF
TYK
u
UV
V
VEGF
w
WT
Sterile  and HEAT-Armadillo Motifs
STAT Binding Site
Sodium Dodecyl Suphate
Sodium Dodecyl Suphate-Polyacrylamide Gel Electrophoresis
Second
Serine
Src Homology 2
Src Homology 2 domain Phosphatase
Small Interfering RNA
Systemic Lupus Erythematous
Suppressor of Cytokine Signalling
Surfactant Protein
Signal Transducer and Activator of Transcription
TAK binding protein
TNF  Converting Enzyme
TGF Activated Protein Kinase
Tumour Associated Macrophage
TRAF family member Associated NF-B
Tris Borate EDTA
Tris Buffered Saline
T Cell Protein Tyrosine Phosphatase
The Tissue Culture Infectious Dose
T Cell Receptor
Thioglycolate
Transforming Growth Factor
T Helper
TIR-Domain Containing Molecule
Tumour Infiltrating Lymphocyte
Toll Interleukin Receptor
TIR-Associated Protein
Toll Like Receptor
Tumour Necrotic Factor
TNF Receptor
N--Tosyl-L-Phenylalanine Chloromethyl Ketone
TNF Receptor Associated Death Domain
TNFR-Associated Factor
TRIF Related Adaptor Molecule
Regulatory T cells
TIR-Domain Containing Adaptor Protein Inducing IFN
Tyrosine kinase
Unit
Ultraviolet
Voltage
Vascular Endothelial Growth Factor
Watt
Wild Type
22
1. Introduction
1.1 Macrophages and their roles in innate and adaptive immunity
Macrophages have pleiotropic functions in the immune system (Gordon 2003;
Gordon and Taylor 2005); they are classified as professional phagocytes
responsible for microbe recognition and phagocytosis, macrophages also produce
reactive
oxygen
intermediates
and
lysosomal
enzymes
to
destroy
microorganisms. Furthermore, macrophage activation results in the release of
proinflammatory cytokines interleukin (IL) -1, IL-6 and tumour necrotic factor
(TNF) , these proinflammatory cytokines are important in host defence and the
development of adaptive immune responses. In addition to the production of
proinflammatory cytokines, macrophages can also act as antigen presenting cells
by providing costimulatory molecules and major histocompatibility complex
(MHC) II-antigen complex for T cell activation (Janeway 2001; Goldsby R
2003).
Apart from recognising ‘non-self’, macrophages can also recognise
‘altered self’ such as necrotic and apoptotic cells. This is important in the
immune system to maintain homeostasis and prevent prolonged inflammation
(Fadok, Bratton et al. 1998).
1.2 Macrophage heterogeneity
Different
macrophage
macrophages.
population
represents
the
diverse
functions
of
Circulating monocytes migrate into different tissues and
differentiate into resident macrophages.
Under the influence of the
microenvironment, macrophages acquire specific phenotype and distinct effector
functions (Gordon and Taylor 2005; Mosser and Edwards 2008). During
23
microbial infection, macrophages activated by microbial products and/or
proinflammatory cytokine such as interferon (IFN)  and become the ‘classically’
activated macrophages or M1 macrophages. These cells are proinflammatory
cells of the immune system that are responsible in the killing intracellular
pathogens and tumour cells. In addition, they also secrete proinflammatory
cytokines such as IL-12 and act as antigen presenting cells that promote the
development of TH1 immune responses (Gordon and Taylor 2005). In 1992,
Stein et al. described another type of macrophage; "alternatively" activated
macrophages or M2 macrophages that can be induced by TH2 cytokines, such as
IL-4, IL-13 (Stein, Keshav et al. 1992). To date, the precise roles of M2
macrophages in the immune system are still not clear, but these cells have been
associated with parasitic diseases (Wim Noël 2004), anti-inflammation (Goerdt S
1999; Gordon 2003) and wound healing (Song, Ouyang et al. 2000).
M1 and M2 macrophages have distinct characteristic phenotypes in terms of
receptor expression, effector function and cytokine production. The cytokine
profile of M1 macrophages includes predominantly proinflammatory cytokines;
such as IL-12, IL-6 and TNF α, whereas M2 macrophages typically produce antiinflammatory cytokines - IL-10, IL-1 receptor antagonist (IL-1ra), the type II IL1 decoy receptor and transforming growth factor (TGF) β (Goerdt S 1999;
Gordon 2003). In terms of chemokine receptors and ligands, M1 macrophages
predominantly express the IFN inducible chemokine IP-10 (CXCL10) and MIG
(CXCL9), which preferentially attract TH1 cells.
On the other hand, IL-4
selectively induces TARC (CCL17) (Imai, Nagira et al. 1999), CCL18 (also
known as AMAC1) and MDC (CCL22) (Imai, Nagira et al. 1999).
The up-
24
regulation of these chemokines preferentially recruiting CCR4 positive T cells,
thereby amplifying TH2 responses (Bonecchi, Sozzani et al. 1998).
The inducible nitric oxide synthase (iNOS)-arginase balance has also been
demonstrated as a marker to distinguish between M1 and M2 macrophages
(Munder, Eichmann et al. 1998). M1 macrophages produce elevated nitric oxide
(NO) and L-citrulline by upregulating iNOS, which uses L-arginine as a
substrate. In M2 macrophages, arginase is up-regulated instead of iNOS (Hesse,
Modolell et al. 2001), the induction of arginase hydrolyses L-arginine into Lornithine and urea. L-ornithine is a necessary metabolite for the production of
polyamines and prolines, polyamines are involved in the cell growth and division
and proline is a key component of collagen. Consistent with this observation,
Song et al. 2000, have shown that M2 macrophages enhance fibrogenesis while
M1 macrophages release anti-fibrotic and fibrolytic factors (Song, Ouyang et al.
2000). M2 macrophages also actively inhibit proliferation of peripheral blood
lymphocytes and CD4+ T cells in vitro (Kodelja, Muller et al. 1997). Two
transcription factors, FIZZ1 and YM1 have also been shown to be distinctly
induced in M2 macrophages although their function is not clear (Raes, De
Baetselier et al. 2002).
Tumour associated macrophages (TAM) or myeloid derived suppressor cells
(MDSC) are another type of macrophage that is abundantly found within
tumours. The roles of TAM in tumour development including angiogenesis,
metastasis and tumour growth are well demonstrated (L. Bingle 2002) and poor
prognosis is often correlated to the increased TAM density (Lewis and Pollard
25
2006). It has been suggested that tumour cells polarize infiltrating macrophages
to a tumour promoting phenotype (Hagemann, Wilson et al. 2006) and because
of the similar biological functions of TAMs and M2 macrophages, TAMs are
also described as M2 macrophages (Mantovani, Sozzani et al. 2002). Like the
phenotype of M2 macrophage, TAMs produce suppressive mediators and growth
factors that facilitate tumour development and suppress anti-tumour immunity
(Loercher, Nash et al. 1999; Sica, Saccani et al. 2000; Mantovani, Sozzani et al.
2002). Over-expression of arginase in macrophages have shown enhanced
tumour cell proliferation and suppress NO mediated tumour cytotoxicity (Chang,
Liao et al. 2001). Furthermore, arginase production by TAM impairs T cell
receptor CD3 chain expression and antigen specific T cell proliferation
(Rodriguez, Quiceno et al. 2004). TAMs are poor antigen presenting cells and
they suppress T cell responses by producing IL-10 and TGF (Loercher, Nash et
al. 1999; Sica, Saccani et al. 2000; Mantovani, Sozzani et al. 2002).
Furthermore, TAMs synthesis epidermal growth factor (EGF), vascular
endothelial growth factor (VEGF), and fibroblast growth factor (FGF) to
promote angiogenesis and tumour cell proliferation (Mantovani, Sozzani et al.
2002).
Macrophages are capable to switch their phenotype after differentiation (Stout
and Suttles 2004); in response to lipopolysaccharide (LPS) and IFNγ, M2
macrophages generated in the TH2 environment of helminth infection were
capable to switch to a more classical activated phenotype. This was illustrated
by the switch in the enzymatic pathway for arginine metabolism from arginase to
iNOS and the reduced expression of YM-1 (Mylonas, Nair et al. 2009). These
26
studies clearly illustrated the plasticity of macrophages and their ability in
phenotype switching to adapt the changing environments.
1.3 Pathogen recognition
Macrophages recognize pathogens by their cell surface receptors termed pattern
recognition receptors (PRR), these receptors recognize pathogen associated
molecular
patterns
(PAMPs)
that
are
selectively
conserved
among
microorganisms. Examples of PAMPs include LPS (Hoshino, Takeuchi et al.
1999; Qureshi, Lariviere et al. 1999), peptidoglycan (PGN) (Schwandner,
Dziarski et al. 1999) and flagellin (Hayashi, Smith et al. 2001). Macrophages are
professional phagocytes that express a wide range of PRRs to recognise, bind
and internalise invading pathogens in the innate immune response. A number of
different PRRs have been described, they can be divided into two main types;
receptors that mediate antigen uptake, including phagocytic receptors, and
receptors that lead to activation of proinflammatory signalling pathways such as
Toll like receptors (TLRs).
1.4 Non signalling pattern recognition receptors
Examples of phagocytic receptors include C-type lectins, scavenger receptors
and opsonic receptors. C-type lectin receptors recognise a broad range of
carbohydrate structures in a calcium dependent manner. Interaction of these
carbohydrate structures with different lectin receptors is dependent on
carbohydrate branching, spacing and multivalency. C-type lectins are either
produced as transmembrane proteins (Type II receptors) or secreted as soluble
27
proteins (collectins). Examples of membrane C-type lectins include selectins
(Ley and Kansas 2004), the mannose receptor (MR) family (Isacke 2002) and the
dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) (Yeunis B. H.
Geijtenbeek 1 2000).
Membrane C-type lectins are designed to capture
pathogens for intracellular destruction, degradation and antigen loading on MHC
molecules. In addition, C type lectins have been shown to act as adhesion
receptors. DC-SIGN mediates the contact between dendritic cells and T cells, by
binding to ICAM-3 (Yeunis B. H. Geijtenbeek 1 2000). Soluble collectins, like
mannose binding protein (MBP) (Bohlson, Fraser et al. 2007), lung surfactant
protein A (SP-A) (Heinrich, Hartl et al. 2006) and SP-D (Hartl and Griese 2006)
function by opsonising microorganisms.
The scavenger receptors are a family of cell surface glycoproteins that are able to
bind modified lipoproteins (mLDLs) such as oxidised and acetylated LDLs and
polyanonic molecules like lipid A (Hampton, Golenbock et al. 1991; Maxeiner,
Husemann et al. 1998). Some of these receptors are also implicated in the
phagocytosis of apoptotic cells and bacteria (Platt, Suzuki et al. 1996; Thomas,
Li et al. 2000), as well as in cell adhesion (Khoury, Hickman et al. 1996; van
Velzen, Suzuki et al. 1999). These transmembrane receptors vary markedly in
structure, for example, they can be cysteine rich, collagenous, C type lectins (S
2001). The high diversity of receptor allows them to bind a wider range of
modified host molecules and microorganisms.
In addition to PRR, opsonic
phagocyte receptors are another type of non-signalling receptor expressed on the
cell surface of macrophages, examples of these are Fc and complement receptors
(CR) that recognise antibody and complement coated pathogens or apoptotic
28
cells and contribute to their enhanced uptake and destruction (Cairns, Crockard
et al. 2003; Hart SP 2004).
1.5 Phagocytosis
Once antigen is bound, the phagocytes extend their pseudopodia around the
antigen and create a membrane-bound structure called phagosome. Cytosolic
vesicles called lysozomes containing hydrolytic enzymes and defensins then fuse
with the phagosomes to become phagolysosomes where the antigen is digested
(Janeway 2001; Goldsby R 2003).
In addition to hydrolytic enzymes,
phagocytes can also eliminate microorganisms by the respiratory burst
generating reactive oxygen intermediates (ROIs) and reactive nitrogen
intermediates (RNI) (Table 1) (Janeway 2001; Goldsby R 2003).
Table 1. ROI, RNI
Reactive oxygen
intermediates
O2- (superoxide anion)
Reactive nitrogen
intermediates
NO (nitric oxide)
OH (hydroxyl radicals)
NO2 (nitrogen dioxide)
H2O2 (hydrogen peroxide)
HNO2 (nitrous acid)
Others
NH2Cl (monocloramine)
ClO- (hypochlorite anion)
1.6 Toll like receptors signalling
Toll was originally discovered in Drosophila melanogaster and described as a
protein involved in embryonic development and only later, an additional role in
innate immunity was defined (Bruno Lemaitre 1996; Keshishian 1998).
Currently, 10 human TLRs and 12 mouse TLRs are described (TLRs 1-9 and 1113) (Takeda and Akira 2005).
TLRs are differentially distributed within cells;
29
most of them are present on the cell surface, whereas others like TLR3, TLR7,
TLR8 and TLR9 are expressed in intracellular compartments, such as endosomes
(Kaisho and Akira 2006) (Figure 1.6.1).
Figure 1.6 1 Location of TLRs
TLRs play a critical role in spotting ‘non-self’ by recognising the unique
molecular patterns associated with different classes of pathogens, and a series of
genetic studies have revealed their ligands (Table 2) (Takeda and Akira 2005).
For examples, TLR2 is required for the response to microbial lipoproteins
(Takeuchi, Kaufmann et al. 2000) and yeast cell wall (Underhill, Ozinsky et al.
1999). TLR3 binds double stranded RNA that is produced from viruses during
replication (Alexopoulou, Holt et al. 2001). TLR4 binds the exogenous ligands
LPS (Gram-negative bacteria) (Qureshi, Lariviere et al. 1999) and fusion protein
of respiratory syncytial virus (RSV) (Kurt-Jones, Popova et al. 2000) as well as
the endogenous ligands such as hsp60, hsp70, hsp96 (Vabulas, Ahmad-Nejad et
al. 2001; Vabulas, Ahmad-Nejad et al. 2002; Vabulas, Braedel et al. 2002),
fibronectin (Okamura, Watari et al. 2001), polysaccharides of hyaluronic acid
(Termeer, Benedix et al. 2002) and fibrinogen (Smiley, King et al. 2001). TLR5
binds to the flagella element flagellin (Hayashi, Smith et al. 2001). TLR7 and
TLR8 bind to ssRNA from viruses (Lund, Alexopoulou et al. 2004) as well as
30
small nuclear RNAs, TLR9 binds unmethylated CpG DNA from bacteria
(Hemmi, Takeuchi et al. 2000) and TLR11 for the response to unknown
components of uropathogenic bacteria (Zhang D 2004) and a profiling-like
molecule of the protozoan parasite Toxoplasma gondii (Yarovinsky F 2005). In
addition to function individually, TLRs can also cooperate and recognise
different components of pathogens (Ozinsky, Underhill et al. 2000; Wyllie, KissToth et al. 2000).
In the combination with TLR6, TLR2 recognises
peptidoglycan (Ozinsky, Underhill et al. 2000) and furthermore, TLR2 can also
pair with TLR1 to recognise soluble factors released by Neisseria meningitis
(Wyllie, Kiss-Toth et al. 2000).
31
Table 2. Exogenous and endogenous TLR ligands
TLR
TLR1/2
TLR2/6
Exogenous ligands
and Lipoproteins/lipopeptides
Endogenous ligands
Hsp60
Peptidoglycan
Lipteichoic acid
Lipoarabinomannan
Glycosylphosphatidylinositol
anchors
Porins
Zymonsan
Hsp70
Gp96
TLR3
TLR4
Double stranded RNA
Lipopolysaccharide
Taxol
Respiratory syncytial virus (RSV)
Envelop proteins (MMTV)
Hsp60 (Chlamydia pneumoniae
mRNA
Hsp60
Hsp70
Gp96
Fibronectin
Oligosaccharides of
hyaluronic acid
Heparan sulphate
Fibrinogen
Saturated fatty acid
TLR5
TLR7
TLR8
TLR9
Flagellin
ssRNA
ssRNA
Unmethylated CpG DNA
Small nuclear RNAs
Small nuclear RNAs
Chromatin-Ig complexes
32
TLR signalling can lead to activation of several transcription factors, including
nuclear factor kappa B (NF-κB), activating protein (AP) 1 and IFN responsive
factors (IRFs) (Kaisho and Akira 2006).
These transcription factors
subsequently activate a wide variety of genes that results in the induction of
inflammatory cytokines, chemokines and costimulatory molecules expression.
All TLRs use similar signal transduction mechanisms and TLRs interact with
different combinations of adaptor proteins to activate various signalling cascade.
To date, five adaptor molecules have been characterised (O'Neill and Bowie
2007); myeloid differentiation primary response gene 88 (MyD88), TIRassociated
protein
(TIRAP)/MyD88-adaptor-like
(MAL),
TIR-domain-
containing adaptor protein-inducing IFN-β (TRIF)/TIR-domain containing
molecule 1(TICAM1), and TRIF-related adaptor molecule (TRAM)/TIR-domain
containing molecule 2 (TICAM2).
A fifth TIR domain containing adaptor
molecule SARM (sterile α and HEAT-Armadillo motifs) was shown to be a
TRIF-specific inhibitory protein (O'Neill and Bowie 2007). MyD88 is utilised
by all TLRs except TLR3 and is unique by the presence of a death domain (DD)
at its N-terminus.
For TLR2 and TLR4 signalling, an additional adaptor
molecule - MAL is required that acts as a bridge for recruiting MyD88 to the
receptor (Kagan and Medzhitov 2006). The adaptor molecule TRIF is recruited
to TLR3 and TLR4 (MyD88 independent pathway) while TRAM binds to the
TIR domain of TLR4 and serves as a bridge between the receptor and TRIF
(Figure 1.6.1).
33
TLRs are characterised structurally by the cytoplasmic Toll/IL-1 receptor (TIR)
domain and extracellular leucine rich repeats. The presence of a TIR domain in
the cytoplasmic region of the IL-1R suggests that both receptors share a common
molecular framework for signalling (Figure 1.6.2). Upon ligand binding, TLR
and IL-1R recruit the adaptor protein MyD88 through homotypic interactions
(Wesche H 1997), MyD88 in turn recruits the interleukin receptor associated
kinase (IRAK) 1 and IRAK-2 by its death domain. After binding, IRAK-1 and
IRAK-2 become activated, phosphorylated and subsequently associate with
TNFR-associated factor 6 (TRAF6).
TRAF6 activates the kinases TGF-
activated protein kinase (TAK1) and TAK1 binding protein 1 (TAB1) (Wang,
Deng et al. 2001). TAK1 then phosphorylates the IB kinase (IKK) complex
that leads to nuclear factor kappa B (NF-кB) activation. At the same time,
TAK1 can also phosphorylates mitogen activated protein (MAP) kinase kinase
(MKK)3 and MMK6, which in turn activate c-Jun NH2 terminal kinase (JNK)
and p38 MAP kinase (Daun and Fenton 2000; Akira and Takeda 2004). In
addition to the MyD88 dependent pathway, MyD88 independent pathway has
also been described and is associated with type I IFN response to LPS (Kawai,
Takeuchi et al. 2001). Upon LPS stimulation, Toll/IL-1 receptor domain
containing adaptor inducing IFN (TRIF) activates TRAF family member
associated NF-B activator (TANK) binding kinase 1 (TBK1) via TRAF3
(Hacker, Redecke et al. 2006; Oganesyan, Saha et al. 2006). TBK1 together with
inducible IB kinase (IKK/IKKi) activate IRF3 and IRF7 (Fitzgerald,
McWhirter et al. 2003) that subsequently translocate into the nucleus, and bind to
the interferon stimulated response element (ISRE), resulting in the expression of
34
IFN that can initiate type I IFN responses (Doyle S 2002; Toshchakov, Jones et
al. 2002).
Figure 1.6 2 TNF/IL-1/TLR signalling
35
1.7 TNF signalling pathway
TNF is a pro-inflammatory cytokine that can be expressed as a 26kDa
transmembrane protein and as a soluble form by metalloprotease TNF
converting enzyme (TACE) (Black, Rauch et al. 1997; Moss, Jin et al. 1997).
TNF induces cellular responses upon binding to specific cell surface receptors;
TNF receptor type -1 (TNFR-1; p55) and TNFR-2 (p75) (Vandenabeele,
Declercq et al. 1995). These receptors share 28% homology in their extracellular
domains and are characterised by the presence of cysteine repeats (MacEwan
2002).
However, p55 and p75 are structurally different in the intracellular
region, suggesting distinct biological functions between the two receptors.
TNF is pleiotropic cytokine that regulates various aspects of immune and
inflammatory reactions by activate multiple signalling pathways including Fas
associated death domain (FADD), JNK and NF-B pathways. Engagement of
TNF to TNFR1 results in receptor trimerization and the recruitment of TNF
receptor associated death domain (TRADD). In turn, TRADD interacts with
TRAF2 and receptor interacting protein (RIP), which subsequently activates the
IKK complex and initiate the NF-B signalling event (Hailing Hsu 1996).
Alternatively, TRAF2 can also induce the activation of MAPK cascade that
results in the activation of JNK, a kinase that phosphorylates AP-1 and increases
its transcriptional activity (Chen and Goeddel 2002). These signalling pathways
are important for the gene expression involved in inflammation and cell survival.
On the other hand, TRADD can also interact with FADD. This adaptor protein
which in turn recruit caspase 8 and initiates a protease cascade that leads to
apoptosis (Hailing Hsu 1996). In contrast to TNFR1, the expression of TNFR2
is more restricted and its expression are only found on hematopoietic and
36
endothelial cells (Aggarwal 2003). Occupancy of TNFR2 results in direct
recruitment of TRAF2 (Rothe M 1994) in which subsequently activate
transcription factors AP1, p38 and NF-B (Aggarwal 2003).
1.8 NF-кB signalling pathway
NF-кB transcription factors regulate expression of a wide range of genes
involved in the control of immune responses, inflammation, cellular proliferation
and survival (Caamano and Hunter 2002). NF-кB consists of homo- and heterodimeric complexes of Rel proteins; Rel-A (p65), c-Rel, Rel-B, p105/p50 (NFкB1) and p100/p52 (NF-кB2). NF-кB1 and NF-кB2 are expressed as precursor
proteins p105 and p100 that are post-transcriptionally cleaved to generate DNAbinding subunits p50, and p52.
All NF-кB proteins carry a Rel-homology
domain (RHD) that contains a nuclear localization sequence (NLS) that is
involved in dimerisation, sequence-specific DNA binding and interaction with
the inhibitory IкB proteins (Li and Verma 2002). However, only Rel-A, c-Rel
and Rel-B posses transcription domains that regulate transcription.
Activation of the NF-кB pathway is dependent on the IKK complex that consists
of 2 catalytic subunits; IKKβ, IKKα, and a regulatory subunit IKKγ (NEMO). In
unstimulated cells, NF-κB dimers are retained in the cytoplasm as a consequence
of the interaction between the RHD and ankyrin repeats of IκB (inhibitor of κB)
α protein. Two distinct NF-κB pathways have been described; the canonical and
the alternative pathways.
Activation of canonical pathway is dependent on
IKKβ, this pathway can be triggered by PAMP and pro-inflammatory cytokines
such as TNFα and IL-1 and leads to activation of RelA/p50 dimers. Upon
37
activation, IKK phosphorylates IκBα at amino terminal serine residues Ser32 and
Ser36, the phosphorylated IκBα is then ubiquitylated at Lys21 and Lys22 which
targets it for degradation by the 26s proteasome (Karin and Ben-Neriah 2000),
the released NF-κB dimers translocate to the nucleus and activate gene
transcription (Ghosh and Karin 2002).
TNF-related cytokines lymphotoxin (LT) β (Dejardin, Droin et al. 2002), CD40
ligand (CD40L)(O'Sullivan and Thomas 2002) and receptor activator of NF-B
ligand (RANKL) (Novack, Yin et al. 2003), but not TNFα (Matsushima, Kaisho
et al. 2001) have been shown to be “inflammatory triggers” of the alternative
NF-κB signalling pathway. In contrast to the canonical pathway, the alternative
pathway is dependent on IKKα, and not IKKβ or NEMO. Stimulation of this
pathway leads to activation of NF-κB inducing kinase (NIK), this in turn
phosphorylates and activates IKKα. IKKα then selectively phosphorylates p100
resulting in the release of active p52/RelB and its translocation to the nucleus.
Accumulating evidence suggests the IKKβ-dependent canonical pathway is
important for innate immunity (Alcamo, Mizgerd et al. 2001; Senftleben, Li et al.
2001) whereas the IKKα-dependent alternative NF-κB pathway is essential in
development of lymphoid organs and adaptive immunity (Senftleben, Cao et al.
2001).
38
1.9 IFN signalling pathway
IFNs are widely expressed cytokines that play an important role in viral infection
(Stark, Kerr et al. 1998). The IFN family includes two main classes termed type
I and II interferons; type I interferons includes of the IFN-α family (IFNαI,
IFNαII, or IFNω and IFN-τ) and IFNβ.
The type II interferon (IFNγ) is
structurally unrelated to IFNα/β, and the mechanisms that regulate its production
are also different. Type I IFN is secreted by virus infected cells, whereas type II
IFN is secreted by activated T cells and NK cells.
Both type I and II IFN receptors are composed of two subunits. Type I interferon
(IFNα/β) transmits signals through its homologous receptor complex IFNAR1
and IFNAR2, whereas the type II IFN signals through a different receptor
complex IFNGR1 and IFNGR2. Each of these receptor subunits interact with a
member of the Janus activated kinase (JAK) family. For the type I IFN receptor,
IFNAR1 and IFNAR2 associate with tyrosine kinase 2 (TYK2) and JAK2
(Darnell, Kerr et al. 1994). In the case of the type II IFN receptor, IFNGR1 and
IFNGR2 associate with JAK1 and JAK2 respectively (Bach, Aguet et al. 1997).
39
Upon ligand binding to the type I IFN receptor, a signalling complex consisting
IFNAR1, IFNAR2, TYK2 and JAK1 is formed. This complex formation leads to
TYK2 and JAK1 auto-phosphorylation and activation that results in the
subsequent tyrosine phosphorylation of signal transducer and activator of
transcription (STAT) 1 and STAT2. Following downstream of the signalling
cascade, phosphorylated STAT1 and STAT2 then create a heterotrimeric
transcriptional complex, termed IFN stimulated gene factor 3 (ISGF3) together
with IRF-9 (p48/ISGF3γ) (Bluyssen, Durbin et al. 1996), which can then
translocate to the nucleus and bind IFN-stimulated response elements (ISREsAGTTTCNNTTTCNC/T) to initiate gene transcription. The transcription of
type II IFN (IFN-γ) dependent genes is regulated by gamma interferon activation
site (GAS) elements, and STAT1 is the most important IFN- γ activated
transcription factor for the regulation of these transcriptional responses. After
engagement of the type II IFN receptor by IFN-γ, JAK1 and JAK2 are activated
and regulate downstream phosphorylation of STAT1 on the tyrosine residue at
position 701 (Tyr 701). Such phosphorylation results in rapid dissociation of the
receptor:STAT complex and leads to the formation of STAT homodimers.
These dimers translocate to the nucleus and bind to gamma activated site - GAS
elements (TTNCNNNAA) to initiate transcription (Platanias 2005). Examples of
STAT1 regulated genes include iNOS, IRF1, IL-12 and MHCII indirectly
through CTIIA. IRF1 itself is also a transcription factor that recognises ISRE,
and acts like STAT1 to induce IFN gene expression (Kamijo, Harada et al. 1994;
Briken, Ruffner et al. 1995), and thus further amplify the response.
40
1.10 Resolution of Inflammation
During inflammation, leukocytes accumulate and amplify the response, however,
excessive or prolonged inflammation can be damaging to the host. In normal
circumstances, the immune system has several mechanisms to resolve the
inflammatory responses (Lawrence and Gilroy 2007).
The resolution of
inflammation requires the termination of pro-inflammatory signalling pathways
and clearance of inflammatory cells, allowing the restoration of normal tissue
function, a failure of these mechanisms may lead to chronic inflammation and
disease.
1.11 Negative regulation of signalling
The inflammatory response is a complex process involving many different
signalling pathways.
Most of our knowledge of signalling is gained from
studying members of TNFR, IL1R, and TLRs.
Although these signalling
pathways are well characterised, most of these studies are limited to a single
pathway, which is unlikely to represent the complexity of the inflammatory
responses. There are many examples of synergistic effects of mediators and
signalling pathways. The production of TNFα and IFNβ by LPS can activate
other signalling pathways in an autocrine loop through receptors on the cell
surface and amplify immune responses.
Cooperation between cytokines is
another way to enhance the immune response. IFNγ and TNFα have been shown
to synergize in the induction pro-inflammatory genes (Ohmori, Schreiber et al.
1997), such as RANTES (Lee AH 2000). This cooperation is dependent on the
presence of binding sites for STAT1 and NF-кB on the gene promoter (Ohmori
and Hamilton 1995; Ohmori, Schreiber et al. 1997). These synergistic effects
41
permit a more efficient clearance of infection.
However, excessive and
prolonged expression of pro-inflammatory mediators could be harmful to the
host. Therefore, a variety of negative regulatory mechanisms have evolved to
prevent prolonged inflammation. Inducible suppressors of cytokine signalling
(SOCS), characterised by a central Src homology domain (SH) 2 domain and a
SOCS box, function as a ubiquitin ligases to induce proteasomal degradation or a
competitor for the activation loop of JAK, are responsible for negative feedback
control of JAK-STAT signalling (Alexander and Hilton 2004). A20, a direct
target gene for NF-кB signalling can also function as a deubiquitinating enzyme
to negative regulates TLR and TNFR signalling via interacting with TRAF6
(Heyninck and Beyaert 1999; Boone, Turer et al. 2004). MyD88s, an alternative
spliced form of MyD88 that blocks recruitment of IRAK4 has also been shown
to act as a negative regulator of Toll and IL-1 signalling (Janssens, Burns et al.
2002; Janssens, Burns et al. 2003). Protein–tyrosine phosphatases (PTPs) are
another type of inhibitor, these include src-homology 2 domain phosphatase
(SHP-1), SHP-2, CD45, plasma membrane intrinsic protein (PIP) 1B, T cell
protein tyrosine phosphatase (TC-PTP) and PTP-BL). PTPs bind to the tyrosine
phosphorylated residues in the cytoplasmic domain of receptors, and catalyses
their dephosphorylation (Jiao, Berrada et al. 1996), thereby blocking downstream
signalling.
1.12 Apoptosis
Programme cell death or apoptosis, is critical in the resolution of inflammation
(Savill, Dransfield et al. 2002), this non-phlogistic mechanism of cell death is
required to clear inflammatory cells and furthermore the phagocytosis of
42
apoptotic cells is an important mechanism to switch off macrophage activation
(Fadok, Bratton et al. 1998).
During apoptosis, the plasma membrane of
apoptotic cells undergoes dramatic rearrangement and expresses specific markers
for phagocyte recognition (Savill and Fadok 2000; Savill, Dransfield et al. 2002).
One of the key markers of apoptotic cells is the exposure of phospholipids, the
intracellular portion of the membrane of viable cells.
Externalization of
membrane phospholipids triggers recognition of phosphatidyserine (PS) by
macrophages and facilitates phagocytosis (Fadok, de Cathelineau et al. 2001).
Increasing evidences suggest defective clearance of apoptotic cells could lead to
chronic inflammatory diseases.
For example, systemic lupus erythematous
(SLE) patients express autoantibodies that are able to opsonise apoptotic cells
and facilitate the binding of macrophage Fc receptors (Reefman Esther 2007).
Anti-phospholipid autoantibodies were reported as one of the autoantibodies.
They recognise and bind PS exposed on apoptotic cells and facilitate the
production of TNF-α by macrophages (Angelo A. Manfredi 1998). In addition to
the removal of inflammatory cells by phagocytosis, the phagocytic clearance of
apoptotic cells by macrophages can also promote the release of antiinflammatory cytokine TGF-1 to suppress the pro-inflammatory activity and
promote wound healing (Huynh ML 2002; Reidy and Wright 2003).
1.13 NF-ĸB and the resolution of inflammation
NF-B transcription factors regulate genes involved in many aspects the
inflammatory response (Bonizzi and Karin 2004). In response to a diverse array
of pro-inflammatory stimuli; cytokines, PAMPs and stress (oxidative stress or
UV radiation), NF-ĸB transcription factors induce the pro-inflammatory genes;
43
including cytokines, chemokines and adhesion molecules which are essential for
both the innate and adaptive immune response (Ghosh and Karin 2002). NF-ĸB
activation is also widely implicated in inflammatory diseases and much attention
has focused on the development of anti-inflammatory drugs targeting NF-ĸB
(Karin, Yamamoto et al. 2004). However, recent research has revealed another
side of NF-ĸB and a role in the resolution of inflammation (Lawrence, Gilroy et
al. 2001; Greten, Arkan et al. 2007; Fong, Bebien et al. 2008). The NF-ĸB family
composes of 5 members NFB1 (p50), NFB2 (p52), Rel-A, Rel-B and c-Rel.
Different combinations of NF-ĸB subunits generate different homodimers and
heterodimers with distinct roles in the immune response. Homodimers of the
p50 subunit of NF-ĸB, which lack transactivation domains have been shown to
repress expression of NF-ĸB target genes. A homodimeric complex of p50 was
found in resting T cells and reduced p50 expression was observed after T cell
activation, furthermore, over-expression of p50 was shown to repress IL-2
expression in T cells (Kang SM 1992). Increased p50 expression was reported to
suppress TNF production in LPS tolerance (Kastenbauer and Ziegler-Heitbrock
1999). However, Gadjeva et al. have shown that p50-/-p65+/- mice were extremely
sensitive to LPS induced shock (Gadjeva, Tomczak et al. 2004). These studies
suggest anti-inflammatory roles of p50 homodimer and p50p65 heterodimers in
septic shock. Apart from sepsis, an anti-inflammatory role of NF-ĸB was also
reported in inflammatory bowel disease; p50-/-p65+/- mice were more susceptible
to Helicobacter hepaticus induced colitis (Erdman, Fox et al. 2001).
Later
studies have shown that colitis was associated with increased IL-12p40
expression in the colon (Tomczak, Erdman et al. 2003), and a further study has
shown administration of IL-10 Ig fusion protein inhibited IL-12p40 production
44
and H. hepaticus induced colitis, which was dependent on p50/p105 expression
in macrophages (Tomczak, Erdman et al. 2006). These studies suggest NF-B
can have anti-inflammatory roles by directly inhibiting expression of proinflammatory genes and by manipulating the expression or activity of antiinflammatory cytokines such as IL-10.
1.14 NF-ĸB and apoptosis
As described above, apoptosis is an essential mechanism to prevent prolonged
inflammation; neutrophil apoptosis during acute inflammation and activation
induced cell death (AICD) of antigen-specific T cells are important mechanisms
to limit inflammatory and immune responses. Studies from Teixeiro et al. and
Kasibhatla et al. have shown that inhibition of NF-ĸB activation decreases Fas
(CD95) ligand expression on T cells that is required for AICD (Ju, Panka et al.
1995; Emma Teixeiro 1999; Kasibhatla, Genestier et al. 1999). Over-expression
of the endogenous NF-B inhibitor, IB in T cells also suggests a pro-apoptotic
role for NF-B in double positive thymocytes (Hettmann, DiDonato et al. 1999).
These studies contradict the anti-apoptotic role of NF-ĸB in inducing expression
Bcl-xL, TRAF1, TRAF2, c-IAP1 and cIAP2 (Martin SJ 1995; Wang, Mayo et al.
1998). Studies from Lin et al 1999 have shown the involvement of NF-ĸB in
both pro- and anti-apoptotic function in T cells. Inhibiting NF-ĸB reduces
phorbol myristate acetate (PMA)/ionomycin mediated induction of FasL and
apoptosis while inhibition of NF-ĸB increase the glucocorticoid mediated
apoptosis. Glucocorticoids are produced in the thymus and function to induce
thymocyte apoptosis during positive selection. However, Fas and FasL
interaction is important in AICD and peripheral T cell deletion. These data
45
suggest NF-ĸB inhibits glucocorticoid-mediated apoptosis and survival during
positive selection. On the other hand, NF-ĸB has the opposite role in mature
peripheral T cells, promoting apoptosis by increasing FasL expression (Lin B
1999). These studies suggest NF-ĸB activation can have distinct roles in different
cell lineages, physiological contexts or even throughout cell differentiation.
In 2001, Lawrence and colleagues showed the involvement of NF-ĸB in both the
onset and resolution of acute inflammation. These studies confirmed the
expected role of NF-ĸB in pro-inflammatory gene induction during the onset of
inflammation, but also demonstrated a role for NF-ĸB in expression of antiinflammatory genes and induction of leukocyte apoptosis during the resolution of
inflammation.
Inhibition of NF-ĸB during the resolution of inflammation
prolonged inflammatory response and inhibited apoptosis (Lawrence, Gilroy et
al. 2001). In 2007, Greten et al. have also shown an anti-inflammatory role for
IKK, an essential activator of NF-B, in sepsis. Conditional deletion of IKK
in myeloid cells increased sensitivity of mice to endotoxin-induced shock
associated with elevated plasma IL-1 as a result of increased pro-IL-1
processing in macrophages and neutrophils (Greten, Arkan et al. 2007).
In
addition, Greten et al. confirmed a pro-apoptotic role for NF-B in neutrophils,
which may also contribute to the anti-inflammatory role of NF-B as previously
described by Lawrence et al.
46
1.15 NF-B and macrophage activation
Although many macrophage sub-types and their specific effector roles have been
described (Gordon 2003; Gordon and Taylor 2005), the signalling pathways that
regulate their activation is limited. Recent studies published by our group have
shown the tissue specific role of IKK during infection and a role for IKK in
inhibition of M1 macrophages (Fong, Bebien et al. 2008). Using Cre-lox gene
targeting, IKK was deleted in either macrophages or lung epithelial cells, in a
model of Streptococcal pneumonia, neutrophil recruitment and bacterial
clearance was inhibited in mice lacking IKK in lung epithelial cells (IKKEpi)
but were enhanced in mice with IKK deletion in macrophages (IKKMye). In
addition, IKKMye macrophages showed increased MHC II, iNOS and IL-12
expression, which are hallmarks of M1 macrophage activation. Furthermore,
CD124 (IL-4 receptor) was absent on IKKMye macrophages suggesting these
cells have less ability to respond to IL-4 and develop a M2 phenotype. Arginase
1 (Arg-1) expression was also decreased in IKKMye macrophages, the balance
of iNOS and Arg-1 expression has also been demonstrated as a marker to
discriminate M1 macrophages from M2 macrophages, M2 macrophages upregulate Arg-1 while M1 macrophages express high levels of iNOS. Arg-1
activity contributes to the immunosuppressive activity of M2 macrophages and
suppresses iNOS expression (Kropf, Fuentes et al. 2005). Taken together, these
data suggest that IKK is involved in the suppression of the pro-inflammatory
M1 phenotype, and favours the development of anti-inflammatory M2
macrophages. M2 macrophages are also thought to be important in promoting
cancer, Hagemann et al. 2008 have shown in a parallel study that inhibiting
IKK in TAM switched the phenotype from M2 to M1, characterized by
47
increased IL-12, iNOS and MHC II, but decreased IL-10 and Arg-1.
Furthermore, adoptive transfer of IKK targeted macrophages inhibited tumour
growth in a mouse model of ovarian cancer. These studies suggest another antiinflammatory role for NF-B to promote the immunosuppressive phenotype of
macrophages that limits their tumoricidal and bactericidal function.
1.16 Macrophages bridge innate and adaptive immunity
The development of adaptive immunity is initiated by the interaction between
antigen presenting cells and naïve T cells. Optimal T cell activation requires two
signals; the engagement of T cell receptors with MHC II-peptide complex (signal
1) and signal from the accessory costimulatory molecules (signal 2). In the
absence of signal 2, T cell undergoes anergy or apoptosis (Jenkins, Chen et al.
1990). Resting macrophages have low or no costimulatory molecules and MHC
class II molecules express on their surface (Janeway 2001). The expression of
these molecules is induced by the ingestion of microorganism and recognition of
PAMPs.
Macrophages, like dendritic cells (DCs) express a wide range of
receptors including mannose receptor, scavenger receptor, complement receptor
and TLRs. Uptake of microorganisms via any of these receptors lead to the
degradation in the endosomes and lysosomes, generating peptides that can be
presented by MHC class II molecules.
At the same time, the receptors
recognising these microorganisms transmit a signal that leads to expression of
MHC II and costimulatory molecules and subsequently present the antigen to T
cells. Activated T cells differentiate into TH1 cells and migrate to infected sites
and secrete a variety of cytokines and chemokines to amplify the immune
response (Janeway 2001). IFNγ, one of the proinflammatory cytokines secreted
48
by TH1 cells, drive ‘classical’ macrophage activation in which increased levels of
phagocytosis and their ability to kill microorganisms through various cytotoxic
mediators.
1.17 The effector functions of T cells
Differentiation of naïve T cells into effector T helper cells plays a crucial part in
the development of the adaptive immunity. The most well studied T helper cells
are TH1 and TH2 cells. Different T cell lineage secretes specific cytokines that
contribute to their characteristic effector functions. IFN is the principal TH1
effector cytokine and is important in the cellular mediated immunity against
intracellular pathogens and tumours. For example, IFNγ activates macrophages
and enhance their microbicidal activity, upregulate the level of MHC II
expression and IL-12 production.
IFNγ is also important in antibody class
switching to IgG classes, which is important in facilitating opsonisation and
phagocytosis. Furthermore, IFNγ and IL-2 produced by TH1 cells can also
induce cytotoxicity indirectly by promoting CD8+ T cells differentiation into
active cytotoxic cells (Abbas, Murphy et al. 1996; O'Garra 1998). TH2 cells
secrete IL-4, IL-5 and IL-13 and are responsible for immunity against
extracellular parasites (Sher and Coffman 1992). IL-4 is the major inducer of on
antibody class switching from IgM to IgE and therefore important in inducing
IgE B cell dependent reactions mediated by mast cells (Galli 1993). IL-5 is an
eosinophil-activating cytokine (Rothenberg and Hogan 2006). Mice knockout
for IL-5 receptor or treated with neutralising antibody for IL-5 showed marked
defects in eosinophil responses to helminths (Yoshida, Ikuta et al. 1996).
49
1.18 T cell polarisation
When antigen presenting cells (APC) interact with the naïve T cells, the
surrounding cytokine microenvironment has a great impact on determining TH1
or TH2 cell development (Seder and Paul 1994). IL-12 and IFNγ are the wellknown cytokines for driving TH1 polarisation. Mice deficient in IL-12 or STAT4
have markedly reduced TH1 responses (Kaplan, Sun et al. 1996; Magram,
Connaughton et al. 1996; Thierfelder, van Deursen et al. 1996). In response to
intracellular bacteria, DCs or macrophages produce IL-12, which provide the
primary source of IL-12 for driving TH1 cell differentiation. IFNγ is another
important cytokine for TH1 development. Both activated NK cells or TH1 cells
are the producers of IFN in the immune system (Abbas, Murphy et al. 1996; He
XS 2004).
IL-4 is an important differentiation factor for TH2 cells (Swain,
Weinberg et al. 1990) and its critical role in TH2 cell polarisation is illustrated
from the impairment of TH2 cell development in IL-4 knockout mice (Kopf,
Gros et al. 1993). Other evidence comes from the reduced TH2 responses in mice
deficient for STAT6, a factor required for IL-4 receptor signalling (Kaplan,
Schindler et al. 1996; Shimoda, van Deursent et al. 1996; Takeda, Tanaka et al.
1996). The initial source of IL-4 has been controversial. TH2 cells are the key cell
population in the immune system for IL-4 production. However, IL-4 is only
produced after TH2 differentiation, so the initial source of IL-4 has always been
an open question. Basophils and mast cells have been also suggested as the
source of IL-4 production (Seder RA 1991) (Plaut, Pierce et al. 1989). However,
later study by Schmitz et al. has shown TH2 differentiation does not depend on
IL-4 produced by non-T cells (Schmitz J 1994), suggesting signals other than IL4 might contribute to TH2 differentiation. The existence of IL-4 independent
50
signals is suggested by the observation that strong TH2 responses occur in
STAT6-/- mice infected with parasites (Jankovic, Kullberg et al. 2000).
Similarly, dendritic cells incubated with parasite products, cholera toxin, or
Prostaglandin E2 (PGE2) induce IL-4 independent TH2 responses (Maria Cristina
Gagliardi 2000; de Jong, Vieira et al. 2002).
Notch pathway has increasingly been recognised as another factor that
contributes to T cell polarisation (Amsen, Blander et al. 2004). Different Notch
ligands were shown to be express on DCs under different conditions. Upon
stimulation with LPS, a typical stimulus for TH1 promotion, Delta was induced
whereas Jagged was induced when stimulated with PGE2 and cholera toxin,
conditions that have previously been shown to induce TH2 responses (Amsen,
Blander et al. 2004).
The direct influence of the Notch ligands on T cell
differentiation was further demonstrated by APC cell line engineered to express
either Delta or Jagged. APC expressing Delta strongly promoted the generation
of TH1 cells as demonstrated by the enhanced IFNγ, reduced IL-4 and IL-5
production.
Opposite results were observed with APC expressing Jagged
(Amsen, Blander et al. 2004). RBPJĸ was reported as the key mediator in Notch
signalling. These papers demonstrate that Jagged and Delta play as an instructive
signal for T cell differentiation.
Much effort has been made in identifying the transcription factors that control
the transition of naïve T cells into effector T cells.
In 2000, Szabo et al.
discovered a novel TH1 specific T box transcription factor T-bet and have shown
its role in directing TH1 lineage development (Szabo, Kim et al. 2000). For
51
example, T-bet induces IFNγ production by directly activating IFNγ reporter
activity, inducing IL-12Rβ2 expression and stabilising its own expression
(Mullen, High et al. 2001; Mullen, Hutchins et al. 2002). Furthermore, T-bet
suppresses TH2 responses by repressing IL-4 and IL-5 production (Szabo, Kim et
al. 2000). IRF1, another transcription factor that has been shown to be involved
in TH1 cell development (Lohoff, Ferrick et al. 1997; Kano, Sato et al. 2008)
(Taki, Sato et al. 1997). CD4+ T cells from IRF-/- DO11.10 TCR transgenic mice
fail to differentiate into IFNγ producing TH1 cells when stimulated with
ovalbumin (OVA) in the present of wild type (WT) APC. It was also shown that
in response to IFNγ, IRF1 directly regulates the gene expression of IL-12Rβ1,
which consequently defect IL-12-STAT4 signalling.
As TH1 cells, numerous transcriptional factors have been reported and used as
the markers of TH2 cells. GATA3 is a transcription factor that can be induced by
IL-4 (Zhang, Cohn et al. 1997; Ouyang, Ranganath et al. 1998). In addition to its
selective expression in TH2 cells, GATA3 has also been shown regulating TH2
cytokines. Zhang et al. has shown that GATA3 is important in transactivation of
the IL-5 promoter in which mutation in GATA3 site abolished IL-5 promoter
activation (Zhang, Cohn et al. 1997). Later study by Zhu et al. has further
confirmed the role of GATA3 in IL-5 expression and illustrated its regulatory
role on other TH2 cytokine, IL-13 (Zhu, Min et al. 2004). Furthermore, ectopic
expression of GATA3 in STAT6-/- T cells induces TH2 cell specific cytokines
(Ouyang, Ranganath et al. 1998; Ouyang, Lˆhning et al. 2000). GATA3 also
plays negative regulatory role in TH1 development. Ouyang et al. has shown an
IL-4 independent inhibitory role of GATA3 (Ouyang, Ranganath et al. 1998).
52
Retroviral expression of GATA3 in TH1 cells inhibited IL-12R2 expression and
IL-12 induced IFN production was also suppressed. GATA3 has also been
shown in participating in cell population expansion. Deletion of GATA3 caused
a substantial reduction in the expansion of the TH2 cell population while no
effect was observed when GATA 3 was deleted in TH1 cells (Zhu, Min et al.
2004). C-Maf is another TH2 specific transcription factor that was identified in
1996 by Ho et al. c-Maf belongs to the b-ZIP protein family and was shown to
regulate the expression of IL-4 by direct interaction with the IL-4 promoter at the
MARE element (Ho, Hodge et al. 1996). Transgenic mice over-expressing c-Maf
increased skewing towards TH2 responses as demonstrated by the increased TH2
cytokines and Ig isotype - IgG1 and IgE (Ho, Lo et al. 1998). In addition to the
factors mentioned above, many other factors have also been reported to have an
influence in the balance between TH1 and TH2 subsets. For example, antigen
dose, the form of the antigen, costimulatory molecules, the affinity of the peptide
antigen-TCR interaction (Constant and Bottomly 1997). Taken together, these
studies illustrate that polarisation of TH cells from a naïve T cell is a complex
process that is not dependent on single mechanism but the balance from many
factors occur at the moment of T cell-APC interaction.
1.19 NF-B, inflammation and carcinogenesis
The idea on linking inflammation and cancer was first proposed by Virchow in
1863 (Balkwill and Mantovani 2001).
This assumption was based on the
observations of the frequent occurrence of malignant neoplasm at the sites of
inflammation.
Since then, mounting evidences have arisen to support this
hypothesis. From epidemiological studies, approximately 20% of total cancer
53
death is related to infection and inflammatory responses (H. Kuper 2000;
Mantovani, Allavena et al. 2008) (Schottenfeld and Beebe-Dimmer 2006).
Helicobacter pylori is a microbial agent that have been reported in association
with stomach cancer. Other examples included Herpes viruses in cervical cancer
(Castellsague, Bosch et al. 2002) and Schistosomes in bladder cancer (Rosin,
Anwar et al. 1994; Mostafa, Sheweita et al. 1999). Autoimmune disease such as
ulcerative colitis has been reported as an increased risk on the development of
colorectal cancer (Kornfeld, Ekbom et al. 1997).
Tumours have also been described as ‘wounds that do not heal’ (Dvorak 1986).
Throughout the process of wound healing, leukocytes infiltrate to the injured site
and secrete a variety of cytokines, chemokines and growth factors to support the
growth of connective tissues.
This process is similar to the tumour
microenvironment that tumour cells and infiltrating leukocytes provide the
necessary factors to facilitate the proliferation and invasion of the neoplastic
cells.
NF-B signalling pathway is well known as the regulator of
proinflammatory and anti-apoptotic gene, hence it is proposed as the molecular
player in inflammatory associated tumour development. Study of inflammationassociated colon cancer with conditional knockout mice supports this hypothesis.
Deletion of IKKβ in enterocytes decreased colitis associated tumour incidence by
enhanced epithelial apoptosis. On the other hand, reduction of tumour size was
observed when IKKβ is deleted in myeloid cells and the reduction of tumour size
was suggested to be the consequence of reduced inflammatory mediators
produced by the myeloid cells (Greten, Eckmann et al. 2004). In addition to
tumour progression, IkappaB kinase has also been related to metastasis. In 2007,
54
Luo et al. has demonstrated the role of IKKα in metastasis. IKKαAA/AA TRAMP
mice developed fewer secondary site metastases compared to WT/TRAMP mice.
Subsequent analysis has shown that IKKα regulates the expression of maspin at
the transcriptional level.
These data provide a new insight for the promotion of
metastasis in which tumour infiltrating macrophages expressing RANKL activate
IKKα in prostatic epithelial tumour cells that subsequently repress maspin and
induce metastasis (Luo, Tan et al. 2007). Within solid tumours, non-malignant
cells including immune cells, fibroblasts, adipocytes, and blood vessel cells are
often found and reported to be participating in tumour development (Balkwill
and Mantovani 2001). TAM is one of the key cell populations found in solid
tumour. Much evidence has demonstrated the roles of TAM in angiogenesis,
metastasis, and tumour growth, however the molecular mechanism behind TAM
in tumour biology is still not clear. A recent study by Hagemann et al. 2008 has
reported a role of IKK in maintaining the immunosuppressive phenotype of
TAM (Hagemann, Lawrence et al. 2008). Conditional knockout of IKKβ in
TAM switches its phenotype from immunosuppressive (IL-10 high, arginase
high, IL-12 low) to cytotoxic as the classical activated macrophages (IL-12 high,
IL-10 Low, MHC II high)(Hagemann, Lawrence et al. 2008). Other member of
NF-B pathway, p50 has also been reported in maintaining the phenotype of
TAM. TAM isolated from p50-/- mice shows production of M1 cytokines, and it
was associated with reduced tumour growth (Saccani, Schioppa et al. 2006).
Taken together, these studies illustrate different roles of NF-B pathway in
tumour development. One of its ways is to prevent cell death and second, by
production of pro-inflammatory cytokines in inflammatory cells in the tumour
mass and thirdly by promoting metastasis.
55
1.20 Aim of the thesis
1. Determine the effect of IKKβ deletion on macrophage activation in vitro
2. Investigate cross talk between TNF signalling and STAT1 activation
3. Investigate the inhibitory roles of IKKβ in the adaptive immunity
4. Investigate the effect of IKKβ deletion in anti-tumour immunity
56
2. Methods and Materials
2.1 Mice
All mice were housed in negative pressure isolation at the Cancer Research UK
containment facility. p55 (TNFR1) and p75 (TNFR2) knockout mice and age
match WT on a C57BL/6 background were provided by Jackson labs. 8-10 week
old mice were used for all experiments.
2.2 Cell lines and primary cells
All glassware, used for cell culture, was baked at 220oC for 12 h to remove
contaminating endotoxin. All media was prepared at Claire Hall using endotoxin
free water and glassware.
2.2.1 Cell lines
Murine bladder (MB) 49 tumour cells (a kind gift from Dr Jian-Guo Chai,
Imperial College, London, UK) and JH293 cells (a kind gift from Iain McNeish)
were grown in RPMI supplemented with L-glutamine (2mM), penicillin
(10,000U/ml)/streptomycin (10,000µg/ml), 20mM HEPES, 10% heat-inactivated
fetal bovine serum (FBS) at 37oC, 5% carbon dioxide (CO2). The adherent cells
were passaged when reaching 80% confluent in a T175 flask.
Cells were
trypsinised and 3x106 cells were seeded in a fresh T175 flask in 25ml RPMI.
Cells were routinely checked for the presence of mycoplasma.
57
2.2.2 Macrophage Colony-Stimulating Factor derived bone marrow derived
macrophages
8-10 week old mice were killed; tibias and femurs were removed from both legs.
The remaining muscle was removed and the top and bottom of the bones were
cut with scissors. A 26g needle was attached to a 10ml syringe filled with
complete DMEM and the needle inserted into the bone marrow cavity. The bone
marrow was flushed into a 50ml falcon and collected cells were centrifuged at
453xg for 5 min at 4oC. The cells were resuspended in 1-2ml of red blood cell
lysis buffer (Sigma) and centrifuged at 453xg, 4oC, 5 min. Supernatant was
discarded, cells were resuspended in 20ml of complete medium and centrifuged
again at 453xg, 4oC, 5 min. Bone marrow cells were cultured at a density of
1x106
cells/ml
in
DMEM
supplemented
with
L-glutamine
(2mM),
penicillin/streptomycin, 20mM HEPES, 10% heat inactivated FBS and 10ng/ml
recombinant macrophage colony stimulating factor (MCSF) (Peprotech) at 37oC,
5% CO2. After 7 days, adherent cells were removed by adding cell dissociation
buffer (Invitrogen) and left at 37oC for 5-10 min. Cell viability was measured
using the ViCell XR Counter (Beckman Coulter), and was typically >90%.
58
2.2.3 Thioglycolate elicited macrophages
Thioglycolate (tg) (BD Pharmingen) induced peritoneal macrophages were
collected via peritoneal lavage on day 3 after 1.5ml injection of 3% tg. 3x106 tg
elicited peritoneal macrophage were cultured in 6 well tissue culture plates at
37oC and 5% CO2 in RPMI supplemented with 10% heat-inactivated FBS,
penicillin/streptomycin, and 2mM glutamine. For different experiments, cells
were treated as indicated in Table 3.
Table 3 Stimulus used in experiments
Treatment
Final concentration
Company
LPS (E.coli)
100ng/ml
Sigma
Recombinant murine IFNγ
100U/ml
Preprotec
Recombinant murine TNFα
20ng/ml
Preprotec
Recombinant murine IL-1
10ng/ml
Preprotec
Recombinant murine M- 10ng/ml
Preprotec
CSF
59
2.3 Delayed-type hypersensitivity
2.3.1 Immunisation
Fresh reagents were prepared for each experiment; mBSA/ complete Freund’s
adjuvant (CFA) solution was prepared by mixing solutions A and B. Solution A;
50mg of mBSA (Sigma) was dissolved in 5ml of distilled water and 45mg of
sodium chloride (Sigma) was added. Solution B; 5ml of incomplete Freund’s
adjuvant (IFA) (Sigma) was added dropwise to 20mg Mycobacterium Butyricum
(Difco) and mixed by passing in and out with 21g needle attached to a 10 ml
syringe. Solution B was placed on ice and sonicated for 1 min, then solution A
was added.
The mixture was again sonicated until an emulsion was obtained.
For primary immunisation, 0.1ml of mBSA/CFA was injected intradermally.
After 14 days, a subsequent boost with mBSA/IFA was given.
60
2.3.2 CD4+ T cell isolation
CD4+ T cells were purified by negative selection using CD4+ T cell isolation kit
(Miltenyi Biotech) following the manufacturer’s instructions. 8-10 week old
C57BL/6 mice were killed and lymph nodes (superficial cervical nodes, brachial
nodes, mesenteric nodes, inguinal nodes) collected. Lymph nodes were mashed
through a 70μm cell strainer to obtain single cell suspension.
Cells were
centrifuged at 453xg, 10 min, 4oC and supernatant was aspirated. Cell pellet was
resuspended in 40μl MACS buffer (phosphate buffered saline (PBS), 0.5%
bovine serum albumin (BSA), 2mM ethylenediaminetetraacetic acid (EDTA))
per 107 cells and incubated with 10μl of biotin-antibody cocktail per 107 cells at
4oC. After 10 min, 30μl of MACS buffer and 20μl of anti-biotin microbeads
were added per 107 cells. Cells were incubated at 4oC. After 15 min, 40ml of
MACS buffer was added and cells were centrifuged at 453xg for 10 min at 4 oC.
Cell pellets were resuspended in 500μl of MACS buffer per 108 cells. LS
columns (Miltenyi Biotech) were placed on a magnetic MACS separator and
prepared by adding 3ml of MACS buffer. For each column, 500μl of cell
suspension was applied and columns were washed three times with 3ml of
MACS buffer. Unbound CD4+ T cells were obtained from the washout and
centrifuged at 453xg, 5 min, 4oC. CD4+ T cells were resuspended in 15ml of
PBS, cell number and viability were measured using ViCell XR Counter and
viability was typically >90%.
61
2.3.3 CFSE labelling
Carboxyfluorescein succinimidyl ester (CFSE) solution was prepared in prewarmed PBS to give a final concentration of 2μM. CD4+ T cells were purified
from lymph nodes as described in section 2.3.2. After purification, T cells were
washed with 15ml of PBS by centrifugation at 453xg, 5 min, 4oC. After 2
washes, cells were stained by adding 1ml of CFSE solution per 107 cells and
placed in a 37oC water bath for 4 min. After incubation, 10ml of cold PBS was
added and cells were centrifuged at 453xg, 4oC, 5 min.
Supernatant was
aspirated and cells were resuspended in 15ml cold PBS and centrifuged at 453xg,
4oC, 5 min. The cell pellet was resuspended in pre-warmed RPMI medium
supplemented with 10% heat-inactivated FBS, penicillin/streptomycin, and 2mM
glutamine medium.
2.3.4 Allogenic mixed lymphocyte reaction
8-10 week old Balb/c mice were killed and lymph nodes (superficial cervical
nodes, brachial nodes, mesenteric nodes, inguinal nodes) collected. CD4+ T cells
were isolated and stained with CFSE as described in section 2.3.3. To determine
the optimum ratio of T cells and macrophages, 3x105 T cells were cultured with
2x104, 4x104 and 6x104 allogenic tg elicited macrophages from C57BL/6 mice.
At indicated timepoints, non-adherent CD4+ T cells were collected and washed
with 200μl FACS buffer by centrifugation at 453xg, 4oC, 5 min. After two
washes, T cells were resuspended in 150μl FACS buffer and 10μl 40g/ml 7amino-actinomycin D (AAD) (Sigma) was added. CFSE dilution was measured
by FACS as a measure of T cell proliferation.
62
2.3.5 Characterisation of OTII specific CD4+ T cells
1.6x106 macrophages were treated with 100ng/ml LPS with or without 20nM
OVA323-339. After 24 h, 8x106 CD4+ T cells isolated from OTII mice were added.
At indicated timepoints, non-adherent CD4+ T cells were collected and washed
with 200μl of PBS by centrifugation at 453xg, 4oC, 5 min. After two washes,
total RNA was isolated and analysed by quantitative real time polymerase chain
reaction (qRT-PCR). Culture supernatant was collected and IFN production
was measured by enzyme-linked immunosorbent assay (ELISA).
2.3.6 Characterisation of CD4+ T cells isolated from mBSA immunised mice
IKKF/F, IKKMye or WT mice were immunised with mBSA as described in
section 2.3.1. After 21 days, CD4+ T cells were isolated from the lymph nodes
of immunised mice and stained with CFSE (section 2.3.3). 6x104 WT BMDM or
IKKF/F, IKKMye tg elicited macrophages were treated with 100ng/ml LPS and
pre-loaded with or without 20g/ml mBSA. After 24 h, 3x105 CFSE labelled
CD4+ T cells were added. At indicate timepoint, tissue culture supernatant was
collected and IFN production was measured by ELISA.
2.3.7 mBSA recall assay
mBSA immunised mice were killed 21 days post-immunisation (Section 2.3.1).
Spleens were collected and single cell suspensions prepared by mashing the
spleens through a 70μm cell strainer. Splenocytes were centrifuged at 453xg for
10 min at 4oC and 2-3ml of red blood cell lysis buffer (Sigma) was added per
spleen. Splenocytes were incubated at room temperature for 5 min, an equal
volume (2-3ml per spleen) of complete RPMI medium was added and the cells
63
were centrifuged at 453xg, 4oC, 10 min.
Splenocytes were resuspended in
complete RPMI medium and counted with ViCell XR Counter.
3x106
splenocytes were plated in a 24 well plate in 2ml of complete RPMI medium
with or without 0.1, 10, 50, and 100μg/ml mBSA. Cells were incubated at 37oC,
5% CO2, culture supernatant was collected on day 4, IFNγ and IL-10 were
measured by ELISA.
2.4 MB49 tumour model
2.4.1 Preparation of MB49 tumour cells for injection
MB49 tumour cells at 80% confluence were used for all experiments. Cells were
trypsinised and centrifuged at 453xg for 5 min, then resuspended in RPMI
without FBS and penicillin/streptomycin. Single cell suspensions were prepared
by passing cells through a 21g needle and 0.5x106 cells in 0.1ml were injected
subcutaneously in the right flank of each mouse. Tumour size was measured
every other day with electronic callipers.
2.4.2 Tumour digestion
Tumours were collected on day 7 under sterile conditions, placed in small glass
bottles and cut into pieces with scissor. 2ml of digestion buffer (RPMI, 5% FBS,
2.5mg/ml collagenase II (Invitrogen), 2.5mg/ml collagenase IV (Invitrogen) and
0.5mg/ml deoxribonuclease (Sigma)) was added and tumours were placed at
37oC on a magnetic stirrer for 10-15 min. After digestion, tumour cells were
collected and passed through a 70μm cell strainer. Cell strainers were flushed
with complete RPMI and tumour cells collected by centrifugation at 453xg, 5
min, 4oC and then resuspended in complete RPMI. Cell number and viability
64
were measured using ViCell XR Counter and viability was typically >80%.
RNA was extracted with Tri-reagent as described in section 2.5.2 and analysed
by qRT-PCR (section 2.5.4).
2.4.3 H-Y recall assay
IKKβF/F and IKKβ∆Mye female mice were injected subcutaneously in the right
flank with 0.5x106 MB49 tumour cells. After two weeks, mice were sacrificed
and spleens were collected. Single cell suspensions were obtained by mashing
spleens through a 70μm cell strainer. Splenocytes were centrifuged at 453xg for
10 min at 4oC and 2-3ml of red blood cell lysis buffer was added per spleen and
incubated at room temperature. After 5 min, an equal volume (2-3ml per spleen)
of complete RPMI medium was added and centrifuged at 453xg, 4oC, 10 min.
Cells were resuspended in complete RPMI medium and counted with ViCell XR
Counter. 7x106 splenocytes from tumour bearing mice were co-cultured with
3x106 syngenic female or male splenocytes in triplicate wells of 24 well plates in
a final volume of 2ml complete RPMI. Cells were cultured at 37 oC, 5% CO2.
Culture supernatant was collected after 24 h and IFNγ production was measured
by ELISA.
65
2.5 RNA
2.5.1 RNA extraction with QIAamp RNA blood mini kit
All ribonucleic acid (RNA) was extracted with QIAamp RNA blood mini kit
unless stated. Procedures were followed according to the instructions of the
manufacturer. 3x106 cells were washed twice with PBS and lysed with 350μl of
buffer RLT supplemented with 10μl -mercaptoethanol per 1ml. Lysate was
pipetted repeatedly until the sample became homogenous and transferred to
QIAshredder spin column and centrifuged at 7826xg, 2 min, room temperature.
After centrifugation, 350μl of 70% ethanol was added to the lysate and mixed by
pipetting, then transferred to QIAamp spin column, followed by centrifugation at
7826xg, 15 sec, room temperature. The flow through was discarded and 350μl of
RW1 buffer was added to the column. The tube was centrifuged at 7826xg for
15 sec, 80μl of DNase reaction mix (10μl DNase, 70μl RDD buffer) was added
to the membrane of the column and left at room temperature. After 15 min,
350μl RW1 was added and centrifuged at 7826xg for 15 sec. After spinning, the
QIAamp spin column was placed into a new 2ml collection tube and 500μl of
buffer RPE buffer was added to the column, followed by centrifugation at
7826xg for 15 sec. The flow through was discarded and another 500μl of RPE
buffer was added. The tube was centrifuged at 13226xg for 3 min. The flow
through was discarded and the centrifugation was repeated to remove the
remaining buffer. After spinning, the QIAamp spin column was placed into a
fresh 1.5ml collection tube and 35μl of diethylpyrocarbonate (DEPC)-treated
water was added directly to the membrane, after 1 min, the tube was centrifuged
at 7826xg for 1 min.
66
2.5.2 RNA extraction with Tri-reagent
MB49 tumour cells were prepared as described in section 2.4.2. Tumour cells
were washed 3 times with PBS by centrifugation at 453xg, 4oC, 5 min. After the
last wash, PBS was aspirated and cell pellet was resuspended with 1ml of TriReagent (Sigma) per 1x107 cells. Cells were left at room temperature to allow
complete nucleoprotein complex dissociation. After 5 min, 200μl of chloroform
(Sigma) was added and samples were immediately shaken for 15 sec. The RNA
mixture was left at room temperature for 5 min and centrifuged at 13226xg, 15
min, room temperature. The upper aqueous phase was transferred to fresh tube
and 500μl of isopropanol (Sigma) was added, followed by vortexing for 15 sec.
The RNA mixture was incubated at room temperature for 30 min, followed by
centrifugation at 13226xg, 15 min, room temperature. The supernatant was
removed and the pellet was washed by vortexing with 0.5ml 75% ethanol
(Merck/BDH made with DEPC-treated water). The tube was centrifuged at
13226xg for 5 min at 4oC. The supernatant was removed and the pellet was airdried and resuspended in an appropriate volume of DEPC water.
2.5.3 DNase treatment of RNA
Total RNA was DNase-treated to remove contaminating genomic DNA. 1l
RNasin ribonuclease inhibitor (Promega), 10l transcription optimised 5x
buffer (Promega), 10g RNA, 2l DNaseI (Amersham), DEPC-treated water to a
final volume of 50l. The reagent mixture was incubated at 37oC for 1 h.
67
The RNA mixture was phenol: chloroform extracted as follows: 50l of DEPCtreated water was added to the RNA mixture, followed by 100l of citratebuffered phenol:chloroform (5:1 ratio Sigma). The contents were vortexed for
15 sec, then centrifuged at 13226xg for 5 min at 4oC. The upper aqueous phase
was transferred to fresh tube and 100l of chloroform: isoamyl alcohol (24:1
ratio, Merck/BDH) was added, followed by vortexing for 15 sec and
centrifugation at 13226xg for 5 min at 4oC. The upper aqueous phase was
transferred to a fresh tube. 1l glycogen (Roche), 1/5 volume (20l) of 5M
ammonium acetate (Sigma) and 2.5 volumes (250l) of absolute alcohol
(Merck/BDH) were added. The tube was placed on dry ice for 30 min and
centrifuged at 13226xg for 15 min at 4oC. The supernatant was removed and the
pellet was washed by vortexing with 0.5ml 75% ethanol (Merck/BDH made with
DEPC-treated water). The tube was centrifuged at 13226xg for 5 min at 4oC and
the supernatant was removed, the pellet was air-dried and resuspended in an
appropriate volume of DEPC water.
2.5.4 Quantitative real-time polymerase chain reaction
Total RNA was extracted with QIAamp RNA Blood Mini Kit (Qiagen) or Trireagent as described in sections 2.5.1-2.5.3 and the concentration of RNA was
quantified by Nanodrop 1000 UV-vis spectrophotometer (Thermo Scientific).
cDNA was syntheized from 0.5g of DNase treated total RNA using Moloney
murine leukaemia virus (M-MLV) reverse transcriptase kit (Promega) according
to the manufacturer’s instruction.
The following reagents were added to a
ribonuclease-free 1.5ml eppendoff tube, followed by incubation at 75oC for 5
min: 2l of random hexamer primers, 0.5g DNase treated RNA, DEPC-treated
68
water to a final volume 12l. The tube was cooled on ice for 5 min and the
following reagents (5l deoxyribonucleotide triphosphate (dNTP), 5l M-MLV
RT 5 x reaction buffer, 1l M-MLV RT enzyme), followed by incubation at
room temperature for 10 min and then 40oC for 50 min. The cDNA was made up
to the volume of 200µl with DEPC-treated water.
Ten micro-litres of cDNA was used as a template for qRT-PCR. Sequence
specific primers were used to analyse expression of specific mRNA (Table 4).
Cycle conditions were as follows: 50oC for 2 min, 10 min at 95oC, 15 sec at
95oC and 1 min at 60oC repeated 50x. Experiments were performed in triplicate
for each sample. Gene expression was normalized to the level of cyclophillin
RNA.
69
Table 4 Primer sequences for qRT-PCR
Gene
Cyclophillin For
Rev
Primer sequences
ATG GTC AAC CCC ACC GTG
TTC TTG CTG TCT TTG GAA CTT TGT C
TNF
For
Rev
ACA GAA AGC ATG ATC CGC G
GCC CCC CAT CTT TTG GG
iNOS
For
Rev
CCC TCC TGA TCT TGT GTT GGA
CCA CCC GAG CTC CTG GAA C
IFN
For
Rev
ACT AGA GGA AAA GCA AGA GGA
CTG GTA AGT CTT CGA ATG ATG
IFN
For
Rev
ACT GGC AAA AGG ATG GTG AC
TGA GCT CAT TGA GAA TGC TTG G
T-bet
For
Rev
CCT CTT CTA TCC AAC CAG TAT C
GTC CAC CAA ACT TCC ACC GA
IRF1
For
Rev
CAG AGG AAA GAG AGA AAG TCC
CAC ACG GTG ACA GTG CTG G
IL-12R1
For
Rev
GGG AGG CAA CAT GAC ATC
TTG GCA GGA TCT TCT TCT TC
IL-12R2
For
Rev
GGC ATT TAC TCT CCT GTC
GAG ATT ATC CGT AGG TAG C
IP10
For
Rev
GGA GTG AAG CCA CGC ACA C
ATG GAG AGA GGC TCT CTG CTG T
GATA
For
Rev
CGA GAT GGT ACC GGG CAC TA
GAC AGT TCG CGC AGG ATG T
IL-4
For
Rev
ACA GGA GAA GGG ACG CCAT
GAA GCC CTA CAG ACG AGC TCA
Arginase-1
For
Rev
CAG AAG AAT GGA AGA GTC AG
CAG ATA TGC AGG GAG TCA CC
CD206
For
Rev
ATG CCA AGT GGG AAA ATC TG
TGT AGC AGT GGC CTG CAT AG
70
2.5.5 Ribonuclease protection assay
0.5g of RNA was dried in MV-100 vacuum evaporator (Tomy Tech) by
centrifugation for 20 min. RNA was reconstituted with 8l of hybridization
buffer (BD Biosciences). Following reconstitution, RNA was hybridized with
RNA probes.
RNA probes were synthesized using RiboquantTM Multiprobe RPA system (BD
Biosciences) according to the manufacturer’s instructions. A reaction mix
containing 1l RNasin, 1l GACU pool, 1l DTT, 4l 5x transcription buffer,
1l multi-probe template set, 10l [-32P], 1l T7 RNA polymerase was
prepared and incubated at 37oC for 1 h. 1l of DNase was added and RNA
probe was incubated at 37oC for 15 min. In order to remove the enzymes from
the RNA probe, the reaction mix was passed through MicroSpin G-25 column
(Amersham). 79l of DEPC-treated water was added to make up the final
volume of 100l and ratio of 1:1 volume of phenol:chloroform (Sigma) was
added.
RNA probe was vortexed and centrifuged with 15,700xg at room
temperature for 5 min. After centrifugation, the aqueous phase was collected and
transferred to the column. RNA probe was centrifuged at 0.8rcf for 1 min, and
1l of purified RNA probe was diluted in 2ml of scintillant for cpm reading
(Scintillation Counter LS6500, Beckman Coulter).
71
2l of 40,000cpm probe was added to each RNA sample and mixed by pipetting.
A drop of mineral oil was added to each tube. The mixture was heated at 90oC
and temperature was allowed to decrease to 56oC gradually incubated for 12-16
h. Samples were placed at room temperature for 15 min to allow the temperature
equilibrate slowly. After hybridization, non-protected fragments of RNA were
treated with RNase, RNase cocktail for 20 samples was prepared: 2.5ml RNase
buffer and 6l RNase A/T1, 100l of RNase cocktail was added to each sample.
RNA samples were centrifuged for 10 sec and incubated at 37 oC for 30 min.
During the digestion, Proteinase K cocktail containing 390l Proteinase K
buffer, 20l Proteinase K, 30l yeast tRNA was prepared (for 20 samples) and
18l was aliquot into new eppendorf tubes. After digestion, RNase digests
underneath the oil were transferred to the tube containing the Proteinase K
solution, samples were vortexed, centrifuged and incubated for 15 min at 37oC.
100l of phenol:chloroform was added to each sample and vortexed into
emulsion.
Samples were again centrifuged at 15,700xg for 5 min at room
temperature. After centrifugation, the upper aqueous phase (approx. 100l) was
collected and transferred to a new tube, 100l of 4M ammonium acetate and
500l ice cold 100% ethanol were added, samples were mixed by inverting and
incubated at -20oC for 1 h. Samples were centrifuged at 15,700xg for 15 min at
4oC, the supernatant was carefully removed from the RNA pellet and 100l of
ice cold 90% ethanol was added. Samples were centrifuged again for 15 min at
4oC, the supernatant was removed and the pellet was air dried for 5 min. 10l of
1x loading buffer was added, and each sample was vortexed for 2-3 min,
centrifudged and heated at 90oC for 3 min, then immediately placed on ice. In
parallel, 50,000cpm unprotected labelled probe was prepared with the samples.
72
Samples were run on a 6% denaturing polyacrylamide gel at 0.2 A until the
leading edge of the bromophenol blue disappeared from the gel (approx. 2-3 h),
the gel was placed on a filter paper and dried in a gel drier (Bio-Rad Model 583)
under vacuum for 2 h at 80oC.
2.5.6 Polyacryamide gel preparation
A set of gel plates (>40cm in length) was cleaned thoroughly with water
followed by ethanol. Plates were assembled together with a 0.5mm spacer and a
gel comb. Acrylamide solution was prepared and poured into the plate cassette
and allowed to polymerize at room temperature for 1 h. After polymerization,
gel was pre-run with 0.5% tris-borate-EDTA (TBE) buffer at 0.2 A for 45 min.
Before sample loading, wells were flushed with 0.5% TBE buffer to remove the
excessive urea.
73
2.6 Genotyping for IKKβF/F and IKKβΔMye mice
2.6.1 Polymerase chain reaction
Ear biopsies were collected from IKKβF/F and IKKβΔMye mice and digested with
digestion buffer (100mM Tris, pH8, 500mM KCl, 0.1mg/ml gelatin, 0.45% NP40, 0.4% Tween 20) containing 500g/ml proteinase K (Ambion) at 55oC for 5
h. Samples were then heat-inactivated at 95oC for 10 min.
A master mix was prepared with reagents shown in Tables 5 and 6. Master mix
was added to 1l genomic DNA, and the final volume was adjusted to 25l with
DEPC-treated water. PCR was performed using a GeneAmp PCR System 9700
thermal cycler (PerkinElmer). The following cycle conditions were used for
IKKβF/F: 94oC (5 min); 35 cycles 94oC (1 min), 56oC (1 min), 72oC (1 min);
72oC (10 min). LysMCre: 94oC (3 min); 35 cycles 94oC (30 sec), 63.oC (1 min),
72oC (1 min); 72oC (2 min).
After PCR, products were analysed by electrophoresis, 3l loading buffer
(10mM TrisHCl pH 7.5, 60mM EDTA, 0.03% [w/v] bromophenol blue, 60%
[v/v] glycerol) was added to the PCR reaction sample and electrophoresed
through a 1.5% agarose gel containing 0.5g/ml ethidium bromide (Sigma).
Bands were visualised by UV transillumination and analysed with Kodak digital
science 1D software (Eastman Kodak Company).
74
Table 5 PCR primer sequences
Primers
Sequences
IKKβF/F
LysMCre
For
CCT TGT CCT ATA GAA GCA CAA C
Rev
GTC ATT TCC ACA GCC CTG TGA
For
CCC AGA AAT GCC AGA TTA CG
Rev
CCT GGG CTG CCA GAA TTT CTC
Table 6 Master mix of PCR reagents
IKKβlox-P
LysMCre
PCR reagents
Volume
10x PCR buffer
2.5l
2.4l
25mM MgCl2
2.5l
1.9l
10mM dNTPs
2.5l
0.5l
5U/l
Taq
DNA 0.25l
0.2l
polymerase
Primer forward
1l
1l
Primer reverse
1l
1l
75
2.7 Adenovirus production
2.7.1 Initial adenoviral amplification
30l of virus stock was diluted into 30ml of DMEM media containing 2% FBS
and used to infect a 90% confluent T175cm2 flask of AD-293 cells. At full
cytopathic effect (CPE), the crude lysate from the T175 cm2 flask was collected
and stored at -80oC until required.
2.7.2 Cell factory -10TM preparation and infection
Four T175 cm2 flasks of >80% confluent AD-293 cells were required to seed a
single cell factory (CF)-10TM (Fisher Scientific). Five millilitres of trysin was
added to each T175 cm2 flasks of AD-293 cells and left at 37oC for 1-3 min.
Once the cells detached, 5ml of DMEM 10% FBS was added to each flask and
cells were centrifuged for 5 min at 453xg, room temperature. Supernatant was
discarded and collected cells were resuspended in 40ml of DMEM 10% FBS.
From the 40ml cell suspension, 1ml was transferred to a fresh T175 flask
containing 30ml of DMEM 10% FBS and the remaining cells were transferred to
400ml of fresh DMEM 10% FBS. Using a funnel, the 439ml of cell suspension
was slowly added to CF-10TM via a sterile tube. Once all the cell suspension had
passed into the unit, 600ml of fresh DMEM 10% FBS was added to flush any
remaining cells from the tubing into the CF-10TM. The unit was allowed to stand
for a few minutes to ensure even distribution of the medium. A filter (VWR
international) was inserted into the CF-10TM and cells were incubated at 37oC,
5% CO2 for 48-72 h. The confluency of cells in CF-10TM was determined by the
parallel culture of the T175cm2 flask.
76
Infection was performed once the cells reached >80% confluence; crude lysate
from -80oC was defrosted at 37oC, and the freeze and thaw cycle was repeated
twice. 7ml of crude lysate was added to 400ml of infection medium (DMEM,
2% FBS). Medium from the CF-10TM was discarded and 407ml of virus
suspension was slowly transferred to CF-10TM through a sterile tube. 600ml of
fresh DMEM 2% FBS was added to flush any remaining virus from the tubing
into the CF-10TM. Once all medium has transferred, the unit was allowed to
stand for a few minutes to ensure even distribution of the medium. Cells were
left at 37oC, 5% CO2 until cells detached (Approx. 48-72 h).
Once the cells detached (approx. 48-72 h), cells were harvested and centrifuged
at 805xg for 10 min at 4oC. Supernatant was aspirated and cell pellets were
resuspended in 15ml of cold PBS. Cell suspensions were transferred to 50ml
falcon and centrifuged at 200xg for 10 min at 4oC, supernatant was aspirated and
cell pellets were resuspended in 12ml cold 10mM Tris.HCl (pH8).
Cell
suspension was then transferred to a single 50ml falcon and stored at -80oC until
ready for Cesium Chloride (CsCl) purification.
77
2.7.3 Cesium Chloride purification
Virus concentrate was defrosted at 37oC and the freeze and thaw cycle was
repeated twice. After defrosting, virus suspension was centrifuged for 10 min at
6000rpm at room temperature, and virus solution was collected.
Cesium
Chloride gradient was prepared by placing 11.4ml of a 1.25g/ml CsCl solution
into a 1x3.5in ultra centrifuge tube (Beckman Coulter) followed by underlay
7.6ml of 1.4g/ml CsCl solution. Once the CsCl gradient was generated, virus
solution was slowly loaded and this was then centrifuged at 25,000rpm for 2 h at
15oC using Beckman SW28 swing out rotor in an Optima LE-80K centrifuge.
Following ultracentrifugation, the tube was fixed in a clamp and pierced using a
19G needle fitted to a 10ml syringe. The virus bands were collected and pooled
in a 15ml tube. Once all bands were collected, the virus concentrate was layered
onto 3-4ml of 1.35g/ml CsCl solution in 0.5 x 2in centrifuge tubes (Beckman
Coulter). Tubes were then centrifuged at 40,000rpm at 15oC for 15 h using
Beckman SW50.1 swing out rotor in an Optima LE-80K ultracentrifuge. Virus
bands were collected using 19G needle as described above and transferred to a
15ml tube. The volume was made up to 12ml with TSG buffer (96mM NaCl,
0.5mM NaHPO4, 2.8mM KCl, 0.3mM MgCl2, 0.5mM CaCl2, 30% glycerol
[v/v]). Virus solution was injected into a dialysis cassette (Perbio) using the 18G
needle and the dialysis cassette was placed on a float in a 5L beaker containing
2L of dialysis solution (10mM Tris HCl (pH7.4), 1mM MgCl2, 150mM NaCl,
10% glycerol [v/v]). The beaker was placed on a magnetic stirrer in a cold room
and the virus was left to dialyze for 24 h. After dialysis, the virus solution was
collected using a syringe and the solution transferred to a 15ml tube. A small
78
aliquot was removed for particle count and the remaining virus solution was
aliquoted and stored at -80oC.
2.7.4 Determination of particle counts
Table 7 Determination of particle counts
Tube
Virus stock (l)
Dialysis buffer (l)
Lysis buffer (l)
1
100
100
200
2
200
/
200
3
/
200
200
Three tubes were prepared and labelled as shown in Table 7. All tubes were
vortexed and heated at 56oC. After 10 min of incubation, tubes were removed,
vortexed and allowed to cool at room temperature for 5-10 min.
600l of
sterile water was added to all tubes to make to the final volume of 1ml, vortexed,
and samples were read in a spectrophotometer at 260nm twice. Particle count
per ml was calculated with the equation shown below –
Particle count/ml = average reading x dilution factor x 1.12E12 (extinction
coefficient).
79
2.7.5 The tissue culture infectious dose 50 Assay
In order to titre how many viral particles (per ml of the stock) are able to infect
cells, tissue culture infectious dose (TCID) 50 Assay was performed. A single
T175cm2 flask of JH293 cells was prepared.
Once cells reached 80%
confluence, cells were trypsinized and plated at 10,000 cells/well in 200l of
DMEM 10% FBS in four 96 well plates. From the defrosted virus stock, a range
of dilutions was prepared in eppendoff tubes as shown in Table 8. From the 10-5
dilution, 22l of virus was added to cells along row A and mixed, using a multichannel pipette, 22l of diluted virus from row A was added to row B, mixed
and so on until virus dilution reached 10-11. Cells were left at 37oC, 5% CO2 for
10 days, and plates were scored under light microscope by counting wells with
full CPE.
Table 8 TCID 50 Assay
Dilutions
Volume of virus stock
TrisHCl (pH8) (l)
10-1
10l of virus stock
90
10-2
10l from 10-1
90
10-3
10l from 10-2
90
10-4
30l from 10-3
270
10-5
200l from 10-4
1800
80
Final virus dilution along each row of 96 well plates
22l of virus of 10-5
Row A final = 10-6
22l of 10-6 from A
Row B final = 10-7
22l of 10-7 from B
Row C final = 10-8
22l of 10-8 from C
Row D final = 10-9
22l of 10-9 from D
Row E final = 10-10
22l of 10-10 from E
Row F final = 10-11
2.7.6 Infecting bone marrow derived macrophages with adenovirus
Bone marrow cells were infected with adenovirus expressing IKK dominant
negative (Ad-IKKdn) or green fluorescence protein (Ad-GFP) with multiplicity
of infection (M.O.I) 200:1 on day 3 of macrophage differentiation for 24 h in the
presence of 10ng/ml recombinant M-CSF (Preprotec). After 24 h of infection,
cells were washed and fresh complete DMEM with 10ng/ml recombinant mouse
MCSF was added.
81
2.8 Immunoblotting
2.8.1 Preparation and quantification of whole cell protein lysates
3x106 macrophages were washed in 1ml ice cold PBS and lysed for 20 min on
ice in 80l of lysis buffer (50mM Hepes, pH 7.9, 250mM NaCl, 3mM EDTA,
pH8, 3mM EGTA, 1% Triton X100 and 10% Glycerol) containing proteases and
phosphatase inhibitors (Table 9).
Lysates were cleared of cell debris by
centrifugation at 4oC for 15 min at 14,263xg, lysates were collected and stored at
-80oC until further use. Protein concentration was quantified by Bradford assay
(Bio-Rad), 1µl of protein extract was diluted in 99µl of PBS, 900µl of Bradford
reagent was added and the absorbance was measured at 595nm on a
spectrophotometer. Protein concentration was determined against a standard
curve using BSA.
82
Table 9 Supplement per ml of whole cell lysis buffer
Reagent
Volume
Final
Company
Concentration
1M DTT
2μl
2μl
Sigma
100mM PMSF
10μl
1mM
Sigma
100mM NaVO4
1μl
0.1mM
Sigma
1M p-Nitrophenyl
2μl
2μl
Sigma
1M β-glycerophosphate
10μl
10μM
Sigma
Protease inhibitor cocktail -
10μl
Phosphate (PNPP)
Sigma
AEBSF (104mM)
1.04mM
Aprotinin (80μM)
0.8μM
Leupeptin (2mM)
0.02mM
Bestatin (4mM)
0.04mM
Pepstatin A (1.5mM)
15μM
E-64 (1.4mM)
14μM
83
2.8.2 Sodium dodecyl suphate-polyacrylamide gel electrophoresis
Protein samples were denatured prior electrophoresis by addition of laemmli
buffer (50 mM Tris-HCl; pH 6.8, 2% SDS, 10% Glycerol, 1% bMercaptoethanol, 12.5 mM EDTA, 0.02 % Bromophenol Blue) followed by
boiling at 95oC for 5 min. 10μg of lysate was fractionated on 10% sodium
dodecyl
suphate-polyacrylamide
gel
electrophoresis
(SDS-PAGE)
gels
(Invitrogen) and electro-transferred to polyvinylidene difluoride (PVDF)
membrane (Perkin Elmer Life Science inc) with transfer buffer (25mM Tris base,
192mM Glycine, 20% [v/v] methanol) at 0.5 A for 2 h. Membranes were
blocked in 5% [w/v] dried milk powder in Tris Buffered Saline (TBS)0.05%Tween20 for 1 h at room temperature and incubated with the primary
antibodies (Table 10) at 4oC overnight.
To visualize proteins, the membrane was incubated with appropriate secondary
HRP-conjugated antibody (Table 10) for 1 h at room temperature. Proteins were
detected using the chemiluminecence substrate solution ECL (Amersham
Biosciences) and visualized on Super RX-Fujifilm (Fujifilm).
Films were
developed by automatic film processor IGP Compact 2 Developer. In most
cases, the membranes were stripped with stripping buffer (Chemicon
International) and re-probed with different antibodies; 3 ml of 1X stripping
buffer was added on the membranes at room temperature for 15 min.
Membranes were washed with TBS-0.05% Tween20 twice for 15 min and
blocked again with 5% [w/v] milk in TBS-0.05% tween20 for 1 h at room
temperature before incubated with primary antibodies at 4oC overnight as
described above.
84
Table 10 Antibodies for Western Blotting
Antibodies
Dilution
Company
Rabbit anti-mouse IRF1 (M-20)
1:1000
Santa Cruz Biotechnology
Mouse anti-beta actin
1:1000
abcam
Mouse anti-mouse IKKβ
1:1000
Cell signaling
Rabbit anti-mouse NOS2 (M-19)
1:2000
Santa Cruz Biotechnology
Rabbit anti-mouse STAT1, CT
1:1000
Upstate
Rabbit anti- mouse phospho-STAT1
(Tyr701)
1:1000
Upstate
Anti-goat IgG F (ab)2 HRP conjugate
1:5000
Amersham Biosciences
Anti-mouse IgG F(ab)2 HRP conjugate
1:5000
Amersham Biosciences
Anti-rabbit IgG F(ab)2 HRP conjugate
1:5000
Amersham Biosciences
85
2.9 ELISA
Nunc MaxiSorp 96 well plates were coated with 100µl of capture antibody
(Table 11) diluted in PBS at 4oC overnight. Wells were washed three times with
washing buffer (PBS, 0.05% Tween-20) and blocked in 250µl dilution buffer
(PBS, 10% FBS [v/v]) for 1 h at room temperature. Following another 3 washes,
100µl of samples and serial dilution of recombinant protein (standard) were
added to the plates and incubated for 2 h at room temperature or overnight at
4oC. The plates were washed three times and incubated with 100µl of biotin
conjugated secondary antibody for 1 h at room temperature. Plates were washed
three times and 100µl of streptavidin-HRP conjugate was added to each well.
After 1 h incubation, plates were washed three times, TMB peroxidase substrate
(TMB (3,3’,5,5’-tetramethylbenzidine) (KPL Inc) was added and the reaction
was stopped with 2N H2SO4 between 5-15 min. Absorbance 490nm was read on
a spectrophotometer plate reader and analysed using Acent 2.4.2 labsystems
software program.
86
Table 11 Antibodies for ELISA
Primary antibodies
Final Concentration
Company
Goat anti-mouse TNF-α
1μg/ml
R & D systems
Rat anti-mouse IL-10
4μg/ml
BD Bioscience
Secondary antibodies
Final Concentration
Company
Biotinylated anti-mouse TNF-α
250ng/ml
R & D systems
Biotinylated rat anti-mouse IL-10
1:2000
BD Bioscience
ELISA kit
Company
Anti-mouse IL-12p40
BD Bioscience
Anti-mouse IL-12p70
BD Bioscience
Anti-mouse IL-6
BD Bioscience
Anti-mouse IFN
BD Bioscience
87
2.10 Fluorescence activated cell sorting analysis
2.10.1 Cell surface staining
2.5x105 cells were washed twice with 2ml ice-cold (fluorescence activated cell
sorting) FACS buffer (PBS, 1% BSA [w/v], 0.01% sodium azide [v/v]) and
centrifuged at 453xg for 5 min at 4oC. Fc receptors were blocked with antiCD16.2/32.2 for 10 min on ice. Cells were incubated with 0.1-0.5μg/ml of
primary antibody as listed in Table 12 for 30 min on ice. The cells were washed
three times with FACS buffer and fixed by 0.1% formaldehyde. Cells were
analysed by flow cytometry using FACScalibur and CellQuest software (Becton
Dickinson).
2.10.2 Intracellular staining
1x106 cells were washed twice with 2ml ice-cold FACS buffer and centrifuged at
453xg for 5 min at 4oC. Cells were fixed by incubation in 100l of fixing buffer
(6% formaldehyde, PBS) for 15 min at room temperature, followed by 3 washes
with FACS buffer.
Non-specific binding was blocked by adding 100l of
blocking buffer (0.5% saponin [w/v], 10% FBS [v/v], PBS) and cells were left at
room temperature.
After 20 min, cells were washed 3 times with
permeabilisation buffer (0.5% saponin [w/v], PBS). After washing, 1.25μg/ml of
rat anti-mouse IFN-PE and hamster anti-mouse CD3-FITC (1:100) diluted in
permeabilisation buffer were added and cells were incubated at room
temperature for 20 min. The cells were washed three times with permeabilise
buffer and resuspended in FACS buffer after the final wash, cells were analysed
by flow cytometry using FACScalibur and CellQuest software (Becton
Dickinson).
88
Table 12 Antibodies used for FACS analysis
Antibodies
Dilution
Company
Rat anti-mouse CD16.2/32.2
1:100
eBioscience
Rat anti-mouse CD11b : APC
1:200
eBioscience
Rat anti mouse CD11b - PE
1:300
eBioscience
Rat anti mouse MHC II -PE
1:300
eBioscience
Rat anti-mouse MHC - FITC
1:100
eBioscience
Rat anti-mouse CD86 - PE
1:100
eBioscience
Rat anti-mouse CD86 - APC
1:100
eBioscience
Rat anti-mouse CD80 - PE
1:100
eBioscience
Rat anti-mouse IL-4R - PE
1:100
eBioscience
Rat anti-mouse B220 - FITC
1:100
eBioscience
Rat anti-mouse CD4 - FITC
1:100
eBioscience
Rat anti-mouse CD8 - PE
1:100
eBioscience
Hamster anti-mouse CD11c - 1:100
BD Pharmingen
PE
Rat anti-mouse IFN - PE
1:150
Hamster anti-mouse CD3 - 1:100
BD Pharmingen
eBioscience
FITC
Rat IgG1 - PE
1:100
BD Pharmingen
Rat IgG2a - FITC
1:100
eBioscience
Rat IgG2b - FITC
1:100
eBioscience
Rat-IgG2a - PE
1:100
eBioscience
Rat IgG2b - PE
1:100
eBioscience
Rat IgG2a - APC
1:100
eBioscience
Rat IgG2b - APC
1:100
eBioscience
Hamster IgG - FITC
1:100
eBioscience
Hamster IgG - PE
1:100
eBioscience
89
2.11 Statistical analysis
Standard error mean (SEM), student’s T test, one way ANOVA, two way
ANOVA and post-hoc Bonferroni tests, Log-rank test were calculated using
GraphPad Prism version 4 (GraphPad Software. Inc)
90
2.12 Full company name and location
Company name
Location
Abcam
Cambridge, UK
Amersham International plc.
Buckinghamshire, UK
Applied Biosystems
Cheshire, UK
BD Pharmingen
Oxford, UK
Beckman Coulter
High Wycombe, UK
BioRad Laboratories Ltd
Hertfordshire, UK
Cell Signalling Technology
Hertfordshire, UK
Fisher Scientific
Leicestershire, UK
Fujifilm
Bedfordshire, UK
Invitrogen
Paisley, UK
KPL Inc.
Maryland, USA
Merck/BDH
West Sussex, UK
Millipore
Watford, UK
Miltenyl Biotec
Bergisch Gladbach, Germany
PeproTech Inc.
Rocky Hill, UK
PerkinElmer
Beaconsfield, UK
Promega
Hampshire, UK
Qiagen
West Sussex, UK
Roche
Hertfordshire, UK
Santa Cruz Biotechnology
Heidelberg, Germany
Sigma-Aldrich
Dorset, UK
Thermo Scientific
Wilmington, USA
Tomy Tech
California, USA
VWR International
West Sussex, UK
91
3. Results
3.1 Negative regulatory role of IKK in macrophage activation
It has long been known that NF-B transcription factors are important in the
onset of inflammation. However, recent research also suggests a role for NF-B
in the resolution of inflammation. Earlier studies by our group have revealed a
tissue specific role for IKK in inflammation in vivo (Fong, Bebien et al. 2008).
Deletion of IKK in airway epithelial cells reduce neutrophil recruitment during
lung infection, however, inflammation was increased when IKKβ was deleted in
myeloid cells. These data illustrate a tissue specific role for IKK in
inflammation and suggest IKKβ activity in macrophages inhibits the
inflammatory response during infection. In this chapter, I extended these studies
and investigated the possible mechanisms of the inhibitory effect of IKKβ in
macrophage activation.
92
3.1.1 Genotyping of IKKF/F and IKKMye mice
IKK knock out (Ikbkb-/-) mice die in utero due to TNF induced apoptosis of
liver progenitors (Li, Antwerp et al. 1999; Tanaka, Fuentes et al. 1999). To
characterise the possible roles of IKKβ in inflammatory resolution, a Cre-lox
recombinase system (Sauer 1998; Clausen, Burkhardt et al. 1999) was used to
generate tissue specific IKK knockout mice. To specifically knock out IKK in
macrophages, mice expressing a floxed Ikbkb allele (IkkF/F) were generated and
bred with LysMCre mice that utilise the lysozome M promoter to express Cre
recombinase in myeloid cells (Figure 3.1.1.1). Tissue specific targeting of IKKβ
in these mice has been previously established (Greten, Eckmann et al. 2004;
Broide, Lawrence et al. 2005; Greten, Arkan et al. 2007). Ear biopsies were
collected from mice and genomic DNA was prepared and analysed for the
IkkF/F allele and Cre transgene expression by PCR (Figure 3.1.1.2).
93
loxP
loxP
loxP
Figure 3.1.1 1 Selective deletion of IKK in macrophages using Cre-lox
recombinase
(1-3) A targeting vector with three loxP sites and a positive selective marker, neomycin
(neo) was used to insert loxP sites into the flanking region of exon 3 (IKK) through
homologous recombination.
(4) Cre recombinase was transiently expressed in
embryonic stem (ES) cells resulting in the combination of loxP sites, two loxP sites
remain flanking exon 3 in the target gene (IKKloxP). (5) IKKloxP mice were bred
with mice expressing Cre recombinase in the lysosome M promoter to generate myeloid
cell specific IKK knockout mice.
94
B
A
Ikbkb
Cre
bp
bp
700
310
220
Figure 3.1.1 2 PCR analysis of Ikbkb and Cre transgenic mice
Genomic DNA was prepared from ear biopsies and analysed for the floxed Ikk allele
and Cre transgene expression by PCR. (A) The 310bp band represents the Ikk allele
with the presence of the loxP sites. The 220bp band represents the Ikk allele without
the loxP sites. (B) The 700bp band represents the presence of Cre transgenic gene.
The level of IKK deletion in the IKKF/F and IKK∆Mye macrophages was
determined by western blot. An example of the IKKβ level in IKKβF/F and
IKKβ∆Mye thioglycolate elicited peritoneal macrophages is shown in Figure
3.1.1.3.
IKKβF/F IKKβ∆Mye
Mice
1
2
1
2
IKKβ
87kDa
α-Actin
43kDa
Figure 3.1.1 3 Level of IKK expression in IKKβF/F and IKKβ∆Mye peritoneal
macrophages
1.5ml of 3% tg was injected into peritoneum of two IKKβF/F and IKKβ∆Mye mice labelled
as 1 and 2. Thioglycolate elicited peritoneal macrophages were harvested 3 days after
injection. Whole cell lysates were prepared and IKK expression was analysed by
western blot. Alpha actin was used as a loading control.
95
3.1.2 IKK inhibits STAT1 activation
Previous studies from our group have shown that knocking out IKKβ in
macrophages increased resistance to Group B Streptococcus (GBS) infection and
this was associated with increased iNOS expression in alveolar macrophages
(Fong, Bebien et al. 2008). STAT1 activation is important for innate immunity
and anti-microbial activity of macrophages, which are severely impaired in
STAT1 knockout mice (Matsukawa 2007). Multiple transcription factor binding
sites including; GAS, ISRE and B were identified in the promoter of iNOS
(Gao, Morrison et al. 1997). STAT1 is the transcription factor that binds GAS
and increases iNOS gene expression (Gao, Filla et al. 1998; Ganster, Taylor et al.
2001), I first investigated the inhibitory roles of IKKβ on STAT1 signalling.
IKKβF/F and IKKβΔMye thioglycolate elicited peritoneal macrophages were
stimulated with LPS, the cell wall component of gram-negative bacteria, that
activates NF-B signalling via TLR4 (Takeda and Akira 2004), or in
combination of recombinant IFN in vitro, STAT1 phosphorylation was analysed
by immunoblotting. As shown in Figure 3.1.2.1A, the expression of total STAT1
and STAT1 phosphorylation were increased in IKKβΔMye macrophages when
compared to IKKβF/F macrophages. STAT1 phosphorylation was induced after 8
hours of LPS stimulation in IKKβF/F macrophages. In contrast, the basal level of
STAT1 phosphorylation was high even in unstimulated IKKβΔMye macrophages
and phosphorylation was increased further after 2 h of LPS treatment. iNOS
expression was markedly increased in LPS stimulated IKKβΔMye macrophages at
8 h.
In response to the combination of LPS and IFN treatment, STAT1
phosphorylation was detected at 30 min and remained present at 2 h in IKKβF/F
96
macrophages (Figure 3.1.2.1B).
In comparison, higher levels of STAT1
phosphorylation were detected from IKKβΔMye macrophages at both 30 min and
2 h. Furthermore, iNOS expression was detected only after 8 h of LPS/IFN
stimulation in IKKβF/F macrophages but iNOS expression was detected at 4 h and
further increased at 8 h in IKKβΔMye macrophages. These data suggest negative
cross talk between IKK and STAT1 signalling pathways.
97
A
B
Figure 3.1.2 1 IKK inhibits STAT1 activation and iNOS expression
3x106 tg elicited macrophages/well were cultured in 6 well plates and stimulated with
(A) 100ng/ml LPS or (B) in combination with 100U/ml IFN in vitro. Whole cell lysates
were collected at the indicated time points and the expression of total STAT1, pSTAT1
and iNOS were analysed by immunoblotting. Alpha-actin was used as a loading control.
Experiments were repeated at least 3 times and representative data are shown.
98
3.1.3 IKKβ suppresses expression of iNOS and IRF1 mRNA
Total RNA from LPS stimulated tg elicited peritoneal macrophages was isolated
at indicated timepoints and analysed by qRT-PCR. TNF is a typical NF-κB
dependent gene that does not contain STAT1 binding sites, I first tested if
knocking out IKK decreased the mRNA levels of TNF. After 4 h of LPS
stimulation, the mRNA level of TNF was 40% lower in IKKβΔMye macrophages
as compared to IKKF/F macrophages (Figure 3.1.3.1A). IFN, another NF-κB
regulated gene (Honda, Yanai et al. 2005) was also analysed.
IFN mRNA
level was reduced by 50% from IKKΔMye macrophages after 4 h of LPS
stimulation (Figure 3.1.3.1B). However, the mRNA level of iNOS was 2 fold
higher in IKKβΔMye macrophages when compared to IKKF/F macrophages after
8 h of LPS stimulation (Figure 3.1.3.1C) in keeping with iNOS protein
expression (Figure 3.1.2.1). Another STAT1 dependent gene, IRF1 (Durbin,
Hackenmiller et al. 1996; Meraz MA 1996) was increased by 3 fold at 8 h in LPS
stimulated IKKβΔMye macrophages (Figure 3.1.3.1D).
99
A
TNF
C
B
IFN
D
iNOS
IRF1
Figure 3.1.3 1 IKK inhibits STAT-dependent gene expression
3x106 tg elicited macrophages/well were plated in 6 well plates and stimulated with
100ng/ml LPS in vitro. Total RNA was isolated at the indicated time points for qRTPCR analysis of TNF, IFN, iNOS and IRF1 mRNA. Experiments were repeated 3
times and representative data are shown.
100
Increased iNOS expression is characteristic of M1 macrophage activation, MHC
II is also a marker of M1 macrophage (Gordon and Taylor 2005). IFN mediated
JAK/STAT signalling has been shown to regulate MHC II expression CIITA
(Chang CH 1994; Steimle, Siegrist et al. 1994). To test if deleting IKK had a
negative influence on MHC II expression, tg elicited macrophages from IKKF/F
and IKKβΔMye mice were stimulated with IFNγ in vitro for 24 h and the
expression levels of MHC II were measured by FACS. Figure 3.1.3.2 shows that
IKKβΔMye macrophages have 2.5 fold higher MHC II expression (mean
fluorescence intensity (MFI); IKKβΔMye 1829 vs IKKF/F 720) when compared to
IKKβF/F macrophages upon IFNγ stimulation.
100
MHC II
% of Max
80
MFI-720
MFI-1829
60
IKKF/F
IKKMye
40
20
0
100
101
102
FL1-H
103
104
Figure 3.1.3 2 IKKβ inhibits MHC II expression
3x106 tg elicited macrophages/well were plated in a 6 well plate and stimulated with
100U/ml IFN for 24 h. MHC II level was measured by FACS and the expression of
MHC II was gated on CD11b positive cells. Black – isotype control. Red - IKK F/F.
Blue -IKK Mye Experiment was repeated at least 3 times and representative data are
shown.
101
3.1.4 IKK inhibits IL-12 production
IL-12 is a typical proinflammatory cytokine produced by M1 macrophages and
its expression is regulated by IFN/STAT1 signalling (Scharton-Kersten, Wynn
et al. 1996; Gautier, Humbert et al. 2005), I therefore tested if the inhibitory
effect of IKK also applied to IL-12 production. After 8 h of LPS and IFN
stimulation, IL-12p40 production was 2 fold higher from IKKMye macrophages
(Figure 3.1.4.1A). IL-12p70, the active form of IL-12 (Watford, Moriguchi et al.
2003) was also measured. Consisting to IL-12p40, 7.5 fold increase in IL-12p70
levels detected in macrophages isolated from IKKMye macrophages when
compared to IKKF/F macrophages after 8 h of LPS and IFN stimulation (Figure
3.1.4.1B).
A
B
***
***
Figure 3.1.4 1 IKK inhibits IL-12 production
0.5x106 tg elicited macrophages/well were plated in a 24 well plate and stimulated with
100ng/ml LPS and 100U/ml IFN.
Culture supernatant was collected at indicated
timepoints and cytokine production was analysed by ELISA. Data represents mean+sem
of triplicates. Two way ANOVA and post-hoc Bonferroni tests were used for statistical
analysis; *** P<0.001
102
3.1.5 TNFα inhibits STAT1 activation in macrophages in vivo
It is known that IKKβ is required for TNFα expression (Figure 3.1.3.1A) and
TNFα signalling (Collart, Baeuerle et al. 1990; Beg, Finco et al. 1993; Caamano
and Hunter 2002), I therefore explored the hypothesis that TNFα modulates cross
talk with IFN signalling through IKKβ activation. To test the role of TNFα in
vivo, anti-TNFα neutralising antibody or rabbit IgG was injected together with
Tg into C57BL/6 mice. Anti-TNFα treatment resulted in increased total STAT1,
pSTAT1 and iNOS expression upon stimulation of tg elicited macrophages with
LPS in vitro (Figure 3.1.5.1). STAT1 phosphorylation was induced from 2 h and
increased further at 8 h in macrophages of IgG treated mice. As observed in tg
elicited macrophages of IKKMye mice, the basal level of pSTAT was higher in
peritoneal macrophages from anti-TNFα treated mice and the level of pSTAT
was further increased upon LPS stimulation. Similar to data from IKKMye
macrophages, the level of iNOS expression was detected from 4 h and further
increased at 8 h in macrophages from mice treated with anti-TNF neutralizing
antibody while iNOS expression was only detected at 8 h from macrophages of
control mice.
103
Figure 3.1.5 1 Blocking TNF in vivo increased STAT1 phosphorylation and iNOS
expression
1.5ml of 3% tg was injected i.p with 750g of neutralizing anti-TNF antibody or rabbit
IgG into C57BL/6 mice and peritoneal macrophages were harvested after 3 day. 3x106
Tg elicited macrophages/well were plated in 6 well plates and stimulated with 100ng/ml
LPS. At indicated time points, protein lysates were prepared and total STAT1, pSTAT1
and iNOS expression were analysed by immunoblotting. Alpha-actin was used as
loading control.
104
I next tested if the inhibitory effect of TNF also applied to IL-12 production.
After 8 h of LPS stimulation, IL-12p40 production was 2.5 fold higher from
macrophages of anti-TNFα treated C57BL/6 mice (Figure 3.1.5.2A). Correlating
to IL-12p40, IL-12p70, the active form of IL-12 (Watford, Moriguchi et al.
2003) was 3 fold higher after 8 h LPS stimulation from macrophages isolated
from anti-TNFα treated C57BL/6 mice (Figure 3.1.5.2B). To test if the inhibitory
effect of TNFα was selective to IL-12, I measured IL-6, a proinflammatory
cytokine that is regulated independently of STAT1 but induced by TNF
(Kishimoto, Akira et al. 1995; Vanden Berghe W 2000; Yu, Kennedy et al.
2003). Figure 3.1.5.2C shows a more than 80% reduction in the levels of IL-6
from the macrophages of anti-TNFα treated mice compared to non-treated
macrophages. This data shows the inhibitory effect of TNFα is selective to IL12.
105
A
B
***
**
C
***
C
Figure 3.1.5 2 TNF selectively inhibits IL-12 production
C57BL/6 mice were injected with 1.5ml 3% tg i.p with 750g of neutralizing anti-TNF
antibodies or rabbit IgG. Peritoneal macrophages were harvested after 3 days. 0.5x10 6
tg elicited macrophages/well were plated in a 24 well plate and stimulated with
100ng/ml LPS. Culture supernatant was collected at indicated timepoints and cytokine
production was analysed by ELISA. Data represents mean+sem of triplicates. Two way
ANOVA and post-hoc Bonferroni tests were used for statistical analysis; ** P<0.01, ***
P<0.001
106
3.1.6 TNF inhibits STAT1 activation in macrophages in vitro
The experiments described in sections 3.1.5.1 and 3.1.5.2 showed that
neutralizing TNF in vivo increased STAT1 activation, iNOS and IL-12
production in LPS stimulated macrophages. To further confirm the inhibitory
effect of TNFα, I pre-treated BMDM with recombinant TNFα prior to
stimulation with LPS alone or in combination with IFNγ. Upon LPS stimulation,
STAT1 phosphorylation was detected at 4-8 h from both BMDM with and
without TNF treatment, but the level of pSTAT1 was reduced in TNF pretreated BMDM (Figure 3.1.6.1A).
For BMDM stimulated with LPS and IFN,
pSTAT1 expression was detected from 30 min and the expression level was
reduced at 4 h in TNF pre-treated BMDM. However, the reduction was modest
at 30 min and 8 h when compared to untreated BMDM (Figure 3.1.6.1B). To
determine if these inhibitory effects were selective to TNF, BMDM were pretreated with IL-1β, a cytokine that can also activate the IKKβ dependent
canonical NF-κB pathway. Figures 3.1.6.1A & B shows that pre-treatment of
BMDM with IL-1β has no inhibitory effect on pSTAT1 upon LPS or LPS/IFN
stimulation in fact IL-1β treatment increased pSTAT1 activation.
Taken
together, these results illustrate that TNFα specifically inhibits STAT1 activation
in macrophages.
107
A
B
Figure 3.1.6 1 Inhibition of STAT1 phosphorylation in BMDM pre-treated with
TNF
Bone marrow cells were cultured with 10ng/ml MCSF, on day 4, 20ng/ml TNF or
10ng/ml IL-1 was added to the culture as indicated. On day 7, 3x106 BMDM/well
were plated in 6 well plates and stimulated with 100ng/ml LPS alone or in combination
with 100U/ml IFN. Protein lysates were collected at indicated time points and the
expression of pSTAT1 was analysed by immunoblotting. Alpha-actin was used as
loading control. Experiments were repeated 3 times and representative data are shown.
108
In parallel, tissue culture supernatant was collected for cytokine analysis. Figure
3.1.6.2A shows TNF pre-treated BMDM produced 73% less IL-12p40 after 4 h
LPS/IFN stimulation while 29% reduction was obtained at 8 h compared to the
untreated group.
Consistent with the IL-12p40 production, pre-treatment of
BMDM with TNFα dramatically reduced IL-12p70 production by more than
90% (Figure 3.1.6.2B) at 8 h. The inhibitory effect was not seen with IL-1β
treatment.
A
***
B
***
*
***
Figure 3.1.6 2 Reduction of IL-12 production by TNF pre-treated MCSF-BMDM
Bone marrow cells were cultured with 10ng/ml MCSF, with or without TNF or IL-1
as described.
On day 7, 0.5x106 BMDM/well were plated in 24 well plates and
stimulated with 100ng/ml LPS and 100U/ml IFN. Tissue culture supernatant was
collected at 4 and 8 h. The levels of IL-12p40 and IL-12p70 were measured by ELISA.
Data represents mean+sem of triplicates. Experiments were repeated 3 times and
representative data are shown. Two way ANOVA and post-hoc Bonferroni tests were
used for statistic analysis; *P<0.05, ** P<0.01, *** P<0.001
109
To test if pre-treatment of TNF has an inhibitory effect at the transcriptional
level of IL-12, ribonuclease protection assay (RPA) was performed to measure
mRNA levels.
In BMDM stimulated with LPS/IFN, IL-12p40 mRNA
expression was detected from 4 h and increased further at 8 h, comparable levels
of IL-12p40 were obtained from BMDM pre-treated with IL-1 (Figure 3.1.6.3A
& B). In response to TNF pre-treatment, there was an 85% reduction of IL12p40 mRNA expression after 4 h of LPS/IFN stimulation. IP10 (CXCL10),
another IFNγ regulated gene, was reduced by 80% in BMDM pre-treated with
TNF (Fig 3.1.6.3A & C).
Again, the inhibitory effect was not seen with
BMDM pre-treated with IL-1.
110
A
B
C
Figure 3.1.6 3 Reduced mRNA level of IL-12p40 and IP10 by TNF pre-treated
MCSF-BMDM
Bone marrow cells were cultured with 10ng/ml MCSF, with or without TNF or IL-1
as described. On day 7, 3x106 BMDM/well were plated in a 6 well plate and stimulated
with 100ng/ml LPS and 100U/ml IFN. Total RNA was isolated at the time points
indicated and analysed by ribonuclease protection assay using Pharmingen’s multiprobe
template. Protected RNA fragments were resolved on a 6% polyacrylamide gel (A).
The gel was dried and exposed to a PhosphoImager screen for quantification indicated
as specific activity (B & C).
111
3.1.7 TNF inhibits STAT1 through p55
To study which TNF receptor was responsible for the inhibitory effect of TNF
in macrophages, TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2;
p75) deficient mice were injected with 1.5ml of 3% tg as described and
peritoneal macrophages were stimulated with LPS and IFNγ in vitro. In control
mice, STAT1 phosphorylation was detected at 30 min and was almost
undetectable from 4-8 h (Figure 3.1.7.1). In comparison, pSTAT1 expression in
p55-/- macrophages was greatly increased at all time points tested, although the
basal level of pSTAT1 was also increased in p55-/- macrophages.
In p75-/-
macrophages, pSTAT1 levels were not increased compared to WT BMDM.
Immunoblotting also showed iNOS expression was significantly higher in p55-/macrophages at 4 h compared to both WT and p75-/- macrophages.
112
Figure 3.1.7 1 The inhibitory effect of TNF on STAT1 was mediated by p55
WT, p55-/- and p75-/- mice were injected with 1.5ml 3% tg i.p and peritoneal
macrophages were harvested after 3 days. 3x106 tg elicited macrophages/well were
plated in 6 well plates. Macrophages were stimulated with 100ng/ml LPS and 100U/ml
IFN. At indicated time points, protein lysates were prepared and the level of pSTAT1
and the iNOS expression were analysed by immunoblotting. Alpha-actin was used as
loading control. Experiments were repeated 3 times and representative data are shown.
113
To test if the inhibitory effect of TNF on IL-12 production is also mediated by
p55, tissue culture supernatant was collected and the levels of IL-12 were
measured by ELISA. As shown in Figures 3.1.7.2A and B, p55-/- macrophages
showed 50% increase in IL-12p40 and 40% increase in IL-12p70 production
after 24 h of LPS/IFN stimulation when compared to WT macrophages. To test
if the inhibitory effect was specific for IL-12, IL-6 was measured in parallel, and
no significant difference was detected between WT and p55-/- macrophages
(Figure 3.1.7.2C).
114
A
*
*
B
*
C
Figure 3.1.7 2 Increased IL-12 production from p55-/- peritoneal macrophages
0.5x106 tg elicited macrophages/well were plated in 24 well plates and stimulated with
100ng/ml LPS and 100U/ml IFN. At indicated time points, culture supernatant was
collected and analysed for IL-12p70, IL-12p40 and IL-6 production by ELISA. Data
represents mean+sem of triplicates. Experiments were repeated 3 times and
representative data are shown. Two way ANOVA and post-hoc Bonferroni tests were
used for statistical analysis; *P<0.05, ** P<0.01
115
3.1.8 Inhibition of IKK activity in vitro with adenovirus expressing IKK
dominant negative inhibitor
To confirm the inhibitory role of IKK in IFN signalling seen in IKKMye
macrophages, BMDM were infected with recombinant adenovirus expressing
IKKβ dominant negative inhibitor (Ad-IKKβdn) or control adenovirus expressing
green fluorescent protein (Ad-GFP). Infection efficiency was determined by the
percentage of the cells with GFP expression. An example of BMDM infected
with Ad-GFP is shown in Figure 3.1.8.1A & B, the efficiency of virus infection
was approximately 60%.
A
B
Figure 3.1.8 1 Efficiency of adenovirus infection
Bone marrow cells were cultured with 10ng/ml MCSF and infected with Ad-GFP with
M.O.I of 200:1 on day 3. Virus was removed after 24 h and fresh complete
DMEM+MCSF was replaced. Infection efficiency was determined on day 7 by the
percentages of GFP positive cells. A) Phase contrast image B) GFP fluorescence image.
116
3.1.9 Ad-IKKdn infected BMDM increased STAT1 phosphorylation and iNOS
expression
To test if BMDM infected with Ad-IKKβdn also increase STAT1 activation as
IKKMye macrophages, BMDM infected with Ad-GFP or Ad-IKKβdn were
stimulated with LPS and STAT1 phosphorylation was measured by
immunoblotting. Consistent to the results from IKKMye macrophages, STAT1
phosphorylation and iNOS expression were both increased from BMDM infected
with Ad-IKKβdn (Figure 3.1.9.1). Upon LPS stimulation, pSTAT1 was detected
from 8 h in Ad-GFP infected BMDM while it was detected at 4 h from AdIKKβdn infected BMDM and the expression level remained high up to 24 h
compared to Ad-GFP infected cells. Increased STAT1 activation correlated with
elevated expression of iNOS from 4 to 24 h.
117
Figure 3.1.9 1 Ad-IKK dn infected BMDM increased STAT1 phosphorylation
Bone marrow cells were cultured with 10ng/ml MCSF and infected as described in
Figure 3.1.8.1.
On Day 7, 3x106 BMDM/well were plated in 6 well plates and
stimulated with 100ng/ml LPS. Whole cell lysates were collected at indicate time
points. Expression of iNOS and STAT1 phosphorylation were analysed by immunoblot.
Alpha-actin was used as loading control. Experiments were repeated at least 3 times and
representative data are shown.
118
I next measured IL-12p40 production from BMDM infected with Ad-GFP or AdIKKβdn after stimulation with LPS and IFN by ELISA. As shown in Figure
3.1.9.2, IL-12p40 production was 7.5 fold higher from macrophages infected
with Ad-IKKβdn compared to macrophages infected with control virus.
***
Figure 3.1.9 2 IKK dn infected BMDM increased IL-12p40 production
Bone marrow cells were cultured with 10ng/ml MCSF and infected as described. On
Day 7, 0.5x106 BMDM/well were plated in 24 well plates and stimulated with 100ng/ml
LPS and 100U/ml IFN. Culture supernatant was collected at indicated time points and
IL-12p40 production was analysed by ELISA. Data represents mean+sem of triplicates.
Experiments were repeated 3 times and representative data are shown.
Two way
ANOVA and post-hoc Bonferroni tests were used for statistical analysis; *** P<0.001
119
3.1.10 TNFα inhibits IL-12p40 production through activation of IKKβ
To investigate if the inhibitory effect of TNFα on IL-12 production is mediated
by IKKβ, BMDM were infected with Ad-IKKβGFP or Ad-IKKβdn before treating
cells with TNFα. Tissue culture supernatant was collected and IL-12p40 was
measured by ELISA (Figure 3.1.10.1). Compared to the untreated group, TNFαtreated BMDM produced 30% less IL-12p40 at 8 h. On the other hand, infection
of TNFα treated-BMDM with Ad-IKKβdn prevented the inhibition of IL-12
production and in fact further increased IL-12p40 level by 2.5 fold.
***
**
***
Figure 3.1.10 1 IKKβ inhibition rescued TNFα mediated suppression of IL-12p40
production
Bone marrow cells were cultured with 10ng/ml MCSF and infected as described. Virus
was removed after 24 h and fresh complete DMEM+MCSF with or without 20ng/ml
TNFα was added. On day 7, 0.5x106 BMDM/well were plated in 24 well plates and
stimulated with 100ng/ml LPS and 100U/ml IFNγ.
Cell culture supernatant was
collected at 4 and 8 h and IL-12p40 was measured by ELISA. Data represents
mean+sem of triplicates. Experiments were repeated 3 times and representative data are
shown. Two way ANOVA and post-hoc Bonferroni tests were used for statistical
analysis; ** P<0.01, *** P<0.001
120
IL-10 is a well known inhibitor of IL-12 production (Aste-Amezaga, Ma et al.
1998). I tested whether TNFα mediated inhibition of IL-12 production was due
to increased IL-10 expression.
Figure 3.1.10.2 shows TNFα pre-treatment
inhibited IL-10 production by 50% and the inhibition was blocked by adenovirus
expressing IKKβ dominant negative inhibitor.
***
**
*
Figure 3.1.10 2 TNF and IKK inhibits IL-10 production
Bone marrow cells were cultured with 10ng/ml MCSF and infected as described. On
day 7, 0.5x106 BMDM/well were plated in 24 well plates and stimulated with 100ng/ml
LPS and 100U/ml IFNγ. Cell culture supernatant was collected at 4 and 8 h and IL-10
was measured by ELISA. Data represents mean+sem of triplicates. Experiments were
repeated 3 times and representative data are shown. Two way ANOVA and post-hoc
Bonferroni tests were used for statistical analysis; *P<0.05, ** P<0.01, *** P<0.001
121
3.1.11 Summary
Earlier studies from our group have shown that knocking out IKK in
macrophages increased iNOS expression in alveolar macrophages, with
increased bacterial clearance in a lung infection model (Fong, Bebien et al.
2008).
To further investigate the inhibitory role of IKK in macrophage
activation, Cre-lox mediated gene targeting was used to specifically delete IKK
expression in macrophages (IKKMye) and macrophage activation was
investigated in vitro.
Upon LPS stimulation, IKKβ∆Mye macrophages show
increased iNOS, IL-12 and MHC II expression.
Immunoblotting showed
enhanced STAT1 phosphorylation in IKK knockout macrophages, further
experiments with adenovirus expressing IKK dominant negative inhibitor also
showed increased STAT1 phosphorylation, iNOS and IL-12 expression. These
data confirm the results obtained from IKKβ∆Mye macrophages and suggest that
negative cross talk between IKK and STAT1 signalling might be a mechanism
for inhibition of ‘classical’ macrophage activation. Because IKK is required for
TNF gene expression and TNF signalling, I investigated the hypothesis that
TNF inhibits ‘classical’ macrophage activation through activation of IKK. I
used macrophages from TNF receptor I knockout (p55-/-) mice and blocked
TNF in vivo with neutralising antibody. p55-/- macrophages and macrophages
from mice treated with anti-TNF antibody show increased STAT1 activation
and IL-12 expression when stimulated in vitro with LPS and IFN. In addition,
pre-treatment of macrophages with recombinant TNF prior to LPS and IFN
stimulation inhibited both STAT1 activation and IL-12 expression. Furthermore,
bone marrow derived macrophages infected with recombinant adenovirus
122
expressing IKKβ dominant negative inhibitor blocked the inhibitory effect of
TNFα on IL-12 production, suggesting TNF mediated IKKβ activation is
required for inhibition of IL-12.
3.1.12 Discussion
3.1.12.1 Mechanisms of IKK inhibition of classical macrophage activation
My studies show increased expression of MHC II, IL-12, iNOS and IRF1 when
IKK is deleted in macrophages.
This was also associated with increased
activation of STAT1. IFN is a NF-B regulated gene in response to LPS
stimulation (Honda, Yanai et al. 2005).
It has been shown that
autocrine/paracrine IFN production is required for LPS mediated iNOS
expression through activation of STAT1 (Gao, Filla et al. 1998), neutralising
IFN with anti-IFN antibody abolished STAT1 phosphorylation and the
subsequent iNOS expression (Gao, Filla et al. 1998). My studies show a
reduction of IFN mRNA expression from LPS stimulated IKKMye
macrophages (Figure 3.1.3.1B), suggesting the elevated iNOS expression was
not related to the increased IFN production and suggests IKK inhibits STAT1
signalling. Earlier studies from our group used luciferase reporter assays to
confirm the role of IKK in the suppression of STAT1 transcription activity
(Fong, Bebien et al. 2008); transient over-expression of IKK in RAW 264.7
macrophages suppressed GAS reporter activity, whereas transfection of
endogenous NF-B inhibitor IB increased GAS reporter activation (Fong,
Bebien et al. 2008), these studies illustrate an inhibitory role of IKK in STAT1
transcription activity and suggest increased expression of M1-associated genes in
123
IKKMye macrophages is, at least in part, associated with the elevated GAS
activity.
3.1.12.2 TNF and inhibition of classical macrophage activation
TNF is a proto-typical proinflammatory cytokine thought to be a key mediator
of chronic inflammation. Because IKK is required for TNF gene expression
and TNF signalling (Collart, Baeuerle et al. 1990; Beg, Finco et al. 1993;
Caamano and Hunter 2002), I tested the hypothesis TNF inhibits ‘classical’
macrophage activation through IKK. First, I tested if TNF can suppress
STAT1 signalling in macrophages. Macrophages from mice administered with
anti-TNF neutralising antibody show increased STAT1 activation when
stimulated in vitro (Figure 3.1.5.1). Furthermore, IL-12 production was also
increased from these macrophages (Figure 3.1.5.2).
These data illustrate the
inhibitory effect of endogenous TNF in STAT1 activation. The effect of TNFα
can be mediated via two receptors; TNF receptors p55 and p75 (Vandenabeele,
Declercq et al. 1995). In order to determine which receptor was involved,
macrophages were isolated from p55 and p75 knockout mice and stimulated with
LPS and IFN in vitro. These experiments showed the inhibitory effect of TNFα
was mediated by p55 and not p75 (Figures 3.1.7.1 & 3.1.7.2). Moreover, the
inhibitory effect of TNF on STAT1 signalling was further tested with
exogenous TNF treatment. Pre-treatment of macrophages with recombinant
TNF prior to LPS and IFN stimulation inhibits both STAT1 activation and IL12 expression while suppression was not seen with IL-1 pre-treatment. Since
IKK can be activated by both TNF and IL-1, these data indicate the effect of
IKK on STAT1 is dependent on other elements that are not involved in IL-1
124
signalling. TNF is a well known inducer of cell apoptosis, so it is possible that
reduced IL-12 production was associated with reduced cell viability. However,
no changes in cell viability were observed microscopically in the experiments
performed, hence cell death is unlikely the explanation for the reduced IL-12.
Consistent with current study, the negative regulatory effect of TNF on IL-12
has been reported elsewhere (Marino, Dunn et al. 1997) (Hodge-Dufour, Marino
et al. 1998; Zakharova and Ziegler 2005). Marino et al. 1997 suggested a role for
TNF in limiting the inflammatory response to heat killed C.parvum in mice.
From their studies, WT mice developed prompt granulomatous response
followed by resolution of the granuloma whereas TNF-/- mice showed little or
no initial response, but later developed a vigorous inflammatory responses
leading to death (Marino, Dunn et al. 1997). Later, Hodge-Dufour et al. showed
increased serum levels of IL-12 in TNF-/- mice (Hodge-Dufour, Marino et al.
1998). Furthermore, administration of anti-IL-12 neutralising antibody led to the
resolution of granulomatous responses to C.parvum in the livers and spleens of
TNF-/- mice (Hodge-Dufour, Marino et al. 1998). These studies together with
the current study clearly demonstrate the inhibitory effect of TNF on IL-12
production and a role in resolving inflammation. IFN/STAT1 signalling is a key
pathway involved in IL-12 production. Similar to the current study, an inhibitory
effect of TNF on IFN/STAT1 signalling has been reported by other studies
(Wang, Contursi et al. 2000). Elevated TNF is found in viral hepatitis patients
and was suggested to be associated with resistance to IFN therapy (Larrea,
Garcia et al. 1996; Hong, Nguyen et al. 2001). In 2001, Hong et al. have shown
TNF inhibits STAT1 Tyr 701 phosphorylation in the liver and further analysis
indicated the inhibitory effect was associated with SOCS3 and SHP2 induction
125
(Hong, Nguyen et al. 2001). Taken together, the inhibitory effect of TNF on
IL-12 expression could be mediated by direct STAT1 inhibition, or alternatively
through the production of suppressing proteins such as SOCS3 and SHP2. From
the later section of this chapter, BMDM infected with recombinant adenovirus
expressing IKKβ dominant negative inhibitor blocked the inhibitory effect of
TNFα on IL-12 production (Figure 3.1.10.1), demonstrating TNF inhibits IL-12
production through IKK activation.
However, the increase in IL-12 p40
production by Ad-IKKdn infected BMDM was greater than the level of
inhibition with TNF. This suggests other mechanisms regulated by IKK/NFB inhibits IL-12 expression in the context of viral infection.
3.1.12.3 Experimental limitations
3.1.12.3.1 Targeting IKK in macrophages
Knockout of the Ikbkb gene is lethal, IKK-/- embryos die at E13 due to
hepatocyte apoptosis (Li, Antwerp et al. 1999; Li, Chu et al. 1999; Tanaka,
Fuentes et al. 1999), this phenotype is also seen in RelA-/- mice (Beg, Sha et al.
1995). Cell death was attributed to TNF signalling as double knockout mice
(RelA-/-TNFR1-/-, RelA-/-TNF-/-) were rescued from embryonic lethality (Doi,
Marino et al. 1999; Alcamo, Mizgerd et al. 2001). The Cre-lox recombinase
system was utilised to generate mice with tissue specific IKK deficiency.
LysMCre transgenic mice utilise the lysosyme M promoter to express Cre
recombinase in myeloid cells, thus crossing LysMCre mice with IKK floxed
(F/F) mice selectively targeted the IKK gene in the myeloid cell lineage. This
method allows investigation of IKK function selectively in macrophages.
126
Although the Cre-lox recombinase system is a powerful tool to create conditional
knock out in specific tissues, there are several limitations of the LysMCre mouse
to study gene function selectively in macrophages; the LysM-promoter drives
Cre expression in both granulocytes and macrophages and higher deletion
efficiency is obtained in the granulocyte lineage (Clausen, Burkhardt et al. 1999).
In fact, due to the cell heterogeneity, there is still lack of specific gene markers
for macrophages and M1/M2 sub-types and this is an area that needs focus for
the field to move forward. Another potential limitation of the Cre-lox
recombination system is the variation of deletion efficiency between mice. This
problem was overcome by increasing repetition of experiments. Furthermore,
deletion efficiency is not 100% using the Cre-lox system, typically between 8095% deletion of IKKMye mice, however this may offer some advantages
including less compensation through increased expression of other genes, and a
more comparable effect expected with pharmacological targeting of IKK.
According to earlier studies from our group, tg elicited peritoneal macrophages
exhibit the most efficient IKK deletion in IKKMye mice. The peritoneal
cavity provides an easily accessible site to harvest a large numbers of
macrophages. Upon tg injection, tg elicited an inflammatory response,
neutrophils are the predominant cell population recruited initially followed by
monocytes that differentiate into macrophages. A disadvantage of this system is
tg is an inflammatory irritant, so tg elicited macrophages are exposed to
proinflammatory cytokines before harvesting. As observed in IKKMye and p55/-
mice, a high basal level of STAT1 phosphorylation was detected suggesting
macrophages were activated in vivo before harvesting (Figures 3.1.2.1 &
127
3.1.7.1).
To further confirm the data obtained from IKKMye tg elicited
peritoneal macrophages, different experiment approach was used to block IKK
activation, MCSF derived BMDM were infected with recombinant adenovirus
expressing IKK dominant negative. In the absent of IKK activity, similar
results were obtained compared to experiments with IKKMye tg elicited
macrophages, thus further confirm the inhibitory role of IKK on STAT1
signalling. Despite the consistent data obtained from the current studies, other
studies have reported opposing results using different approaches to target IKK.
Shultz et al. 2007 has shown that IKK is required for the activation of IFN
regulated genes (Shultz, Fuller et al. 2007), knocking down IKK by
recombinant retrovirus in mouse embryonic fibroblast (MEF) showed
impairment of IP10 induction after IFN stimulation. Similar observations were
obtained from siRNA mediated targeting of IKK in RAW 264.7 cells, a mouse
macrophage cell line (Shultz, Fuller et al. 2007). Furthermore, STAT1 activation
was not affected by IKK inhibition as illustrated by equivalent STAT1 tyrosine
and serine phosphorylation between scrambled siRNA and siRNA targeting
IKK in RAW 264.7 cells. Different approaches to target IKK might be
responsible for the inconsistent data, siRNA is a common in vitro system to
induce specific gene silencing, however there are limitations of this technique.
Off-target effects are often an issue with siRNA technology and different
mechanisms were reported. For example, high concentration of siRNA has
reported to induce global up/down regulation of genes (Persengiev SP 2004),
partial sequence complementation could lead to unspecific mRNA degradation
(Saxena S 2003). Furthermore, it has also been reported that transfection of
siRNA results in IFN mediated activation of JAK/STAT pathway and global
128
upregulation of IFN genes (Sledz, Holko et al. 2003), suggesting siRNA
technology might not be the ideal method when studying JAK/STAT1 signalling.
Lastly, the use of RAW 264.7 macrophage cell line may not reflected studies in
primary cells due to changes in cell signalling pathways during transformation.
129
3.2 Inhibitory roles of IKK in adaptive immunity
Classically activated, or M1 macrophages are important in T cell activation and
the development of the adaptive immune responses (Gordon 2003). From earlier
data, I have shown an inhibitory effect of IKK on MHC II expression and IL-12
production by macrophages. MHC II is required for antigen presentation to CD4+
T cells while IL-12 is important in TH1 cell development. Based on these results,
it is plausible to suggest knocking out IKK in macrophages will increase T cell
activation and subsequently promote TH1 responses. The aim of this section was
to investigate the inhibitory roles of IKK in the adaptive immunity and T cell
activation.
130
3.2.1 IKKMye macrophages express higher levels of CD80, CD86, MHCII and
low CD124/IL-4 receptor 
Knocking out IKKβ in macrophages enhanced MHC II expression upon IFNγ
stimulation in vitro (Figure 3.1.3.2). In addition to antigen presentation by MHC
II, CD80 and CD86 are costimulatory molecules that are important for successful
T cell activation, I thus measured CD80 and CD86 on tg elicited peritoneal
macrophages from IKKF/F and IKKΔMye mice by FACS. As shown in Figures
3.2.1.1A, unstimulated tg elicited peritoneal macrophages from IKKΔMye mice
expressed higher basal level of CD80 when compared IKKF/F macrophages.
Upon LPS stimulation, approximately 1.5 fold increased of CD80 was detected
on both IKKF/F and IKKΔMye macrophages.
Compare to CD80, the induction
of CD86 expression was more profound on IKKΔMye macrophages after LPS
stimulation (MFI; IKKF/F 343 vs IKKΔMye 734) (Figure 3.2.1.1B), there was
approximately 4 fold increased in CD86 expression on IKKΔMye macrophages
while there was only 2.5 fold induction on IKKF/F macrophages. MHC II
expression was also measured, and as expected MHC II levels were higher on
IKKΔMye macrophages with or without LPS stimulation (Figure 3.2.1.1C). The
basal level of MHC II of tg elicited macrophages from IKKΔMye mice was very
high compared to IKKF/F macrophages (MFI; IKKF/F 742 vs IKKΔMye 2245)
and because of the high basal, there was no significant increase in MHC II
expression after LPS stimulation (MFI; IKKF/F 806 vs IKKΔMye 2325).
131
A
Unstimulated
100
CD80
80
% of Max
80
% of Max
LPS
100
MFI-66.9
MFI-99.1
60
40
20
20
0
100
B
101
102
CD80
103
100
104
100
102
CD80
103
MFI-343
MFI-734
60
40
40
20
20
0
104
CD86
80
% of Max
60
101
100
MFI-150
MFI-155
80
% of Max
MFI-109
MFI-131
60
40
0
IKKF/F
IKKMye
0
100
101
102
CD86
103
104
100
101
102
CD86
103
104
C
100
2325
80
100
MFI-742
MFI-2245
MHC II
MFI-806
MFI-2325
80
% of Max
% of Max
742
60
60
40
40
20
20
0
0
100
101
102
MHC II
103
104
100
101
102
MHC II
103
104
Figure 3.2.1 1 IKK Mye macrophages express higher level of CD80, CD86 and
MHCII
3x106 tg elicited macrophages/well were plated in 6 well plates and stimulated with or
without 100ng/ml LPS. After 24 h, non-adherent cells were removed and only adherent
cells were collected, the expression of CD80, CD86 and MHC II gated on CD11b+ cells
were measured by FACS. Data are represented as histogram and MFI is indicated.
Black – isoptype control. Red - IKK F/F macrophages. Blue - IKK Mye macrophages.
Experiments were repeated at least 3 times and representative data are shown.
132
According to the previous data, IKKMye macrophages have high MHC II
expression and IL-12 production, which are markers of M1 macrophages. IL-4
is important for M2 macrophages development (Gordon 2003; Herbert, Hˆlscher
et al. 2004), so I sought to test for the presence of IL-4 receptor (IL-4R)  on
IKKMye macrophages. Thioglycolate elicited macrophages were stimulated
with LPS for 24 h and IL-4R was measured by FACS. As shown in Figure
3.2.1.2A & B, there were 20% of IKKF/F macrophages expressed IL-4R, by
comparison, only 2% of IKKMye macrophages were IL-4R positive.
IKKF/F
A
B
IKKΔMye
100
% of Max
80
60
40
20
0
10
0
10
1
2
10
FL2-H
Figure 3.2.1 2 Thioglycolate elicited peritoneal IKK Mye macrophages express
reduced IL-4R
3x106 tg elicited macrophages/well were plated in 6 well plates and stimulated with
100ng/ml LPS. After 24 h, non-adherent cells were removed and only adherent cells
were collected, the expression of CD11b and IL-4R by FACS. CD11b and IL-4R
double positive cells are illustrated by (A) dot plot and (B) histogram. Experiments
were repeated at least 3 times and representative data are shown.
133
10
3
10
4
3.2.2 Bacteria elicited peritoneal macrophages from IKKMye mice express high
levels of CD80, CD86, MHC II and low IL-4R
To confirm the previous data obtained from LPS stimulated tg elicited
macrophages, IKKF/F and IKKΔMye mice were injected i.p with heat killed
Group B Streptococcus (GBS - Gram positive bacteria), costimulatory
molecules, MHC II and IL-4R expression levels were measured by FACS.
Consistent with Tg elicited IKKΔMye macrophages, GBS elicited macrophages
from IKKΔMye mice expressed higher levels of CD80, CD86, MHC II and lower
IL-4R. Compared to IKKF/F macrophages, 3 fold of CD80 (MFI; IKKF/F 179
vs IKKΔMye 512) and 2 fold of CD86 (MFI; IKKF/F 140 vs IKKΔMye 277) were
detected on IKKΔMye macrophages (Figures 3.2.2.1A & B). Both IKKF/F and
IKKΔMye macrophages express high levels of MHC II, but 30% higher MHC II
expression (MFI; IKKF/F 1046 vs IKKΔMye 1478) was detected on IKKΔMye
macrophages (Figure 3.2.2.1C). IL-4R was present on GBS elicited IKKΔMye
macrophages, however the expression level was 70% lower compared to the
IKKF/F macrophages (MFI; IKKF/F 56.5 vs IKKΔMye 15.6) (Figure 3.2.2.1D).
134
A
B
100
MFI-179
MFI-512
60
MFI-140
MFI-277
IKKF/F
IKKMye
60
40
40
20
20
0
0
100
101
102
FL2-H
103
100
104
C
101
102
FL5-H
103
104
D
100
100
MHC II
MFI-1046
MFI-1478
60
IL4R
MFI-56.5
MFI-15.6
80
% of Max
80
% of Max
CD86
80
% of Max
80
% of Max
100
CD80
60
40
40
20
20
0
0
10 0
10 1
10 2
FL5-H
10 3
10 4
100
101
102
FL2-H
103
104
Figure 3.2.2 1 Bacteria elicited peritoneal macrophages from IKK Mye mice
express high costimulatory molecules, MHC II and low IL-4R
1x108 heat killed GBS were injected i.p into IKKF/F and IKKMye mice and peritoneal
macrophages were harvested after 3 days. 3x106 peritoneal macrophages/well were
plated with 6 well plates. After 24 h, non-adherent cells were removed and IL-4R,
CD80, CD86 and MHC II expression were measured on adherent cells by FACS. The
expression of IL-4R, CD80, CD86 and MHC II were gated on CD11b positive cells
and the expression levels are indicated with the mean fluoresce intensity. Black –
isotype control. Red - IKK F/F macrophages.
Blue - IKK Mye macrophages.
Experiments were repeated 3 times and representative data are shown.
135
3.2.3 Allogenic mix lymphocyte reaction
In an allogenic mix lymphocyte reaction (MLR), the proliferation of T cells is
dependent on the recognition of mismatched human leukocyte antigen (HLA
class II antigen). This assay is routinely used to measure the stimulatory activity
of antigen presenting cells in T cell activation. As shown in earlier data, MHC II
and costimulatory molecules expression were increased in
IKKΔMye
macrophages, thus I hypothesised that IKKΔMye macrophages could enhance
allogenic T cell proliferation.
3.2.3.1 CD4+ T cell isolation
CD4+ T cells were collected from lymph nodes of Balb/c mice (H-2d haplotype)
and cultured with allogenic macrophages of C57BL/6 mice (H-2b haplotype).
CD4+ T cells were purified by negative immunogenic cell separation using CD4+
T cell isolation kit. To confirm the efficiency of the isolation, CD4, CD8, B220,
CD11b and CD11c were stained on purified cells by FACS. As shown from
Figure 3.2.3.1.1, the purity of the isolated CD4+ T cell population was
approximately 90%, and there were 8% of CD8+ T cells. No contaminating
antigen presenting cells were found as indicated by CD11b, CD11c, B220
negative staining.
136
CD4
100
CD8
100
92%
80
% of Max
% of Max
80
8%
60
60
40
40
20
20
0
0
100
101
102
FL1-H
103
104
100
101
CD11b
103
104
CD11c
100
B220
0%
100
0%
80
% of Max
60
60
60
40
40
40
20
20
20
0
0
100
101
102
FL2-H
103
104
0%
100
80
% of Max
80
% of Max
102
FL2-H
0
100
101
102
FL2-H
103
104
100
101
102
FL1-H
103
Figure 3.2.3.1 1 Isolation of CD4+ T cells with magnetic bead separation
Inguinal, brachial, superficial cervical, and mesenteric lymph nodes were collected from
Balb/c mice. CD4+ T cells were isolated by negative selection with MACS separation
kit (Miltenyi Biotec).
Selected cells were collected and stained with CD11b-PE,
CD11c-PE, CD4-FITC, CD8-PE, B220-FITC and analysed by FACS. Blue – isotype
control. Red – different surface markers as indicated. Experiments were performed
twice and representative data are shown.
137
104
3.2.3.2 Optimisation of allogenic MLR
To determine the optimal ratio of T cells: macrophages for allogenic MLR, a
dose response assay with different numbers of macrophages was performed.
2x104, 4x104 and 6x104 tg elicited peritoneal macrophages were cultured with
3x105 CFSE labelled T cells. T cell proliferation was measured with FACS by
dilution of CFSE. As shown in Figure 3.2.3.2.1, ratio of 1:5 and 1:7 provided the
greatest percentage of T cell proliferation and the allogenic response was further
increased with macrophages stimulated with LPS alone or in combination with
IFN.
138
10
2
10
1
10
0
8.95%
8.95
10
4
10
3
10
2
10
1
10
0
91
10
1
2
10
CFSE
10
3
0
10
4
10
3
10
2
10
1
10
0
10
90.8
10
1
2
10
CFSE
10
3
0
10
FL5-H
94.6
10
1
2
10
CFSE
10
3
10
10
2
10
1
10
0
10
4
10
3
10
2
10
1
10
0
4
0
14.6%
14.6
0
85.4
10
1
2
10
CFSE
10
3
0
10
4
10
3
10
2
10
1
10
0
10
87.3
10
1
2
10
CFSE
10
3
0
10
77.8
10
1
2
10
CFSE
10
3
10
3
10
2
10
1
10
0
4
0
Ratio
5.3%
1:15
5.3
10
4
10
3
10
2
10
1
10
0
0
94.7
10
1
2
10
CFSE
10
3
0
10
4
10
3
10
2
10
1
10
0
10
4
0
26..4%
1:7
26.4
10
0
22.2
0
10
LPS/IFN
0
4
22.2%
10
4
10
0
12.7
0
10
4
12.7%
10
0
5.38
0
3
4
5.38%
10
10
LPS
0
10
0
9.21
0
4
4
9.21%
10
FL5-H
0
FL5-H
FL5-H
10
10
FL5-H
3
0
FL5-H
10
Unstimulated
0
FL5-H
4
FL5-H
FL5-H
10
0
73.6
10
1
2
10
CFSE
10
3
0
10
4
0
29.9%
1:5
29.9
10
0
70.1
10
1
2
10
CFSE
10
3
10
4
Figure 3.2.3.2 1 Optimization of the ratio of T cells and macrophages in allogenic
MLR
2x104, 4x104 and 6x104 tg elicited macrophages/well (C57BL/6) were plated in 96 well
plates and stimulated 100ng/ml LPS or in combination with 100U/ml IFN. After 24 h,
3x105 CFSE labelled CD4+ T cells purified from lymph nodes of Balb/c mice were
added. Non-adherent T cells were collected after 4 days and T cell proliferation was
measured by FACS. Only viable cells were gated and dead cells were excluded by
staining with 7-Amino-actinomycin D. Percentage of proliferating cells is indicated.
Experiments were performed twice and representative data are shown.
139
3.2.3.3 Capacity of different macrophages in promoting T cell proliferation
Different sources of macrophages have different activation states and therefore
may induce different levels of T cell activation. I tested three different types of
macrophages; tg elicited, Biogel elicited peritoneal macrophages and BMDM. tg
elicited peritoneal macrophages had the greatest capacity in the induction of T
cell proliferation compared to Biogel elicited cells and BMDM (Figures
3.2.3.3.1A, B & C). Between the 3 timepoints tested, highest allogenic T cell
proliferation was detected on day 5 with 30% proliferation detected from T cells
co-cultured with tg elicited macrophages stimulated with LPS and IFN (Figure
3.2.3.3.1C). In comparison, 15% T cell proliferation was induced by BMDM
and even less (5%) was detected from macrophages elicited by Biogel (Figure
3.2.3.3.1A & B). Taken together, these experiments establish that ratio of 1:5 tg
elicited macrophages: T cells was optimal for allogenic T cell proliferation.
140
A
B
C
Figure 3.2.3.3 1 Activity of different macrophages in allogenic MLR
1.5ml of 3% Biogel or tg was injected i.p to C57BL/6 mice, after 3 days, peritoneal
macrophages were harvested. 6x104 macrophages/well were plated in 96 well plates and
stimulated with 100ng/ml LPS or in combination with 100U/ml IFN. BMDM were
tested in parallel. After 24 h of stimulation, 3x105 CFSE labelled CD4+ T cells purified
from lymph nodes of Balb/c mice were added to the macrophages and T cell
proliferation was measured at indicated timepoints by FACS. Only viable cells were
gated and dead cells were excluded by staining with 7-Amino-actinomycin D. Data
represents mean+sem of triplicates. Experiments were performed twice and
representative data are shown.
141
3.2.3.4 IKKMye macrophages increase allogenic T cell proliferation
I next investigated the ability of IKKF/F and IKKMye macrophages to induce
allogenic T cell proliferation. As shown in Figure 3.2.3.4.1, allogenic T cell
proliferation was more than 50% higher from T cells co-cultured with IKK∆Mye
macrophages compared to IKKF/F macrophages.
***
Figure 3.2.3.4 1 IKK Mye macrophages induced enhanced allogenic T cell
proliferation
6x104 tg elicited macrophages/well were plated in a 96 well plate and stimulated with
100ng/ml LPS for 24 h. 3x105 CFSE labelled CD4+ T cells purified from lymph nodes
of Balb/c mice were added and T cell proliferation was measured by FACS on day 4.
Only viable cells were gated and dead cells were excluded by staining with 7-Aminoactinomycin D. Data represents mean+sem of triplicates. Experiments were repeated 3
times and representative data are shown. Student’s T test was used to test statistical
significance; *** P<0.001
142
3.2.4 Delayed type hypersensitivity
Delayed type hypersensitivity (DTH) is an in vivo model to measure cellmediated immune responses. I used this model as an in vivo system to study the
roles of IKK in T cell activation. In DTH, mice are immunised to a specific
antigen and then subsequently challenged with the same antigen to measure
antigen-specific T cell activation. This model tests the ability to ‘prime’ T cell
responses and the activation of secondary responses by APCs. By using this
model, the capacity of IKKβ∆Mye macrophages to generate antigen specific T
cells in vivo was tested.
3.2.4.1 Immunisation
C57BL/6 mice were immunised with mBSA and complete Freund adjuvant
(CFA). On day 14, mice were boosted with mBSA and incomplete Freund
adjuvant (IFA). To determine the efficiency of immunisation, a recall assay was
preformed. Spleens were collected on day 21 from the mBSA immunised mice
and splenocytes were stimulated with mBSA in vitro. Tissue culture supernatant
was collected on day 4, IFN and IL-10 were measured by ELISA. Both IFN
and IL-10 production were dose dependently induced with mBSA up to 50g/ml
(Figures 3.2.4.1.1A & B). A positive control was performed in parallel with an
antigen independent T cell activator concanavalin A (ConA) and both IFN and
IL-10 were induced upon stimulation (Figures 3.2.4.1.1C & D).
143
A
B
C
D
Figure 3.2.4.1 1 Splenocytes from mBSA immunised mice elicited antigen
dependent cytokine production ex-vivo
C57BL/6 mice were immunised with mBSA and CFA. After 21 days, spleens were
collected and splenocytes were cultured with 0-100g/ml mBSA in vitro. Supernatant
was collected on day 4, IFN and IL-10 production was tested by ELISA. Data
represents mean+sem of triplicates. Experiments were repeated 3 times and
representative data are shown.
144
3.2.4.2 Dose response assay for mBSA induced T cell proliferation
To determine the optimal loading dose of mBSA for macrophages, a dose
response assay was preformed. Macrophages pre-loaded with different doses of
mBSA were cultured with CFSE labelled CD4+ T cells and T cell proliferation
was measured by FACS. As shown in Figure 3.2.4.2.1, 30% of T cell
proliferation was induced with 10μg/ml of mBSA and proliferation was further
increased with increased antigen dose.
Figure 3.2.4.2 1 Optimisation of the dose of mBSA for T cell proliferation assay
6x104 tg elicited macrophages/well were plated in 96 well plates and macrophages were
loaded with different concentration of mBSA as indicated and stimulated with or
without 100ng/ml LPS. After 24 h, 3x105 CFSE labelled CD4+ T cells from mBSA
immunised C57BL/6 mice were added and T cell proliferation was measured by FACS
on day 4. Only viable cells were gated and dead cells were excluded by staining with 7Amino-actinomycin D.
Data represents mean+sem of triplicates. Experiment was
repeated 3 times and representative data are shown.
145
3.2.4.3 IKKβ∆Mye splenocytes increase mBSA specific IFNγ production exvivo
Earlier data showed increased IL-12 production in IKKΔMye macrophages, thus I
predicted that knocking out IKK in macrophages could promote the
development of TH1 responses. To study the development of mBSA specific T
cell responses elicited by IKKβ∆Mye macrophages in vivo, splenocytes were
prepared from IKKβF/F and IKKβ∆Mye mBSA immunised mice and re-stimulated
with mBSA in vitro. In response to mBSA, IFNγ production was significantly
increased from splenocytes of IKKβ∆Mye mice when compared to IKKβF/F
splenocytes (Figure 3.2.4.3.1).
***
Figure 3.2.4.3 1 IKKβ∆Mye splenocytes increased mBSA specific IFNγ production
ex-vivo
IKKβF/F and IKKβ∆Mye mice were immunised with mBSA and CFA. After 21 days,
spleens were collected and splenocytes were cultured in vitro with 0-100g/ml mBSA.
Supernatant was collected on day 4 and IFN was measured by ELISA. Data represents
mean+sem of triplicates. Experiments were repeated 3 times and representative data are
shown. Two way ANOVA and post-hoc Bonferroni tests were used for statistical
analysis; *** P<0.001
146
IL-10 is a well known anti-inflammatory cytokine that can be produced by TH2
cells and antagonises TH1 responses (Abbas, Murphy et al. 1996). I therefore
measured IL-10 in parallel. In contrast to IFN, IL-10 production was reduced
from IKKβ∆Mye splenocytes in response to mBSA (Figure 3.2.4.3.2). Taken
together, these data indicate IKKβ∆Mye mice are more prone to develop TH1
responses.
*
Figure 3.2.4.3 2 IKKβ∆Mye splenocytes decreased mBSA specific IL-10 production
ex-vivo
IKKβF/F and IKKβ∆Mye mice were immunised with mBSA and CFA. After 21 days,
spleens were collected and splenocytes were cultured with 0-100g/ml mBSA in vitro.
Supernatant was collected on day 4 and IL-10 was measured by ELISA. Data represents
mean+sem of triplicates. Experiments were repeated 3 times and representative data are
shown. Two way ANOVA and post-hoc Bonferroni tests were used for statistical
analysis; *P<0.05
147
3.2.4.4 Priming mBSA specific T cell responses in IKK ∆Mye mice
B cells, DCs and macrophages are antigen-presenting cells in the immune system
that can activate T cells and develop antigen specific T cell responses.
It is
generally considered DCs are responsible for priming naïve T cells although
macrophages have also been reported to have this ability (Moser.M 2001; T.
Rothoeft 2003). To study the ability of IKKMye macrophages to prime T cells
and develop antigen specific responses in vivo, IKKF/F and IKKMye mice were
immunised with mBSA in the combination with CFA and lymph nodes were
collected on day 21. CD4+ T cells were purified from lymph nodes by negative
selection and cultured with mBSA loaded wild type BMDM. Tissue culture
supernatant was collected on day 4 and IFN production was measured by
ELISA (Figure 3.2.4.4.1).
In this experiment, a high basal level of IFN
production was detected in the absence of mBSA. Although mBSA only slightly
increased IFN production from lymph node T cells, by comparison IFN
production was much greater in T cells from IKKMye lymph nodes (IKKF/F –
12,000pg/ml vs IKKMye – 25,000pg/ml)
148
**
**
Figure 3.2.4.4 1 Increased TH1 cell development in IKK Mye mice
6x104 BMDM/well were plated in 96 well plates and stimulated with 100ng/ml LPS
with or without 20g/ml mBSA for 24 h. Inguinal, brachial, superficial cervical, and
mesenteric lymph nodes were collected from IKK F/F and IKK Mye mBSA immunised
mice. CD4+ T cells were isolated by negative selection with MACS separation kit
(Miltenyi Biotec) and 3x105 CD4+ T cells were added to the mBSA loaded BMDM.
Tissue culture supernatant was collected on day 4 and IFN was measured by ELISA.
Data represents mean+sem of triplicates.
Experiments were repeated 3 times and
representative data are shown. One way ANOVA and post-hoc bonferroni tests were
used for statistic analysis; ** P<0.01
149
3.2.4.5
Secondary
mBSA
specific
response
elicited
by
IKKMye
macrophages in vitro
I next tested the APC activity of IKKMye macrophages in activating mBSA
specific T cells.
mBSA loaded tg elicited macrophages from IKKF/F and
IKKMye mice were co-cultured with CD4+ T cells from mBSA immunised
C57BL/6 mice. Tissue culture supernatant was collected on day 4 and IFN was
measured by ELISA (Figure 3.2.4.5.1). Similar to earlier data, basal level of
IFN was detected from CD4+ T cells co-cultured with IKKF/F and IKKMye
macrophages in the absent of antigen. However, higher IFN production was
detected from T cells cultured with IKKMye macrophages.
150
***
**
*
NS
Figure 3.2.4.5 1 IKK ∆Mye macrophages increase mBSA specific IFN production in
vitro
IKK F/F and IKKMye mice were injected with 1.5ml of 3% tg and peritoneal
macrophages were harvested after 3 days. 6x104 tg elicited macrophages/well were
plated in 96 well plates with or without 20g/ml mBSA for 24 h. Inguinal, brachial,
superficial cervical, and mesenteric lymph nodes were collected from mBSA immunised
C57BL/6 mice. CD4+ T cells were isolated by negative selection with MACS separation
kit (Miltenyi Biotec) and 3x105 T cells were added to the macrophages. Tissue culture
supernatant was collected on day 4 and IFN was analysed by ELISA.
NS – not
significant. Data represents mean+sem of triplicates. Experiments were repeated 3
times and representative data are shown. One way ANOVA and post-hoc bonferroni
tests were used for statistic analysis; ** P<0.01, *** P<0.001
151
3.2.4.6 IKKMye macrophages increase antigen specific IFN production by
OTII T cells
Previous experiments suggested IKKMye macrophages increased priming of
antigen-specific T cells as well as activation of effector T cells upon secondary
stimulation.
To further confirm the ability of IKKMye macrophages in
activating naïve T cells, I used CD4+ T cells from OTII T cell receptor (TCR)
transgenic mice (H-2b) that have been genetically engineered to express
ovalbumin peptide (OVA323-339)-specific TCR. Co-culture of CD4+ OTII T cells
with antigen (OVA323-339) loaded IKKMye macrophages in vitro showed 7 fold
higher antigen dependent IFN production than T cells co-cultured with IKKF/F
cells (Figure 3.2.4.6.1). In the absence of antigen, no IFN production was
detected from the OTII T cells co-cultured with IKKMye or IKKF/F
macrophages, which confirms IFN production was antigen-dependent. This
data is consistent with the previous results and confirms the capacity of
IKKMye macrophages to activate naïve T cells and the development of TH1
responses.
152
***
***
Figure 3.2.4.6 1 IKK ∆Mye macrophages enhance OVA323-339 specific T cell activation
in vitro
6x104 tg elicited macrophages/well were plated in 96 well plates with or without 20nM
OVA323-339. After 24 h, inguinal, brachial, superficial cervical, and mesenteric lymph
nodes were collected from OTII TCR transgenic mice (H-2b). 3x105 CD4+ T cells were
isolated by negative selection with MACS separation kit (Miltenyi Biotec) and added to
the macrophages. Tissue culture supernatant was collected on day 4 and IFN was
measured by ELISA. Data represents mean+sem of triplicates. Experiments were
performed twice and representative data are shown. One way ANOVA and post-hoc
bonferroni tests were used for statistic analysis; *** P<0.001
153
3.2.4.7 IKKMye macrophages increase TH1 specific gene expression in OTII
cells
To confirm the CD4+ OTII T cell phenotype, RNA was isolated from T cells cocultured with IKKF/F or IKKMye macrophages and analysed by qRT-PCR. As
shown in Figure 3.2.4.7.1, the expression of IFN mRNA was antigen dependent.
Compared to the control, the mRNA level of IFN was increased by almost 20
fold from the T cells co-cultured with IKKMye macrophages (Figure
3.2.4.7.1A). In addition to IFN, the transcription factors T-bet and IRF1 have
been shown to participate in the development of TH1 responses (Taki, Sato et al.
1997; Szabo, Kim et al. 2000), I therefore tested the influence of IKKMye
macrophages on the expression of these transcription factors. In the presence of
OVA peptide, IRF1 expression was elevated by 4 fold from OTII T cells cocultured with IKKMye macrophages whereas the levels of IRF1 mRNA was
only increased by 2 fold from OTII T cells cultured with IKKF/F macrophages
(Figure 3.2.4.7.1B).
However, T bet mRNA was slightly reduced in the
presence of OVA peptide and no significant differences were observed in the
mRNA level of T-bet between T cells co-cultured with IKKF/F or IKKMye
macrophages (Figure 3.2.4.7.1C).
In parallel, the mRNA of IL-12 receptor
subunits IL-12R1 and IL-12R2 require for TH1 cell development (Szabo,
Dighe et al. 1997) were measured on OTII cells from co-culture experiments. As
shown in Figures 3.2.4.7.1 D & E, the mRNA levels of IL-12R1 and IL-12R2
were higher in OTII cells co-cultured with IKKMye macrophages in the
presence of OVA peptide. In response to antigen, IL-12R1 mRNA expression
increased by 2 fold in OTII cells cultured with IKKF/F macrophages and there
154
were 3 fold from the knockout group. IL-12R2, another subunit of IL-12R was
more than 2 fold higher in OTII cells cultured with IKKMye macrophages
compared to IKKF/F macrophages.
TH1 cell commitment can antagonise
development of TH2 cells by inhibiting IL-4 expression (Szabo, Kim et al. 2000).
Earlier data showed IKKMye macrophages promote TH1 cell responses, so I
predicted that TH2 specific genes would be inhibited by IKKMye macrophages.
In response to OVA peptide, the mRNA expression of IL-4 was increased by 3
fold in OTII cells co-cultured with IKKF/F macrophages while IL-4 mRNA was
reduced in OTII T cells cultured with IKKMye macrophages (Figure
3.2.4.7.1F). The mRNA of GATA3 was induced by 2 fold in response to OVA
peptide in OTII cells co-cultured with IKKF/F macrophages whereas slightly less
GATA3 mRNA was induced by IKKMye macrophages (Figure 3.2.4.7.1G).
155
A
IFN
B
IRF1
IL-12R1
D
F
IL-4
C
E
G
T-bet
IL-12R2
GATA3
156
Figure 3.2.4.7 1 IKK ∆Mye macrophages increase expression of TH1 specific genes in
OTII cells
IKK F/F and IKKMye mice were injected with 1.5ml of 3% tg and peritoneal
macrophages were harvested after 3 days, 1.6x106 tg elicited macrophages/well were
plated in 6 well plates together with or without 20nM OVA peptide for 24 h. Inguinal,
brachial, superficial cervical, and mesenteric lymph nodes were collected from OTII
TCR transgenic mice (H-2b) and CD4+ T cells were isolated by negative selection with
MACS separation kit (Miltenyi Biotec).
8x106 OTII T cells were added to
macrophages, non-adherent T cells were collected on day 4 and total RNA was isolated
and analysed by qRT-PCR.
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3.2.5 Inhibitory roles of TNF on macrophage APC activity
3.2.5.1 TNF inhibits MHC II and CD80 expression on BMDM
Data from the previous sections suggest TNFα inhibits ‘classical’ macrophage
activation through IKKβ activation. I therefore predicted that TNFα would have
an inhibitory effect on promoting TH1 cell responses. I first studied the effect of
TNFα on MHC II and costimulatory molecule expression. BMDM were pretreated with recombinant TNFα prior stimulation with LPS/IFNγ and CD80,
CD86 and MHC II were measured by FACS. Upon LPS/IFNγ stimulation,
CD80 expression (MFI; 45) was 40% lower on TNFα pre-treated BMDM as
compared with the untreated group (MFI; 74.4) (Figure 3.2.5.1.1A). Similarly,
35% reduction of MHC II was detected on BMDM pre-treated with TNFα (MFI;
BMDM 97.8 vs TNFα treated BMDM 62.3) (Figure 3.2.5.1.1B). However, the
inhibitory effect did not apply to CD86 and compared to the untreated BMDM
(MFI; 14), TNFα treatment actually increased CD86 expression (MFI; 35.8)
(Figure 3.2.5.1.1C).
158
A
C
100
MFI-74.4
MFI-45
60
MFI-14
MFI-35.8
BMDM
BMDM+TNF
60
40
40
20
20
0
CD86
80
% of Max
80
% of Max
100
CD80
0
100
101
102
FL2-H
103
104
100
101
102
FL5-H
103
104
B
MHC II
100
MFI-97.8
MFI-62.3
% of Max
80
60
40
20
0
100
101
102
FL1-H
103
104
Figure 3.2.5.1 1 Inhibition of CD80 and MHC II by TNFα in BMDM
Bone marrow cells were cultured with 10ng/ml MCSF, on day 4, 20ng/ml TNF was
added to the culture. On day 7, 3x106 BMDM/well were plated in a 6 well plate and
stimulated with 100ng/ml LPS and 100U/ml IFN for 24 h. BMDM were stained with
CD80-PE, CD86-APC, MHC II-FITC and the expression levels were measured by
FACS and MFI is indicated. Blue – BMDM. Red – BMDM treated with TNF. Black
– isotype controls. Experiments were repeated 3 times and representative data are
shown.
159
3.2.5.2 TNF inhibits allogenic T cell proliferation
I next investigated the affect of TNFα on allogenic T cell proliferation. BMDM
of C57BL/6 mice were pre-treated with or without TNFα and stimulated with
LPS, after 24 h BMDM were co-cultured with CFSE labelled CD4+ T cells of
Balb/c mice, allogenic T cell proliferation was measured by FACS. As shown in
Figure 3.2.5.2.1, allogenic T cell proliferation was reduced by 50% when
BMDM were pre-treated with TNFα.
**
Figure 3.2.5.2 1 TNFα treatment of BMDM reduced allogenic T cell proliferation
Bone marrow cells were cultured with 10ng/ml MCSF, on day 4, 20ng/ml TNF was
added to the culture. On day 7, 6x104 BMDM/well were plated in a 96 well plate and
stimulated with 100ng/ml LPS. After 24 h, 3x105 cells/well of CD4+ T cells purified
from Balb/c mice were stained with CFSE and added to the macrophages. After 4 days,
non-adherent T cells were collected and the dilution of CFSE was measured by FACS.
Only viable cells were gated and dead cells were excluded by staining with 7-Aminoactinomycin D. Data represents mean+sem of triplicates. Experiments were repeated 3
times and representative data are shown. Student T test was used to test statistical
significance; ** P<0.01
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3.2.5.3 TNFα treatment inhibits BMDM induced IFN production by OTII
cells
From the previous data, knocking out IKK in macrophages increased classical
macrophage activation and TH1 responses, further experiments show TNF
inhibits the ability of macrophages to activate T cells. So I next tested if TNF
treated BMDM could inhibit IFN production, a characteristic cytokine produce
by TH1 cells. BMDM with or without TNF pre-treatment were cultured with
CD4+ T cells from OTII transgenic mice in the presence of OVA peptide. Tissue
culture supernatant was collected on day 4 and IFN was measured by ELISA.
As shown in Figure 3.2.5.3.1, antigen induced IFN production was completely
inhibited from OTII cells co-cultured with TNF pre-treated BMDM.
161
***
ND
***
ND
Figure 3.2.5.3 1 TNFα treatment inhibits BMDM induced IFN production by
OTII cells
Bone marrow cells were cultured with 10ng/ml MCSF, and 20ng/ml TNF was added
on day 4. On day 7, 6x104 BMDM/well were plated in a 96 well plate and stimulated
with 100ng/ml LPS with or without 20nM OVA peptide. After 24 h, 3x105cells/well
CD4+ T cells from OTII TCR transgenic mice were added to the BMDM.
Tissue
culture supernatant was collected on day 4 and IFN was measured by ELISA. ND –
not detected. Data represents mean+sem of triplicates. One way ANOVA and post-hoc
bonferroni tests were used for statistic analysis; *** P<0.001
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3.2.5.4 TNFα inhibits TH1 specific gene expression
To further study the inhibitory role of TNF on TH1 cell development, BMDM
with or without TNF pre-treatment were cultured with OTII T cells and TH1
characteristic genes were analysed by qRT-PCR. From untreated BMDM, the
mRNA levels of IFN were increased by 40 fold in response to OVA peptide and
only basal level of IFN mRNA was detected in OTII cells co-cultured with
TNF pre-treated BMDM (Figure 3.2.5.4.1A). This data correlates with ELISA
data (Figure 3.2.5.3.1) and showed TNF inhibits IFN at the transcription level.
In the case of IRF1, 2 fold induction in mRNA was seen in OTII cells cultured
with OVA peptide loaded BMDM and 1.5 fold from OTII cells with TNF
treated BMDM (Figure 3.2.5.4.1B). In the presence of OVA peptide, IL12-R2
mRNA was induced by more than 2 fold in OTII cells cultured with BMDM, but
with TNF treated BMDM the mRNA levels of IL-12R2 was almost remained
at the basal levels (Figure 3.2.5.4.1D). By comparison, less profound induction
of IL-12R1 mRNA was detected from both groups (Figure 3.2.5.4.1C). In
response to OVA peptide, the mRNA levels of IL-12R1 were increased by 50%
from OTII cells co-cultured with untreated BMDM whereas there was 30% IL12R1 mRNA induction detected from OTII cells cultured with TNF treated
BMDM.
163
A
B
IFN
C
IL-12R1
IRF1
D
IL-12R2
Figure 3.2.5.4 1 TNF treatment of BMDM inhibits characteristic TH1 gene
expression in OTII cells
Bone marrow cells were cultured with 10ng/ml MCSF, on day 4, 20ng/ml TNF was
added to the culture. On day 7, 1.6x106 BMDM/well were plated in 6 well plates and
stimulated with 100ng/ml LPS with or without 20nM OVA peptide for 24 h. Lymph
nodes were collected from OTII TCR transgenic mice and 8x106 OTII cells were added
164
to the BMDM. Total RNA was isolated on day 4 from non-adherent OTII T cells and
analysed by qRT-PCR.
3.2.6 Summary
In this section, I have shown the role of IKK in macrophages in inhibiting TH1
cell development and activation. Upon LPS stimulation, Tg elicited IKKMye
macrophages increased expression of MHC II, CD80 and CD86 but reduced IL4R levels. Consistent results were obtained from bacteria elicited peritoneal
macrophages from IKKMye mice. In addition, IKKMye macrophages showed
an increased ability to drive T cell proliferation in MLR, indicating the APC
function of IKKMye macrophages. To further investigate the inhibitory role of
IKK in T cell mediated responses, a model of DTH was used. From a recall
assay with splenocytes from mBSA immunised mice, IKKMye mice showed
increased antigen specific IFN production ex-vivo, but antigen induced IL-10
production was reduced. The inhibitory effects of IKK in priming naïve T cells
and TH1 responses were further confirmed using OTII TCR transgenic mice.
OVA peptide dependent IFN production was greatly enhanced by CD4+ OTII T
cells co-cultured with IKKMye macrophages, indicating the inhibitory role of
IKK in priming naïve CD4+ T cells and promoting TH1 responses. Furthermore,
qRT-PCR analysis showed expression of characteristic TH1 genes such as; IRF1,
IFN, IL-12R1 and IL-12R2, were increased in OTII cells co-cultured with
IKKMye macrophages, but the TH2 marker IL-4 was reduced. These genes are
important for the development of TH1 responses and the commitment of TH1 cell
lineage. From sections 3.1.5-3.1.7, I have shown the inhibitory role of TNF
mediated IKK activation in IL-12 production by macrophages, so I sought to
further characterise the regulatory role of TNF in TH1 responses.
TNF
165
inhibited TH1 cell development in vitro; BMDM treated with TNF had reduced
MHC II and CD80 but not CD86 expression, TNF treatment of BMDM also
reduced allogenic T cell proliferation. These data indicate the inhibitory effect of
TNF on APC function.
Further experiments showed TNF treatment of
BMDM inhibits priming CD4+ T cells; this was illustrated by the marked
reduction on IFN production from OTII T cells co-cultured with TNF treated
BMDM loaded with antigen peptide.
Furthermore, the expression of
characteristic TH1 genes; IFN, IRF1 and IL-122 were reduced during T cell
priming by TNF treatment of BMDM.
166
3.2.7 Discussion
3.2.7.1 NF-B and APC function of macrophages
NF-B has been shown to have an important role in adaptive immunity, for
example, mutation of IB in T cells leads to a reduction of T cell proliferation
and IL-4, IL-10 and IFN production (Ferreira, Sidenius et al. 1999). Mora et al.
2001 also showed that T cells expressing mutated IB failed to activate STAT5a,
a transcription factor required for IL-2 production (Mora, Youn et al. 2001).
These studies demonstrated an intrinsic role for NF-B signalling in T cell
activation.
My studies have shown NF-B can regulate T cell activation
indirectly through modulation of macrophage APC activity. For naïve T cell
activation, two signals are required from the APC; signal 1 is engagement of
MHC II-peptide complex and the second signal is activation of costimulatory
molecules, e.g. CD80 and CD86. Failure to provide signal 2 often leads to T cell
apoptosis or anergy (Jenkins, Chen et al. 1990). In my experiments, FACS
analysis demonstrated increased expression of MHC II, CD80 and CD86 on LPS
stimulated IKKβ∆Mye macrophages when compared to control cells (Figure
3.2.1.1). Consistent with the enhanced MHC II expression, increased allogenic T
cell proliferation was obtained with IKKβ∆Mye macrophages (Figure 3.2.3.4.1).
These data suggest a role of IKKβ in suppressing the APC activity of
macrophages. This is in contrast to studies with DCs that suggest APC activity is
positively regulated by NF-B signalling (Rescigno, Martino et al. 1998;
167
Yoshimura, Bondeson et al. 2001; Yoshimura, Bondeson et al. 2003). These
studies showed inhibiting NF-B activation with protease inhibitor or adenovirus
expressing IB in DCs impaired MHC II and B7 costimulatory molecule
expression (Rescigno, Martino et al. 1998; Yoshimura, Bondeson et al. 2001).
Furthermore, blocking IKK in DCs with Ad-IKK dominant negative inhibited
T cell proliferation in an allogenic MLR assay (Yoshimura, Bondeson et al.
2001; Yoshimura, Bondeson et al. 2003). This is the exact opposite to what I
have observed in macrophage that lack IKK expression.
The discrepancy
between these observations may be due to different roles for NF-B in DCs and
macrophages. Alternatively, these differences may due to the use of different
experimental systems. Over expression of IB has been reported to affect the
activity of several proteins including p53 (Chang 2002; Dreyfus, Nagasawa et al.
2005), cyclin-dependent kinase 4 (Li, Joo et al. 2003) and histone
acetyltransferases and deacetylases (HDACs) (Viatour, Legrand-Poels et al.
2003; Aguilera, Hoya-Arias et al. 2004). These off target effects may have an
influence on the experimental outcome. Other studies have used the serine
protease inhibitor N--tosyl-L-phenylalanine chloromethyl ketone (TPCK) that
blocks nuclear translocation of NF-B by stabilising IB and preventing its
degradation (Finco TS 1994). It has been shown that TPCK blocked NF-B
dependent nitric oxide synthase in murine macrophages (Kang BY 1999) and
consistent with experiments using adenovirus expressing IB (Yoshimura,
Bondeson et al. 2001; Yoshimura, Bondeson et al. 2003), DCs treated with
TPCK showed reduction of CD86 and MHC II (Rescigno, Martino et al. 1998).
Furthermore, TPCK inhibit IL-12 production in LPS stimulated macrophages
(Kang BY 1999).
This is again in contrast to my data using IKK deficient
168
macrophages or adeno-IKKdn. Although NF-B activation can be blocked by
TPCK, this inhibitor was also shown to affect other pathways, including
proteolytic loss of STAT6 (Perez-G, Cortes et al. 2008) and disrupting of 3phosphoinositide-dependent kinase 1 (PDKI) signalling (Ballif, Shimamura et al.
2001). The NF-B-independent effects may also impact this data. However,
studies from Ouaaz et al. that show no impairment of MHC II and CD86
expression and allogenic T cell proliferation with DCs isolated from RelA-/-, p50/-
, c-Rel-/- and p50-/-c-Rel-/- chimeric mice (Ouaaz, Arron et al. 2002), also suggest
over expression of IB and use of protease inhibitors have resulted in NF-B
independent effects on DCs function.
A cell-specific role of NF-B could be another explanation for the contradictory
observations between my studies and those of Yoshimura et al. (Yoshimura,
Bondeson et al. 2001; Yoshimura, Bondeson et al. 2003). Earlier studies from
our group showed enhanced bacterial clearance when IKKβ was ablated in
macrophages but not in epithelial cells (Fong, Bebien et al. 2008), illustrating a
cell specific role in inflammation.
Other studies have shown NF-B from
different cells, NF-B may also have a differential function within the same cell
lineage, for example Lin et al 1999 have shown the involvement of NF-ĸB in
both pro- and anti-apoptotic function in T cells. Inhibition of NF-ĸB reduced
PMA/ionomycin mediated induction of FasL and apoptosis but increased
glucocorticoid-mediated apoptosis. Glucocorticoids are produced in the thymus
and function to induce thymocyte apoptosis during positive selection (Vacchio
MS 1997) whereas Fas and FasL interaction is important in AICD and peripheral
T cell deletion (Lin B 1999). These data suggest NF-ĸB inhibits glucocorticoid169
mediated apoptosis and survival during positive selection. On the other hand,
NF-ĸB has the opposite role in mature peripheral T cells, promoting apoptosis by
increasing FasL expression. Collectively, these studies suggest NF-ĸB activation
can have distinct roles in different cell lineages, physiological contexts or even
throughout cell differentiation.
3.2.7.2 IKK activation in macrophages and generation of antigen-specific T
cell responses
In cell-mediated immunity, antigen presentation by APC is critical for T cell
priming and subsequent antigen-specific T cell activation. Using a model of
DTH, I tested the role of IKK activation in macrophages in generation of
antigen-specific T cell responses in vivo. Splenocytes isolated from mBSA
immunised IKKΔMye mice showed increased antigen-specific IFNγ production
when restimulated with mBSA in vitro (Figure 3.2.4.3.1), suggesting IKK in
macrophages inhibits the development of TH1 responses in vivo.
However,
splenocytes are a mixed cell population, to specifically measure CD4+ T cell
responses, CD4+ T cells were purified from mBSA immunised IKKΔMye mice
and co-cultured with mBSA pre-loaded BMDM. Increased IFN production was
detected from T cells isolated from IKKΔMye mice, although high basal IFN
production made it difficult to conclude the effect of IKK on antigen-specific
IFNγ production (Figure 3.2.4.4.1). These data provide an indication that IKK
in macrophages modulates development of TH1 responses, however, the
limitations of the experimental system leave some unanswered questions.
170
I have used CD4+ T cells isolated from immunised mice, purified CD4+ T cells
can be heterogenous and consist of naïve cells, effector cells, memory and
regulatory T cells.
Although my studies show IKK inhibits ability of
macrophage to activate TH1 cells in vitro, it isn’t clear what affect IKK on T
cell responses in vivo. Mice were immunised with mBSA/CFA and CD4+ T cells
were collected on day 21. Naïve T cells are ‘primed’ during immunisation with
mBSA/CFA. In this model, mBSA specific T cells develop a TH1 phenotype
dependent on production of IL-12 by APC. Once naïve T cells develop into TH1
lineage, IL-12 receptor (IL-12R) expression is increased and consequently
creates a positive feedback loop to reinforce the responsiveness to IL-12 (Rogge,
Barberis-Maino et al. 1997; Szabo, Dighe et al. 1997). In addition to IL-12R, IL18 receptor is also selectively induced on TH1 cells after activation (Xu, Chan et
al. 1998). Cooperation between IL-12 and IL-18 has been reported as an antigenindependent means of inducing IFN production by T cells (Jianfei Yang 1999).
This provides a boost for persistent IFN production for and complete clearance
of infection. The background of antigen-independent IFNγ production observed
in some of my experiments may be due to increased IL-12 and IL-18 produced
by macrophages in co-culture. Differentiated TH1 cells isolated from mBSA
immunised mice express both IL-12 receptor and IL-18 receptor (Xu, Chan et al.
1998) (Szabo, Dighe et al. 1997) and responded to these cytokines to produce
IFNγ without the requirement of antigen.
171
It is difficult to conclude from my experiments if IKK in macrophages inhibits
T cell priming or maintenance of the TH1 response in vivo. Immunisation was
performed intradermally, although macrophages have been demonstrated as a
major cell population in the mouse dermis (Dupasquier, Stoitzner et al. 2004),
other cells such as Langerhans cells and dermal dendritic cells are also abundant.
It is possible that Langerhan cells and dermis DCs are responsible for CD4+ T
cell priming and macrophages trigger activation of primed CD4+ T cell as APC.
To address this question, I have taken advantage of OTII transgenic mice with
TCR specific for ovalbumin peptide (OVA323-339), T cells from OTII mice have
not encountered antigen (OVA323-339) previously and therefore display a naïve
phenotype, thus OTII mice provide an ideal source of naïve T cells with defined
antigen-specificity.
Using OTII mice, the inhibitory role of IKKβ in the
development of TH1 responses was confirmed; IKK∆Mye macrophages were able
to activate naïve OTII T cells and enhance antigen specific IFN production
(Figure 3.2.4.6.1). This data suggests IKK activation in macrophages could
inhibit T cell priming at least in vitro and confirms an inhibitory role for IKK in
the development of TH1 cells and subsequent TH1 responses.
However, to
further confirm the capacity of macrophage on priming naïve CD4+ T cells,
further experiments will be required; naïve T cells (CD4+CD62L+CD44-CD25-)
can be selected from OTII cells by cell sorting and co-cultured with IKK∆Mye
172
and IKKF/F macrophages with or without antigen and IFN production can be
measured by ELISA as described.
In normal circumstances, TH1 and TH2 cells produce specific cytokines to
promote and maintain the cell lineage (Abbas, Murphy et al. 1996) (O'Garra
1998). At the same time, cytokines produce by TH1 or TH2 cells inhibit the
development of the opposing subset (Abbas, Murphy et al. 1996). Quantitative
RT-PCR analysis of the OTII T cells co-cultured with IKK∆Mye macrophages
showed increased expression of TH1 characteristic genes; IFN, IRF1, IL-12R1
and IL-12R2 but reduced expression of the TH2 gene, IL-4. IRF1 has been
identified as a transcription factor involved in TH1 cell development (Lohoff,
Ferrick et al. 1997; Kano, Sato et al. 2008) (Taki, Sato et al. 1997), CD4+ T cells
from IRF-/- DO11.10 TCR transgenic mice fail to differentiate into IFNγ
producing TH1 cells when stimulated with OVA in the presence of APC. IL12R1 and IL-12R2 are subunits of IL-12 receptor that are selectively induced
by TH1 cells (Rogge, Barberis-Maino et al. 1997; Szabo, Dighe et al. 1997).
These data support the conclusion that IKKβ activation in macrophages
negatively regulates TH1 cell development. The experiments described above
with OTII T cells suggest IKKMye macrophages can increase priming of naïve
T cells. The role of macrophages in priming naïve T cells is controversial and is
in conflict with the general belief that DCs are unique in this capacity
(Banchereau and Steinman 1998).
However, accumulating evidence has
revealed this may not always be the case and macrophages can also prime naïve
T cells. Direct comparison of naïve T cell activation by DCs and macrophages
173
was performed by Rothoeft et al. 2003 who showed that both cell populations are
able to activate naïve T cells (T. Rothoeft 2003). DCs were shown to be more
efficient in inducing TH1 responses, as demonstrated by the enhanced IL-2 and
IFNγ production, while the macrophages selectively induced IL-4 production (T.
Rothoeft 2003). Similarly, Moser et al reported that adoptive transfer of antigen
pulsed macrophages could prime T cells in vivo and TH2 cell development was
selectively elicited by macrophages when compared to DCs that have skewed
cells to TH1 differentiation (Moser.M 2001). These data are consistent with the
idea macrophages can prime naïve T cells and favour TH2 responses in normal
circumstances.
Deleting IKK in macrophages not only increases T cell
activation but favours TH1 responses rather than TH2, it may be possible that
IKKMye macrophages are unable to promote TH2 responses and therefore
favour TH1 cell activation.
3.2.7.3 The role of TNF in TH1 responses
Despite the fact TNF is a major pro-inflammatory cytokine, increasing
evidence suggests a negative regulatory role of TNF in TH1 responses. For
example, TNF was shown to limit T cell activation by promoting apoptosis
(Kanaly, Nashleanas et al. 1999) and TNF can dampen TH1 cell activation by
inhibiting IL-12 production by APC (Hodge-Dufour, Marino et al. 1998; Notley,
Inglis et al. 2008). Previous studies have also shown increased lethality of
TNFR1-/- mice after infection with Mycobacterium avium, associated with
elevated systemic IL-12 levels and increased CD4 and CD8 T cell accumulation
(Ehlers, Kutsch et al. 2000). Similar observations were made in a lung infection
174
model; TNF-/- mice succumbed to lung failure after infection with M.bovis
BCG. These mice suffered a severe type I immune response, illustrated by
elevated IL-12 and uncontrolled CD4 and CD8 T cell expansion. Recently, the
inhibitory effect of TNF on TH1 cells was further demonstrated in an
autoimmune disease (collagen induced arthritis - CIA) by Notley et al 2008.
Expanded TH1 cells were found in the lymph nodes of TNFR1 deficient mice,
further analysis with TNFR1 deficient mice showed increased IL-12p40
expression. Collectively, these studies suggest a negative regulatory role for
TNF in the type I inflammatory responses. Although it has been shown IL-12
expression was inhibited by TNF in macrophages and dendritic cells (Ma, Sun
et al. 2000; Zakharova and Ziegler 2005; Notley, Inglis et al. 2008), the
inhibitory effect of TNF on APC activity and the influence of T cell activation
have not previously been studied. My studies have shown TNF treatment
suppresses MHC II and CD80 expression on BMDM (Figure 3.2.5.1.1),
furthermore decreased allogenic T cell proliferation (Figure 3.2.5.2.1) and
antigen-specific IFN production by OTII T cells (Figure 3.2.5.3.1) were
obtained from BMDM pre-treated with TNF. Quantitative RT-PCR analysis of
OTII T cells showed a reduction of TH1 genes including IFN, IRF1, and IL122 when compared with T cells co-cultured with BMDM without TNF pretreatment. Taken together, these results clearly demonstrate the inhibitory effect
of TNF on APC function and the subsequent influence on T cell activity.
However, these data appear to be in contradiction with the success of anti-TNF
therapy in patients with autoimmune diseases (Feldmann and Maini 2003). But
recent study of collagen induced arthritis by Notley et al 2008 has shown TNF
175
inhibition can expand TH1 cells, this was limited to the lymph nodes and not in
the joint, which may explain how anti-TNF therapy ameliorates arthritis despite
increasing numbers of potentially pathogenic T cells.
3.3 IKKMye macrophages increase anti-tumour immunity
Chronic inflammation has been shown to be associated with the development of
human malignant diseases (H. Kuper 2000; Mantovani, Allavena et al. 2008)
(Schottenfeld and Beebe-Dimmer 2006). The immune system is capable to
recognise and remove malignant cells (Ostrand-Rosenberg 2008), however,
tumours generate a microenvironment that prevents the development of
protective anti-tumour immunity (Halak, Maguire et al. 1999; Yang and Lattime
2003). Tumours frequently have a significant macrophage infiltrate, but TAM
acquire a M2 phenotype that promotes immunosupression through increased IL10 production and reduced IL-12 expression (Yang and Lattime 2003;
Hagemann, Lawrence et al. 2008). From the previous data, I have shown that
knocking out IKK in macrophages increases iNOS production and TH1 cell
development. TH1 cytokines; IL-12 and IFN are critical cytokines for protective
anti-tumour immunity, I therefore predicted that knocking out IKK in TAM
would reverse the M2 phenotype and enhance the activity of effector T cells and
consequently increase anti-tumour immunity.
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3.3.1 Targeting IKK in macrophages inhibits MB49 tumour growth in female
mice
MB49 is a bladder carcinoma cell line derived from a male C57BL/6 mouse.
MB49 cells express the male minor histocompatibility antigen H-Y, and
therefore when grown in syngenic female mice, H-Y provides a surrogate
tumour-specific antigen which can be used to study anti-tumour immune
responses. To investigate the role of IKKβ in TAM, MB49 cells were injected
s.c to IKKF/F and IKK∆Mye female mice, and tumour growth was measured
every other day for 13 days. As shown in Figure 3.3.1.1, tumour growth was
similar between IKKF/F and IKK∆Mye female mice up to 7 days, however
tumour growth completely regressed between 7-12 days in IKK∆Mye female
mice. In contrast, the growth of tumours in IKKF/F female mice continued to
progress until experimental limits were reached.
***
**
Figure 3.3.1 1 Targeting IKK in macrophages promotes rejection of MB49
tumours in female mice
177
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye female
mice. Tumour size was measured every other day with electronic callipers for 13 days.
Data represents mean+sem of n=10.
Experiments were repeated 3 times and
representative data are shown. Two way ANOVA and post-hoc Bonferroni were used
for statistical analysis; ** P< 0.01, *** P<0.001
3.3.2 Targeting IKK in macrophages does not inhibit MB49 tumour growth in
male mice
The growth of MB49 tumours was completely suppressed in IKK∆Mye female
mice. To determine if the inhibition of tumour growth in IKK∆Mye female mice
was due to H-Y specific anti-tumour immunity, MB49 cells were injected s.c to
IKK∆Mye and IKKF/F male mice and tumour size was measured every other day
for 12 days. As shown in Figure 3.3.2.1, tumour growth rate was consistent
between IKKF/F and IKKMye male mice throughout the period monitored.
Between days 7-12, the size of tumour was slightly smaller in IKK∆Mye mice
when compared to the control mice.
However, tumours from both groups
continue to progress and no suppression was observed in IKKMye male mice,
this data suggested rejection of MB49 tumours in IKKMye female mice is
dependent on immune response to H-Y antigen.
178
Figure 3.3.2 1 Targeting IKK in macrophages does not affect MB49 tumour
growth in male mice
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye male
mice. Tumour size was monitored every other day for 12 days.
Data represents
mean+sem of n=10. Experiments were repeated 3 times and representative data are
shown.
179
3.3.3 IKKMye splenocytes increase H-Y specific IFN production ex-vivo
Previous data shows MB49 tumours were rejected in IKKβ∆Mye female mice but
not in IKKβ∆Mye male mice.
These observations suggest that IKKβ in
macrophages might play a role in developing antigen (H-Y) specific anti-tumour
immunity. To test for H-Y specific immune responses, splenocytes from tumour
bearing IKKβ∆Mye female mice were co-cultured with naïve splenocytes from WT
syngeneic female or H-Y bearing male mice, and IFNγ was measured in culture
supernatant by ELISA. As shown in Figure 3.3.3.1, 8 fold of IFNγ production
was detected from IKKβ∆Mye splenocytes co-cultured with H-Y bearing male
splenocytes compared to female splenocytes.
No IFNγ was detected from
IKKβF/F splenocytes co-cultured with either male or female splenocytes.
180
***
***
ND
ND
Figure 3.3.3 1 H-Y restimulation assay with splenocytes from MB49 tumourbearing female mice
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye female
mice. Spleens were collected on day 12 and splenocytes were co-cultured with H-Y
bearing splenocytes from male mice or H-Y negative female splenocytes. Tissue culture
supernatant was collected after 24 h and IFN production was measured by ELISA. ND
– not detected. Data represents mean+sem of triplicates. Experiments were repeated 3
times and representative data are shown. Two way ANOVA and post-hoc Bonferroni
tests were used for statistical analysis; *** P<0.001
181
3.3.4 Targeting IKK in macrophages inhibits the immunosuppressive tumour
microenvironment
Solid tumours generate a microenvironment to suppress immune responses and
promote their growth (Halak, Maguire et al. 1999; Yang and Lattime 2003). To
characterise the MB49 tumour microenvironment in IKKF/F and IKKMye mice,
tumours were collected on 7 days from IKKF/F and IKKMye female mice and
total RNA was isolated for qRT-PCR analysis. As shown in Figure 3.3.4.1A, the
mRNA levels of IFNγ were 2 fold higher in tumours of IKKβ∆Mye compared to
the control group. IP10, a chemokine for TH1 cells and has been shown to have
anti-tumour activity (Angiolillo AL 1995; Dufour, Dziejman et al. 2002). I thus
tested if IKK inhibited IP10 expression in MB49 tumours, as shown in Figure
3.3.4.1B, mRNA expression of IP10 was 30% higher in tumours of IKKβ∆Mye
mice when compared to the control group. In contrast, the expression of CD206
(MR) and arginase-1 – both established markers of TAM and M2 macrophages,
were reduced in IKK∆Mye tumours. In the case of CD206, 50% reduction of
CD206 mRNA expression was detected in tumours of IKKβ∆Mye mice (Figure
3.3.4.1D) and slightly lower arginase-1 mRNA expression was obtained although
this was not statistically significant (Figure 3.3.4.1C).
182
A
IFN
B
*
C
Arginase-1
IP10
*
D
CD206
*
Figure 3.3.4 1 Gene expression in MB49 tumours from IKKF/F and IKKMye
female mice
183
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye female
mice. Tumours were collected on day 7 and total RNA was isolated for qRT-PCR
analysis. Data represents mean±SEM of 8 animals. Student T test was used to test
statistical significance; * P<0.05
To determine if the enhanced IFN RNA level was related to tumour infiltrating
T cells (TIL). Tumours were collected from IKKβF/F and IKKβ∆Mye female mice
after 7 days and intracellular IFNγ was measured from CD3+ T cells by FACS.
In tumours isolated from IKKF/F mice, 9.49% of CD3+ T cells were IFNγ
positive (MFI; 12.9). In comparison, there were 22.3% IFNγ positive CD3+ T
cells (MFI; 24.8) detected in tumours isolated from IKKMye mice, suggesting
the enhanced IFN mRNA was related to CD3+ T cells (Figure 3.3.4.2).
100
IFN
% of Max
80
MFI-12.9
MFI-24.8
% positive cells
IKKβF/F
IKKβ∆Mye
60
40
20
0
10
0
10
1
2
10
FL2-H
10
3
10
4
Figure 3.3.4 2 CD3+ tumour infiltrating lymphocytes from IKKβ∆Mye mice
expressed higher intracellular IFNγ
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye female
mice. Tumours were collected on day 7 and intracellular IFN was measured on CD3+ T
184
cells by FACS. MFI is indicated. Black – isotype control, Red – IKKβF/F mice Blue IKKβ∆Mye mice.
TAM express the phenotype of M2 macrophages which are predominantly
involved in anti-inflammation and tissue repair (Mantovani, Sozzani et al. 2002).
Previous studies from our group showed that knocking out IKKβ altered the M2
phenotype of TAM into a pro-inflammatory M1 phenotype (Hagemann,
Lawrence et al. 2008).
To determine if knocking out IKKβ in macrophages
could alter the immunsuppressive phenotype of TAM in MB49 tumours, tumours
were collected on day 7 and the TAM phenotype was analysed by FACS. IL4R is required for the development of M2 macrophages (Gordon 2003), hence
IL-4R expression was studied on tumour associated F4/80+ cells (TAM). As
shown in Figure 3.3.4.3A, the expression of IL-4R (MFI; IKKβF/F 24 vs
IKKβ∆Mye 7.5) was 70% lower on F4/80+ cells from IKKβ∆Mye mice while MHC
II expression (MFI; IKKβF/F 770.0 vs IKKβ∆Mye 1116) (Figure 3.3.4.3B), a
marker of M1 macrophage activation was increased. These data suggest a role of
IKK in skewing the M2 phenotype of TAM and blockade of IKK could
restore the proinflammatory M1 phenotype.
185
A
B
100
100
IL-4R
MFI-24
MFI-7.5
60
40
MFI-770.9
MFI-1116
80
% of Max
% of Max
80
IKKβF/F
IKKβ∆Mye
MHC II
60
40
20
20
0
100
101
102
IL-4 receptor
103
104
0
100
101
102
MHC II
103
104
Figure 3.3.4 3 IL-4R and MHC II expression on TAM from MB49 tumour
0.5x106 MB49 cells were injected s.c to the right flank of IKK F/F and IKK Mye female
mice. Tumours were collected on day 7 and IL-4R and MHC II were measured on
F4/80+ cells by FACS. Black – isotype control, Red – IKKβF/F mice Blue - IKKβ∆Mye
mice.
3.3.5 MB49 tumour rejection in IKKMye female mice is CD8 dependent
From the previous data, I have shown that IFN expression was increased in the
MB49 tumour from IKKMye female mice. FACS analysis showed increased
IFN production by TIL. CD8+ cytotoxic T lymphocytes (CTL) are critical
effector cells in tumour regression, to determine if MB49 tumour rejection
observed in IKKMye female mice was mediated by CTL, anti-CD8 antibody
186
was administered to IKKMye female mice before injection of MB49 tumour
cells. Mice were culled when the tumour size reached experimental limits and
data are represented as percentage survival. As expected, tumour rejection was
observed in untreated IKKMye female mice, however treatment with anti-CD8
antibody resulted in a dramatic reduction in survival (Figure 3.3.5.1), indicating
CD8 T cells are required for MB49 tumour rejection.
+IgG
**
Figure 3.3.5 1 Tumour rejection in IKKMye female mice was mediated by CD8+ T
cells
On day 1 and 3, IKK Mye female mice were injected i.p with or without 0.2ml of antiCD8 antibody or IgG isotype control. 0.5x106 MB49 cells were injected s.c to the right
flank on day 4 and tumour size was monitored every other days. Mice were culled when
the tumour size reached the experimental limit and data are representing as survival of
mice in each group over time.
n=8.
Log-rank test was used to test statistical
significance; ** P<0.01
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3.3.6 MB49 tumour rejection in male mice after Dxr treatment is CD8 T cell
dependent
Chemotherapy and radiotherapy are conventional treatments for many cancers,
recently it has been shown the response to chemo- and radio-therapies is
dependent on adaptive immune responses (Apetoh, Ghiringhelli et al. 2007;
Zitvogel, Apetoh et al. 2008). In response to chemotherapy, it was reported that
dying tumour cells release high mobility group box 1 (HMGB1) that can trigger
TLR4 on APC and elicit the activation of anti-tumour immunity (Apetoh,
Ghiringhelli et al. 2007). From earlier data, IKKMye macrophages were shown
to promote TH1 cell development, furthermore CD8+ T cell-associated tumour
rejection was observed in IKKMye female mice and enhanced anti-tumour
immunity was associated with increased response to tumour antigen.
To
determine if IKKMye macrophages can also enhance immune responses against
other tumour antigens revealed during chemotherapy, I administrated
doxorubicin (Dxr) to IKKF/F and IKKMye male mice with established tumours.
According to the previous experiment, MB49 tumours reached experimental
limits after 14 days from both IKKF/F and IKKMye male mice (Figure 3.3.2.1).
188
As shown in Figure 3.3.6.1, Dxr treatment significantly prolonged the survival
time of IKKF/F mice to 36 days however 80% of mice eventually reached
experimental limits.
But 70% of IKKMye mice receiving Dxr remained
survived the course of the experiment, showing a significant increase in survival.
To test if the response to Dxr in IKKMye mice requires CD8+ T cells, anti-CD8
antibody was administered to IKKMye male mice twice before MB49 tumour
cells injection and Dxr was given every other day as described. 90% of IKKMye
male mice reached experimental limits after 23 days when treated with Dxr and
anti-CD8 antibody.
Taken together, these data suggest inhibition of IKK in
macrophages enhances anti-tumour immunity and increase immune responses to
antigens revealed by chemotherapy.
***
*
Figure 3.3.6 1 Targeting IKK in TAM increased response to chemotherapy
On day 1 and 3, IKKMye male mice were injected with 0.2ml of IgG or anti-CD8
antibody i.p. On day 4, 0.5x106 MB49 tumour cells were injected s.c to the right flank
of all mice. When the tumour size reached 0.5cm2, mice were injected with 0.2ml
2mg/kg doxorubicin i.p three times per week. Mice were culled when the tumour size
189
reached the experimental limit and data are indicated as percentage survival. n=10 Logrank test was used to test statistical significance; * P<0.05, *** P<0.001
3.3.7 Summary
NF-B is a ubiquitous transcription factor that plays an important role in both
immunity and cancer (Karin, Cao et al. 2002; Karin 2006). Earlier data have
shown that knocking out IKKβ in macrophages increases IL-12, MHC II, and
costimulatory
molecule
expression,
furthermore,
IKKMye
macrophages
enhanced TH1 cell development and activation. These factors are critical in the
development of protective anti-tumour immunity, therefore the aim of this
section was to evaluate the hypothesis that blocking IKKβ in TAM will enhance
protective anti-tumour immunity.
MB49 tumour cells were injected s.c to
IKKF/F and IKKMye female mice and tumour growth was measured for 2
weeks. From days 0-7, both animal groups display no difference in tumour
growth. However, tumour regression was observed from day 7 in IKKβMye
female mice and tumours were completely rejected after day 13.
To investigate
if tumour rejection in IKKMye female mice was due to H-Y specific antitumour immunity, the experiment was repeated in IKKF/F and IKKMye H-Y
bearing male mice. Both groups had similar rates of tumour growth and reached
experimental limits within 14 days. To test if H-Y specific tumour immunity had
190
developed in IKKβMye female mice, a H-Y re-stimulation assay was preformed.
Splenocytes from tumour bearing IKKF/F and IKKMye female mice were
cultured with splenocytes from WT syngeneic female or H-Y bearing male mice.
Splenocytes of IKKMye mice had dramatically increased IFN production when
co-cultured with H-Y bearing male splenocytes. These data support the
hypothesis that IKKβ activation in TAM is important in the regulation of antigen
specific tumour immunity.
Characterisation of the tumour microenvironment in IKKMye female mice
suggested a switch from an immunosuppressive (CD206High Arginase-1High
IFNLow IP10Low) phenotype to a more proinflammatory and immuno-stimulatory
environment (CD206Low Arginase-1Low IFNHigh IP10High). Intracellular staining
also revealed increased IFN expression by TIL.
Finally, chemotherapy of
MB49 tumours was greatly enhanced in IKKMye male mice that were
dependent on the presence of CD8+ T cells, despite the absence of anti-H-Y
immune responses. These data suggest inhibition of IKK in macrophages can
increase immune responses to tumour antigens revealed by chemotherapy.
191
3.3.8 Discussion
3.3.8.1 IKK deletion in macrophages enhanced H-Y antigen specific antitumour immunity
Previous studies by our group have shown targeting IKK in macrophages
increased resistance to infection (Fong, Bebien et al. 2008). In vitro analysis
confirmed enhanced iNOS production (Figurer 3.1.2.1) and bacterial killing by
IKKMye macrophages.
In addition to anti-bacterial activity, iNOS/NO
production also contributes to the tumoricidal activity of macrophages (Chang,
Liao et al. 2001). Earlier studies from our lab showed IKKβ deletion in TAM
enhanced tumoricidal activity through increased NO production (Hagemann,
Lawrence et al. 2008), furthermore these studies also showed targeting IKK in
TAM increased IL-12 expression and NK cell mediated cytotoxicity towards
tumour cells. Collectively, these data suggest targeting IKKβ in TAM increased
innate immunity to cancer cells. Adaptive immunity is also important for tumour
suppression, I have shown IKK activation in macrophages inhibits cellmediated immunity in the previous sections, to study the role of IKK in the
development of anti-tumour immune responses, I used the MB49 bladder
192
carcinoma cell line; MB49 tumour cells express the male H-Y antigen, therefore
when grown in syngeneic female mice, H-Y antigen provides a surrogate
tumour-specific Ag to track anti-tumour immune responses. MB49 tumours were
completely rejected by IKKMye female mice after 13 days (Figure 3.3.1.1). On
the other hand, no tumour rejection was observed from IKKMye male mice
(Figure 3.3.2.1). MB49 tumour rejection in IKKMye female mice and not male
mice indicates tumour rejection is H-Y dependent and suggest the development
of anti-tumour immunity to H-Y antigen.
Increased H-Y specific immune
responses was confirmed by further analysis with antigen-recall assay;
splenocytes from MB49 tumour bearing IKK∆Mye female mice showed
significantly enhanced IFNγ production when co-cultured with H-Y positive
male splenocytes (Figure 3.3.3.1). This is consistent with the conclusion MB49
rejection in IKK∆Mye female mice is due to increased anti-H-Y immune
responses. Intracellular cytokine staining of MB49 tumour cell suspensions
showed increased IFN expression in CD3+ tumour infiltrating lymphocytes
from IKKβ∆Mye female mice (Figure 3.3.4.2).
This also suggests tumour
rejection might be dependent or partially dependent on enhanced cytotoxic T
lymphocyte activity. CD8+ T cell depletion in IKKβ∆Mye female mice prior to
MB49 tumour growth confirmed a role for CD8+ T cells in tumour rejection; no
tumour rejection was observed in IKKβ∆Mye female mice receiving anti-CD8
depleting antibody (Figure 3.3.5.1). These data indicate IKKβ activation in TAM
inhibits the activation of tumour-specific immune responses.
193
3.3.8.2 Mechanisms for tumour rejection in IKKβ∆Mye mice
Several mechanisms might be lead to enhanced CTL activity in IKKβ∆Mye mice.
First, knocking out IKK in macrophages could enhance CTL activity directly,
as observed in CD4+ T cells in previous sections, the capacity of macrophages to
increase CTL activity has previously been demonstrated (Pozzi, Maciaszek et al.
2005). Pozzi et al compared the ability of Ag-pulsed BMDM and DCs to trigger
CTL activation in vivo and macrophages were comparable with DCs in their
ability to stimulate CTL proliferation, express effector functions and mature into
memory cells (Pozzi, Maciaszek et al. 2005). Accumulating evidence has also
shown the importance of CD4+ T cell in CTL-mediated anti-tumour immunity
(Marzo, Kinnear et al. 2000; Gao, Khammanivong et al. 2002; Wang and
Livingstone 2003). Marzo et al 2000 have shown that CD4+ T cells are not only
required for the infiltration of CTL into tumours, but also the induction of CTL
effector function (Marzo, Kinnear et al. 2000). Furthermore, a study from Gao et
al 2002 has shown the requirement of CD4+ T cells in activating memory CD8+
T cells to facilitate tumour killing (Gao, Khammanivong et al. 2002). CD4+ T
cell help for CTL activity is also dependent on interactions with APC (Bennett,
Carbone et al. 1998; Ridge, Di Rosa et al. 1998; Schoenberger SP 1998). Thus,
CD4+ T cells may contribute to tumour rejection in the IKKMye female mice by
enhancing CTL activation. Another possibility is elevated IFN produced by
CD4+ T cells or CTLs may increase cytotoxic activity of NK cells and TAM.
Further experiments are required to elucidate the exact mechanism for increased
CD8+ dependent tumour regression in IKKMye mice.
194
3.3.8.3 IKK deletion in macrophages response to chemotherapy
In response to chemotherapy tumour cells undergo apoptosis and necrosis,
apoptosis is silent cell death that is characterised by chromatin condensation
(pyknosis) and nuclear fragmentation (karyorhexis) occurring within an intact
plasma membrane (Zamzami and Kroemer 1999), in contrast necrotic cells
release damage associated molecular patterns (DAMPs) including; hyaluronans,
heat shock proteins (HSPs), fibronectin and high mobility group box 1 (HMGB1)
(Marshak-Rothstein 2006). DAMPs have been suggested to activate TLRs and
trigger immune responses (Marshak-Rothstein 2006) (Apetoh, Ghiringhelli et al.
2007). Apetoh et al. have shown that HMGB1 released by dying tumour cells
activates DCs via TLR4 to elicit activation of antigen specific T cell immunity.
To test the role of IKK in this context I used chemotherapy treatment of MB49
tumours. IKKMye male mice treated with doxorubicin showed significantly
prolonged survival and tumours were rejected in 70% of the mice compared to
control mice that all eventually reached experimental limits (Figure 3.3.6.1).
Depleting CD8+ T cells in these mice reversed this effect as demonstrated by
reduced survival and tumour rejection was decreased to just 10%.
These
experiments suggest targeting IKK in TAM not only increases immune
responses specific to H-Y in female mice as shown previously, but also increased
the immune response to other antigen revealed upon chemotherapy treatment.
3.3.8.4 IKK maintains tumour-induced tolerance
From the previous studies, Halak et al. have shown MB49 tumour cells inhibit
priming of H-Y specific type I responses in female mice (Halak, Maguire et al.
195
1999). However, in my experiments MB49 tumours were rejected in female mice
when IKK is deleted in macrophages (Figure 3.3.1.1) and H-Y specific type I
responses developed as illustrated from the antigen-recall assay (Figure 3.3.3.1).
These studies suggest a role for IKK in tumour-induced immune tolerance. It
was reported that increased production of anti-inflammatory cytokines such as
IL-10 are found in the tumour microenvironment that suppress anti-tumour
immunity (Halak, Maguire et al. 1999; Jarnicki, Lysaght et al. 2006). Halak and
colleagues have shown that MB49 failed to prime an HY specific type I (IFN)
responses in WT female mice while HY specific type I (IFN) responses were
able to induce from IL-10-/- mice. Furthermore, IL-10-/- mice show prolonged
survival and increased capacity to reject MB49 tumours.
Because IL-10 production was measured in MB49 tumours in WT mice but not
IL-10-/- mice, whose infiltrating cells are unable to produce IL-10 (Halak,
Maguire et al. 1999), authors suggested that MB49 tumour induces infiltrating
cells to produce IL-10, which consequently suppress the development of antitumour immunity. Elevated IL-10 has been found in cancer patients and IL-10
production is often correlated with the induction of T cell anergy (De Vita,
Orditura et al. 2000) (Ferdinando De Vita 1999). It has also been shown that
DCs cultured with IL-10 have reduced capacity to stimulate T cells and instead
promote anergy (Steinbrink, Wolfl et al. 1997). IL-10 can also promote the
development of regulatory T cells that actively suppress immune responses
(Groux, O'Garra et al. 1997) (Asseman C 1998). TAMs are potential sources for
the IL-10 production in MB49 tumours (Hagemann, Lawrence et al. 2008),
previous studies from our group showed deleting IKK in TAM could switch the
196
TAM phenotype (IL-10 high, IL-12 low) to a M1 phenotype (IL-10 low, IL-12
high) (Hagemann, Lawrence et al. 2008). These studies suggest IKK activation
in TAM may promote tumour-induced tolerance, possibly mediated by IL-10
production, blocking IKK activity can reverse tolerance by switching off IL-10
production. Further experiments targeting IL-10 in IKKMye mice are required
to confirm this hypothesis.
4. General discussion
4.1 IKK/NF-B inhibits classical macrophage activation
NF-B transcription factors regulate expression of many genes important in the
inflammatory response (Li and Verma 2002), the NF-B signalling pathway is
thus considered a potential target for new anti-inflammatory drugs (Li and
Verma 2002; Karin, Yamamoto et al. 2004). However, recent research has
revealed roles for NF-B in the resolution of inflammation. Lawrence et al.
2001 reported that NF-B activity is required for leukocyte apoptosis during the
resolving phase of inflammation (Lawrence, Gilroy et al. 2001). More recently,
mice with IKK deletion in myeloid cells were shown to have elevated plasma
IL-1 and increased sensitivity to endotoxin-induced shock (Greten, Arkan et al.
2007). Moreover, recent studies published by our group have shown enhanced
bacterial clearance in mice with a myeloid-specific deletion of IKK, but the
opposite was observed in mice with IKK deletion in lung epithelial cells,
197
suggesting this anti-inflammatory role for IKK is tissue specific (Fong, Bebien
et al. 2008).
4.2 NF-B-STAT signalling and regulation of macrophage activation
NF-B mediated inflammatory responses are crucial during infection, however
fine-tuning the amplitude of the response to balance effective pathogen clearance
and avoiding excessive inflammation are equally important. Effective pathogen
clearance requires cooperation between innate and acquired immune responses.
For example, LPS, a component of gram-negative bacteria and the TH1-cytokine
IFN elicit M1 macrophage activation. M1 macrophages are important effector
cells that are characterised by increased production of NO, reactive oxygen
intermediates and inflammatory cytokines such as IL-12 that promote TH1
responses.
On the other hand, IL-4 promotes the development of M2
macrophages, characterised by production of anti-inflammatory cytokines such
as TGF and IL-10. Moreover, arginase 1 (Arg1) expression is also considered a
typical marker for M2 macrophages, which not only counteracts NO production
by iNOS but also inhibits T cell activation. In general, M1 macrophages are
potent effector cells that kill microorganisms, tumour cells, produce
proinflammatory cytokines and promote the development of adaptive immune
responses. In contrast, macrophages with M2 phenotype are more prone to
198
produce anti-inflammatory cytokines, suppress adaptive TH1 immunity and
promote angiogenesis (Mantovani, Sozzani et al. 2002; Gordon 2003; Gordon
and Taylor 2005).
Macrophages are able to switch their phenotype in response to changes in the
environment (Stout and Suttles 2004), a recent study showed M2 macrophages
elicited by helminth infection can be reprogrammed to an M1 phenotype by LPS
and IFNγ stimulation (Mylonas, Nair et al. 2009).
These studies demonstrate
the plasticity of macrophage phenotype, however the molecular mechanisms
involved are still unknown. A role for NF-B signalling in the M2-like TAM
phenotype was indicated in a study by Biswas et al. 2006. Microarray analysis
showed several NF-B regulated genes including IL-6, TNF-alpha and CCL3
were reduced in LPS stimulated TAMs compared to peritoneal macrophages
(Biswas SK 2006). In a mouse ovarian cancer model, Hagemann and colleagues
have also shown a role for IKK in maintenance of the IL-10High/IL-12Low TAM
phenotype (Hagemann, Lawrence et al. 2008). Furthermore, enhanced
tumoricidal activity was observed associated by increased iNOS expression and
IL-12 dependent activation of NK cells. Previous studies from our group have
shown increased activation of M1 macrophages in IKKMye mice infected with
Cryptococcus neoformans (Cn), a pathogen that promotes an M2-phenotype
(Fong, Bebien et al. 2008). These studies indicate a role for IKK/NF-B
activation in M2/TAM phenotype and activity.
I have shown that knocking out IKK in macrophages promotes a M1 phenotype
based on the following observations;
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4.2.1 Cytokine production
Specific cytokine production is a key feature of different macrophage subtypes;
M1 macrophage activation is characterized by proinflammatory cytokine
production, such as IL-12, IL-6 and TNF whereas M2 macrophages favour the
synthesis of anti-inflammatory cytokines IL-10 and TGF (Fenton, Buras et al.
1992; Schebesch, Kodelja et al. 1997). I have shown that blocking IKK with
adenovirus expressing dominant negative inhibitor in macrophages increased IL12 production and conditional deletion of IKK in macrophages increased IL-12,
iNOS and MHC II expression. Consistent with the current study, conditional
knock down IKK in TAM also showed elevated IL-12 production and reduction
in IL-10 (Hagemann, Lawrence et al. 2008). These studies suggest a role of
IKK in suppressing the proinflammatory phenotype of M1 macrophages.
4.2.2 iNOS-Arginase balance
From an earlier in vivo study (Fong, Bebien et al. 2008), immunohistochemical
analysis showed increase iNOS expression in alveolar macrophages from
IKKMye mice. Increased iNOS expression by IKKMye macrophages was
further confirmed in vitro (Figure 3.1.2.1).
The iNOS-arginase balance
represents one of the features to distinguish M1 and M2 macrophages (Munder,
Eichmann et al. 1998). M1 macrophages produce elevated NO by upregulating
iNOS, which uses L-arginine as a substrate for NO production.
In M2
macrophages, arginase is upregulated instead of iNOS (Hesse, Modolell et al.
2001), arginase hydrolyses L-arginine into L-ornithine and urea, therefore
depleting the substrate for iNOS-mediated NO production. L-ornithine is also a
200
metabolite for polyamine and proline production.
The upregulation of NO
production contributes to microbicidal and tumoricidal activity of M1
macrophages (Fong, Bebien et al. 2008; Hagemann, Lawrence et al. 2008),
whereas prolines and polyamines produced by M2 macrophages and TAM are
important for wound healing and promotion of tumour growth respectively
(Song, Ouyang et al. 2000; Chang, Liao et al. 2001).
Over-expression of
arginase in macrophages can directly enhance tumour cell proliferation (Chang,
Liao et al. 2001). Furthermore, increased arginase activity can attenuate NO
production by LPS activated macrophages and reduce its cytotoxic activity
towards co-cultured tumour cells (Chang, Liao et al. 2001). These studies
suggest an important role of iNOS-Arginase balance in the effector functions of
M1 and M2 macrophages. My data shows a role for IKK in skewing the iNOSArginase balance by inhibiting iNOS expression that may have important
implications for macrophage effector function in infection and cancer.
4.2.3 APC activity
Induction of TH1 responses and CTL activation are other important roles of M1
macrophages in combating infection and tumour development.
In contrast,
TAMs or M2 macrophages have poor APC function and in fact express several
mechanisms to suppress T cell responses that may contribute to evasion of
protective immune responses.
For example; TGF-, an immunosuppressive
cytokine produced by TAMs, has been shown to interfere with the development
of anti-tumour immune responses (Mantovani, Sozzani et al. 2002). Microarray
analysis has shown TGF- suppresses genes encoding key effector proteins of
CTLs; perforin, granzymes A and B, Fas ligand, and IFN (Trapani 2005). TAM
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can also suppress T cell responses through induction of Treg cells. Neutralizing
IL-10 in vivo indicates the association of IL-10 production by TAM and Treg cell
development. (Kusmartsev, Nagaraj et al. 2005; Huang, Pan et al. 2006). In a
murine lung carcinoma model, arginase production by M2-like TAMs was
shown to decrease expression of T cell receptor CD3 chain and impair T cell
function (Rodriguez, Quiceno et al. 2004). My data demonstrate that knocking
out IKK in macrophages enhances the development of TH1 responses and antitumour immunity. Similar studies have shown conditional knock down IKK or
inhibition of IKK with adenovirus expressing dominant negative inhibitor in
TAMs increased the innate anti-tumour immune responses through increased
NO-mediated tumouricidal activity and IL-12 dependent NK cell recruitment
(Hagemann, Lawrence et al. 2008). These data suggest IKK inhibits innate and
adaptive protective immunity through blocking M1 macrophage activation.
4.3 Molecular mechanisms of macrophage regulation by IKK
Here I have further explored the inhibitory role of IKK in macrophage
activation and revealed negative cross-talk between IKK and STAT1 signalling.
Negative cross-talk between STAT1 and NF-B pathways was also indicated by
other studies of LPS tolerance. Pre-treatment with LPS results in a dramatic
reduction of IFN-induced DNA binding of STAT1 (Crespo, Filla et al. 2000).
Further experiments suggested accumulation of SOCS1 in response to LPS
contributed to STAT1 inhibition. LPS does not induce SOCS1 expression
directly, this was dependent on autocrine or paracrine IFN production (Crespo,
Filla et al. 2000). In addition to the indirect SOCS1 production, LPS can also
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directly induce expression of SOCS3 (Bode, Nimmesgern et al. 1999; Stoiber,
Kovarik et al. 1999), another SOCS family member that can suppress IFN
responses. The inhibitory effects of SOCS1 and SOCS3 has been reported on
iNOS (Ana Crespo 2002), IL-12 (Eyles, Metcalf et al. 2002) and MHC II
expression (Stoiber, Kovarik et al. 1999). The current study has shown that
IKK deficient (IKKMye) macrophages have increased STAT1 tyrosine
phosphorylation, MHC II expression, iNOS and IL-12 production in response to
bacteria, LPS alone or LPS plus IFN.
Quantitative RT-PCR has shown that
the mRNA of IFN was reduced in IKKMye macrophages and there were no
differences in expression of SOCS1/SOCS3 (Fong, Bebien et al. 2008), this
suggests IKK may inhibit STAT1 signalling by regulating the activity of SOCS
proteins rather than their expression. There are three ways that SOCS proteins
inhibit JAK-STAT signalling; SOCS1 and SOCS 3 directly inhibit JAK kinase
activity through their kinase inhibitory region (KIR) (Yasukawa H 1999). SOCSs
proteins also inhibit STAT signalling by competing with STATs for common
phosphotyrosine-binding (PTB) sites within the cytoplasmic domains of cytokine
receptors. The third mechanism of SOCS action is by promoting the degradation
of target signalling proteins through ubiquitin ligase activity (Krebs and Hilton
2001). IKK/NF-B could inhibit STAT1 activation by affecting one of these
mechanisms.
For example; the stability of SOCS proteins is regulated by
phosphorylation (Chen XP 2002; Haan, Ferguson et al. 2003) and degradation
through the 26S proteasome and this involves the interaction between SOCS box
and Elongin B and C (Zhang, Farley et al. 1999), IKK could modulate
phosphorylation of SOCS1 or its interaction to the regulatory proteins Elongin B
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and C. Further experiments are required to determine the affects of IKK on
SOCS1 activity and interaction with other regulatory proteins.
4.4 IL-4 signalling and STAT6
4.4.1 STAT1 and STAT6
IFN and IL-4 are the master regulators of TH1 and TH2 differentiation and their
distinctive roles in driving cellular and humoral immune responses. In addition to
driving TH1 and TH2 cell differentiation, IFN and IL-4 also antagonize each
other to ensure continuous development of the signature lineage (Abbas, Murphy
et al. 1996). Apart from T cells, IFN and IL-4 also regulate macrophage
phenotype; IFN signalling is required for M1 macrophage activation, suggesting
STAT1 is important for the M1 phenotype. On the other hand, IL-4 is critical for
M2 differentiation and thus STAT6 is likely the transcription factor required for
M2 activation. Several studies have demonstrated a suppressive role of IL-4 on
IFN/STAT1 regulated gene expression in mouse macrophages (Gautam, Tebo et
al. 1992; Deng, Ohmori et al. 1994; Ohmori and Hamilton 1997; Paludan,
Lovmand et al. 1997). However, the exact molecular mechanisms still remain
unclear. Ohmori and Hamilton have suggested a possible inhibitory mechanism
for IL-4 on STAT-mediated IRF1 expression (Ohmori and Hamilton 1997).
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They showed IL-4 treatment inhibited IRF1 induction when the IFN
concentration was low, this was suggest to be mediated by competition between
STAT6 and STAT1 for the binding of STAT binding element (SBE). STAT6
does not have trans-activation activity at the IRF1 promoter thus inhibits gene
transcription. However, this mechanism did not apply when the concentration of
IFN was high. From my studies, increased STAT1 phosphorylation was
observed in IKK deficient macrophages, it is possible the elevated level of
activated STAT1 might overcome STAT6 activity and promote M1 macrophage
phenotype (Figure 4.4.1.1).
Figure 4.4.1 1 Competition between STAT1 and STAT6 for SBE
Blocking IKK increased STAT1 phosphorylation which competes with STAT6 for
IRF1 SBE binding.
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4.4.2 STAT6 and NF-B
Interaction between STAT6 and NF-B could be another molecular mechanism
regulating macrophage phenotype. It has been shown that STAT6 and NF-B
cooperate and synergistically activate IgE promoter in B cells (Messner, Stutz et
al. 1997; Shen and Stavnezer 1998).
Shen and Stavnezer 1998 provided
evidence that STAT6 and NF-B interact directly and cooperatively bind their
cognate DNA binding sites (Shen and Stavnezer 1998).
Ablating IKK in
macrophages might limit the interaction between STAT6 and NF-B and
subsequently impair the expression of IL-4 responsive genes and consequently
suppress the M2 phenotype of macrophages.
4.4.3 IKK/NF-B and IL-4R expression
Two types of IL-4 receptors have been reported; type 1 IL-4 receptor consists of
IL-4 chain and c chain whereas type 2 receptor is composed of IL-4R and
IL-13R (Nelms, Keegan et al. 1999). The critical role of IL-4 on TH2 cell
development has resulted in much attention on IL-4R regulation on T cells, but
its regulation in macrophages is not well understood.
In T cells, several
intracellular signalling pathways have been reported to control IL-4R expression.
Human T cells stimulated with phorbol myristate acetate (PMA), the protein
kinase C (PKC) activator or the calcium ionophore A23187, increased IL-4R
206
expression at mRNA and protein level (Dokter, Borger et al. 1992). In addition,
IL-4 stimulated T cells show increased in IL-4R expression (Renz, Domenico et
al. 1991) and later studies have shown the role of STAT6 for IL-4R induction in
response to IL-4 (Kotanides and Reich 1996). In 1994, De Wit and colleagues
investigated the regulation of IL-4R in human monocytic cells and showed PKC
and c-AMP dependent pathways are involved in IL-4R gene expression while
calcium ionophore and IL-4 had no effect (de Wit, Hendriks et al. 1994). These
studies indicate IL-4R expression is differentially regulated between cells. In the
current study I have shown that IL-4R is absent on LPS stimulated IKKMye
macrophages, indicating the involvement of IKK/NF-B in the regulation of
IL-4R expression in macrophages. In the absence of IL-4R macrophages may
fail to respond to IL-4 and develop into M2 macrophages (Figure 4.4.3.1),
indicating IKK could promote M2 macrophage development by inducing IL4R expression.
207
Figure 4.4.1 2 IKK activation promotes IL-4R expression
IL-4/IL-13 mediated STAT6 activation antagonists STAT1 induced by IFN and
therefore inhibits M1 associated gene expression.
Deleting IKK in macrophages
suppress the expression of IL-4R that may limit their response to IL-4 or IL-13 and the
development of a M2 phenotype.
4.5 Inhibition of classical macrophage activation by TNF
Synergistic action between proinflammatory cytokines is an important facet of
the immune system. Both synergistic and antagonistic activities between IFN
(STAT1) and TNF signalling pathways have been documented. The
coordinated binding of TNF activated NF-B and IFN activated STAT1 or
IRF1, to specific promoters is a typical example for the enhancement of gene
transcription (Ohmori and Hamilton 1995; Ohmori, Schreiber et al. 1997).
Cross-talk between IFN and TNF also occurs at the receptor level of the
signalling pathways, an antibody screen for STAT1 binding protein found
208
TRADD to interact with STAT1 in a TNF dependent way in Hela cells
(Wang, Wu et al. 2000). Further experiments showed binding of STAT1 to the
TNFR1-TRADD signalling complex promoted TNF-induced apoptosis while
preventing NF-B activation (Wang, Wu et al. 2000). TNFR1 and STAT1
interaction was also shown by others as a negative regulator of TNF mediated
NF-B activation (Wesemann and Benveniste 2003). In STAT1 deficient cells,
TNF treatment leads to a more robust NF-B activation while restoration of
STAT1 inhibits TNF dependent NF-B activation. Although these studies
clearly demonstrated physical interaction between TNFR1/TRADD and STAT1
and their influence on NF-B activation, no experiments were performed to
investigate the effect of TRADD/STAT1 complex on IFN signalling. In 2004,
Wesemann et al. demonstrated that IFN induces the formation of a
TRADD/STAT1 complex. In TRADD-/- macrophages, IFN induced a stronger
and prolonged STAT1 activation. Furthermore, nuclear translocation, STAT1
DNA binding activity and transcription were enhanced in the TRADD-/- cells
(Wesemann, Qin et al. 2004). These data clearly indicate the formation of
TRADD/STAT1 complex negatively regulates IFN mediated STAT1 activation.
From the current study, I have shown that TNF inhibits STAT1
phosphorylation in BMDM. According to the study by Wesemann et al. 2004,
one possible mechanism may be the formation of a TRADD-STAT1 complex
that restricts the amount of STAT1 available for phosphorylation by JAK
signalling (Figure 4.5.1).
Furthermore, STAT1 phosphorylation was only
increased from TNFR1-/- (p55-/-) peritoneal macrophages and not TNFR2-/- (p75-/) macrophages (Figure 3.1.7.1). Upon TNF binding, the first protein recruited
209
to TNFR1 is TNFR1-associated death domain protein (TRADD), which serves a
platform to recruit FADD, RIP1, TRAF2 and subsequently initiate the signalling
cascade.
On the other hand, TNF binding to TNFR2 results in direct
recruitment of TRAF2, which in turn recruits TRAF1 (Rothe, Wong et al. 1994).
The absence of TRADD downstream of TNFR2 signalling pathway failed to
increase STAT1 activation from p75-/- macrophages as obtained from p55-/macrophages, hence suggesting this is a mechanism for TNFR1-specific
inhibition of STAT1 activation.
210
Figure 4.5 1 Possible mechanisms of the inhibitory effect of IKK/TNF on STAT1
signalling
1) IKK regulates the induction or the activity STAT inhibitors, such as SOCS1 and/or
SOCS3 that can inhibit STAT1 signalling. 2) IKK inhibits STAT1 activity directly. 3)
TNF induces TRADD-STAT1 complex formation and subsequently reduced free
STAT1 for phosphorylation that are available for JAK signalling.
211
5. Future work
5.1 Regulation of SOCS by IKK
The stability of SOCS proteins is regulated by phosphorylation (Chen XP 2002;
Haan, Ferguson et al. 2003) and degradation through the 26S proteasome and
this involves the interaction between SOCS box and Elongin B and C (Zhang,
Farley et al. 1999).
To investigate the possibility that IKK mediates the
interaction of SOCS1 with regulatory proteins such as Elongin B and C, I plan to
infect IKKF/F and IKKMye macrophages with adenovirus expressing SOCS1Flag and co-immunoprecipitate (co-IP) with anti-Flag antibody to detect IKKdependent interactions between SOCS1 and elongin B/C.
To test the hypothesis that SOCS1 is a target for IKK-mediated phosphorylation,
I will elicit peritoneal macrophages from IKKF/F and IKKMye macrophages
and stimulated with LPS in vitro, following IP with anti-SOCS3 and anti-SOCS1
antibody.
Phosphorylation will be detected with anti-phosphotyrosine
immunoblotting.
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5.2 Antigen processing and presentation
I have shown IKKMye macrophages increased antigen specific IFN production
by OTII T cells.
In these experiments, OVA323-339 peptide was used and
therefore measures the antigen presentation and co-stimulation capacity of
macrophages but effects on antigen processing were not measured. To test the
role of IKK in antigen processing, I will load whole OVA protein on IKKF/F
and IKKMye Tg elicited macrophages and co-culture with OTII T cells and
measure T cell proliferation and cytokine production.
5.3 T cell priming in vivo
To measure the role of IKK activation in T cell priming in vivo, I will pulse
IKKF/F and IKKMye Tg elicited macrophages with OVA peptide in vitro.
Antigen loaded macrophages will be injected i.v into syngeneic naive mice and
un-treated macrophages will be used as a negative control. Mice will then be
injected with CFSE-labelled OTII cells intravenously. Lymph nodes and spleen
will be collected after 1-2 days to measure OVA specific T cell expansion.
These experiments will be done with and without LPS injection as an adjuvant.
5.4 Humoral immune responses
Type I immune responses are not limited to the production of TH1 cytokines by
CD4+ T cells but also characterised by specific antibodies secreted by B cells.
Immune responses dominated by TH1 cells produce IFN and trigger B cells
secreting IgG2a antibody whereas TH2 cells are excellent helpers for B cells, and
stimulate the production of IgM and IgG1 (Abbas, Murphy et al. 1996). I have
213
shown that deleting IKK in macrophages promotes the development of TH1
responses, thus I would predict high titres IgG2a, but low IgM and IgG1
antibodies will be detected in IKKMye mice after OVA immunisation. To test
this I will immunise IKKF/F and IKKMye mice with OVA protein in CFA.
After 14 days, mice will be bled to measure the titre of OVA specific antibodies
in the serum.
5.5 STAT6 signalling
I have shown knocking out IKK suppress IL4R expression (Figure 3.2.1.2).
To test the role of IKK in IL-4/STAT6 signalling, macrophages elicited from
IKKF/F and IKKMye mice will be stimulated with IL-4 in vitro and STAT6
phosphorylation will be studied by IB. Arginase, FIZZ1 and YM1 are IL-4
regulated genes in macrophages (Gordon 2003), therefore I will measure their
expression by qRTPCR from RNA isolated from IL-4 stimulated IKKF/F and
IKKMye macrophages. Previous studies have shown that STAT6 and NF-B
cooperate and synergistically activate gene transcription in B cells (Messner,
Stutz et al. 1997; Shen and Stavnezer 1998). I will also test the influence of
IKK in STAT6-NF-B interactions by co-IP and IB with protein lysate
collected from IL-4 stimulated IKKF/F and IKKMye macrophages.
214
5.6 Tumour-induced tolerance
As I have discussed, in addition to CD8 T cells, CD4 T cells are also important
in anti-tumour immunity. To investigate their possible role in rejection of MB49
tumours in IKKMye female mice, depleting antibodies to CD4 T cells will be
injected 3 days prior to s.c. injection of MB49 cells. I will also confirm the
dependency of tumour rejection on IFN with neutralising IFN antibodies.
To investigate the role of IKKβ in the development of antigen (HY)-specific T
cells, tetramers will be used to track for the presence of HY specific CD4+ and
CD8+ T cells in vivo. Spleen, lymph nodes T cells and TIL will be collected and
stained with anti-Uty - PE (CD4) and anti-Dby - APC (CD8) the proportion of
antigen specific cells will be determined with FACS.
To assess the role of IKKβ in TAM on HY-specific T cell proliferation, I will
isolate TAM from MB49 tumours of IKKβ∆Mye and IKKβF/F mice and co-culture
with CD4 and/or CD8 T cells from Marylin (CD8) and MataHari (CD4) HY
TCR transgenic mice in the presence of Uty or Dby peptide. I will extend the
study in vivo by performing competitive in vivo killing assays with a mixture of
male and female splenocytes labelled with 0.2 and 2μM CFSE respectively.
After injected into MB49 tumour bearing IKKMye and IKKF/F mice. Spleen
and the tumour draining lymph nodes (inguinal and axillary) will be collected
and killing of CFSE+ cells will be detected by FACS.
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6. References
Abbas, A. K., K. M. Murphy, et al. (1996). "Functional diversity of helper T
lymphocytes." Nature 383(6603): 787-793.
Aggarwal, B. B. (2003). "SIGNALLING PATHWAYS OF THE TNF
SUPERFAMILY: A DOUBLE-EDGED SWORD." Nature Reviews
Immunology 3(9): 745-756.
Aguilera, C., R. Hoya-Arias, et al. (2004). "Recruitment of IκBα to the hes1
promoter is associated with transcriptional repression." Proceedings of
the National Academy of Sciences of the United States of America
101(47): 16537-16542.
Akira, S. and K. Takeda (2004). "TOLL-LIKE RECEPTOR SIGNALLING."
Nature Reviews Immunology 4(7): 499-511.
Alcamo, E., J. P. Mizgerd, et al. (2001). "Targeted mutation of TNF receptor I
rescues the RelA-deficient mouse and reveals a critical role for NF-kappa
B in leukocyte recruitment." J Immunol 167(3): 1592-600.
Alexander, W. S. and D. J. Hilton (2004). "The Role of Suppressors of Cytokine
Signaling (SOCS) Proteins in Regulation of the Immune Response."
Annual Review of Immunology 22(1): 503-529.
Alexopoulou, L., A. C. Holt, et al. (2001). "Recognition of double-stranded RNA
and activation of NF-[kappa]B by Toll-like receptor 3." Nature
413(6857): 732-738.
Amsen, D., J. M. Blander, et al. (2004). "Instruction of Distinct CD4 T Helper
Cell Fates by Different Notch Ligands on Antigen-Presenting Cells." Cell
117(4): 515-526.
Ana Crespo, M. B. F. W. J. M. (2002). "Low responsiveness to IFN-γ;, after
pretreatment of mouse macrophages with lipopolysaccharides, develops
via diverse regulatory pathways." European Journal of Immunology
32(3): 710-719.
Angelo A. Manfredi (1998). "Apoptotic cell clearance in systemic lupus
erythematosus: I. Opsonization by antiphospholipid antibodies." Arthritis
& Rheumatism 41(2): 205-214.
Angiolillo AL, S. C., Taub DD, Liao F, Farber JM, Maheshwari S, Kleinman
HK, Reaman GH, Tosato G. (1995). "Human interferon-inducible protein
10 is a potent inhibitor of angiogenesis in vivo." J Exp Med 182(1): 15562.
Apetoh, L., F. Ghiringhelli, et al. (2007). "Toll-like receptor 4-dependent
contribution of the immune system to anticancer chemotherapy and
radiotherapy." Nat Med 13(9): 1050-1059.
Asseman C, P. F. (1998). "Interleukin 10 is a growth factor for a population of
regulatory T cells." Gut 42(2): 157-8.
Aste-Amezaga, M., X. Ma, et al. (1998). "Molecular Mechanisms of the
Induction of IL-12 and Its Inhibition by IL-10." J Immunol 160(12):
5936-5944.
Bach, E. A., M. Aguet, et al. (1997). "THE IFNγ; RECEPTOR:A Paradigm for
Cytokine Receptor Signaling." Annual Review of Immunology 15(1):
563-591.
Balkwill, F. and A. Mantovani (2001). "Inflammation and cancer: back to
Virchow?" The Lancet 357(9255): 539-545.
216
Ballif, B. A., A. Shimamura, et al. (2001). "Disruption of 3-Phosphoinositidedependent Kinase 1 (PDK1) Signaling by the Anti-tumorigenic and Antiproliferative Agent N-alpha -tosyl-L-phenylalanyl Chloromethyl
Ketone." J. Biol. Chem. 276(15): 12466-12475.
Banchereau, J. and R. M. Steinman (1998). "Dendritic cells and the control of
immunity." Nature 392(6673): 245-52.
Beg, A. A., T. S. Finco, et al. (1993). "Tumor necrosis factor and interleukin-1
lead to phosphorylation and loss of I kappa B alpha: a mechanism for NFkappa B activation." Mol. Cell. Biol. 13(6): 3301-3310.
Beg, A. A., W. C. Sha, et al. (1995). "Embryonic lethality and liver degeneration
in mice lacking the RelA component of NF-κB." Nature 376(6536): 167170.
Bennett, S. R. M., F. R. Carbone, et al. (1998). "Help for cytotoxic-T-cell
responses is mediated by CD40 signalling." Nature 393(6684): 478-480.
Biswas SK, G. L., Paul S, Schioppa T, Saccani A, Sironi M, Bottazzi B, Doni A,
Vincenzo B, Pasqualini F, Vago L, Nebuloni M, Mantovani A, Sica A.
(2006). "A distinct and unique transcriptional program expressed by
tumor-associated macrophages (defective NF-kappaB and enhanced IRF3/STAT1 activation)." Blood 107(5): 2112-22.
Black, R. A., C. T. Rauch, et al. (1997). "A metalloproteinase disintegrin that
releases tumour-necrosis factor-α from cells." Nature 385(6618): 729733.
Bluyssen, H. A. R., J. E. Durbin, et al. (1996). "ISGF3γ p48, a specificity switch
for interferon activated transcription factors." Cytokine & Growth Factor
Reviews 7(1): 11-17.
Bode, J. G., A. Nimmesgern, et al. (1999). "LPS and TNFα induce SOCS3
mRNA and inhibit IL-6-induced activation of STAT3 in macrophages."
FEBS Letters 463(3): 365-370.
Bohlson, S. S., D. A. Fraser, et al. (2007). "Complement proteins C1q and MBL
are pattern recognition molecules that signal immediate and long-term
protective immune functions." Molecular Immunology 44(1-3): 33-43.
Bonecchi, R., S. Sozzani, et al. (1998). "Divergent Effects of Interleukin-4 and
Interferon-gamma on Macrophage-Derived Chemokine Production: An
Amplification Circuit of Polarized T Helper 2 Responses." Blood 92(8):
2668-2671.
Bonizzi, G. and M. Karin (2004). "The two NF-kappaB activation pathways and
their role in innate and adaptive immunity." Trends Immunol 25(6): 2808.
Boone, D. L., E. E. Turer, et al. (2004). "The ubiquitin-modifying enzyme A20 is
required for termination of Toll-like receptor responses." Nat Immunol
5(10): 1052-1060.
Briken, V., H. Ruffner, et al. (1995). "Interferon regulatory factor 1 is required
for mouse Gbp gene activation by gamma interferon." Mol. Cell. Biol.
15(2): 975-982.
Broide, D. H., T. Lawrence, et al. (2005). "Allergen-induced peribronchial
fibrosis and mucus production mediated by IκB kinase β-dependent genes
in airway epithelium." PNAS 102(49): 17723-17728.
Bruno Lemaitre, E. N., Lydia Michaut, Jean-Marc Reichhart and Jules A.
Hoffmann (1996). "The Dorsoventral Regulatory Gene Cassette
217
spätzle/Toll/cactus Controls the Potent Antifungal Response in
Drosophila Adults." Cell 86(6): 973-983.
Caamano, J. and C. A. Hunter (2002). "NF-kappaB family of transcription
factors: central regulators of innate and adaptive immune functions." Clin
Microbiol Rev 15(3): 414-29.
Cairns, A. P., A. D. Crockard, et al. (2003). "Complement receptor expression of
relevance to apoptotic cell clearance in SLE." Rheumatology 42(3): 487488.
Castellsague, X., F. X. Bosch, et al. (2002). "Environmental co-factors in HPV
carcinogenesis." Virus Research 89(2): 191-199.
Chang, C.-I., J. C. Liao, et al. (2001). "Macrophage Arginase Promotes Tumor
Cell Growth and Suppresses Nitric Oxide-mediated Tumor Cytotoxicity."
Cancer Res 61(3): 1100-1106.
Chang CH, F. J., Peterlin M, Flavell RA. (1994). "Class II transactivator (CIITA)
is sufficient for the inducible expression of major histocompatibility
complex class II genes." J Exp Med 180(4): 1367-74.
Chang, N.-S. (2002). "The Non-ankyrin C Terminus of Ikappa Balpha Physically
Interacts with p53 in Vivo and Dissociates in Response to Apoptotic
Stress, Hypoxia, DNA Damage, and Transforming Growth Factor-beta 1mediated Growth Suppression." J. Biol. Chem. 277(12): 10323-10331.
Chen, G. and D. V. Goeddel (2002). "TNF-R1 signaling: a beautiful pathway."
Science 296(5573): 1634-5.
Chen XP, L. J., Cowan S, Donahue E, Fay S, Vuong BQ, Nawijn MC, Capece D,
Cohan VL, Rothman P. (2002). "Pim serine/threonine kinases regulate
the stability of Socs-1 protein." Proc Natl Acad Sci U S A. 99(4): 217580.
Clausen, B. E., C. Burkhardt, et al. (1999). "Conditional gene targeting in
macrophages and granulocytes using LysMcre mice." Transgenic
Research 8(4): 265-277.
Collart, M. A., P. Baeuerle, et al. (1990). "Regulation of tumor necrosis factor
alpha transcription in macrophages: involvement of four kappa B-like
motifs and of constitutive and inducible forms of NF-kappa B." Mol.
Cell. Biol. 10(4): 1498-1506.
Constant, S. L. and K. Bottomly (1997). "INDUCTION OF TH1 AND TH2
CD4+ T CELL RESPONSES:The Alternative Approaches." Annual
Review of Immunology 15(1): 297-322.
Crespo, A., M. B. Filla, et al. (2000). "Indirect induction of suppressor of
cytokine signalling-1 in macrophages stimulated with bacterial
lipopolysaccharide: partial role of autocrine/paracrine interferonalpha/beta." Biochem J 349(Pt 1): 99-104.
Darnell, J. E., Jr., I. M. Kerr, et al. (1994). "Jak-STAT pathways and
transcriptional activation in response to IFNs and other extracellular
signaling proteins." Science 264(5164): 1415-1421.
Daun, J. M. and M. J. Fenton (2000). "Interleukin-1/Toll Receptor Family
Members: Receptor Structure and Signal Transduction Pathways."
Journal of Interferon & Cytokine Research 20(10): 843-855.
de Jong, E. C., P. L. Vieira, et al. (2002). "Microbial Compounds Selectively
Induce Th1 Cell-Promoting or Th2 Cell-Promoting Dendritic Cells In
Vitro with Diverse Th Cell-Polarizing Signals." J Immunol 168(4): 17041709.
218
De Vita, F., M. Orditura, et al. (2000). "Serum Interleukin-10 Levels as a
Prognostic Factor in Advanced Non-small Cell Lung Cancer Patients*."
Chest 117(2): 365-373.
de Wit, H., D. Hendriks, et al. (1994). "Interleukin-4 receptor regulation in
human monocytic cells." Blood 84(2): 608-615.
Dejardin, E., N. M. Droin, et al. (2002). "The lymphotoxin-beta receptor induces
different patterns of gene expression via two NF-kappaB pathways."
Immunity 17(4): 525-35.
Deng, W., Y. Ohmori, et al. (1994). "Mechanisms of IL-4-mediated suppression
of IP-10 gene expression in murine macrophages." J Immunol 153(5):
2130-2136.
Doi, T. S., M. W. Marino, et al. (1999). "Absence of tumor necrosis factor
rescues RelA-deficient mice from embryonic lethality." Proc Natl Acad
Sci U S A 96(6): 2994-9.
Dokter, W., P. Borger, et al. (1992). "Interleukin-4 (IL-4) receptor expression on
human T cells is affected by different intracellular signaling pathways
and by IL-4 at transcriptional and posttranscriptional level." Blood
80(11): 2721-2728.
Doyle S, V. S., O'Connell R, Dadgostar H, Dempsey P, Wu T, Rao G, Sun R,
Haberland M, Modlin R, Cheng G. (2002). "IRF3 mediates a
TLR3/TLR4-specific antiviral gene program." Immunity 17(3): 251-63.
Dreyfus, D., M. Nagasawa, et al. (2005). "Modulation of p53 activity by
IkappaBalpha: Evidence suggesting a common phylogeny between NFkappaB and p53 transcription factors." BMC Immunology 6(1): 12.
Dufour, J. H., M. Dziejman, et al. (2002). "IFN-γ-Inducible Protein 10 (IP-10;
CXCL10)-Deficient Mice Reveal a Role for IP-10 in Effector T Cell
Generation and Trafficking." J Immunol 168(7): 3195-3204.
Dupasquier, M., P. Stoitzner, et al. (2004). "Macrophages and Dendritic Cells
Constitute a Major Subpopulation of Cells in the Mouse Dermis." J
Investig Dermatol 123(5): 876-879.
Durbin, J. E., R. Hackenmiller, et al. (1996). "Targeted Disruption of the Mouse
Stat1 Gene Results in Compromised Innate Immunity to Viral Disease."
Cell 84(3): 443-450.
Dvorak, H. (1986). "Tumors: wounds that do not heal. Similarities between
tumor stroma generation and wound healing." N Engl J Med 315(26):
1650-9.
Ehlers, S., S. Kutsch, et al. (2000). "Lethal Granuloma Disintegration in
Mycobacteria-Infected TNFRp55-/- Mice Is Dependent on T Cells and
IL-12." J Immunol 165(1): 483-492.
Emma Teixeiro, A. G.-S. B. A. R. B. (1999). "Apoptosis-resistant T cells have a
deficiency in NF-κB-mediated induction of Fas ligand transcription."
European Journal of Immunology 29(3): 745-754.
Erdman, S. E., J. G. Fox, et al. (2001). "Cutting Edge: Typhlocolitis in NF-κB
Deficient Mice." J Immunol 166(3): 1443-1447.
Esther Reefman. (2007). "Opsonization of late apoptotic cells by systemic lupus
erythematosus autoantibodies inhibits their uptake via an Fcgamma
receptor-dependent mechanism." Arthritis & Rheumatism 56(10): 33993411.
219
Eyles, J. L., D. Metcalf, et al. (2002). "Negative Regulation of Interleukin-12
Signaling by Suppressor of Cytokine Signaling-1." J. Biol. Chem.
277(46): 43735-43740.
Fadok, V. A., D. L. Bratton, et al. (1998). "Macrophages that have ingested
apoptotic cells in vitro inhibit proinflammatory cytokine production
through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and
PAF." J Clin Invest 101(4): 890-8.
Fadok, V. A., A. de Cathelineau, et al. (2001). "Loss of Phospholipid Asymmetry
and Surface Exposure of Phosphatidylserine Is Required for Phagocytosis
of Apoptotic Cells by Macrophages and Fibroblasts." J. Biol. Chem.
276(2): 1071-1077.
Feldmann, M. and R. N. Maini (2003). "TNF defined as a therapeutic target for
rheumatoid arthritis and other autoimmune diseases." Nat Med 9(10):
1245-1250.
Fenton, M. J., J. A. Buras, et al. (1992). "IL-4 reciprocally regulates IL-1 and IL1 receptor antagonist expression in human monocytes." J Immunol
149(4): 1283-1288.
Ferdinando De Vita. (1999). "Serum interleukin-10 levels in patients with
advanced gastrointestinal malignancies." Cancer 86(10): 1936-1943.
Ferreira, V., N. Sidenius, et al. (1999). "In Vivo Inhibition of NF-κB in TLineage Cells Leads to a Dramatic Decrease in Cell Proliferation and
Cytokine Production and to Increased Cell Apoptosis in Response to
Mitogenic Stimuli, But Not to Abnormal Thymopoiesis." J Immunol
162(11): 6442-6450.
Finco TS, B. A., Baldwin AS Jr. (1994). "Inducible phosphorylation of I kappa B
alpha is not sufficient for its dissociation from NF-kappa B and is
inhibited by protease inhibitors." Proc Natl Acad Sci U S A. 91(25):
11884-8.
Fitzgerald, K. A., S. M. McWhirter, et al. (2003). "IKKε and TBK1 are essential
components of the IRF3 signaling pathway." Nat Immunol 4(5): 491-496.
Fong, C. H. Y., M. Bebien, et al. (2008). "An antiinflammatory role for
IKK{beta} through the inhibition of "classical" macrophage activation."
J. Exp. Med. 205(6): 1269-1276.
Gadjeva, M., M. F. Tomczak, et al. (2004). "A Role for NF-κB Subunits p50 and
p65 in the Inhibition of Lipopolysaccharide-Induced Shock." J Immunol
173(9): 5786-5793.
Galli, S. J. (1993). "New Concepts about the Mast Cell." N Engl J Med 328(4):
257-265.
Ganster, R. W., B. S. Taylor, et al. (2001). "Complex regulation of human
inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB."
Proceedings of the National Academy of Sciences of the United States of
America 98(15): 8638-8643.
Gao, F. G., V. Khammanivong, et al. (2002). "Antigen-specific CD4+ T-Cell
Help Is Required to Activate a Memory CD8+ T Cell to a Fully
Functional Tumor Killer Cell." Cancer Res 62(22): 6438-6441.
Gao, J., D. C. Morrison, et al. (1997). "An Interferon-gamma -activated Site
(GAS) Is Necessary for Full Expression of the Mouse iNOS Gene in
Response to Interferon-gamma and Lipopolysaccharide." J. Biol. Chem.
272(2): 1226-1230.
220
Gao, J. J., M. B. Filla, et al. (1998). "Autocrine/Paracrine IFN-αβ Mediates the
Lipopolysaccharide-Induced Activation of Transcription Factor
Stat1{alpha} in Mouse Macrophages: Pivotal Role of Stat1α in Induction
of the Inducible Nitric Oxide Synthase Gene." J Immunol 161(9): 48034810.
Gautam, S., J. M. Tebo, et al. (1992). "IL-4 suppresses cytokine gene expression
induced by IFN-gamma and/or IL- 2 in murine peritoneal macrophages."
J Immunol 148(6): 1725-1730.
Gautier, G., M. Humbert, et al. (2005). "A type I interferon autocrine-paracrine
loop is involved in Toll-like receptor-induced interleukin-12p70 secretion
by dendritic cells." J Exp Med 201(9): 1435-46.
Ghosh, S. and M. Karin (2002). "Missing pieces in the NF-kappaB puzzle." Cell
109 Suppl: S81-96.
Goerdt S, O. C. (1999). "Other functions, other genes: alternative activation of
antigen-presenting cells.
." Immunity 10(2): 137-142.
Goldsby R, K. T., Osborne B, Kuby J (2003). Immunology 5th Edition.
Gordon, S. (2003). "ALTERNATIVE ACTIVATION OF MACROPHAGES."
Nature Reviews Immunology 3(1): 23-35.
Gordon, S. and P. R. Taylor (2005). "Monocyte and macrophage heterogeneity."
Nat Rev Immunol 5(12): 953-964.
Greten, F. R., M. C. Arkan, et al. (2007). "NF-κB Is a Negative Regulator of IL1[beta] Secretion as Revealed by Genetic and Pharmacological Inhibition
of IKKβ." Cell 130(5): 918-931.
Greten, F. R., L. Eckmann, et al. (2004). "IKKβ Links Inflammation and
Tumorigenesis in a Mouse Model of Colitis-Associated Cancer." Cell
118(3): 285-296.
Groux, H., A. O'Garra, et al. (1997). "A CD4+T-cell subset inhibits antigenspecific T-cell responses and prevents colitis." Nature 389(6652): 737742.
H. Kuper, H. O. A. D. T. (2000). "Infections as a major preventable cause of
human cancer." Journal of Internal Medicine 248(3): 171-183.
Haan, S., P. Ferguson, et al. (2003). "Tyrosine Phosphorylation Disrupts Elongin
Interaction and Accelerates SOCS3 Degradation." J. Biol. Chem.
278(34): 31972-31979.
Hacker, H., V. Redecke, et al. (2006). "Specificity in Toll-like receptor signalling
through distinct effector functions of TRAF3 and TRAF6." Nature
439(7073): 204-207.
Hagemann, T., T. Lawrence, et al. (2008). ""Re-educating" tumor-associated
macrophages by targeting NF-κB." J. Exp. Med. 205(6): 1261-1268.
Hagemann, T., J. Wilson, et al. (2006). "Ovarian Cancer Cells Polarize
Macrophages Toward A Tumor-Associated Phenotype." J Immunol
176(8): 5023-5032.
Hailing Hsu, H.-B. S., Ming-Gui Pan and David V. Goeddel, (1996). "TRADDTRAF2 and TRADD-FADD interactions define two distinct TNF
receptor 1 signal transduction pathways." Cell 84: 299-308.
Halak, B. K., H. C. Maguire, Jr., et al. (1999). "Tumor-induced Interleukin-10
Inhibits Type 1 Immune Responses Directed at a Tumor Antigen As Well
As a Non-Tumor Antigen Present at the Tumor Site." Cancer Res 59(4):
911-917.
221
Hampton, R. Y., D. T. Golenbock, et al. (1991). "Recognition and plasma
clearance of endotoxin by scavenger receptors." Nature 352(6333): 342344.
Hart SP, S. J., Dransfield I (2004). "Phagocytosis of opsonized apoptotic cells:
roles for 'old-fashioned' receptors for antibody and complement." Clin
Exp Immunol 135(2): 181-5.
Hartl, D. and M. Griese (2006). "Surfactant protein D in human lung diseases."
European Journal of Clinical Investigation 36(6): 423-435.
Hayashi, F., K. D. Smith, et al. (2001). "The innate immune response to bacterial
flagellin is mediated by Toll-like receptor 5." Nature 410(6832): 10991103.
He XS, D. M., Mahmood K, Holmes TH, Kemble GW, Dekker CL, Arvin AM,
Parham P, Greenberg HB. (2004). "T cell-dependent production of IFNgamma by NK cells in response to influenza A virus." J Clin Invest
114(12): 1812-9.
Heinrich, S., D. Hartl, et al. (2006). "Surfactant Protein A - From Genes to
Human Lung Diseases." Current Medicinal Chemistry 13: 3239-3252.
Hemmi, H., O. Takeuchi, et al. (2000). "A Toll-like receptor recognizes bacterial
DNA." Nature 408(6813): 740-745.
Herbert, D. B. R., C. Hˆlscher, et al. (2004). "Alternative Macrophage Activation
Is Essential for Survival during Schistosomiasis and Downmodulates T
Helper 1 Responses and Immunopathology." Immunity 20(5): 623-635.
Hesse, M., M. Modolell, et al. (2001). "Differential Regulation of Nitric Oxide
Synthase-2 and Arginase-1 by Type 1/Type 2 Cytokines In Vivo:
Granulomatous Pathology Is Shaped by the Pattern of L-Arginine
Metabolism." J Immunol 167(11): 6533-6544.
Hettmann, T., J. DiDonato, et al. (1999). "An Essential Role for Nuclear Factor
kappa B in Promoting Double Positive Thymocyte Apoptosis." J. Exp.
Med. 189(1): 145-158.
Heyninck, K. and R. Beyaert (1999). "The cytokine-inducible zinc finger protein
A20 inhibits IL-1-induced NF-κB activation at the level of TRAF6."
FEBS Letters 442(2-3): 147-150.
Ho, I. C., M. R. Hodge, et al. (1996). "The Proto-Oncogene c-maf Is Responsible
for Tissue-Specific Expression of Interleukin-4." Cell 85(7): 973-983.
Ho, I. C., D. Lo, et al. (1998). "c-maf Promotes T Helper Cell Type 2 (Th2) and
Attenuates Th1 Differentiation by Both Interleukin 4-dependent and independent Mechanisms." J. Exp. Med. 188(10): 1859-1866.
Hodge-Dufour, J., M. W. Marino, et al. (1998). "Inhibition of interferon gamma
†induced interleukin 12 production: A potential mechanism for the antiinflammatory activities of tumor necrosis factor." PNAS 95(23): 1380613811.
Honda, K., H. Yanai, et al. (2005). "Regulation of the type I IFN induction: a
current view." Int. Immunol. 17(11): 1367-1378.
Hong, F., V.-A. Nguyen, et al. (2001). "Tumor necrosis factor α attenuates
interferon α signaling in the liver: involvement of SOCS3 and SHP2 and
implication in resistance to interferon therapy." FASEB J. 15(9): 15951597.
Hoshino, K., O. Takeuchi, et al. (1999). "Cutting Edge: Toll-Like Receptor 4
(TLR4)-Deficient Mice Are Hyporesponsive to Lipopolysaccharide:
222
Evidence for TLR4 as the Lps Gene Product." J Immunol 162(7): 37493752.
Huang, B., P.-Y. Pan, et al. (2006). "Gr-1+CD115+ Immature Myeloid
Suppressor Cells Mediate the Development of Tumor-Induced T
Regulatory Cells and T-Cell Anergy in Tumor-Bearing Host." Cancer
Res 66(2): 1123-1131.
Huynh ML, F. V., Henson PM (2002). "Phosphatidylserine-dependent ingestion
of apoptotic cells promotes TGF-beta1 secretion and the resolution of
inflammation." J Clin Invest 109(1): 41-50.
Imai, T., M. Nagira, et al. (1999). "Selective recruitment of CCR4-bearing Th2
cells toward antigen-presenting cells by the CC chemokines thymus and
activation-regulated chemokine and macrophage-derived chemokine."
Int. Immunol. 11(1): 81-88.
Isacke, L. E. a. C. M. (2002). "The mannose receptor family." Biochimica et
Biophysica Acta 1572(2-3): 364-386.
Janeway, C. A. T. P. W. M. S. M. (2001). Immunobiology, 5th Edition.
Jankovic, D., M. C. Kullberg, et al. (2000). "Single Cell Analysis Reveals That
IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro
Development of CD4+ Lymphocytes with a Th2 Cytokine Profile." J
Immunol 164(6): 3047-3055.
Janssens, S., K. Burns, et al. (2002). "Regulation of Interleukin-1- and
Lipopolysaccharide-Induced NF-κB Activation by Alternative Splicing of
MyD88." Current Biology 12(6): 467-471.
Janssens, S., K. Burns, et al. (2003). "MyD88S, a splice variant of MyD88,
differentially modulates NF-κB- and AP-1-dependent gene expression."
FEBS Letters 548(1-3): 103-107.
Jarnicki, A. G., J. Lysaght, et al. (2006). "Suppression of Antitumor Immunity by
IL-10 and TGF-beta-Producing T Cells Infiltrating the Growing Tumor:
Influence of Tumor Environment on the Induction of CD4+ and CD8+
Regulatory T Cells." J Immunol 177(2): 896-904.
Jenkins, M. K., C. A. Chen, et al. (1990). "Inhibition of antigen-specific
proliferation of type 1 murine T cell clones after stimulation with
immobilized anti-CD3 monoclonal antibody." J Immunol 144(1): 16-22.
Jianfei Yang, T. L. M. W. O. K. M. M. (1999). "Induction of interferon-gamma
production in Th1 CD4 positive T cells: evidence for two distinct
pathways for promoter activation." European Journal of Immunology
29(2): 548-555.
Jiao, H., K. Berrada, et al. (1996). "Direct association with and
dephosphorylation of Jak2 kinase by the SH2- domain-containing protein
tyrosine phosphatase SHP-1." Mol. Cell. Biol. 16(12): 6985-6992.
Ju, S.-T., D. J. Panka, et al. (1995). "Fas(CD95)/FasL interactions required for
programmed cell death after T-cell activation." Nature 373(6513): 444448.
Kagan, J. C. and R. Medzhitov (2006). "Phosphoinositide-Mediated Adaptor
Recruitment Controls Toll-like Receptor Signaling." Cell 125(5): 943955.
Kaisho and Akira (2006). "Toll-like receptor function and signaling." The
Journal of Allergy and Clinical Immunology 117(5): 979-987.
223
Kamijo, R., H. Harada, et al. (1994). "Requirement for transcription factor IRF-1
in NO synthase induction in macrophages." Science 263(5153): 16121615.
Kanaly, S. T., M. Nashleanas, et al. (1999). "TNF Receptor p55 Is Required for
Elimination of Inflammatory Cells Following Control of Intracellular
Pathogens." J Immunol 163(7): 3883-3889.
Kang BY, C. S., Im SY, Hwang SY, Kim TS. (1999). "Chloromethyl ketones
inhibit interleukin-12 production in mouse macrophages stimulated with
lipopolysaccharide." Immunol Lett. 70(2): 135-8.
Kang SM, T. A., Grilli M, Lenardo MJ. (1992). "NF-kappa B subunit regulation
in nontransformed CD4+ T lymphocytes." Science 5(256): 1452-1456.
Kano, S.-i., K. Sato, et al. (2008). "The contribution of transcription factor IRF1
to the interferon-γ-interleukin 12 signaling axis and TH1 versus TH-17
differentiation of CD4+ T cells." Nat Immunol 9(1): 34-41.
Kaplan, M. H., U. Schindler, et al. (1996). "Stat6 Is Required for Mediating
Responses to IL-4 and for the Development of Th2 Cells." Immunity
4(3): 313-319.
Kaplan, M. H., Y.-L. Sun, et al. (1996). "Impaired IL-12 responses and enhanced
development of Th2 cells in Stat4-deficient mice." Nature 382(6587):
174-177.
Karin, M. (2006). "Nuclear factor-κB in cancer development and progression."
Nature 441(7092): 431-436.
Karin, M. and Y. Ben-Neriah (2000). "Phosphorylation meets ubiquitination: the
control of NF-κB activity." Annu Rev Immunol 18: 621-63.
Karin, M., Y. Cao, et al. (2002). "NF-κB in cancer: from innocent bystander to
major culprit." Nat Rev Cancer 2(4): 301-310.
Karin, M., Y. Yamamoto, et al. (2004). "THE IKK NF-κB SYSTEM: A
TREASURE TROVE FOR DRUG DEVELOPMENT." Nature Reviews
Drug Discovery 3(1): 17-26.
Kasibhatla, S., L. Genestier, et al. (1999). "Regulation of Fas-Ligand Expression
during Activation-induced Cell Death in T Lymphocytes via Nuclear
Factor kappa B." J. Biol. Chem. 274(2): 987-992.
Kastenbauer, S. and H. W. L. Ziegler-Heitbrock (1999). "NF-kappa B1 (p50) Is
Upregulated in Lipopolysaccharide Tolerance and Can Block Tumor
Necrosis Factor Gene Expression." Infect. Immun. 67(4): 1553-1559.
Kawai, T., O. Takeuchi, et al. (2001). "Lipopolysaccharide Stimulates the
MyD88-Independent Pathway and Results in Activation of IFNRegulatory Factor 3 and the Expression of a Subset of
Lipopolysaccharide-Inducible Genes." J Immunol 167(10): 5887-5894.
Keshishian, M. S. H. a. H. (1998). "TheTollPathway Is Required in the
Epidermis for Muscle Development in theDrosophilaEmbryo."
Developmental Biology 199(1): 164-174.
Khoury, J. E., S. E. Hickman, et al. (1996). "Scavenger receptor-mediated
adhesion of microglia to [beta]-amyloid fibrils." Nature 382(6593): 716719.
Kishimoto, T., S. Akira, et al. (1995). "Interleukin-6 family of cytokines and
gp130." Blood 86(4): 1243-1254.
Kodelja, V., C. Muller, et al. (1997). "Differences in angiogenic potential of
classically vs alternatively activated macrophages." Immunobiology
197(5): 478-93.
224
Kopf, M., G. L. Gros, et al. (1993). "Disruption of the murine IL-4 gene blocks
Th2 cytokine responses." Nature 362(6417): 245-248.
Kornfeld, D., A. Ekbom, et al. (1997). "Is there an excess risk for colorectal
cancer in patients with ulcerative colitis and concomitant primary
sclerosing cholangitis? A population based study." Gut 41(4): 522-525.
Kotanides, H. and N. C. Reich (1996). "Interleukin-4-induced STAT6
Recognizes and Activates a Target Site in the Promoter of the
Interleukin-4 Receptor Gene." J. Biol. Chem. 271(41): 25555-25561.
Krebs, D. L. and D. J. Hilton (2001). "SOCS Proteins: Negative Regulators of
Cytokine Signaling." Stem Cells 19(5): 378-387.
Kropf, P., J. M. Fuentes, et al. (2005). "Arginase and polyamine synthesis are
key factors in the regulation of experimental leishmaniasis in vivo."
FASEB J.: 04-3416fje.
Kurt-Jones, E. A., L. Popova, et al. (2000). "Pattern recognition receptors TLR4
and CD14 mediate response to respiratory syncytial virus." Nat Immunol
1(5): 398-401.
Kusmartsev, S., S. Nagaraj, et al. (2005). "Tumor-Associated CD8+ T Cell
Tolerance Induced by Bone Marrow-Derived Immature Myeloid Cells." J
Immunol 175(7): 4583-4592.
L. Bingle, N. J. B. C. E. L. (2002). "The role of tumour-associated macrophages
in tumour progression: implications for new anticancer therapies." The
Journal of Pathology 196(3): 254-265.
Larrea, E., N. Garcia, et al. (1996). "Tumor necrosis factor alpha gene expression
and the response to interferon in chronic hepatitis C." Hepatology 23(2):
210-217.
Lawrence, T. and D. W. Gilroy (2007). "Chronic inflammation: a failure of
resolution?" International Journal of Experimental Pathology 88(2): 8594.
Lawrence, T., D. W. Gilroy, et al. (2001). "Possible new role for NF-κB in the
resolution of inflammation." Nat Med 7(12): 1291-1297.
Lee AH, H. J., Seo YS. (2000). "Tumour necrosis factor-alpha and interferongamma synergistically activate the RANTES promoter through nuclear
factor kappaB and interferon regulatory factor 1 (IRF-1) transcription
factors." Biochem. J. 350(1): 131-8.
Lewis, C. E. and J. W. Pollard (2006). "Distinct Role of Macrophages in
Different Tumor Microenvironments." Cancer Res 66(2): 605-612.
Ley, K. and G. S. Kansas (2004). "SELECTINS IN T-CELL RECRUITMENT
TO NON-LYMPHOID TISSUES AND SITES OF INFLAMMATION."
Nature Reviews Immunology 4(5): 325-336.
Li, J., S. H. Joo, et al. (2003). "An NF-kB-Specific Inhibitor, Binds to and
Inhibits Cyclin-Dependent Kinase 4†." Biochemistry 42(46): 1347613483.
Li, Q., D. V. Antwerp, et al. (1999). "Severe Liver Degeneration in Mice
Lacking the IB Kinase 2 Gene." Science 284(5412): 321-325.
Li, Q. and I. M. Verma (2002). "NF-κB REGULATION IN THE IMMUNE
SYSTEM." Nature Reviews Immunology 2(10): 725-734.
Li, Z. W., W. Chu, et al. (1999). "The IKKbeta subunit of IkappaB kinase (IKK)
is essential for nuclear factor kappaB activation and prevention of
apoptosis." J Exp Med 189(11): 1839-45.
225
Lin B, W.-S. C., Tao Y, Schleicher MS, Cano LL, Duke RC, Scheinman RI.
(1999). "NF-kappaB functions as both a proapoptotic and antiapoptotic
regulatory factor within a single cell " 6(6): 570-582.
Loercher, A. E., M. A. Nash, et al. (1999). "Identification of an IL-10-Producing
HLA-DR-Negative Monocyte Subset in the Malignant Ascites of Patients
with Ovarian Carcinoma That Inhibits Cytokine Protein Expression and
Proliferation of Autologous T Cells." J Immunol 163(11): 6251-6260.
Lohoff, M., D. Ferrick, et al. (1997). "Interferon Regulatory Factor-1 Is Required
for a T Helper 1 Immune Response In Vivo." 6(6): 681-689.
Lund, J. M., L. Alexopoulou, et al. (2004). "Recognition of single-stranded RNA
viruses by Toll-like receptor 7." Proceedings of the National Academy of
Sciences of the United States of America 101(15): 5598-5603.
Luo, J.-L., W. Tan, et al. (2007). "Nuclear cytokine-activated IKKα controls
prostate cancer metastasis by repressing Maspin." Nature 446(7136):
690-694.
Ma, X., J. Sun, et al. (2000). "Inhibition of IL-12 Production in Human
Monocyte-Derived Macrophages by TNF." J Immunol 164(4): 17221729.
MacEwan, D. J. (2002). "TNF ligands and receptors, a matter of life and death."
Br J Pharmacol 135(4): 855-875.
Magram, J., S. E. Connaughton, et al. (1996). "IL-12-Deficient Mice Are
Defective in IFNγ Production and Type 1 Cytokine Responses."
Immunity 4(5): 471-481.
Mantovani, A., P. Allavena, et al. (2008). "Cancer-related inflammation." Nature
454(7203): 436-444.
Mantovani, A., S. Sozzani, et al. (2002). "Macrophage polarization: tumorassociated macrophages as a paradigm for polarized M2 mononuclear
phagocytes." Trends in Immunology 23(11): 549-555.
Maria Cristina Gagliardi, F. S. M. M. A. L. A. L. M. T. D. M. (2000). "Cholera
toxin induces maturation of human dendritic cells and licences them for
Th2 priming." European Journal of Immunology 30(8): 2394-2403.
Marino, M. W., A. Dunn, et al. (1997). "Characterization of tumor necrosis
factor-deficient mice." Proceedings of the National Academy of Sciences
of the United States of America 94(15): 8093-8098.
Marshak-Rothstein, A. (2006). "Toll-like receptors in systemic autoimmune
disease." Nat Rev Immunol 6(11): 823-835.
Martin SJ, R. C., McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR
(1995). "Early redistribution of plasma membrane phosphatidylserine is a
general feature of apoptosis regardless of the initiating stimulus:
inhibition by overexpression of Bcl-2 and Abl." J Exp Med 182(2): 15451556.
Marzo, A. L., B. F. Kinnear, et al. (2000). "Tumor-Specific CD4+ T Cells Have
a Major "Post-Licensing" Role in CTL Mediated Anti-Tumor Immunity."
J Immunol 165(11): 6047-6055.
Matsukawa, A. (2007). "STAT proteins in innate immunity during sepsis:
lessons from gene knockout mice." Acta Med Okayama 61(5): 239-45.
Matsushima, A., T. Kaisho, et al. (2001). "Essential role of nuclear factor (NF)kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha
in NF-kappaB activation through lymphotoxin beta receptor, but not
through tumor necrosis factor receptor I." J Exp Med 193(5): 631-6.
226
Maxeiner, H., J. Husemann, et al. (1998). "Complementary Roles for Scavenger
Receptor A and CD36 of Human Monocyte-derived Macrophages in
Adhesion to Surfaces Coated with Oxidized Low-Density Lipoproteins
and in Secretion of H2O2." J. Exp. Med. 188(12): 2257-2265.
Meraz MA, W. J., Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH,
Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN,
Clark R, Aguet M, Schreiber RD. (1996). "Targeted disruption of the
Stat1 gene in mice reveals unexpected physiologic specificity in the JAKSTAT signaling pathway." Cell 84(3): 431-42.
Messner, B., A. M. Stutz, et al. (1997). "Cooperation of binding sites for STAT6
and NF kappa B/rel in the IL-4- induced up-regulation of the human IgE
germline promoter." J Immunol 159(7): 3330-3337.
Mora, A. L., J. Youn, et al. (2001). "NF-κB/Rel Participation in the LymphokineDependent Proliferation of T Lymphoid Cells." J Immunol 166(4): 22182227.
Moser.M (2001). "Regulation of Th1/Th2 development by antigen-presenting
cells in vivo." Immunobiology 204(5): 551-7.
Moss, M. L., S. L. C. Jin, et al. (1997). "Cloning of a disintegrin
metalloproteinase that processes precursor tumour-necrosis factor[alpha]." Nature 385(6618): 733-736.
Mosser, D. M. and J. P. Edwards (2008). "Exploring the full spectrum of
macrophage activation." Nat Rev Immunol 8(12): 958-969.
Mostafa, M. H., S. A. Sheweita, et al. (1999). "Relationship between
Schistosomiasis and Bladder Cancer." Clin. Microbiol. Rev. 12(1): 97111.
Mullen, A. C., F. A. High, et al. (2001). "Role of T-bet in Commitment of TH1
Cells Before IL-12-Dependent Selection." Science 292(5523): 19071910.
Mullen, A. C., A. S. Hutchins, et al. (2002). "Hlx is induced by and genetically
interacts with T-bet to promote heritable TH1 gene induction." Nat
Immunol 3(7): 652-658.
Munder, M., K. Eichmann, et al. (1998). "Alternative Metabolic States in Murine
Macrophages Reflected by the Nitric Oxide Synthase/Arginase Balance:
Competitive Regulation by CD4+ T Cells Correlates with Th1/Th2
Phenotype." J Immunol 160(11): 5347-5354.
Mylonas, K. J., M. G. Nair, et al. (2009). "Alternatively Activated Macrophages
Elicited by Helminth Infection Can Be Reprogrammed to Enable
Microbial Killing." J Immunol 182(5): 3084-3094.
Nelms, K., A. D. Keegan, et al. (1999). "THE IL-4 RECEPTOR: Signaling
Mechanisms and Biologic Functions." Annual Review of Immunology
17(1): 701-738.
Notley, C. A., J. J. Inglis, et al. (2008). "Blockade of tumor necrosis factor in
collagen-induced arthritis reveals a novel immunoregulatory pathway for
Th1 and Th17 cells." J. Exp. Med. 205(11): 2491-2497.
Novack, D. V., L. Yin, et al. (2003). "The IkappaB function of NF-kappaB2
p100 controls stimulated osteoclastogenesis." J Exp Med 198(5): 771-81.
O'Garra, A. (1998). "Cytokines Induce the Development of Functionally
Heterogeneous T Helper Cell Subsets." Immunity 8(3): 275-283.
227
O'Neill, L. A. J. and A. G. Bowie (2007). "The family of five: TIR-domaincontaining adaptors in Toll-like receptor signalling." Nat Rev Immunol
7(5): 353-364.
O'Sullivan, B. J. and R. Thomas (2002). "CD40 ligation conditions dendritic cell
antigen-presenting function through sustained activation of NF-kappaB."
J Immunol 168(11): 5491-8.
Oganesyan, G., S. K. Saha, et al. (2006). "Critical role of TRAF3 in the Toll-like
receptor-dependent and -independent antiviral response." Nature
439(7073): 208-211.
Ohmori, Y. and T. A. Hamilton (1995). "The interferon-stimulated response
element and a kappa B site mediate synergistic induction of murine IP-10
gene transcription by IFN-gamma and TNF-alpha." J Immunol 154(10):
5235-5244.
Ohmori, Y. and T. A. Hamilton (1997). "IL-4-induced STAT6 suppresses IFNgamma-stimulated
STAT1-dependent
transcription
in
mouse
macrophages." J Immunol 159(11): 5474-5482.
Ohmori, Y., R. D. Schreiber, et al. (1997). "Synergy between interferon-gamma
and tumor necrosis factor-alpha in transcriptional activation is mediated
by cooperation between signal transducer and activator of transcription 1
and nuclear factor kappaB." J Biol Chem 272(23): 14899-907.
Ohmori, Y., R. D. Schreiber, et al. (1997). "Synergy between Interferon-gamma
and Tumor Necrosis Factor-alpha in Transcriptional Activation Is
Mediated by Cooperation between Signal Transducer and Activator of
Transcription 1†and Nuclear Factor kappa B." J. Biol. Chem. 272(23):
14899-14907.
Okamura, Y., M. Watari, et al. (2001). "The Extra Domain A of Fibronectin
Activates Toll-like Receptor 4." J. Biol. Chem. 276(13): 10229-10233.
Ostrand-Rosenberg, S. (2008). "Immune surveillance: a balance between
protumor and antitumor immunity." Current Opinion in Genetics &
Development 18(1): 11-18.
Ouaaz, F., J. Arron, et al. (2002). "Dendritic Cell Development and Survival
Require Distinct NF-κB Subunits." Immunity 16(2): 257-270.
Ouyang, W., M. Lˆhning, et al. (2000). "Stat6-Independent GATA-3
Autoactivation Directs IL-4-Independent Th2 Development and
Commitment." Immunity 12(1): 27-37.
Ouyang, W., S. H. Ranganath, et al. (1998). "Inhibition of Th1 Development
Mediated by GATA-3 through an IL-4-Independent Mechanism."
Immunity 9(5): 745-755.
Ozinsky, A., D. M. Underhill, et al. (2000). "The repertoire for pattern
recognition of pathogens by the innate immune system is defined by
cooperation between Toll-like receptors." Proceedings of the National
Academy of Sciences of the United States of America 97(25): 1376613771.
Paludan, S. r. R., J. Lovmand, et al. (1997). "Effect of IL-4 and IL-13 on IFN[gamma]-induced production of nitric oxide in mouse macrophages
infected with herpes simplex virus type 2." FEBS Letters 414(1): 61-64.
Perez-G, M., J. R. Cortes, et al. (2008). "Treatment of cells with n-alpha-tosyl-lphenylalanine-chloromethyl ketone induces the proteolytic loss of
STAT6 transcription factor." Molecular Immunology 45(15): 3896-3901.
228
Persengiev SP, Z. X., Green MR. (2004). "Nonspecific, concentration-dependent
stimulation and repression of mammalian gene expression by small
interfering RNAs (siRNAs)." RNA 10(1): 12-18.
Platanias, L. C. (2005). "MECHANISMS OF TYPE-I- AND TYPE-IIINTERFERON-MEDIATED
SIGNALLING."
Nature
Reviews
Immunology 5(5): 375-386.
Platt, N., H. Suzuki, et al. (1996). "Role for the class A macrophage scavenger
receptor in the phagocytosis of apoptotic thymocytes in vitro." Proc Natl
Acad Sci USA 93: 12456 - 12460.
Plaut, M., J. H. Pierce, et al. (1989). "Mast cell lines produce lymphokines in
response to cross-linkage of FcεRI or to calcium ionophores." Nature
339(6219): 64-67.
Pozzi, L.-A. M., J. W. Maciaszek, et al. (2005). "Both Dendritic Cells and
Macrophages Can Stimulate Naive CD8 T Cells In Vivo to Proliferate,
Develop Effector Function, and Differentiate into Memory Cells." J
Immunol 175(4): 2071-2081.
Qureshi, S. T., L. Lariviere, et al. (1999). "Endotoxin-tolerant Mice Have
Mutations in Toll-like Receptor 4†(Tlr4)." J. Exp. Med. 189(4): 615-625.
Raes, G., P. De Baetselier, et al. (2002). "Differential expression of FIZZ1 and
Ym1 in alternatively versus classically activated macrophages." J Leukoc
Biol 71(4): 597-602.
Reefman Esther. (2007). "Opsonization of late apoptotic cells by systemic lupus
erythematosus autoantibodies inhibits their uptake via an Fcgamma
receptor-dependent mechanism." Arthritis & Rheumatism 56(10): 33993411.
Reidy, M. F. and J. R. Wright (2003). "Surfactant protein A enhances apoptotic
cell uptake and TGF-β1 release by inflammatory alveolar macrophages."
Am J Physiol Lung Cell Mol Physiol 285(4): L854-861.
Renz, H., J. Domenico, et al. (1991). "IL-4-dependent up-regulation of IL-4
receptor expression in murine T and B cells." J Immunol 146(9): 30493055.
Rescigno, M., M. Martino, et al. (1998). "Dendritic Cell Survival and Maturation
Are Regulated by Different Signaling Pathways." J. Exp. Med. 188(11):
2175-2180.
Ridge, J. P., F. Di Rosa, et al. (1998). "A conditioned dendritic cell can be a
temporal bridge between a CD4+ T-helper and a T-killer cell." Nature
393(6684): 474-478.
Rodriguez, P. C., D. G. Quiceno, et al. (2004). "Arginase I Production in the
Tumor Microenvironment by Mature Myeloid Cells Inhibits T-Cell
Receptor Expression and Antigen-Specific T-Cell Responses." Cancer
Res 64(16): 5839-5849.
Rogge, L., L. Barberis-Maino, et al. (1997). "Selective Expression of an
Interleukin-12 Receptor Component by Human T Helper 1†Cells." J.
Exp. Med. 185(5): 825-832.
Rosin, M. P., W. A. Anwar, et al. (1994). "Inflammation, Chromosomal
Instability, and Cancer: The Schistosomiasis Model." Cancer Res
54(7_Supplement): 1929s-1933.
Rothe, M., S. C. Wong, et al. (1994). "A novel family of putative signal
transducers associated with the cytoplasmic domain of the 75 kDa tumor
necrosis factor receptor." Cell 78(4): 681-692.
229
Rothe M, W. S., Henzel WJ, Goeddel DV. (1994). "A novel family of putative
signal transducers associated with the cytoplasmic domain of the 75 kDa
tumor necrosis factor receptor." Cell 78(4): 681-92.
Rothenberg, M. E. and S. P. Hogan (2006). "THE EOSINOPHIL." Annual
Review of Immunology 24(1): 147-174.
S, P. L. a. G. (2001). "The function of scavenger receptorsexpressed by
macrophages and their role in the regulation of inflammation." Microbes
and Infection 3(2): 149-159.
Saccani, A., T. Schioppa, et al. (2006). "p50 Nuclear Factor-κB Overexpression
in Tumor-Associated Macrophages Inhibits M1 Inflammatory Responses
and Antitumor Resistance." Cancer Res 66(23): 11432-11440.
Sauer, B. (1998). "Inducible Gene Targeting in Mice Using the Cre/loxSystem."
Methods 14(4): 381-92.
Savill, J., I. Dransfield, et al. (2002). "A blast from the past: clearance of
apoptotic cells regulates immune responses." Nat Rev Immunol 2(12):
965-975.
Savill, J. and V. Fadok (2000). "Corpse clearance defines the meaning of cell
death." Nature 407(6805): 784-788.
Saxena S, J. Z., Dutta A. (2003). "Small RNAs with imperfect match to
endogenous mRNA repress translation. Implications for off-target
activity of small inhibitory RNA in mammalian cells." J Bio Chem
278(45): 44312-9.
Scharton-Kersten, T. M., T. A. Wynn, et al. (1996). "In the absence of
endogenous IFN-gamma, mice develop unimpaired IL-12 responses to
Toxoplasma gondii while failing to control acute infection." J Immunol
157(9): 4045-4054.
Schebesch, C., V. Kodelja, et al. (1997). "Alternatively activated macrophages
actively inhibit proliferation of peripheral blood lymphocytes and CD4+
T cells in vitro." Immunology 92(4): 478-86.
Schmitz J, T. A., Kühn R, Rajewsky K, Müller W, Assenmacher M, Radbruch A.
(1994). "Induction of interleukin 4 (IL-4) expression in T helper (Th)
cells is not dependent on IL-4 from non-Th cells." J Exp Med 179(4):
1349-53.
Schoenberger SP, T. R., van der Voort EI, Offringa R, Melief CJ. (1998). "T-cell
help for cytotoxic T lymphocytes is mediated by CD40-CD40L
interactions." Nature 393(6684): 480-3.
Schottenfeld, D. and J. Beebe-Dimmer (2006). "Chronic Inflammation: A
Common and Important Factor in the Pathogenesis of Neoplasia." CA
Cancer J Clin 56(2): 69-83.
Schwandner, R., R. Dziarski, et al. (1999). "Peptidoglycan- and Lipoteichoic
Acid-induced Cell Activation Is Mediated by Toll-like Receptor 2." J.
Biol. Chem. 274(25): 17406-17409.
Seder, R. A. and W. E. Paul (1994). "Acquisition of Lymphokine-Producing
Phenotype by CD4+ T Cells." Annual Review of Immunology 12(1):
635-673.
Seder RA, P. W., Dvorak AM, Sharkis SJ, Kagey-Sobotka A, Niv Y, Finkelman
FD, Barbieri SA, Galli SJ, Plaut M. (1991). "Mouse splenic and bone
marrow cell populations that express high-affinity Fc epsilon receptors
and produce interleukin 4 are highly enriched in basophils." 88 7: 2835-9.
230
Senftleben, U., Y. Cao, et al. (2001). "Activation by IKKalpha of a second,
evolutionary conserved, NF-kappa B signaling pathway." Science
293(5534): 1495-9.
Senftleben, U., Z. W. Li, et al. (2001). "IKKbeta is essential for protecting T
cells from TNFalpha-induced apoptosis." Immunity 14(3): 217-30.
Shen, C.-H. and J. Stavnezer (1998). "Interaction of Stat6 and NF-kappa B:
Direct Association and Synergistic Activation of Interleukin-4-Induced
Transcription." Mol. Cell. Biol. 18(6): 3395-3404.
Sher, A. and R. L. Coffman (1992). "Regulation of Immunity to Parasites by T
Cells and T Cell-Derived Cytokines." Annual Review of Immunology
10(1): 385-409.
Shimoda, K., J. van Deursent, et al. (1996). "Lack of IL-4-induced Th2 response
and IgE class switching in mice with disrupted State6 gene." Nature
380(6575): 630-633.
Shultz, D. B., J. D. Fuller, et al. (2007). "Activation of a Subset of Genes by
IFN-gamma requires IKKbeta; but Not Interferon-Dependent Activation
of NF-kappaB." Journal of Interferon & Cytokine Research 27(10): 875884.
Sica, A., A. Saccani, et al. (2000). "Autocrine Production of IL-10 Mediates
Defective IL-12 Production and NF-κB Activation in Tumor-Associated
Macrophages." J Immunol 164(2): 762-767.
Sledz, C. A., M. Holko, et al. (2003). "Activation of the interferon system by
short-interfering RNAs." Nat Cell Biol 5(9): 834-839.
Smiley, S. T., J. A. King, et al. (2001). "Fibrinogen Stimulates Macrophage
Chemokine Secretion Through Toll-Like Receptor 4." J Immunol 167(5):
2887-2894.
Song, E., N. Ouyang, et al. (2000). "Influence of Alternatively and Classically
Activated Macrophages on Fibrogenic Activities of Human Fibroblasts."
Cellular Immunology 204(1): 19-28.
Stark, G. R., I. M. Kerr, et al. (1998). "How cells respond to interferons." Annu
Rev Biochem 67: 227-64.
Steimle, V., C. A. Siegrist, et al. (1994). "Regulation of MHC class II expression
by interferon-gamma mediated by the transactivator gene CIITA."
Science 265(5168): 106-109.
Stein, M., S. Keshav, et al. (1992). "Interleukin 4 potently enhances murine
macrophage mannose receptor activity: a marker of alternative
immunologic macrophage activation." J Exp Med 176(1): 287-92.
Steinbrink, K., M. Wolfl, et al. (1997). "Induction of tolerance by IL-10-treated
dendritic cells." J Immunol 159(10): 4772-4780.
Stoiber, D., P. Kovarik, et al. (1999). "Lipopolysaccharide Induces in
Macrophages the Synthesis of the Suppressor of Cytokine Signaling 3
and Suppresses Signal Transduction in Response to the Activating Factor
IFN-{gamma}." J Immunol 163(5): 2640-2647.
Stout, R. D. and J. Suttles (2004). "Functional plasticity of macrophages:
reversible adaptation to changing microenvironments." J Leukoc Biol
76(3): 509-513.
Swain, S. L., A. D. Weinberg, et al. (1990). "IL-4 directs the development of
Th2-like helper effectors." J Immunol 145(11): 3796-3806.
231
Szabo, S. J., A. S. Dighe, et al. (1997). "Regulation of the Interleukin (IL)-12R
beta 2 Subunit Expression in Developing T Helper 1†(Th1) and Th2
Cells." J. Exp. Med. 185(5): 817-824.
Szabo, S. J., S. T. Kim, et al. (2000). "A Novel Transcription Factor, T-bet,
Directs Th1 Lineage Commitment." Cell 100(6): 655-669.
T. Rothoeft, A. G. H. B. O. A. U. S. (2003). "Antigen dose, type of antigenpresenting cell and time of differentiation contribute to the T helper 1/T
helper 2 polarization of naive T cells." Immunology 110(4): 430-439.
Takeda, K. and S. Akira (2004). "TLR signaling pathways." Semin Immunol
16(1): 3-9.
Takeda, K. and S. Akira (2005). "Toll-like receptors in innate immunity." Int.
Immunol. 17(1): 1-14.
Takeda, K., T. Tanaka, et al. (1996). "Essential role of Stat6 in IL-4 signalling."
Nature 380(6575): 627-630.
Takeuchi, O., A. Kaufmann, et al. (2000). "Cutting Edge: Preferentially the RStereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating
Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2and MyD88-Dependent Signaling Pathway." J Immunol 164(2): 554-557.
Taki, S., T. Sato, et al. (1997). "Multistage Regulation of Th1-Type Immune
Responses by the Transcription Factor IRF-1." Immunity 6(6): 673-679.
Tanaka, M., M. E. Fuentes, et al. (1999). "Embryonic lethality, liver
degeneration, and impaired NF-kappa B activation in IKK-beta-deficient
mice." Immunity 10(4): 421-9.
Termeer, C., F. Benedix, et al. (2002). "Oligosaccharides of Hyaluronan Activate
Dendritic Cells via Toll-like Receptor 4." J. Exp. Med. 195(1): 99-111.
Thierfelder, W. E., J. M. van Deursen, et al. (1996). "Requirement for Stat4 in
interleukin-12-mediated responses of natural killer and T cells." Nature
382(6587): 171-174.
Thomas, C. A., Y. Li, et al. (2000). "Protection from Lethal Gram-positive
Infection by Macrophage Scavenger Receptor-dependent Phagocytosis."
J. Exp. Med. 191(1): 147-156.
Tomczak, M. F., S. E. Erdman, et al. (2006). "Inhibition of Helicobacter
hepaticus-Induced Colitis by IL-10 Requires the p50/p105 Subunit of NF{kappa}B." J Immunol 177(10): 7332-7339.
Tomczak, M. F., S. E. Erdman, et al. (2003). "NF-κB Is Required Within the
Innate Immune System to Inhibit Microflora-Induced Colitis and
Expression of IL-12 p40." J Immunol 171(3): 1484-1492.
Toshchakov, V., B. W. Jones, et al. (2002). "TLR4, but not TLR2, mediates IFN[beta]-induced STAT1α/β-dependent gene expression in macrophages."
Nat Immunol 3(4): 392-398.
Trapani, J. (2005). "The dual adverse effects of TGF-beta secretion on tumor
progression." Cancer Cell 8(5): 349-50.
Underhill, D. M., A. Ozinsky, et al. (1999). "The Toll-like receptor 2 is recruited
to macrophage phagosomes and discriminates between pathogens."
Nature 401(6755): 811-815.
Vabulas, R. M., P. Ahmad-Nejad, et al. (2001). "Endocytosed HSP60s Use Tolllike Receptor 2 (TLR2) and TLR4 to Activate the Toll/Interleukin-1
Receptor Signaling Pathway in Innate Immune Cells." J. Biol. Chem.
276(33): 31332-31339.
232
Vabulas, R. M., P. Ahmad-Nejad, et al. (2002). "HSP70 as Endogenous Stimulus
of the Toll/Interleukin-1 Receptor Signal Pathway." J. Biol. Chem.
277(17): 15107-15112.
Vabulas, R. M., S. Braedel, et al. (2002). "The Endoplasmic Reticulum-resident
Heat Shock Protein Gp96 Activates Dendritic Cells via the Toll-like
Receptor 2/4 Pathway." J. Biol. Chem. 277(23): 20847-20853.
Vacchio MS, A. J. (1997). "Thymus-derived glucocorticoids regulate antigenspecific positive selection." J Exp Med 185(11): 2033-8.
van Velzen, A. G., H. Suzuki, et al. (1999). "The Role of Scavenger Receptor
Class A in the Adhesion of Cells Is Dependent on Cell Type and Cellular
Activation State." Experimental Cell Research 250(1): 264-271.
Vanden Berghe W, V. L., De Wilde G, De Bosscher K, Boone E, Haegeman G.
(2000). "Signal transduction by tumor necrosis factor and gene regulation
of the inflammatory cytokine interleukin-6." Biochemical Pharmacology
60(8): 1185-1195.
Vandenabeele, P., W. Declercq, et al. (1995). "Two tumour necrosis factor
receptors: structure and function." Trends in Cell Biology 5(10): 392-399.
Viatour, P., S. Legrand-Poels, et al. (2003). "Cytoplasmic IκBα Increases NFκB-independent Transcription through Binding to Histone Deacetylase
(HDAC) 1 and HDAC3." J. Biol. Chem. 278(47): 46541-46548.
Wang, C., L. Deng, et al. (2001). "TAK1 is a ubiquitin-dependent kinase of
MKK and IKK." Nature 412(6844): 346-351.
Wang, C.-Y., M. W. Mayo, et al. (1998). "NF-B Antiapoptosis: Induction of
TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to Suppress Caspase-8
Activation." Science 281(5383): 1680-1683.
Wang, I.-M., C. Contursi, et al. (2000). "An IFN-γ-Inducible Transcription
Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates
IL-12 p40 Expression in Macrophages." J Immunol 165(1): 271-279.
Wang, J.-C. E. and A. M. Livingstone (2003). "Cutting Edge: CD4+ T Cell Help
Can Be Essential for Primary CD8+ T Cell Responses In Vivo." J
Immunol 171(12): 6339-6343.
Wang, Y., T. R. Wu, et al. (2000). "Stat1 as a Component of Tumor Necrosis
Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NFkappa B Activation." Mol. Cell. Biol. 20(13): 4505-4512.
Watford, W. T., M. Moriguchi, et al. (2003). "The biology of IL-12: coordinating
innate and adaptive immune responses." Cytokine Growth Factor Rev
14(5): 361-8.
Wesche H, H. W., Shillinglaw W, Li S, Cao Z. (1997). "MyD88: an adapter that
recruits IRAK to the IL-1 receptor complex." Immunity 7(6): 837-847.
Wesemann, D. R. and E. N. Benveniste (2003). "STAT-1α and IFN-γ as
Modulators of TNF-α Signaling in Macrophages: Regulation and
Functional Implications of the TNF Receptor 1:STAT-1α Complex." J
Immunol 171(10): 5313-5319.
Wesemann, D. R., H. Qin, et al. (2004). "TRADD interacts with STAT1-α and
influences interferon-[gamma] signaling." Nat Immunol 5(2): 199-207.
Wim Noël , G. R., Gholamreza Hassanzadeh Ghassabeh , Patrick De Baetselier
and Alain Beschin (2004). "Alternatively activated macrophages during
parasite infections." Trends in Parasitology 20(3): 126-133.
233
Wyllie, D. H., E. Kiss-Toth, et al. (2000). "Evidence for an Accessory Protein
Function for Toll-Like Receptor 1 in Anti-Bacterial Responses." J
Immunol 165(12): 7125-7132.
Xu, D., W. L. Chan, et al. (1998). "Selective Expression and Functions of
Interleukin 18 Receptor on T Helper (Th) Type 1†but not Th2 Cells." J.
Exp. Med. 188(8): 1485-1492.
Yang, A. S. and E. C. Lattime (2003). "Tumor-induced Interleukin 10
Suppresses the Ability of Splenic Dendritic Cells to Stimulate CD4 and
CD8 T-Cell Responses." Cancer Res 63(9): 2150-2157.
Yarovinsky F, Z. D., Andersen JF, Bannenberg GL, Serhan CN, Hayden MS,
Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A. (2005). "TLR11
activation of dendritic cells by a protozoan profilin-like protein." Science
308(5728): 1626-9.
Yasukawa H, M. H., Sakamoto H, Masuhara M, Sasaki A, Wakioka T, Ohtsuka
S, Imaizumi T, Matsuda T, Ihle JN, Yoshimura A. (1999). "The JAKbinding protein JAB inhibits Janus tyrosine kinase activity through
binding in the activation loop." The Embo Journal 18(5): 1309-1320.
Yeunis B. H. Geijtenbeek 1, R. T., Sandra J. van Vliet 1, Gerard C. F. van
Duijnhoven 1, Gosse J. Adema 1, Yvette van Kooyk , 1, * and Carl G.
Figdor 1 (2000). "Identification of DC-SIGN, a Novel Dendritic Cell–
Specific ICAM-3 Receptor that Supports Primary Immune Responses."
Cell 100(5): 575-583.
Yoshida, T., K. Ikuta, et al. (1996). "Defective B-1 Cell Development and
Impaired Immunity against Angiostrongylus cantonensis in IL-5RαDeficient Mice." Immunity 4(5): 483-494.
Yoshimura, S., J. Bondeson, et al. (2003). "Antigen presentation by murine
dendritic cells is nuclear factor-kappa B dependent both in vitro and in
vivo." Scand J Immunol 58(2): 165-72.
Yoshimura, S., J. Bondeson, et al. (2001). "Effective antigen presentation by
dendritic cells is NF-κB dependent: coordinate regulation of MHC, costimulatory molecules and cytokines." Int. Immunol. 13(5): 675-683.
Yu, X., R. H. Kennedy, et al. (2003). "JAK2/STAT3, Not ERK1/2, Mediates
Interleukin-6-induced Activation of Inducible Nitric-oxide Synthase and
Decrease in Contractility of Adult Ventricular Myocytes." J. Biol. Chem.
278(18): 16304-16309.
Zakharova, M. and H. K. Ziegler (2005). "Paradoxical Anti-Inflammatory
Actions of TNF-α: Inhibition of IL-12 and IL-23 via TNF Receptor 1 in
Macrophages and Dendritic Cells." J Immunol 175(8): 5024-5033.
Zamzami, N. and G. Kroemer (1999). "Apoptosis: Condensed matter in cell
death." Nature 401(6749): 127-128.
Zhang D, Z. G., Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S.
(2004). "A toll-like receptor that prevents infection by uropathogenic
bacteria." Science.
Zhang, D.-H., L. Cohn, et al. (1997). "Transcription Factor GATA-3 Is
Differentially Expressed in Murine Th1 and Th2 Cells and Controls Th2specific Expression of the Interleukin-5 Gene." J. Biol. Chem. 272(34):
21597-21603.
Zhang, J.-G., A. Farley, et al. (1999). "The conserved SOCS box motif in
suppressors of cytokine signaling binds to elongins B and C and may
234
couple bound proteins to proteasomal degradation." PNAS 96(5): 20712076.
Zhu, J., B. Min, et al. (2004). "Conditional deletion of Gata3 shows its essential
function in TH1-TH2 responses." Nat Immunol 5(11): 1157-1165.
Zitvogel, L., L. Apetoh, et al. (2008). "Immunological aspects of cancer
chemotherapy." Nat Rev Immunol 8(1): 59-73.
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