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PubMed1983

Catabolites of the third component of complement in urines of hereditary nephritis patients.

Hereditary nephritis protein (HNP), an unusual urine protein from patients with hereditary nephritis (Alport Syndrome), was purified 120-fold to homogeneity. A slightly larger protein, pro-HNP, was similarly purified and was found to be a precursor of HNP. Both pro-HNP and HNP showed immunological identity to the third component of human complement, C3, and to its catabolite C3c. Pro-HNP had a molecular weight of 143,000 and, in equimolar ratio, polypeptide chains or fragments of molecular weights 75,000, 40,000, and 28,000. The largest and smallest chains contained carbohydrate. HNP had a molecular weight of 141,000 and fragments of molecular weights 60,000, 38,000, 26,000, and 17,000 in equimolar ratio; the two smallest fragments contained carbohydrate. Plasmin digestion of pro-HNP showed that the 75,000-Da chain, identical with the intact beta-chain of C3, broke down to the 60,000- and 17,000-Da fragments of HNP. In both pro-HNP and HNP, the polypeptide chains were linked by disulfide bonds, with the exception of the 17,000-Da fragment of HNP. This fragment was readily dissociated from the rest of the HNP molecule in the presence of sodium dodecyl sulfate. Amino acid analyses showed that both pro-HNP and HNP contained approximately 22 half-cystine residues per molecule. Extinction coefficients, epsilon 1% 1cm, at 280 nm were calculated to be 8.5 and 8.8 for pro-HNP and HNP, respectively.

Amino Acids↗

PubMed1983

Functional properties of membrane-associated complement receptor CR1.

It was previously shown that membrane receptors for C3b (CR1) purified from human erythrocytes were powerful inhibitors of the complement cascade and that they encompass the regulatory functions of the serum proteins beta 1H (H) and C4-binding protein (C4bp). In the present report we study the functional properties of membrane-associated CR1. When tonsil lymphocytes, which contain between 30 and 60% of CR1-bearing B cells, are incubated with the red cell complement intermediate EAC14oxy2lim or EAC14oxy23lim, they inhibit both C42 and C423 in a dose-dependent manner. These effects are mediated by membrane-associated molecules. Indeed, mild trypsinization of the lymphocytes abolishes their activity, and formaldehyde-fixed cells are as effective as viable cells. The inhibitory effects are in part mediated by CR1. The lymphocyte activities are reversed about 60% if monoclonal antibodies to CR1 or fluid phase C3b are present in the incubation medium. Moreover, upon addition of C3b-inactivator (l), lymphocytes release C3c fragments from EAC14oxy23b. The release of C3c was also abolished by antibodies to CR1. These results support the idea that CR1, as well as other molecules from the lymphocyte membrane, can function as inhibitor(s) of complement activation in their vicinity.

Animals↗

PubMed1989

C3c concentrations in sera of persons living in areas with different levels of air pollution in Northrhine-Westphalia (Federal Republic of Germany).

A number of air pollutants is known to affect the immune system resulting in alterations of the complement activity in serum. We determined the levels of C3c, one of the activation products of the component C3, in serum samples obtained from various population groups living in areas with different levels of air pollution in Northrhine-Westphalia (FRG). The results show that the serum C3c levels tend to be increased in subjects living in areas with elevated levels of air pollution. By applying a linear regression model it can be demonstrated that a significant part of the variance of the C3c concentrations is explained by the residence area factor after making allowance for the effect of various confounders. The increase of the serum C3c concentrations appears to result from an increased activation of the complement component C3. The underlying mechanisms as well as the pathophysiological significance of these findings are unknown.

Age Factors↗

PubMedDate not supplied

Complement C3 cleavage product in synovial fluids detected by immunofixation.

54 synovial fluids (SFs), 46 of them derived from various inflammatory diseases (30 rheumatoid arthritis (RA) SFs, 8 undefined arthritis (UA) SFs, 8 psoriatic arthritis (PSA) SFs) and 8 SFs from degenerative joint diseases (OA) were tested for C3c split product, using the immunofixation method. There were significant differences in the C3c product between the four groups investigated. In the OA group in the mean the percentage of C3c was low in comparison to the native C3 (C3c = 2.95%). RA SFs and UA SFs showed considerably higher values (20.1% for RA and 23.2% for UA) which were statistically significant in comparison to the OA SFs. With the exception of one SF the PSA SFs exhibited a relatively low percentage of the cleavage product. Despite the one high value the average C3c content of the PSA SFs was not statistically different from the OA SFs. In contrast to this low percentage of the C3c split product the PSA SFs showed the highest C3 concentration of all groups (87.0 +/- 36.5 mg/100 ml). Immunofixation is a simple and effective tool to determine the C3c split product in SFs. It might also be helpful for establishing the differential diagnosis of PSA vs RA on the basis of the C3 level of the SF in those patients where an elevated level of C3 is present.

Arthritis, Psoriatic↗

PubMed1985

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.

Antibodies, Monoclonal↗

PubMed1987

Deposits of immunoglobulins, complement, and immune complexes in inflamed human gingiva.

Gingival biopsy specimens from 20 patients with moderate to advanced periodontitis were obtained from inflamed sites with pockets of 5 mm or more. Sections were studied by an immunofluorescence technique, using polyclonal rabbit or goat anti-IgG, anti-IgM, anti-C1q, anti-C3a, and anti-C3c and mouse monoclonal anti-C9. Prewashed ethanol-fixed and nonwashed ethanol-fixed or frozen specimens showed many plasma cells staining for IgG or C3a, suggesting the possible occurrence of a receptor for C3a in plasma cells. Plasma cells containing IgM were also seen. Deposits of IgG and IgM with C1q, C3a, and C3c, suggesting immune complexes, were demonstrated by a double staining technique, combining fluorescein (FITC) or rhodamine (TRITC)-labeled anti-immunoglobulins with TRITC- or FITC-conjugated antibody to C3a, C3c, and C1q. The complexes were located mainly within or around vessel walls. Deposits of C3a and C1q were found in vessel walls, in the basement membrane zone of oral gingival epithelium, or diffusely distributed in the tissues. Deposits of C3c were found to a lesser extent and only in vessel walls. Mouse monoclonal anti-C9, visualized with FITC-labeled rabbit anti-mouse and swine anti-rabbit antiserum, showed granular deposits of C9, mainly in the basement membrane zone of oral gingival epithelium. The study indicates the involvement of immune complex vasculitis in inflammatory periodontal lesions. Also, our observations of the occurrence of deposits of complement factors support the hypothesis that complement factors play an important role in the immunopathology of the periodontal lesion.

Antigen-Antibody Complex↗

PubMed1983

[Complement components, whole complement activity, and circulating immune complexes in neoplastic diseases].

Serum levels of complement components(cc), whole complement activity (CH 50), and circulating immune complexes (IC) were measured in 41 patients with neoplastic diseases. The level of cc was higher than in healthy controls; the levels of C1q, C1INA, C4, C3c, C3ACT, C5, and C9 were statistically higher. In patients with lung cancer, the levels of cc were correlated with the clinical stage as well as the performance status. Both the IC serum level and the incidence of high serum IC levels in lung cancer were higher in stage III and IV than in stage I and II. Serum CH 50 was higher than in healthy controls, but not correlated with the clinical stage.

Aged↗

PubMedDate not supplied

Quantitation of the third component of complement on stored red cells.

By means of an automated anti-C3c consumption technic, we quantitated the molecules of the third component of human complement (containing the C3c fragment) that accumulate on red cells (RBCs) during liquid whole-blood storage. Calibrated C-3 sensitized zymosan particles ( ZyC3 ) were used as standards for bound C3. Although the dose-response curves of anti-C3c neutralization by ZyC3 and stored RBCs were similar, mathematical analysis showed that the shapes of these two sigmoidal curves were significantly different. This indicated that the large C3 molecules bound to stored RBCs differed antigenically from those bound to ZyC3 . Despite this difference, the former could be quantitated adequately by the automated anti-C3c consumption technic. Red cell-bound large C3 molecules were measured following various periods of storage at 4 degrees C of whole blood samples (n=102). An average of 48 molecules were detected on RBCs stored for 21 days. Statistical analysis of these data indicated that during storage at 4 degrees C there was a continuous accumulation of C3 on RBCs, followed by cleavage of C3c fragments. The degree of agglutination of stored RBCs with anti-C3c was proportional to the number of cell-bound large C3 molecules.

Blood Preservation↗

PubMed1984

Measurement of fragments of the third component of human complement on erythrocytes by a new immunochemical method.

This paper describes a new antiglobulin consumption method to quantitate the fragments of the third component of human complement (C3) on red blood cell (RBC) membranes. Zymosan-bound C3, which can be stored frozen at -80 degrees C for years, was used as a standard in these tests. Using anti-C3c antibody, zymosan-bound C3 could be calibrated against soluble converted C3 (beta 1A), but not against soluble, native C3 (beta 1C). Calibration with several commercial serum standards yielded virtually identical values. Approximately 79.8 +/- 28.2 C3d molecules (mean +/- 1 SD, n = 50) were detected on normal, freshly collected RBC by this method, whereas no C3c fragments were noted. EC43, prepared by dilution of blood samples with low ionic strength solution, had between 650 and 3,100 C3 molecules/RBC when measured with anti-C3c and between 1,140 and 6,500 C3 molecules when measured with anti-C3d. These data indicated that part of the C3b molecules on EC43 had cleaved to C3d. EC43 are reported to have up to 200,000 C3 molecules when measured by other techniques. To resolve this discrepancy, EC43 were prepared by dialysis of blood samples against low ionic strength solution. About 97.5% of C3 remained in plasma after dialysis supporting the results of our tests. The new assay is an accurate and sensitive method of quantitating C3 fragments bound to RBC in vivo and in vitro.

Binding Sites, Antibody↗

PubMed1983

Streptococcal antibodies and complement components in tropical post-streptococcal glomerulonephritis.

Anti-deoxyribonuclease "B' was elevated in 94.9% of 59 cases of post-streptococcal glomerulonephritis studied, whereas ASO was elevated only in 54.4% of cases showing that the ADNB titre is a more sensitive indicator of antecedent streptococcal infection than ASO, in tropical countries with a high prevalence of pyoderma. Serum C3c (beta 1A) levels were significantly decreased, especially in the early phase of the illness and returned to normal with remission. C4 levels were decreased only in the early phase of the illness, suggesting involvement of the alternate pathway subsequent to classical pathway activation.

Adolescent↗

PubMed1983

Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin.

The many different recognized functions of C3 are dependent upon the ability of the activated C3 molecule both to bind covalently to protein and carbohydrate surfaces and to provide binding sites for as many as eleven different proteins. The location of the binding sites for six of these different proteins (factors B and H, complement receptors CR(1), CR(2) and CR(3) and conglutinin) was examined in the naturally occurring C3-fragments generated by C3 activation (C3b) and degradation by Factor I (iC3b, C3c, C3d,g) and trypsin (C3d). Evidence was obtained for at least four distinct binding sites in C3 for these six different C3 ligands. One binding site for B was detectable only in C3b, whereas a second binding site for H and CR(1) was detectable in both C3b and iC3b. The affinity of the binding site for H and CR(1) was charge dependent and considerably reduced in iC3b as compared to C3b. H binding to iC3b-coated sheep erythrocytes (EC3bi) was measurable only in low ionic strength buffer (4 mS). The finding that C3c-coated microspheres bound to CR(1), indicated that this second binding site was still intact in the C3c fragment. However, H binding to C3c was not examined. A third binding site in C3 for CR(2) was exposed in the d region by factor I cleavage of C3b into iC3b, and the activity of this site was unaffected by the further I cleavage of iC3b into C3d,g. Removal of the 8,000-dalton C3g fragment from C3d,g with trypsin forming C3d, resulted in reduced CR2 activity. However, because saturating amounts of monoclonal anti-C3g did not block the CR(2)-binding activity of EC3d,g, it appears unlikely that the g region of C3d,g or iC3b forms a part of the CR(2)-binding site. In addition, detergent-solubilized EC3d (C3d-OR) inhibited the CR(2)-binding activity of EC3d,g. Monocytes and neutrophils, that had been previously thought to lack CR(2) because of their inability to form EC3d rosettes, did bind EC3d,g containing greater than 5 x 10(4) C3d,g molecules per E. The finding that monocyte and neutrophil rosettes with EC3d,g were inhibited by C3d-OR, suggested that these phagocytic cells might indeed express very low numbers of CR(2), and that these CR(2) were detectable with EC3d,g and not with EC3d because C3d,g had a higher affinity for CR2 than did C3d. A fourth C3 binding site for CR(3) and conglutinin (K) was restricted to the iC3b fragment. Because of simultaneous attachment of iC3b to phagocyte CR3 and CR(3), the characteristics of iC3b binding to CR3 could only be examined with phagocytes on which the CR(1) had been blocked with anti-CR(1). Inhibition studies with EDTA and N-acetyl-D-glucosamine demonstrated a requirement for both calcium cations and carbohydrate in the binding of EC3bi to CR3 and to K. However, CR(3) differed from K in that magnesium cations were required in addition to calcium for maximum CR(3) binding activity, and NADG produced less inhibition of CR(3) activity than of K activity.

Animals↗

PubMedDate not supplied

Vitronectin modulates the expression of complement components of the terminal pathway synthesized by human umbilical vein endothelial cells in vitro.

In this study we demonstrate that human endothelial cells (EC) synthesize mRNA for vitronectin by using techniques based on reverse transcriptase (RT) reaction and polymerase chain reaction (PCR). The identification of vitronectin mRNA, shown by sequence analysis of PCR-amplified RT product of RNA extracted from EC, clearly demonstrates that these cells synthesize mRNA for vitronection. We further investigated whether vitronectin in serum-free EC cultures regulates the net expression of the terminal complement pathway, measured as the terminal complement complex (TCC) bound to co-cultured agarose beads which activate the alternative pathway. Presence of polyclonal F(ab')2 anti-human vitronectin (VN) antibodies, regardless of concentration (10-80 micrograms/ml), significantly reduced the binding of monoclonal anti-C3c antibodies to co-cultured beads, whereas the binding of monoclonal anti-TCC antibodies was unaltered or significantly increased compared with controls. Despite some interexperimental variation in the results, addition of vitronectin (10-80 micrograms/ml) to the EC resulted in an inversely related pattern compared with experiments using anti-VN antibodies. The binding indices of anti-C3c are comparable to the controls. On the other hand, there is a steady concentration-dependent (10-80 micrograms of vitronectin added) reduction in binding of anti-TCC up to approximately 60%. The results indicate that vitronectin regulates the expression of synthezised and surface-bound TCC in serum-free EC cultures, comparable to previous findings in serum.

Cells, Cultured↗

PubMed1989

Analysis of the complement C3 fragments associated with hemodialysis membranes.

During hemodialysis with cuprophan membranes, bioactive peptides are generated because the alternative pathway of complement is activated. When cellulose acetate membranes are employed, complement activation is attenuated. The molecular basis for this improved biocompatibility is unknown. It has been postulated, however, that the complement activating potential of dialysis membranes is influenced by the availability of free hydroxyl groups which would provide an acceptor site for activated C3. To investigate this hypothesis, the forms of C3 associated with cellulose acetate and cuprophan membranes have been analyzed. By Western blot, the predominant form of C3 present on both types of membranes is C3c, a degradation product of C3 that lacks the thiolester necessary for covalent bonding. Minimal amounts of C3d (the region of C3 which contains the thiolester) were observed on both membranes; however, by ELISA, there was no difference in the amount bound to cellulose acetate compared to cuprophan. Further, membrane-associated C3d could be removed by urea, suggesting that it was not bound covalently. These studies indicate that the complement activating potential of dialysis membranes is not determined primarily by the availability of potential covalent binding sites for activated C3b.

Blotting, Western↗

PubMed2001

Complement C3b interactions studied with surface plasmon resonance technique.

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Binding Sites↗

PubMed1992

Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1984

Plasma exchange in myasthenia gravis complicated with complement activation and urticarial reactions using fresh-frozen plasma as replacement solution.

6 patients with myasthenia gravis (MG) underwent 26 plasma exchanges (PE) using fresh-frozen plasma (FFP) as replacement solution. After each procedure, complement C3 conversion products (C3c) were detected in the plasma of all the patients. 3 patients had one or more attacks of urticaria during or immediately after the procedure. Using 5% albumin-saline solution as plasma replacement solution, complement activation and urticarial reactions were not seen. It is concluded that infusion of FFP to patients with MG should be avoided as it may induce complement activation and allergic reactions.

Adolescent↗

PubMed1988

Immune complexes in solid tumours precipitable by 3.5% polyethylene glycol: analysis of some nonspecific components.

Some nonspecific components (IgG, C3c, C4,) of circulating immune complexes (IC) precipitable by 3.5% PEG were assayed by laser nephelometry in the sera of 71 patients with solid tumours, and of 39 patients with autoimmune diseases. PEG-IgG, PEG-C3c, and PEG-C4 were higher in cancer and in autoimmune diseases than in controls. In cancer patients, PEG-C4 levels and PEG-C4 positivity rate correlated well with tumour burden. PEG-C3c and PEG-IgG were present at higher levels and PEG-C4 was present at lower concentration in cancer than in autoimmune diseases. The results of the present study, performed on a large population of patients with cancer and autoimmune diseases, indicate a different composition of circulating IC in these diseases. Particularly, these data suggest a preferential involvement of the alternative pathway of complement activation in cancer, as already described by other authors. The aggregation of C3c to IC, mediated by the alternative pathway, seems to be the "key" event in the process of IC solubilization. In cancer patients' sera, the presence of "solubilized" IC could partly explain their peculiar biological behaviour as well as the disagreement observed among the different assay methods based upon the binding with complement factors.

Antigen-Antibody Complex↗

PubMed1983

Complement (C3)-receptor-mediated phagocytosis of agarose beads by mouse macrophages. II. Extracellular activation of macrophage-derived complement on agarose via the alternative pathway.

Phagocytosis of agarose beads by macrophages cultured under serum-free conditions was studied. 48 h was needed before a plateau in the uptake was reached. The ingested agarose beads were coated extracellularly with macrophage-derived protein before attachment and ingestion of the beads. Intracellularly, the agarose-linked protein was removed from the agarose. If the ingested agarose beads were extracted from the macrophages within 24 h after the plateau in the uptake was reached, a fraction of the beads could attach to new macrophages, demonstrating modification of the agarose beads by opsonin(s). Because of binding of anti-human C3c antibodies to beads extracted from the macrophages after 24 h of phagocytosis and the trypsin sensitivity of the protein on the agarose, we conclude that the main opsonin on the agarose beads is C3bi. Requirements for the stimulatory effect of agarose on macrophages are summarized.

Animals↗