Download B cells

1
PHM142 Metabolic Biochemistry and Immunology
II WHITE CELLS
CHEMICAL WARFARE lecture outline
1) Molecular mechanisms of leukocyte function
• Rheumatoid arthritis
• CGD disease
2) Eosinophils
3) Macrophages
• Function
• Growth factors and cytokines
• But – macrophage - induced tissue injury
• Multiple organ failure, ARDS, sepsis
2
Blood Cells
Type of cell
Main function
Red blood cells (erythrocytes)
Transport O2 and CO2
White blood cells (leukocytes)
polymorphonuclear/granular leukocytes
Neutrophils (50  60%)
Eosinophils (1  4%)
Destroy invading bacteria
Basophils (0.5  2%)
mononuclear leukocytes
Lymphocytes (20  40%)
Phagocytose & kill invading antigens
Destroy parasites and modulate allergic
inflammatory reactions
Release histamine, serotonin, bradykinin,
heparin, and cytokines; converts arachidonic
acid to prostaglandins & leukotrienes
(T & B) mediate cytokine release
Phagocytose & kill ingested microbes
Monocytes (2  9%)
macrophages in extravascular tissue)
Killer cells
Megakaryocytes ( platelets)
(
Kill virally infected cells and tumor cells
(offer “natural” immunity as well as
adaptive)
Initiate blood clotting; also release
histamine and serotonin
3
1
2
3
Macrophage
Thin sections of the major types of white blood cells (leukocytes) found
in the circulation, showing the variety of internal structures observed.
4
1 Molecular mechanisms of neutrophil function
Amer J Med. 109, 33-44 (2000)
Neutrophil membrane electron transport
I
n
t
r
a
c
e
G
S
H
l
l
u
l
G
a
r
l
l
u
y
c
o
i
s
e
n
w
h
i
t
e
CYTOSOL
H2O2
NADP+
g
l
u
c
o
s
e
-
6
-
p
h
l
u
c
o
s
e
-
6
-
p
h
o
g
d
G
H2O
S
S
G
e
h
6
NADPH
y
d
P
r
g
o
l
g
u
e
c
n
o
a
n
s
i
c
PHAGOSOME MEMBRANE
2O2.
H2O2
-
NADP+
N
o
A
x
G
D
i
P
d
NADPH
2O2
H
a
l
s
u
c
o
s
e
-
6
-
P
e
G
l
u
c
o p
HOCl
e
r
o
o
n
i
c
a
c
i
d
PHAGOSOME VACUOLE
H2O2
+
Cl- m
y
Fe2+
e
l
B
x
a
i
c
d
t
a
e
s
r
e
i
c
i
5
Inflammation or infection
MPO(myeloperoxidase), HOCl(hypochlorite), SOD (superoxide dismutase).
HOCl + red cell GSH = GSH cyclic sulfonamide (biomarker for HOCl formation in
vivo that reflects inflammation (oxidant formed by neutrophils))
Living with a killer: The effects of hypochlorite on mammalian cells
IUMB Life 50,259-266 (2000) ; Chem.Res.Toxicol.21,1011-6 (2008)
6
7
A CGD disease clinical test for the
Leukocyte Respiratory burst by
phagosome membr. elect. transport. lack of leukocyte respiratory burst.
F 2-
S
+
3
F - e C
5
5
-
5
+
e C
u
y
5
p
y
8
H2O2
F
AD
+
FAD
FADH2
K
b
NADPH
+
NADP
superoxide detected by NBT reduction to
formazan (blue precipitate).
 No O2 consumption by CGD patient’s
leukcoytes.
i
a
O2 level in solution
O2.
O2
e
r
Leukocytes (from normal patient)
*t
t
8
Opsonized bacteria or
N-formyl methionine peptide
b
Oxygen uptake is
insensitive to cyanide
l
c
l
t
Time
Oxygen in solution is measured by an O2
electrode
* Made by bacteria;
recognized by
mammalian cells as
foreign.
8
Trends in Biochem.
Sci. 2, 63 (1977)
9
Activation of leukocyte NADPH oxidase (Nox)
showing assembly of the enzyme and fusion of the oxidase-containing vesicle with the
phagosomal membrane.
Babior, 2000. American Journal of Medicine. 109:33  44.
10
(phosphorylation
by kinase)
Proton Channel
Function
+ H+
membrane
b558
b558
Proton
Channel
Vacuolar space
phox is “phagocyte oxidase”
NADPH oxidase (Nox)
activation
Medicine 79, 170-200 (2000)
gp91phox is b558 with proton channel (or K+?)
11
NADPH OXIDASE DEFICIENCY
Chronic Granulomatous Disease (CGD)
White Cell
bact.
0.1 M Chloride
in blood
• CGD can kill Bacteria that produce H2O2 and have low catalase activity, e.g., streptococci.
• But can’t kill bacteria which have high catalase activity or low H2O2 production, e.g., serratia,
nocardia, and aspergillus; fungal, staphylococcus, burkholderia (pneumonia, sepsis).
12
LEUKOCYTE ELECTRON TRANSPORT
Chronic granulomatous disease (CGD)
1. Two-thirds have mutations in gp91phox gene  Unable to form H2O2.
i.e., no cyanide resistant respiratory burst & don’t live beyond childhood
Have X-linked chromosomal deficiency
Have no cyt. b558 function
b-subunit – 91kD – glycosylated – X chromosome DEFECT
a-subunit – 22kD – normal chromosome 16
CGD patients have mutation in p47phox gene (reach adulthood)
Have autosomal recessive deficiency
2. CGD patients have no cytosol activating protein that phosphorylates and
activates NADPH oxidase which causes translocation of the oxidase from
the endoplasmic reticulum to the plasma membrane.
Medicine 79, 170  200 (2000); J Leukocyte Biol. 69, 191  6, 2001
13
THERAPY FOR CGD DISEASE
1. Prophylaxis with trimethoprim-sulfamethoxazole and interferon gamma
2. Bone marrow transplantation or granulocyte transfusion (with severe
infections)
3. Hemopoietic stem cell gene therapy (retrovirus-mediated expression of gp91phox to
reconstitute NADPH oxidase – Dinauer et al., 1999. Blood. 94(3):914-22)..
4. Possible Pharmaceutical Therapy?
Opsin + Chemoattractant
Polystyrene
Beads
Glucose oxidase
(Fungal)
Glucose + O2
H2O2
NADPH oxidase family: tissue location
14
J.Biol.Chem 283,16961-16965(2008)
• NOX1 colon>prostate,uterus,breast,macrophage?
• NOX2 phagocyte H2O2 for MPO >> hepatocyte,B
lymphocyte, cardiomyocytes,endothelial cells
• NOX3 inner ear,fetal
• NOX4 kidney,blood vessels,cardiomyocytes,endoth.
• NOX5 lymphoid tissue,testis
• Duox1 & 2 (dual oxidase) makes thyroxine hormone
and H2O2 for thyroid peroxidase TPO >lung,GI
Rheumatoid Arthritis is an autoimmune
inflammatory disease that may affect
(skin, blood vessels, heart, lungs,
muscles-- but principally attacks joints
(proliferative synovitis progressing to
cartilage destruction/joint ankylosis.
Pathogenesis (unknown etiology):genetic susceptibility;
joint damage mediated by leukocytes or exogenous
arthritogen (virus, mycobacteria).
15
leukocytes
Synovium
(synovial fluid)
Babior, 2000. American Journal
of Medicine. 109:33  44.
From Am J Med. (2000). 190:33-44
16
NSAID Drug therapy for RA or OA
IBUPROPHEN COX-1 inhib. 300mg 3 times pd
Cardiovascular & GI bleeding risk,inhib platelets, kidney/liver tox.
ALEVE (Naproxen) 200mg otc ; lasts 12h
• Inhibits inflammation , COX-I and COX-2 inhibitor
• Decrease pain, temp, muscle pain, menstrual cramps
• Lower stroke risk than ibuprofen; high dose risk GI bleed.
VIOXX COX-2 inhibitor for OA,RA 1999 introd.
• 2004 (withdrawn due to heart attack,stroke). Late?
CELEBREX (celecoxib)1998 COX-2 inhibitor 400mg
• low cardiorenal tox, platelet effects & GI bleeding
Side effects of COX-1 inhibitors
17
• e.g. Aspirin may cause stomach bleeding and risk GI
ulcer formation.
• molecular mechanism of bleeding/ulcers unknown.
• Aspirin causes stomach cell mitochondrial
uncoupling and acidosis.
• inhibits COX-1 activity thereby increasing tissue
unsat. fatty acid levels and cause acidosis.
• decreases PGE2 levels that protect stomach membr.
• inhibits thromboxane formn. and platelet aggreg.
• unsat.fatty acid + PGS attacks protective mucous layer
18
Mechanism of NSAID induced GI toxicity
• Stage I NSAIDs decrease intestinal mucosal
prostanoids (PGE,TXB2, 6-keto-PGF)
• Mitochondrial uncouplers (e.g.indomethacin,DNP)
compromised intestinal barrier). Resp.inhibited
• Stage 2 mild inflammation + aspirin
• Stage 3 histopathology, ulcers and bleeding
• Rat model: ulcers, intestinal inflammation or gastric
permeability induced by indomethacin or DNP +
Cox inhib (aspirin).
Aliment Pharmacol. Ther. (2000) 14,639-650
Rheumatoid Arthritis Therapy
Repetitive hypoxia in
joints, and
endothelial cells
Monosodium
urate crystals
in joint
ATP
hydrolysis
19
 Hypoxanthine
from ATP
Reperfusion O2 +
endothelial cell
xanthine oxidase
Uric acid
ALLOPURINOLinhib.
ROS
endothelial cell
nitric oxide
Destruction of joint
peroxynitrite
20
21
Anti-Inflammation therapy
• ROS Scavenger Therapy
SOD, selenomethionine/Vit E, 5-aminosalicylate, penicillamine:Cu
• Macrophage inhibitor therapy:
Gold thiomalate or auranofin or zinc or copper salicylate
• Prostaglandin Synthetase Inhibitors:-(NSAIDS)
COX-1 many cells e.g.aspirin,ibuprofen, but GI bleeding,kidney
COX-2 inflammatory cells e.g. VIOXX (withdrawn), CELEBREX
but cardiovascular problems.
• “Biologics” antibodies that inhibit inflammatory cytokines e.g.TNF-α
• Diet: decrease arachidonate intake (meat), increase omega 3 fatty
acids (fish) decreases bad prostaglandins, decrease Fe intake
References: Semin. Arthritis Rheum. 27: 366-70 (1998).
Autoimmunity Reviews 7,1-7(2007) Anti-inflammatory Biologics
22
2 EOSINOPHILS: (chemical warfare cont.)
Accumulate in parasite infection, asthma, rheumatoid diseases,
Hodgkin’s lymphoma and allergic or inflammatory diseases
1)destroy parasitic worms, tumor cells, fungi and bacteria by forming
hypobromite
H2O2 + Br- + H+
HOBr (hypobromite) + H2O
2) cytokine production e.g. PAF, LTC4 unlike neutrophils (leukocytes) .
Biochem J. 358, 233-239 (2001).
23
3
MACROPHAGES - chemical warfare function
1) Endocytosis and exocytosis via specific receptors for IgG and C3
coated in bacteria
2) H2O2 production by NADPH oxidase to kill mycobacteria
3) Arachidonate oxidation to prostaglandin
4) Cytokine production - upon activation by
PDGF
a) lipopolysaccharide (endotoxin)
TNF-a
b) immune system activation BCG infection
IL-1
c) inflammation or interferon (IFN-g)
PAF
TGF a and b
arginine
nitric oxide
kill tumor cells
5) endocytosis and delivery to lysosomes (via scavenger receptor) of
oxidised LDL (low density lipoprotein) - can result in transformation
to foam cell (the basis of the formation of atherosclerotic plaque)
SURGERY TRAUMA
e.g. if intestinal surgery causes prolonged interrupted blood flow
(reperfusion injury)
EARLY ORGAN FAILURE if endotoxin from Gram –ve cell
wall of Gram-ve bacteria in the intestine crosses gut barrier
Endotoxin releases immune cell growth factors &
humoral/cellular mediators (now too late!)
e.g. TNF, IL, arachidonic oxidation production
Attract PMN, macrophages, endothelium cells which further release
TNF,NO,H2O2
anaphylactic shock mast cells release histamine
(e.g. ARDS (adult respiratory distress syndrome), hepatitis
Multiorgan failure, sudden death
Harrison 2007 “Principles of Medicine”
24
25
Tumor Necrosis Factor (TNFα) as the primary trigger
for inflammatory response
Macrophages,monocytes,lymphocytes,keratinocyte
• TNFα incr in chronic inflammatory diseases:
rheumatism,arthritis,encephalitis,tumors
 Rheumatoid arthritis , psoriasis,Crohn’s disease
• Proinflammatory > antiinflammatory cytokines
• Drug therapy: NSAIDs, GC glucocorticoids, Disease Modifying
Antirheumatic Drugs (DMARDs).
26
Drug induced hepatocyte cytotoxicity caused by activated immune
cells releasing cytokines and reactive oxygen species
(Kupffer cells , macrophages ,neutrophils) i
1) Toxic doses of drugs or chemicals injure hepatocytes.
Injured hepatocytes release factors that attract Kupffer cells to specific
regions of the liver.
2) Additional mononuclear phagocytes are also recruited from blood and
bone marrow precursors.
3) Once localised in the liver, the macrophages become activated by
hepatocyte-derived factors of endothelial cells.
4) Activated macrophages and endothelial cells release cytokines
e.g.TNFα & platelet activating factor prime & activate Kupffer cells
which release Reactive Oxygen Species and more cytokines.
5) Some chemoattractants and cytokines can attract and activate
neutrophils that also contribute to hepatocyte injury.
27
Macrophages and Tissue Injury
Toxicant
Target tissues
Gold thiomalate
Gadolinium chloride
(macrophage inhibitor)
Activated macrophages
e.g. Kupffer cells
Amplification
Cytotoxicity
Mediators
Tissue Injury
Model for the role of macrophages in tissue injury by
generating inflammatory mediators.
28
Macrophage killing mechanism
• Macrophages endocytose & present antigens to T
cells as foreign substances
• Exocytose (via specific receptors) IgC and C3
coated bacteria
• Produce H2O2 to kill mycobacteria
• Produce cytokines PDGF,PAF,TNFα & β, IL1
convert arginine to NO
• Prevents foam cell formation by endocytosis of
oxid. LDL by macrophage oxid LDL & fusion with
lysosomes and digestion by cathepsins & Plipases.
INFLAMMATION MODEL FOR IDIOSYNCRATIC
DRUG TOXICITY (Liver injury, bone marrow agranulocytosis,
lung toxicity)
NEUTROPHIL or
KUPFFER CELL
DRUG
H2O2
MPO
H2O2
Peroxidase
Drug
Hepatocyte
Oxidative Stress
Radical
Mitochondria
GSH
NECROSIS
APOPTOSIS
or
GS DRUG
Ascorbate Cooxidation
LIPID PEROXIDATION
O’Brien et. al. Drug Discovery Today 2005 10, 617-625
29
Table 2: Inflammatory mediators implicated in toxicity
Toxicant
Mediator
1) Reactive oxygen intermediates
(H2O2, .OH)
Reactive nitrogen intermediates
(peroxynitrite)
Lung
Ozone
Asbestos
Amiodarone
Bleomycin
2) Hydrolytic enzymes
(collagenase, elastase)
Endotoxin
Silica
3) Lipids
(leukotrienes, prostaglandins,
thromboxanes)
Hyperoxia
4) IL-1
Cigarette smoke
5) TNF-a (mitochondrial toxin,
reactive oxygen species)
Cadmium chloride
Toxicology 160, 111  8 (2001).
Ann. Rev. Pharmacol. Toxicol. 35, 655 (1995).
Liver
Endotoxin
Acetaminophen
Corynebacterium parvum
Galactosamine
Carbon tetrachloride
1,2-dichlorobenzene
Phenobarbital
Endotoxin
Alcohol
30
31
Table 1: Toxicants whose pathophysiology is thought to be associated with
macrophages and inflammatory mediators
Pulmonary toxicants
Ozone
Sulfuric acid
Nitrogen dioxide
Cigarette smoke
Hyperoxia
Bleomycin
Amiodarone
Titanium dioxide
Silica
Asbestos
Cadmium chloride
Hepatotoxicants
Acetaminophen
Carbon tetrachloride
Lipopolysaccharide
Galactosamine
1,2-dichlorobenzene
Cadmium
Allyl alcohol
Endotoxin
Autoimmune disease drug therapy
1. Alkylating Agents (anticancer cytostatic drugs)
32
Inhibit cell division of both T cells and B cell (lymphocytes)
•
Cyclophosphamide, nitrosoureas, platinum compounds
Efficient therapy for systemic lupus, autoimmune hemolytic anemias,
Wegener’s
2. Antimetabolites Interfere with synthesis of nucleic acids
-
a) Folic acid analogues (METHOTREXATE) binds dihydrofolate reductase
and prevents synthesis of tetrahydrofolate. Used in treatment of autoimmune
diseases (rheumatoid arthritis) and in transplantations
b)Purine analogues (AZATHIOPRINE, or MYCOPHENOLATE (CellCept)
inhib. IMP dehydrogenase and GMP synth. Treatment of autoimmune
diseases (Wegeners,psoriasis ) also prevents transplant rejection.
Mercaptopurine (leukemia,Crohns).
c)Pyrimidine analogues LEFLUNOMIDE
3. Cytotoxic anticancer e.g. mithramycin, dactinomycin
inhibits RNA transcription and DNA replication
4. Biologics: anti-cytokine antibodies
33
• TNFalfa blockers1)Etanercept for RA
2)Infliximab (remicade) for Crohns
disease,colon cancer a chimeric human Fab.
3) Adalimumab (Humira) a humanised
monoclonal for colon cancer,macular degen.
4)Stelera NEW for psoriasis, 5 shots p.a.$50K
• Rituximab kills CD20 B cell NH lymphomas,RA
• Abatacept,fusion protein inhib.T cell costim.RA
• Avastin (bevacizumab) inhib.VEGF-A (vascular
endoth.growth factor) glioblastoma,macular
degen.
T lymphocytes formed in b.m.& mature in
thymus
34
1) T helper cells help B cells mature into plasma cells.
CD4+T cells express CD4 protein on their surface
2)19% Cytotoxic T cells destroy viral infective & tumor cells
3)Memory Tcells CD4+(lost in AIDs) or CD8+(cytotoxic)
subset of antigen T cells that persist long after infection.
4) Regulatory T cells (suppressor T cells)
5)Natural killer (NK) largest T cell.Interferons cause
cytotoxic granule release
6) Antigen-presenting cell (APC)
35
B cells bursa of fabricius(birds)
• Plasma cells are large B cells exposed to antigens
which produce antibodies that bind to microbes. In
tissues not plasma.
• Contain rough e.r. & cell rapid apoptosis (short life)
• Memory B cells formed from activ.B cells(long life)
• 23% B-1 cells IgM>IgG in peritoneal & pleural
cavities
• B-2 cells
36
Immunosuppressants and
anti-inflammatories RA
• B cell depleting agents
like rituximab
• tocilizumab human
monoclonal antibody
against IL-6R cytokine
No truly effective therapies
exist
37
Biomarkers for inflammation disease
Pro-inflammatory cytokines
• Elevated CRP,IL-1B,IL-1B,IL-6,IL-8
• Tumor necrosis factor alpha
• c-ANCA antineutrophil cytoplasmic stain
• p-ANCA antineutrophil perinuclear stain
• x-ANCA chronic inflammatory bowel disease
38
Biomarkers for determining oxidative stress
associated with diseases
•
•
•
•
•
•
•
•
•
1) Plasma lipid hydroperoxide
2) Oxidative DNA damage DNA 8-OHdQ
Urinary 8-OHdQ
Thymine oxidation HMdU
DNA strand breaks (Comet assay)
3) Protein carbonyls using dinitrophenylhydrazine
Protein oxidation,cysteine,methionine,histidine
Protein AGEs
4) Fructose oxidation forms toxic glyoxal
39
Celiac disease (dietary autoimmune)
• Small intestine inflammation caused by an immune
reaction to gliadin, a gluten protein, found in
wheat,barley,rye (bread).
• Blunting of villi, damage to inner surface ,
lymphocyte infiltration of crypts.
• Abdominal pain and diarrhea.
• Impaired vitamin uptake (brain,nervous,bone,liver)
• Treatment life long gluten free diet
• 1:>105-1:750 starting middle infancy
40
• White blood cells
within tissue, have a
role in innate and
adaptive immunity
• They engulf
pathogens and debris
via phagocytosis, and
move around via
amoeboid movement
41
• When a pathogen (can be self in this case) is
ingested by a macrophage, pathogen
proteins attach it to class II MHC
• Macrophage activated to deliver signals to
T-cells which produces autocrines and
stimulate their own production
• Helper T cells activate B cells which
produce antibodies that inhibit the
pathogens
42
• Results from a loss of immunological tolerance –
which is the ability to ignore self-antigens
• T and B lymphocytes that recognize self-antigens
are usually destroyed in the Thymus and Bone
marrow, respectively, preventing autoimmunity.
• Infection and overstimulation of APCs can break
tolerance and induce priming of T-cells
• A combination of genetics and environment are
responsible for autoimmune disease
• Human Lymphocyte Antigen (HLA/MHC) is the
best predictor as it enhances antigen presentation
resulting in increased T-cell activation
43
Antibody-mediated (B cells)
•
• Binding of antigens on the
surfaces of B-cells produces
antibodies
•
• Autoantibodies:
– Bind to self-tissue, and
activates the complement
cascade which targets the
self-antigen to be
phagocytosed (opsonized)
by Macrophages
Cell-mediated (T cells)
Immune cells both kill
cells directly and
indirectly via cytokines
(PG, NO, etc.)
Macrophages:
– INITIATE the response as
antigen presenting cells
– PARTAKE in killing cells
through antibody
dependent-dependent cellmediated cytotoxicity and
by releasing cytokines
(TNF and IL-1)
– PRESENT SELF-TISSUE
TO T CELLS
44
II White (Immune) Cells
• THE END