Phagocyte Laboratory, Centre for Inflammation Research, University of Edinburgh, UK
Keywords: apoptosis; autoimmunity; inflammation; lupus; phagocytes
Introduction
During normal tissue homeostasis, the rate of cell death is perfectly balanced by the rate of production of new cells, resulting in a constant cell number. Apoptosis is a critically important mechanism that facilitates deletion of unwanted or damaged cells in various circumstances, including embryogenesis, inflammation and tissue healing. The purpose of this article is to outline a darker side of apoptosis since defects in the apoptotic cell death programme and subsequent clearance of cellular corpses are implicated in the pathogenesis of clinically important autoimmune diseases such as systemic lupus erythematosus (SLE).
What is apoptosis?
Apoptosis is characterized by stereotypical morphological and biochemical changes including the activation of specific intracellular proteolytic enzymes (caspases) that cleave myriad nuclear and cytoplasmic substrates [1,2]. Apoptosis may result from an insufficient supply of survival signals or may be actively induced by various injurious stimuli such as hypoxia, reactive oxygen species, complement attack, nitric oxide, cytokines such as tumour necrosis factor- or ligation of the Fas (CD95) cell surface death receptor. Apoptosis elicits specific cell surface changes, such as the exposure of phosphatidylserine, normally found on the intracellular aspect of the cell membrane, resulting in the swift uptake and degradation of apoptotic cells either by local resident cells or infiltrating phagocytes. This process is very rapid such that apoptotic cells are conspicuously absent in normal tissues. Furthermore, cell deletion by apoptosis leading to clearance by professional phagocytes such as macrophages is not associated with proinflammatory mediator release but rather causes release of anti-inflammatory agents such as transforming growth factor beta 1 (TGFß1) [3,5]. The mechanisms whereby macrophages and semi-professional phagocytes (including mesangial cells) recognise and ingest apoptotic cells are complicated and may involve numerous molecules including the vitronectin receptor (
vß3 integrin), CD36, thrombospondin, the phosphatidylserine receptor, the first component of complement C1q, ß2 thrombomodulin, class A scavenger receptors, etc. [4] (Table 1
).
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The role of apoptosis in the maintenance of self tolerance and the regulation of T cell populations
Self tolerance requires the removal or deactivation of autoreactive T cells with specificity to both central (i.e. T cells reacting to self antigen present in the thymus) and peripheral antigens. Within the developing thymus, engagement of autoreactive T cells by self antigen induces apoptosis and deletion of these potentially injurious T cells. Consequently, defects in this process will facilitate the persistence of T cells capable of recognising self and inducing autoimmunity.
However, the entire repertoire of self antigens is not represented within the thymus and therefore additional mechanisms are needed to maintain adequate peripheral self tolerance. Although this process is incompletely understood apoptosis may also be involved. It has been recognised for some time that certain organs such as the eye and the testes exhibit immunological privilege. It is now apparent that this is at least partly due to the expression of Fas ligand by resident tissue cells at these sites. This tissue self defense mechanism results in the induction of apoptosis in infiltrating Fas-bearing lymphocytes following Fas ligation.
Apoptosis also plays a role in the regulation of T cell populations. Clonally expanded populations of activated T cells that have served their functional purpose are rapidly deleted by activation induced cell death (AICD), a process dependent upon ligation of the cell surface Fas death receptor. It is therefore very pertinent that mice with mutations in the genes encoding either Fas or Fas ligand develop autoimmune disease. Depending upon the genetic background, such mice exhibit lymphadenopathy, splenomegaly, autoantibody formation, joint inflammation and glomerulonephritis with features of SLE [6]. Interestingly, early treatment of gld/gld mice, deficient in functional Fas ligand, with an agonistic anti-Fas antibody protected mice from the subsequent development of autoimmune disease by a mechanism which presumably involves Fas-dependent deletion of autoreactive lymphocytes [7]. Furthermore, treatment of established autoimmune disease with the same agonistic anti-Fas antibody resulted in a significant improvement in disease pathology [7].
A small number of human patients have been described with mutations in the genes encoding either Fas or Fas ligand. These patients exhibit an autoimmune lymphoproliferative syndrome (ALPS or CanaleSmith syndrome) characterized by lymphadenopathy, splenomegaly and autoantibodies directed at blood components such as erythrocytes and platelets [8]. Affected individuals do not typically develop joint or renal disease, and therefore defects in the Fas death receptor or its ligand do not appear vital to the pathology in these organs but can contribute to autoimmune disease.
Apoptotic cells express potential autoantigens on their cell surface
The origin of the autoantibodies typically present in the sera of patients with autoimmune conditions such as SLE (often directed towards intracellular antigens such as DNA, ribonucleoproteins and nucleosomes) has been perplexing as these autoantigens are normally invisible to the immune system because of their localization within the cell. However, seminal work by Casciola-Rosen et al. [9] has indicated that keratinocytes undergoing apoptosis display potential autoantigens upon their cell surface where they are available for interaction with immunologically competent cells. These potential autoantigens on apoptotic cells are likely to have undergone proteolysis by caspases, resulting in the production of immunogenic altered self motifs. Indeed, cleavage of intracellular substrates by the enzyme granzyme B can result in unique modification of potential intracellular autoantigens and raises the possibility that cytotoxic lymphocyte-mediated death of target cells may play a specific role in the development of autoimmunity [10].
Defective clearance of apoptotic cells may induce autoimmunity
Although the surface of apoptotic cells may express potential autoantigens, it is obvious that the majority of the population do not develop autoimmune pathology and therefore apoptosis itself is insufficient to induce autoimmune disease. It is believed that, in normal circumstances, apoptotic cells are rapidly ingested and degraded by phagocytes. Indeed, in vivo studies of both renal development and glomerulonephritis indicate that the vast majority of apoptotic cells evident in tissue sections actually lie within other cells [11,12]. This suggests that autoimmunity may be more likely to arise if there is a defect in the clearance of apoptotic cells, and accumulating evidence suggests that this is indeed the case.
For example, experiments involving the injection of normal mice with irradiated syngeneic apoptotic thymocytes resulted in the transient development of antinuclear autoantibodies and anticardiolipin, and anti-ssDNA antibodies, albeit at relatively low levels [13]. Furthermore, these mice also exhibited mild glomerular immunoglobulin deposition. These data indicate that exposure to large numbers of apoptotic cells, which may exceed the phagocytic capacity of the reticuloendothelial system, is able to elicit an autoantibody response.
As indicated previously, multiple macrophage cell surface receptors and bridging molecules may be involved in the recognition and ingestion of apoptotic cells (Table 1). However, the involvement of C1q, the first component of the classical complement pathway, is of particular interest since C1q deficiency is strongly associated with the development of SLE [14]. It is therefore extremely important that mice targeted for the deletion of the C1q gene spontaneously develop both autoantibodies and glomerulonephritis [15]. Indeed, the glomerular inflammation in the C1q-deficient mice is characterized by an impressive accumulation of apoptotic cells within glomeruli, implying that defective clearance of apoptotic cells is an important aetiological factor in the development of disease [15]. In addition, as in human disease, the genetic background of the C1q-knockout mice plays an important role in modulating disease susceptibility and phenotype. A pathogenetic role of defective apoptotic cell clearance in the development of autoimmunity may also explain why exposure to UV light or intercurrent infections are associated with increased disease activity since they would be predicted to increase the burden of apoptotic keratinocytes or leukocytes requiring phagocytic clearance. Lastly, it is pertinent that monocyte-derived macrophages isolated from patients with SLE exhibit reduced ingestion of apoptotic cells in a quantifiable in vitro assay of phagocytosis, suggesting that a defect in apoptotic cell clearance may be relevant to the pathogenesis of SLE in C1q-replete human patients [16].
Biological systems have evolved to ensure that apoptotic cells do not normally undergo secondary necrosis, which would be predicted to increase the likelihood of autoimmune responses. For example, C reactive protein, a teleologically ancient acute phase reactant protein, can bind to the apoptotic cell surface. Bound CRP stimulates activation of the classical pathway of complement, resulting in opsonization of the cell with complement components that can augment apoptotic cell clearance [17], and also assists the maintenance of cell viability by inhibiting the activation of the cytolytic C5b-9 terminal membrane attack complex. In addition, the pentraxin PTX3 is an acute phase protein generated locally within inflamed tissues that acts to inhibit uptake of apoptotic cells by dendritic cells, which are capable of presenting apoptotic cell-derived antigen to T cells [18,19]. Finally, the acute phase protein serum amyloid P component (SAP) binds chromatin on apoptotic cell surfaces as well as binding, solubilizing and regulating the degradation of free chromatin that results from cell breakdown. The importance of this chromatin scavenging system is underscored by the phenotype of mice targeted for the deletion of the SAP gene, which exhibit antinuclear autoantibodies and glomerulonephritis [20].
Immunological presentation of antigens derived from apoptotic cells
Unlike macrophages, dendritic cells are unique in their ability to stimulate primary immune responses. Immature dendritic cells exist in the periphery and constantly sample their antigenic milieu. In order to become fully functional antigen-presenting cells they must mature and migrate to draining lymph nodes where they can interact with naïve T cells. Dendritic cell maturation is normally provoked by inflammatory stimuli such as TNF- or bacterial products. Recent data indicate that dendritic cells not only ingest apoptotic cells but are capable of presenting apoptotic cell-derived antigen to both CD4 and CD8 T cells if they receive appropriate activation signals [18,19]. Since apoptotic cells are a source of potential autoantigens it is conceivable that the phagocytic immature dendritic cell can induce a primary autoimmune response if it encounters apoptotic cells concurrently with appropriate danger signals. Indeed, it has been suggested that a large load of apoptotic cell material, perhaps due to secondary necrosis following failed clearance, may predispose to the development of autoimmune responses by increasing the potential for dendritic cells to phagocytose apoptotic cell material and subsequently present autoantigens [21]. However, this is a controversial area of research as conflicting data suggests that dendritic cells that have ingested apoptotic cells may play a role in the maintenance of peripheral tolerance [22].
Resolution of inflammation and scarring
Defective clearance of apoptotic cells in patients with SLE may actually represent a double whammy, i.e. it may promote the development of autoimmunity as well as blunt the macrophage deactivating effects of apoptotic cell ingestion at inflamed sites [5]. In theory, this failure to switch off activated proinflammatory macrophages may impair the resolution of inflammation in the kidney and other tissues, with the consequent promotion of scarring and loss of organ function.
Conclusion
Apoptosis and the clearance of apoptotic cells are essential for normal tissue homeostasis, embryogenesis and tissue remodelling. Apoptotic deletion of potentially autoreactive T cells is involved in the maintenance of self tolerance. Furthermore, apoptotic cells are an important and preferential source of many potential autoantigens. Defective apoptotic cell clearance may facilitate the inappropriate presentation of potential autoantigens by dendritic cells, thereby initiating autoimmune responses. These observations confirm an important role for defects in apoptosis and the clearance of apoptotic cells in the development of autoimmunity (Figure 1). Future research will shed further light upon the involvement of apoptosis in autoimmunity and hopefully highlight new targets for potential therapeutic intervention.
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Acknowledgments
L.S. is in receipt of a Wellcome Trust Clinical Training Fellowship (Grant No. 34842). J.H. is a Wellcome Trust Senior Research Fellow in Clinical Science (Grant No. 061139). We are grateful to Professor John Savill for helpful comments.
Notes
Correspondence and offprint requests to: Lynda Stuart, Phagocyte Laboratory, MRC Centre for Inflammation Research, University of Edinburgh Medical School, Teviot Place, Edinburgh, UK. Email: lynda.stuart{at}ed.ac.uk
References