Background/Purpose The complement system has been viewed as a predominantly serum-derived host defense mechanism with multiple functions, including clearance of apoptotic cells. Defective function of the complement pathways have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Recently, intracellular complement C3 storage was demonstrated in many different cell types, and C3 activation products were shown to participate in the survival and effector cell differentiation of murine and human lymphoid cells. Despite the known role of serum-derived C3 activation products in the removal and immunosuppressive properties of dying cells, the role of intracellular C3 in clearance of apoptotic cells has never been explored. Thus, we asked whether human cells expose C3 and/or C3 activation products on their surface upon cell death induction, and whether such exposure functionally participates in their phagocytic clearance, independently of serum complement factors

Methods Apoptosis and secondary necrosis of human lymphoid cells was induced by both UV irradiation and serum starvation. C3/C3b/C3bi surface and intracellular expression on live and apoptotic human T and B primary cells as well as cell lines was monitored by flow cytometry (FACS), western blot and confocal microscopy. Macrophages were derived from circulating CD14+ monocytes by culture with M-CSF. Phagocytosis of apoptotic cells in presence or absence of serum was quantified by microscopy (phagocytic index) and by FACS with the aid of fluorescently labeled apoptotic cells

Results We observed that live human primary T and B cells, and human T and B cell lines expressed intracellular but not cell surface C3/C3bi. However, upon apoptosis induction, C3 activation products were exposed on the surface of dying cells in a time dependent manner. Detection of C3/C3bi correlated with later stages of apoptosis characterized by cell shrinkage and loss of membrane integrity (Annexin V+ PI+ cells). Confocal microscopy of unfixed cells revealed detection of C3/C3bi in a granular distribution, possibly in blebs and/or other endosomal compartments. To determine whether surface exposed C3/C3bi had functional relevance, we blocked the macrophage C3bi receptors CR3 and CR4 with antibodies, and compared phagocytosis of apoptotic cells in the absence of serum. Strikingly, functional blockade of CR3 and CR4 on human macrophages in the absence of serum specifically reduced the uptake of C3bi+ late apoptotic cells, and not that of latex beads (with an inhibition of 19.3% for CR3, 24.1% for CR4, and 48% for CR3+CR4 blockade; p= 0.182, 0.066; and 0.043, respectively)

Conclusion Our results suggest that we have uncovered a novel function of intracellular complement C3 activation products in the removal of dying cells. C3 is a very large protein of 185,000 molecular mass and penetration into tissues is likely to be limited. Cell intrinsic, serum-independent, C3-mediated clearance of apoptotic cells may therefore be of particular relevance in tissues where there is accumulation of dead cells as observed in patients with SLE, as well as under other conditions such as hypoxia reperfusion injury and stroke

ACR Meeting Abstracts - https://acrabstracts.org/abstract/intracellular-complement-c3-is-exposed-on-the-cell-surface-upon-apoptosis-induction-and-participates-in-the-clearance-of-apoptotic-cells-by-phagocytes/