John wiley sons vch apoptosisandautoimmunityfrommechanismstotreatments isbn3527304428 jakarta underground

Part 5 “Altered Self ” in Dying Cells 24314 Autoantigens as Substrates for Apoptotic Proteases: Implications for the Pathogenesis of Systemic Autoimmune Disease 245 Antony Rosen and Livi

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Edited by

M Herrmann and J R Kalden

Apoptosis and Autoimmunity From Mechanisms to Treatments.

Edited by J R Kalden, M Herrmann

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Further titles of interest

Stuhler, G / Walden, P (Eds.)

Cancer Immune Therapy

Current and Future Strategies

Culture of Animal Cells

A Manual of Basic Technique

Krauss, G / Cooper, B L / Schönbrunner, N

Biochemistry of Signal Transduction and RegulationBuilding Blocks and Fine Chemicals

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Edited by

M Herrmann and J R Kalden

Apoptosis and Autoimmunity

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Prof Dr Dr Joachim R Kalden

University of Erlangen-Nuremberg

Department of Internal Medicine III

and Institute for Clinical Immunology

Bibliographic information published by Die Deutsche Bibliothek

Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at

<http://dnb.ddb.de>.

in other languages) No part of this book may be reproduced in any form – nor transmitted or translated into a machine language without writ- ten permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to

be considered unprotected by law.

Printed in the Federal Republic of Germany Printed on acid-free paper

Composition K+V Fotosatz GmbH, Beerfelden

Printing betz-druck gmbh, Darmstadt

Bookbinding Großbuchbinderei J Schäffer GmbH & Co KG, Grünstadt

n This book was carefully produced Nevertheless,

authors, editors and publisher do not warrant the information contained therein to be free of er- rors Readers are advised to keep in mind that statements, data, illustrations, procedural details

or other items may inadvertently be inaccurate.

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More than 100 years ago Paul Ehrlich coined the expression ‘horror autotoxicus’implicating the existence of autoimmune diseases After the first description of anautoimmune disease of the thyroid, it soon became obvious that autoimmunity inprinciple is a self-limiting process, which in certain situations might proceed in

an autoaggressive disease situation, when stringent control mechanisms havefailed or are dysregulated Despite of extensive research activities over the past de-cades, the etiology of autoimmune diseases is still enigmatic Different hypoth-eses have been postulated, although these only partially explain the phenomenon

of ‘autoimmunity’

More recently, the relationship between autoimmunity and apoptosis has beenthe focus of much research activity Apoptosis as a genetically predetermined pro-cess is not only a vital mechanism sustaining homeostasis in the regulation of im-mune reactivity, but, in addition to being an important factor in general cell phy-siology, produces pronounced morphological changes of cells and the breakdown

of cellular constituents by nucleolytic and proteolytic cleavage, resulting in thepersisting presence of potential autoantigens This book presents an up-to-datediscussion on apoptosis and its role in autoimmunity

We would like to express our appreciation and gratitude to the authors for theiroutstanding contributions and cooperation We also gratefully acknowledge thecontinuous support of Andreas Sendtko and his colleagues at Wiley-VCH in therealization of this book

Joachim R Kalden

Preface

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Preface V

List of Contributors XVII

Part 1 General Features of Apoptosis 1

1 Apoptosis and Autoimmunity 3

Keith Elkon

2 Caspase Knockouts: Matters of Life and Death 13

Saquib Lakhani, Binfeng Lu and Richard A Flavell

VII

Contents

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Part 2 Clearance of Apoptotic Cells 37

3 Anti-inflammatory and Immunoregulatory Effects of Apoptotic Cells 39

Reinhard E Voll, Martin Herrmann, Irute Girkontaite, Wasilis Kolowos and Joachim R Kalden

In Vivo 51

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5.1 Introduction 79

6 The Role of ATP-binding Cassette Transporters in the Clearance

of Apoptotic Cells: A Tale of Two Systems 97

Véronique Rigot and Giovanna Chimini

7 Innate Immunity and Apoptosis:

CD14-dependent Clearance of Apoptotic Cells 111

Christopher D Gregory and Andrew Devitt

Contents IX

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Part 3 Autoimmunity Caused by Defective Execution of Apoptosis

or Defective Clearance of Apoptotic Cells 133

8 Autoimmune Lymphoproliferative Syndromes (ALPS) 135

Frédérik Rieux-Laucat, Françoise le Deist and Alain Fischer

9 Infection and Inflammation as Cofactors for Autoimmunity

of Systemic Lupus Erythematosus Patients 157

Hanns-Martin Lorenz

10 Apoptosis in Rheumatoid Arthritis 169

Yuji Yamanishi and Gary S Firestein

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10.5.2 p53 Protein Expression in Inflammation 175

11 Systemic Lupus Erythematosus 187

Thomas D Beyer, Susanne Kuenkele, Udo S Gaipl, Wasilis Kolowos,

Reinhard E Voll, Irith Baumann, Joachim R Kalden

and Martin Herrmann

Contents XI

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11.9.3 Hypothesis: Accumulation of Apoptotic Dell-Derived Nuclear Fragments

Part 4 Immunogenicity of Apoptotic Cells 205

12 Dendritic Cells Pulsed with Apoptotic Tumor Cells as Vaccines 207

Lars Jenne and Birthe Sauter

13 The Immune Response against Apoptotic Cells 227

Angelo A Manfredi

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Part 5 “Altered Self ” in Dying Cells 243

14 Autoantigens as Substrates for Apoptotic Proteases:

Implications for the Pathogenesis of Systemic Autoimmune

Disease 245

Antony Rosen and Livia Casciola-Rosen

during Apoptosis, Becoming Clustered and Concentrated

may be an Important Defect Underlying Systemic

15 Distinct Cleavage Products of Nuclear Autoantigens

in Apoptosis and Necrosis: Implications for Autoimmunity 261

Carlos A Casiano, Christine Molinaro, Sheldon Holder and Xiwei Wu

Contents XIII

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16 ‘Tissue’ Transglutaminase and Autoimmunity 289

Alessandra Amendola and Mauro Piacentini

Part 6 The Role of DNA Binding Proteins for Systemic Autoimmunity 317

18 Nucleosomes and Anti-Nucleosome Autoantibodies

as Mediators of Glomerular Pathology in Systemic

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Institut für Klinische Immunologie

Medizinische Klinik III

Krankenhausstraße 12

91054 Erlangen

Germany

Casciola-Rosen, LiviaThe Johns Hopkins UniversitySchool of Medicine

Departments of Medicineand Dermatology

720 Rutland AvenueBaltimore, MD 21205-2196USA

Casiano, Carlos A

Loma Linda UniversityDepartment of Biochemistryand Microbiology

Division of Microbiologyand Molecular GeneticsCenter for Molecular Biologyand Gene Therapy

and Department of MedicineSection of Rheumatology

11085 Campus StreetMortensen Hall 132Loma Linda, CA 92350USA

Chimini, GiovannaCentre d’ImmunologieINSERM/CNRS de Marseille-LuminyCase 906

Parc Scientifique de Luminy

13288 Marseille Cedex 09France

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Hospital for Special Surgery

Weill Medical College

School of Biomedical Sciences

Queen’s Medical Centre

330 Cedar Street

PO Box 208011New Haven, CT 06520USA

Gaipl, Udo S

Universität Erlangen-NürnbergInstitut für Klinische ImmunologieMedizinische Klinik III

91054 ErlangenGermanyGirkontaite, IruteUniversität Erlangen-NürnbergInstitut für Klinische ImmunologieMedizinische Klinik III

91054 ErlangenGermanyGregory, Christopher D

University of NottinghamMedical School

School of Biomedical SciencesQueen’s Medical Centre

D FloorNottingham NG7 2UHUK

Herrmann, MartinUniversität Erlangen-NürnbergInstitut für Klinische ImmunologieMedizinische Klinik III

91054 ErlangenGermany

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and Molecular Genetics

Center for Molecular Biology

and Gene Therapy

91054 Erlangen

Germany

Lakhani, SaquibYale UniversitySection of Immunobiology

330 Cedar Street

Le Deist, FrançoiseINSERM Unit 429Hopital Necker-Enfants Malades

149 rue de Sevres

75743 Paris Cedex 15France

Lorenz, Hanns-MartinUniversität Erlangen-NürnbergInstitut für Klinische ImmunologieMedizinische Klinik III

91054 ErlangenGermany

Lu, BinfengYale UniversitySchool of MedicineHoward Hughes Medical Institute andSection of Immunobiology

330 Cedar Street

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List of Contributors

XX

Mevorach, Dror

The Hebrew University

Hadassah Medical Center

and Molecular Genetics

Center for Molecular Biology

and Gene Therapy

Departments of Medicineand Pathology

Cell Biology and Anatomy

720 Rutland AvenueBaltimore, MD 21205-2196USA

Rovere-Querini, PatriziaInstituto Scientifico OspedaleSan Raffaele

Department of Internal Medicinevia Olgettina 60

20132 MilanoItaly

Sauter, BirteUniversität Erlangen-NürnbergDermatologische Klinik mit PoliklinikHartmannstraße 14

91052 ErlangenGermanySchuler, GeroldUniversität Erlangen-NürnbergDermatologische Klinik mit PoliklinikHartmannstraße 14

91052 ErlangenGermanyUtz, Paul J

Stanford UniversitySchool of MedicineDepartment of MedicineDivision of Immunology and Rheuma-tology

CCSR BuildingRoom 2215A

300 Pasteur DriveStanford, CA 94305-5166USA

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van der Vlag, Johan

91054 Erlangen

Germany

Wu, XiweiLoma Linda UniversityDepartment of Biochemistryand Microbiology

Division of Microbiologyand Molecular GeneticsCenter for Molecular Biologyand Gene Therapy

11085 Campus StreetMortensen Hall 132Loma Linda, CA 92350USA

Yamanishi, YujiUniversity of CaliforniaMEDCTR/Rheumatology

9500 Gilman Drive BSB 5064

La Jolla, CA 92093-0656USA

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Part 1

General Features of Apoptosis

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Introduction

Whereas the appearance of cells dying by apoptosis has been recognized for cades [1], if not more than a century [2], the understanding that cell survival anddeath are under stringent control is relatively new The detailed elucidation of thebiochemistry of apoptosis that has emerged over the last decade is nothing short

de-of astounding and has altered the way in which we think about disease ally, diseases are classified according to the organ system effected – cardiovascular,endocrine, neurological, etc However, it is much more useful for biomedical in-vestigators to think about the mechanisms responsible for the diseases In this re-spect, the simple reclassification according to whether diseases are associated withtoo little or too much cell death is highly informative [3, 4] Cancers are clearlycaused by enhanced cell growth and survival, whereas a large number of neurode-generative diseases are caused by premature cell death of specific neurons Amyo-trophic lateral sclerosis (ALS), and Alzheimer’s and Parkinson’s diseases as well asdiseases associated with polyglutamate repeats demonstrate intracellular proteinaggregates that, most likely, trigger apoptosis through the mitochondrial pathway[5] As will be discussed below, abnormal cell death is intimately involved in thepathogenesis of many autoimmune disorders, in part because apoptosis is a keyfacet of immunologic homeostasis and immune regulation (reviewed in [6])

Tradition-Descriptions of the biochemistry of apoptosis are detailed elsewhere in thisbook Fig 1.1 shows a simple overview of the process There are four key steps inthe death program: (1) initiation of death either through a professional death re-ceptor or through the mitochondria, (2) activation of effector caspases, (3) execu-tion of death including activation of nucleases and cell membrane changes, and(4) phagocytosis and removal of the corpse Each of these steps is highly regu-lated, and, in most cases, both activators and inhibitors have been identified

The causes and the pathogenesis of autoimmune diseases are complex Insome diseases such as systemic lupus erythematosus (SLE) and insulin-dependentdiabetes mellitus (IDDM), a fairly strong genetic component is modulated by envi-ronmental factors [7, 8] In fact, the increasing prevalence of IDDM in Westerncountries may well be associated with increased hygiene and reduced exposure to

1

Apoptosis and Autoimmunity

Keith Elkon

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1 Apoptosis and Autoimmunity

4

Fig 1.1 Schematic of the major steps in

apoptosis Death is induced through

profes-sional death receptors (e.g Fas/APO-1/CD95)

on the cell surface, or by Bak- or

Bax-mediated damage to the mitochondria (e.g.

by drugs, ultraviolet radiation, genotoxic

in-jury) Caspases are activated and cleave more

than 100 substrates in the cell These

proteo-lytic changes lead to a variety of morphologic

and functional alterations within the cell.

Amongst the most important are cell surface membrane exposure of ligands for phagocytosis, collapse of the nuclear membrane, activation of nucleases, and inactivation transcrip- tional and protein synthesis machinery Final-

ly, the dying cell is recognized by specific ceptors on phagocytes and is degraded Further details are provided in subsequent chapters in this volume.

re-Tab 1.1 Autoimmune diseases associated with targeted cell destruction

? ulcerative gastrointestinal diseases intestinal cells

? primary biliary cirrhosis bile duct cells

Tab 1.2 Autoimmune diseases associated with enhanced cell survival/proliferation

cells of the immune system

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common microorganisms Despite the varied pathogenesis, most of the mune diseases can be compartmentalized into diseases associated with targetedcell death (Tab 1.1), enhanced cell growth (Tab 1.2) or abnormal processing(Tab 1.3).

autoim-1.2

Autoimmune Diseases Associated with Targeted Cell Destruction

The clinical manifestations of the diseases listed in Tab 1.1 are caused by thedeath and subsequent loss of function of specific cells within a tissue or organ.The key questions relevant to this text are as follows

1.2.1

What is the Mode of Cell Death?

In their pure forms, death by apoptosis occurs through defined biochemical ways (programmed cell death), whereas death by necrosis is not programmed.This implies that, through intentional manipulation of the pathways, apoptoticdeath can either be induced or arrested, with obvious therapeutic implications It

path-is therefore vital to define the mode of cell death in organ specific autoimmunediseases

The criteria for distinguishing between apoptosis and necrosis are now well scribed [9] and it would seem relatively straightforward to determine which pro-

de-cess occurs in organ-specific autoimmune diseases In situ staining for DNA

strand breaks (TUNEL) is almost invariably positive in the effected organ in cific autoimmune diseases, but this test cannot be considered specific for apopto-sis A key distinction between apoptosis and necrosis is the lack of inflammationfollowing apoptotic cell death By this criterion alone, most autoimmune diseaseswould be associated with necrosis

spe-Another confounding factor relating to establishing the mode of cell death isthat apoptosis/necrosis cannot simply be inferred by the identification of the celltype within the lesion For example, the cell types most closely associated with adeath effector function, CD8 T cells and natural killer (NK), could cause cell death

by the perforin/granzyme pathway (apoptosis and necrosis), Fas ligand (FasL;apoptosis) or by the release of tumor necrosis factor (TNF)-a (apoptosis or necro-sis) CD4 T cells can kill by FasL, yet expression of FasL may serve as a chemotac-tic signal for neutrophils [10] which, once activated, would almost certainly cause

Tab 1.3 Autoimmune diseases possibly associated with abnormal processing of dying cells

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necrotic cell death Even when a disease is clearly caused by defective apoptosis

(the lpr mouse or humans with ALPS/CSS), is the massive infiltration of

lympho-cytes in multiple organs a failure of normal homeostasis (‘lymphoaccumulation’),

a manifestation of inflammation or both?

While necrotic cell death does occur in established disease, very little tion is available on the mode of cell death in the preclinical stage of human dis-eases It is the earliest changes that are likely to be most informative with regard

informa-to disease pathogenesis Models of the human disease, IDDM in rodents, havebeen particularly informative in this regard At around 2 weeks of age, there is a

this seems to occur at the same rate in non-obese diabetic (NOD) and controlmice, it has been reported that the handling of the apoptotic cells may be differ-ent in the pre-diabetic mice [11] Could this abnormality set in motion the unre-lenting attack of the immune system involving both apoptosis and necrosis? Inanother disease, congenital heart block in the infants of mothers with SLE, couldthe exposure of Ro or La (SSA/SSB) on apoptotic cardiomyocytes in the develop-ing heart allow autoantibodies to initiate disease [12]?

1.2.2

What Cells and What Effector Pathways are Responsible for Cell Death?

It is likely that the ultimate destruction of cells shown in Tab 1.1 and, in somecases, the whole organ results from a concerted attack by all of the components ofthe immune system – macrophages, lymphocytes, neutrophils, dendritic cells, NKcells Efforts to implicate a single cell type have generally been unsuccessful,although immunohistologic studies in humans and adoptive transfer studies inmice indicate that CD8 T cells are important effectors As in infectious diseases, it

is likely that components of the innate immune system (macrophages, dendriticcells, NK cells and, possibly, neutrophils) initiate the recruitment of lymphocytesand that CD4 T cells function in antigen recognition and/or cytokine priming ofCD8 T cells

As discussed above, each cell type can kill by multiple effector pathways andthis topic has been reviewed in the context of organ specific autoimmune diseasespreviously [13] Unfortunately, no clear consensus is available in individual dis-eases In mouse models of IDDM, evidence supporting the involvement of perfor-in/granzyme, FasL, TNF-a and lymphotoxin have all been reported More detaileddiscussion of these effector pathways are discussed elsewhere in this volume Thekey question, at present, is whether attenuation of any one of these pathways will

be sufficient to treat disease in the dramatic way in which TNF-a blockade hasbeen of therapeutic value in RA and Crohn’s disease

6

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Autoimmune Diseases Associated with Enhanced Cell Growth and Survival

Understanding the basic mechanisms responsible for prevention of cell death is

just as important as understanding how cells die in different autoimmune ders Some pertinent examples will be discussed

disor-Graves’ disease is a form of thyrotoxicosis associated with an autoantibody(LATS) that acts as an agonist for the thyroid stimulating hormone (TSH) recep-tor on thyrocytes Reports that TSH exerts an anti-apoptotic effect by downmodu-lating Fas expression and that this effect is mimicked by IgG from patients withGraves’ disease [14, 15] is appealing However, other investigators have failed to

confirm changes in Fas expression following exposure of thyrocytes to TSH in tro More recently, Stassi et al [16] reported that T cell in Graves’ disease express

vi-Th2 cytokines (IL-4 and IL-10) in contrast to T cells in Hashimoto’s disease that press Th1 cytokines [interferon (IFN)-c] These authors propose that the two dia-metrically opposed diseases are a consequence of the effects of the cytokines –IFN-c causes enhanced susceptibility to Fas-mediated apoptosis (Hashimoto’s thyr-oiditis), whereas Th2 cytokines promote Fas resistance through expression of c-

Although the initiating events are poorly understood, two rheumatic diseases sociated with activation and growth of fibroblasts are scleroderma and RA Trans-forming growth factor (TGF)-b is the cytokine most closely associated with theskin thickening and fibrosis observed in scleroderma (reviewed in [17]) It is ofconsiderable importance to determine how a cytokine such as TGF-b can be asso-ciated with growth of cells such as fibroblasts, but with cell cycle arrest and/orapoptosis of lymphocytes [18] RA is characterized by growth of tissue called thepannus that invades cartilage and bone Growth of the pannus is driven by anumber of cytokines and growth factors, but the striking therapeutic effect ofTNF-a blockade, suggests that TNF-a is a necessary component of this pathologi-cal process In most cells, TNF-a activates NF-jB, which in turn activates a cas-cade of inflammatory mediators as well as anti-apoptotic pathways Recent resultssuggest that NF-jB attenuates apoptosis through blockade of the JNK pathwayand may interfere with GADD45 [19, 20] Since TNF-a can also induce cell death(hence its name), elucidating how the signal transduction pathways diverge willhave major implications for therapies of inflammatory disorders

as-1.4

Autoimmune Diseases Associated with Abnormal Processing of Dying Cells

As shown in Fig 1.1, the fourth and final component of the apoptotic pathway isthe ingestion and ‘safe’ disposal of the dying cell The basic mechanisms involved

in the clearance of apoptotic cells and the possible links to autoimmunity are cussed elsewhere in this book The evidence linking SLE to defective clearance ofapoptotic cells (Tab 1.3) is indirect, and derives mostly from knowledge of autoan-

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dis-tibody specificities, the mechanisms responsible for the clearance of dying cellsand information regarding macrophage signal transduction and cytokine release.Autoantibodies may have greater specificity for cellular components that areeither chemically modified or become more accessible to the immune system fol-lowing death of the cell The evidence to support this idea include: (1) phosphati-dylserine, the negatively charged phospholipid that translocates to the outside ofthe cell is antigenic for some anticardiolipin autoantibodies – possibly followingoxidation [21], (2) nucleosomes (a product of activation of DNases during theapoptotic process) are antigenic for B and T cells in SLE [22, 23], nucleosomes aredeposited in the glomeruli (see Chapter 18), (3) some autoantigens are translo-cated to apoptotic blebs, some autoantigens are cleaved by caspases and/or gran-zyme B (see Chapter 15), and (4) hyperimmunization of mice with apoptotic cellsresults in an increase in antiphospholipid autoantibodies [24].

In vitro and in vivo studies support the idea that defective phagocytosis of dying

cells promotes autoimmunity Recent studies have shown that early complementcomponents [25] as well as acute-phase proteins such as CRP [26] bind to, andpromote the phagocytosis of [26], apoptotic cells Mice that have disruption ofgenes encoding C1q [27] or the acute-phase protein serum amyloid P [28] develop

a lupus-like disease Of considerable interest, C1q-deficient mice demonstrate creased numbers of apoptotic cells in the glomeruli [27] Together, these observa-tions make a compelling case that the increased susceptibility of patients withearly complement component deficiencies for the development of lupus is asso-ciated with delayed clearance of dying cells It remains to be determined why thekidney is an important target organ and whether similar defects account for lupusthat is not associated with inherited complement deficiencies

in-Observations relating the quality of macrophage cytokine response to the nature

of the dying cell [29–31] provide the critical link between cell death and the

poten-tial for autoimmunity Specifically, in vitro studies revealed that the uptake of

apoptotic cells induced the expression of immunosuppressive cytokines such as

These cytokines are known to dampen the immune response to self or foreignantigens Additional evidence supporting an anti-inflammatory role of apoptoticcells is that the administration of apoptotic cells promotes the resolution of in-

flammation in vivo [32] In contrast, necrotic cells provoke a pro-inflammatory

re-sponse associated with the release of TNF-a TNF-a not only provokes an immuneresponse to the antigens phagocytosed (self-antigens in this case), but also pro-motes the maturation of macrophages to dendritic cells, the cell type most effi-cient in antigen presentation [33]

The receptors and ligands involved in the recognition of apoptotic cells by gocytes are discussed elsewhere in this book Whereas blockade of many of these

pha-receptor/ligand systems reduce phagocytosis in vitro, deletion of the genes

encod-ing these proteins rarely cause an obvious increase in apoptotic cells or mune diseases in mice This suggests a redundancy in function of many of thereceptor/ligands identified to date In contrast, knockout of the gene encoding theMER kinase, a member of the Tyro 3 receptor tyrosine kinase family, caused both

autoim-1 Apoptosis and Autoimmunity

8

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an increase in the numbers of apoptotic cells in the thymus and spleen as well as

a lupus-like autoimmune disease [34] This mouse model therefore provides haps the most persuasive evidence linking defective clearance of apoptotic cells tolupus-like disease It will be important to determine how MER is linked to apopto-tic cell recognition and to identify its downstream signal transduction pathways

per-As discussed above, macrophages provide signals that dictate the response oflymphocytes Regardless of how the immune response to self-antigens is initiated,the receptors that become engaged on macrophages continue to orchestrate the

immune response For example, Manfredi et al [35] have shown that when

anti-cardiolipin antibodies engage Fcc receptors, they promote a TNF-a dominated inflammatory response Thus, once initiated the antigen antibody complexes mostlikely fuel the inflammatory process and continue to promote immune responses

be translated into therapeutic action The literature contains many examples ofsuccessful blockade of death effectors or receptors with antibodies or soluble re-ceptors as well as attenuation of biochemical pathways of apoptosis (e.g with cell-permeable tetrapeptide inhibitors of caspases [36]) in animal models As exciting

as these observations are in experimental animals, the fact that either ‘too little ortoo much apoptosis’ is associated with diseases, indicates that therapy must either

be short lived or cell specific when considering application to spontaneous immune diseases in humans Furthermore, as emphasized above, it is likely thatmultiple cells and death pathways are operational in established autoimmune dis-eases suggesting that therapy administered early is much more likely to effective.TNF-a inhibitors provide the most striking example of single molecule adminis-tration with therapeutic efficacy in patients with rheumatoid arthritis and Crohn’s

induc-ing apoptosis of monocytes [37, 38]? This very important question should be

re-solved prior to the administration of small molecule inhibitors of TNF in vivo.

Can the TNF success story be replicated in other diseases?

‘Apoptosis and autoimmunity’ has been a bidirectional learning process – each

has informed the other For example, the discovery of Fas mutations in lpr mice

led to the elucidation of the concept of ‘activation-induced cell death’ and the derstanding that activated cells were eliminated at sites of inflammation by sui-cide and fratricide [39] This process is vital to the maintenance of peripheral tol-erance [40] Similarly, the almost invariable association between C1q deficiencyand lupus coupled with elucidation of the responses of phagocytes to their mealsdescribed above have provided fundamental new insight into problem of ‘self/

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un-non-self’ discrimination At this pace, it is likely that additional basic discoveries

in apoptosis will be uncovered, leading to further insight and improved therapy ofmany human autoimmune disorders

5 Guegan C, Vila M, Rosoklija G, Hays

AP, Przedborski S.J Neurosci 2001, 21,

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Ta-hara K, Ishikawa N, Ito K, Nagataki S.

Clin Exp Immunol 1997, 110, 434–9.

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Zeuner A, Ricci-Vitiani L,

Stoppac-ciaro A, Ruco L, Farina F, Zummo G,

De Maria R.Nat Immunol 2000, 1, 483–

Auffre-19 Tang G, Minemoto Y, Dibling B, cell NH, Li Z, Karin M, Lin A.Nature

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Death, Development and Immune Function

Apoptosis has been defined as programmed cell death, a term which, in contrast

to necrosis, implies the active participation of a dying cell in its own death Thisprocess can be initiated in response to a range of intrinsic and extrinsic signals

In developing vertebrates and invertebrates, apoptosis serves to carefully regulatecell number and tissue formation This physiologic program continues in matureorganisms, working to maintain a constant cellular homeostasis There is growingevidence, however, that cell death by apoptosis also plays a role in a variety of dis-ease states Tissue and organ injury in response to environmental stressors, such

as hypoxia, trauma and infection, are thought to be mediated in part throughapoptotic cell death Insufficient apoptosis has been linked to conditions such ascancer, while excess apoptosis is felt to contribute to the pathogenesis of somechronic degenerative diseases

In the immune system, the importance of apoptosis is borne out by tions that dysregulated apoptosis can lead to a variety of disorders including auto-immune disease, lymphoid tumors or immune suppression from excessive lym-phocyte death Experimental evidence has revealed that programmed cell deathplays an important role in several steps throughout both the maturation and sub-sequent functional life of T and B cells During early development, apoptosis con-tributes to the generation of a functional repertoire of mature cells by eliminatinglymphocytes that fail to express an antigen receptor (death by neglect) Expression

observa-of both cytokine receptors and the pre-T cell receptor (TCR) is required for sion and differentiation, and in the absence of survival signals delivered via these

also the method of elimination of T cell during the next phase of development,positive and negative selection During this stage, CD4/CD8 double-positive thy-mocytes that express TCR of intermediate affinity for peptide ligands in the con-text of MHC molecules differentiate into either CD4 or CD8 single-positive cells

T cells that express TCR with low affinity for peptide, however, fail to receive cient survival stimuli and die by neglect On the other end of the spectrum frompositive selection, an overly high-affinity TCR interaction with self-MHC causes

suffi-13

2

Caspase Knockouts: Matters of Life and Death

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negative selection, the clonal deletion of these potentially autoreactive cells viewed in [2]) In a similar fashion, B cells that recognize self-antigens are elimi-nated in the bone marrow by apoptosis Apoptosis, therefore, serves as an impor-tant tool in the development of lymphocytes, ensuring the central elimination ofboth non-functional and potentially autoreactive cells.

(re-Programmed cell death is also a critical component of the mature immune tem The clonal expansion of antigen-specific lymphocytes is a central feature ofthe adaptive immune response However, once an infection has been successfullycleared, it is important to efficiently remove these activated, proliferating lympho-cytes to avoid risking adverse effects such as an autoimmune response or poten-tial malignancy In this situation, apoptosis functions to maintain homeostasis by

activation-induced cell death (AICD), a process of inducing apoptosis through petitive TCR stimulation, or through death by cytokine withdrawal, in which lym-phocytes die from a lack of trophic factors [4] Likewise, activated B cells can beinduced to undergo apoptosis through B cell receptor (BCR) stimulation [5].Our understanding of the mechanisms of apoptosis in these and other para-digms has rapidly advanced over the last few years (Fig 2.1), and although wehave developed a more complete understanding of its molecular events, numer-ous aspects remain to be elucidated Much of our appreciation of the intricacies of

re-apoptosis has evolved from the study of animals lacking specific caspases (cystinyl aspartate proteases) and other related proteins.

Fig 2.1 Apoptotic pathways involving caspases Established pathways appear as solid lines Dashed lines indicate possible pathways.

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Apoptotic Pathways: from Nematode to Mammals

The crucial role of caspases in cell death was first recognized through studies of

their homologues in Caenorhabditis elegans During development, 131 of this atode’s 1090 somatic cells die by apoptosis Screens of C elegans with defective

nem-apoptosis initially revealed loss-of-function mutations of the proteins CED-3 (for

cell death abnormal) and CED-4, demonstrating their necessity for apoptosis [6].

Subsequently, the protein CED-9 has been identified as an inhibitor of apoptosisfunctioning upstream of CED-3 and CED-4 Loss-of-function CED-9 mutationscause embryonic lethality due to excess apoptosis, a phenotype that is reversible

by loss-of-function CED-3 or CED-4 mutations [7] Finally, loss-of-function tions of the protein EGL-1, which binds to CED-9, also suppress apoptosis [8].The scheme that has emerged (Fig 2.2) places CED-9 as a negative regulator ofapoptosis that works by binding to and suppressing CED-4 [9] When EGL-1 is in-duced, it displaces this complex, allowing CED-4 to bind to CED-3 and resulting

muta-in apoptosis

Analysis of these proteins identified CED-3 as a caspase, with homology to themammalian interleukin (IL)-1b-converting enzyme (ICE, also known as caspase-1)[10] The recognition that CED-3 is crucial for apoptosis prompted the search forother mammalian caspases that could play a similar role While the general

scheme for apoptosis that has emerged for higher organisms is conserved from C elegans, there have been 14 mammalian caspases identified to date and the result-

ing pathways are accordingly more complex In addition to the caspases,

mamma-lian counterparts have also been found for the other members of the C elegans

apoptotic pathway (Fig 2.2) [8, 11, 12]

2.2 Apoptotic Pathways: from Nematode to Mammals 15 Fig 2.2 Apoptosis-related molecules inC elegans and

mammals.

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Triggering a Killer: General Aspects of Caspase Activation

Although there are no fixed criteria that define apoptosis, a number of changes incellular morphology can help distinguish it from necrotic cell death In the apop-totic cell, chromosomes condense, the nucleus fragments, cytoplasmic volume de-creases, organelles compact, the cell membrane fuses with the endoplasmic reticu-lum and the cell finally fragments into numerous ‘apoptotic bodies’, which are en-gulfed by surrounding cells [13] These morphologic changes are accompanied byother subcellular indicators of apoptosis, including the exposure of phosphatidyl-serine on the external surface of the cell membrane and a decrease in mitochon-drial transmembrane potential [14] An important characteristic that results fromthis distinctive cellular packaging is that apoptosis lacks the inflammation and po-tential for injury to surrounding cells that is seen with necrosis The subcellularchanges that transpire in apoptosis result, at least in part, from the cleavage ofspecific subcellular proteins by caspases Caspases have among the most stringentsubstrate specificities of all proteases, always cleaving on the carboxyl side of as-partate residues [15] Their name also hints to the presence of a conserved cyste-ine residue found in the peptide motif QACXG, which, along with a conservedglycine and histidine, contribute to the catalytic site [16] Caspases exist in cells asinactive monomeric zymogens (Fig 2.3), consisting of an N-terminal prodomain,

a large subunit and a small subunit Processing occurs at sites between the mains that, consistent with the ability of pro-caspases to autoactivate or to be acti-vated by other caspases, contain aspartate residues Cleavage of two zymogen pre-cursors at these sites releases the subunits, which then form the functional het-erotetrameric enzyme, a complex of two large and two small subunits with twoseparate active sites [17, 18]

do-The nature of the prodomain (Tab 2.1), classified as either short (caspase-3, -6,-7 and -14) or long (the remainder) is thought to influence the route of caspase ac-

Fig 2.3 Caspase zymogen cleavage and assembly of active heterotetramer Asp-X indicates aspartate residues at processing sites; QACXG indicates catalytic cysteine residue; stars indicate lo- cation of enzyme active site.

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tivation The long prodomains have been found to contain either death effectordomains (DED) or caspase recruitment domains (CARD) that are capable of inter-acting with adapter molecules and result in clustering of pro-caspases This closeproximity can enhance a low, intrinsic autocatalytic activity of the zymogen, allow-ing it to cleave and activate itself For example, oligomerization of the Fas receptor(Apo1, CD95) recruits the adapter molecule FADD (Fas-associated death domainprotein, also called Mort-1) through the interaction of their death domains (DD)[19, 20] The other end of FADD contains a DED, which can then interact with aDED on pro-caspase-8, recruiting it to the death-inducing signaling complex(DISC) [21, 22] Once recruited, pro-caspase-8 can activate itself through this ‘in-duced proximity’ [23] Caspase-10, which also has a DED, is similarly thought tointeract with FADD [24] Known interactions of CARD include those mediatingthe association of pro-caspase-9 with Apaf-1 [25] and pro-caspase-2 with RAIDD/CRADD [26, 27] Caspases with long prodomains have frequently been termed

‘initiators’ and, once activated, are thought to cleave caspases with short domains,termed ‘effectors’, which then carry out the apoptotic program It has becomeclear, however, that such a distinction is an oversimplification For example, there

is evidence that caspase-3 can cleave upstream caspase-8 and -9 [28], and also that

it may be capable of autoactivation [29]

2.4

Caspase-1 and -11: More than Mediators of Inflammatory Cytokines?

Caspase-1, or ICE, was the first member of this family to be cloned [30] It is a toplasmic protease that is capable of converting the 34-kDa inactive precursor ofIL-1 to its mature 17-kDa form, and can also process the cytokine precursor of IL-

cy-18 [interferon (IFN)-inducing factor] [31] The discovery of the serine/threoninekinase RIP2/CARDIAK/RICK and demonstration that it can bind and activate cas-pase-1 processing [32] provide a possible mechanism for caspase-1 regulation

2.4 Caspase-1 and -11: More than Mediators of Inflammatory Cytokines? 17 Tab 2.1 Characterization of murine caspases with long prodomains

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Upon receipt of a pro-inflammatory stimulus, RIP2 engages caspase-1 through teraction of their respective CARD, causing oligomerization of pro-caspase-1.These zymogens then undergo autoprocessing to generate large (p20) and small(p10) subunits which combine to form the active protease In addition to its abil-

caspases, is able to induce apoptosis when overexpressed in cultured cells [33].Unlike mice with targeted deletions in some of the other caspase genes, however,

deficient in IL-1b and IL-18 production

Mouse caspase-11 is most homologous to human caspase-4 [36] Overexpression

of caspase-11 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited

by CrmA and Bcl-2 [36] The expression of caspase-11 is highly inducible by LPS,suggesting that it may have a regulatory role in both apoptosis and inflammatoryresponses [36] Caspase-11 does not process pro-IL-1 directly, but overexpression ofcaspase-11 stimulates processing of pro-IL-1 by caspase-1 [36]

Analysis of knockout mice established the critical role of caspase-1 in acute

resistant to the effects of lipopolysaccharide (LPS)-induced shock, showing proved survival and, in addition to the absence of mature IL-1b, decreased produc-tion of IL-1a, IL-6 and tumor necrosis factor (TNF)-a [34, 35] Interestingly, cas-

observations that caspase-11 is absolutely required for caspase-1 activity and thatthey exist in the same protein complex inside cells suggest a mechanism wherebyinteraction of the two molecules is required for activity and cytokine processing

Fas-induced apoptosis [35, 37] This raises the issue of their involvement in tive selection, possibly via CD30, a TNF family member suspected to play a role

nega-in negative selection [38, 39] It has been shown usnega-ing H-Y transgenic mice, nega-inwhich the TCR recognizes a Y chromosome antigen resulting in clonal deletion

by negative selection in the male thymus, that CD30 knockout causes a partial

Ad-ditionally, overexpressing CD30 in the mouse thymus results in enhance programcell death by TCR and CD30 crosslinking reagents In this situation, caspase-1 isactivated by CD30 and may thereby mediate negative selection A critical role ofCD30 in negative selection is arguable, however, due to recent studies showingthat CD30-deficient mice are able to carry out negative selection of both anti-self

back-grounds to generate TCR transgenic mice that recognize endogenous antigen,either a peptide presented by MHC or superantigen, and monitor the deletion of

T cells in such mice Likewise, future study should also address the role of pase-1 and -11 in peripheral deletion of activated cells, although there is no report

cas-of systemic autoimmune conditions in these mice, making any role they mayhave in peripheral tolerance likely to be less significant

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In addition to its involvement in inflammation, caspase-11 has been shown to play

a role in pathological death in neurons In a model of brain injury induced by middlecerebral artery occlusion, caspase-11 knockout mice were found to have a reduction

in caspase-3 activation and brain apoptosis [41] Cultured oligodendrocytes from pase-11 knockout mice also showed reduced caspase-3 activation and cell death inresponse to hypoxia, IFN-c and anti-Fas stimulation [42, 43] Furthermore, cas-pase-11 knockout mice were found to have significant resistance to development

cas-of MOG peptide-induced experimental autoimmune encephalitis [42] In addition,

caspase-11 is able to activate caspase-1 and -3 by direct cleavage in vitro [41].

Although these data may indicate that caspase-11 is important in apoptosis via ulation of caspase-3 activation, it has remained difficult to definitively separate thiseffect from alterations in cytokine levels seen in caspase-11 knockouts The possibil-ity that caspase-11 deficiency protects against apoptosis due to altered cytokine reg-ulation will need to be more conclusively addressed in the future

mod-2.5

Caspase-8 and the FAS Signaling Pathway

A subset of TNF receptor (TNF-R) family members that includes Fas, TNF-RI,DR3 (TRAMP, wsl-1, APO-3, LARD), DR4 (TRAIL-R1, APO-2), DR5 (TRAIL-R2,TRICK2, KILLER) and DR6 (reviewed in [44]) is involved in transducing signalsthat result in cell death and are therefore referred to as ‘death receptors’ Fas-in-duced apoptosis is triggered by binding of its natural ligand, Fas ligand (FasL), re-sulting in the rapid recruitment of FADD to the cytoplasmic membrane FADDthen brings in pro-caspase-8, also called FLICE (FADD-like IL-1-converting en-zyme) or MACH, via their homologous DED, forming the DISC (reviewed in [5]).Next, proteolytic cleavage of pro-caspase-8, presumably an autocatalyzed reactionpromoted by the close proximity of multiple caspase-8 zymogens recruited to theDISC, results in its activation and subsequent release from the DISC into the cy-toplasm [23] Depending on the cell context, the downstream signal of caspase-8

is propagated in one of two ways (Fig 2.1) In so-called type I cells [45], induction

of apoptosis is accompanied by activation of large amounts of caspase-8 by theDISC Caspase-8 then rapidly cleaves and activates caspase-3, leading to the effec-tor stage of apoptosis In type II cells, on the other hand, DISC formation isstrongly reduced and activation of caspase-8 and -3 occurs following the loss ofmitochondrial transmembrane potential (see Chapter 1) In this variant of the cas-pase cascade, caspase-8 cuts and activates the pro-apoptotic Bcl-2 family memberBid [46] Truncated Bid induces mitochondria pore formation via Bak or Bax [47],

resulting in the unleashing of pro-apoptotic molecules such as cytochrome c [48] and Smac/DIABLO [49, 50] Cytochrome c can subsequently form a complex with

Apaf-1 and pro-caspase-9 in the cytoplasm, to form the apoptosome (reviewed in[5]), which can then activate effector caspases such as caspase-3 and -7 Of note,although mitochondrial activation can occur in both type I and II cells, it is notnecessary for apoptosis in type I cells

2.5 Caspase-8 and the FAS Signaling Pathway 19

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Despite diverging at a later stage, both of these mechanisms involve caspase-8

as an initiator caspase in death receptor signaling This linear model has been

TNF-aRII-deficient mice, which all develop normally to adulthood, caspase-8 deficiencycauses prenatal lethality [51] with two particularly striking features: impaired heartmuscle development and congested accumulation of erythrocytes (hyperemia).This indicates that caspase-8 mediates key developmental steps, either throughdeath receptors other than Fas and TNF-a, or through some combination of thesereceptors Wild-type and caspase-8 embryonic fibroblasts were used to show thatthe activation of JNK and NF-jB by death receptors such as Fas and TNF-a recep-tors is not affected by caspase-8 deficiency However, the apoptosis induced bythese receptors is totally blocked in caspase-8 knockout fibroblasts, showing thatcaspase-8 is a critical initiator caspase for transducing apoptosis signals fromthese death receptors Consistent with the model that FADD is a key adaptor mol-

mice develop normally but are resistant to Fas-mediated hepatocyte apoptosis, dicating that the developmental effects of caspase-8 deficiency likely occur in atype I fashion through direct activation of caspase-3 by caspase-8, without involve-ment of Bid [40]

in-Members of death receptors have been implicated in negative selection in thethymus [52] However, deletion of autoreactive thymocytes occurs normally in Lpr

cannot be attributed to ligation of any one of these receptors alone Therefore,conditional deletion of caspase-8 or its partner FADD, a strategy that presumablyblocks the signaling of many death receptors, may be informative in addressingthe role of death receptors in negative selection The role of caspase-8 in lym-phoid development has also not been well established using the knockout modeldue to the prenatal lethality of these mice Interestingly, however, T cell-specificFADD-deficient mice show a proliferative defect of thymocytes between the

ex-pressing a dominant-negative mutant FADD in the thymus provided the ing result of enhanced thymocyte negative selection [58] The effect of the cas-pase-8/FADD module on the homeostasis of peripheral lymphocytes appears to be

surpris-a bsurpris-alsurpris-ance of hypo-responsiveness surpris-and defective surpris-apoptosis FADD deficiency csurpris-ausesdecreased activation in peripheral T cells which is associated with a defective co-stimulatory response [58, 59] On the other hand, it is also observed that activated

with-drawal-mediated death Since activated T cells have prolonged survival [60], theymay potentially accumulate in aging animals, consistent with the autoimmunesyndromes seen in TNF-RI/Fas double knockouts [61] and, in a strain-dependentfashion, Fas and FasL mutants [62] It is likely, therefore, that signaling pathwaysoperating through FADD do not lead exclusively to apoptosis, but under certain

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circumstances can promote cell survival and proliferation Whether such vival pathways would involve caspase-8 is unclear A selective knockout strategythat induces deletion of caspase-8 within cells undergoing negative selection, forexample, would help clarify this issue.

pro-sur-2.6

Caspase-3: The Chief Executioner?

Caspase-3 has assumed a position as a central executioner caspase in mammals,though not as indispensable as its counterpart CED-3 which is absolutely required

for programmed cell death in C elegans This assumed role of caspase-3 is

sup-ported by the convergence of both death-receptor and mitochondrial-mediateddeath pathways at caspase-3 activation as well as by the wide range of potential

peri-natal mortality as a result of defects in brain development that correlate with a crease in levels of apoptosis [63, 64]

de-In contrast to the essential role caspase-3 plays in neuronal development, it

display normal T and B cell development [63, 64] Apoptosis of thymocytes duced by anti-CD3 crosslinking or anti-Fas is unaltered in the absence of caspase-

in-3 [6in-3], suggesting that at least some aspect of negative selection is not affected bycaspase-2 An involvement of caspases in thymocyte apoptosis is supported, how-ever, by previous studies that employed fetal thymic organ culture and the phar-macological pan-caspase inhibitor zVAD-fmk In these experiments, the authorsobserved inhibition of deletion of thymocytes induced either by anti-CD3, dexa-

methasone, or antigenic peptide in vitro Moreover, caspase-3 activation was

de-tected specifically during apoptosis induced by TCR stimulation, but not duringspontaneous cell death [65, 66] It has been established recently [67] that compen-satory caspase activation is a mechanism for mammalian cells to use to induceapoptosis in the absence of a given key caspase The lack of defects in negative se-lection in caspase-3 mice can therefore be due to activation of other alternativecaspases, such as caspase-6 and -7

Both p53 and Bcl-2 have been shown to be important in mediating death by glect during T cell development in the thymus (see Section 2.7) [68, 69] However,

ne-T cell development in caspase-3-deficient mice appears normal Additionally, ies utilizing a strategy that block all caspase activities in the thymus failed to re-veal a deficiency in this type of cell death [70] These data argue that a pathwaythat is not dependent on caspases, but instead on p53 and Bcl-2 may be requiredfor death by neglect in T cell development

stud-After infection, professional antigen-presenting cells, dendritic cells in particular,present antigenic peptides of the infectious agents and drive T cells into clonal expan-sion After a clonal expansion phase and resolution of infection, the number of anti-gen-reactive lymphocytes must decline until the pool of lymphoid cells reaches abaseline level This is achieved by AICD, a balanced fine-tuning between growth/ex-

2.6 Caspase-3: The Chief Executioner? 21

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pansion and death by apoptosis which classically occurs via death receptors typified

deficiency in AICD [64] After challenge with superantigen staphylococcal

an increased viability when treated with anti-CD3 or anti-Fas antibody [64] Theseresults indicate a requirement for caspase-3 for AICD in peripheral T cells The de-ficiency of the Fas signaling pathway has been linked direct to systemic autoimmuneconditions such as lupus (reviewed in [5]) Therefore, further studies should focus onthe effect of caspase deficiency in this kind of autoimmune condition

In addition to AICD, there are many other ways that cause the death of eral T and B cells The death of peripheral T cells may be due to a high intensity

periph-of TCR signaling [72], the withdrawal periph-of growth factors such as IL-2 or IL-15 from

T cells [73, 74], or the absence of a tonic TCR signal on nạve T cells [75] Futurestudies should also address whether these types of cell death are affected by cas-pase-3 mutation

2.7

Caspase-9: Mitochondrial Activation and the Apoptosome

The caspase-9 activation pathway was discovered after the observation that the dition of ATP, or preferably dATP, to cell extracts prepared from normally grow-ing cells initiates an apoptotic program, as measured by caspase-3 activation andDNA fragmentation [48] Subsequent biochemical studies identified a protein

ad-complex consisting of Apaf-1 and cytochrome c that, upon hydrolysis of ATP or

dATP, is able to recruit and activate pro-caspase-9 (reviewed in [76]) Although pases are usually activated by cleavage, proteolytic processing of pro-caspase-9does not significantly increase its catalytic activity [77, 78] Rather, the key require-ment for caspase-9 activation is its association with its protein cofactors, Apaf-1

cas-and cytochrome c Together they form the active holoenzyme, often referred to as

the apoptosome (reviewed in [79])

The in vivo significance of the apoptosome has been demonstrated by studying

mice deficient in either caspase-9 or Apaf-1 [80–82] Both of these knockoutscaused a perinatal lethality starting at post-conception day 16.5 Brain malforma-

sug-gesting that these cells were not proliferating tumors These phenotypes are

cen-tral role of the apoptosome in programmed cell death is the cytochrome c

knock-out [83] These animals die prenatally at embryonic day 8.5, presumably due tothe defect in aerobic metabolism, but analysis of knockout embryo-derived celllines reveal a resistance to apoptosis induced by ultraviolet (UV) irradiation, se-

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rum withdrawal and staurosporine Interestingly, however, cytochrome c–/– cellsappear more sensitive to TNF-a-induced death.

In contrast to the dramatic effect of caspase-9 and Apaf-1 deficiency on nal development, T cell development appears to be normal in these mice How-

sus-ceptible to Fas-mediated apoptosis, suggesting that Fas-mediated cell death in mocytes is independent of the Apaf1/caspase-9 pathway The lack of gross devel-opmental defects in T cell development also suggests that Apaf-1 and caspase-9are not involved in death by neglect in thymus

thy-Due to the timing of the lethality caused by caspase-9 and Apaf-1 mutation, it isnot possible to address the significance of this pathway in peripheral cell deathusing these models Since the lymphoid systems are largely normal, further ex-

role of this pathway in programmed cell death of the peripheral lymphoid system.This is of particular interest because recent studies have shown an involvement of

a positive feedback loop mediated by mitochondria during tumor-induced death ofactivated T cells [84, 85] These studies demonstrate that loss of mitochondrialtransmembrane potential, an event apparently independent of death receptor sig-naling, leads to a series of events including cleavage of Bid, that in turn furtherincrease mitochondrial permeability and lead to apoptosome activation Therefore,

it is important to examine specifically whether the Apaf-1/caspase-9 pathway is volved in the death of activated T cells, despite the demonstration that Fas-mediated thymocyte death is not affected by these mutations

in-In embryonic fibroblasts expressing c-Myc and Ras, Apaf-1 and caspase-9 tions can also block p53-induced cell death, a process which is felt to play a role

muta-in the negative selection of developmuta-ing thymocytes [86], placmuta-ing Apaf-1 and pase-9 downstream in a p53-induced apoptosis pathway [87] Therefore, p53 maymediate negative selection through activation of Apaf1 and caspase-9 Interest-ingly, p53 may also mediate death by neglect at the pre-TCR stage because its de-

pro-apopto-tic Bcl-2 family members results in thymus hyperplasia and thymoma [91–93].The molecular mechanisms underlying apoptosis in the immune system havealso been examined among the Bcl-2 family, whose members modulate the mito-chondrial-directed activation of Apaf1 and caspase-9 Deficiency of the anti-apopto-tic Bcl-2 protein results in early postnatal lethality, but initially normal lymphocytedevelopment [94] At variable times after birth, however, these mice undergo ful-minant lymphocyte apoptosis with involution of lymphoid organs, consistent withsusceptibility to uncontrolled apoptosis in the face of activation In contrast, Bcl-x-

Bcl-xL, are embryonic lethal with severe excessive neuronal apoptosis and reduced

2.7 Caspase-9: Mitochondrial Activation and the Apoptosome 23

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