C4b-binding Protein and Factor H Compensate for the Loss of Membrane-bound Complement Inhibitors to Protect Apoptotic Cells against Excessive Complement Attack

Abstract

Apoptotic cells have been reported to down-regulate membrane-bound complement regulatory proteins (m-C-Reg) and to activate complement. Nonetheless, most apoptotic cells do not undergo complement-mediated lysis. Therefore, we hypothesized that fluid phase complement inhibitors would bind to apoptotic cells and compensate functionally for the loss of m-C-Reg. We observed that m-C-Reg are down-regulated rapidly upon apoptosis but that complement activation follows only after a gap of several hours. Coinciding with, but independent from, complement activation, fluid phase complement inhibitors C4b-binding protein (C4BP) and factor H (fH) bind to the cells. C4BP and fH do not entirely prevent complement activation but strongly limit C3 and C9 deposition. Late apoptotic cells, present in blood of healthy controls and systemic lupus erythematosus patients, are also positive for C4BP and fH. Upon culture, the percentage of late apoptotic cells increases, paralleled by increased C4BP binding. C4BP binds to dead cells mainly via phosphatidylserine, whereas fH binds via multiple interactions with CRP playing no major role for binding of C4BP or fH. In conclusion, during late apoptosis, cells acquire fluid phase complement inhibitors that compensate for the downregulation of m-C-Reg and protect against excessive complement activation and lysis.Cell death via apoptosis is an essential process for development, maintenance of tissue integrity, and resolution of inflammation (1). Several active processes ensure that the cell dies in a way that results in fast removal, to prevent release of its potential pro-inflammatory content as is the case in primary or secondary necrosis (2-4).Although billions of cells die via apoptosis every day, only a few apoptotic cells are present at any given point in time under healthy conditions (5) as the result of a very efficient clearance process. Only when clearance mechanisms are defective or overwhelmed by excessive apoptosis do apoptotic cells accumulate, which is thought to underlie autoimmune diseases such as systemic lupus erythematosus (SLE) 3 (6). Apoptotic cells express several ligands that enhance phagocytosis such as phosphatidylserine and calreticulin, and they down-regulate molecules that may work as "do not eat me" signals (7). In addition, apoptotic cells have been reported to bind complement-initiating molecules such as mannose-binding lectin and C1q that enhance uptake by phagocytes (8 -10). Some discrepancies exist in reports of the stage of cell death necessary for binding of such recognition molecules, which can partly be explained by differences in semantics, experimental design, and readout (9,(11)(12)(13)(14).The complement system is an integral part of the innate immune defense and is also involved in the instruction of adaptive immunity and clearance of waste such as dead cells and immune complexes (15,16). This potent enzyme cascade system has both membrane-bound and fluid phase inhibitors that protect host surfaces and prevent systemic depletion of complement activi...

Factor H uptake regulates intracellular C3 activation during apoptosis and decreases the inflammatory potential of nucleosomes

“…We previously demonstrated that late apoptotic cells bind FH with the strongest intensity whereas the binding to live cells is negligible. 1,5 As anticipated of an active process, early (annexin A5 positive) but not late (annexin A5 and ViaProbe positive) apoptotic cells internalized FH (Figure 1d). …”

“…Apoptotic cells bind FH while triggering complement activation by binding of C1 complex, which ensures efficient opsonization and removal of apoptotic debris but prevents excessive complement activation and inflammation. 1,2 Dysregulation of complement contributes significantly to the pathology of many diseases. 3 Recently, novel roles and intracellular location for complement have been identified suggesting that its functions exceed our current understanding.…”

Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells

“…, and FH have all been proven to bind to apoptotic cells (3,14,15). To clarify whether they influence one another's binding, competition assays were performed.…”

The C-type lectin of the aggrecan G3 domain activates complement

“…FH is able to affect later stages of the classical pathway as well, since the alternative pathway, which it controls, acts as amplification loop to the classical pathway. The simultaneous interactions of ligands with C1q and complement inhibitors FH and C4BP are frequently observed for endogenous ligands, for example fibromodulin [6], osteoadherin [6], C-reactive protein [40], amyloid [41], prions [42], DNA [40] and dying cells [43]. This contrasts to the interaction between C1q and immune complexes, in which case no inhibitors bind, resulting in full complement activation.…”