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PubMed1977

Improved efficiency of mouse, guinea pig and human C3b inactivator at low ionic strength, and reproducible immune-adherence with mouse complement.

Inactivation of homologous C3b by heated guinea pig, mouse and human serum was found to be much more rapid and complete at low ionic strength (0.037) than at micron = 0.15. The C3b inactivator in human and mouse serum was somewhat unstable to heating at 56 degrees C. Heated guinea pig serum showed the greatest ability to inactivate heterologous C3b, and human serum the least. Suramin (1 mg/ml) completely blocked homologous C3b inactivation by heated human, guinea pig and mouse serum, and 0.1 mg/ml was effective with mouse but not with human or guinea pig serum. Immune-adherence reactions with mouse C3 produced somewhat unstable hemagglutination patterns, which were improved by using ovalbumin in the buffer and minimizing EAC exposure to warm temperatures. A prozone phenomenon was frequently observed in immune-adherence hemagglutination with mouse C3, and less frequently with guinea pig and human C3.

Animals↗
PubMed1982

A fluorometric method for determination of C3b inactivator.

A simple method for the determination of C3b inactivator in human plasma was investigated. Fluorescent-labeled C3, which was prepared by treatment with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide, was used as the substrate. Diluted human plasma is incubated with fluorescent C3, reduced with 2-mercaptoethanol, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The fluorescent cleavage product is then determined by scanning the gels with a fluorospectrophotometer. This method is simple and sensitive enough to determine C3b inactivator activity in 1 microliter of human plasma.

Complement C3b Inactivator Proteins↗
PubMed1994

The role of the carbohydrate chains in complement (C3) fixation by solid-phase-bound human IgA.

In contrast to antigen-antibody complexes containing native human IgA, solid-phase-deposited IgA activates the alternative complement pathway and binds C3b. To investigate the role of carbohydrate chains in this, various human IgA preparations were treated with neuraminidase alone or together with N-glycanase or O-glycanase, or with mixed glycosidases from the oral bacterium, Streptococcus mitis. Depletion of oligosaccharides was determined by carbohydrate analysis. Removal of sialic acid and N-linked glycan chains greatly increased the C3b-fixing properties of normal serum IgA1 and IgA2. Myeloma IgA1 and IgA2 proteins and secretory IgA had higher C3b-binding activity than normal serum IgA, and this was further increased by removal of sialic acid and N-linked glycans. Fc alpha and Fc alpha-SC fragments of myeloma and secretory IgA1, respectively, but not Fab alpha fragments, obtained by cleavage with bacterial IgA1 proteases and also free secretory component, fixed C3b by the alternative pathway.

Complement C3b↗
PubMed1996

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that binds iC3b and CD23.

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short consensus repeats (SCR) 1-2, SCR 4-5 and SCR 9-11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1-2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.

B-Lymphocytes↗
PubMed1989

Localization of Fc gamma receptors and complement receptors CR1 on human peripheral nerve fibres by immunoelectron microscopy.

The localization of receptors for the Fc part of IgG (Fc gamma R) and for the complement C3b/C4b components (CR1) on human peripheral nerve fibres was investigated by indirect immunoperoxidase staining of frozen nerve sections with monoclonal antibodies. Transmission electron microscopy revealed that Fc gamma R and CR1 are localized to the entire surface membrane and inner membrane (axolemma) of the Schwann cell. Myelin and axons were not stained. The presence of Fc gamma R and CR1 in human Schwann cells adds further evidence for the immunocompetence of these cells.

Animals↗
PubMed1980

Analysis of receptor-mediated C1q binding to human peripheral blood mononuclear cells.

The binding of C1q to human peripheral blood leukocytes has been investigated. Studies with fluorescein conjugated F(ab')2 anti-C1q show that few (0 to 4%) normal leukocytes isolated in the presence of EDTA have C1q on their surface. However, approximately 26% of the mononuclear cell population is able to bind added C1q. Quantitative binding studies using 125I-C1q show that the binding to mononuclear cells is specific, saturable, and reversible. Scatchard plot analyses indicate an approximate equilibrium constant of 1.2 times 10(7) M-1. C1q binding appears to be mediated via the collagenous portion of the molecule in that 1) type I collagen inhibits this binding; 2) C1 reconstituted from purified C1q, C1r, and C1s does not bind to mononuclear cells, whereas the same amount of free C1q binds avidly; and 3) C1q enhances the binding of aggregated IgG to mononuclear cells.

Binding Sites↗
PubMed1980

Mouse C3b/C4b inactivator: purification and properties.

Mouse C3b/C4b inactivator (C3b/C4bINA) was purified approximately 400 times from mouse serum. It is a beta-globulin and consists of 2 disulfide bonded chains of m.w. 60,000 and 35,000. Under nonreducing conditions, its m.w. is 95,000. It cleaves the alpha'-chain of cell-bound C4b into 3 fragments: alpha 2, alpha 3, alpha 4. The alpha 2 fragments remain bound to the cell surface (C4d), and the rest of the molecule (C4c) is released into the fluid phase. In fluid phase, C3b/C4bINA cleaves the alpha'-chain of C4b in a similar manner but only in the presence of mouse or human C4-binding protein (C4-bp). Mouse C4-bp and human C3b/C4bINA do not cleave human C4b, although mouse C4-bp binds to human C4b. This incompatibility suggests that C4-bp and C3b/C4bINA must interact to cleave fluid phase C4b. Mouse C3b/C4bINA also cleaves the alpha'-chain of human C3b in solution into 2 fragments in the presence of human beta 1H. Therefore, it is likely that mouse and human C3b/C4bINA are homologous proteins. A monospecific antiserum to mouse C3b/C4bINA has been prepared in rabbits. By crossed immunoelectrophoresis, this antiserum detects, in addition to the protein described above, a fast beta-globulin with a m.w. of approximately 200,000 and antigenically identical to C3b/C4bINA but enzymatically inactive. This protein could represent a precursor of C3b/C4bINA.

Animals↗
PubMed1989

[The effect of staphylococci on the manifestation of opsonic cooperation in the complement system].

The influence of the cultures of 14 Staphylococcus aureus and Staphylococcus epidermidis strains on the capacity of factor C3b of the complement for mediating the adhesive reaction of human neutrophils was studied. In experiments with 5 out of 11 S. aureus strains the essential weakening of reactions was registered, while one of the strains considerably enhanced reactions. Out of 3 S. epidermidis strains, the weakening of C3b-dependent reaction was noted in one case. The activity of the cultures did not correlate with the gelatinase properties of staphylococci and was absent in all gelatinase-positive Pseudomonas aeruginosa strains. The model worked out by the authors may be used for studying the influence of bacterial metabolites and biologically active substances on the effector properties of factor C3b of the complement.

Cell Adhesion↗
PubMed1978

Interaction of beta1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding.

Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.

Animals↗
PubMed1986

Use of a high-efficiency expression vector to isolate cDNA clones for factor H and map their positions within the molecule.

A human liver cDNA library, cloned in a novel high-efficiency bacterial expression vector (PEX), was screened with an affinity-purified antibody to human factor H. Four distinct cDNA clones, H-2, H-40, H-46 and H-49, were identified. Of these, H-2 also reacted with two monoclonal antibodies to H, MAH-4 and OX-24, which were previously shown to recognize the 38,000 N-terminal tryptic fragment of H, carrying the binding site for C3b. By using polyclonal antibodies specific for the domains in H coded for by these cDNA-clones, it could be established that H-2 codes only for the 38,000 N-terminal tryptic fragment of H, whereas H-40, H-46 and H-49 are derived from the 142,000 C-terminal fragment of H. By subcloning H-2 the epitope for OX-24 could be localized as being coded near the central Sma-site of H-2.

Antibodies↗
PubMed1985

Studies on the binding of C3b-coated microspheres to human neutrophils.

A method is described for the quantitation of C3b receptors on human neutrophils using a mixture of C3b-coated fluorescent and C3b-coated non-fluorescent microspheres. The method measures the "sterically available' C3b receptors on the cells, for example, the receptors available to opsonized bacteria. The use of mixtures of fluorescent and non-fluorescent microspheres resulted in lowered fluorescence intensities of the microsphere-coated neutrophils that were well within the fluorescence limitations of fluorescence activated cell analyzers or sorters used in the assay procedure. These mixtures also allowed the distribution of the C3b-coated microspheres around the neutrophils to be easily visualized in the fluorescence microscope. The binding of the C3b-coated microspheres to the neutrophils was shown to be receptor mediated by typical saturable binding kinetics, by complete inhibition by fluid phase C3b, but not by other proteins and by nearly complete inhibition by anti-C3b receptor antibody. Several parameters that could affect the binding of C3b-coated microspheres to neutrophils were studied; these included time and temperature of incubation of the microspheres with the cells, the diameter of the microspheres, the C3b content of the C3b-coated microspheres, the presence of metal ions, azide, EDTA, protein (BSA, IgG), soybean trypsin inhibitor in the buffers, and the method of isolation of the neutrophils. The C3b-coated microspheres were evenly distributed around the neutrophils in almost all of the cases; however, the neutrophils used in these studies were not activated and were not phagocytosing. The method is extremely reproducible and sensitive in detecting small changes in number of C3b receptors on cells.

Complement C3b↗
PubMed1987

C3-mediated release of prostaglandin from human monocytes: evidence for independent regulation of thromboxane and prostaglandin synthesis.

Human mononuclear phagocytes (HMP) lose the capacity to respond to C3b, exogenous arachidonic acid (AA), and other stimuli of arachidonic acid (AA) metabolism when precultured for 24 hours prior to the addition of stimulus. Data presented in this report suggest that this reduced capacity is not due to loss of the cyclooxygenase enzyme, to autoinactivation of cyclooxygenase, or to release by the cells of inhibitors of PGE and TxB2 production. Additional metabolism of PGE or a shift of AA metabolism from the cyclooxygenase pathway to the lipoxygenase pathways does not occur and hence cannot account for the observation. HMP precultured for 24 hours with gamma interferon (IFN-gamma) retain the capacity to metabolize AA in response to subsequent exposure to C3b and LPS. HMP precultured with LPS or C3b respond to exogenous AA, but not to restimulation with LPS or C3b, by releasing PGE and to a lesser extent TxB2. The amounts of PGE and TxB2 produced under these conditions are reversed when compared to HMP treated with IFN-gamma LPS, or C3b at the initiation of culture. These data suggest that the production of TxB2 and PGE may be independently regulated in HMP and that IFN-gamma affects HMP AA metabolism by different mechanisms than LPS or C3b.

Arachidonic Acid↗
PubMed1982

Partial H (beta 1H) deficiency and glomerulonephritis in two families.

H (beta 1H) controls the C3b amplification loop by its ability to displace Bb from the alternative pathway convertase, C3b,Bb, and acts as a cofactor with I (C3b inactivator) to produce inactive C3b. Serum C3 levels are dependent to a large extent on the levels of H and I. Partial H deficiency was found in two families. The index case in Family 1 had vasculitis, thrombocytopenia, proteinuria, and depressed serum H and C3 levels. The index case in Family 2 had depressed serum H and B (Factor B) levels and IgA nephropathy which progressed to renal failure. His sister also had IgA nephropathy and depressed serum H and C3 levels. The depressed serum C3 level, B level, and H level could be responsible for the development of the immune diseases found in some members of these families.

Adolescent↗
PubMed1978

Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator.

We recently described the isolation from human serum of a high molecular weight protein with specific binding affinity for fluid-phase activated C4. We show here that the C4-binding protein (C4-Bp) functions as an essential cofactor in the proteolysis of C4b in the presence of C3b-inactivator (C3bINA). C4-bp, together with C3bINA, cleave the alpha'-chain of C4b into three fragments called alpha2, alpha3, and alpha4, with mol wt of 47,000, 25,000, and 17,000 daltons, respectively. The alpha2 fragment was dissociated from C4b without reduction, whereas the alpha3 and alpha4 fragments were disulfide bonded the other chains of C4b. The reaction did not occur when either C4-bp or C3bINA were omitted, nor in the presence of either protein in combination with beta1H. Native C4 was not affected by C3bINA aand C4-bp. C4b was not cleaved when incubated in serum of a patient with genetic deficiency of C3bINA. However, when purified C3bINA was added, the alpha'-chain of C4b was cleaved and fragments with the same molecular weight as alpha2, alpha3, and alpha4 were generated.

Carrier Proteins↗
PubMed1986

Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells.

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.

Antigen-Antibody Complex↗