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APOPTOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS
SYSTEMIC LUPUS ERYTHEMATOSUS
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APOPTOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS Clinical Implications Felipe Andrade, MD, Livia Casciola-Rosen, PhD, and Antony Rosen, MBChB, BSc (Hons)
Systemic lupus erythematosus (SLE) is a heterogeneous and complex group of disorders of unclear cause. The complexity arises from three major sources (genes, environment, and time) and makes determining disease pathogenesis difficult, particularly in the outbred human population. The genetic heterogeneity may affect immune response, target tissue, and disease expression in the patient p~pulation.~ Numerous different environmental agents have been proposed to interact with the host during the initiation and propagation of SLE.'*, 32 There is considerable kinetic complexity in individual patients and in the population (with great variability in the intervals between exposure to initiating stimuli and onset of the disease process and in recurrence). This article provides an apoptosis-centric hypothesis for the initiation and propagation of SLE, which is able to explain much of this complexity. Although there are several aspects of the hypothesis that have not yet been proven, These studies were supported by N M Grants AR44684 (LC-R) and DE12354 (AR), the SLE Foundation, the Donald B. and Dorothy L. Stabler Foundation, and the Schauman Lupus Research Fund. Antony Rosen is a Pew Scholar in the Biomedical Sciences and is supported by a Burroughs Wellcome Fund Translational Research award. Felipe Andrade is supported by a Fullbright/ Consejo Nacional de Ciencia y Tecnologia, Mexico scholarship.
From the Departments of Dermatology and Cell Biology and Anatomy (LC-R); and Division of Rheumatology (AR, FA), Johns Hopkins University School of Medicine, Baltimore, Maryland
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the questions raised can be addressed both in vitro and in vivo. The answers provided are likely to furnish important insights into the normal immune consequences of different forms of apoptosis in tissues and about whether defects in the signaling, execution, and clearance phases of the apoptotic process are susceptibility factors for the development of systemic autoimmune disease. This novel view of SLE focuses therapeutic thinking on a process of potentially central relevance in the pathogenesis of SLE. AUTOANTIBODIES: PROBES OF THE PERTURBED STATE
There is a growing consensus that the highly specific humoral immune response to autoantigens in systemic autoimmune diseases is T-cell-dependent and that flares of disease result when this primed immune system is rechallenged with self-antigen.'" 22, 5o Although the mechanisms responsible for initiation and propagation of the immune response to specific autoantigens remain ~nclear,~, 45 autoantigens are unified by being selected for an immune response. The targeted molecules must have satisfied the stringent criteria for initiation of a primary immune response at the onset of disease (i.e., presentation of suprathreshold concentrations of nontolerized structure in a proimmune context).14 Because tolerance is only induced to dominant determinants in autoantigens (which are generated and presented at suprathreshold concentrations during natural processing of whole-protein antigens), a potential exists for T-cell-autoreactivity directed against cryptic determinants (which are not generated at all or are generated at subthreshold 58 The hypothesis that autoimmunity arises when usually cryptic determinants become visible to the immune system has received increased attention in the past several years. Indeed, several experimental systems have now clearly demonstrated circumstances that allow previously cryptic determinants to be presented.% These include novel autoantigen cleavage9,39 and altered autoantigen processing induced by high-affinity ligand binding (e.g., to an antibody or receptor molecule).54,60, 65 The authors proposed that autoantigens in systemic autoimmune diseases are unified by the fact that they satisfy the stringent criteria for initiation of a primary immune response.14,l5 The corollary of this proposal is that if the perturbed state can be recreated in vitro, these common features of autoantigens (i.e., similar alterations of concentration, distribution, and structure) can be observed. The authors and others have used high-titer autoantibodies as probes of cell biology and biochemistry of autoantigens during different clinically relevant perturbed states to search for those circumstances in which autoantigens become clustered, concentrated, and structurally m ~ d i f i e d . ' 19-~1 ~ ~ ~These . studies have highlighted apoptosis as the potential perturbed state underlying the initiation and propagation of systemic autoimmune diseases
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and focus attention on the alterations in the cell biology and biochemistry that unify autoantigens during this form of cell death. LUPUS AUTOANTIGENS UNDERGO A STRIKING REDISTRIBUTION DURING APOPTOSIS, BECOMING CLUSTERED AND CONCENTRATED IN THE SURFACE BLEBS OF APOPTOTIC CELLS
Studies to delineate the potential perturbed states that might initiate systemic autoimmune diseases have been based on a striking clinical observation in SLE, that is, that ultraviolet irradiation has a marked propensity to induce flares of both systemic and skin disease in lupus patients.35In this regard, the epidermis seems to be an important target of the immunopathologic response in lupus and constitutes an appropriate in vitro model with which to address the effects of flare-inducing stimuli (e.g., ultraviolet B)." Earlier studies demonstrated that intracellular autoantigens can normally be stained at the exterior surface of keratinocytes incubated in vitro for 20 to 24 hours after irradiation with UVB, although the mechanism of this redistribution of antigens was not dete1mined.2~.37 To determine how this phenomenon might arise, the authors studied the subcellular distribution of lupus autoantigens at increasing times after UVB irradiation in both intact and permeabilized cells.I4The authors' initial studies made several observations. First, UVBirradiated keratinocytes undergo apoptosis beginning a few hours after irradiation. Apoptotic keratinocytes manifest the classic morphologic hallmarks of this process, including prominent surface blebbing (early event) and nuclear condensation and fragmentation into apoptotic bodies (later event). In addition, lupus autoantigens, which are not restricted to any specific subcellular compartment in control cells, are strikingly redistributed in apoptotic cells such that they become clustered and concentrated within small surface blebs and apoptotic bodies (Fig. 1). Small surface blebs (which contain fragmented rough endoplasmic reticulum; see Fig. 1) are highly enriched in 52-kd Ro, ribosomal autoantigens, and those autoantigens found within the endoplasmic reticulum lumen (e.g., calreticulin). This marked enrichment of autoantigens in small surface blebs is accompanied by a concomitant depletion from the cytosol. Nuclear autoantigens also undergo a striking redistribution and concentration during apoptosis. 60-kd Ro, La, the snRNPs, Ku, and poly(adenosine diphosphoribose)polymerase, which normally have a diffuse nuclear distribution, initially become concentrated as a rim around the condensing chromatin in early apoptosis. As apoptosis progresses and the nucleus becomes fragmented into multiple membranebound apoptotic bodies, nuclear autoantigens remain as a rim around l5 Many of these lupus autoantigens are also the condensed ~hromatin.'~, structurally modified during apoptosis, undergoing cleavage, phosphorylation, and novel complex formation, among other processes.18,19,21, =, 53, 62,63 It is likely that these structural changes alter the fate of the molecules
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Figure 1. Autoantigens are highly enriched in the surface blebs of apoptotic cells. Keratinocytes are irradiated to induce apoptosis and then stained with propidium iodide to mark the small suiface blebs containing fragmented rough endoplasmic reticulum (ER) (arrows). These blebs are highly enriched in 52kDa Ro, phospholipid, ER, and ribosomal autoantigens. (From Rosen A, Casciola-Rosen L Autoantigens as substrates for apoptic proteases: Implications for the pathogenesis of systemic autoimmune disease. Cell Death Differ 6:6, 1999; with permission.)
when they enter the major histocompatibility complex class I1 processing pathway, generating suprathreshold amounts of determinants not normally generated when the unmodified molecule is similarly processed.6O Although in many cases, autoantibodies preferentially recognize modi63 the altered immunogenicity of these fied forms of a~toantigens,5~, modified forms of autoantigens remains to be directly demonstrated. AUTOANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS ALSO RECOGNIZE PROTEIN COMPLEXES ON THE APOPTOTIC CELL SURFACE
Several recent studies demonstrate that the surface of apoptotic surface blebs also has the capacity to concentrate potential autoantigens. For example, phosphatidylserine (PS)becomes concentrated at this site early in the apoptotic process,lg,33, where it is accessible to phospholipid-binding protein^.'^, 33, PS, normally restricted to the inner surface of the plasma membrane bilayer, becomes rapidly redistributed early in apoptosis and appears at the external membrane surface. This generates a procoagulant external cell surface which has the capacity to bind several autoantigenic PS-binding proteins, including P,-glycoprotein-1 and annexin V.I9, 26, 33, 49 The demonstration that these phospholipidbinding proteins decorate the surface of apoptotic cells strongly suggests that the immunogenic phospholipid-protein complexes form at the surface of apoptotic cells in vivo. This is further underscored by the obser-
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vation that IgG purified from the plasma of patients with antiphospholipid syndrome binds to the surface of apoptotic cells, and inhibits their procoagulant activity. Apoptosis thus has the potential to explain several clinical associations of antiphospholipid antibodies. In the setting of decreased clearance of apoptotic cells, the apoptotic cell surface may favor accumulation of phospholipid-binding proteins, and immunization with the resultant complexes may ensue, generating antiphospholipid antibodies. Where the apoptotic surface is in contact with plasma, initiation of the clotting cascade may also occur. This may account in part for the striking association of antiphospholipid antibodies with pathologic coagulation events.51Although clearance of apoptotic cells is generally rapid and noninflammatory, the presence of antiphospholipid antibodies markedly alters this fate.41Antiphospholipid antibodies (APL) opsonize apoptotic cells for phagocytosis by macrophages, which secrete proinflammatory cytokines rather than the anti-inflammatory cytokines that normally accompany phagocytosis of apoptotic It is not yet known whether and how the presence of antiphospholipid antibodies influences coagulation events when endothelial cells undergo apoptosis in vivo, but this is important to establish. There have been several recent reports that a subset of antiphospholipid antibodies, anti-endothelial cell antibodies, and anti-DNA autoantibodies can themselves induce apoptosis.lO,38, This phenomenon creates the potential for an autoamplifying loop of vascular damage and intravascular coagulation as might potentially occur during the antiphospholipid syndrome, particularly the catastrophic variant: The ability of autoantibodies to initiate a cycle of damage that further drives the immune response may be a general feature of SLE, and where identified, it should be a target of therapeutic intervention. It is also of interest that the surface blebs of apoptotic keratinocytes bind Clq,% whose collagen-like domains are the frequent (-47%) target of a high-titer autoantibody response in patients with SLE.66 The exact binding partner for Clq at the apoptotic surface is not yet known, but recent studies suggest that Clq binding to apoptotic cells may have a critical function in clearance of apoptotic cells in some microenvironments." Interestingly, Clq deficiency is strongly associated with the development of SLE in both human beings and mice.I2 In the Clq-null mouse, Botto et a1 noted a marked accumulation of apoptotic cells in the kidney, suggesting that clearance of apoptotic cells is defective in this microenvironment" and focusing attention on clearance of apoptotic cells as an important potential defect underlying the development of systemic autoimmunity. The generation of autoantibodies that recognize the surface of the apoptotic cell may play an important role in the propagation and amplification of the autoimmune response to apoptotic cells in several ways: (1) they may enhance capture and presentation of apoptotic surface blebs by B cells, favoring spreading of the immune response to other modified autoantigens in the blebs that may be present at lower concentrations; (2) they change the nature of the response on phagocytosis from
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noninflammatory to inflammatory; (3) they may themselves induce cell damage and death and thereby autoamplify any injury; and (4)they may enhance the procoagulant consequences of endothelial cell apoptosis. UNIQUE APOPTOTIC EVENTS ARE THE LIKELY INITIATOR OF SYSTEMIC LUPUS ERYTHEMATOSUS It is important to note that autoantigen clustering and modification occur in almost all forms of apoptosis described to date, which often takes place in nonimmune contexts.16,17, 21, 28, The marked frequency of apoptosis in normal development and homeostasis coupled with the infrequency of systemic autoimmunity in the population strongly suggests that only a restricted subset of apoptotic events (e.g., those occurring in a proimmune setting) in individuals who are genetically predisposed to allowing the generation of novel autoantigen structure (e.g., from abnormalities in the clearance and degradation of apoptotic material in tissues) initiate a self-sustaining autoimmune response. In this regard, it is of great interest that intravenous immunization of nonautoimmune mice with y-irradiated, syngeneic, apoptotic thymocytes results in only the transient production of low levels of autoantibodies (anticardiolipin and anti-single stranded DNA).& It is of interest to define whether unique forms of apoptotic cell death and defects in the clearance of apoptotic material might alter the response to immunization with apoptotic material in this model. NOVEL AUTOANTIGEN FRAGMENTS ARE PRODUCED DURING CYTOTOXIC LYMPHOCYTE-INDUCED TARGET CELL APOPTOSIS One form of apoptosis that frequently occurs in a proimmune setting is the death of virally infected target cells, which is induced by cytotoxic T lymphocytes (CTLs). CTLs use several pathways to induce target cell apoptosis, including Fas-ligation and granule e x o c y t ~ s i s . ~ ~ Recent studies have shown that granzyme B plays an important role in inducing apoptotic changes in target cells during granule exocytosisinduced cytotoxicity;l, 59 partly by catalyzing the cleavage and activation B of several apoptosis-specific cysteine proteases ( c a s ~ a s e s )Granzyme .~~ also initiates caspase-independent pathways that contribute to target cell death, although the nature of these additional pathways remains undefined.56,61 One candidate process is the direct proteolysis of death substrates by granzyme B. In recent studies, the authors demonstrated that the catalytic subunit of DNA-dependent protein kinase (DNA PKcs) and nuclear mitotic apparatus protein (NuMA) are directly and efficiently cleaved by granzyme B both in vitro and in cells undergoing granule-induced cytotoxicity.2Granzyme B-mediated cleavage of these substrates generates unique fragments not generated during any other form of apoptosis studied to date (Fig. 2). Efficient cleavage by granzyme
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Figure 2. Different autoantigen fragments are generated during cytotoxic lymphocyte granule-induced apoptosis. Caspase-3 fragments of autoantigens are generated during all forms of nonimmune, noninflammatory apoptotic death, including those that occur during lymphocyte selection. In contrast, unique grantyme B fragments (with novel N- and Ctermini) are generated during CTL-granule-induced death. PARP and DNA-PK are shown as representative examples.
B (with the generation of distinct fragments) has recently been observed for numerous other systemic disease autoantigens, including several that are not cleaved by caspases (L. Casciola-Rosen et al, unpublished results). This striking common feature of many autoantigens together with their clustering at the same site in apoptotic cells focuses attention on the process of granule-induced apoptosis as a potential initiator of autoimmunity in the susceptible host. It is extremely important to evaluate whether the immunogenicity of these novel forms of autoantigen is indeed increased over that of uncleaved forms of these molecules. DEFECTS IN CLEARANCE AND DEGRADATION OF THE APOPTOTIC CORPSE IN TISSUES MAY BE AN IMPORTANT DEFECT UNDERLYING SYSTEMIC LUPUS ERYTHEMATOSUS
Recent studies have emphasized that apoptotic cells are not immunologically inert but have either positive or negative immune effects depending on the antigen-presenting cell with which they interact.’, 52 For example, human dendritic cells (but not macrophages) efficiently present antigen derived from apoptotic (but not nonapoptotic) cells, stimulating class I restricted CTLs.l When macrophages are present together with dendritic cells in this assay, the presentation of apoptotic
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material is markedly inhibited. Several potential mechanisms might account for this observation. First, phagocytosis and degradation of apoptotic cells by macrophages are highly effi~ient,~~ thereby minimizing access of apoptotic material to dendritic cells. Phagocytosis of apoptotic cells by macrophages induces the production of anti-inflammatory cytokines (e.g., tumor growth factor-p,) and inhibits the production of several proinflammatory cytokines (e.g., interleukin-1P, tumor necrosis a It is possible that an abnormality in any of these pathways involved with rapid clearance of apoptotic material or expression of its anti-inflammatory activities may initiate autoimmunity. The recent observation of increased numbers of uncleared apoptotic cells in the kidneys of an SLE-susceptible Clq-null mouse reinforces the theory that abnormal clearance of apoptotic cells may play a role in the pathogenesis of SLE in this animal." Although there are no data at present that directly address whether and by what mechanism impaired clearance of apoptotic cells may initiate or exacerbate the autoimmune state in vivo, delayed clearance might change the compartmentalization of autoantigens (allowing leakage of autoantigen during secondary necrosis and access of soluble molecules to enable efficient macropinocytotic and endocytic antigen capture by dendritic cells) or provide apoptotic cells and membrane-bound fragments access to (proimmune) populations of antigen-presenting cells from which they are normally exc1uded.l A recent study in human beings has demonstrated that the clearance of apoptotic lymphocytes and fragments by macrophages is impaired in some patients with SLE.3O There was significant heterogeneity in the level of impairment among patients, which did not appear to correlate with disease activity or therapy.30It remains unclear whether (1) this phenomenon results from abnormalities in recognition, binding, or phagocytosis of apoptotic cells by SLE macrophages; (2) the phagocytosis of all types of apoptotic cells is similarly affected; and (3) the degree of impairment in a particular patient varies with disease activity. Recent advances in understanding the molecular basis for recognition and phagocytosis of apoptotic cellsz6,48, 52 should begin to allow the molecular basis of impaired clearance of apoptotic material in patients with autoimmune diseases to be defined. The marked heterogeneity of the degree of impairment observed in different patients predicts significant complexity in this population resulting from the contribution of multiple different clearance pathways. Understanding the immunologic consequences of apoptosis is likely to be simiIarly complex, because the form of apoptotic cell, the phenotype and physiologic state of the antigen-presenting cells, and the relevant tissue microenvironment may contribute.
MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS The studies discussed above have focused attention on unique forms of apoptosis as a candidate process that initiates and propagates systemic
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Figure 3. Model of SLE. A, Initiation phase. Autoantigens are clustered and concentrated in the surface blebs of apoptotic cells where they are modified by apoptosis-specific proteolysis and phosphorylation. When this event (1) occurs in a genetically susceptible individual; (2) involves a microenvironment containing high concentrations of the targeted antigens; (3)is pro-immune in nature (e.g., during CTL-induced killing of virally infected cells); and (4) allows suprathreshold concentrations of antigen with non-tolerized structure to enter the MHC class II processing pathway, a primary immune response directed against the non-tolerizedstructure is initiated. Defective clearance, reduced anti-inflammatoryconsequences of apoptotic material, and abnormal regulation of the immune response are likely to be important susceptibility factors in this group of diseases. 6,Propagation phase. Once the primary immune response to apoptotic antigens is initiated, other apoptotic events (occurring in the course of homeostasis or damage) may stimulate the secondary immune response with less stringency, resulting in flares.
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autoimmune diseases. In the genetically susceptible individual (e.g., someone who has a defect in the ability to efficiently phagocytose and degrade apoptotic cells and debris or to mount an adequate antiinflammatory response on ingestion of apoptotic material), the confluence of several forces allows the generation of suprathreshold concentrations of nontolerized structure in the presence of costimulatory signals (Fig. 3A) and the access of this material to the major histocompatibility complex class I1 pathway of a population of antigen-presenting cells that efficiently initiate a primary immune response. The low frequency of this form of autoimmunity in the population likely reflects this need to simultaneously satisfy several stringent criteria to initiate the primary immune response. The molecules targeted are unified by their susceptibility to modification during the perturbing process, likely revealing a previously cryptic structure. Available data demonstrate that many (but not all) autoantigens are specifically cleaved by caspases during apoptosis. A similar (but not identical) subset of autoantigens is directly cleaved by granzyme B, generating unique fragments not observed during any other forms of apoptosis studied to date. Once primary immunization has occurred, the repeated generation of apoptotic material (e.g., during sun exposure, viral infection, or drug exposure) might efficiently rechallenge the primed immune system (the stringency of this secondary response being significantly lower than that of the primary response) (see Fig. 3B). The effector pathways activated by the primed immune system include several that generate loads of apoptotic material (e.g., cellular cytotoxicity and inflammatory cell apoptosis).52The opsonization of apoptotic material by antiphospholipid and anti-p,-glycoprotein I antibodies6,7, **, may increase the efficiency of apoptotic antigen capture and induce the production of pro- rather than anti-inflammatory cytokines, potentially further driving the immune response. This capacity for immune-driven autoamplification may be one of the critical principles underlying severe systemic autoimmune disease (see Fig. 3B). An important target of future therapeutic intervention may include interrupting the autoamplifymg loop by decreasing apoptosis during flares. Identifying the factors that have an impact on the production of unique forms and proper clearance of apoptotic cells may suggest other novel pathways for intervention. References 1. Albert ML, Sauter 8,Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells
and induce class I restricted CTLs. Nature 392:86, 1998 2. Andrade F, Roy S, Nicholson D, et al: Granzyme B directly and efficiently cleaves
several downstream caspase substrates: Implications for CTL-induced apoptosis. Immunity 8:451, 1998 3. Amett FC, Reveille JD:Genetics of systemic lupus erythematosus. Rheum Dis Clin North Am 18:865, 1992 4. Asherson RA: The catastrophic antiphospholipid syndrome [editorial]. J Rheumatol 19:508, 1992 5. Bach JF, Koutouzov S New clues to systemic lupus. Lancet 350:11, 1997
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28. Greidinger EL, Miller DK, Yamin T-T, et al: Sequential activation of three distinct ICElike activities in Fas-ligated Jurkat cells. FEBS Lett 390:299, 1996 29. Henkart PA: Lymphocyte-mediated cytotoxicity: Two pathways and multiple effector molecules. Immunity 1343, 1994 30. Herrmann M, Voll RE, Zoller OM, et al: Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum 41:1241, 1998 Wesselschmidt RL, Shresta S, et al: Cytotoxic lymphocytes require gran31. Heusel JW, zyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977, 1994 32. Isenberg DA: Systemic lupus erythematosus: Immunopathogenesis and the card game analogy. J Rheumatol Suppl48:62, 1997 33. Koopman G, Reutelingsperger CP, Kuitjen GA, et al: Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415, 1994 34. Korb LC, Ahearn JM: C l q binds directly and specifically to surface blebs of apoptotic human keratinocytes-complementdeficiency and systemic lupus erythematosus revisited. J Immunol 158:4525, 1997 35. Laman SD, Provost Cutaneous manifestations of lupus erythematosus. Rheum Dis Clin North Am 20:195, 1994 36. Lanzavecchia A: How can cryptic epitopes trigger autoimmunity? J Exp Med 181:1945, 1995 37. LeFeber WP, Norris DA, Ryan SR, et al: Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes. J Clin Invest 741545, 1984 38. Madaio MP, Fabbi M, Tiso M, et al: Spontaneously produced anti-DNA/DNase I autoantibodies modulate nuclear apoptosis in living cells. Eur J Immunol26:3035, 1996 39. Mamula MJ: The inability to process a self-peptide allows autoreactive T cells to escape tolerance. J Exp Med 177567, 1993 40. Mancini M, Nicholson DW, Roy S, et al: The caspase-3 precursor has a cytosolic and mitochondria1 distribution: Implications for apoptotic signaling. J Cell Biol 140:1485, 1998 41. Manfredi AA, Rovere P, Galati G, et al: Apoptotic cell clearance in systemic lupus erythematosus1. Opsonization by antiphospholipid antibodies. Arthritis Rheum 41: 205, 1998 42. Manfredi AA, Rovere P, Heltai S, et al: Apoptotic cell clearance in systemic lupus erythematosus-11. Role of P1-glycoprotein I. Arthritis Rheum 41215, 1998 43. Martin SJ, Finucane DM, Amarante-Mendes GP, et a 1 Phosphatidylserine extemalization during CD95-induced apoptosis of cells and cytoplasts requires ICE/ CED-3 protease activity. J Biol Chem 271:28753, 1996 44. Mevorach D, Zhou JL, Song X, et a1 Systemic exposure to irradiated apoptotic cells induced autoantibody production. J Exp Med 188:387,1998 45. Mohan C, Datta SK: Lupus: Key pathogenic mechanisms and contributing factors. Clin Immunol Immunopathol77:209, 1995 46. Nakamura N, Ban T, Yamaji K, et al: Localization of the apoptosis-inducing activity of lupus anticoagulant in an annexin V-binding antibody subset. J Clin Invest 101:1951, 1998 47. Norris DA: Pathomechanisms of photosensitive lupus erythematosus. J Invest Dermato1 100:58, 1993 48. Platt N, Da Silva RP, Gordon S Recognizing death: The phagocytosis of apoptotic cells. Trends in Cell Biology 8:365, 1998 49. Price BE, Rauch J, Shia MA, et al: Anti-phospholipid autoantibodies bind to apoptotic, but not viable, thymocytes in a P,-glycoprotein I-dependent manner. J Immunol 1572201, 1996 50. Radic MZ, Weigert M Origins of anti-DNA antibodies and their implications for Bcell tolerance. Ann NY Acad Sci 764:384, 1995 51. Rand JH: Antiphospholipid antibody syndrome: New insights on thrombogenic mechanisms. Am J Med Sci 316:142, 1998
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52. Ren Y, Savill J: Apoptosis: The importance of being eaten. Cell Death Differentiation 5:563, 1998 53. Rosen A, Casciola-Rosen L: Autoantigens as substrates for apoptotic proteases: Implications for the pathogenesis of systemic autoimmune disease. Cell Death Differentiation 6:6, 1999 54. Salemi S, Caporossi AP, Boffa L, et al: HIVgpl20 activates autoreactive CD4-specific T cell responses by unveiling of hidden CD4 peptides during processing. J Exp Med 181:2253, 1995 55. Salvesen GS, Dixit VM: Caspases: Intracellular signaling by proteolysis. Cell 91:443, 1997 56. Sarin A, Williams MS, Alexander-Miller MA, et al: Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 6209, 1997 57. Satoh M, Ajmani AK, Ogasawara T, et al: Autoantibodies to RNA polymerase I1 are common in systemic lupus erythematosus and overlap syndrome. Specific recognition of the phosphorylated (110) form by a subset of human sera. J Clin Invest 941981,1994 58. Sercarz EE, Lehrnann PV, Ametani A, et al: Dominance and crypticity of T cell antigenic determinants. Ann Rev Immunol 11:729, 1993 59. Shi L, Kam C-M, Powers JC, et al: Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med 1761521,1992 60. Simitsek PD, Campbell DG, Lanzavecchia A, et al: Modulation of antigen processing by bound antibodies can boost or suppress class I1 major histocompatibility complex presentation of different T cell determinants. J Exp Med 181:1957, 1995 61. Talanian RV, Yang XH, Turbov J, et al: Granule-mediated killing: Pathways for granzyme B-initiated apoptosis. J Exp Med 186:1323,1997 62. Utz PJ, Hottelet M, van Venrooij WJ, et a1 Association of phosphorylated serine/ arginine (SR) splicing factors with the U1-small ribonucleoprotein (snRNP) autoantigen complex accompanies apoptotic cell death. J Exp Med 187547, 1998 63. Utz PJ, Hottelet M, Schur PH, et al: Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus. J Exp Med 185343, 1997 64. Voll RE, Herrrnann M, Roth EA, et al: Immunosuppressive effects of apoptotic cells. Nature 390350, 1997 65. Watts C, Lanzavecchia A. Suppressive effect of an antibody on processing of T cell epitopes. J Exp Med 178:1459, 1993 66. Wener MH, Uwatoko S, Mannik M Antibodies to the collagen-like region of Clq in sera of patients with autoimmune rheumatic diseases. Arthritis Rheum 32:544, 1989 Address reprint requests to Antony Rosen, MBChB, BSc (Hons) Johns Hopkins University School of Medicine Division of Rheumatology 720 Rutland Avenue, Room 1059 Baltimore, MD 21205 e-mail: arosenQhmi.edu
0889-857X/OO $15.00
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APOPTOSIS IN SYSTEMIC LUPUS ERYTHEMATOSUS Clinical Implications Felipe Andrade, MD, Livia Casciola-Rosen, PhD, and Antony Rosen, MBChB, BSc (Hons)
Systemic lupus erythematosus (SLE) is a heterogeneous and complex group of disorders of unclear cause. The complexity arises from three major sources (genes, environment, and time) and makes determining disease pathogenesis difficult, particularly in the outbred human population. The genetic heterogeneity may affect immune response, target tissue, and disease expression in the patient p~pulation.~ Numerous different environmental agents have been proposed to interact with the host during the initiation and propagation of SLE.'*, 32 There is considerable kinetic complexity in individual patients and in the population (with great variability in the intervals between exposure to initiating stimuli and onset of the disease process and in recurrence). This article provides an apoptosis-centric hypothesis for the initiation and propagation of SLE, which is able to explain much of this complexity. Although there are several aspects of the hypothesis that have not yet been proven, These studies were supported by N M Grants AR44684 (LC-R) and DE12354 (AR), the SLE Foundation, the Donald B. and Dorothy L. Stabler Foundation, and the Schauman Lupus Research Fund. Antony Rosen is a Pew Scholar in the Biomedical Sciences and is supported by a Burroughs Wellcome Fund Translational Research award. Felipe Andrade is supported by a Fullbright/ Consejo Nacional de Ciencia y Tecnologia, Mexico scholarship.
From the Departments of Dermatology and Cell Biology and Anatomy (LC-R); and Division of Rheumatology (AR, FA), Johns Hopkins University School of Medicine, Baltimore, Maryland
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the questions raised can be addressed both in vitro and in vivo. The answers provided are likely to furnish important insights into the normal immune consequences of different forms of apoptosis in tissues and about whether defects in the signaling, execution, and clearance phases of the apoptotic process are susceptibility factors for the development of systemic autoimmune disease. This novel view of SLE focuses therapeutic thinking on a process of potentially central relevance in the pathogenesis of SLE. AUTOANTIBODIES: PROBES OF THE PERTURBED STATE
There is a growing consensus that the highly specific humoral immune response to autoantigens in systemic autoimmune diseases is T-cell-dependent and that flares of disease result when this primed immune system is rechallenged with self-antigen.'" 22, 5o Although the mechanisms responsible for initiation and propagation of the immune response to specific autoantigens remain ~nclear,~, 45 autoantigens are unified by being selected for an immune response. The targeted molecules must have satisfied the stringent criteria for initiation of a primary immune response at the onset of disease (i.e., presentation of suprathreshold concentrations of nontolerized structure in a proimmune context).14 Because tolerance is only induced to dominant determinants in autoantigens (which are generated and presented at suprathreshold concentrations during natural processing of whole-protein antigens), a potential exists for T-cell-autoreactivity directed against cryptic determinants (which are not generated at all or are generated at subthreshold 58 The hypothesis that autoimmunity arises when usually cryptic determinants become visible to the immune system has received increased attention in the past several years. Indeed, several experimental systems have now clearly demonstrated circumstances that allow previously cryptic determinants to be presented.% These include novel autoantigen cleavage9,39 and altered autoantigen processing induced by high-affinity ligand binding (e.g., to an antibody or receptor molecule).54,60, 65 The authors proposed that autoantigens in systemic autoimmune diseases are unified by the fact that they satisfy the stringent criteria for initiation of a primary immune response.14,l5 The corollary of this proposal is that if the perturbed state can be recreated in vitro, these common features of autoantigens (i.e., similar alterations of concentration, distribution, and structure) can be observed. The authors and others have used high-titer autoantibodies as probes of cell biology and biochemistry of autoantigens during different clinically relevant perturbed states to search for those circumstances in which autoantigens become clustered, concentrated, and structurally m ~ d i f i e d . ' 19-~1 ~ ~ ~These . studies have highlighted apoptosis as the potential perturbed state underlying the initiation and propagation of systemic autoimmune diseases
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and focus attention on the alterations in the cell biology and biochemistry that unify autoantigens during this form of cell death. LUPUS AUTOANTIGENS UNDERGO A STRIKING REDISTRIBUTION DURING APOPTOSIS, BECOMING CLUSTERED AND CONCENTRATED IN THE SURFACE BLEBS OF APOPTOTIC CELLS
Studies to delineate the potential perturbed states that might initiate systemic autoimmune diseases have been based on a striking clinical observation in SLE, that is, that ultraviolet irradiation has a marked propensity to induce flares of both systemic and skin disease in lupus patients.35In this regard, the epidermis seems to be an important target of the immunopathologic response in lupus and constitutes an appropriate in vitro model with which to address the effects of flare-inducing stimuli (e.g., ultraviolet B)." Earlier studies demonstrated that intracellular autoantigens can normally be stained at the exterior surface of keratinocytes incubated in vitro for 20 to 24 hours after irradiation with UVB, although the mechanism of this redistribution of antigens was not dete1mined.2~.37 To determine how this phenomenon might arise, the authors studied the subcellular distribution of lupus autoantigens at increasing times after UVB irradiation in both intact and permeabilized cells.I4The authors' initial studies made several observations. First, UVBirradiated keratinocytes undergo apoptosis beginning a few hours after irradiation. Apoptotic keratinocytes manifest the classic morphologic hallmarks of this process, including prominent surface blebbing (early event) and nuclear condensation and fragmentation into apoptotic bodies (later event). In addition, lupus autoantigens, which are not restricted to any specific subcellular compartment in control cells, are strikingly redistributed in apoptotic cells such that they become clustered and concentrated within small surface blebs and apoptotic bodies (Fig. 1). Small surface blebs (which contain fragmented rough endoplasmic reticulum; see Fig. 1) are highly enriched in 52-kd Ro, ribosomal autoantigens, and those autoantigens found within the endoplasmic reticulum lumen (e.g., calreticulin). This marked enrichment of autoantigens in small surface blebs is accompanied by a concomitant depletion from the cytosol. Nuclear autoantigens also undergo a striking redistribution and concentration during apoptosis. 60-kd Ro, La, the snRNPs, Ku, and poly(adenosine diphosphoribose)polymerase, which normally have a diffuse nuclear distribution, initially become concentrated as a rim around the condensing chromatin in early apoptosis. As apoptosis progresses and the nucleus becomes fragmented into multiple membranebound apoptotic bodies, nuclear autoantigens remain as a rim around l5 Many of these lupus autoantigens are also the condensed ~hromatin.'~, structurally modified during apoptosis, undergoing cleavage, phosphorylation, and novel complex formation, among other processes.18,19,21, =, 53, 62,63 It is likely that these structural changes alter the fate of the molecules
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Figure 1. Autoantigens are highly enriched in the surface blebs of apoptotic cells. Keratinocytes are irradiated to induce apoptosis and then stained with propidium iodide to mark the small suiface blebs containing fragmented rough endoplasmic reticulum (ER) (arrows). These blebs are highly enriched in 52kDa Ro, phospholipid, ER, and ribosomal autoantigens. (From Rosen A, Casciola-Rosen L Autoantigens as substrates for apoptic proteases: Implications for the pathogenesis of systemic autoimmune disease. Cell Death Differ 6:6, 1999; with permission.)
when they enter the major histocompatibility complex class I1 processing pathway, generating suprathreshold amounts of determinants not normally generated when the unmodified molecule is similarly processed.6O Although in many cases, autoantibodies preferentially recognize modi63 the altered immunogenicity of these fied forms of a~toantigens,5~, modified forms of autoantigens remains to be directly demonstrated. AUTOANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS ALSO RECOGNIZE PROTEIN COMPLEXES ON THE APOPTOTIC CELL SURFACE
Several recent studies demonstrate that the surface of apoptotic surface blebs also has the capacity to concentrate potential autoantigens. For example, phosphatidylserine (PS)becomes concentrated at this site early in the apoptotic process,lg,33, where it is accessible to phospholipid-binding protein^.'^, 33, PS, normally restricted to the inner surface of the plasma membrane bilayer, becomes rapidly redistributed early in apoptosis and appears at the external membrane surface. This generates a procoagulant external cell surface which has the capacity to bind several autoantigenic PS-binding proteins, including P,-glycoprotein-1 and annexin V.I9, 26, 33, 49 The demonstration that these phospholipidbinding proteins decorate the surface of apoptotic cells strongly suggests that the immunogenic phospholipid-protein complexes form at the surface of apoptotic cells in vivo. This is further underscored by the obser-
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vation that IgG purified from the plasma of patients with antiphospholipid syndrome binds to the surface of apoptotic cells, and inhibits their procoagulant activity. Apoptosis thus has the potential to explain several clinical associations of antiphospholipid antibodies. In the setting of decreased clearance of apoptotic cells, the apoptotic cell surface may favor accumulation of phospholipid-binding proteins, and immunization with the resultant complexes may ensue, generating antiphospholipid antibodies. Where the apoptotic surface is in contact with plasma, initiation of the clotting cascade may also occur. This may account in part for the striking association of antiphospholipid antibodies with pathologic coagulation events.51Although clearance of apoptotic cells is generally rapid and noninflammatory, the presence of antiphospholipid antibodies markedly alters this fate.41Antiphospholipid antibodies (APL) opsonize apoptotic cells for phagocytosis by macrophages, which secrete proinflammatory cytokines rather than the anti-inflammatory cytokines that normally accompany phagocytosis of apoptotic It is not yet known whether and how the presence of antiphospholipid antibodies influences coagulation events when endothelial cells undergo apoptosis in vivo, but this is important to establish. There have been several recent reports that a subset of antiphospholipid antibodies, anti-endothelial cell antibodies, and anti-DNA autoantibodies can themselves induce apoptosis.lO,38, This phenomenon creates the potential for an autoamplifying loop of vascular damage and intravascular coagulation as might potentially occur during the antiphospholipid syndrome, particularly the catastrophic variant: The ability of autoantibodies to initiate a cycle of damage that further drives the immune response may be a general feature of SLE, and where identified, it should be a target of therapeutic intervention. It is also of interest that the surface blebs of apoptotic keratinocytes bind Clq,% whose collagen-like domains are the frequent (-47%) target of a high-titer autoantibody response in patients with SLE.66 The exact binding partner for Clq at the apoptotic surface is not yet known, but recent studies suggest that Clq binding to apoptotic cells may have a critical function in clearance of apoptotic cells in some microenvironments." Interestingly, Clq deficiency is strongly associated with the development of SLE in both human beings and mice.I2 In the Clq-null mouse, Botto et a1 noted a marked accumulation of apoptotic cells in the kidney, suggesting that clearance of apoptotic cells is defective in this microenvironment" and focusing attention on clearance of apoptotic cells as an important potential defect underlying the development of systemic autoimmunity. The generation of autoantibodies that recognize the surface of the apoptotic cell may play an important role in the propagation and amplification of the autoimmune response to apoptotic cells in several ways: (1) they may enhance capture and presentation of apoptotic surface blebs by B cells, favoring spreading of the immune response to other modified autoantigens in the blebs that may be present at lower concentrations; (2) they change the nature of the response on phagocytosis from
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noninflammatory to inflammatory; (3) they may themselves induce cell damage and death and thereby autoamplify any injury; and (4)they may enhance the procoagulant consequences of endothelial cell apoptosis. UNIQUE APOPTOTIC EVENTS ARE THE LIKELY INITIATOR OF SYSTEMIC LUPUS ERYTHEMATOSUS It is important to note that autoantigen clustering and modification occur in almost all forms of apoptosis described to date, which often takes place in nonimmune contexts.16,17, 21, 28, The marked frequency of apoptosis in normal development and homeostasis coupled with the infrequency of systemic autoimmunity in the population strongly suggests that only a restricted subset of apoptotic events (e.g., those occurring in a proimmune setting) in individuals who are genetically predisposed to allowing the generation of novel autoantigen structure (e.g., from abnormalities in the clearance and degradation of apoptotic material in tissues) initiate a self-sustaining autoimmune response. In this regard, it is of great interest that intravenous immunization of nonautoimmune mice with y-irradiated, syngeneic, apoptotic thymocytes results in only the transient production of low levels of autoantibodies (anticardiolipin and anti-single stranded DNA).& It is of interest to define whether unique forms of apoptotic cell death and defects in the clearance of apoptotic material might alter the response to immunization with apoptotic material in this model. NOVEL AUTOANTIGEN FRAGMENTS ARE PRODUCED DURING CYTOTOXIC LYMPHOCYTE-INDUCED TARGET CELL APOPTOSIS One form of apoptosis that frequently occurs in a proimmune setting is the death of virally infected target cells, which is induced by cytotoxic T lymphocytes (CTLs). CTLs use several pathways to induce target cell apoptosis, including Fas-ligation and granule e x o c y t ~ s i s . ~ ~ Recent studies have shown that granzyme B plays an important role in inducing apoptotic changes in target cells during granule exocytosisinduced cytotoxicity;l, 59 partly by catalyzing the cleavage and activation B of several apoptosis-specific cysteine proteases ( c a s ~ a s e s )Granzyme .~~ also initiates caspase-independent pathways that contribute to target cell death, although the nature of these additional pathways remains undefined.56,61 One candidate process is the direct proteolysis of death substrates by granzyme B. In recent studies, the authors demonstrated that the catalytic subunit of DNA-dependent protein kinase (DNA PKcs) and nuclear mitotic apparatus protein (NuMA) are directly and efficiently cleaved by granzyme B both in vitro and in cells undergoing granule-induced cytotoxicity.2Granzyme B-mediated cleavage of these substrates generates unique fragments not generated during any other form of apoptosis studied to date (Fig. 2). Efficient cleavage by granzyme
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Figure 2. Different autoantigen fragments are generated during cytotoxic lymphocyte granule-induced apoptosis. Caspase-3 fragments of autoantigens are generated during all forms of nonimmune, noninflammatory apoptotic death, including those that occur during lymphocyte selection. In contrast, unique grantyme B fragments (with novel N- and Ctermini) are generated during CTL-granule-induced death. PARP and DNA-PK are shown as representative examples.
B (with the generation of distinct fragments) has recently been observed for numerous other systemic disease autoantigens, including several that are not cleaved by caspases (L. Casciola-Rosen et al, unpublished results). This striking common feature of many autoantigens together with their clustering at the same site in apoptotic cells focuses attention on the process of granule-induced apoptosis as a potential initiator of autoimmunity in the susceptible host. It is extremely important to evaluate whether the immunogenicity of these novel forms of autoantigen is indeed increased over that of uncleaved forms of these molecules. DEFECTS IN CLEARANCE AND DEGRADATION OF THE APOPTOTIC CORPSE IN TISSUES MAY BE AN IMPORTANT DEFECT UNDERLYING SYSTEMIC LUPUS ERYTHEMATOSUS
Recent studies have emphasized that apoptotic cells are not immunologically inert but have either positive or negative immune effects depending on the antigen-presenting cell with which they interact.’, 52 For example, human dendritic cells (but not macrophages) efficiently present antigen derived from apoptotic (but not nonapoptotic) cells, stimulating class I restricted CTLs.l When macrophages are present together with dendritic cells in this assay, the presentation of apoptotic
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material is markedly inhibited. Several potential mechanisms might account for this observation. First, phagocytosis and degradation of apoptotic cells by macrophages are highly effi~ient,~~ thereby minimizing access of apoptotic material to dendritic cells. Phagocytosis of apoptotic cells by macrophages induces the production of anti-inflammatory cytokines (e.g., tumor growth factor-p,) and inhibits the production of several proinflammatory cytokines (e.g., interleukin-1P, tumor necrosis a It is possible that an abnormality in any of these pathways involved with rapid clearance of apoptotic material or expression of its anti-inflammatory activities may initiate autoimmunity. The recent observation of increased numbers of uncleared apoptotic cells in the kidneys of an SLE-susceptible Clq-null mouse reinforces the theory that abnormal clearance of apoptotic cells may play a role in the pathogenesis of SLE in this animal." Although there are no data at present that directly address whether and by what mechanism impaired clearance of apoptotic cells may initiate or exacerbate the autoimmune state in vivo, delayed clearance might change the compartmentalization of autoantigens (allowing leakage of autoantigen during secondary necrosis and access of soluble molecules to enable efficient macropinocytotic and endocytic antigen capture by dendritic cells) or provide apoptotic cells and membrane-bound fragments access to (proimmune) populations of antigen-presenting cells from which they are normally exc1uded.l A recent study in human beings has demonstrated that the clearance of apoptotic lymphocytes and fragments by macrophages is impaired in some patients with SLE.3O There was significant heterogeneity in the level of impairment among patients, which did not appear to correlate with disease activity or therapy.30It remains unclear whether (1) this phenomenon results from abnormalities in recognition, binding, or phagocytosis of apoptotic cells by SLE macrophages; (2) the phagocytosis of all types of apoptotic cells is similarly affected; and (3) the degree of impairment in a particular patient varies with disease activity. Recent advances in understanding the molecular basis for recognition and phagocytosis of apoptotic cellsz6,48, 52 should begin to allow the molecular basis of impaired clearance of apoptotic material in patients with autoimmune diseases to be defined. The marked heterogeneity of the degree of impairment observed in different patients predicts significant complexity in this population resulting from the contribution of multiple different clearance pathways. Understanding the immunologic consequences of apoptosis is likely to be simiIarly complex, because the form of apoptotic cell, the phenotype and physiologic state of the antigen-presenting cells, and the relevant tissue microenvironment may contribute.
MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS The studies discussed above have focused attention on unique forms of apoptosis as a candidate process that initiates and propagates systemic
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Drug
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Figure 3. Model of SLE. A, Initiation phase. Autoantigens are clustered and concentrated in the surface blebs of apoptotic cells where they are modified by apoptosis-specific proteolysis and phosphorylation. When this event (1) occurs in a genetically susceptible individual; (2) involves a microenvironment containing high concentrations of the targeted antigens; (3)is pro-immune in nature (e.g., during CTL-induced killing of virally infected cells); and (4) allows suprathreshold concentrations of antigen with non-tolerized structure to enter the MHC class II processing pathway, a primary immune response directed against the non-tolerizedstructure is initiated. Defective clearance, reduced anti-inflammatoryconsequences of apoptotic material, and abnormal regulation of the immune response are likely to be important susceptibility factors in this group of diseases. 6,Propagation phase. Once the primary immune response to apoptotic antigens is initiated, other apoptotic events (occurring in the course of homeostasis or damage) may stimulate the secondary immune response with less stringency, resulting in flares.
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autoimmune diseases. In the genetically susceptible individual (e.g., someone who has a defect in the ability to efficiently phagocytose and degrade apoptotic cells and debris or to mount an adequate antiinflammatory response on ingestion of apoptotic material), the confluence of several forces allows the generation of suprathreshold concentrations of nontolerized structure in the presence of costimulatory signals (Fig. 3A) and the access of this material to the major histocompatibility complex class I1 pathway of a population of antigen-presenting cells that efficiently initiate a primary immune response. The low frequency of this form of autoimmunity in the population likely reflects this need to simultaneously satisfy several stringent criteria to initiate the primary immune response. The molecules targeted are unified by their susceptibility to modification during the perturbing process, likely revealing a previously cryptic structure. Available data demonstrate that many (but not all) autoantigens are specifically cleaved by caspases during apoptosis. A similar (but not identical) subset of autoantigens is directly cleaved by granzyme B, generating unique fragments not observed during any other forms of apoptosis studied to date. Once primary immunization has occurred, the repeated generation of apoptotic material (e.g., during sun exposure, viral infection, or drug exposure) might efficiently rechallenge the primed immune system (the stringency of this secondary response being significantly lower than that of the primary response) (see Fig. 3B). The effector pathways activated by the primed immune system include several that generate loads of apoptotic material (e.g., cellular cytotoxicity and inflammatory cell apoptosis).52The opsonization of apoptotic material by antiphospholipid and anti-p,-glycoprotein I antibodies6,7, **, may increase the efficiency of apoptotic antigen capture and induce the production of pro- rather than anti-inflammatory cytokines, potentially further driving the immune response. This capacity for immune-driven autoamplification may be one of the critical principles underlying severe systemic autoimmune disease (see Fig. 3B). An important target of future therapeutic intervention may include interrupting the autoamplifymg loop by decreasing apoptosis during flares. Identifying the factors that have an impact on the production of unique forms and proper clearance of apoptotic cells may suggest other novel pathways for intervention. References 1. Albert ML, Sauter 8,Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells
and induce class I restricted CTLs. Nature 392:86, 1998 2. Andrade F, Roy S, Nicholson D, et al: Granzyme B directly and efficiently cleaves
several downstream caspase substrates: Implications for CTL-induced apoptosis. Immunity 8:451, 1998 3. Amett FC, Reveille JD:Genetics of systemic lupus erythematosus. Rheum Dis Clin North Am 18:865, 1992 4. Asherson RA: The catastrophic antiphospholipid syndrome [editorial]. J Rheumatol 19:508, 1992 5. Bach JF, Koutouzov S New clues to systemic lupus. Lancet 350:11, 1997
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6. Balasubramanian K, Chandra J, Schroit AJ: Immune clearance of phosphatidylserineexpressing cells by phagocytes. J Biol Chem 27231113, 1997 7. Balasubramanian K, Schroit AJ: Characterization of phosphatidylserine-dependent pzglycoprotein I macrophage interactions-implications for apoptotic cell clearance by phagocytes. J Biol Chem 27329272, 1998 8. Bellone M, Iezzi G, Rovere P, et al: Processing of engulfed apoptotic bodies yields T cell epitopes. J Immunol 159:5391, 1997 9. Bockenstedt LK, Gee RJ, Mamula MJ: Self-peptides in the initiation of lupus autoimmunity. J Immunol 154.3516, 1995 10. Bordron A, Dueymes M, Levy Y, et al: The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis. J Clin Invest 101:2029, 1998 11. Botto M, Dell'Agnola C, Bygrave AE, et al: Homozygous C l q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 19:56, 1998 12. Bown'ess P, Davies KA, Norsworthy PJ; et' al: Hereditary Clq deficiency and systemic lupus erythematosus. QJM 87455, 1994 13. Burlingame RW, Rubin RL, Balderas RS, et al: Genesis and evolution of antichromatin autoantibodies in murine lupus implicates T-dependent immunization with self-antigen. J Clin Invest 91:1687, 1993 14. Casciola-Rosen LA, Anhalt G, Rosen A Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 179:1317, 1994 15. Casciola-Rosen LA, Anhalt G, Rosen A: DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis. J Exp Med 1821625, 1995 16. Casciola-Rosen LA, Miller DK, Anhalt GJ, et al: Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J Biol Chem 26930757, 1994 17. Casciola-Rosen LA, Nicholson DW, Chong T, et al: Apopain/CPP32 cleaves proteins that are essential for cellular repair: A fundamental principle of apoptotic death. J Exp Med 183:1957,1996 18. Casciola-Rosen LA, Rosen A Ultraviolet light-induced keratinocyte apoptosis: A potential mechanism for the induction of skin lesions and auotantibody production in Lupus Erythematosus. Lupus 6175, 1997 19. Casciola-Rosen L, Rosen A, Petri M, et al: Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: Implications for coagulation events and antigenic spread in systemic lupus erythematosus. Proc Natl Acad Sci USA 93:1624, 1996 20. Casciola-Rosen L Wigley F. Rosen A Sclerodenna autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis. J Exp Med 185:71, 1997 21. Casiano CA, Martin SJ, Green DR, et al: Selective cleavage of nuclear autoantigens during CD95 (Fas/APO-1)-mediated T cell apoptosis. J Exp Med 184:765, 1996 22. Diamond B, Katz JB, Paul E, et a1 The role of somatic mutation in the pathogenic antiDNA response. Ann Rev Immunol 10:731, 1992 23. Dong X, Hamilton KJ, Satoh M, et al: Initiation of autoimmunity to the p53 tumor suppressor protein by complexes of p53 and SV40 large T antigen. J Exp Med 1791243, 1994 24. Fadok VA, Voelker DR, Campbell PA, et al: Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1482207, 1992 25. Fadok VA, Bratton DL, Frasch SC, et al: The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differentiation 5551, 1998 26. Fadok VA, Bratton DL, Konowal A, et al: Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-P, PGE, and PAF. J Clin Invest 101:890, 1998 27. Golan TD, Elkon KB, Gharavi AE, et al: Enhanced membrane binding of autoantibodies to cultured keratinocytes of systemic lupus erythematosus patients after ultraviolet B / ultraviolet A irradiation. J Clin Invest 901067, 1992
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