The role of death-associated molecular patterns in the pathogenesis of systemic lupus erythematosus

Rheum Dis Clin N Am 30 (2004) 487 – 504

The role of death-associated molecular patterns in the pathogenesis of systemic lupus erythematosus Dror Mevorach, MD Laboratory for Cellular and Molecular Immunology, Rheumatology Unit, Department of Medicine, Hadassah-Hebrew University, POB 12000, Kiryat Hadassah, Jerusalem 91200, Israel

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease of unknown causes characterized by the presence of pathogenic high-titer autoantibodies to a diverse group of autoantigens. Recently, it was shown that, in 88% of patients, autoantibodies are present an average of 3.3 years before diagnosis [1]. Of the characteristic panel of autoantibodies, antinuclear, anti-Ro, anti-La, and antiphospholipid antibodies appear first, followed by anti – double-strand DNA, anti-Sm, and anti-RNP. These autoantibodies have features of an antigen-driven, T cell – dependent immune response [2,3]. Once present, the course of SLE is characterized by disease flares and autoimmune dysregulation. Treatment is based on immunosuppressive drugs, such as corticosteroids, azathioprine, and cyclophosphamide. The prognosis depends on the organs involved, the outcome of treatment, and the extent of adverse effects from immunosuppressive drugs. Programmed cell death (apoptosis), an essential developmental and homeostatic mechanism, is the preferred physiologic death process for cells and an important immune-response regulator. The appropriate clearance of apoptotic material completes the apoptotic process and is essential for regulation of inflammation and maintaining self-tolerance. This article describes apoptotic cell clearance mechanisms and discusses altered mechanisms for clearance of dying material, which represents a central pathogenic process in the development and acceleration of SLE. In addition, the article attempts to use this perspective to glimpse a potential direction for future treatments.

E-mail address: [email protected] 0889-857X/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.rdc.2004.04.011

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The normal innate immunity response to dying cells Apoptotic cell clearance mechanisms Integrins, scavenger receptors, phosphatidylserine receptors (PSRs), CD14, ABC1 cassette transporter, CD44, and C1q/CD91 receptors have specific roles in tethering or uptake of apoptotic cells by macrophages and immature dendritic cells (iDCs) in mammals (Fig. 1A) [4]. As suggested by the many receptors involved [4,5], the mechanisms underlying the recognition, engulfment, and phagocytosis of apoptotic cells by macrophages are complex. In addition, serum factors have been shown to increase mammalian macrophages and iDC uptake of apoptotic cells by three- to tenfold [6,7]. The complement system has been shown to be activated by human and murine apoptotic material (reviewed in reference [8]). The current author and colleagues first showed activation of both the classic and alternative complement pathways by apoptotic cells with opsonization by iC3b [6]. Taylor et al [9] confirmed this observation and suggested a hierarchical role for C1q in the ex vivo clearance of apoptotic thymocytes by thioglycolatederived peritoneal murine macrophages. Ogden et al [10] showed a role for CD91 and calreticulin in the uptake of apoptotic cells opsonized by mannose-binding lectin. C1q [10] and complement factors were suggested to mediate late apoptotic or necrotic cell clearance [11]. Clearance by iDCs also was shown to be facilitated by opsonization with iC3b [7]. Thus, complement opsonins and degradation products, such as C1q, mannose-binding lectin, and iC3b, seem to have a role in the uptake of apoptotic cells and cell debris, possibly by interaction with C1qR/CD91, CR3 (CD11b/CD18), or CR4 (CD11c/CD18) on phagocytes [6,7,10,12 – 14]. Other opsonins, such as IgM, IgG, serum amyloid protein (SAP), C-reactive protein (CRP), protein S, and other serum proteins, may also play a role in apoptotic cell clearance, whether or not they are related to complement activation [13,15 – 18]. The receptors and opsonin/bridging molecules that mediate uptake of apoptotic cells are summarized in Fig. 1A. Signaling cascade following interaction with apoptotic cells Apoptotic cells signal their neighbors in various ways. ‘‘Eat me’’ signals, which can appear on the membranes of apoptotic cells, serve as markers for phagocytes to recognize specific cells and subsequently ingest them. Direct signals include alteration in cell surface phospholipid composition [19] and changes in cell-surface glycoproteins or in surface charge [20]. Alternatively, certain serum proteins can opsonize an apoptotic cell surface and signal to phagocytes to engulf the opsonized apoptotic cells [6,14]. Similarly, viable cells express ‘‘do not eat me’’ signals by restriction of phosphatidylserine (PS) to the inner leaflet of the membrane [21] or CD31 expression [22]. Apoptotic cells can also secrete molecules that are important for recruitment of phagocytic cells, phagocytosis, and immune response into the immediate milieu. Transforming growth factor b (TGF-b) [23] and phosphoisocholine [24] are examples of immune suppression

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Fig. 1. Mechanisms of apoptotic cell clearance. (A) The different receptors shown to be important in apoptotic cell clearance are presented. Bridging molecules, or opsonins, which are either presented in serum or secreted by phagocytes, are indicated. (see Savill et al [5] for further reading). (B) The signaling cascade following interaction with apoptotic cells has been studied primarily during the engulfment process. Activation of Rac-1 is a common intersection following the ‘‘eat me’’ signal, which leads to cytoskeleton movement toward engulfment. Integrins may have some opposing effect on Rac-1 by means of Rho-A (see Grimsley et al [27] for further reading). ABC1, ABC1 cassette transporter; b2GPI, b2 glycoprotein I, MBL, mannose binding lectin; PSr, phosphatydilserine receptor; R, receptor; TSP-I, thrombospondin I.

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and phagocyte recruitment molecules secreted from cells undergoing apoptosis. Most of these mechanisms enable efficient identification of a cell undergoing apoptosis and efficient clearance, with appropriate noninflammatory and nonautoimmune consequences (see Fig. 1A) [7,25,26]. The signaling cascade following interaction with apoptotic cells has been studied primarily in Caenorhabditis elegans. It seems that activation of Rac-1, which leads to cytoskeletal movement and engulfment, is a common pathway resulting from an ‘‘eat me’’ signal [27]. The role of integrins is not yet clear but may have some opposing effect on Rac-1 [28]. The signaling cascade during engulfment is summarized in Fig. 1B. Phagocytosis of apoptotic cells is considered noninflammatory and has been shown to suppress the release of granulocyte-macrophage colony – stimulating factor, interleukin 1b (IL-1b), IL-8, tumor necrosis factor a (TNF-a), and thromboxane B2, but not TGF-b and prostaglandin E2 (PGE2) [25]. IL-10 has anti-inflammatory properties, which are manifested by suppression of the release of proinflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-a. Contradictory results, however, were obtained in two studies when the production of IL-10 following phagocytosis of apoptotic cells was evaluated. One study reported increased production [29] and the second suppressed release [25] of IL-10 following ingestion of apoptotic cells by human macrophages. These studies were performed in the absence of serum; therefore, the effect of complement and other opsonins on IL-10 release could not be evaluated. IL-10 is secreted in the presence of serum and complement opsonization (G. Amarylio, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003). Tagging the surfaces of apoptotic cells with C3b and iC3b may not only promote efficient clearance of these cells but may also induce anti-inflammatory responses. The binding and phagocytosis by means of macrophage CD11b/CD18 does not trigger a leukotriene release [30] or a respiratory burst [31,32]. Furthermore, ligation of CD11b/CD18 and other complement receptors may actually be immunosuppressive because of the resulting down-regulation of IL-12 and interferon g (IFN-g) production by human monocytes [33,34]. It seems likely, therefore, that the pro- and anti-inflammatory consequences of complement activation depend on the specific ligands involved and the coreceptors engaged. Macrophages ingesting iC3b-opsonized apoptotic cells downregulated their IL-1b and IL-6 secretion after triggering by zymosan, which stimulates Toll-like receptor 4 (TLR4) (G. Amarylio, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003). The general immunosuppressive or immune nonactivation consequences that are induced following ligation of apoptotic cells to specific and diverse receptors suggest at least partial common downstream pathways that the author calls by the general name death-associated molecular patterns (DAMPs), because these pathways bear some resemblance to pathogen-associated molecular patterns (PAMPs; see later). The author prefers the term DAMPs to apoptotic-associated molecular patterns because, contrary to accepted assumptions, some forms of necrotic cell death result in cell clearance processes that resemble those for

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apoptotic cells (D. Mevorach, MD, unpublished data, September 2002) [35], whereas some apoptotic patterns induce inflammation (see later discussion). DAMPs and PAMPs are associated with different receptors and, in general, lead to opposing immune consequences. Apoptotic cells generally induce immune suppression, although exceptions to this rule exist. In some conditions, proinflammatory cytokines are produced by cells undergoing apoptosis. Earlier studies led to the hypothesis that Fas ligand (FasL) expression enabled cells to counterattack the immune system, and that transplant rejection, for example, could be prevented by expressing FasL on transplanted organs. More recent studies have indicated that the notion of FasL as a mediator of immune privilege should be reconsidered, and Fas ligation may even be proinflammatory [36]. Furthermore, Fas was proposed to mediate proinflammatory cytokines such as IL-1b [37], and Park et al [38] suggested that following anti-Fas (CH11-)-induced apoptosis, human monocytes triggered Fas-dependent IL-8 and TNF-a secretion, which was shown to occur in macrophages even in the absence of apoptosis and was associated with nuclear factor kB (NF-kB) activation. Recently, the author showed a novel, non– Fas-dependent pattern of proinflammatory cytokine/chemokine secretion associated with mitogen activated protein kinase (MAPK) activation in monocytes undergoing apoptosis (D. Mevorach, MD, unpublished data, September 2002). Despite being doomed to die, monocytes showed transcriptional and translational activity and generated interleukin (IL)-1b, IL-8, and macrophage inflammatory protein (MIP)1a. Thus, certain modes of apoptosis in specific cells may induce proinflammatory milieu. Clearance of apoptotic cells by immature dendritic cells Dendritic cells (DCs) are professional antigen-presenting cells (APCs) within the immune system. They are continuously produced from hematopoietic stem cells in the bone marrow and are widely distributed, initially as iDCs, into lymphoid and nonlymphoid tissues [39,40]. When pathogens trigger iDCexpressed pattern-recognition and other receptors, iDCs phagocytose the pathogen and transform to mature DCs (mDCs). These mDCs lose much of their phagocytotic capacity, but they acquire their APC capability, and thus can initiate primary T cell – mediated immune responses. Their APC capability stems, in large part, from the pathogen antigens they express together with large quantities of cell surface major histocompatibility complexes (MHCs) and costimulatory molecules. DCs are not only excellent immune-response stimulators but are also potent immune inhibitors. The iDCs, including epidermal Langerhans cells, splenic marginal zone DCs, and interstitial DCs within nonlymphoid tissues, continuously sample self-antigen to maintain T cell self-tolerance (for review, see Steinman et al [41]). Do iDCs use specific receptors for engulfing apoptotic cells (see Fig. 1A)? The iDCs engulf apoptotic cells and are able to acquire antigens found in the

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Fig. 2. Stimulatory and tolerogenic DCs. PAMPs and their counterparts, DAMPs, are associated with different receptors and lead, in general, to opposite immune consequences. (A) Following interaction in the periphery of iDCs with a pathogen that triggers PAMPs, iDCs undergo classic maturation with upregulation of costimulatory molecules MHC class II and CCR7, and migrate to regional lymph nodes in the T cell area, to stimulate T cells. (B) Following interaction with opsonized apoptotic cells, DAMPs are similarly triggered and iDCs undergo maturation type II where no up-regulation of costimulatory molecules and MHC class II is observed but migration capacity is maintained. With arrival at the regional lymph nodes, interaction with T cells causes apoptosis or anergy to possible autoimmune clones (see Verbovetski et al [7] for further reading).

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dying cells [42 – 46]. Rubartelli et al [47] suggested that iDCs may use an integrin, the vitronectin receptor avb3, for apoptotic cell uptake. Albert et al [42] showed that another integrin, avb5, and the scavenger receptor CD36 both mediate apoptotic cell uptake by iDCs. Later, Nouri-Shirazi et al [45] suggested that iDCs may have subpopulations that use different integrins. The current author and colleagues recently showed that iDCs increase apoptotic cell uptake in the presence of complement degradation products [7]. They were also able to show generation of tolerizing DCs following interaction with iC3b-opsonized apoptotic cells. The tolerizing DCs were able to migrate to lymph nodes as they expressed CCR7, but they were resistant to Toll-like and CD40 stimulation and inhibited NF-kB activation (G. Amarylio, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003). Thus, clearance of apoptotic cells was shown to down-regulate the DC immune response following PAMPs triggering. The two proposed patterns of DC APC function are presented in Fig. 2.

Death-associated molecular patterns and pathogen-associated molecular patterns regulate inflammation and immune responses A year after the discovery of the role of the Drosophila Toll in the host defense against fungal infection, a mammalian Toll homolog was identified [48]. Subsequently, a family of proteins structurally related to Drosophila Toll was identified, collectively referred to as the Toll-like receptors (TLRs). The TLR family now consists of 10 members (TLR1 – TLR10; for review, see Takeda et al [49]). TLR recognition of microbial components triggers PAMPs, which leads to activation of innate and adaptive immunity. The signals for adaptive immunity activation are largely provided by DCs. The iDCs residing in the periphery are activated by various microbial components, undergo maturation, and express many of the TLRs, including TLR1, TLR2, TLR4, and TLR5 [50]. Various microbial components also elicit DC maturation through TLRs, including lipopolysaccharide, CpG DNA, peptidoglycan, lipoprotein, and the cell wall skeleton of Mycobacteria [51 –55]. TLR-mediated recognition of microbial components by DCs induces the expression of costimulatory molecules, such as CD80/CD86 and CD40, and production of inflammatory cytokines, such as IL-12 [56]. Following maturation, DCs migrate into the draining lymph nodes. Here they present microorganism-derived peptide antigens expressed on their cell surfaces with MHC class II antigen to naive T cells, thereby initiating an antigen-specific adaptive immune response [39,40]. The involvement of TLRs in the regulation of the adaptive immune response has been demonstrated in vivo. MyD88-deficient mice immunized with Ag mixed with complete Freund’s adjuvant exhibit defective production of both IFN-g from CD4+ T cells and Ag-specific IgG2a [55,57]. Furthermore, the Th1 immune response provoked by a protozoan parasite is abolished in these mice [58]. Thus, the Th1 immune response is regulated by the MyD88-dependent

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signaling pathway. TLRs have been described as immune system sentinels (Fig. 2A) that are believed to function in detecting and sampling invading pathogenic microorganisms, thus enabling discrimination between self and nonself. Endogenous ligands have been shown to activate TLRs [59 – 61], however, which may instead indicate that they discriminate between ‘‘danger’’ and homeostasis [62], rather than between self and nonself. In contrast to PAMPs, DAMPs, represented by receptors like CR3 and PSR, trigger an immune inhibitory response [7,25]. In normal circumstances, DAMPs are triggered during everyday apoptosis, and noninflammatory, nonautoimmune clearance is the rule. This noninflammatory response represents an efficient lifetime mechanism for clearance and peripheral tolerance induction [7,63]. In some circumstances, which are not completely understood, PAMPs may be triggered and become the dominant mechanism when cells phagocytose selfmaterial from dying cells. Their ligands may include the following: endogenous ligands derived from apoptotic, secondary necrotic, or primary necrotic dying cells; exogenous ligands derived from a virus; or any other microorganism. When both DAMPs and PAMPs are triggered, the consequences may favor immune stimulation and conversion from tolerance or ignorance to autoimmunity, as exemplified by heat-shock protein 70 (Hsp70), in the model of cytotoxic lymphocyte –induced autoimmune diabetes [64]. Thus, the balance between PAMPs and DAMPS is a crucial determinant whether the immune response will be tolerant or inflammatory/autoimmune.

Clearance of dying cells in systemic lupus erythematosus Apoptotic and necrotic cells are the source of antigens that derive an autoimmune response in systemic lupus erythematosus SLE is characterized by the production of various autoantibodies, such as nucleosomes, U1-RNP, Sm, SSA, SSB, PARP, NuMA, cardiolipin, and several others. These are heterogeneous autoantibodies. They are targeted against various structures such as DNA, RNA, and acidic phospholipids, which are found in diverse locations within the cell, including the nucleus, cytoplasm, and membrane. DNA and histones are major autoantigens in SLE. More recently, however, it has become clear that DNA-histone complexes (ie, nucleosomes) are preferred targets for autoantibodies in SLE [65]. How do nucleosomes and several other intracellular antigens become immunogenic in patients with SLE? In the preface to a 1992 issue of this journal, the editor, D. Pisetsky, wrote, ‘‘The mechanisms by which autoantibodies to ubiquitous intracellular antigens arise remain a central mystery’’ [66]. The first article that suggested the relationship between antigen selection and apoptosis appeared 2 years later [67]. Casciola-Rosen et al [67] showed that exposure of keratinocytes to ultraviolet B – mediated apoptosis induces cell surface expression of Ro and La, nucleosomes, and ribosomes, possibly because of translocation of certain

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intracellular particles to the apoptotic surface blebs. Subsequent work showed that PS, which is restricted to the inner membrane leaflet in viable cells, translocates to the cell surface during apoptosis [21], and that b2 glycoprotein I (b2GPI), a major autoantigen for antiphospholipid antibodies in SLE, binds to PS on apoptotic cells [68]. In vivo experiments in mice showed transient elevations of antiphospholipid, antinuclear antibodies, and anti – single-stranded DNA autoantibodies following high-dose injections of apoptotic cells [13]. It has therefore been hypothesized that SLE is triggered by immune responses to exposed intracellular macromolecules that are translocated to cell surfaces during apoptosis [67]. The current author would argue that necrotic cell death is also a source for autoantigens because, with necrosis, DNAse1 enters the cell and cleaves its chromatin to nucleosomes that are the antigens toward which antiDNA is generated [69]. Abnormal clearance of apoptotic or necrotic material in systemic lupus erythematosus Why do patients with SLE mount an immune response to the apoptotic material? One possible explanation is their impaired capacity to clear apoptotic cells in the normal manner. Apoptosis usually occurs when apoptotic cell membranes are intact so that nucleosomes are unable to leak out. In patients with SLE, however, there are high levels of nucleosomes circulating in peripheral blood [70], indicating accelerated apoptosis, inefficient clearance [71,72], or both. The nucleosomes are cleaved and derived from the apoptotic process. Necrotic cell death is another source of nucleosomes, and accelerated primary necrosis in SLE is possible. Furthermore, reduction of DNAse1 and increased blood nucleosome levels are suggested to have a critical role in the initiation of SLE [69]. Increased spontaneous lymphocyte apoptosis [73,74] and higher rates of spontaneous monocyte apoptosis [72] have been documented in patients with SLE. These observations suggest that an accelerated mononuclear apoptosis rate in SLE is one possible source of increased nucleosome levels. These observations are ex vivo, however. In vivo, these cells may be alive, and undergo accelerated apoptosis caused by withdrawal of cytokines or growth factors. Nevertheless, leukopenia, typical in patients with SLE, may result from this accelerated apoptosis, as it does in patients with AIDS [75]. In addition, SLE sera in vitro may induce leukocyte apoptosis. Is there convincing evidence for impaired apoptotic or necrotic cell clearance in SLE? In vitro studies have suggested impaired apoptotic cell clearance by monocyte-derived macrophages [71,72,76], which is at least partially related to accelerated in vitro monocyte apoptosis [72]. This accelerated apoptosis causes both increased apoptotic material and impairment of the ability of the remaining diluted macrophages to phagocytose apoptotic cells because of loss of ‘‘a community effect’’ [72]. DCs have a similar, albeit perhaps milder decrease in their capacity to phagocytose apoptotic cells in these patients (D. Mevorach, MD,

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unpublished data, October 2003). Although it remains difficult to conclude that there is altered clearance of apoptotic cells in patients with SLE in vivo based solely on these in vitro studies, Baumann et al [77] have shown that, in one subgroup of patients with SLE, apoptotic cells are not properly cleared by tingible body macrophages of the germinal centers. Impaired dying cell clearance therefore probably occurs in SLE, and this finding may have been documented as early as 1948. In the lupus erythematosus (LE) cell, nuclei are phagocytosed and digested by polymorphonuclear leukocytes [78] following opsonization by antichromatin/antihistone antibodies [79,80]. The LE cell represents the first documentation of abnormal chromatin clearance, that is, opsonization by an autoantibody, and clearance by a polymorphonuclear leukocyte. How is complement involved in this process? Homozygous C1q deficiency in humans and mice shows perhaps the strongest single-gene association with development of an SLE-like disease [81,82]. Furthermore, multiple TUNELpositive (terminal-deoxynucleotidyl-transferase-mediated dUTP nick end-labeling) cells are found in the kidneys of C1q-deficient mice developing an SLE-like disease [81]. Is it impaired clearance of apoptotic cells because of C1q deficiency [6] that causes SLE-like autoimmunity in the presence of other background genes? The previously described results provide the basis for the hypothesis that early classic pathway components together with phagocytic cells are involved in disposal of potentially hazardous immunogens from the body. Early complement deficiencies acting together with genetic and environmental susceptibility factors could thus increase susceptibility to an SLE-like disease because of diminished clearance of dying cells. This process is not as clear as physicians would like it to be, however. For instance, C1q is not required for in vivo clearance of sunburn apoptotic cells [83]. Furthermore, injection of large doses of apoptotic cells to C1q-deficient mice does not accelerate disease or autoantibody generation. [14] An alternative hypothesis, which is not mutually exclusive, proposes a role for the early classic pathway components in maintaining self-tolerance by removing or silencing selfAPS [84]. The current author proposes that C1q plays a significant role in the clearance of chromatin released from apoptotic cells that are not cleared efficiently. Furthermore, only apoptotic cell lysate was found to be immunogenic in C1q-deficient mice [14]. Patients with SLE have reduced levels of other important apoptotic cell opsonins and complement. Complement may be reduced because of genetic defect in a minority of patients or to complement consumption in most patients. CRP, recently shown to be genetically controlled [85], is another example. Patients with SLE also have increased levels of potentially dangerous opsonin-like autoantibodies [86], abnormal monocytes (Y. Berkun, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003) and DCs [87]. Tolerogenic DC generation following apoptotic cell –DC interaction is also altered (Y. Berkun, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003). In summary, altered clearance of dying cells most likely occurs in patients with SLE, and this process might have a role in drug-induced lupus as well

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(J. Ablyn, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003). Accelerated leukocyte apoptosis, genetic or functional deficiencies of natural opsonins such as complement and CRP, the presence of abnormal opsonin-like T cell – derived autoantibodies, and phagocyte, monocyte, macrophage, and DC dysfunction may lead to increased levels of apoptotic debris and immune complexes (ICs) of chromatin and other components of dying cells. Consequences of altered apoptotic cell clearance Autoimmunity develops in murine models only in response to some receptor or opsonin deficiencies involved in apoptotic cell clearance (see Fig. 1A). Deficiencies of C1q, C3, C4, C2, IgM, ABC1, Mer, and SAP may lead to autoimmunity, but deficiencies of CD36, vitronectin receptor, CD14, and others do not. In addition, autoimmunities will appear only in the presence of certain background genes, emphasizing that disturbed clearance by itself is not sufficient to trigger autoimmunity. Although apoptotic and necrotic debris may have an effect on PAMPs, a hyperstimulatory state of B and T cells is also required. Furthermore, since Casciola-Rosen et al [67] suggested that apoptotic cells are the autoantigen source for the autoimmune response seen in SLE, there have been no clear and direct studies proving isolated apoptotic cell pathogenicity. Furthermore, as indicated previously, most interactions with apoptotic cells are immunosuppressive. In the current author’s laboratory, the author and others have tried to immunize normal mice strains [13], and, apart from a transient IgM response by natural autoantibodies, they have been unable to document either generation of a significant autoimmune response or clinical disease. Only with very high loads of late apoptotic cells in very active autoimmune mice like the MRL/MpJ-Faslpr were they able to show autoimmunity acceleration; in proautoimmune strains with milder disease, like MRL/MpJ, no acceleration was seen [88]. Others have been able to generate autoimmunity only using foreign antigens [89]. Although these observations do not disprove the Casciola-Rosen hypothesis, they do provide strong evidence that, in most conditions, apoptotic cells are tolerogenic and not immunogenic [7,25], and that an apoptotic load of thymocytes, for example, is not sufficient to render them immunogenic in most cases. Something has to occur in the interface between the apoptotic cell and innate immunity, and perhaps also adaptive immunity, to overcome the tolerogenic character of the apoptotic cell. It is also misleading to regard apoptotic cells as a homogenous population. Is there a difference between a cell dying by means of the extrinsic pathway or the intrinsic apoptotic pathway? Is there a difference between a cell that dies by means of Fas or by means of granzyme B? Is there a difference between the death of a monocyte, a neutrophil, resting T cell, or activated T cell? Will some or all of these differences influence the microenvironment? The answers to most of these questions are probably positive. For example, spontaneous serum and contactwithdrawal monocyte apoptosis generates proinflammatory cytokines and chemo-

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kines, such as IL-1b, IL-8, and MIP1a (I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2002), and monocyte apoptosis is altered in SLE (Y. Berkun, MD, I. Verbovetski, PhD, D. Mevorach, MD, unpublished data, October 2003) [72]. Furthermore, Gergely et al [90] have shown mitochondrial hyperpolarization and adenosine triphosphate (ATP) depletion in patients with SLE. Thus, the problem in SLE is not with apoptosis in general but rather in specific modes of apoptosis or necrosis and in inadequate clearance, leading to the formation of free cell debris and resulting ICs of cell debris autoantibodies. Another point is localization. Apoptotic and necrotic debris may have critical importance at the germinal centers, where such debris could act as a natural adjuvant to TLR9 and additional TLRs, and trigger PAMPs, with resulting activation of autoimmune B- and T cell clones. The paradox of tolerogenic versus immunogenic apoptotic material is shown in regard to B cells. Triggering autoimmune B-cell receptor (BCR) with a chromatin and antichromatin complex leads to a marked autoimmune response that involves BCR and TLR9 [91,92]. Viglianti et al [92] showed that apoptotic mammalian chromatin IC is an effective ligand in the context of BCR-mediated

Fig. 3. Dying cells and the development of SLE. In a host with susceptible genes, whether related or unrelated to clearance of apoptotic cells, loss of tolerance to dying cells is a crucial factor in the development of autoantibodies. Loss of tolerance is related to aberrant function of interacting cells, such as monocytes, macrophages, DCs, and B cells, on interaction with apoptotic cells and blebs. Specific programmed cell death, specific apoptotic cells, apoptotic cell lysate, and the load of dying cells may determine immunogenicity. Additional unknown factors are necessary for the development of clinical SLE. Once autoimmunity is established, apoptotic material could either be tolerogenic, expressing PS and opsonized by iC3b, as is normally the case in healthy individuals, or immunogenic. Mechanisms of immunogenicity are not well understood but may include decreased uptake, opsonization by autoantibodies, cytokine exposure, stimulation of TLRs and PAMPs, accelerated apoptosis or necrosis, and dysfunction of DNAse1. Once the disease is established, it is characterized by remissions and exacerbations. Dying material may exacerbate the disease but also has the potential to re-educate the immune system and reinduce tolerance.

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delivery to TLR9 and can stimulate autoantibody generation. This finding is all the more remarkable because mammalian chromatin is considered a very weak adjuvant [93], which perhaps may become a stronger adjuvant in the context of apoptotic chromatin or IC [92] or internalization by means of BCR [91,92]. Li et al [94], however, now have shown that developing B cells expressing PS-reactive 3H9 are regulated by receptor editing in a manner similar to the anti-DNAreactive 3H9. The authors propose that the apoptotic cell surface is an active B-cell tolerogen in vivo. The results of this research provide an opportunity to better understand the effect of old drugs such as hydroxychloroquine, which abrogates the stimulatory effect of chromatin in IC on autoimmune BCR [91,92], or corticosteroids, which ameliorate apoptotic cell clearance [95]. At the same time, could physicians also look forward to possible novel treatments? Could they ameliorate apoptotic cell clearance or use related parameters to measure disease activity? Is re-education of the dysregulated immune system possible? If lupus autoantigens are derived from apoptotic cells, and these same apoptotic cells could potentially generate a strong immunosuppressive effect and peripheral tolerance, could they induce tolerance using autologous apoptotic cells [7,63]? Understanding better the pathogenesis of SLE (Fig. 3) could lead to accurate and focused questions that may show the way, if not the cure, for long-term remissions.

Acknowledgments The author thanks Mrs. Shifra Fraifeld for her assistance in the preparation of this article.

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