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PubMed1991

A flow cytometric assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes.

A flow cytometric assay (FCA) was developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It was possible to measure these parameters accurately with intra- and interassay coefficients of variation of 2.0% and 6.5% respectively. The method was able to discriminate between low and high levels of erythrocyte CR1, C3d and C4d. Comparison with a previously described RIA method gave excellent correlation coefficients with r2 values of 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay was used to measure CR1, C3d and C4d on the erythrocytes of 98 healthy individuals and the 95% upper limits for C3d and C4d were established. There was a wide distribution of CR1 levels amongst these individuals but their C3d and C4d levels were low and often not above background. The possible application of this method in clinical medicine is discussed.

Antigens, CD↗

PubMed1988

The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions.

Complement Activation↗

PubMed1981

Complement receptor is an inhibitor of the complement cascade.

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

Complement C3-C5 Convertases↗

PubMed1988

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

CD55 Antigens↗

PubMed1975

Complement-mediated bactericidal system: evidence for a new pathway of complement action.

The early components of human complement (C1, C4, and C2) plus certain serum euglobulins will kill pathogenic strains of Shigella sonnei. Serum from patients with hereditary C3 deficiencies and specific antiserums to C3, C5, and C6 were utilized to demonstrate the absence of requirements for late-acting complement components in this unusual bactericidal system.

Antibodies, Bacterial↗

PubMed1985

C56 formation in the reaction mixture of isolated complement components through the classical complement pathway.

The mechanism of hemolysis of unsensitized erythrocytes by a mixture of 9 isolated, human-derived complement components, C1s, C4, C2, C3, C5, C6, C7, C8 and C9 (C1s-C9) was studied. Of the tested erythrocytes, guinea pig erythrocytes (Egp) were the most susceptible to lysis by C1s-C9, followed by human and sheep erythrocytes. Contamination of the isolated complement components by C56 was ruled out. It was determined that a factor was generated in the reaction mixture of C1s, C4, C2, C3, C5 and C6 (C1s-C6), which had lytic activity against Egp when C7, C8 and C9 were added. We found that the lytic factor was similar to C56 in the following properties: (1) the activity of the lytic factor decreased when incubated with isolated C7 prior to its reaction with Egp; (2) the lytic factor did not bind to Egp by itself but it did bind in the presence of C7; (3) EDTA did not have any inhibitory effect on the lytic factor; (4) the activity of the lytic factor decreased by treatment with anti-C5 and anti-C6 but not by treatment with anti-C3 and anti-C4, and (5) gel filtration of the reaction mixture (C1s-C6) indicated that the elution volumes of the lytic factor and of isolated C56 were similar. Thus, it is likely that C56 is generated in the reaction mixture of C1s-C6 and the lytic factor binds to unsensitized erythrocytes together with C7, to form an intermediate EC567 which is susceptible to lysis by the action of C8 and C9.

Animals↗

PubMed1981

The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.

Binding, Competitive↗

PubMed1982

Complement activation and complement control proteins in acute pancreatitis.

Serum levels of the complement proteins C3, C4, C1 inhibitor (C1 INH), factor I (C3b inactivator) and factor H (BIH) and plasma levels of cleavage products of C3 (C3c) and factor B were measured in 26 patients with acute pancreatitis. Breakdown of C3 occurred in 19 patients, as shown by a reduction in C3 level and the presence of C3c. C4 levels, however, did not fall and factor B breakdown products were not detected, thus suggesting that enzymatic cleavage of C3 occurred without significant involvement of either the early classical pathway or the alternative pathway. C1 INH and factor H both showed increases, presumably reflecting an acute phase response. Factor I showed an initial fall followed by a rise. There was no correlation between the presence or extent of C3 breakdown and the clinical condition of the patients. It is concluded that C3 cleavage in pancreatitis probably results from tryptic activity and that the measurement of complement components has no part to play in the management of the disease.

Acute Disease↗

PubMed1984

Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9.

The cytolytic efficiency of the terminal complement complex (C5b-9) against erythrocytes of different species is, in part, dependent on the species of C9 origin. In the present study, we have examined the interaction of C9 with erythrocytes in terms of the binding, dimerization, and insertion of C9 into the membranes of sheep and human erythrocytes lysed by human complement (C). The membranes of these C-lysed erythrocytes were analyzed for bound, dimerized, and inserted C9 by a combination of photolabeling, SDS-PAGE, electroblotting, and immunostaining techniques. We found that neither binding nor dimerization of C9 could be correlated with the relative hemolytic efficiency of human C on these erythrocytes, but that C9 insertion into the membranes of these cells varied in direct relation to the extent of lysis. Interestingly, the binding of C3 to these cells under conditions of equivalent C1 fixation also correlated with lytic efficiency. These data indicate that the C9-related differences in the cytolytic efficiency of C against erythrocytes from different species is primarily due to the efficiency of C9 insertion into these cells. Moreover, these data emphasize that neither the binding of C9 to a target membrane nor the formation of C9 dimers necessarily leads to the insertion of C9 into the membrane, suggesting the presence of membrane-bound but inactive C5b-9 complexes. Because the extent of C3 binding also correlated with the relative degree of lysis of sheep vs human erythrocytes, the possibility exists that surface-bound C3 may regulate hemolysis by directing the insertion of C9 in terminal complexes into cells.

Animals↗

PubMed1989

Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems.

The cDNA sequence encoding the human complement-associated protein, SP-40,40, is reported. The two chains of SP-40,40 are coded in a single open reading frame on the same mRNA molecule, indicating the existence of a biosynthetic precursor protein which matures post-synthetically by the proteolysis of at least one peptide bond. The precursor is preceded by a signal sequence for vectorial export and contains six N-linked glycosylation sites distributed equally between the two chains of the structure. The sequence of the SP-40,40 precursor bears a 77% identity to a rat sulphated glycoprotein-2 (SGP-2) which is the major secreted product of Sertoli cells. The presence of SP-40,40 within human seminal plasma at levels comparable to those in serum was demonstrated, indicating that SP-40,40 and SGP-2 are serum and seminal forms of the same protein. A sequence of 23 amino acids within the beta-chain of SP-40,40 exhibited significant homology to corresponding segments located within complement components C7, C8 and C9. The short cysteine-containing motif represented the only evidence of a possible vestigial relationship between SP-40,40 and other complement components. The precise role of SP-40,40 is not known in either blood or semen but the present findings document an intriguing link between the immune and the reproductive systems.

Amino Acid Sequence↗

PubMed1988

Complement-mediated cytolytic activity associated with cytomegalovirus complement-fixing IgM antibody.

Cytomegalovirus-specific complement-fixing IgM (CMV CF-IgM) antibody was detected in 12/84 (14.3 percent) sera containing CMV-specific IgM. Higher titres were obtained by using a purified antigen preparation containing enveloped virus particles and membrane fragments when compared with a crude extract of infected cells, suggesting that the CF component of CMV IgM reacts predominantly with antigens on the surface of the infected cells. This also accounts for its complement-mediated cytolytic activity. Neither CMV-specific non-complement-fixing IgM nor CMV-specific IgG demonstrates cytolytic activity.

Antibodies, Viral↗

PubMed1989

Lack of complementation between xeroderma pigmentosum complementation groups D and H.

The construction of permanent hybrid cell lines between xeroderma pigmentosum (XP) cells from different complementation groups allows analysis not only of the degree of repair correction but also of the restoration of biological activity to the UV-irradiated cells. With use of an immortal human cell line (HD2) that expresses excision repair defects typical of XP group D, a series of permanent hybrid cells has been produced with XP cells from groups A to H. Excision repair, as measured by incision analysis and unscheduled DNA synthesis, is restored to normal or near normal levels in crosses involving HD2 and cells from XP groups A, B, C, E, F, G, and I. All these hybrids show complementation for the recovery of normal UV resistance. As expected, hybrids expressing poor incision and hypersensitivity to UV were produced in crosses between HD2 and XPD fibroblasts, but they were also produced without exception when XPH was the partner. In the permanent HD2 x XPD or XPH hybrids, analysis of incision capacity reveals abnormally low activity and therefore that there has been no complementation. The true hybrid nature of HD2 x XPH cells has been confirmed by HL-A and -B tissue typing; moreover, detailed kinetic analysis of incision in these cells shows that the XPH phenotype, rather than the XPD, is expressed, i.e. breaks accumulate at low UV fluence of 1 J/m2. To help confirm these findings, another immortal XPD cell line was used in fusions involving HD2, XPH, or XPI. Cells resistant to ultraviolet were produced only with XPI fibroblasts. These data are discussed in terms of whether XPD and H mutations are likely to be allelic with respect to incision.

DNA Damage↗

PubMed1985

Hemolytic plate assay for quantification of active human complement component C3 using methylamine-treated plasma as complement source.

A hemolytic plate assay specific for active human complement component C3 is described. The method is well suited for tracing active C3 during preparative purification or for screening of plasma samples. The assay is based on activation of the alternative pathway of complement by unmodified rabbit erythrocytes. Plasma treated with methylamine supplies the essential complement components other than C3. The lytic reaction is complete in 5 h at 37 degrees C and is unchanged by incubation overnight. The dose-response curve, i.e., lysis diameter versus logarithm of C3 concentration, is linear within 0.1-10 times normal plasma concentrations of C3. The standard deviation is below 10%. The hemolytic agarose plates are easy and inexpensive to prepare, and they can be stored at 4 degrees C for 2 weeks before use. This paper describes the optimal conditions of the assay and proves its specificity. Its use in C3 preparation and plasma screening for C3 is discussed.

Animals↗

PubMed1983

Single radial complement fixation test using complement film. Assay of the antibody response to strain and type specific antigens of influenza virus.

A stabilized modification of the single radial complement fixation test in gel (SRCF) was developed for detecting influenza antibodies. The principle of the test is the use of a single-step procedure with the following reagents: (1) Agarose plate containing influenza antigen and antibody coated erythrocytes (EA). (2) Thin plastic film coated with dried complement. By filling the wells cut in the agar with the heat inactivated serum samples and covering the agar surface with the complement film, a zone of unlysed cells surrounded by a haemolytic area appears after overnight incubation for 16-18 h at 4 degrees C and 1-2 h at 37 degrees C. The squares of the zone diameter were measured for estimating the antibody quantity by using CF(S) and virion antigen of influenza virus, and the type-specific antibody was demonstrated by using CF(S) antigen, while the strain-specific antibody was demonstrated by using virion antigen. An excellent correlation was demonstrated for antibody titres between conventional CF and SRCF with CF(S) antigen, on the one hand, and, between conventional HI and SRCF with virion antigen, on the other.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1974

A study of foot-and-mouth disease virus strains by complement fixation. I. A model for the fixation of complement by antigen-antibody mixtures.

An examination was made of the relations between antigen, antibody and fixation of complement with foot-and-mouth disease virus (FMDV). It was found that complement fixation in this system follows the same principles as models developed in other antigen/antibody systems. The assumption that there is a relation of direct proportionality between the amount of complement fixed and the amount of antiserum reacting with constant antigen was found to be incorrect. An alternative method was proposed for the quantitative differentiation of FMDV strains by comparing the titres of an antiserum when reacting with optimum amounts of homologous or heterologous antigens.

Animals↗

PubMed1998

Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I.

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1987

Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity.

Cells infected with herpes simplex virus type 1 (HSV-1) form rosettes with C3b-coated erythrocytes, whereas cells infected with herpes simplex virus type 2 (HSV-2) or other herpes viruses do not. It was reported that glycoprotein C of HSV-1 (gC-1) mediates the binding of C3b-coated erythrocytes to infected cells and has regulatory (decay-accelerating) activity for the alternative pathway C3 convertase of human complement. We show here that solubilized gC-1 binds to iC3-Sepharose affinity columns. We also report that solubilized gC-2, the genetically related glycoprotein specified by HSV-2, binds to iC3-Sepharose. mAb specific for gC-1 or gC-2 and mutant viral strains were used to identify the C3-binding glycoproteins. In other experiments, HSV-1 mutant strains and recombinants, differing only in their expression of gC, were tested for sensitivity to neutralization by human complement in the presence or absence of antibodies specific for HSV gD. In either case the gC- strain was most sensitive. Expression of gC-1 or gC-2 by isogenic insertion mutants provided protection against complement-mediated neutralization. These results indicate that the genetically and structurally related gC-1 and gC-2 share the functional activity of binding to human C3 and enhance viral infectivity.

Chromatography, Affinity↗

PubMed1998

High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone.

High-level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement-mediated injury in an ex vivo perfusion model. In this study we examine whether co-expression of CD55 provides additional protection. CD55/CD59 double-transgenic mice were generated by co-injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM-2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild-type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high-level endothelial co-expression of CD55 and CD59 provides greater protection from human complement-mediated injury in this model than expression of CD59 alone.

Animals↗