Defective Clearance of Apoptotic Cells and Autoimmunity

The role of impaired phagocytosis of apoptotic cells in the development of autoantibodies in systemic autoimmunity has attracted considerable attention in recent years. Under normal circumstances, apoptotic bodies are recognized and engulfed by phagocytic cells. Professional phagocytic cells such as macrophages and DCs clear apoptotic cells swiftly, whereas nonprofessional phagocytes appear to take up apoptotic cells when they reach the later stages of the dying process [163]. This clearance process is facilitated by the presence of "eat me" signals exposed in apoptotic cells (e.g., phosphatidylserine), apoptotic cell recognition receptors in phagocytic cells (e.g., phosphatidylserine receptor, /52-glycopro-tein 1 receptor, vitronectin receptor, complement receptors, and tyrosine kinase Mer receptor), and serum proteins (e.g., complement cascade components such as C1q, C-reactive protein (CRP), and serum amyloid protein) [164]. It is widely accepted that the efficient clearance of apoptotic cells controls inflammatory responses by preventing the release of danger signals from dying cells and by suppressing proinflammatory cytokines, such as TNF, that may be expressed by macrophages [27, 28, 30, 49, 164]. This suppression is mediated by the production of anti-inflammatory mediators such as TGF-/5, prostaglandin E, and IL-10 [164].

The efficient clearance of apoptotic cells is crucial for the avoidance of autoimmune responses to intracellular antigens [27, 28, 30, 34, 49, 164, 165]. This clearance results in the exposure of intracellular self-antigens to the immune system under non-inflammatory conditions, leading to tolerization of these antigens, regardless of whether or not they are modified by caspases. It has been proposed that under these conditions circulating DC precursors take up apopto-tic cells that they encounter in the various tissues and travel to lymphoid organs, where they present self-antigens from apoptotic cells to T cells in the absence of costimulatory molecules [137, 138, 166]. However, under certain circumstances these intracellular self-antigens could be processed and presented to the immune system under proinflammatory conditions, potentially leading to a pathogenic autoimmune response. These circumstances may include increased secondary necrosis due to inefficient clearance of apoptotic cells, enhanced apoptosis rates, infections causing cell death (both apoptotic and necrotic), or the presence of proinflammatory molecules in the environment in which cell death and clearance occurs [20, 21, 27-30, 163, 165].

Apoptotic cells that are not removed efficiently by phagocytosis ultimately lose their cytoplasmic membrane integrity and undergo secondary necrosis (also referred to as late apoptotic stage, post-apoptotic necrosis, or post-apoptotic cell lysis) [34, 165]. Secondary necrosis occurs as a consequence of the disruption of mitochondrial function, ATP depletion, and the activation of lysosomal enzymes. It is becoming evident that deficiencies in proteins involved in the phagocytic clearance of dying cells and immune complexes, including C1q, C-reac-tive protein, serum amyloid P (SAP), and the Mer tyrosine kinase, may lead to impairment of phagocytic function, with resulting excessive accumulation of cells in different stages of the cell death continuum, particularly in secondary necrosis [167-172].

The accumulation of cells in secondary necrosis would facilitate the release of proinflammatory signals that induce DC maturation and presentation of modified self-antigens from the dying cells. Evidence for this comes from studies by Manfredi and colleagues [173, 174], who demonstrated in vitro that excessive apoptosis or delayed apoptosis leading to secondary necrosis, mimicking a failure of their in vivo clearance, was sufficient to trigger DC maturation and presentation of intracellular antigens. This could skew the outcome of cross-presentation of intracellular antigens to autoimmunity if intracellular antigens, proteins, and nucleic acids are presented to autoreactive lymphocytes under the appropriate cytokine environment. As mentioned previously, we have reported that the transition from apoptosis to secondary necrosis is associated with proteolysis of specific autoantigens [34]. In these studies, various cell lines were exposed for up to 60 hours to specific apoptosis inducers. Under these conditions, cells underwent a rapid apoptosis that gradually progressed to secondary necrosis. This progression coincided with the loss of cytoplasmic membrane integrity, as assessed by trypan blue exclusion, and irregular cellular fragmentation characteristic of late necrotic cell death. Immunoblotting analysis indicated that the progression to secondary necrosis was associated with a second wave of proteolysis of specific intracellular autoantigens that are cleaved during apoptosis, including LEDGF/p75, PARP, SSB/La, topo I, and U1-70 kDa. Interestingly, although some of the cleavage fragments produced during secondary necrosis were also detected in primary necrosis, identical cleavage patterns were not observed in these pathways for all the autoantigens tested. This could be attributed to differential compartmentalization of the proteases mediating these cleavages during upstream events leading to primary and secondary necrosis.

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