Apoptosis

Chapter 14 Apoptosis





KINETICS OF AUTOANTIBODY APPEARANCE IN SLE HIGHLIGHT TWO-STEP PATHOGENIC MODEL


A centrally important element in the pathogenesis of SLE has become apparent through kinetic studies on individuals during the evolution of autoantibodies and clinical SLE.1,2 The appearance of antiphospholipid and antinuclear antibodies can be detected several years prior to development of SLE, clearly separating initiation of autoimmunity from the propagation phase that generates the clinical phenotype. Coincident with or slightly prior to development of clinical SLE, the antibody profile of individuals broadens to include antibodies to ribonucleoproteins and components of the spliceosome. In contrast, additional autoantibody specificities tend not to appear after development of disease. The asymptomatic autoantibody-positive state can thus be viewed as representing the precursor of the amplified disease phenotype, with targeting of ribonucleoproteins playing a potentially important and direct role in disease amplification and clinical manifestations in the second phase. Importantly, while initiation can be viewed as the generation of the immune response by antigen, the amplifying character of the propagation phase is better viewed as the generation of antigen by the immune response that can further drive the process. It is becoming increasingly evident that once self-antigen becomes the target of an active immune response, lymphoid, inflammatory, and target tissues may contribute to the self-sustaining loop underlying disease propagation through a cycle of proliferation and apoptosis.


It is within this kinetic construct of SLE that the roles of abnormalities in apoptotic pathways will be explored. In particular, impaired tolerance induction by apoptotic cells, dysfunctional clearance of apoptotic material, and novel pro-immune forms of apoptotic death will be examined in terms of possible roles in initiation of the autoimmune response. The role of changes in the availability or context of self-antigen, ligation of Toll-like receptors by self-nucleic acid, and opsonization of apoptotic cells by autoantibodies will be emphasized as possible drivers of the amplification phase.



SLE AUTOANTIGENS CLUSTER INTO CELL SURFACE BLEBS DURING APOPTOSIS


Apoptosis is a form of programmed cell death in which a highly specific and orderly set of biochemical changes underlie the unique morphologic changes and the ultimate disposition of the dying cell and its contents. Apoptotic cells undergo a striking, orderly fragmentation and disassembly, and are strong inducers of immune tolerance (see below). Several specific biochemical pathways form the apoptotic framework. In addition to a specialized signaling apparatus (which transduces proapoptotic signals from a variety of subcellular domains), apoptosis is mediated through the activation of a proteolytic caspase cascade, in which a restricted subset of cellular targets are cleaved after aspartic acid residues. Substrates include proteins whose fragments function directly in generating the apoptotic phenotype, as well as a variety of molecules in which cleavage abolishes critical, antiapoptotic functions.3


Reorganization of multiple cellular components occurs during apoptosis, with clustering of a variety of otherwise intracellular molecules in blebs at the surface of the apoptotic cell.4 Prior to nuclear fragmentation, DNA, RNA, ribosomes, and ER components such as Ro can be found clustered at the surface of apoptotic blebs.46 Redistribution of histones to the surface of apoptotic blebs and release from the nucleus early in apoptosis has also been documented.5,6 More than a decade ago, it was noted that many of these redistributed intracellular components are autoantigens in lupus and other systemic autoimmune diseases, suggesting that apoptotic blebs might be an important source of autoantigens for both tolerance induction and systemic autoimmunity.



APOPTOSIS INDUCES TOLERANCE


Significant data demonstrate that when apoptosis results in the removal of dead cells, it is associated with induction of immune tolerance to the contained antigens.712 Although the mechanisms underlying tolerance induction are numerous and are not fully elucidated, signals at the apoptotic cell surface (e.g. phosphatidyl serine) and cytokine secretion patterns in phagocytic cells (increased secretion of IL-10 and TGF-α and decreased secretion of IL-12 and TNF-β) are clearly important in this regard.1317 This pattern of cytokines is associated with induction of peripheral tolerance to antigens contained in apoptotic cells. Unlike engulfment of apoptotic cells, necrotic cells trigger phagocytic maturation, proinflammatory cytokine secretion, and up-regulation of co-stimulatory molecules.18,19


Apoptosis is vital in providing self-antigen to induce the central and peripheral tolerance of naive lymphocytes. During development and maturation, T cells20 and B cells,21 which have high affinity for self-antigens, are clonally deleted. The source of this antigen largely comes from the phagocytosed apoptotic bodies of dying lymphocytes present in the thymus,22 bone marrow,23,24 and lymph nodes.10,25,26 Recent data have shown that nucleosome-specific T cells undergo selection in the thymus, as evident by clonal deletion of autoreactive T cells in normal mice. In lupus-prone SNF1 mice, however, a defect exists in the ability of thymic dendritic cells to successfully present an array of endogenous nucleosome antigens to developing thymocytes, allowing the survival of potentially autoreactive T cells.27 Dendritic cells are also important in maintaining peripheral T-cell tolerance to apoptotic antigen by presenting phagocytosed apoptotic material on MHC class-II molecules and migrating to the lymph node. Defects in B-cell tolerance checkpoints in the bone marrow and periphery have been observed in human and mouse SLE, which allow for the production of autoantibodies.2833 It has been proposed that defects in the tolerance-inducing clearance of apoptotic material, with the resulting incomplete deletion of autoreactive lymphocytes, might result in susceptibility to initiation of autoimmunity to autoantigens normally presented from apoptotic cells.34,35


It is important to note that, under some circumstances, apoptotic death may not follow the typical biochemical and morphologic patterns generally observed, which may provide an opportunity for novel processing and presentation of self-antigens. For example, the organization of nuclear antigens during apoptosis can be altered under intense death stimuli such as exposure to high doses of UVB. While low doses of UVB cause translocation of nuclear antigens to the plasma membrane in keratinocytes, high-dose UVB causes release of antigen from the cell. This UV-induced release of nuclear autoantigen has been postulated to contribute to initiation of disease observed in some lupus patients after prolonged sun exposure.36



IMPAIRED CLEARANCE OF APOPTOTIC CELLS RENDERS INDIVIDUALS SUSCEPTIBLE TO SYSTEMIC AUTOIMMUNITY


The first indication that defects in the clearance and anti-inflammatory effects of apoptotic cells rendered individuals susceptible to systemic autoimmunity came from studies on C1q deficiency. C1q-deficient humans or mice have a striking propensity to develop systemic autoimmunity with lupus-like features.3741 When Botto and colleagues41 examined the phenotype that developed in C1q null mice, they observed a striking accumulation of apoptotic bodies in the kidneys of affected animals. They subsequently noted that clearance of apoptotic cells from the peritoneal cavity was diminished in C1q-deficient animals, and could be corrected by C1q administration.40,41 Interestingly, C1q-deficient animals have a high prevalence of autoantibodies, but frequently have milder autoimmune phenotypes.41 In contrast, deficiencies in several other receptor—ligand pairs that are also associated with impaired clearance of apoptotic cells, and in which animals develop features of SLE, tend to be strongly associated with prominent autoimmune phenotypes. For example, mice deficient for the receptor tyrosine kinase Mer or milk fat globule epidermal growth factor 8 (MFG-E8) exhibit dramatically impaired phagocytosis of apoptotic cells and spontaneously develop autoantibodies. Mer tyrosine kinase appears to be important for the intracellular signaling necessary to trigger the uptake of apoptotic cells, but not bacteria or opsonized particles, by phagocytes.42,43 Mer deficiency causes a lupus-like phenotype in mice with development of rheumatoid factor, antichromatin, and anti-dsDNA antibodies.42 The glycoprotein MFG-E8 is secreted by tingible body macrophages in the spleen and lymph node, and mediates clearance of the apoptotic debris.44 In germinal centers, tingible body macrophages play an important role in removing low-affinity B cells that have undergone apoptosis.45 Mice lacking MFG-E8 show accumulation of apoptotic material around germinal center macrophages and develop anti-dsDNA and antinuclear antibodies with age, leading to immune complex deposition and proteinuria.44,46 Interestingly, similar features have been observed in the lymph nodes of a small group of human SLE patients in which reduced numbers of germinal center macrophages, increased apoptotic cells, and apoptotic cell accumulation around follicular dendritic cells was observed.47 The mechanisms underlying reduced clearance of apoptotic cells in these patients remain unclear. While it is possible that deficiency in tingible body macrophage function may be genetic in some patients, it is also possible that the defect is acquired, potentially through antibodies that block relevant receptor—ligand interactions. This latter category of defect, where abnormalities in apoptotic cell clearance affect immune cells during processes of selection in an ongoing immune response, may be particularly relevant to disease amplification. Defining such defects and pathways in human SLE remains a high priority.



DISEASE INITIATION: RECRUITMENT OF FEED-FORWARD LOOP INVOLVING TOLL-LIKE RECEPTORS


Although the mechanisms underlying the synchronized change in autoantigen targets and the onset of clinical symptoms in SLE remain unknown, this change in specificity strongly suggests recruitment of a feed-forward amplification loop focused on the splicing ribonucleoproteins. Several recent discoveries are particularly tantalizing in this regard. For example, there is now striking data demonstrating that Toll-like receptors (TLRs) recognize self-nucleic acid in the context of splicing ribonucleoproteins and nucleosomes, and induce proinflammatory signals in dendritic cells (DCs) and B cells.4855 TLRs recognize invariant, repeating, pathogen-associated patterns, and trigger the innate immune response to microbial infections. Most TLRs, including TLR 7, 8, and 9, signal through the adapter molecule MyD88 to trigger proinflammatory cytokine secretion, up-regulation of co-stimulatory molecules, and increased antigen presentation. TLR 9 is known to bind hypomethylated CpG DNA found in bacteria and mammalian DNA promoter regions while TLRs 7 and 8 bind ssRNA (reviewed in Ishii et al.56). Recent data demonstrate that splicing RNAs are highly effective activators of TLR7 and possibly TLR8.55 The emerging consensus from these studies is that a group of frequently targeted autoantigens in SLE have the unusual capacity to co-ligate antigen receptors and TLRs, and that this may be important pathogenetically in the feed-forward loop of SLE.57 It is important to note that eukaryotic nucleic acids are poor ligands for the TLRs, suggesting that additional modifications of nucleic acids may be relevant for recruiting the amplifying properties of these autoantigens51 (see below).




MODIFIED PROTEIN STRUCTURE DURING CELL DEATH


Many cellular proteins are altered during apoptosis by proteolytic cleavage and other posttranslational modifications including phosphorylation,62,63 transglutamination,64 oxidation,65,66 citrullination,67,68 and ubiquitination.69 Antigen processing by cellular proteases is highly dependent on protein structure, sequence, and post-translational modifications. While lymphocytes are exposed to the surface and contents of apoptotic cells in the bone marrow and thymus, it is likely that they only encounter forms of antigens that represent the “core” apoptotic process. Novel antigenic modifications not normally presented to lymphocytes during development and selection might be generated under some nonhomeostatic circumstances, exposing cryptic epitopes (reviewed in Hall et al.70).


Although each of the modifications noted above may be relevant in terms of providing novel forms of antigens not previously seen and tolerized by the immune system, much attention has been paid to the apoptosis-specific proteolytic modifications of autoantigens. Two observations are most pertinent in this regard: (1) many autoantigens are unified by their susceptibility to cleavage by the caspases, which are themselves responsible for generating the apoptotic phenotype; and (2) the relevance of caspase cleavage to immunogenicity still remains unclear. It has been proposed that caspase-cleaved autoantigens are the predominant tolerance-inducing forms of these molecules, and that abnormalities in the execution of apoptotic death (e.g., antiapoptotic Bcl-2 family member expression) may allow uncleaved forms of the molecules to be presented with selection of different epitopes.66,7173 The effects of caspase cleavage on processing and presentation of endogenous autoantigens remain to be directly determined.


There are also data demonstrating novel proteolytic cleavage events affecting a large number of autoantigens during non—caspase-mediated cell death processes, including cytotoxic lymphocyte granule—mediated death71 or necrotic death.74,75 Of particular relevance in this regard is granzyme-induced death of target cells, which is not usually homeostatic or developmental in nature, and is generally focused on virus-infected or transformed cells. Autoantigens in systemic autoimmunity are frequently cleaved by granzyme B (GrB), generating unique fragments.71 GrB is a serine protease found in the cytolytic granules of CD8+ cytotoxic T lymphocytes (CTLs) and NK cells. These cells primarily kill virally infected or transformed cells by triggering apoptosis through cleavage of intracellular substrates, including caspases, at aspartate residues. Although both GrB and caspases cleave at aspartate residues, they prefer different consensus sequences and can generate different peptides from the same substrate.76,77 Furthermore, granzyme-induced apoptosis does not occur in the thymus and bone marrow, and thus peptides generated by granzyme cleavage are not likely presented to developing lymphocytes.7881 Lastly, the granule pathway is able to kill target cells through caspase-independent pathways,82,83 thus increasing the chances of generating non—caspase-cleaved, novel autoantigen fragments.


Taken together, the available data suggest that autoantigens are preferentially modified during specific forms of nonhomeostatic death, in which caspase- independent structural modifications predominate. Such uniquely modified molecules may form the substrate for systemic autoimmunity.

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Jul 12, 2016 | Posted by in RHEUMATOLOGY | Comments Off on Apoptosis

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