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PubMed1989

Absence of induction of IL-1 production in human monocytes by complement fragments.

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.

Antigen-Antibody Complex↗
PubMed1989

[A study on complement activation in cardiopulmonary bypass--experimental and clinical evaluations].

Complement activation in patients undergoing cardiopulmonary bypass (CPB) was studied, with particular reference to anaphylatoxins. Complement levels were compared between a bubble oxygenator group (BOG) and a membrane oxygenator group (MOG). The influence of primed homologous blood on complement activation was also evaluated. C4a levels gradually increased during CPB in the BOG, whereas in the MOG they changed little. There was a marked initial rise of C4a levels in the BOG of high H/A (primed homologous vs. circulating autologous blood volume ratio). C3a levels increased more steeply in the BOG than in the MOG. In general C5a levels remained low in both groups, but were slightly higher in the BOG than in the MOG. Complement levels were determined in monkeys (Macaca fascicularis) undergoing continuous intraaortic infusion of 1) autologous blood extracorporeally bubbled with O2, 2) blood contacted with nylon, or 3) O2 microbubbles. The results indicated 1) elevation of C4a and C3a in the bubbled blood group, 2) elevation of C3a alone in the nylon group, and 3) no elevation of either anaphylatoxin. It was thus inferred that 1) during CPB, the complement might be rather dominantly activated via the classical pathway in the BOG, and via the alternative pathway in the MOG, 2) higher anaphylatoxin levels in the BOG than in the MOG might be related to mode and grade of blood trauma including plasma denaturation, and 3) the H/A ratio might be a factor able to modify complement activation.

Adolescent↗
PubMed1989

Phylogeny of regulatory proteins of the complement system. Isolation and characterization of a C4b/C3b inhibitor and a cofactor from sand bass plasma.

We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.

Amino Acids↗
PubMed1985

Influence of genetically inherited complement deficiencies on humoral immune response in guinea pigs.

To assess the role of complement in the induction of the humoral immune response, we studied the antibody response of guinea pigs genetically deficient in the second component of the classical complement pathway (C2D-GP) to bacteriophage phi X 174--a T cell-dependent antigen--in comparison with normal guinea pigs and C4D-GP, for which a disturbance in induction of antibody response has been described. We were able to establish a clear dose-response relationship: with low doses of antigen (1 X 10(9) PFU/kg), the antibody response of both complement-deficient strains was grossly impaired as compared with normal guinea pigs. After primary immunization, the peak antibody titer was diminished (1 log10) and declined rapidly; after secondary immunization, the diminution became even more distinct. Both complement-deficient strains had unusual secondary antibody responses almost identical to their primary ones, and amplification of antibody titer, as well as regular isotype switch from IgM to IgG, was absent. By increasing the antigen dose (2 X 10(9) PFU/kg), the antibody responses of the complement-deficient guinea pigs tend to normalize, and when high doses of antigen (1 X 10(10) PFU/kg) were used, the behavior of the complement-deficient animals was nearly indistinguishable from that of normal animals. Partial restoration of the immune response was seen when substituting the genetic complement deficiency by giving serum as source of the missing complement component. The important contribution of the C2 deficiency is given by the now compelling evidence that it is not the missing individual component itself, but rather the common block in sequential activation of C3 via the classical pathway in both complement deficiencies, that is responsible for the impaired humoral immune response, especially at low antigen doses. We therefore postulate that an intact classical pathway contributes to reaching a normal humoral immune response.

Animals↗
PubMed1986

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2.

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.

Amino Acid Sequence↗
PubMed1986

Functional and immunologic protein S levels are decreased during pregnancy.

Protein S, is a natural anticoagulant protein which serves as a cofactor for activated protein C. During pregnancy and in the postpartum period, functional protein S levels are significantly reduced (38% +/- 17.3%, mean +/- 1 SD) when compared to nonpregnant females (97% +/- 31.6%) (P less than 0.001). In plasma an equilibrium exists between functionally active free protein S and protein S complexed with C4b-binding protein, which is functionally inactive. As a result of this equilibrium either a decreased level of total protein S antigen or an elevation of C4b-binding protein could lead to reduced protein S activity. C4b-binding protein levels measured by enzyme-linked immunoassay are not significantly different in pregnant women versus nonpregnant controls (103.5% +/- 21.2% v 100% +/- 16.9%). However, during pregnancy and in the postpartum period, total protein S levels are reduced (68% +/- 10.7%) compared to nonpregnant controls (100% +/- 17.0%). This difference is significant at P less than 0.001. These data demonstrated that the reduction in protein S activity observed during pregnancy is a result of reduced total protein S antigen.

Carrier Proteins↗
PubMed1979

L-asparaginase-induced hypocomplementemia in acute lymphocytic leukemia (ALL) of childhood.

Serum complement studies were carried out in five children with acute lymphocytic leukemia. During therapy with L-asparaginase, prednisone and vincristine, hypocomplementemia developed in all patients, and disappeared within 2 weeks after the discontinuation of L-asparaginase. Complement breakdown products were not present in plasma. The changes of serum complement levels paralleled those of plasma fibrinogen. These findings suggest that the hypocomplementemia observed in these patients may be related to impaired protein synthesis induced by L-asparaginase.

Adolescent↗
PubMed1988

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD.

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.

Animals↗
PubMed1988

Novel subunit in C4b-binding protein required for protein S binding.

C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.

Amino Acid Sequence↗
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↗
PubMed1987

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement.

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.

Calcium↗