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PubMed1979

C3b binding immune complexes and immunoconglutinins in human sera. Detection by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) designed to measure autoantibodies against C3b (immunoconglutinins: IK) also detects immune complexes (IC). Solid phase C3b, in addition to binding IK of IgG, IgM and IgA classes, bound aggregated human IgG, IgA and aggregated immunologically purified anti-tetanus toxid antibodies as well as model complexes of tetanus toxoid-human anti-toxoid. Significant C3b binding IgG, IgM and IgA activities were seen in the sera of 20 SLE patients but not in sera from healthy blood donors. Ultracentrifugation analysis of two SLE sera revealed C3b binding IgG and IgA activities in both light (7S) and heavy (greater than or equal to 11S) fractions. This indicates simultaneous presence of IK and IC in these sera. On the basis of the known relationship between IK and IC formation we suggest that the solid phase C3b ELISA may be of value in evaluating immune reactions in patients.

Antigen-Antibody Complex↗
PubMed1987

Suppressive effects of C3b on monocyte-dependent T cell proliferation.

The effect of C3b treatment of human monocytes on secondary antigen-dependent T cell response was studied. When antigen-specific T cell blasts were cultivated together with C3b-treated monocytes the proliferative response was inhibited in a dose-dependent fashion. This suppressive effect was specific for C3b because heat-inactivated C3b or buffer alone had no influence on T cell proliferation. In part, this suppressive effect is mediated through a C3b-induced decreased expression of class II antigens on the surface of treated monocytes, but another suppressive mechanism exists because the C3b pretreatment of monocytes also led to an inhibition of the proliferative response in a class II antigen-independent T cell proliferation system. In addition to the C3b data, our finding that treatment of monocytes with C3d resulted in a lower T cell proliferation, while C3c has no effect, suggested that C3d, which could be generated from C3b in the culture, may induce the second inhibitory mechanism.

Cells, Cultured↗
PubMed2003

Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Recent studies have identified a factor H-associated form of HUS, caused by gene mutations that cluster in the C-terminal region of the complement regulator factor H. Here we report how three mutations (E1172Stop, R1210C, and R1215G; each of the latter two identified in three independent cases from different, unrelated families) affect protein function. All three mutations cause reduced binding to the central complement component C3b/C3d to heparin, as well as to endothelial cells. These defective features of the mutant factor H proteins explain progression of endothelial cell and microvascular damage in factor H-associated genetic HUS and indicate a protective role of factor H for tissue integrity during thrombus formation.

Animals↗
PubMed1988

Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

Antigen-Presenting Cells↗
PubMed1984

Characterization of the interaction of human C4b-binding protein with physiological ligands.

The binding of C4b to C4b-binding protein (C4BP) was demonstrated at physiological ionic strength by analytical ultracentrifugation. The sedimentation rate of C4BP gradually increased from 9.4 S to a maximum of 18.5 S with increasing C4b concentration. The stoichiometry of different C4BP X C4b complexes was calculated from the sedimentation-velocity data. A linear relationship was established between the number of C4b bound per C4BP and the sedimentation rate of the complex. In order to define further the C4BP-C4b interaction, sucrose density gradient ultracentrifugation was also used. Trace amounts of 125I-C4BP were centrifuged through 12 sucrose density gradients, each of which contained a different concentration of C4b throughout the gradient. The sedimentation rate of the C4BP increased with increasing C4b input to a maximum of 19.5 S. These binding data, in conjunction with the stoichiometry measurements determined in the analytical ultracentrifuge, were analyzed by the methods of Scatchard and Hill. At physiological ionic strength, C4BP exhibited four binding sites for C4b, each having an association constant of 1.2 X 10(7) M-1. A Hill coefficient of 1.1 was calculated, indicating that the four binding sites were independent. At reduced ionic strength, two additional sites were detected. The sedimentation coefficient of C4BP(C4b)6 was 24 S. The hydrodynamic data suggest that after four C4b molecules have bound to C4BP, the binding of additional C4b is sterically hindered. This interpretation implies that all six binding sites on C4BP are identical. C4BP also bound C4(H2O) (the product resulting from spontaneous hydrolysis of the thiol ester bond in native C4) and weakly bound C4c, but had no measurable affinity for native C4 or C4d at physiological ionic strength. A low-affinity interaction between C3b and C4BP was also demonstrated in the analytical ultracentrifuge. The C4BP X C3b complex was specific because C4BP mediated the cleavage of C3b by Factor I to C3bi with concomitant dissociation of the complex.

Carrier Proteins↗
PubMed1995

A recombinant polypeptide, composed of the alpha-helical neck region and the carbohydrate recognition domain of conglutinin, self-associates to give a functionally intact homotrimer.

A recombinant polypeptide composed of the alpha-helical neck region and carbohydrate recognition domain (CRD) of bovine conglutinin was expressed in Escherichia coli. The recombinant protein formed inclusion bodies but could be solubilised using a denaturation-renaturation cycle based on urea and then purified by affinity chromatography on a TSK-N-acetylglucosamide column. The purified product behaved as a homotrimer in non-dissociating conditions, with three CRDs held together by the alpha-helical neck regions. The trimer, although lacking the N-terminal and collagen regions of the native conglutinin, showed the same binding carbohydrate specificities as the native molecule, for the complement fragment C3b and for lipopolysaccharides derived from Gram-negative bacteria.

Animals↗
PubMed1983

C3b deposition during activation of the alternative complement pathway and the effect of deposition on the activating surface.

Examination of C3b deposition on the surface of activators during alternative pathway activation revealed three temporal phases: a lag phase, an amplification phase, and a heretofore uncharacterized plateau phase. During the plateau phase no C3b deposition appeared to occur even in the presence of an excess of alternative pathway components. Double label experiments, however, revealed that the plateau was a steady state between continued C3b deposition and release of C3b or C3bi from the activator. Under conditions of excess complement it was found that deposition of increasing numbers of C3b molecules caused a gradual increase in the ability of Factors H and I to inactivate newly deposited C3b; i.e., the deposited C3b converted the activator into a nonactivator. The data indicate that the surface of rabbit erythrocytes is rendered completely nonactivating when 2.4 X 10(6) molecules of C3b plus C3bi are bound per cell. The plateau of C3b deposition appears to represent the maximum steady state level maintainable by a given concentration of complement components, and it also reflects conversion of an activating surface to one resembling a nonactivator.

Animals↗
PubMed2001

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.

Antigen-Antibody Complex↗
PubMed1982

Membrane stimulation and intracellular killing by monocytes.

The present paper reviews our recent studies concerning the intracellular killing of bacteria by human monocytes and compares the results with those found for circulating granulocytes. The findings show that optimal intracellular killing of catalase-positive and catalase-negative bacteria is only reached during interaction of the Fc part of IgG and complement component C3b with their specific receptors in the membrane of the phagocytes. Similar stimulation was found during interaction of plant lectins and their specific sugar moieties in the cell membrane. The studies permit the general conclusion that optimal intracellular killing of micro-organisms requires membrane stimulation, which occurs after triggering of certain receptors.

Blood Bactericidal Activity↗
PubMed1993

Evidence for three binding sites for C3 (hemolytically inactive), C3b and C3d on a CR2-positive Burkitt lymphoma-derived cell line (Raji).

The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2-positive Burkitt lymphoma-derived cell line. Pretreatment of the cells with OKB-7 inhibited the binding of C3, whereas pretreatment with HB-5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB-7 or HB-5 bound high amounts of C3d. TPA-treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti-CR2 MoAbs and TPA.

Antibodies, Monoclonal↗
PubMed1994

Binding of C3b and C4b by the CR1-like site in murine CR1.

We determined whether the six short consensus repeats (SCRs) that are appended to the amino terminus of murine CR2 to form murine CR1 contain a binding site for C4b in addition to that for C3b, and whether these sites overlap or are distinct. Human K562 transfectant cell lines were established that stably expressed constructs encoding variable combinations of these six murine SCRs attached to the amino terminus of a truncated form of human CR2 lacking its iC3b/C3dg binding site. These cell lines, and two others expressing full-length human CR1 and SCRs lacking its iC3b/C3dg binding site. These cell lines, and two others expressing full-length human CR1 and SCRs 8-11 of the C3b binding site of human CR1, respectively, were assessed for their capacity to form rosettes with sheep E bearing rat C4b or guinea pig C3b. K562 cells with full length human CR1 formed rosettes with both EC3b and EC4b, and the cells expressing the construct with human CR1 SCRs 8-11 bound only EC3b. The murine CR1/human CR2 chimera containing murine SCRs 1-6 resembled the full length human CR1 in binding both EC3b and EC4b. Deletion of SCRs 5-6 from the murine CR1/human CR2 chimera diminished in parallel, but did not abolish, binding of EC3b and EC4b. Constructs containing SCRs 2-5, SCRs 3-6, or SCRs 2-6 lacked activity, indicating an absolute requirement for SCR-1 for binding of both C3b and C4b. Therefore, murine CR1 binds both C3b and C4b, and the sites for these ligands have similar, if not identical, amino- and carboxyl-terminal boundaries.

Animals↗
PubMed1985

Normal function of CR1 on affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which affected erythrocytes (E) are abnormally sensitive to lysis by autologous complement. Affected E from patients with PNH (PNH-E) are deficient in an E membrane regulatory protein of complement, decay-accelerating factor (DAF). Because a functional defect in a second membrane regulatory protein of complement, CR1 (C3b receptor), has also been hypothesized, severely affected PNH-E (type III PNH-E) were tested for abnormalities in CR1 by four methods. E from two patients with 100% type III PNH-E had 3201 and 6783 sites per cell for binding of 125I-labeled rabbit polyclonal F(ab')2 anti-CR1. These values fall within the normal range of CR1 antigenic sites per cell (1267 to 7915, mean = 5,014 +/- 155 SEM) established by assaying the E from 113 healthy donors. The Ka of CR1 on type III PNH-E for 125I-labeled C3b dimer was 2.06 X 10(7) M-1, and the Ka values for the binding of the same ligand to the E from two healthy individuals were 2.45 X 10(7) M-1 and 1.58 X 10(7) M-1. In an assay designed to measure the capacity of human E (Eh) to accelerate the decay of the classical C3 convertase deposited on 1 X 10(7) bystander sheep E (EAC1gp,4bh,2agp), the half-life (t 1/2) of this convertase was diminished from 18.1 min (range 15.2 to 22.9) to 8.1 min (range 7.4 to 8.5) by the addition of 1 X 10(7) normal Eh, to 6.2 min by 100% type III PNH-E, and to 7.5 min by Eh pretreated with an IgG fraction of human antiserum directed against the D antigen of the Rh system. In contrast, Eh (t 1/2 = 7.4) pretreated with a saturating dose of F(ab')2 anti-CR1, and CR1-deficient Eh (less than 10 CR1 molecules/E) from a patient with systemic lupus erythematosus, showed a loss of convertase decay-accelerating capacity to t 1/2 = 11.6 and t 1/2 = 12.4, respectively. Type III PNH-E pretreated with anti-CR1 demonstrated a total loss of their decay-accelerating capacity (t 1/2 = 19.9). In an assay of I cofactor activity, soluble C3b was rapidly converted to iC3b by purified I plus Eh or type III PNH-E, whereas CR1-deficient Eh exhibited less than 5% the I cofactor activity of normal Eh.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-Antibody Complex↗
PubMed1991

Glycoprotein C of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) is a receptor for the complement component C3b. We have previously isolated HSV-1 gC- strains (TN1, TN2 and TN3) from a patient with recurrent keratitis at three different times. These are very rare isolates because gC was thought to be essential for the virus in vivo. To determine whether gC modifies the interaction of complement with cell-free virus or virus-infected cells, we constructed gC+ recombinant viruses in which the intact gC gene of strain KOS was inserted into the TN1 virus genome. TN1 virus was inactivated by complement and TN1 virus-infected cells were lysed by complement; however, gC+ recombinant viruses became resistant to these effects of complement. These results suggest a role for gC in protection of both the virion envelope and the infected cell surface against damage by complement. TN1 virus was inactivated by complement from rats (Wistar, WKA, F344 and SD), guinea-pigs (Hartley) and humans, but not by complement from mice (C3H, DDD and BALB/c), which indicates that mice seem to be inappropriate as an experimental model for the study of HSV infection in which complement factors need to be considered.

Animals↗
PubMed1987

Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b.

Upon incubation at 37 degrees C with purified human C3b (500 micrograms/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up-regulation of CR1, assessed by the binding of radiolabeled CR1-specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10-20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1-dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 micrograms/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor-mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion-exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti-C3b-Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.

Antibodies, Monoclonal↗
PubMedDate not supplied

Assembly and regulation of the complement amplification loop in blood: the role of C3b-C3b-IgG complexes.

Amplification of complement activation in blood and serum starts on multi-protein complexes that act as precursors of an alternative C3 convertase. Among these covalently linked C4b-, C3b-, and IgG-containing complexes C3b-C3b-IgG complexes represent the major species containing C3b and IgG. Recent work on their purification and characterization is discussed. Special emphasis is placed on the arrangement of ester bonds in these complexes and their dual type of partial protection from inactivation. Partial protection from inactivation is mediated by properdin which binds to these complexes in the complete absence of any other complement protein. High dose IgG, known to stimulate inactivation of these complexes, appears to lower properdin binding in a process that also involves factor H. Properdin stimulates factor B binding to these complexes and renders them far better precursors of a C3 convertase than C3b. The available information allows a suggestion for a new scheme on how the amplification loop is assembled and regulated in blood and serum.

Antibody Formation↗
PubMed1989

The central segment of herpes simplex virus type 1 glycoprotein C (gC) is not involved in C3b binding: demonstration by using monoclonal antibodies and recombinant gC expressed in Escherichia coli.

Three monoclonal antibodies (MAbs) have been raised against cell membrane-derived herpes simplex virus type 1 glycoprotein C (gC-1). By using different DNA constructs of gC-1 expressed in Escherichia coli the sites recognized by these antibodies could be assigned to a peptide in the more hydrophobic and probably non-glycosylated middle third of the gC-1 molecule. This peptide segment corresponds to a 571 bp segment on the gC-1 gene located between the NcoI and the NruI restriction sites. None of the three MAbs interfered with the binding of the human serum complement component C3b to gC, which has been shown to be rather glycosylation-dependent. Since the data of other groups have suggested that antibodies directed against all regions of gC-1 inhibit C3b binding to gC-1, whereas our results suggest that the central part of gC-1 is not actually involved in C3b-binding activity, it can be inferred that the N- and C-terminal segments are involved in C3b binding by gC-1.

Animals↗