Factors influencing the interaction of herpes simplex virus glycoprotein C with the third component of complement

Summary The factors influencing the interaction of herpes simplex virus (HSV) glycoprotein C (gC) with the third component of complement (C3) were investigated in this study. The ability of gC of HSV type 1 (gC-1) to bind to the C3b fragment of C3 was found to be influenced by cell specific processing of gC-1 in a different manner, binding being remarkably enhanced in some cell lines following removal of sialic acid residues. Testing several intertypic recombinants of HSV we found that only strains expressing gC-1 exhibited binding to C3b, even though their genome consisted mainly of HSV-2 sequences in some recombinants. Expression of type-2 glycoproteins gB, gD, gE, gG, gH, and gI did not alter the ability of gC-1 to bind to C3b. Rosetting of HSV-1 infected Vero cells with C3b-coated red blood cells (EAC) was found to be temperature dependent and could be inhibited with purified C3b and anti-C3 antibodies. Polyanions like heparin or dextran sulfate were also inhibitory in a dose dependent manner, whereas C3d, neomycin and other aminoglycoside antibiotics failed to block. As the tested polyanions are also known to inhibit the infectivity of HSV, it could be speculated, that the complement binding function and the heparin-binding/attachment function of gC might be related.     

Structural and functional relationships among receptors and regulators of the complement system

The classical and alternative pathway of complement activation are regulated by a series of fluid phase and cell-bound factors, some of which at the same time serve as receptors for fragments of C3 and C4. These molecules are factor H, CR1 (C3b/C4b receptor), CR2 (C3d/EBV receptor), C4BP (C4b binding protein), DAF (decay accelerating factor), MCP (membrane cofactor protein; earlier designated p45/70), CR3 (iC3b receptor or Mac-1) and CR4 (protein 150/95). Due to structural, genetic and functional features these factors are members of one or several newly recognized large families of proteins: (1) molecules with 60 amino acids long repeats (H, CR1, CR2, C4BP, DAF); (2) proteins with 1,2-diacylglycerol membrane anchoring (DAF); (3) proteins with a heterodimer structure and preference for ligands containing the tripeptide arginine-glycine-asparagine (CR3, CR4). Recognizing the above mentioned regulators and receptors of the complement system as belonging to these protein families opens new perspectives for further genetic and functional research of mutual interest to complement and noncomplement scientists.     

Kinetic analysis of glycoprotein C of herpes simplex virus types 1 and 2 binding to heparin,heparan sulfate,and complement component C3b

Glycoprotein C (gC) from herpes simplex virus (HSV) facilitates virus entry by attaching the virion to host cell-surface heparan sulfate (HS). Although gC from HSV-1 (gC1) and from HSV-2 (gC2) bind to heparin, gC2 is believed to play a less significant role than gC1 in attachment of virus to cells. This attachment step is followed by the binding of gD to one of several cellular receptors. gC also plays an important role in immune evasion by binding to the C3b fragment of the third component of the host complement system. Yet, although both gC1 and gC2 protect HSV against complement-mediated neutralization, only gC on HSV-1-infected cells acts as a receptor for C3b. We used optical biosensor technology to quantitate the affinities (K(D)) and the stabilities (k(off)) between both serotypes of gC with heparin, HS, and C3b to address three questions concerning gC interactions. First, can differences in affinity or stability account for differences between the contributions of HSV-1 and HSV-2 gC in attachment? Our data show that the gC2-HS complex is highly unstable (k(off) = 0.2 s(-1)) compared to the gC1-HS complex (k(off) = 0.003 s(-1)), suggesting why gC2 may not play an important role in attachment of virus to cells as does gC1. Second, does gC2 have a lower affinity for C3b than does gC1, thereby explaining the lack of C3b-receptor activity on HSV-2 infected cells? Surprisingly, gC2 had a 10-fold higher affinity for C3b compared to gC1, so this functional difference in serotypes cannot be accounted for by affinity. Third, do differences in gC-HS and gD-receptor affinities support a model of HSV entry in which the gC-HS interaction is of lower affinity than the gD-receptor interaction? Our biosensor results indicate that gC has a higher affinity for HS than gD does for cellular receptors HveA (HVEM) and HveC (nectin-1).     

Phosphorylation of C3 by a casein kinase released from activated human platelets increases opsonization of immune complexes and binding to complement receptor type 1

We have previously demonstrated that complement component C3 is phosphorylated both in vitro and in vivo by a casein kinase released from activated human platelets. In vitro, the studies have shown that cleavage of C3b by factor I is decreased, and binding to various target surfaces is enhanced by affecting the thiol ester. In the present study we have examined the effect of phosphorylation on the binding of C3b to complement receptor 1 (CR1, CD35). Upon phosphorylation by platelet casein kinase, C3b covalently bound to activated thiol Sepharose bound higher amounts of soluble recombinant CR1. Similar effects were demonstrated with two ELISA systems in which microtiter plates were coated with phosphorylated or unphosphorylated purified C3b or with C3 activated by the alternative pathway convertase. Phosphorylated C3b was also four times more efficient than unphosphorylated C3b in inhibiting the binding of complement-opsonized human aggregated gammaglobulin to erythrocytes. A similar increase in binding was found at low serum concentrations when the C3 activation occurred in C3-deficient serum reconstituted with phosphorylated or unphosphorylated C3. In this serum system, using a monoclonal antibody specific for iC3b, we also demonstrated that the phosphorylated C3b was protected against cleavage to iC3b. Corresponding experiments using factor H showed a decrease in binding of both fluid-phase and bound C3b to factor H. We postulate that phosphorylation of C3 by activated platelets amplifies the complement-mediated binding of immune complexes to CR1 by three different mechanisms: decreased cleavage of C3b to iC3b, increased deposition of C3b to immune complexes, and increased binding of C3b to CR1.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Human Alveolar Macrophages Cultured on Fibronectin in Vitro

We studied the phagocytosis of agarose beads by human alveolar macrophages in terms of the morphology, the receptors involved, and the cellular substrates (plastic or fibronectin) used. Beads coated with C3b (58%) and iC3b (42%) by treatment with serum, were ingested during 45 min by CR1 and CR3 on the macrophages. This ingestion was inhibited 80-90% by the presence of polyclonal F(ab')2 anti-C3 fragments. Since the phagocytosis of both C3b- and iC3b-coated beads was about threefold stronger than for C3b-coated beads (trypsinized serum-treated beads), the results indicate that the CR3 is more phagocytic than the CR1. The phagocytosis of initially complement uncoated beads, which are slowly opsonized with macrophage-produced C3b and iC3b in vitro, was also strongly inhibited (70-80%) by the presence of anti-human C3 F(ab')2 fragments. There was an increased phagocytosis (10-17%) of complement precoated beads by macrophages cultured on the fibronectin substrate versus the plastic substrate. The morphology and rapid phagocytosis of the complement precoated beads was demonstrated by SEM. The general impression was that membranous protrusions stretched towards the beads, which became increasingly enclosed by plasma membrane.     

Direct binding of herpes simplex virus type 1 virions to complement C3

Glycoprotein C (gC) of type 1 herpes simplex virus (HSV-1) binds the human complement C3 as purified proteins, or when expressed on the surface of infected cells. However, it is not clear whether the purified HSV virion binds directly to C3. In this study, direct binding of purified virions, HSV-1(KOS) or HSV-1(hrR3), to C3-coated plate was demonstrated by an enzyme-linked immunosorbent assay (ELISA). Captured virions on C3-coated plates were still infectious as determined by adding Vero cells to allow for infection to occur. The binding of virions to C3 was abolished if C3 was heat-inactivated, confirming a requirement for complement. In addition, the interaction was inhibited by preincubation of purified virions with heparin. In conclusion, a direct interaction of C3 with the HSV-1 virions was demonstrated.     

Characteristics of iC3b binding to human polymorphonuclear leucocytes.

We determined in binding assays using monomeric fluid-phase iC3b and Scatchard analysis that iC3b binds to human polymorphonuclear leucocyte type 3 complement receptor (CR3), a low-density/high-affinity receptor (28,200 binding sites, affinity constant (Ka) = 2.1 +/- 0.47 X 10(6) L/M), and to the C3b receptor (CR1), a high-density/low-affinity receptor (54,700 binding sites, Ka = 1.7 +/- 2.04 X 10(5) L/M. Binding of iC3b to CR1 was confirmed by blocking experiments with polyclonal F(ab')2 antibody against CR1, and competitive binding experiments with C3b. Binding of iC3b to CR3 was demonstrated by blocking experiments with the monoclonal antibody OKM10 against the ligand binding site of CR3. Inhibition of both CR1 and CR3 did not completely reduce iC3b binding, indicating the existence of additional iC3b-binding sites on PMN. Using flow cytometric analysis of receptor expression, no positive or negative co-operativity was observed between CR1 and CR3. Expression of both receptors increased in a dose-dependent manner after incubation with f-met-leu-phe or phorbol myristate acetate; however, only CR3 expression was enhanced at very low concentrations of these stimuli. iC3b/CR3 interactions probably play a central role in host defence against microorganisms.     

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.     

Cleavage of complement C3b to iC3b on the surface of Staphylococcus aureus is mediated by serum complement factor I

Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.     

Importance of factors H and I for the adherence of C3b-coated erythrocytes to cells

The role of cell membrane-associated human factor H for the binding of cell-bound C3b to complement receptor-carrying (CR+) cells was investigated. Pretreatment of CR+ cells with antibodies to factor H inhibited the adherence of C3b-coated red cells to human tonsil lymphocytes (TL) and peripheral blood monocytes (M phi). The C3b receptor reactivity of human polymorphonuclear leucocytes (PMN) was not influenced and the one of Raji lymphoblastoid cells only slightly influenced; iC3b and C3d receptor reactivity was in no case affected. When diisopropylfluorophosphate (DFP) in a concentration of 0.1 mM was present during pretreatment of the CR+ cells with anti H, the antibodies gained the capacity to inhibit the adherence of C3b-coated erythrocytes to Raji cells; this effect was dose-dependent with respect to DFP. In contrast, there was no influence of DFP on the inhibition pattern of anti H in the case of TL and M phi. The adherence of C3b-coated erythrocytes to PMN remained unaffected by anti-H antibodies in the presence of DFP. Polyclonal as well as monoclonal antibodies directed against human factor I inhibited the binding of C3b cells to Raji cells but not to TL. Additionally, when anti I and anti H antibodies were both present, C3b receptor reactivity of Raji cells was inhibited to a larger extent than with either antibody alone; again, TL remained unaffected. Results obtained by washing the Raji cells before and after treatment with anti H and anti I suggest that the respective antibodies act on factor H primarily on the level of the cell membrane and on factor I in the fluid phase.     

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.     

Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.     

Family studies of erythrocyte complement receptor type 1 levels: reduced levels in patients with SLE are acquired, not inherited.

It has been claimed that patients with systemic lupus erythematosus (SLE) have an inherited deficiency of erythrocyte complement receptor type 1 (CR1, with ligand binding specificity for C3b, iC3b and C4b). CR1 functions as the only cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg. The activity of this receptor on red cells may be an important mechanism for handling immune complexes which have bound C3b or iC3b. Radioligand binding studies were performed using a monoclonal antibody to CR1, E11, to enumerate these receptors accurately. The results confirmed that patients with SLE have a reduced number of CR1 molecules per red cell, but showed no reduction in CR1 levels amongst their consanguineous relatives. Study of 13 normal families suggested the presence of heritable factors controlling the numbers of erythrocyte CR1 molecules; in particular there was a correlation between mean parental CR1 numbers and CR1 numbers in their children. However, amongst 17 families of 19 patients with SLE, four families were identified in which genotypically 'high CR1' SLE patients had persistently low phenotypes. This is not compatible with the hypothesis that the reduction in erythrocyte CR1 numbers in these patients is inherited.     

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors.

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.     

Purification and characterization of IgG immunoconglutinins from patients with systemic lupus erythematosus: implications for a regulatory function.

The levels of IgG immunoconglutinins in plasma from patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis were monitored by ELISA. High levels of IgG immunoconglutinins were found mainly in plasma from patients with systemic lupus erythematosus. These immunoconglutinins bound to microtitre plate-fixed C3, C3b and C3c but poorly to C3d. This binding was inhibited by particle-bound C3b and iC3b but not by the corresponding soluble fragments. Furthermore, Western blot analysis revealed no immunoconglutinin-binding to reduced C3 peptides and no binding was shown to soluble C3 alpha and beta chain by ELISA. IgG immunoconglutinins were purified from three plasma specimens by affinity chromatography on activated thiol sepharose ATS/C3 fragments. Two immunoconglutinin preparations that preferentially recognize ATS-C3b, inhibited C5-convertase function by 50-100% while one immunoconglutinin that recognized ATS-C3d,g had no effect. The two former immunoconglutinins also inhibited all three factor I cleavages in C3 alpha chain but the latter inhibited only the third cleavage. None of the immunoconglutinins affected the binding of complement-coated anti-BSA/BSA complexes to CR1 (CD35) on human erythrocytes, but the two immunoconglutinins that inhibited all factor I cleavages also inhibited the factor I-induced release of anti-BSA/BSA complexes from CR1. The results show that immunoconglutinins recognize specific epitopes on bound C3 fragments and that they are able to modulate C3-mediated functions.     

Complement fragments in patients with bronchial asthma]

In this study, we investigated the pharmacological reactions induced by ibudilast to the complement system with the aim of clarifying the functional relation of the complement system to allergic reactions and pathology in patients with bronchial asthma. Complement hemolytic activities (CH50 and ACH50), complement profile, anaphylatoxins (C3a and C5a) and complement fragments (Bb, iC3b and C4d) were measured in 20 patients with bronchial asthma. One of antiasthmatic activities induced by ibudilast was concluded to be brought about though inactivation of the alternative complement pathway working on type III allergic reaction. Ibudilast increased the complement fragment Bb in the patients' plasma with the fairly controlled bronchial asthma. This increase in circulating Bb was suspected to be a result of inactivation of intermediate complement complexes, for example C3b.Bb.P, because the amounts in plasma of C3 and C5 showed no changes, while those of factor, B, P, H and I were decreased by ibudilast administration in patients with fairly controlled bronchial asthma. This antiasthmatic ability of ibudilast was restrained in those patients whose peripheral leukocytopenia was advanced before ibudilast administration, and in those whom ibudilast did not provoke an increase in the plasma level of iC3b, or did not prevent the serum level of C5 from increasing. In those unfairly controlled cases, enough anaphylatoxins, especially C5a might be produced to make the margination of peripheral neutrophils to the lung and increase CR3 on neutrophils binding with iC3b.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

The pathogenesis of hyaline arteriolosclerosis

Although hyaline arteriolosclerosis is very common and has been of interest to pathologists for well over 100 years, its pathogenesis has never been determined. This study demonstrates that iC3b bound via an ester linkage to hydroxyl groups on the repeating disaccharide units of hyaluronic acid is a major component of arteriolar hyaline. The deposition of iC3b within the walls of arterioles appears to be due to slow spontaneous activation of the alternative complement pathway and random binding of metastable C3b to proximate hyaluronic acid within the arteriolar wall. Since hyaluronic acid does not activate the alternative complement pathway, bound C3b is rapidly inactivated by factors I and H to iC3b, which, along with factor H, remains bound to hyaluronic acid. The hyaline in some hyalinized arterioles also contains IgM and early and late classical complement pathway components. Indirect evidence suggests that the IgM represents immunoconglutinin, an autoantibody to neoantigens on iC3b and that their interaction results in activation of the classical complement pathway. The gradual accumulation of iC3b, factor H, and, at times, IgM and classical complement pathway components within the walls of arterioles is considered to be a physiologic consequence of aging and probably cannot be prevented, because interruption of the initial binding of metastable C3b to hyaluronic acid would require abrogation of the critically important functions of the alternative complement pathway.     

Characterization of alternative pathway inhibition by a serum derived, low molecular weight complement inhibitor.

Normal human serum contains an inhibitor of complement which is distinguished by its small size of 500 daltons, the low molecular weight inhibitor (LMWI). When LMWI was present during incubation of zymosan or cobra venom factor with serum, formation of complement reactive complexes was blocked as measured by failure of these mixtures to lyse susceptible erythrocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH). Addition of LMWI to pre-formed complexes had no effect on their subsequent haemolytic activity. When dialysis was used to remove LMWI from reaction mixtures, it was shown that LMWI had not irreversibly altered any of the complement components. Purified components were used to demonstrate that LMWI prevented factor D activation of cobra venom factor-factor B complexes. LMWI also strongly inhibited binding of 125I-factor B to human erythrocytes bearing C3b and had little or nor effect on binding of 125I-factor H to the C3b bearing cells. Factor B binding to C3b was equally inhibited on normal and PNH erythrocytes. Thus, a dialysable fraction from normal human serum prevents activation of human complement by blocking formation, but not the activity of the C3/C5 convertase. These low molecular weight inhibitors result in inhibition of factor B binding to C3b and inhibition of factor D activation of C3bB complexes.     

Complement in removal of the dead – balancing inflammation

Recognition and removal of apoptotic and necrotic cells must be efficient and highly controlled to avoid excessive inflammation and autoimmune responses to self. The complement system, a crucial part of innate immunity, plays an important role in this process. Thus, apoptotic and necrotic cells are recognized by complement initiators such as C1q, mannose binding lectin, ficolins, and properdin. This triggers complement activation and opsonization of cells with fragments of C3b, which enhances phagocytosis and thus ensures silent removal. Importantly, the process is tightly controlled by the binding of complement inhibitors C4b-binding protein and factor H, which attenuates late steps of complement activation and inflammation. Furthermore, factor H becomes actively internalized by apoptotic cells, where it catalyzes the cleavage of intracellular C3 to C3b. The intracellularly derived C3b additionally opsonizes the cell surface further supporting safe and fast clearance and thereby aids to prevent autoimmunity. Internalized factor H also binds nucleosomes and directs monocytes into production of anti-inflammatory cytokines upon phagocytosis of such complexes. Disturbances in the complement-mediated clearance of dying cells result in persistence of autoantigens and development of autoimmune diseases like systemic lupus erythematosus, and may also be involved in development of age-related macula degeneration.