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PubMed1998

Inhibition of activation of the classical pathway of complement by human neutrophil defensins.

Defensins are small, cationic antimicrobial peptides that are present in the azurophilic granules of neutrophils. Earlier studies have shown that defensins may influence complement activation by specific interaction with activated C1, C1q, and C1-inhibitor. In the present study, we show that the defensin human neutrophil peptide-1 (HNP-1) is able to inhibit activation of the classical complement pathway by inhibition of C1q hemolytic activity. The binding site for HNP-1 on C1q is most likely located on the collagen-like stalks, as a clear, dose-dependent binding of HNP-1 to either intact C1q or to the collagen-like stalks of C1q was demonstrated using enzyme-linked immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a limited binding to C1 and to a mixture of C1r and C1s was observed, whereas no binding to C1-inhibitor was found. Because binding of HNP-1 to C1-inhibitor has been suggested in earlier studies, we also assessed the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s or C1. No binding was found. Using a competition ELISA, it was found that HNP-1, but not protamine, inhibited binding of biotin-labeled HNP-1 to C1q in a dose-dependent fashion. In the fluid phase, preincubation of HNP-1 with C1q resulted in complex formation of HNP-1 and C1q and generation of stable complexes. In conclusion, HNP-1 is able to bind to C1q in the fluid phase and inhibits the classical complement pathway. This mechanism may be involved in the control of an inflammatory response in vivo.

Binding Sites↗

PubMed1990

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

Animals↗

PubMed1982

Hemolytic and antigenic measurements of complement. A comparison of serum and plasma samples in normal individuals and patients.

Over a 2-year period we evaluated 10 patients with a discrepancy between functional and antigenic assays in the routinely employed clinical assays for measuring serum complement concentrations. These differences were shown to be secondary to cold-dependent activation of the classical complement pathway in vitro, in some cases by cryoglobulins and in others by unknown means. Plasma samples were procured with commonly used anticoagulants (EDTA, citrate, or heparin) that prevent in vitro complement activation. To assess whether plasma samples were suitable for complement determination, a comparison of serum vs. plasma samples for THC, C4, C2, C3, factor B, and C6 levels in normals and patient populations was undertaken. Only modest differences were found between serum and plasma samples for these functional and antigenic assays except for heparinized samples in which determinations by rate nephelometry produced falsely elevated C4, C3, and factor B antigenic levels. Thus plasma samples, especially EDTA or citrate, are suitable for complement determinations and could be used either routinely or, more selectively, in patients in whom there is a discrepancy between functional and antigenic determinations.

Adult↗

PubMed1985

A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide.

Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.

Animals↗

PubMed1987

Preparation and characterization of monoclonal antibodies against the fifth component of rabbit complement (C5).

By immunizing mice genetically deficient in C5 we were able to obtain a group of monoclonal antibodies to rabbit C5 that cross-react with C5 from a wide variety of mammalian sera, including mouse. The specificity of the monoclonal antibodies was against native C5 and C5b but not C5a. The antibodies strongly inhibit the expression of C5 hemolytic activity. We suggest that these monoclonal antibodies will be useful for studying C5 as well as providing a way to selectively deplete C5 from plasma in vitro or in vivo.

Animals↗

PubMed1988

Normal polymorphic variations and transcription of the decay accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells.

In paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic anemia, deficiency of decay accelerating factor (DAF) renders blood cells susceptible to increased deposition of autologous complement activation fragments (C3b) and complemented-mediated injury. To investigate the mechanism of the DAF defect, DNA and mRNA from normal and PNH leukocytes were compared in blot hybridization assays by using DAF cDNA and oligonucleotide probes. Southern analyses of DNA from normal cells revealed a single gene spanning approximately equal to 35 kilobases of DNA. Six HindIII banding patterns were distinguishable among normal individuals. In family studies, the patterns segregated as three homozygous and three heterozygous genotypes deriving from three haplotypes: A, B, and C with frequencies of 0.47, 0.36, and 0.17, respectively. Oligonucleotide mapping localized the polymorphic HindIII sites to two noncoding regions in the vicinity of exons encoding (i) the protein oligosaccharide-rich domain and (ii) the mRNA 3'-untranslated region. Analyses of DNA from DAF-negative leukocytes of eight PNH patients demonstrated restriction fragment profiles identical to those of normal individuals for all enzymes studied. Three patients had the BC (normals = 3/32), three patients had the AA (normals = 6/32), and two patients had the AC (normals = 8/32) HindIII genotype. Of the three PNH patients exhibiting the BC genotype, family studies of two demonstrated the expected inheritance patterns, and RNA gel blot analyses of two showed mRNA transcripts indistinguishable from those in normal cells. The absence of DAF gene or mRNA alterations in affected PNH cells that lack other glycolipid-anchored proteins as well as DAF argues that the lesion underlying PNH cells resides in the glycolipid-anchor pathway.

Blood Proteins↗

PubMed1984

Fixation of complement by anti-F(ab')2 antibodies or ssDNA when added to systemic lupus erythematosus sera.

A solid phase total hemolytic complement assay was used to quantitatively determine the % complement fixation by anti-F(ab')2 antibodies or ssDNA when added to serum from patients with systemic lupus erythematosus (SLE). Both autologous and homologous anti-F(ab')2 antibodies with demonstrated anti-anti-ssDNA activity fixed complement when added to SLE sera and normal human complement. Furthermore, comparable amounts of anti-F(ab')2 antibodies with minimal anti-anti-ssDNA activity fixed zero or minimal amounts of complement when added to the same lupus sera. When measured by the same assay with the same sera, ssDNA fixed greater amounts of complement than did comparable amounts of any of the anti-F(ab')2 antibodies.

Antibodies, Anti-Idiotypic↗

PubMed1985

Rat Clq: isolation and purification from normal serum and development of a sensitive hemolytic assay.

Rat Clq was isolated and purified from normal rat serum by a two-step procedure: affinity chromatography on a human IgG-Sepharose 4B column and Bio-Gel A 5m column chromatography. From 1.8 l of normal serum, the procedure yielded 4.5 mg of homogeneous and hemolytically active rat Clq as shown by three bands of peptide in SDS polyacrylamide gel electrophoresis under reducing conditions and by a single precipitin line between rat Clq and rabbit anti-rat Clq antibody. Rabbit anti-rat Clq antibody prepared by immunizing a rabbit with 225 micrograms of rat Clq was specific as shown by a single precipitin line between the antibody and normal rat serum and rat Clq with complete identity. The amino acid composition of rat Clq was very similar to that of human Clq. The purification procedure also yielded Clq-depleted rat serum which was used with the homogeneous Clq to establish a sensitive hemolytic assay: 4.5-13.5 ng of rat Clq can be reproducibly quantitated. The concentration of Clq in Brown Norway rats was estimated to be 41.5 +/- 3.0 micrograms/ml serum.

Amino Acids↗

PubMed2001

Comparative analysis of human serum albumin adsorption and complement activation for intraocular lenses.

Intraocular liquid, in contrast to blood, has no cellular components; therefore, proteins (human serum albumin [HSA], and [alpha, beta, gamma] globulins) are the major components that determine patients' response to the intraocular lens (IOL) surface. In addition to the amount of adsorbed proteins, the possibility of its conformational changes, including conformational changes of globulins C1 and C3 that respond for the activation of the complements system by the classical and alternative pathways, cannot be excluded. The interaction between IOLs and protein components of intraocular liquid directly influences the ocular exudative reaction in the early postoperational period, the intensity of cellular and pigmental scurf on the surface of the IOLs, and the state of endothelial cells of the cornea in the distant postoperational period. Our goal was to compare the interaction of commercial IOLs made from polymethylmethacrylate, silicone, poly-2-hydroxyethyl methacrylate (p-HEMA), and copolymer p-HEMA with collagen with HSA and the complement system. The total internal reflection fluorescence (TIRF) method and hemolytic assay were used for this task, respectively. It has been demonstrated that the probability of biocompatibility of commercially produced IOLs on the stage of protein adsorption can be evaluated using the kinetic of HSA-fluorescein isothiocyanate adsorption onto the IOL surface by the TIRF METHOD: In the case of IOLs from p-HEMA, a negative correlation was shown between the degree of irreversible adsorption of HSA and the minimum relative rate constant of the surface-induced complement activation. We did not find any correlation between hydrophilicity/hydrophobicity of lenses and their adsorptional properties including complement activation. From suggested adsorptional criteria in vitro for biocompatible surfaces, the hydrogel lens from p-HEMA has a lower probability of biocompatibility in comparison with other IOLs.

Adsorption↗

PubMed1978

Studies on the murine Ss protein: demonstration that the Ss protein is functionally the fourth component of complement.

This report describes a simple and reproducible assay for the fourth component of murine complement (C4). Utilizing this assay, experiments are described which conclusively demonstrate that the murine Ss protein (serum substance) is functional C4 and that ascitic fluid may be used as a source for isolating Ss. The evidence that Ss is functional C4 is: (i) pretreatment of mouse plasma with F(ab')2 anti-Ss completely abrogated the C4 hemolytic titer; and (ii) in the isolation of the Ss protein from ascitic fluid, Ss immunochemical activity and C4 functional activity coeluted in each step of a four-step purification procedure.

Animals↗

PubMed1985

Serum IgG and IgM rheumatoid factors and complement activation in extraarticular rheumatoid disease.

Rheumatoid factors (RF) may participate in both synovial and extraarticular (EA) inflammation in rheumatoid disease (RD). The relative roles of serum IgG RF and IgM RF in extraarticular rheumatoid disease (EARD) are unclear, as is the importance of complement (C) activation by these proteins. To investigate the relation of C activating properties of IgM RF (RF CAP) and total IgG RF and IgM RF to EARD we compared 18 patients with only articular disease to 27 patients with various EA manifestations (nodules, cutaneous vasculitis, neuropathy, Felty's syndrome) using radioimmunoassays for IgG and IgM RF and an established hemolytic assay for C activation by IgM RF. We calculated RF CAP by determining the mean hemolysis of sensitized SRBC/ml of RF serum (MH/ml). Normal volunteers and patients with other inflammatory arthritides served as controls. Controls had negligible amounts of IgG RF, IgM RF, and RF CAP. Mean IgG and IgM RF levels and RF CAP values were significantly higher in patients with EARD than those in the arthritis-only (AO) group. Mean IgM RF concentrations and IgM RF CAP correlated with each other and EARD. IgG RF also correlated with IgM RF and EARD, but did not contribute to RF CAP or EARD when adjusted for IgM RF. Further, some patients had high RF CAP values despite modest IgM RF levels. These data suggest that quantitative differences in IgM RF CAP and total IgM RF may be more important than IgG RF as determinants of EARD.

Arthritis, Rheumatoid↗

PubMed2006

Antinucleosome antibodies in patients with juvenile systemic lupus erythematosus.

Our objective was to evaluate the frequency of antinucleosome antibodies (anti-Ncs) in juvenile systemic lupus erythematosus (JSLE) comparing it to that observed for anti-DNA and to correlate the presence of these antibodies with clinical manifestations and disease activity. Anti-Ncs and anti-DNA were detected by ELISA in 74 patients with JSLE and 64 normal controls. Clinical records were reviewed. Disease activity was assessed by SLEDAI score. Anti-Ncs and anti-DNA showed sensitivity of 52.7% and 54% and specificity of 98.4% and 95.3%, respectively. Disagreement between the two assays was found in 25.7% of the cases: isolated positive Anti-Ncs in nine cases (12.2%) and isolated positive anti-DNA in 10 cases (13.5%). Agreement was found in 74.3%: both positive antibodies in 30 cases and both negative in 25. The presence of anti-Ncs was significantly associated with malar erythema, hemolytic anemia, anti-DNA and low complement levels, but not with renal manifestations. The presence of anti-Ncs was associated with a higher SLEDAI median (P < 0.001) and its titers correlated with the SLEDAI score (r = 0.504; P < 0.001). The frequency, sensitivity and specificity values were similar between anti-Ncs and anti-DNA antibodies in patients with JSLE. Nevertheless, the discordance of 25.7% between the two assays suggests that both antibodies may have a complementary diagnostic role. The association and correlation between anti-Ncs and several disease activity parameters demonstrated its usufulness in the follow-up of these patients.

Adolescent↗

PubMed1988

The complement system in sclera.

Sclera from donor globes was eluted in phosphate-buffered saline at 4 degrees C for 24 hr. Hemolytic assays were used to measure functional C1, C4, C2, C3, C5 and C6 in scleral eluates. Radial immunodiffusion was used to measure Factor B, a component of the alternative complement pathway, and albumin in scleral eluates. Molecular weight appeared to be a factor in determining the hemolytic activities of complement components C1-C6 in the sclera, because complement components with higher molecular weights had lower percentages of hemolytic activity in sclera relative to serum, while complement components with lower molecular weights had higher levels of hemolytic activity in sclera relative to serum. The sclera contains complement which could be activated by immune complexes that may be important in the pathogenesis of scleritis.

Adult↗

PubMed1987

Complement activation in peritonitis. Association with hepatic and renal perfusion abnormalities. First place winner: Conrad Jobst award.

The authors have shown that systemic activation of the complement system with either zymosan or cobra venom factor produces some of the hemodynamic changes characteristic of sepsis, specifically, a reduction in hepatic perfusion despite a normal or hyperdynamic systemic circulation. This study was undertaken to determine whether complement activation accompanied reductions in effective hepatic and renal blood flow (EHBF and ERBF, respectively) in a septic murine model previously demonstrated to be associated with flow redistribution. Rats underwent either cecal ligation and puncture (CLP) or sham laparotomy after a baseline blood sample was collected for complement assay. Eighteen hours later, thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, EHBF by galactose clearance, and ERBF by p-aminohippurate (PAH) clearance were determined. A second blood sample was collected for measurement of total hemolytic complement (CH50) by immune hemolysis of sheep erythrocytes and was compared to the t = 0 sample for calculation of per cent change in CH50. The cardiac output and hematocrit were normal in the CLP group relative to sham. The septic animals were tachycardic and slightly hypotensive, suggesting a diminished systemic vascular resistance. EHBF and ERBF fell dramatically in the septic group despite the normal cardiac output. Residual hemolytic complement activity was reduced to less than 40% of preseptic levels in the CLP group while sham values were no different than baseline, indicating massive complement activation in the septic animals. This study demonstrates an association between complement activation and hepatic and renal perfusion abnormalities in murine peritonitis. Work is underway to establish the temporal relationship between complement activation and visceral flow changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Isolation and characterization of a 92-KD surface molecule of Trypanosoma cruzi amastigotes recognized by a monoclonal antibody that induces complement-mediated killing.

The presence of lytic antibodies in the circulation of patients with chronic Chagas' disease might lead to their cure. It has been shown that amastigotes of Trypanosoma cruzi activate complement and accumulate large amounts of the terminal complement components, but without killing the parasites. One plausible explanation for this observation is that the insertion of the membrane attack complex of complement is prevented by inhibitors present in the parasite membrane. To explore this possibility, we raised a panel of monoclonal antibodies (MAbs) against the surface molecules of T. cruzi amastigotes. One of these, MAb M4C12, induced complement-mediated lysis of amastigotes as detected with a 86Rb-release assay. The antigen molecule from the membrane lysate of amastigotes that was recognized by MAb M4C12 was purified, characterized, and designated M4C12Ag. It is a 92-kD molecule structurally related to Ssp4, a previously characterized amastigote surface molecule. However, M4C12Ag is more basic (pI 6.9-7.1) than Ssp4 (pI 5.2-6.0), and is larger (92 kD) than Ssp4 (70 kD and 84 kD). Purified M4C12Ag did not inhibit the terminal components of complement when tested in hemolytic assays. This molecule may not be an inhibitor of complement, yet it is certainly an amastigote-specific surface molecule with immunologic importance because it is the only molecule that could induce formation of a lytic antibody.

Animals↗

PubMed1977

[Application of CH50 assay to the detection of circulating immune complexes (author's transl)].

Detection and titration of circulating immune complexes appear as an essential tool for the diagnosis and prognostic evaluation of immune complex diseases. Authors propose a simple, rapid, senstive and quantitative method based on the evaluation of complement by soluble immune complexes measured by 50% hemolytic activity. This technique was compared to the I125 C1q binding test and may be recommended for clinical laboratory use.

Antigen-Antibody Complex↗

PubMedDate not supplied

The role of complement in urticaria and angioedema.

Complement may play a primary or exacerbating factor in the production of urticaria and/or angioedema. As we have seen, the absence of inhibitor proteins, such as ClINH, C3bINA, or Carboxypeptidase N Anaphylatoxin Inactivator) can permit small amounts of active complement fragments, liberated "normally" through the action of nonspecific effector pathways, to produce generalized urticaria or angioedema. Abnormal degrees of complement activation, produced by circulating immune complexes, cryoglobulins, paraproteins, may also cause angioedema and/or urticaria. Autoimmune diseases, lymphoreticular malignancy, infections, drug reactions, and syndromes currently termed idioipathic may be associated with urticaria and angioedema due to pathologic complement activation. Direct effects of complement on blood vessels, either by anaphylatoxins or by the deposition of complement fixing immune complexes, may produce the vascular permeability requisite to these syndromes. Secondary effects, produced by the recruitment of inflammatory cells and/or the liberation of other vasoactive mediators from these cells may also occur. Finally, complement may act in a potent synergistic fashion with mediators such as prostaglandins to produce vascular permeability. Current studies, using antigenic, hemolytic, and immunofluorescent techniques for detecting complement involvement have identified a relatively limited number of syndromes in which complement is involved. It is anticipated that the use of more sensitive assays for anaphylatoxins, such as radioimmunoassay, neutrophil aggregation, and others, coupled with an understanding of the potential synergism between these and other mediators, will lead to the recognition of a wider role for complement in urticaria and angioedema.

Angioedema↗

PubMed1981

Detection of antibodies to Herpesvirus simiae and Herpesvirus hominis in nonhuman primates.

Sera from nonhuman primates, predominantly Macaca species, were assayed by a serum neutralization test for antibodies to antigenically related Herpesvirus simiae (B virus) and Herpesvirus hominis type 1. The data indicate that there would have been approximately 50% error in the diagnosis of Herpesvirus simiae infection if these sera had been tested only against Herpesvirus hominis antigen. The role of active guinea pig complement in the serum neutralization test was also evaluated and found to be required by many of the sera for reproducible and enhanced virus neutralization, particularly for B virus antibody determination. A plaque reduction assay was found to be highly sensitive, especially when complement (2.5-5.0 hemolytic units) was added, but impractical for large-scale serum surveys.

Animals↗