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PubMed1989

Activation of the alternative pathway of complement by cellulosic hemodialysis membranes.

Compared to cellulose acetate, hemodialysis with cuprophan membranes is associated with greater activation of the alternative pathway of complement. Previous studies have shown that this difference is not due to a greater number of potential covalent binding sites for activated C3 on cuprophan. To investigate further the factors that influence complement activation by hemodialysis membranes, proteins were eluted from serum-treated cuprophan and cellulose acetate membranes with hydroxylamine at alkaline pH and analyzed by SDS-PAGE and Western blot. Approximately 23 times more total protein was removed from cellulose acetate. Virtually all the C3 in the cellulose acetate eluate was in the form of inactive fragments C3c and C3dg. In contrast, the functionally active form of C3 (C3b) was a prominent constituent of the cuprophan eluate. The binding of factor B (precursor of the catalytic subunit of the C3 convertase) and factor H (regulatory protein of C3 activation) to serum-treated membranes was also analyzed. By Scatchard's method, the affinity constant at equilibrium for factor B binding (KB) to the two types of membranes was not significantly different; however, there were approximately four times more factor B binding sites on the cuprophan than on the cellulose acetate. For cuprophan, the number of factor B binding sites was 1.6 times greater than the number of factor H binding sites. These studies demonstrate that a portion of the C3b molecules that bind to cuprophan are protected from degradation, and suggest that the complement activating capacity of hemodialysis membranes is determined by biochemical properties that modulate both the binding of serum proteins to the membrane and the interactions of the endogenous regulatory proteins with membrane-associated C3b.

Cellulose↗
PubMed1982

Complement: activation, consequences, and control.

The activation of complement provides the humoral (fluid-phase) effector mechanism most responsible for immune-mediated injury. The classical pathway is activated by an antigen-antibody reaction. The binding of C1q initiates the sequential activation of the eleven proteins. The classical pathway has a calcium-dependent step (C1q, C1r, C1s) and a magnesium-dependent reaction (the enzymatic action of C1s on C4 and C2). The alternative pathway appears to be spontaneously activated, but the perpetuation of that activation is dependent upon the availability of an activating (or protective) surface which interferes with the inactivation of C3b by control proteins. The alternative pathway has a magnesium-dependent step, the binding of B to C3b to form the C3 convertase. Once initiated, the alternative pathway activation results in the sequential activation of nine proteins, six of which are common to both pathways. The activation of complement results in a variety of biologic consequences which can result in injury to the host. The potential destructiveness of the effects of complement activation is modulated by a series of control proteins.

Carrier Proteins↗
PubMed1989

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

Alleles↗
PubMedDate not supplied

Complement receptors and regulatory proteins in human atherosclerotic lesions.

Complement activation in human atherosclerotic lesions is indicated by the presence of C5b-9 terminal complexes. By using monoclonal antibodies to the complement C3b receptor (CR1) and the iC3b receptor (CR3), it was observed that approximately 20% of the cells in complicated human carotid lesions express CR1 and CR3 antigens. One to five percent of complement receptor-positive cells stained for smooth muscle cell-specific myosin, and the remainder were determined to be predominantly macrophages, based on their reactivity to anti-LeuM3 (CD14) monoclonal antibody. No C3dg receptor (CR2)-positive cells were observed in any of the eight lesions examined. The complement regulatory glycoprotein decay accelerating factor (DAF) was widely distributed extracellularly, in addition to being present on 20% to 60% of the total cell population. Factor H, a plasma protein that regulates alternative pathway C3 convertase formation, was observed extracellularly in 70% of the lesions examined. C1 inhibitor was present in a few plaque specimens, was relatively sparse, and appeared largely cell associated. Terminal C5b-9 complement complexes were pervasive in all lesions. Both the complement regulatory proteins and the activation products were limited to the area of lesion involvement and were absent from normal arterial wall. The results demonstrate that molecules involved in complement regulation and complement ligand binding are present in atherosclerotic lesions, where they may function to modulate the activities of complement.

Arteriosclerosis↗
PubMed1992

Hereditary dysfunction of the third component of complement associated with a systemic lupus erythematosus-like syndrome and meningococcal meningitis.

OBJECTIVE: We describe a dysfunction of C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. Alternative pathway complement function was absent, but classical pathway complement function was partially intact. METHODS: We used functional, preparative, and immunochemical techniques in the study. RESULTS: The patient's C3 proved normally susceptible to trypsin proteolysis and partially resistant to classical pathway, but completely resistant to alternative pathway, convertase-dependent cleavage. CONCLUSION: The dysfunction, thus, was caused by a failure of C3 to interact with the C3 convertases, rather than by a lack of a proteinase-sensitive cleavage site in the deficient protein.

Adult↗
PubMed1976

Modulation of the alternative complement pathways by beta 1 H globulin.

C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to C3b inactivator, it increases the rate of loss of hemolytic sites from EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.

Beta-Globulins↗
PubMed1995

Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein.

Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

Animals↗
PubMedDate not supplied

Inhibition of the classical and alternative pathways of the human complement system by glycosaminoglycan polysulfate.

Glycosaminoglycan polysulfate (GAGPS) concentration-dependently inhibited the activation of the classical and alternative pathways of the human complement system in vitro. Concentrations of > or = 0.2 mg/ml GAGPS prevented the cleavage of C4 by human aggregated gammaglobulin as evidence of inhibition of the classical pathway. At concentrations of > or = 0.15 mg/ml a concentration-dependent inhibition of the cleavage of factor B, the major step in the activation of the alternative pathway, was seen in the presence of inulin. Concentrations < 0.05 mg/ml did not have a measurable effect on either pathway. The lysis of sheep red blood cells, which is mediated largely by the classical pathway, was significantly inhibited at 3.84 mg/ml GAGPS, with a mean inhibition of 45.7%. On the other hand, the same concentration of GAGPS almost completely inhibited the lysis of rabbit red blood cells, which is mediated by the alternative pathway of complement. Our results suggest that the inhibition by GAGPS is an early event in the activation of complement, occurring before the assembly of the C3 convertases of either pathway. The possible use of this drug in acute life-threatening situations where complement is thought to have a pathogenic role is discussed.

Animals↗
PubMed1986

C3 activation by a new factor B-dependent enzyme detected in culture supernatant from guinea-pig peritoneal macrophages.

Culture supernatants (c.s.) collected from thioglycollate-elicited macrophages were concentrated and incubated with purified C3. In this reaction mixture loss of haemolytic C3 according to classical enzyme kinetics was observed. As revealed by SDS-PAGE, c.s.-catalysed fragmentation of the C3 alpha-chain occurred. The cleavage products were identified by size and function as C3a and C3b. The apparent molecular weight of this C3-activating enzyme in c.s. was approximately 220,000 according to ultracentrifugation studies. This large enzyme showed the following characteristics: it had no activity against C5; it was inhibited by EDTA; Mg2+ was required for its optimal function; its half-life at 37 degrees was approximately 35 min; it was completely inhibited by anti-B IgG. Thus, we were able to detect a C3-activating enzyme in c.s. containing B but differing otherwise from a preformed C3 convertase of the alternative pathway. The exact component composition of this new enzyme is under further investigation.

Animals↗
PubMed1986

Guinea pig complement fixation by tissues from hypocomplementaemic renal diseases.

Recently many studies have been done to identify complement pathway activation in renal tissue from patients with renal disease. We examined whether tissues obtained by renal biopsy from such patients would fix guinea pig complement. Nine out of 15 patients with systemic lupus erythematosus (SLE), 4 out of 7 patients with mesangiocapillary glomerulonephritis (MCGN), and 2 out of 7 cases with acute glomerulonephritis (AGN) fixed guinea pig C3. We found that tissues from 5 out of 9 guinea pig C3-positive SLE cases fixed guinea pig C4, while none of the guinea pig C3-positive tissues from patients with MCGN or AGN fixed guinea pig C4. These guinea pig C3-positive renal tissues were further studied for interaction with C4-deficient guinea pig serum, EDTA guinea pig serum, heated guinea pig serum, and EGTA Mg2+ guinea pig serum. The results indicated that activation of both the alternate and classical complement pathways occurred with tissues from patients with SLE, while activation of the alternate pathway occurred with MCGN and AGN. Results for tissues from AGN and MCGN patients indicated the presence of C3 convertase and protease which interacted with guinea pig C3.

Biopsy↗
PubMed2003

Prolonged retention of vaccinia virus complement control protein following IP injection: implications in blocking xenorejection.

The vaccinia virus complement control protein (VCP) blocks classic and alternate complement pathways by binding to the third and fourth complement components and by blocking the formation of the C3-convertase as well as by accelerating the decay of the C3 and C4 convertase. The therapeutic potential of VCP has been extensively studied for brain injury, xenotransplantation, Alzheimer's disease, and spinal cord injury. We investigated the pharmacokinetic behavior of rVCP in mice. Dosage of rVCP was studied by injecting different concentrations of rVCP. A 25 mg/kg or greater dose injected intraperitoneally was found to be adequate to suppress complement for more than 8 hours.

Animals↗
PubMed1984

Activation of vertebrate complement by Helix pomatia haemolymph.

Haemolymph plasma from the pulmonate snail Helix pomatia contains a constituent, not yet identified, which causes activation of vertebrate complement via the alternative complement pathway in fluid phase. The activation of vertebrate complement by snail plasma is closely analogous to the activation caused by cobra venom factor (CVF), the snake's C3b, with one notable exception; the snail factor requires vertebrate C3 for the formation of C3 convertase which cobra venom factor does not. Our results do not allow any definite conclusion on the exact mechanism but we favour the idea that the haemolymph contains a complement-like protein which functions as an opsonin in the snail, and which can interact with vertebrate alternative complement pathway components.

Animals↗
PubMed2005

Binding of C3 fragments on top of adsorbed plasma proteins during complement activation on a model biomaterial surface.

In the present study we investigate whether complement activation in blood in contact with a model biomaterial surface (polystyrene) occurs directly on the material surface or on top of an adsorbed plasma protein layer. Quartz crystal microbalance-dissipation analysis (QCM-D) complemented with enzyme immunoassays and Western blotting were used. QCM-D showed that the surface was immediately covered with a plasma protein film of approximately 8 nm. Complement activation that started concomitantly with the adsorption of the protein film was triggered by a self-limiting classical pathway activation. After adsorption of the protein film, alternative pathway activation provided the bulk of the C3b deposition that added 25% more mass to the surface. The build up of alternative pathway convertase complexes using purified C3 and factors B and D on different protein films as monitored by QCM-D showed that only adsorbed albumin, IgG, but not fibrinogen, allowed C3b binding, convertase assembly and amplification. Western blotting of eluted proteins from the material surface demonstrated that the C3 fragments were covalently bound to other proteins. This is consistent with a model in which the activation is triggered by initiating convertases formed by means of the initially adsorbed proteins and the main C3b binding is mediated by the alternative pathway on top of the adsorbed protein film.

Adsorption↗
PubMed1987

C3b generation is affected by the structure of the O-antigen polysaccharide in lipopolysaccharide from salmonellae.

Salmonellae differing in the O-antigen side chain of their lipopolysaccharide were previously shown to activate the alternative pathway of complement to different extents. We now examine the generation of the major cleavage fragment of the complement component C3 (C3b) on these bacteria in a system that contains the purified components C3, B, D, and P but lacks the regulatory proteins H and I. The deposition of C3b in this system reproduces the same pattern obtained earlier with the use of whole serum, with the expected differences among the strains bearing different O-antigen. However, two distinct mechanisms for these differences in C3b generation became apparent. The intermediate activating strain showed 3 to 4 times less initial deposition of C3b than the other two strains. In contrast, the least activating strain showed adequate initial deposition but poor amplification, as shown by 2 to 3.4 lower amplification indexes as compared with those on the other two strains. Binding studies with factor B showed that decreased C3 convertase formation was responsible for the low amplification on this strain. Only 25% of the C3b bound to its surface was able to bind factor B with a high affinity, in comparison with 90% on the other two strains. No differences were found for the binding of factor H among the strains. These studies identify the molecular mechanisms by which these bacteria avoid complement activation.

Antigens, Bacterial↗
PubMed1984

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B.

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.

Animals↗
PubMed1981

Complement receptor is an inhibitor of the complement cascade.

A glycoprotein from the membrane of human erythrocytes has been identified as a receptor for C3b (CR1). It promotes the dissociation of the alternative pathway C3 convertase C3b,Bb and the cleavage of C3b by C3b/C4b inactivator. We find that CR1 also inactivates the C3 and C5 convertases of the classical pathway. CR1 inhibits the consumption of C3 by C3 convertase EAC142 and enhances the decay of C4b,2a sites. On a weight basis, CR1 is approximately 5-10 times more active than C4 binding protein, a serum inhibitor of C4b,2a. The binding of 125I-CR1 to EAC14 cells is inhibited by C2. Therefore, it is likely that CR1 and C2 compete for a site on C4b. CR1 inhibited C5 convertase even more effectively, but had no effect on the assembly of the late complement components. At high concentrations, CR1 alone has no irreversible effects on cell-bound C4b. In the fluid phase, CR1 can function as a cofactor for the cleavage of the alpha' chain of C4b by C3b/C4b inactivator. A well-known function of CR1 is to promote adherence of microbes or immune complexes bearing C3b and C4b to cells. This interaction could result in a microenvironment damaging to the plasma membrane of the responding cell because the extrinsic C3b and C4b fragments can serve as additional sites of assembly of enzymes of the cascade. We therefore wish to propose that CR1 on the surface of cells supplies an increased local concentration of a strong inhibitor of the amplifying enzymes of the complement system and provides cells with a mechanism for circumventing damage when they bind C3b- and C4b-bearing substrates.

Complement C3-C5 Convertases↗