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Rheumatology 1999;38:1177–1183 Systemic Lupus Erythematosus/Series Editors: D. Isenberg and C. Gordon The role of apoptosis in systemic lupus erythematosus M. Salmon and C. Gordon Division of Immunity and Infection, The University of Birmingham, Birmingham B15 2TT, UK When it was described by Kerr et al. [1], the phenomenon of apoptosis, or programmed cell death, was greeted with a resounding lack of interest. The importance of this phenomenon is such that it is now very difficult to pick up any journal in a biomedical field without finding at least one paper on the subject. It has become apparent that the process is ubiquitous, representing a vital part of growth and differentiation in all tissues. The fundamental mechanisms of apoptosis and the genes that control it are highly conserved between species as diverse as worms and humans [2, 3]. Immunological research in apoptosis has focused on three areas in particular: (1) the selection of new lymphocytes, both B and T cells, before antigen challenge [4–6 ]; (2) the regulation of clonal expansion and resolution, including the elimination of apoptotic cells [7–10]; (3) the selection of memory cells [7–9, 11–14]. About 10 million new lymphocytes are produced in the bone marrow each day [11] and during a viral infection the total number of lymphocytes may easily double, mostly with a vast number of activated cytotoxic T cells [11]. These cells must be eliminated at the end of the response, to avoid an exponential increase in the size of the immune system [7]. However, this homeostatic balance must be achieved within a framework that accommodates the generation and persistence of memory cells, to permit a more potent response on subsequent re-challenge. Rescue from apoptosis is the crucial selection event in immunological memory for both B and T lymphocytes. Apoptosis Apoptosis is an active process that leads to the ordered destruction of cells, avoiding the release of intracellular contents into the extracellular microenvironment, where they have a powerful inflammatory effect. Apoptotic cells undergo a series of distinct physical changes, including alteration of the surface lipid membrane, cytoskeletal disruption, cell shrinkage and a characteristic pattern of DNA fragmentation. Submitted 1 June 1999; revised version accepted 17 June 1999. Correspondence to: M. Salmon, Division of Immunity and Infection, Rheumatology Research Group, The Medical School, The University of Birmingham, Birmingham B15 2TT, UK. Apoptosis can be induced actively, through ligation of specific receptors such as Fas or TNFR, or passively, through lack of essential survival signals [8]. All cells in the body require continuous positive signals to stay alive [15]. In the case of T lymphocytes, for example, these signals include interleukin (IL)-2 and interferon beta (IFN-b), which act by upregulating expression of antiapoptotic molecules such as Bcl-2 and Bcl-x [16, 17]. L These molecules, in turn, suppress the release of cytochrome c from mitochondria [18, 19]. In the absence of a positive signal for survival, cytochrome c forms a complex with APAF-1 and caspase 9, to form a caspase 3 activating unit [20]. Caspase 3 is the fulcrum between regulation of apoptosis and execution of the cell. When the activation of caspase 3 reaches a threshold level, its autocatalytic activity sets off a catastrophic chain reaction that leads to the activation of all the caspase 3 in the cell [21]. This is the point of irrevocable commitment to apoptosis. The active caspase 3 subsequently activates a range of downstream mediators that co-ordinate the ordered destruction of the cell [22]. Active induction of apoptosis may either facilitate the release of cytochrome c, or directly activate caspase 3 by a different route. In each case, caspase 3 activation defines the point of commitment and the convergence point for all apoptosis induction pathways. In vitro, the terminal phase of apoptosis is ‘blebbing’. Minute balloon-like membrane extrusions bud off from the surface of the cell, incorporating nuclear and cytoplasmic material. In vivo, this rarely happens, because apoptotic cells are very effectively removed by phagocytes, which use a range of recognition systems to identify them [10, 23]. A question of tolerance Immunological tolerance is a compromise that works for most people most of the time. Tolerance to self operates through a number of mechanisms, but is principally the domain of T lymphocytes. T cells are selected in the thymus from a population of immature thymocytes expressing randomly generated antigen receptors. Unlike B cells, which use surface-expressed antibody as a receptor and consequently can recognize native antigens, T cells only recognize short peptide antigens presented in the groove of class I (CD8 cells) or class II 1177 © 1999 British Society for Rheumatology 1178 M. Salmon and C. Gordon (CD4 cells) MHC molecules. T cells are selected in the thymus so that they will bind to self-MHC with selfpeptides in the groove (positive selection), but not sufficiently strongly to activate the cell (negative selection). The cells which fail these tests are, predictably, deleted by apoptosis [24]. Cells which fail positive selection are useless, those which fail negative selection are autoimmune and consequently dangerous. The importance of negative selection is ably illustrated by graft rejection reactions. Positive selection is a highly effective process, but negative selection is limited by the impracticality of presenting all possible peptides from every protein expressed in the body, in all available MHC molecules (anywhere from six to about 14) to every thymocyte produced throughout life. The reality seems to be that many antigens are not used for selection in the thymus, so central (thymic) tolerance is only partially effective. In the periphery, if antigens are presented by nonprofessional antigen-presenting cells (APC ) which lack appropriate co-stimulatory molecules such as CD80/86 or CD40, then T cells are switched off (anergy) or deleted by apoptosis. Systemic lupus erythematosus as a disease of defective apoptosis B-cell differentiation and antibody production are highly dependent on T-cell help, as we shall discuss later, so the crucial role of apoptosis in immunological tolerance (somewhat overestimated until quite recently) led to the first model to link apoptosis and systemic lupus erythematosus (SLE ). For many years, SLE was considered to be a disease of B-cell hyper-reactivity [25]. Autoantibodies are produced in patients with this disease to a bewildering variety of cellular components, at least 40 and probably more like 2000 target antigens in the nucleus, cytoplasm and membranes [26 ]. The MRL lpr mouse, which likewise produces autoantibodies to such antigens, was considered by many to be an excellent model for the disease [27, 28]. The discovery that the lpr gene is a defective form of Fas (CD95), which is a surface receptor on the surface of cells (particularly lymphocytes) that transduces an active signal for apoptosis [29], led to much speculation that SLE would similarly have defective Fas-mediated apoptosis at the core of the failure of self-tolerance [30]. Support for this hypothesis came when the gld abnormality (another mouse gene that produces a similar phenotype to lpr) was identified as a non-functional Fas ligand gene [31]. Unfortunately, there were severe problems with this model: firstly, the lpr and gld mice suffer from a severe lymphoproliferative condition. When these mice die, at a few months of age, up to half their body weight consists of abnormal lymphocytes [27, 28]. This is not a characteristic of patients with SLE, who are often profoundly lymphopenic. Secondly, both the expression and function of Fas and its ligand are normal in SLE [32–34]. Indeed, some reports suggest that Fas expression is a little higher than normal [32]. Actually, Fas- mediated apoptosis does not play a significant role in thymic selection of T cells anyway [35]. So, when a small number of individuals were found who had an lpr-type genetic abnormality and they had a lymphoproliferative disease, much like the MRL mouse, but not much like SLE, no one was too surprised [36–38]. Too much apoptosis? In fact, far from being a disease of too little apoptosis, the evidence has been available for a long time that SLE is quite the opposite. Under normal physiological circumstances, apoptotic cells are cleared very effectively by phagocytes [10, 23]. If this were not the case, we would all be in quite serious trouble. Up to 1011 neutrophils are produced in the bone marrow and subsequently die each day. These cells have a half-life in the blood of about 8 h, yet they contain toxic granules that produce significant inflammation if they are released into the extracellular microenvironment. Patients with SLE often have high levels of circulating DNA. This is usually present as short (162 base pair) stretches, wound around a histone core, i.e. as nucleosomes [39] which are the classical cleavage product of apoptosis. Furthermore, haematoxylin bodies, which are often found in the glomeruli of patients with lupus nephritis, are actually ‘blebs’ from fragmented apoptotic cells [46 ]. The LE cell, which has only recently been removed from the ACR criteria for SLE (because no one tests for it anymore, not because it was not specific), is itself a neutrophil that has phagocytosed apoptotic material of this sort. Apoptotic blebs can be processed by professional APC as foreign antigen and directed to class II MHC molecules. In addition to nucleosomes, they contain proteins such as Ro and La, which are clearly recognized autoantigens in SLE [40, 41]. Unfortunately, the major compromise operating in the generation of immunological tolerance means that we are not really functionally tolerant to the inside of our cells, just ignorant. Obligate intracellular proteins do not encounter the immune system very often in significant quantities, because phagocytes are so good at removing them intact. However in SLE, where this process appears to be mildly deficient, the peripheral immune system is challenged by these proteins. The Tcell dependence of autoantibodies in SLE is suggested very strongly by their high-affinity, isotype-switched nature, as we shall discuss in the next section. However, the evidence at this point suggests a new model for antigen-driven autoantibody production in SLE. Deficient phagocyte-mediated clearance of intact apoptotic cells leads to their fragmentation and the release of intracellular antigens (to which the immune system is not tolerant) in a form that can trigger an immune response. Before we can consider the implications of this further, we need to discuss recent developments in our understanding of T-cell-dependent antibody production. Role of apoptosis in SLE Somatic hypermutation and B-cell memory Primary antibody responses are exclusively IgM and predominantly low affinity. Athymic individuals are very good at producing such responses, but repeated challenge does not yield the characteristic features of a secondary antibody response, which are high-affinity IgG antibodies. These features are T-cell dependent. As the antibodies characteristic of SLE are mostly highaffinity IgG antibodies, they are very likely to result from T-cell-dependent responses. High-affinity memory B cells are produced in germinal centres by somatic hypermutation [9] ( Fig. 1). A germinal centre requires B cells and T cells activated in a primary response in the paracortex of secondary lymphoid tissue, and follicular dendritic cells that have the relevant antigen bound to their surface. The B cells (centroblasts) start to divide rapidly and spontaneously mutate the variable region of their antibody genes. As long as they keep dividing, they are safe from apoptosis, but as soon as they leave the dark zone and stop dividing, they have ~8 h to live. This is rather important, because random mutations to the immunoglobulin variable genes are as likely to produce an improvement in affinity as random tinkering with a spanner is to improve the performance of a Formula 1 racing engine. In both cases, blind chance will occasionally do the trick. However, in both cases, most such changes will be either ineffective or dangerous. Spontaneous mutation may well turn a useful anti-pathogen antibody into an autoantibody, but it is much more likely to produce a useless immunoglobulin. The germinal centre functions to select the fortuitous beneficial mutation from the garbage. To do this, the somatically hypermutated B cell, now termed a centrocyte, must pass a series of tests (Fig. 2). Firstly, the centrocytes must remove surface- 1179 bound antigen from the follicular dendritic cells in the light zone, then they must pinocytose the antigen, process it and incorporate it into class II MHC molecules on their surface. As they try to escape the germinal centre, the centrocytes encounter T cells which recognize the same antigen. If a T cell recognizes the antigen presented by a centrocyte, it releases onto its surface a preformed CD40 ligand, which binds to CD40 on the surface of the centrocyte. This cognate interaction overrides the apoptosis programme in the centrocyte, which then becomes a stable mature memory B cell or, with appropriate signals, it can differentiate into a plasma cell and produce large quantities of antibody. Clearly, only a centrocyte that has produced a high-affinity mutation will be able to retrieve antigen from the follicular dendritic cells and thereby survive. This process defines the central role of T cells in self-tolerance. A conundrum Improved understanding of the mechanisms leading to the selection of memory B cells in germinal centres poses an obvious problem. Somatically hypermutated, classswitched B cells can only survive if they can present a T-cell epitope from their pinocytosed antigen to a T cell. The conundrum is this: T cells can only recognize peptides, but the most characteristic antibodies in SLE are to DNA and phospholipids. This is easily solved in the context of the model we describe. Nucleosomes, as described above, consist of short stretches of DNA wound around a histone core, rather like a cotton reel. In an SLE patient, in whom high levels of circulating nucleosomes are found, a germinal centre can form in which the B cells are specific for double-stranded DNA, but effectively mutated centrocytes will use their anti-DNA antibody to remove F. 1. Somatic hypermutation and selection of B-cell antigen receptors (antibody) in a germinal centre. CB, centroblast; CC, centrocyte; mB, memory B cell; PC, plasma cell; T, T cell; FDC, follicular dendritic cell. 1180 M. Salmon and C. Gordon F. 2. The process of selecting memory B cells in a germinal centre. This interaction, where mutated antigen receptors are used by centrocytes (B cells) to retrieve antigen from follicular dendritic cells, which they process and present to T cells, allows the selection of high-affinity clones for the initiating antigen. It also defines the fundamental interaction responsible for tolerance in T-cell-dependent antibody responses. whole nucleosomes from the follicular dendritic cells. When this is processed, the protein core will be incorporated into the grooves of class II MHC molecules and presented to histone-specific T cells ( Fig. 3). Experimental evidence for this model has now been reported [42]. Similarly, membrane phospholipids such as phosphatidyl-serine are linked to lipid-binding proteins which can act as carriers. This provides a simple hapten-carrier model similar to that used in the production of conjugate vaccines. Why is the clearance of apoptotic cells deficient in patients with SLE? A complete failure of the clearance mechanisms that remove apoptotic cells would be fatal in days, so any such deficiency in patients with SLE could only be partial, reducing the threshold for overload of the system. Patients with homozygous deficiencies in the early complement proteins (C2, C4 and especially C1q) develop a severe lupus-like disease early in life [43]. Strangely, deficiencies of C3 produce a much less severe phenotype than deficiencies in C1q [43]. Yet C3 is the first point of convergence for all three complementactivating pathways (rather like caspase 3 in apoptosis) and consequently will have the most marked effect on complement activation. Deficiencies in components of the membrane attack complex are relatively minor. Recent data have shown that C1q receptors on the surface of phagocytes are an extremely important mechanism in clearing apoptotic cells [44]. Patients, or mice, with homozygous C1q deficiency develop autoantibodies and a lupus-like syndrome. This is apparently because they cannot clear apoptotic cells effectively and con- sequently present antigens derived from them to the immune system [45–47]. However, the levels of antinuclear and anti-dsDNA antibodies are not particularly high, and the clinical manifestations tend to be restricted to the skin and kidneys. Quite why patients with SLE develop defective clearance of apoptotic cells is less clear. Antibodies to C1q are present in a large proportion of patients, particularly those with renal disease [48–50]. This probably results in a functional deficiency of the protein. It is unlikely that C1q antibodies represent the primary abnormality in most patients with SLE, but it would certainly provide a mechanism for persistent disease and flaring. Exposure to UV irradiation and infection are the most common triggers of flares in lupus patients. Both will produce a sharp increase in the number of apoptotic cells in the skin [39, 40, 51] and in the blood [8, 11], respectively. If this overloads the inefficient clearance mechanisms of the reticuloendothelial system, large amounts of antigen will be released. This will have two effects: firstly, to drive the immune response to produce more antibodies and, secondly, to bind existing antibodies in the form of immune complexes. This will produce inflammatory reactions in highly vascularized tissues, such as the skin, kidneys and lungs. The immune complexes will also cause massive consumption of classical pathway complement, including C1q, thus exacerbating the initial problem. The relationship between complement deficiency and SLE will be developed further in a future article in this series. A vicious circle of C1q In summary, the most plausible current model for the perpetuation of SLE involves deficient clearance of Role of apoptosis in SLE 1181 F. 3. The hapten-carrier mechanism responsible for selecting high-affinity IgG antibodies to non-protein autoantigens in SLE. Nucleosomes derived from apoptotic cells are pinocytosed by centrocytes, which bind to the DNA wound around the outside. The protein core is processed and presented to the T cell, which delivers a rescue signal through CD40. apoptotic cells under conditions of stress, at least partly due to a functional deficiency of C1q. This produces large amounts of antigen to which the immune system is not tolerant. Periodic episodes of infection or UV exposure produce high levels of apoptotic cells which will fragment and drive the disease process by the formation of immune complexes, thus causing localized microvascular inflammation and consumption of C1q, rendering the protein even more deficient and less able to clear apoptotic cells. In individuals with homozygous C1q deficiency, provision of recombinant C1q may have therapeutic benefit, but in SLE patients with antibodymediated functional deficit, provision of C1q in this way may well exacerbate the disease by providing antigen to drive the specific immune response. The model we describe here is useful for explaining the persistence of SLE and also the strong association between sunlight/infection and disease flares, but it does not really address the causes of the disease. Functional C1q deficiency is unlikely to be a primary factor in causing SLE. It is far more likely to be a secondary development of the disease process that leads to persistence, analogous to stromal proliferation in rheumatoid synovitis [52]. Intriguingly, in severe infections such as infectious mononucleosis, where the reticuloendothelial system is overloaded by a vast excess of apoptotic immune cells [11], autoantibodies are very commonly found, but they resolve when the infection is eliminated. In contrast, an autoimmune disease process such as SLE that actively inhibits the clearance of apoptotic cells is likely to be very difficult to resolve. 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