Download Regulation of the immune response by programmed cell death

Regulation of the immune response
by programmed cell death (apoptosis)
LIVE AND LET DIE
within the Immune System
LIVE AND LET DIE
within the Immune System
Historical aspects
Morphological aspects
Methodological aspects
Molecular aspects
Immunological aspects
•
autoimmunity
•
AICD
•
T cell effector function
History
Apoptosis = Programmed Cell Necrosis
1842
Carl Vogt (1817-1895) published that programmed
cell death is a regulated developmental process.
Untersuchungen über die Entwicklungsgeschichte der Geburtshelferkröte (Alytes obstetricans). Jent und Gassman, Solothurn 1842,
S. 130
Carl Vogt
1885
The anatomist Walter Flemming (Kiel) delivered a
more precise description of nuclear processes of
programmed cell death (chromatolysis).
1965
John Foxton Ross Kerr (Queensland) distinguished
programmed cell necrosis from traumatic cell death.
Walter Flemming
A histochemical study of hypertrophy and ischaemic injury of rat liver
with special reference to changes in lysosomes". J. Pathol and
Bacteriol 90: 419–35.
John FR Kerr
History
Apoptosis = Programmed Cell Death
1972
John FR Kerr joined Alastair R Currie, and Andrew Wyllie in
Aberdeen. In 1972 they published a milestone paper in which they
described that ‚programmed cell death‘ is morphologically
distinct from ‚accidental cell death = necrosis‘.
In this paper, they coined the term „Apoptosis“.
Kerr JF, Wyllie AH, Currie AR (1972) "Apoptosis: a basic biological phenomenon with
wide-ranging implications in tissue kinetics". Br. J. Cancer 26: 239–57.
John FR Kerr
Andrew Wyllie
History
Apoptosis = Programmed Cell Death
In the original Kerr, Wyllie and Currie paper, there is a footnote regarding
the pronunciation:
"We are most grateful to Professor James Cormack of the Department of Greek, University
of Aberdeen, for suggesting this term. The word "Α
Αποπτω
ποπτωσις"
ις (Greek spelling of apoptosis)
is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or
leaves from trees. To show the derivation clearly, we propose that the stress should be on
the penultimate syllable, the second half of the word being pronounced like "ptosis" (with
the "p" silent), which comes from the same root "to fall", and is already used to describe
the drooping of the upper eyelid."
Kerr et al., Brit J Cancer, 1972; 26:239-57
History
Apoptosis = Programmed Cell Death
1980
Wyllie described endonuclease activation during apoptosis.
Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation. Nature 284: 555-556.
Andrew Wyllie
Morphology
Hallmarks: Apoptois versus Necrosis
induction
response
passive
accidental
inflammatory
active
programmed
antiinflammatory
Roche Applied Science - Apoptosis, Cell Death and Cell Proliferation, 3rd edition
History
„Apoptosis“
entering Science and Immunology – the first two decades
120
12
Apoptosis
DNA
10
100
8
80
6
60
4
40
2
20
0
0
ced-1
until 1990:
378 papers on Apoptosis,
51 in an immunological context
ced-3, -4
Fas, APO-1
"The Nobel Prize in Physiology or Medicine 2002". Nobelprize.org. 24 Sep
2010 http://nobelprize.org/nobel_prizes/medicine/laureates/2002/
History
The Nobel Prize in Physiology or Medicine 2002
was awarded "for their discoveries concerning the
'genetic regulation of organ development and programmed cell death'".
Brenner established the nematode Caenorhabditis elegans as a model organism and studied
organ and neuronal development. The first genes and gene products that control apoptosis were
identified in C. elegans. It was noted that 131 of the 1090 worm cells died by programmed cell
death. The team deciphered the genetic regulation of programmed cell death in C. elegans.
Sydney Brenner
Robert Horvitz
John E. Sulston
Photos: Copyright © The Nobel Foundation
History
The C. elegans cell death program
1983
decreased survival
gene
mutagen
ethyl methane
sulfonate
increased survival
Complete lineage description of all 1090 cells born
- 113 cells die during embryogenesis
- 18 cells die later in life
- mutants reveal defects in death induction or removal of dead cells
Hedgecock EM, Sulston JE, Thomson JN (1983) Mutations affecting programmed cell deaths in the
nematode Caenorhabditis elegans. Science 220, 1277-1279 (description of ced-1)
http://www.wormbase.org/ - ced: “CEll Death abnormality”
History
The C. elegans cell death program
1986
decreased survival
gene
mutagen
ethyl methane
sulfonate
increased survival
Mutated Gene
Phenotype
Function
ced-3
blocked all 131 cell deaths
protease
ced-4
blocked all 131 cell deaths
ced-3 adapter/activator
ced-9
absence of cell death, embryonic lethality
anti-apoptotic protein
egl-1
reduced cell death “egg laying defective”
blocks ced-9
Ellis HM, Horvitz HR (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell. 44:817-29
Foghsgaard et al., J. Cell Biol. 153:9991009, 2001
History
The C. elegans cell death program
The Nobel Prize in Physiology or Medicine 2002
Regulator
C. elegans
ced-9
Adapter
ced-4
Effector
ced-3
cell death
Lettre et al. Nat Rev Mol Cell Biol 7:97–108, 2006
History
Anti-Fas mAb induces apoptosis in fibroblasts
1989
Shin Yonehara
To obtain a monoclonal antibody against the
human IFN receptor, Dr. Yonehara immunized
mice with the human fibroblast cell line FS-7,
and then screened an expression library for the
anti-human IFN receptor.
He accidentally found an mAb that had a
cytopathic activity against human FL cells.
The antigen recognized by the antibody was
designated as the Fas antigen (FS-7-associated
surface antigen).
J Exp Med. 169:1747 – 1756, 1989
History
Anti-APO-1 mAb induces apoptosis in B lymphoblasts
1989
Peter H Krammer
To characterize cell surface molecules involved
in control of growth of malignant lymphocytes,
mAb were raised against the human B
lymphoblast cell line SKW6.4.
The mAb anti-APO-1, reacted with a 52kilodalton antigen (APO-1) on a set of
activated human lymphocytes, on malignant
human lymphocyte lines, and on some patientderived leukemic cells.
Science 245:301-305, 1989
History
„Apoptosis“
entering Science and Immunology – the third decade
12000
Fas, AICD
lpr
FasL
gld
FasL/Fas
AICD DISC
Typ I
Typ II
10000
8000
6000
4000
2000
0
1990-1999:
30435 papers on Apoptosis,
5879 in an immunological context
History
Fas = APO-1
1991
Shigekazu Nagata
The Mr of the molecule recognized by the APO1 antibody was reported to be 52 kDa. This Mr
was quite different from that initially predicted
for the Fas antigen (200 kDa).
However, the cloned human Fas cDNA encoded
a glycoprotein of 52 kDa.
Fas, like APO-1, was found to be expressed
in various transformed hemopoietic cells,
including lymphoma, myeloid leukemia, and
HTLV-1-transformed cells. The identity of
Fas with APO-1 was then shown by cloning
of the APO-1 cDNA.
Cell 66:233-243, 1991
History
Cloning of FasL
1993
Shigekazu Nagata
The Fas antigen (Fas) belongs to the tumor
necrosis factor (TNF)/nerve growth factor
receptor family, and it mediates apoptosis.
Fas ligand is a type II transmembrane protein
that belongs to the TNF family.
Recombinant Fas ligand expressed in COS cells
induces apoptosis in Fas-expressing target cells.
Fas ligand is expressed in activated splenocytes
and thymocytes.
Cell 75:1169-1178, 1993
History
Fas/FasL mutations cause lymphoproliferative syndromes
1992/1994
Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S.
Lymphoproliferation disorder in mice explained by defects in Fas antigen that
mediates apoptosis. Nature. 356:314–317, 1992
Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, Nagata
S. Generalized lymphoproliferative disease in mice, caused by a point mutation in
the Fas ligand. Cell. 76:969–976,1994
History
Fas/FasL mutations cause lymphoproliferative syndromes
1992/1994
Phenotype of lpr and gld mice:
accumulation of immature T cells
(CD4-CD8-B220+CD3+TCRαβ)
lymphadenopathy
splenomegaly
IgG/IgM autoantibodies (anti-DNA, RF)
nephritis and/or arthritis
more severe: FasL KO mice
Karrey et al., J Immunol.172:2118-2125, 2004
premature death, massive lymphoproliferation
patients:
ALPS (autoimmune lymphoproliferative syndrom)
per definition - mutation or malfunction of apoptosis
pathways - frequently Fas, rarely FasL mutations
History
Activation-induced T cell death (AICD)
1991/1993
Sebastian Wesselborg
Dieter Kabelitz
History
Fas/FasL mediate AICD – the NATURE Triple
1995
Dhein J, Walczak H, Bäumler C, Debatin
KM, Krammer PH. Autocrine T-cell
suicide mediated by APO-1/(Fas/CD95).
Nature. 373:438–441, 1995
Brunner T, Mogil RJ, LaFace D, Yoo NJ,
Mahboubi A, Echeverri F, Martin SJ, Force
WR, Lynch DH, Ware CF, Green D. Cellautonomous
Fas
(CD95)/Fas-ligand
interaction mediates activation-induced
apoptosis in T-cell hybridomas. Nature.
373:441–444, 1995
Ju ST, Panka DJ, Cui H, Ettinger R, elKhatib M, Sherr DH, Stanger BZ, MarshakRothstein A. Fas(CD95)/FasL interactions
required for programmed cell death after
T-cell activation. Nature. 373:444–448,
1995
Methods to detect apoptotic cells
microscopical
inspection
agarose gel
PI-staining
Nicoletti-assay
TUNEL
caspase assays
annexin staining
mitochondrial
membrane potential
Roche Applied Science - Apoptosis, Cell Death and Cell Proliferation, 3rd edition
Purdue Cytometry CD-ROM Series,volume 4
APOPTOSIS vs. NECROSIS
Marco Vitale, Giorgio Zauli and Elisabetta Falcieri
Morphological features
TEM:
disruption of plasma membrane
disruption of organelles
preservation of nuclear morphology
(x 10,000)
TEM:
apoptotic (A) and normal (N) cell
chromatin rearrangement in A
preservation of membrane
preservation of organelles
(x 8,000)
SEM:
numerous surface lesions
(x 5,000)
SEM:
surface blebbing
(x 5,000)
FF:
nuclear envelope with regular
distribution of nuclear pores
(x 30,000)
FF:
nuclear envelope with
characteristic clustering of
nuclear pores
(x 35,000)
TEM: transmission electron microscopy – SEM: scanning electron microscopy – FF: Freeze Fracture electron microscopy
DNA fragmentation
1.8-2% agarose gel
1991
‚Nicoletti“-assay
1992
TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992). "Identification
of programmed cell death in situ via specific labeling of nuclear
DNA fragmentation". J Cell Biol 119 (3): 493–501.
Caspase activation – state-of-the-art 2010
Caspase activity
Caspase processing
Fluorometric Immunosorbent Enzyme Assay (FIENA) for
the specific, quantitative in vitro determination of Caspase
3 activity in microplates
Cleaved Caspase-3 (Asp175) Antibody #9661
Cell membrane alterations
PI staining
(dead cells)
Annexin V staining
(externalization of Phosphatidylserine)
1994
Blood, 84 :1415-1420, 1994
J Immunol. 150:4338-4345, 1993
7-AAD = 7-Aminoactinomycin D
cpt = camptothecin
Mitochondrial membrane potential
1995
Guido Kroemer
Different cationic dyes are now
available as „Mitochondrial Membrane
Potential Detection Kits“
J Exp Med.182:367-377, 1995
Mol Cell Biochem. 174:185-188, 1997
Jurkat cells were stimulated with (A) DMSO or (B) 1.5 µM staurosporine
for 2 hours. The cells were then labeled with the JC-1 reagent for 15
minutes.
Methods to detect apoptotic cells
microscopical
inspection
agarose gel
PI-staining
Nicoletti-assay
TUNEL
caspase assays
annexin staining
mitochondrial
membrane potential
Roche Applied Science - Apoptosis, Cell Death and Cell Proliferation, 3rd edition
History
„Apoptosis“
entering Science and Immunology – the new millennium
25000
20000
15000
10000
5000
0
2000 - 2009:
155107 papers on Apoptosis,
22835 in an immunological context
A simplified scenario of apoptotic pathways in normal cells
Molecular aspects – from C. elegans to mammals
Regulator
Adapter
Effector
C. elegans
ced-9
ced-4
ced-3
Mammals
Bcl-2
Apaf-1
Casp 3
Mammalian Bcl-2 can substitute for ced-9 in C. elegans !
Intrinsic (mitochondrial) pathway
Anti-APO-1/Anti-Fas induce programmed cell death or apoptosis
in a variety of different cell types
Extrinsic (receptor-driven) pathway
cell death
The C. elegans mitochondrial pathway to apoptosis
Regulator
C. elegans
ced-9
Adapter
ced-4
Effector
ced-3
cell death
Lettre et al. Nat Rev Mol Cell Biol 7, 97–108 (February 2006)
The vertebrate mitochondrial pathway to apoptosis
Proteins/factors that move to and affect mitochondrial membranes
Brenner & Kroemer, Science 289:1150-1151, 2000
The vertebrate mitochondrial pathway to apoptosis
Bcl-2 family proteins in mammals
19 members; the relative abundance of individual
species dictates effect on cells.
• Bcl-2 (homologous to ced-9) is the prototype and
was first discovered in 1985 in a translocation
found in follicular lymphoma
• All have the BH3 domain (Bcl-2 Homology 3)
BH3 is the pro-apoptotic domain exposed on
activation
• Act as hetero- or homodimers
Anti-apoptotic members (Bcl-2, Bcl-XL) form
dimers with pro-apoptotic members to
inactivate them and block cytochrome c release
Pro-apoptotic dimers (e.g. Bax) increase
mitochondrial permeability
Ranger et al., Nature Genetics 28:113-118, 2001
The vertebrate mitochondrial pathway to apoptosis
3 (non-exclusive) models proposed to explain Bcl-2’s function
1.
As ion-channels
crystal structure resemble membrane
insertion domain of bacterial toxins
2.
As proteins that modulate the
activity of caspases
In C. elegans ced-9 (Bcl-2) interacts
with ced-4 (Apaf-1) inhibiting
activation of ced-3 (caspase 9)
3.
Hengartner, Nature 407:770-776, 2000
As inhibitors of cytochrome c
export
Bcl-2 may act to block the release of
cytochrome c
The vertebrate mitochondrial pathway to apoptosis
The intrinsic apoptotic pathway in T cells.
BAX (B-cell lymphoma 2 (BCL-2)-associated
protein X) and BAK (BCL-2 antagonist/killer) are
sequestered in an inactive state at the outer
mitochondrial membrane by binding to
members of the anti-apoptotic BCL-2 family.
Upon various stimuli, BH3-only proteins BIM
(BCL-2-interacting mediator cell death) or PUMA
(p53-upregulated modulator of apoptosis) get
activated and discplace anti-apoptotic BCL-2
proteins
This displacement of anti-apoptotic BCL-2
family members from BAX and BAK proteins
leads to the release of cytochrome c from
mitochondria.
After formation of the apoptosome, a caspasecascade is initated that leads to cell death.
Krammer, Arnold and Lavrik . Nat Rev Immunol. 7: 532-542, 2007
Apaf-1 Apoptotic protease activating factor 1
Caspases
Normal Cells
Cysteine proteases cleave Asp in P1 position of
Apoptosis
Necrosis
tetrameric recognition sequence
Synthesised as inactive zymogens
Activated by Asp cleavage
removes N-terminal prodomain
active tetramer formed
14 mammalian caspases identified to date
caspase 1, 4, 5 and 11(12L), non-apoptotic
function (cytokine processing)
10 caspases implicated in apoptosis
Activated by 3 mechanisms:
autoactivation (caspase 8)
trans-activation (caspase 3, 6 and 7)
conformational change (caspase 9)
RC Taylor, SP Cullen & SJ Martin. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9, 231-241,2008
Foghsgaard et al., J. Cell Biol. 153:9991009, 2001
Caspase cascade
Normal Cells
Apoptosis
Necrosis
Initiator (apical) caspases (group II
caspases: CASP2, CASP8, CASP9, CASP10)
cleave inactive pro-forms of effector
(executioner) caspases (group III caspases:
CASP3, CASP6, CASP7) to activate them.
Effector caspases cleave other protein
substrates to trigger the apoptotic process.
(CASP1, CASP4 and CASP5 (group I) are not
classified as initiator or effector caspases,
because they are inflammatory enzymes that
are involved in cytokine maturation. CASP14 is
involved in skin cell development.)
Krammer, Arnold and Lavrik . Nat Rev Immunol. 7: 532-542, 2007
Foghsgaard et al., J. Cell Biol. 153:9991009, 2001
Caspase Substrates:
• cleaved by executor caspases only (caspase 3, 6 and 7)
Group
Substrates
Signalling Proteins
RasGAD, D4-GDI, PKCδ, FAK,
PAK2, RIP
Structural Proteins
Lamin A and B, G-actin, fodrin,
gelsolin
Cell cycle/DNA repair
PARP, pRb, DNA-PK
DNA fragmentation factor
Anti-Apoptotic Proteins
Bcl-2, Bcl-xL
Pro-apoptotic Proteins
Bid
Nucleases
CAD/ICAD complex
Caspase substrates in humans: > 400 and still counting
Table 1. Caspase substrates:
structural proteins
Table 2. Caspase substrates:
regulators of transcription/translation
http://bioinf.gen.tcd.ie/casbah/
Table 3. Caspase substrates:
kinases and signalling intermediaries
The vertebrate extrinsic pathway to apoptosis
Fas/Apo-1/CD95/TNFRSF6
The vertebrate extrinsic pathway to apoptosis
Fas/Apo-1/CD95/TNFRSF6
1995
DISC
Markus E Peter
Peter H Krammer
Taken from: Lavrik & Krammer, Acta Naturae 2009
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, Peter ME. Cytotoxicity-dependent APO-1
(Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J.
1995 14:5579-88.
How the intrinsic and extrinsic pathways are linked
Fas/Apo-1/CD95/TNFRSF6
1998
Type I and Type II cells show similar
kinetics of CD95-mediated apoptosis
Type I cells
caspase-8 activated in seconds, caspase-3 in
30 min
overexpression of Bcl-2 or Bcl-xL does not
block caspase-8 and caspase-3 activation and
death
DISC formation – caspase-8 activation
Carsten Watzl
(Scaffidi)
Type II cells
cleavage of both caspases delayed for 60 min.
overexpression of Bcl-2 or Bcl-xL blocks
caspase-8 and caspase-3 activation and death
Reduced DISC formation - caspase-8
activation only after loss of mitochondria
potential
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME.Two CD95
(APO-1/Fas) signaling pathways. EMBO J. 1998 7:1675-87.
How the intrinsic and extrinsic pathways are linked
Type II cells
Type I cells
Mac Farlane & Williams, EMBO Rep. 5:674–678, 2004
Apoptosis is essential for immune function
Programmed cell death is part of the
thymic selection of the immune repertoire
silent removal of useless cells and cell debris
termination of immune responses (AICD)
establishment of immune privilege
effector mechanism of killer cells
Consequences of disturbed programmed cell death are
autoimmunity/lymphoproliferation
tumor development
immunodeficiency (e.g. defect T cell effector function)
Apoptosis is essential for immune function
thymic selection of the immune repertoire
Alberts et al., Molecular Biology of the Cell, 4th edition
Apoptosis is essential for immune function
silent removal of useless cells and cell debris
Macrophage ingesting apoptotic bodies
Sebastian Wesselborg
Savill & Fadok, Nature 407:784-788, 2000
Removal of apoptotic
cells:
attraction
recognition
ingestion
anti-inflammation
Lauber, Blumenthal, Waibel and Wesselborg. 2004 Molecular Cell 14:277-287
Apoptosis is essential for immune function
silent removal of useless cells and cell debris
“Eat-Me” signals at the engulfment synapse
Lauber, Blumenthal, Waibel and Wesselborg. 2004 Molecular Cell 14:277-287
Apoptosis is essential for immune function
termination of immune responses (AICD and ACAD)
Krammer et al. Nat Rev Immunol. 7: 532-542, 2007
Apoptosis is essential for immune function
termination of immune responses (AICD and ACAD)
D Brenner, PH Krammer and R Arnold. Critical Rev Oncol/Hematol. 66:52-64, 2008
Consequences of disturbed programmed cell death
autoimmunity/lymphoproliferation
Phenotype of lpr and gld mice:
accumulation of immature T cells
(CD4-CD8-B220+CD3+TCRαβ)
lymphadenopathy
splenomegaly
IgG/IgM autoantibodies (anti-DNA, RF)
nephritis and/or arthritis
more severe: FasL KO mice
Karrey et al., J Immunol.172:2118-2125, 2004
premature death, massive lymphoproliferation
patients:
ALPS (autoimmune lymphoproliferative syndrom)
per definition - mutation or malfunction of apoptosis
pathways - frequently Fas, rarely FasL mutations
Apoptosis is essential for immune function
establishment of immune privilege
Fas/FasL and „immune privilege“
Definition:
Site where immune responses are limited or
prevented (graft survival is much higher in these
sites than in any other site in the body)
Tissues/Organs:
FasL+ cell
immune
privileged tissue
FasL
Fas
brain, eye, testis, ovaries, uterus (pregnancy)
FasL:
constitutively expressed on eye, testis, trophoblasts
Fas:
on «target cells» of lymphoid system,
e.g. placental leukocytes
activated
T cell
D. Green & TA Ferguson. The role of fas ligand in immune privilege. Nat Rev Mol Cell Biol 2: 917-924, 2001
Consequences of disturbed programmed cell death
tumor îmmune privilege
The „tumor counterattack“
Definition:
Malignant cells express mFasL, produce sFasL or
vFasL They generate their «immune privilege»
to escape tumor infiltrating T cells.
At the same time, many tumor cells display
apoptosis resistance.
Cell types:
ovarian cancer, pancreatic cancer, breast cancer,
colon cancer, liver cancer, renal cancer, etc
FasL:
constitutive expression or production of FasL
Fas:
on tumor cells and on activated tumor infiltrating
lymphocytes (TIL)
Igney and Krammer, Nat Rev Cancer, 2002
Apoptosis is essential for immune function
effector mechanism of killer cells
FasL- loaded T cell
M Lettau & O Janssen, 2008
Taken from Scientific American
Apoptosis is essential for immune function
effector mechanism of killer cells
staining: „Lysotracker“
de Saint Basile G, Ménasché G, Fischer A.
Nat Rev Immunol 11: 568-579 2010 (August)
J Qian & O Janssen, 2003
Apoptosis is essential for the survival of an organism
Regulation of development (e.g. interdigital webs)
Tissue skulpting and homeostasis
Immune homeostasis and effector function
Silent removal of useless cells and cell debris
Walensky, Cell Death Differ 13:1339-1350, 2006
Perspective:
No (scientific) life without deathP
Just a few „hot topics“:
T cells:
Cytolytic effector granules and their killing mechanisms
Death ligands as receptors – reverse signaling
Pro- and anti-apoptotic functions of death receptors
Tumor cells:
Overcoming death receptor resistance
Tailored induction of cell death
Cell death in general:
Regulation of death receptors by miRNA
Autophagy (5000 papers in the last decade)
Perspective:
No (scientific) life without deathP
200000
175234
cell death
150000
necrosis
apoptosis
157975
apoptosis+immunology
100000
121530
50000
22593
0
To be continued...
LIVE AND LET DIE
within the Immune System