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PubMed1990

Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.

The alternative complement pathway is capable of discriminating human cells and tissues from a wide variety of potential pathogens. It has been recently demonstrated that attachment of complement component C3b to activator-derived molecules (e.g., small polysaccharides) restricts inactivation of C3b by factors H and I in a manner similar to activator surfaces. It is now shown that restriction is reversed by certain soluble polyanions (e.g., sialoglycopeptides, heparin, or dextran sulfate) that mimic the effects of sialic acid and glycosaminoglycans on human cells and tissues. Fluid-phase polyanions enhanced binding of factor H to C3b attached to activating particles, indicating that the effect resulted from increased affinity between C3b and factor H. The enhancement was specific for activator-bound C3b since no enhancement was observed on nonactivating particles. While several polyanions could cause this effect, some polyanions could not, indicating specificity. The active polyanions also inhibited lysis of cells via the alternative pathway. The binding site for sialic acid appears to reside on factor H, since factor H bound to heparin-agarose and to sialic acid-bearing fetuinagarose, whereas C3b bound to neither under the same conditions. These observations suggest that occupation of a specific site on factor H by polyanions induces an increase in the C3b-H affinity, resulting in discrimination of host cells and tissues from alternative pathway-activating foreign cells.

Animals↗
PubMed1985

Human complement receptors for C3b (CR1) and C3d (CR2).

The human C3b receptor (CR1) is a polymorphic glycoprotein comprised of a single polypeptide chain. Of the 4 allotype forms of CR1 that have been described, the 2 most common have Mr's of 250,000 and 260,000, and are regulated by alleles having frequencies in a Caucasian population of 81.5% and 18.5%, respectively. CR1 is present on erythrocytes, neutrophils, eosinophils, monocytes, macrophages, B lymphocytes, some T lymphocytes, mast cells, and glomerular podocytes. CR1 number on erythrocytes is genetically regulated, and ranges from less than 100 sites per cell to greater than 1000 sites per cell, the average in the normal population being 500-600 sites per cell. A model accounting for this wide distribution proposes the existence of 2 codominant alleles determining low and high receptor number respectively; CR1 number is not affected by the structural polymorphism, so that the loci for these two phenotypic characteristics are distinct. The function of CR1 on erythrocytes may be to promote the clearance of immune complexes from the circulation. CR1 number on myelomonocytic cells is regulated by chemotactic factors which can rapidly transfer CR1 sites from a latent, presumably intracellular, site to the plasma membrane of these cells, thereby enhancing their ability to interact with opsonized foreign material. The receptor is involved in the endocytic reactions of these cells, and recent findings have demonstrated that this function can be modulated by T cell-derived factors, fibronectin, and phorbol esters. The role of CR1 on lymphocytes remains to be fully explored, although the receptor may enhance the differentiation of B cells into antibody-secreting cells.

Alleles↗
PubMed1996

Immune evasion properties of herpes simplex virus type 1 glycoprotein gC.

Herpes simplex virus type I (HSV-1) glycoprotein gC binds complement component C3b, and purified gC inhibits complement activation. Two HSV strains carrying mutations in the gC gene which rendered them unable to bind C3b were compared with wild-type and marker-rescued viruses to evaluate the role of gC on the virion in protecting HSV-1 from complement-mediated neutralization. The gC mutant viruses were markedly susceptible to neutralization by nonimmune human serum, showing up to a 5,000-fold decline in titer after 1 h of incubation with serum. In contrast, wild-type or marker-rescued viruses showed a twofold reduction in titer. Studies with hypogammaglobulinemic and immunoglobulin G-depleted serum supported the observation that neutralization occurred in the absence of antibody. Neutralization of gC mutant strains by nonimmune serum was rapid; their half-life was 2 to 2.5 min, compared with 1 h for wild-type virus. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated human serum or C4-deficient guinea pig serum failed to neutralize gC mutant strains, indicating a role for components of the classical complement pathway. gC had little additional effect on neutralization by the combination of antibody plus complement compared with complement alone. The results indicate that the magnitude of the protection offered by gC-1 is larger than previously recognized; that in the absence of gC-1, complement neutralization is rapid and is mediated by components of the classical complement pathway; and that gC mainly protects against antibody-independent complement neutralization, suggesting a probable role for gC early in infection, before antibodies develop.

Animals↗
PubMed1988

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity.

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.

Afibrinogenemia↗
PubMed1986

Inhibition of complement by culture supernatants of Aspergillus fumigatus.

Aspergillus fumigatus produced a water-soluble extracellular material that inhibited the opsonization of fungal cells by normal human serum. Blastospores of Cryptococcus neoformans and conidia from A. fumigatus or Aspergillus niger were used as fungal targets for ingestion by human monocytes. The opsonic inhibitor from A. fumigatus was found to decrease binding of complement component C3b to fungal surfaces by selectively interfering with activation of the alternative complement pathway. Inhibition of complement did not appear to result simply from proteolysis, as judged by the minimal degradation of casein and of purified C3 and C4. The complement-inhibiting activity was partially labile to heating at 100 C and could be concentrated by using dialysis tubing with a 10,000-dalton exclusion limit. Aspergillus flavus, which is also a pathogen for humans, also produced this activity; A. niger did not. We speculate that this material may represent a pathogenesis factor for Aspergillus species.

Aspergillus fumigatus↗
PubMed1985

[Antibody-dependent cell-mediated cytotoxicity of peripheral blood neutrophils in vitro in acute leukemia].

A study was made of in vitro antibody-dependent cellular cytotoxicity of neutrophils of peripheral blood in 14 cases of acute myeloblastic leukemia and in 20 healthy patients (control group). A decrease in the number of neutrophils, carrying receptors to Fc-IgG and to C3b-component of complement was registered compared to the results obtained for healthy people. Besides, the number of cells restoring Nitro-blue tetrazolium and myeloperoxidase activity decreased. The decrease in antibody-dependent cellular cytotoxicity of neutrophils in the cases of acute myeloblastic leukaemia is explained by the deficiency of the cell receptor apparatus and by the decrease in the intracellular metabolism in them.

Adult↗
PubMed1979

Complement receptor binding of C3b-coated cells treated with C3b inactivator, beta 1H globulin and trypsin.

Treatment of 125I-C3b bound to EAC1423b with C3b inactivator (C3bINA) and beta 1H globulin (beta 1H) cleaved the alpha-chain of C3b into 65,000- and 42,000-dalton fragments, both of which remained disulfide-bonded to the intact beta-chain (C3bi). Subsequent treatment with trypsin (0.1 microgram/ml) released 125I into the supernatant and yielded cells coated with a 33,000-dalton fragment of alpha-chain, presumably C3d. These results are in agreement with those obtained by others using fluid phase C3b. C3b-coated cells (EAC1423b) adhered to complement (C) receptors on human erythrocytes, glomeruli, and monocytes. C3bi-coated cells adhered to the receptors on glomeruli and monocytes, but not to those on human erythrocytes. C3d-coated cells adhered only to the monocyte receptors. The findings suggest that the glomerular C receptor recognizes portions of the C3 molecule different from those recognized by either the erythrocyte or monocyte receptors.

Animals↗
PubMed2003

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H.

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.

Animals↗
PubMed2003

Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents.

Despite the success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about their mechanism(s) of action. Here, we show that certain of these reagents (rituximab and 1F5), which redistribute CD20 into membrane rafts, are bound efficiently by C1q, deposit C3b, and result in complement-dependent cytotoxicity (CDC). This activity is important in vivo, because complement depletion using cobra venom factor (CVF) markedly reduced the efficacy of rituximab and 1F5 in 2 lymphoma xenograft models. However, complement depletion had no effect on the potent therapeutic activity of B1, a mAb that does not redistribute CD20 into membrane rafts, bind C1q, or cause efficient CDC. Equivalent immunotherapy also occurred in the presence or absence of natural killer (NK) cells. Perhaps most surprising was the observation that F(ab')2 fragments of B1 but not 1F5 were able to provide substantial immunotherapy, indicating that non-Fc-dependent mechanisms are involved with B1. In accordance with this, B1 was shown to induce much higher levels of apoptosis than rituximab and 1F5. Thus, although complement is important for the action of rituximab and 1F5, this is not so for B1, which more likely functions through its ability to signal apoptosis.

Animals↗
PubMed1984

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb.

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.

Antibodies, Monoclonal↗
PubMed1998

Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Arginine↗
PubMed1979

Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator.

We recently described the isolation from human serum of a serum protein (C4 binding protein) that functions as an essential cofactor for C3b inactivator in the proteolysis of fluid-phase C4b and to a much lesser extent, C3b. We show here the role of C4 binding protein in the formation and function of the classical pathway C3 convertase (C42). C4 binding protein interferes with the assembly of the membrane-bound C3 convertase of the classical pathway and accelerates the decay of C42 in a dose-dependent fashion. Its removal from serum by means of specific immune absorption promotes the vigorous consumption of C3 after addition of C1; this effect is abolished by reconstitution with purified C4 binding protein. Although C4 binding protein inhibits the hemolytic function of cell-bound C4b, we did not detect any change in the structure of C4b even after prolonged incubations of EAC14 with C4 binding protein. For this reason, and on the basis of studies of the time required for maximal reactivity (Tmax) of cellular intermediates generated in the presence of C4 binding protein and limited amounts of C2, we conclude that the effects of C4 binding protein are probably mediated by displacing C2a from specific binding sites on C4b. In addition, C4 binding protein enhances the cleavage by C3b inactivator of the alpha' chain of cell-bound C4b. When EAC14 cells were incubated with both control proteins, the Tmax of the cells was prolonged and the lysis was markedly diminished. We conclude that C4 binding protein and C3b inactivator control the C3 convertase of the classical pathway in a fashion similar to that described for beta 1H and C3b inactivator in the alternative pathway.

Animals↗
PubMed2004

C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury.

Although the complement system has been implicated in liver regeneration after toxic injury and partial hepatectomy, the mechanism or mechanisms through which it participates in these processes remains ill-defined. In this study, we demonstrate that complement activation products (C3a, C3b/iC3b) are generated in the serum of experimental mice after CCl(4) injection and that complement activation is required for normal liver regeneration. Decomplementation by cobra venom factor resulted in impaired entry of hepatocytes into S phase of the cell cycle. In addition, livers from C3-deficient (C3(-/-)) mice showed similarly impaired proliferation of hepatocytes, along with delayed kinetics of both hepatocyte hyperplasia and removal of injured liver parenchyma. Restoration of hepatocyte proliferative capabilities of C3(-/-) mice through C3a reconstitution, as well as the impaired regeneration of C3a receptor-deficient mice, demonstrated that C3a promotes liver cell proliferation via the C3a receptor. These findings, together with data showing two waves of complement activation, indicate that C3 activation is a pivotal mechanism for liver regeneration after CCl(4) injury, which fulfills multiple roles; C3a generated early after toxin injection is relevant during the priming of hepatocytes, whereas C3 activation at later times after CCl(4) treatment contributes to the clearance of injured tissue.

Animals↗
PubMed1985

A circulating inhibitor of fluid-phase amplification. C3 convertase formation in systemic lupus erythematosus.

C3 nephritic factor (C3NeF) was used to assess the formation of the fluid-phase amplification convertase, C3b,Bb, in 37 serum specimens from 24 patients with systemic lupus erythematosus (SLE). C3b,Bb formation was measured by the concentration of Ba, released when C3b,B is activated. Incubation of normal human serum (NHS) with C3NeF accelerates C3b amplification loop turnover with the formation of large quantities of C3b,Bb. In contrast, sera from 22 of 24 patients with SLE formed little or no convertase when incubated with C3NeF. C3 conversion to C3b was commensurately reduced. The inhibition could not be attributed to depressed serum concentrations of C3, factor B, or classical pathway components. Inhibitor present in excess could be demonstrated in 23 of 34 specimens of SLE serum by mixing experiments. The spontaneous convertase formation that occurs when a portion of the serum H is inactivated with F(ab')2 anti-H was also shown to be inhibited in SLE serum. The inhibition was found, however, to be H dependent in that convertase formation was normal in SLE serum depleted of H. It is concluded that the C3b in most SLE sera is unusually susceptible to inactivation by H, but a functional abnormality was not demonstrable in either C3 or H isolated from SLE serum. The inhibition could be simulated in NHS by addition of heparin, 100 micrograms/ml. In vivo, inhibition of convertase formation could interfere with the solubilization and disposal of immune complexes by reducing the deposition of C3b on the immune complex lattice.

Complement Activating Enzymes↗
PubMedDate not supplied

Binding of fluid phase C3b to nonsensitized bystander human red cells. A model for in vivo effects of complement activation on blood cells.

There is evidence that activated complement fragments (C3b) can bind to human red cells (RBCs) serving as adsorbing surfaces, but not as antigens. This evidence prompted the present in vitro experiments. Using the standard antiglobulin test (AGT), the radioimmune antiglobulin test (RIAT), and the immune adherence test, we found that C3 can indeed be attached to "bystander" human RBCs if complement is activated either through the classical pathway (anti-A hemolysins plus blood group A1 RBCs) or the alternative pathway (activator surfaces, i.e., Escherichia coli bacteria, rabbit RBCs, or inulin). On Scatchard plot analysis, between 24,000 and 44,000 radiolabeled anti-C3d molecules were bound per one adjacent "bystander" RBC, while untreated control RBCs, or RBCs preincubated with fresh compatible serum, bound only 200 to 300, and 600 to 800 molecules, respectively. Despite strong coating with C3 fragments, only "bystander" paroxysmal nocturnal hemoglobinuria, but not normal, RBCs were hemolyzed by complement activation, i.e., through E. coli at pH 8.0. RBCs were not coated with C3 when complement was activated in the fluid phase by heat-aggregated IgG or a staphylococcal "decomplementation antigen." We conclude that the findings of our in vitro experiments accurately mimic some old, but as yet insufficiently understood, in vivo effects of complement activation on circulating blood cells.

Binding Sites, Antibody↗
PubMed1983

Enzymic assay of C3b receptor on intact cells and solubilized cells.

The C3b receptor of human erythrocytes is known to act as a cofactor for the cleavage of the complement protein C3b by the serine proteinase C3b/C4b Ina. The same cofactor activity is shown to be present on human tonsil B-lymphocytes. The cofactor activity of the C3b receptor can be assayed, on intact cells or in solubilized extracts of cells, by determining the rate of C3b cleavage in the presence of fixed concentrations of C3b and of C3b/C4b Ina. This assay method was used to compare the characteristics and relative quantities of C3b receptors on erythrocytes and lymphocytes. The cofactor activities associated with these two cell types resemble each other, but are distinct from the serum cofactor proteins, C4bp and Factor H, in antigenicity and in pH- and ionic-strength-dependence, and are distinct from Factor H in substrate specificity. Assay of cofactor activity in intact cells indicates that there are about 80-fold more receptors per cell on the lymphocyte surface than on erythrocytes. Assays with cells made permeable by detergent show that, whereas essentially all of the receptors on erythrocytes are on the cell surface, B-lymphocytes contain a large internal receptor pool, which makes up more than 80% of the total cofactor activity of the cell.

B-Lymphocytes↗
PubMed1985

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues.

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.

Antigen-Antibody Complex↗