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PubMed1986

Schistosoma mansoni: surface membrane stability in vitro and in vivo.

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.

Animals↗
PubMed1989

In-vitro lysis of sensitized Trypanosoma cruzi by platelets: role of C3b receptors.

Incubation of Trypanosoma cruzi bloodstream trypomastigotes (Btrys) with C5-deficient blood in the presence of anti-T. cruzi immune mouse serum (IMS) or its IgG fraction resulted in an immediate formation of small clumps in which one could easily see platelets adhered to the parasites. After 4 h of incubation most of the clumps had disappeared and the number of the parasites was considerably reduced. Twenty-four hours later there were only a few remaining parasites. This same sequence of events was also observed when the parasites were incubated with isolated platelets in presence of IMS or its IgG fraction. When the parasites were incubated with plasma in the presence of IMS or its IgG fraction but in the absence of platelets there was strong agglutination of the parasites but no reduction in their number. Incubation of the parasites with platelets and IMS or its IgG fraction in C3-depleted plasma prevented adherence of the platelets to the parasites and their subsequent lysis. It is concluded that platelets are able to induce in-vitro lysis of the Btrys and that this phenomenon is dependent on the platelet' receptor for C3b.

Agglutination↗
PubMed1986

G6PD-deficiency infectious haemolysis: a complement dependent innocent bystander phenomenon.

Dramatic haemolysis may accompany viral hepatitis and pneumococcal pneumonia in G6PD-deficient patients. Since red blood cells (RBCs) are richly endowed with receptors for activated complement, particularly C3b, we hypothesized that bulky, complement-activating immune complexes (IC) consisting of microbes and antibody might attract granulocytes (PMNs), facilitating oxidative 'innocent bystander' RBC damage. Indeed, opsonization with only two type-2 pneumococcus (PN3)/anti-PN3/C3b complexes per RBC caused agglutination of RBC, a phenomenon termed immune adherence. Addition of as few as one PMN per 20 opsonized RBCs caused the glutathione (GSH) levels of co-incubated G6PD-deficient RBCs to fall by 30% (from 3.5 to 1.8 +/- 0.8 mumoles GSH/g Hb) compared to identically incubated, but nonopsonized, G6PD-deficient RBCs. GSH levels remained normal (5.2 +/- 0.4 mumoles/g Hb) in PMN-exposed opsonized normal RBCs. GSH depletion in G6PD-deficient RBC was directly related to disease severity--falling a mean 33% in RBCs from two Black G6PD A- subjects but 59% in two Caucasian G6PD deficient RBCs. Prevention of C3b generation (with 10 mM EDTA) during opsonization abrogated both immune adherence and PMN-mediated GSH decline in oxidant-sensitive cells. Similarly, removal of C3b receptors by brief trypsin incubation of RBCs eliminated immune adherence and GSH decline. Thus, both phenomena are dependent on IC complement activation and subsequent binding of the bacterial IC to the RBC complement receptors. Although clearance of IC by RBCs may be beneficial in protecting other tissues from inflammatory damage, G6PD-deficient RBCs are vulnerable to oxidants generated by juxtaposed phagocytes--cells attracted to, and stimulated by, the immune complex/C3b combination. It is suggested that this 'Good Samaritan' activity of RBCs may lead to haemolysis during periods of exuberant antibody response to microbes.

Anemia, Hemolytic↗
PubMed1991

Formation of covalent C3b-tetanus toxin complexes: a tool for the in vitro study of antigen presentation.

A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin, TT), using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage of C3 after co-precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation; under conditions which minimized the formation of covalent C3b multimers, 30% and 8% respectively of C3b and TT were incorporated into covalent one-to-one complexes which were purified using gel filtration chromatography. The linkage was localized between the alpha' chain of C3b and either the H or L chain of TT; it required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl2, a thiol reagent which inhibits the esterase-like activity of bound C3b. These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT. Such C3b-TT complexes provide a defined tool to analyse the influence of antigen-bound C3b on antigen addressing and intracellular processing by antigen-presenting cells.

Animals↗
PubMed1988

C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.

Animals↗
PubMed2001

Expression and regulation of complement factors H and I in rat and human cells: some critical notes.

The complement factors I (FI) and H (FH) are complement regulatory proteins. FI, a highly glycosylated serine protease of 88 kDa cleaves the alpha-chains of both complement components C3b and C4b, thereby inactivating them. Complement FH, a glycoprotein of 150 kDa which is composed of 20 short consensus repeats synergizes with FI by increasing the affinity of FI for C3b in the C3b/FH complex by about 15-fold as compared to free C3b. Furthermore, FH prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. Both, FI and FH are mainly synthesized in the liver. According to the quantification of specific mRNA of both factors, various amounts are produced by different liver cell types, i.e. hepatocytes (HC) and Kupffer cells (KC). Investigations of cultured primary HC and KC from rat liver showed that FI is exclusively synthesized and secreted by HC whereas FH is synthesized by both HC and KC. Using quantitative-competitive PCR for the quantification of FH-specific mRNA, its constitutive rate of synthesis was found to be nearly ten times higher in KC than in HC. An extrahepatic source of both proteins are human umbilical vein endothelial cells (HUVEC) in which the synthesis of FI is upregulated by IL-6 which is in accord with the upregulation observed in rat HC and two rat hepatoma cell lines (FAO and H4IIE). Three other proinflammatory cytokines, IL-1beta, IFN-gamma and TNF-alpha, were alone or in combination, without any effect on the regulation of FI. This demonstrates that the regulation of FI is similar in HUVEC and HC. These results are in contrast to a previously described IFN-gamma-mediated upregulation of FI in HUVEC and suggest, in accordance with other investigations on extrahepatic sources of FI (e.g. myoblasts), that IFN-gamma has probably no prominent role in the regulation of FI. Instead, IL-6 appears to be the main upregulating cytokine of FI mRNA and of FI protein synthesis in HC as well as in rat and human hepatoma cells and in HUVEC. Of note are experiments by others and us who could not identify FI-specific mRNA in peripheral blood-derived monocytes, granulocytes, or B- and T-cells of man or rat and in rat peritoneal macrophages. FI-specific mRNA could also not be detected in B- or T-cell lymphoma cells, whereas FH-specific mRNA was easily detectable in both human and rat monocytes, and in rat peritoneal macrophages. These data support the notion that FI in contrast to FH is not expressed by cells of the monocyte-macrophage lineage or by other leukocytes of peripheral blood, at least in the absence of additional stimulants.

Animals↗
PubMed1987

Complement fixation by small, DNase-resistant DNA-anti-DNA immune complexes.

125I-ds DNA-anti-DNA immune complexes (IC) formed at antibody excess and containing DNA of 300-350 base pairs (bp) fixed complement, incorporated C3b and bound to the C3b receptors (CR1) on human red blood cells (RBC). When the IC were treated with DNase to generate small, DNase-resistant IC, some of the IC incorporated C3b, but did not bind to RBC. In order to examine C3b incorporation and RBC binding by IC of specific sizes, the DNase treated IC were fractionated by sucrose density gradient (SDG) ultracentrifugation. Small IC containing one, two, three or four IgG molecules per fragment of 125I-ds DNA were identified by autoradiography after electrophoresis of the SDG fractions on 3-12% linear polyacrylamide gradient gels. The SDG fractions were tested for C3b incorporation and RBC binding ability. There was neither C3b incorporation nor RBC binding activity in fractions which corresponded to 9-11S (containing IC with one IgG/DNA). Fractions which corresponded to 12-22S (containing IC with up to four IgG/DNA fragment) demonstrated increased C3b incorporation with increased size, but did not show significant RBC binding activity. Fractions with IC containing four or more IgGs (22-24S) incorporated C3b and bound to RBC at approximately the same level. It is concluded that DNase digested IC which contain three-four IgG/DNA fragment are large enough to activate complement and incorporate C3b, but are too small to bind to RBC CR1. These IC could therefore escape rapid clearance from the circulation via the erythrocyte CR1 clearance mechanism. Such IC could persist in the circulation and potentially elicit pathogenic effects in patients with systemic lupus erythematosus.

Antibodies, Antinuclear↗
PubMed1990

Glomerular CR1 express in situ cofactor activity for degradation of C3b.

Adherence of sheep erythrocytes (E) sensitized with IgM antibodies (A) and C3b (EAC3b) to C3b/C4b receptors (CR1) in cryostat sections of human renal glomeruli was studied using the closed chamber technique. The adsorption was stable for at least 3 h at 37 degrees C. In the presence of purified factor I, the indicator cells, however, detached from the sections after 30 min at 37 degrees C. Factor H was not required. The release was not due to loss of CR1 activity in the tissue. The detached indicator cells were negative in the immune adherence test and were agglutinated by antibody to C3d, but not by antibody to C3c. Western blot of the detached indicator cells revealed the presence of C3d and C3c was found in the chamber fluid. Accordingly, detachment of the indicator cells was due to degradation of C3b to C3d with the release of C3c into the chamber fluid. Protease inhibitors did not prevent the detachment of the indicator cells. EAC3b incubated with sections of renal glomeruli preincubated with anti-CR1 antibody were not degraded. The results therefore indicate that CR1 in situ in renal glomeruli can provide the necessary cofactor activity for factor I-mediated degradation of C3b to C3d and C3c.

Complement C3b↗
PubMed1992

Genetics of the complement system and rheumatic diseases.

The complement system, especially the early components of the classic pathway, are critically involved in immune complex processing. The deposition of clusters of complement component C3b on a target marks it for elimination, primarily through an interaction with complement receptors. Not surprisingly, total and partial deficiencies of certain complement components and C3b receptors are associated with rheumatic diseases, particularly systemic lupus erythematosus. This predisposition is explicable, based on the critical role complement plays in immune complex handling.

Complement Factor B↗
PubMed1978

Initiation of the alternative pathway of complement: recognition of activators by bound C3b and assembly of the entire pathway from six isolated proteins.

An intact alternative pathway of complement activation was assembled from six isolated proteins present at their respective physiological concentrations (C3, 1200 microgram/ml: factor B, 200 microgram/ml; factor D, 2 microgram/ml; beta1H, 560 microgram/ml; C3b inactivator, 34 microgram/ml; and native properdin, 20 microgram/ml). Initiation of the pathway required the presence of five of these proteins not including properdin. The initial C3 convertase of the system was shown to be a fluid-phase rather than a surface-bound enzyme. The ability of the pathway to discriminate between activator and nonactivator was found to reside in the bound C3b molecule. When bound to the surface of an activator through its labile binding site, C3b interacts with surface structures of the activator through another site on the molecule. This interaction results in diminished beta1H binding to C3b and thereby allows the bound C3b molecule to escape control and participate in C3 convertase formation. Thus, initiation of the alternative pathway is a two-step process, the first being non-specific and the second being discriminatory.

Binding Sites↗
PubMed1986

Control of C1 activation by nascent C3b and C4b: a mechanism of feedback inhibition.

We have demonstrated that immune complexes turn over C1, i.e., limiting quantities of immune complexes activate an excess of C1. This was readily apparent in a system of purified C1 and C1-inhibitor (C1-In) but not in normal human serum (NHS). The following results indicate that C3 and C4 are the serum factors responsible for the inhibition of C1 turnover by immune complexes. 1) In a purified protein system composed of C1 and C1-In at pH 7.5, ionic strength 0.14 M, doses of immune complexes that activated all the C1 in 60 min at 37 degrees C yielded no detectable C1 activation when C2, C3, and C4 were also present. All proteins were at their physiologic concentrations. Activation was quantified by SDS-PAGE analysis and hemolytic titration 2) In order to inactivate C3 and C4, NHS was treated with 50 mM methylamine (MeAm) for 15 min at 37 degrees C, after which the MeAm was removed by dialysis. The activities of C1, C2, and C1-In were unaffected by this treatment. Doses of immune complexes that consumed no C1 in NHS, consumed all the C1 in MeAm-treated NHS (MeAm-NHS). 3) Reconstitution of MeAm-NHS with physiologic concentrations of C3 and C4 rendered the serum again resistant to excessive C1 consumption by immune complexes. Immune complexes used in these studies included EA-IgG, EA-IgM, tetanus-human anti-tetanus, and aggregated human IgG. There appeared to be specificity to the inhibition reaction since C4 by itself could inhibit C1 consumption by EA-IgM, whereas the presence of C3 was also required to control EA-IgG. Finally, N-acetyl-L-tyrosine was added to NHS at a final concentration of 30 mM. This nucleophile did not interact with native C3 or C4, nor did it directly activate C1. However, upon the addition of low doses of immune complexes, acetyl tyrosine did yield uncontrolled C1 activation, presumably by binding nascent C3b and C4b and thereby blocking their attachment to the immune complexes. We conclude that in NHS there is a mechanism of feedback inhibition by which nascent C3b and C4b inhibit C1 turnover by immune complexes. This mechanism of control might be physiologically important in that it prevents excessive complement activation by low concentrations of immune complexes.

Antigen-Antibody Complex↗
PubMed1987

Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

Animals↗
PubMed1980

Metabolism of C4 and factor B in rheumatoid arthritis. Relation to rheumatoid factor.

Metabolic turnover determined by radioiodide labeled C4 and Factor B was studied in 18 patients with rheumatoid arthritis (RA) and 19 normal control subjects as a means of estimating the relative ratio of consumption of components in the classical and alternative pathways of complement activation. Predominance of fractional catabolic rate (FCR) of C4 over Factor B was demonstrated with differentially labeled C4 and Factor B. The hypercatabolism occurred in the extravascular space. C4 FCR correlated significantly with rheumatoid factor (RF) determined in a hemolytic assay (rs = 0.72), measured as IgG RF (rs = 0.57), and as IgM RF (rs = 0.45). There were no significant correlations with several other antibodies measured. These results are consistent with the hypothesis that RA is a systemic, extravascular immune complex disease, in which RF immune complexes play a significant pathogenetic role principally via activation of the classical pathway of complement.

Adult↗
PubMed1984

Mechanism of activation of the classical pathway of complement by monoclonal IgE (DES). Restricted regulation of C4b by C4b-binding protein.

A human monoclonal IgE from patient DES, IgE (DES), has been shown to activate the classical pathway of complement. The mechanism of this activation has been investigated and can be summarized as follows: (a) IgE (DES) is able to bind and activate C1 in a dose-dependent fashion. This activation increases with the size of the aggregates used, but the affinity of C1 for IgE (DES) is weaker than for IgG. (b) A classical pathway C3 convertase can be assembled on IgE (DES) using purified C1, C4 and C2. The formation decay of this convertase is similar to that formed on IgG with an half-life of 9 min at 37 degrees C. (c) The extrinsic regulation of the C3 convertase by C4bp is restricted on IgE (DES) as compared to IgG. This restriction is shown on both the formation and the decay of the convertase. The mechanism of activation of the classical pathway of complement by IgE (DES) thus present some similarities with the assembly of the C3 convertase by the alternative pathway.

Antibodies, Monoclonal↗
PubMed1987

Human complement factor H: expression of an additional truncated gene product of 43 kDa in human liver.

The human complement factor H is an important factor in the control of the alternative pathway and also induces the stimulation of B cells and macrophages in vitro. Using a human factor H cDNA clone as probe, two factor H-specific transcripts of 4.4 and 1.8 kb were detected in four human livers. Both mRNAs were found independently of the expression of an acute phase marker SAA, in these livers, indicating that their presence is not linked to an acute phase state. The shorter transcript was cloned in two cDNA plasmids H-19 and H-20, lacking only seven amino acids of the N-terminus of the mature factor H protein. The deduced protein sequence showed that this protein is identical to the N-terminal portion of the large classical factor H of 150 kDa mol. mass. Parallel to the finding that the N-terminal sequence of factor H is expressed by two distinct mRNA species, evidence is presented that the C-terminal sequence is also contained on two different transcripts, the common 4.4-kb mRNA and an additional 1.0-kb mRNA. A novel, short form of human complement factor H of 43 kDa was detected in human sera which represents the translation product of the 1.8-kb factor H-specific mRNA detected in human liver. Five distinct epitopes detected with 6 monoclonal antibodies are present on both the 150-kDa factor H protein and the truncated 43-kDa molecule. We conclude that additional H-specific mRNAs are found in human liver and that at least one of them is translated yielding a truncated form of factor H.

Amino Acid Sequence↗
PubMed1990

Expression of complement alternative pathway proteins by endothelial cells. Differential regulation by interleukin 1 and glucocorticoids.

We have studied the secretion of proteins of the alternative pathway of complement C3, factor B and factor H by human umbilical vein endothelial cells (HUVEC). Results showed that factor H and factor B are quantitatively secreted in abundance whereas C3 could only be detected when the cells are maintained in culture during long periods of time. Interferon-gamma stimulated factor H, factor B and, to a lesser extent, C3 secretions. Interleukin (IL) 1 had a differential effect on spontaneous C3, factor B and factor H secretions. In the presence of IL 1, there was a significant secretion of C3 occurring within a short period of culture. IL 1 also stimulated factor B secretion. There was a synergistic stimulating effect between IL 1 and interferon-gamma to bring C3 and factor B productions by HUVEC to very high levels. In contrast, factor H secretion was consistently inhibited by IL 1. Local increase in C3 and factor B secretions by endothelial cells in the presence of IL 1 may have important implications in the inflammatory reaction. In striking contrast, the glucocorticoid dexamethasone (DXM) had modulatory effects which are consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, decreased C3 and factor B secretions and increased factor H secretion. Local modulation of complement protein secretion by DXM appears to be a new mechanism by which this glucocorticoid may control inflammation.

Complement C3b Inactivator Proteins↗
PubMed1991

Human complement factor H: two factor H proteins are derived from alternatively spliced transcripts.

The human complement factor H is an important component in the control of the alternative pathway of complement activation. We have previously shown that a least three factor H homologous mRNA species of 4.3 kb, 1.8 kb and 1.4 kb in length are constitutively expressed in human liver. In addition, several factor H-related proteins have been detected in human sera using antibodies directed against the classical human factor H glycoprotein of 150 kDa. The structure of the additional polypeptides has not been shown so far. Circumstantial evidence suggests that the 1.8-kb mRNA might encode the 43-kDa factor H-like polypeptide. Here we report the isolation, characterization and eukaryotic expression of the first full-length cDNA representing the major 4.3-kb mRNA and the 1.8-kb mRNA of human factor H. We show that the 4.3-kb transcript encodes the 150-kDa-factor H glycoprotein and the 1.8-kb mRNA the 43-kDa factor H polypeptide. The identity of the two cDNA in a region of 1400 nucleotides suggests that the two factor H-related transcripts are derived from one gene by a process of alternative splicing.

Amino Acid Sequence↗