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PubMed1989

Antigenic changes of complement components: relevance for the analysis of complement activation.

The present paper briefly reviews some general principles in the field of activation-dependent epitopes in the complement cascade. The advantage of monoclonal antibodies in the study of these epitopes is pointed out and available antibodies are listed. Application of such antibodies in reliable and valid assays to detect activation at all levels of the cascade is addressed. The importance of these assay in the study of various pathophysiological and clinical conditions is briefly discussed.

Antibodies, Monoclonal↗
PubMed1980

Purification and partial characterization of the third component of the complement system from porcine serum (C3) and of a crystallizable degradation product of the fourth component of the complement system from human serum (C4). Study of the tryptic digestion products of human and porcine C3 and of human C4.

The third component of the porcine complement system (C3) was isolated. The protein with 190000 molecular weight is composed of two chains alpha and beta of Mr 116000 and 74000. The amino-acid composition is very similar to that of human C3. A degradation product of human C4 was also isolated and characterized. This protein was crystallized. The degradation product is characterized by a molecular weight of 128000; the major part of the alpha chain is lost. Native human and porcine C3 and human C4 were submitted to tryptic digestion. The digestion products were isolated and characterized in terms of molecular weight, subunit organization and amino-acid composition. The present study confirms the high homology among these proteins.

Amino Acids↗
PubMed1999

Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation.

BACKGROUND: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. METHODS: Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. RESULTS: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. CONCLUSION: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Adult↗
PubMed1958

Reiter protein complement fixation test; a preliminary comparison of the TPI test with the complement fixation test employing a soluble protein antigen derived from the Reiter strain.

A comparative study of the specificity of the Reiter protein complement fixation (RPCF) test and the Treponema pallidum immobilization (TPI) test on 180 sera showed that the results in 178 instances or 98.9 per cent were in agreement. The sensitivity of the RPCF test, when compared to the TPI test, on 189 sera from patients known to have syphilis was in agreement in 182 or 96.3 per cent, assuming a correlation exists between positive TPI and both the "reactive" or "weakly reactive" RPCF test results. As to reproducibility of results, the RPCF test results agreed in 84 of the 87 sera tested (95.4 per cent). The sera were tested at least three times on different days. Anticomplementary reactions were observed in three of 91 normal sera.

Complement Fixation Tests↗
PubMed1992

Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18).

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.

Amino Acid Sequence↗
PubMed1989

Platelet-activating factor enhances complement-dependent phagocytosis of diamide-treated erythrocytes by human monocytes through activation of protein kinase C and phosphorylation of complement receptor type one (CR1).

Oligomerization of band 3 protein has been recently indicated as an early event in senescent or damaged red cell membrane followed by specific deposition of anti-band 3 antibodies and binding of complement C3 fragments. The band 3-anti-band 3-C3b complex is recognized by homologous monocytes, and phagocytosis ensues. This study shows that recognition of the anti-band 3-C3b complex by the monocyte C3b receptor type one (CR1) plays a crucial role in the process of removal of damaged red cells. Indeed, blocking of monocyte CR1 with an anti-CR1 monoclonal antibody abrogated phagocytosis of diamide-treated red cells. Platelet-activating factor (PAF) is a phospholipid mediator involved in inflammatory processes. Nanomolar (R)-PAF enhanced the CR1-dependent phagocytosis of diamide-treated human red cell and of sheep red cells coated with C3b, induced the fast translocation of protein kinase C to monocyte membrane compartment, and stimulated the phosphorylation of monocyte CR1. The biologically inert lyso-PAF and the enantiomer (S)-PAF were inactive. PAF receptor antagonists and inhibitors of protein kinase C blocked the enhancement of phagocytosis induced by PAF. Protein kinase C translocation, phosphorylation of CR1, and stimulation of this receptor to an active state capable of mediating phagocytosis represent a novel pathway by which PAF interferes with red cell homeostasis and possibly modulates inflammatory reactions and host mechanisms against infections.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Complement-mediated inhibition of function in complement-resistant Escherichia coli.

C-mediated inhibition of function in C-sensitive strains of Escherichia coli involves the assembly of the membrane attack complex (MAC) on the outer membrane with subsequent inhibition of inner membrane function. The inhibition of inner membrane function is critical for effective cell killing as damage to the outer membrane alone is insufficient to kill a cell in the absence of serum lysozyme. Studies on the measurement of oxygen consumption for cells under complement attack showed that C-sensitive cells were inhibited by assembly of the MAC, and that this represents damage to some component of the respiring inner membrane. Mechanisms of cellular resistance to C attack could include 1) inhibition of the assembly of the MAC, 2) inhibition of effective activation of the assembled MAC, or 3) reversal of the inhibitory effects of the MAC. Demonstration of a transient C-mediated inhibition of inner membrane function in C-resistant cells implies that the latter case should be considered as one possible component of cellular resistance to C attack.

Bacteriolysis↗
PubMed1988

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms.

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.

Adult↗
PubMed1985

A DNA restriction fragment length polymorphism in the complement region of the human MHC shows an absolute correlation with polymorphism of complement factor B(Bf) defined by isoelectric focusing.

The gene coding for properdin factor Bf is located in the human major histocompatibility complex and is closely linked to the genes coding for the complement components C2 and C4. Recently, by Southern blotting techniques, a restriction fragment length polymorphism was identified using the endonuclease Taq I, which subdivides haplotypes carrying the F allele of factor Bf. The F allotype has also been subdivided at the protein level by isoelectric focusing into two subtypes Fa and Fb. We have investigated the DNA of 41 healthy unrelated individuals with known BfF subtypes using the 2.3 kb factor Bf cDNA probe to determine if there is any correlation between the Taq I polymorphism and F subtype. We have found that in 23 individuals who carried the Fb subtype a 6.6 kb Taq I fragment was present. The remaining 18 individuals carried the Fa subtype and showed only the 4.5 kb Taq I fragment on Southern blotting (P = 10(-12). This striking correlation (r = 1) between the Fb protein and DNA polymorphism is surprising especially as the 4.5 kb and 6.6 kb Taq I fragments overlap the Bf and C2 genes and the polymorphic Taq I site is located within the C2 gene.

Alleles↗
PubMed1986

Sheep serum complement sensitisation of sheep erythrocyte-rabbit antibody complexes for haemolysis by guinea-pig complement plus EDTA or Mg2+-EGTA.

Sheep erythrocyte (E)-rabbit antibody (A) complexes incubated with sheep serum diluted up to 1:5120 or 1:20480 and washed can be haemolysed by guinea-pig (g-p) serum (complement, C) containing EDTA or Mg2+-EGTA respectively as haemolytic finishing reagents. Sheep E carrying a high dose of rabbit A were necessary for this reaction, particularly with g-p C-EDTA. G-p serum (stored by freezing) was active as a haemolytic finishing reagent with both EDTA and Mg2+-EGTA. Reconstituted freeze-dried g-p serum (also stored by freezing) was haemolytically active with Mg2+-EGTA only. G-p serum preserved by Richardson's method did not function as a finishing reagent with EDTA or Mg2+-EGTA. A non-haemolytic prozone occurred with sheep E-rabbit A treated with dilutions of sheep serum or body fluid up to 1:160, particularly when g-p C (frozen)-EDTA was used as the finishing reagent. Sheep E-rabbit A were sensitized by serum, foetal lamb serum, pericardiac-, synovial- or ovarian follicle-fluids colostrum or milk for haemolysis by g-p C (frozen)-EDTA or -Mg2+-EGTA. With the C3 inhibitors cobra venom factor or salicylaldoxime, serum sensitisation of sheep E-rabbit A for haemolysis by g-p C (frozen)-EDTA or -Mg2+-EGTA was not blocked. Sensitisation by serum heated at 50 degrees C for 30 min (partial inactivation of C2) was incomplete. Inhibitors of C1 (antrypol, chelators of Ca2+ or heating serum at 56 degrees C for 30 min) partially or fully blocked sensitisation for haemolysis by both g-p C (frozen)-EDTA or -Mg2+-EGTA. These results show that at a minimum, components C1, C4 and C2 are present and functionally active in serum and some body fluids of sheep.

Animals↗
PubMed1976

Complement activation in semi-solid medium: Insolubilization of properdin and the third component of complement (C3) in agar gels.

Although the role of properdin in the alternative pathway of complement activation remains unclear, evidence has recently been obtained for the formation of complexes between properdin and other components, including C3. In this study such complexes have apparently been directly visualized. When normal human serum and properdin were allowed to diffuse toward each other in agar gel for 16 hr, a line of precipitation could be seen when stained with Coomassie brilliant blue. The reaction occured at pH 8.6 in 0.05 M Veronal buffer at room temperature but not under physiologic conditions of pH or tonicity. Like the alternative pathway, the reaction was Me++ dependent, occurred with C2- or C5-deficient or hypogammaglobulinemic serum, and did not occur with aged, 52 degrees C-inactivated, C3b inactivator-deficient, or C3-deficient serum. 125I-labeled C3 and properdin but not Factor B were incorporated in the precipitate. Eleven sera containing the C3 nephritic factor failed to produce a precipitate with properdin, but a line of precipitation occurred between seven of these sera and normal serum. This line showed identity with the line occurring between properdin and normal serum. The phenomenon appears to result from formation of insoluble complexes between proteins of the alternative pathway and agar.

Agar↗
PubMed1977

Complement activation by interaction of polyanions and polycations. III. Complement activation by interaction of multiple polyanious and polycations is the presence of C-reactive protein.

Interactions between heparin and protamine previously were found to result in activation of the complement (C) system. In the present investigation, this interaction was shown to result in the binding of purified C1, and this was markedly enhanced in the presence of C-reactive protein (CRP). CRP also enhanced C consumption during heparin-protamine interactions in whole serum, and in the presence of CRP depletion of C components C1-3 was observed. Similar C1 binding and C consumption in the presence of CRP were seen upon the interaction of multiple additional polyanions including DNA, ENA, hyaluronic acid, chondroitin sulfate, and dextran sulfate with the polycations protamine sulfate and poly-L-lysine. These effects were observed with CRP concentrations well within the range found in normal human sera and considerably less than those found in most acute phase sera. We suggest, therefore, C activation by polyanion-polycation interactions in the presence of CRP may be important to certain reactions of host defense and inflammation.

Anions↗
PubMed1988

The C4 and C2 but not C1 components of complement are responsible for the complement activation triggered by the Ra-reactive factor.

Ra-reactive factors (RaRF) are the name of a group of C-dependent bactericidal factors that bind specifically to Ra chemotype strains of Salmonella. These factors are present in the sera of a wide variety of vertebrates and have common characteristics. Here we investigate the C components required for the C activation induced by mouse RaRF, by using hemolysis of Ra LPS-coated E (ELPS) as a model system. It was found that C1-depleted and C1q-depleted sera were as effective as the undepleted serum in the lysis of ELPS sensitized with RaRF. Addition of the C1 component or C1q subcomponent to the depleted sera did not increase the effect. On the other hand, C4 and C2 components were found to be essential for the lysis of RaRF-sensitized ELPS. Activities of C4 and C2 remained on the sensitized cells even after washing the cells, suggesting that the classical C3 convertase, C4b2a, is generated on the RaRF-sensitized ELPS.

Animals↗
PubMed2003

Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule.

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.

Antibodies, Blocking↗