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PubMed1988

Immunoblot analysis of the eighth component of human complement. Demonstration of subunits and detection of C8 alpha-gamma double and triple bands.

Using SDS-PAGE/immunoblot analysis of the eighth component of human complement, C8, we have been able to demonstrate an 85 kDa C8 alpha-gamma and a 62 kDa C8 beta subunit in normal human serum. Serum from an undiagnosed patient who presented undetectable hemolytic C8 activity possessed only the 85 kDa subunit, suggesting a defect in the C8 beta subunit. Serum of a patient with known C8 alpha-gamma deficiency possessed only the complementary 62 kDa subunit. Both sera used together were able to lyse antibody-sensitized sheep erythrocytes, whereas individual sera could not. Optimum conditions for C8 immunoblotting were determined using small amounts of serum or plasma, during low voltage electrophoresis and a sensitive staining technique (nitrobluetetrazolium/bromochloroindoxylphosphate). Using these conditions, the C8 alpha-gamma subunit was found to be composed of up to three bands, termed C8 alpha-gamma 1, -2 and -3. All three bands were found in pooled normal sera. Individual sera had at least the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. Two C8 beta-deficient sera from two unrelated patients exhibited only the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. We conclude that immunoblotting of C8 permits a detailed analysis of the molecular composition of this component and helps to establish a precise diagnosis in inherited C8 deficiencies.

Collodion↗
PubMed1987

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement.

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.

Binding Sites↗
PubMed1992

Genetic polymorphism of complement component C6, C7 and C8(1) in Chinese Han population in northeast China.

Distributions of complement phenotypes, C6, C7, and C8(1) were studied using thin agarose gel isoelectric focusing (AGIEF) or ultra-thin polyacrylamide gel isoelectric focusing (PAGIEF) and subsequent immunoblotting techniques in 203 Chinese Han population in Liaoning Province of northeast China. The gene frequencies were as follows: C6*A 0.4704, C6*B 0.5049, C6*B2 0.0148, C6*B3 0.0049, and C6*M 0.0049; C7*1 0.8251, C7*2 0.1108, C7*3 0.0320, and C7*4 0.0320; C8(1)*A 0.5567 and C8(1)B 0.4433, respectively. All the observed numbers of the phenotypes were in agreement with the expected numbers under the Hardy-Weinberg equilibrium. The gene frequencies among Chinese subpopulations and other various populations were compared.

Asian People↗
PubMed1989

Inhibition of immune haemolysis by a serum factor found in C3-deficient subjects.

A serum factor, which inhibits haemolysis of the buffer control used in a C3 haemolytic assay, was found in a C3-deficient subject (C3D). Since the buffer control consisted of EAC142, C5 and C6-9 reagent (C6-9R, prepared by treatment of guinea-pig serum with KSCN and hydrazine hydrate), the factor seems to be an inhibitor of C3-independent immune haemolysis. Gel filtration and CM cellulose column chromatography of C3D serum suggested that the inhibitor may be C8. The inhibition was not observed in C8-depleted C3D serum. Furthermore, isolated C8 was found to inhibit haemolysis of EAC142 by C5 and C6-9R in a dose-dependent fashion. Thus, C8 was found to be an inhibitor of C3-independent immune haemolysis in the assay. Further studies revealed that C8 also inhibits haemolysis of EAC142 by C3, C5 and C6-9R (C3 assay system) or that of EAC1423 by C5 and C6-9R (C5 assay system), indicating that C3 or C5 haemolytic activity can be underestimated by the presence of C8 in a sample. C8 did not inhibit haemolysis in the assay system when isolated C6-C9 of human origin were used, but did inhibit haemolysis when isolated C6-C9 of guinea-pig origin was used instead of C6-9R. Thus, it was suggested that the incompatibility of human C8 with guinea-pig C6-C9 might be responsible for this phenomenon. Additional experiments for the mechanism clearly showed that human C8 inhibits the haemolysis of EAC1-7 (EA bearing human C1-C5 and guinea-pig C6 and C7) by guinea-pig C8 and C9 by binding to EAC1-7 prior to guinea-pig C8.

Animals↗
PubMed1986

Synthesis of complement components C5, C6, C7, C8 and C9 in vitro by human monocytes and assembly of the terminal complement complex.

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.

Antibodies, Monoclonal↗
PubMed1987

[Recurrent meningitis in familial deficiency of the 8th component of the complement system].

An 18-year-old man suffered from recurrent bacterial meningitis. Investigation of the complement system revealed deficiency of the 8th complement component (C8) in the patient and his sister. Genetic defects of the terminal complement components C5 to C8 predispose to Neisseria infections, probably due to a lack in bacteriolytic activity. It is to be noted that 1 year ago the patient had been hospitalized for a culture-proved pneumococcal meningitis.

Adolescent↗
PubMed1990

Purification of C8 and C9 from rat serum.

A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).

Animals↗
PubMed1991

Human umbilical vein endothelial cells synthesize functional C3, C5, C6, C8 and C9 in vitro.

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.

Antibodies, Monoclonal↗
PubMed1989

Deficiency of the eighth component of complement. Evidence for linkage of C8 alpha-gamma pattern with C8 beta deficiency in sera of twelve patients.

The C8 alpha-gamma subunit of the eighth component of complement was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting in sera from 68 normal individuals, 12 C8 beta-deficient patients (from seven unrelated families), and 10 of the parents of the latter. Three different forms of the C8 alpha-gamma subunit were observed: 34/68 normal individuals were found to have a C8 alpha-gamma triple band (termed C8 alpha-gamma 1, C8 alpha-gamma 2, C8 alpha-gamma 3 variants), 23/68 the C8 alpha-gamma 2 and C8 alpha-gamma 3 variants, and 11/68 the C8 alpha-gamma 1 and C8 alpha-gamma 3 variants. In contrast, all C8 beta-deficient patients had detectable C8 alpha-gamma 2 and C8 alpha-gamma 3 variants but lacked the C8 alpha-gamma 1 variant in addition to the C8 beta subunit. Three out of ten parents of the C8 beta-deficient patients were found to have the C8 alpha-gamma triple band, whereas 7/10, like their children, had the C8 alpha-gamma 2 and C8 alpha-gamma 3 variants only. We conclude that there is a linkage between the C8 alpha-gamma pattern and C8 beta deficiency. These data may support earlier findings that in humans the genes encoding for C8 alpha-gamma and C8 beta are closely linked on chromosome 1.

Adult↗
PubMed1992

Complement component deficiencies and infection: C5, C8 and C3 deficiencies in three families.

Three families are described with complement component deficiencies. In one family, five children had C5 deficiency; in a second family, two children had C8 deficiency and one child in a third family had C3 deficiency. The index cases were identified during screening of patients with recurrent pyogenic infections, recurrent meningitis and meningococcaemia. Two of the five C5 deficient patients had recurrent meningitis and meningococcaemia, two had recurrent respiratory tract infections and otitis and one was healthy. One of the C8 deficient patients had meningitis, meningococcaemia and pneumonia, whereas his sibling with the same deficiency was healthy. The patient with C3 deficiency had four episodes of meningitis and recurrent otitis.

Child↗
PubMed1989

Secretion of the terminal complement proteins, C5-C9, by human platelets.

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.

Blood Platelets↗
PubMed1988

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins.

Animals↗
PubMed1987

Human alveolar macrophages synthesize active complement components C6, C7, and C8 in vitro.

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.

Adult↗
PubMedDate not supplied

C8 beta subunit deficiency in a patient with recurrent neisserial infections.

A 15-year-old woman with a history of recurrent episodes of meningococcal infections was admitted to our hospital with signs and symptoms indicating a meningeal inflammation. Since in the last few years some of the patients affected by recurrent meningococcal infections have been recognized to have selective complement deficiencies, the patient's serum was studied for determining the complement function. C8 was found to be present only in traces, with a pattern of partial identity as compared with that of the normal human serum. Moreover, total hemolytic complement was undetectable and could be completely restored with purified C8, but not with other complement components; thus, we concluded for the presence of a C8 deficiency state. Further reconstitution experiments carried out with sera having selective deficiencies of either C8 beta or C8 alpha-gamma subunits allowed us to recognize the presence of a dysfunctional C8 molecule lacking the beta chain, but possessing the alpha-gamma subunit. The clinical history of the patient, characterized by recurrent meningococcal infections, further supports the current concept of an increased susceptibility of the C8 beta-deficient patients to Neisseria meningitidis infections.

Adolescent↗
PubMed1986

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.

Blood Proteins↗
PubMed1991

Gene responsible for deficient activity of the beta subunit of C8, the eighth component of complement, is located on mouse chromosome 4.

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radial hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had no lytic activity, and Ouchterlony analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored by the addition of partially purified human C8 or human serum deficient in C8 alpha-gamma, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the beta subunit of C8. Typing of (DBA/2J X MSM)F1 hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a single recessive gene, designated C8b; the allele with hemolytic activity is C8b1; and the allele with no activity C8b0. Because of synteny homologies in mouse and human, we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.

Animals↗
PubMed1986

The C8A and C8B loci are closely linked on chromosome 1.

Close linkage was demonstrated between the loci governing the polymorphisms of complement component C8 alpha-gamma (C8A) and beta (C8B). Both C8 loci were linked to the chromosome 1 marker loci PGM1 and Rh. The distance between the two C8 loci and PGM1 appeared identical in males and females. A female/male ratio of 1.6 was observed between the two C8 loci and Rh. No evidence for linkage between the C8 loci and Fy was found. Preliminary results of this study were presented at the Eighth International Workshop on Human Gene Mapping, Helsinki, August 1985 (Rogde et al. 1985b).

Chromosomes, Human, Pair 1↗