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PubMed1991

The effect of antibody isotype and antigenic epitope density on the complement-fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions.

A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.

Animals↗
PubMed1996

Genetic polymorphism of the fourth component of human complement: population study and proposal for a revised nomenclature based on genomic PCR typing of Rodgers and Chido determinants.

The fourth component of human complement (C4) is coded for by two homologous genes, C4A and C4B, located in the class III region of the major histocompatibility complex (MHC). Genetic typing of C4A and B alleles is routinely carried out by high-voltage agarose gel electrophoresis. The electrophoretic C4 polymorphism can be further subdivided by the Rodgers (Rg) and Chido (Ch) blood groups, which are antigenic determinants of the C4A and B alpha-chains, respectively. We have used a recently described direct PCR typing method using sequence-specific primers (PCR-SSP) in combination with electrophoretic C4 typing as well as genomic RFLP analysis to determine the frequency of C4 allotypes, Rg/Ch subtypes and C4A-B haplotypes in a family study of the German population. As the current C4 allele designation does not provide any information about the presence or absence of Rodgers and Chido antigens, we have developed an extension to the existing C4 nomenclature. This revised allele designation combines the existing numerical allotypes defined by electrophoretic mobility with eight subtypes (01-08) based on Rg/Ch PCR genotyping results. Using this approach, most electrophoretic allotypes could be subdivided. Among the C4A allotypes, the most common allele was A*0301 (59.9%), and the most common subtype among all electrophoretic allotypes was 01 (85.1%; = Rg1,2-positive, Ch-negative). For C4B, the most common allele was B*0101 (64.3%), and the most common subtype was 01 (79.6%; = Ch1,2,3,4,5,6-positive, Rg-negative). The subtypes 03, 04, 07 and 08 of the C4A allotypes, and the subtypes 03, 07 and 08 of the C4B allotypes, were not detected in this study. The analysis of duplicated C4 alleles revealed considerable heterogeneity of their subtypes. The results demonstrate that all known C4 allotypes can now be assigned unambiguously, which facilitates the identification of MHC haplotypes relevant for transplantation and disease association studies.

Complement C4↗
PubMed1989

Possible association of sudden infant death with partial complement C4 deficiency revealed by post-mortem DNA typing of HLA class II and III genes.

Based on evidence of an increased rate of respiratory infections in sudden infant death (SID) infants as well as the observation of familial occurrence, we analysed in a retrospective study class II and class II genes of the major histocompatibility complex in 40 cases of SID by Southern blot analysis of DNA obtained post mortem from tissue samples. In 24 cases, the parents were interviewed and confirmatory human lymphocyte antigen (HLA) and DNA typing was carried out. Using HLA-DR beta and -DQ beta probes, no evidence of an abnormal HLA-DR frequency distribution in SID infants was detected (P = 0.97). Using DNA probes for the tandemly arranged complement C4 and steroid 21-hydroxylase genes, an increased number of C4B gene deletions in SID cases was found. The increase in C4 gene deletions was significant (P = 0.0125) in infants with recurrent infections. These data indicate a possible role of partial C4 deficiency as a genetically predisposing risk factor in SID.

Complement C4↗
PubMed2003

A CD40 bridge between innate and adaptive immunity.

In this issue of Immunity, Brodeur et al. show that C4b binding protein (C4BP), a regulator component of the classical complement (C) pathway, can bind to CD40 receptors on B cells and activate them. This suggests a novel way by which CD40 may function to bridge innate and adaptive immune responses.

Animals↗
PubMed2003

Evasion of phagocytosis through cooperation between two ligand-binding regions in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes is a major bacterial virulence factor that confers resistance to phagocytosis. To analyze how M protein allows evasion of phagocytosis, we used the M22 protein, which has features typical of many M proteins and has two well-characterized regions binding human plasma proteins: the hypervariable NH2-terminal region binds C4b-binding protein (C4BP), which inhibits the classical pathway of complement activation; and an adjacent semivariable region binds IgA-Fc. Characterization of chromosomal S. pyogenes mutants demonstrated that each of the ligand-binding regions contributed to phagocytosis resistance, which could be fully explained as cooperation between the two regions. Deposition of complement on S. pyogenes occurred almost exclusively via the classical pathway, even under nonimmune conditions, but was down-regulated by bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis. Different opsonizing antisera shared the ability to block binding of both C4BP and IgA, suggesting that the two regions in M22 play important roles also under immune conditions, as targets for protective antibodies. These data indicate that M22 and similar M proteins confer resistance to phagocytosis through ability to bind two components of the human immune system.

Amino Acid Sequence↗
PubMed1995

Isoforms of human C4b-binding protein. I. Molecular basis for the C4BP isoform pattern and its variations in human plasma.

Human C4b-binding protein (C4BP) is an important regulator of the complement system that also binds and inactivates the anticoagulant vitamin K-dependent protein S. These two activities are performed by two distinct polypeptides of 70 kDa and 45 kDa known as alpha- and beta-chains, respectively. C4BP is present in plasma in various isoforms with different alpha beta composition. Here we report multiple discrete variations of the relative levels of the C4BP isoforms among normal individuals and provide evidence that they are determined by genetic factors that segregate with the regulator of complement activation gene cluster. We also report the characterization of the C4BP molecules secreted by HepG2 and Hep3B cells, as well as transfection experiments in COS cells, to illustrate that the relative levels of expression of the C4BPA and C4BPB genes play a major role in determining the proportion in which the different C4BP isoforms are synthesized. Altogether, the data indicate that the human C4BP isoform pattern is genetically determined, but can be modified by factors with a differential effect on the expression of the C4BPA and C4BPB genes. These observations provide a new way to explore the possible association between elevated levels of C4BP and an increased risk to thromboembolic disorders.

Animals↗
PubMed1980

Interaction between the labile binding sites of the fourth (C4) and fifth (C5) human complement proteins and erythrocyte cell membranes.

We have shown that the labile binding site of C3b interacts covalently with receptive surfaces. We report here an analogous study of the interaction between the labile binding sites of the closely related complement proteins, C4 and C5, with sheep erythrocyte membranes. We find that i) C4b binds covalently to cell surface components; ii) the bond between C4b and receptive molecules is hydroxylamine sensitive; iii) the alpha-polypeptide of C4b binds to receptive molecules; and iv) C5b does not interact covalently with cell surfaces.

Autoradiography↗
PubMed2000

Evolutionary relationships among proteins encoded by the regulator of complement activation gene cluster.

Evolutionary relationships among members of the regulator of complement activation (RCA) gene cluster were analyzed using neighbor-joining and parsimony methods of phylogenetic tree inference. We investigated the structural and functional similarities among short consensus repeats (SCRs) of the following human proteins: the alpha chain of the C4b-binding protein (C4bpalpha), factor H (FH), factor H-related proteins (FHR-1 through FHR-4), complement receptors type 1 (CR1) and type 2 (CR2), the CR1-like protein (CR1L), membrane cofactor protein (MCP), decay accelerating factor (DAF), and the sand bass proteins, the cofactor protein (SBP1) and its homolog, the cofactor-related protein (SBCRP-1). Also included are the beta chain of the human C4b-binding protein (C4bpbeta) and the b subunit of human blood-clotting factor XIII (FXIIIb). Our results indicate that the human plasma complement regulators, FH and C4bpalpha, fall into two distinct groups on the basis of their sequence divergence. Homology among RCA proteins is in agreement with their chromosomal location, with the exception of C4bpbeta. The evolutionary relationships among individual short consensus repeats are confirmed by the exon/intron structure of the RCA members. Structural similarities among repeats of the RCA proteins correlate with their functional activities and demonstrate the importance of the N-terminal SCRs.

Complement Activation↗
PubMed1988

Structural superfamilies of the complement system.

The complete primary structures of nearly all the components, regulatory proteins and membrane-associated receptors of the complement system have now been determined by a combination of protein and cDNA sequence analysis. Based on homologies at the amino acid sequence level, three distinct classes of protein domain are evident. They are the C3, C4, C5 family; the serine proteases, and the short consensus repeat family, which is characterized by the widespread occurrence of a novel 60 amino acid repeat structure. A particular feature of the repeat structures in this last family is that they form the structural and functional domains of a range of complement proteins of distinct function, all of which interact with C3b and/or C4b. Detailed analysis of the gene structures of some of these proteins has been carried out. The results have shown that the intron/exon organization of the serine protease domains of factor B and C2 are related to the classical serine proteases at the DNA level. Each short consensus repeat in factor B, C2, C4b-binding protein, murine factor H and also the functionally unrelated interleukin-2 receptor in all except one case is encoded exactly by a single exon, which defines this novel structural unit at the DNA level.

Amino Acid Sequence↗
PubMed1981

Complete primary structure of human C4a anaphylatoxin.

C4a anaphylatoxin is derived from the fourth component (C4) of the blood complement system. The C4 alpha-chain is selectively cleaved between positions 77 and 78 by the protease C1s, a subcomponent of C1, generating the fragments C4a and C4b. Human C4a was isolated directly from fresh serum after C1 of the classical pathway of complement was activated by heat-aggregated gamma-globulin. The C4a anaphylatoxin is a cationic polypeptide of Mr = 9000 composed of 77 residues and devoid of histidine, tryptophan, and carbohydrate. The primary structure of human C4a was deduced from sequence analysis of two cyanogen bromide fragments and of peptides obtained after chymotryptic digestion of the COOH-terminal cyanogen bromide fragment. The proposed sequence is: (formula, see text) Manual alignment of the linear structures of human C3a, C4a, and C5a, based primarily on the location of two Cys-Cys sequences in each indicate a 30% homology between C3a and C4a and a 36% homology between C5a and C4a. It was concluded from the sequence comparison that C3a, C4a, and C5a are a family of bioactive factors derived from precursor molecules that share a common genetic origin. Although the human anaphylatoxins share a partial structural identity and express similar biological activities, these factors ae immunologically distinct molecules having no antigenic determinants in common as judged by radioimmunoassay.

Amino Acid Sequence↗
PubMed2004

Immunological analysis in paediatric HIV patients at different stages of the disease.

There are only few clinical studies on complement in well-defined (or characterized) paediatric HIV patients. Aim of this study was to evaluate the complement system and immunoglobulins in HIV-infected children and to correlate data to stage of disease. Blood samples of 127 HIV-infected children (11-134 months; 62 male : 65 female) were collected in order to evaluate humoral immunity. The patients were classified according to CDC clinical (N-asymptomatic; A-mild symptoms such as common recurrent infections; B-moderate symptoms such as Candidiasis and herpes infections, meningitis, sepsis and anaemia; C-severe symptoms such as opportunistic infections and neoplasia) and with respect to immunological criteria (T CD4(+) cell count). Analysis of complement system included the classical (CH50), alternative (APH50) pathway activities and plasma concentrations of mannan-binding lectin (MBL), of the C4 allotypic variants C4A and C4B. (ELISA), and of the C3 split product C3d (rocket immunoeletrophoresis). Immunodiagnosis also included CD4(+) and CD8(+) lymphocyte count and immunoglobulin concentrations. Complement activation and consumption was observed in all patients correlating with disease activity. Activated classical and alternative pathways and elevated C3d were significantly correlated with immunologic category 3. C3d levels were also significantly correlated with immunologic category 1. Undetectable CH50 and APH50 were found in two (group C) and 10 patients (n = 2, A = 2, B = 2, C = 4), respectively. Low MBL values were found in 13/127 but without correlation to disease severity. Undetectable C4B levels were observed in three patients, favouring the diagnosis of a complete deficiency. Although not related to clinical symptomatology, a strong ongoing complement activation can be observed in all stages of HIV infection. In contrast to earlier reports MBL could not be considered as a risk factor for HIV.

Antibodies↗
PubMed1981

Treatment of human complement components C4 and C3 with amines or chaotropic ions. Evidence of a functional and structural change that provides uncleaved C4 and C3 with properties of their soluble activated froms, C4b and C3b.

Treatment of human components C4 and C3 with amines like hydrazine, ammonium hydroxide, and neutral ammonium salts or with chaotropic salts like KSCN and NaBr leads to complete loss of haemolytic activity. The pretreated components are, however, still active in formation of soluble C3 convertases. This activity pattern is reminiscent of the activities of C4 and C3 that have been activated by cleavage in the fluid phase. Indeed, the antigenic properties of pretreated C4 and C3 are similar to soluble C4b and C3b. The polypeptide chain structure of pretreated C4 and C3, is, however, identical to that of the untreated components when investigated by SDS gel electrophoresis. Pretreatment even reduces greatly the susceptibility of C4 to cleavage by C1s and of C3 to cleavage by classical and alternative pathway C3 convertases. Pretreated components have lost the ability to combine with EAC1 and EAC142, respectively; this fact explains their failure to exhibit haemolytic activity. In serum, pretreated C4 and C3 are cleaved in a manner similar to C4b and C3b. Amines and chaotropic ions cause the same functional and structural alterations, which are best explained by assumption of a conformational change. A similar transformation can also occur in C4 and C3 during preparation or storage.

Amines↗
PubMed1995

DNase I hypersensitivity mapping and promoter polymorphism analysis of human C4.

Human complement component C4 is encoded by two structurally distinct loci in the major histocompatibility complex (MHC) class III region. The two isotypes, C4A and C4B, differ at only four residues in the C4d fragment, but C4 constitutes the most polymorphic of the complement components. It is not known, however, whether the regions involved in the regulation of C4 expression also display polymorphic variation. By using the technique of DNase I hypersensitivity mapping, we established that the only area of transcriptional activity for C4 in the hepatocyte cell line, HepG2, occurs approximately 500 base pairs upstream of the transcriptional start site. This region was found to be remarkably constant in sequence when analyzed in the context of differing MHC haplotypes including HLA B57, C4A6, C4B1, DR7, which has been correlated with reduced expression of the C4A isotype. Similarly, polymerase chain reaction followed by single-strand conformation polymorphism analysis failed to demonstrate any promoter polymorphisms in 103 individuals comprising 52 systemic lupus erythematosus patients and 51 healthy controls.

Base Sequence↗
PubMed1987

The protein S-binding site localized to the central core of C4b-binding protein.

Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.

Animals↗
PubMed1990

Complement genetics and host defence.

There is a surprisingly high frequency of allelic variation in complement proteins. The best candidate for a true selective polymorphism is that of C3. For C4 and factor B within the MHC it is more difficult to identify the effects of individual alleles. No evidence suggests other alleles of C4 (and C2) than the null alleles (or the two isoproteins C4A and C4B) to have any functional differences with resistance. Thus, the major functions of complement, as shown by the effects of deficiency, are to resist infection against bacteria and particularly against Neisseria, and to prevent immune complex disease. There are also undoubtedly balancing contributions to the pathogenesis of some infections and to all immune complex disease. Data from studies of C4 polymorphism and from the presence of control proteins on micro-organisms suggest there may be in addition more subtle contributions to immunity against a variety of other infections.

Alleles↗
PubMed1989

Drug-induced immune-complex disease.

A range of drugs including hydralazine, isoniazid, procainamide and penicillamine cause toxic side effects which resemble systemic lupus erythematosus (SLE). Deficiencies of C1, C4 and C2 are associated with idiopathic SLE, and these defects may compromise the ability of the patient to deal with immune complexes. Immune complexes with protein as antigen, such as has been reported to be diagnostic of procainamide-induced SLE, interact more with the C4A isotype of C4 than the C4B isotype. It is shown that hydralazine, isoniazid and penicillamine inhibit the covalent binding of C4 to a complement-activating surface and that the drugs themselves become covalently bound to C4. For each of these drugs, C4A is inhibited more than C4B, and it is suggested that this is an important contributory factor in the development of the toxic side effects to these drugs involving immune-complex deposition. For procainamide, it is shown that the hydroxylamine metabolite rather than the drug itself inhibits the covalent binding reaction of C4. Hydralazine, isoniazid and procainamide are metabolised by the polymorphic N-acetyltransferase, and slow acetylators are at increased risk of drug-induced lupus. For procainamide, oxidation to the hydroxylamine form is an alternative metabolic route of increased importance in slow acetylators, and it is suggested that investigation of C4 type in susceptible patients could provide a means of identifying those at greatest risk of immunotoxicity.

Humans↗
PubMed1996

Specific complement inhibition with heparin-coated extracorporeal circuits.

BACKGROUND: Although it is well established that heparin-coated extracorporeal circuits reduce complement activation during cardiac operations, little in vivo information is available on the reduction in alternative and classic pathway activation. METHODS: In a prospective, randomized study involving patients undergoing coronary artery bypass grafting with standard full heparinization, we compared heparin-coated circuits (Duraflo II) (10 patients) with uncoated circuits (10 patients) and assessed the extent of initiation of complement activation by detecting iC3 (C3b-like C3) concentrations, classic pathway activation by C4b/c (C4b, iC4b, C4c) concentrations, terminal pathway activation by soluble C5b-9 concentrations, and C3 activation by C3a (C3a desArg) and C3b/c (C3b, iC3b, C3c) concentrations. RESULTS: Heparin-coated extracorporeal circuits significantly reduced circulating complement activation product C3b/c and soluble C5b-9 concentrations at the end of cardiopulmonary bypass and after protamine sulfate administration compared with the uncoated circuits, but not iC3, C4b/c, or C3a concentrations. CONCLUSIONS: Heparin-coated extracorporeal circuits reduce complement activation through the alternative complement pathway, probably at the C3 convertase level, and, consequently, the terminal pathway. C3b/c seems to be a more sensitive marker than C3a to assess complement activation during cardiac operations.

Aged↗
PubMed1988

Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes.

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.

Animals↗