Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RL, UK
The recent surge of interest in apoptosis is not without reason, least of all for those involved in the care of patients with systemic lupus erythematosus (SLE). Although it has long been suspected that a problem in the waste-disposal mechanism of the body may account for phenomena such as the LE cell and haematoxylin bodies associated with SLE, insights into the tightly controlled process of apoptosis have provided a more unified understanding of how the protean manifestations of lupus may occur. Animal models of lupus have been linked directly to genetic abnormalities in apoptosis genes, apoptotic cells provide the fuel for unwanted and potentially pathogenic autoimmune responses, and defects in specific clearance of apoptotic debris suggest a rationale for current and future approaches to treatment.
Apoptosis is a genetically controlled sequence of events that culminates in the death and efficient disposal of a cell. Characteristic features include condensation of chromatin, DNA fragmentation, membrane blebbing and externalization of phosphatidylserine. Both extrinsic (e.g. receptor activation via Fas ligation) and intrinsic (e.g. DNA damage) events may commit a cell to a death pathway [1]. An intricate series of receptor mechanisms on reticuloendothelial cells are important for sequestering material released from apoptotic cells so as to prevent an inflammatory response [2]. A number of the key molecules regulating checkpoints in the apoptotic process have been implicated in lupus and are the subject of ongoing study.
The first piece of support for SLE as a primary disorder of dysregulated apoptosis comes from genetic studies of relevant murine models, some of which have been very instructive. For instance, the MRL lpr and MRL gld mouse models of lupus are caused by single point mutations in either Fas (CD95) or the Fas ligand respectively, leading to massive lymphoproliferation and autoimmune nephritis [3]. On the other hand, mice that have had the DNAase 1 gene deleted show classical symptoms of lupus, including the presence of antinuclear antibodies and immune complex-positive nephritis [4]. DNase I is a major nuclease responsible for the removal of DNA from nuclear antigens at sites of high cell turnover. Similar phenotypes arise in mice deficient in either C1q [5] or serum amyloid P [6], molecules thought to be involved in clearance of apoptotic material. Therefore, there is persuasive evidence that impairment of the waste-disposal mechanism is a major predisposition to lupus, at least in mice.
So do these genetic defects in animal models have any counterparts in human lupus? Intriguingly, they do, although very uncommonlyonly one patient with SLE who has a point mutation in FasL has been described [7]. With respect to mutations of Fas, the association is with a rather uncommon familial condition that has come to be termed autoimmune lymphoproliferative syndrome (ALPS) [8]. Sixty or so index cases of ALPS have been reported, the main clinical features being non-malignant lymphoma, splenomegaly, lymphadenopathy, pancytopenia and skin rashes, although other complications have been described, such as nephritis, GuillainBarré syndrome, autoimmune hepatitis, uveitis and vasculitis. The most common mutation reported in patients with ALPS is in the death domain of Fas. A mutation in the death domain is associated with a higher penetrance of ALPS within families. In families, the clinical features can be highly variable and occasionally include lymphoma and other haematological malignancies. However, generally speaking, defects in Fas itself have not been found in human SLE. Rather, the reverse seems to apply, in that serum levels of soluble Fas, when studied, are increased in SLE [9], probably reflecting primed T-cell responsiveness. It would also seem that, rather than being reduced, the rate of apoptosis is increased in SLE lymphocytes when cultured [10].
So if not Fas or FasL, what other apoptotic genes may be associated with lupus? The most striking observation is the remarkably high incidence of SLE in individuals with deficiency in early components of complement, which reaches more than 90% in individuals with C1q deficiency [11]. The structurally related mannose-binding protein, which is thought also to regulate immune complex clearance, has functional polymorphisms associated with lupus [12]. Furthermore, polymorphisms affecting the affinity of type 2 and 3 Fc receptors (Fc
RII and Fc
RIII) on macrophages have been the focus of many recent investigations. For instance, a recent study found an association between the low-affinity Fc
RIIIA-158F allele and the risk of nephritis among Caucasians, but not among non-Caucasians [13]. Cytokines that influence Fas levels and lymphocyte activation, such as interleukin (IL)-10, have functional polymorphisms in promoter regions that may be associated with lupus [14]. And large linkage studies using genome scans have implicated another effector molecule for apoptosis: poly-ADP-ribose polymerase, located on chromosome 1 [15]. Therefore, there is an increasing amount of evidence to suggest that genes regulating apoptosis, especially those concerned in clearing apoptotic debris, contribute to the genetic causation of lupus.
Aside from genetic factors, it is highly likely that environmental triggers exert their influence via apoptosis. Thus, ultraviolet light, a potent inducer of apoptosis, is an important provoking factor in cutaneous lupus. It is hardly coincidental that a common site to find apoptotic cells is at the dermo-epidermal junction, the primary lesional site in cutaneous lupus. Lupus-inducing drugs, such as sulphasalazine [16] and chlorpromazine [17], have been shown capable of inducing apoptosis, as have other environmental toxins, such as silica dust [18]. These and other extrinsic factors may contribute to the increased amounts of apoptotic material found specifically in lupus, such as circulating nucleosomes [19].
As well as circulating nucleosomes, apoptosis provides a mechanism for the release of other intracellular antigens that are often the target for autoimmune responses in lupus. Very elegant work from Rosen's group convincingly demonstrated that keratinocytes, when irradiated with UV light, develop small surface blebs containing lupus autoantigens such as the 52-kDa Ro particle [20]. Together with later-forming apoptotic bodies, these small surface blebs, which are induced early on in apoptosis, contain the major autoantigens recognized by lupus sera. Also, the entire membrane of the apoptotic cell provides a surface to which phospholipid-binding proteins such as ß2 GP-1 and C1q can attach. Therefore, apoptosis provides the mechanism for how intracellular proteins become accessible targets for autoantibodies.
Cytotoxic T cells have an important apoptosis-inducing role, including that in relation to killing cells infected with virus. Several pathways are used by cytotoxic T cells, including Fas-ligation and granule exocytosis. One of the effector molecules of cell-mediated pathways is granzyme B, a serine protease released by cytotoxic T cells and NK cells. Granzyme B promotes apoptosis via caspase-dependent and -independent pathways. Therefore, it is of considerable interest that an exclusive property of autoantigens is the capacity to be cleaved by granzyme in a manner that releases unique autoantigen fragments [21]. Rosen's group hypothesize that these autoantigen fragments may behave as non-tolerized neoantigens that, in circumstances favouring an inflammatory reaction, propagate autoimmune disease.
An important determinant of whether an immune response proceeds is the type and relative maturity of the antigen-presenting cell that a T lymphocyte comes in contact with [22]. Normally, macrophages provide appropriate down-regulatory cytokine signals to nearby cells on antigen ingestion, such as those given by transforming growth factor (TGF) ß and IL-10. However, under certain circumstances, such as in a proinflammatory environment driven by cytokines such as tumour necrosis factor and IL-1, mature dendritic cells gain the necessary signalling machinery to drive an immune response (Fig. 1
). Autoantibodies may alter this natural homeostasis and turn macrophages from pacifiers to activators. For instance, it has been shown that antiphospholipid antibodies are able to opsonize apoptotic cells, which promotes their uptake by macrophages; this mechanism (rather than sending down-regulatory signals) enables antigens to be presented in a proinflammatory manner to T cells [23, 24]. Such evidence suggests that autoantibodies may play an active role in accelerating autoimmune disease, and provides further rationale for modes of treatment centred on autoantibody depletion.
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