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PubMed1979

Complement components detected on normal red blood cells taken into EDTA and CPD.

Normal red blood cells (RBC) from fresh EDTA and CPD blood and from stored CPD blood were examined for the presence of bound subcomponents of C3 and C4. By serologic agglutination tests, only C3d was detectable on the cells. Incubation in compatible fresh normal serum (FNS) at 37 degrees C appeared to increase the amount of 3Cd on the RBC. C3b was serologically detectable only on stored CPD cells and only after incubation in compatible FNS. No. C4 components were detected on the cell surfaces in agglutination tests. Using an indirect labeling technique, small, but significant, amounts of C3d and C4d were found on all three types of untreated cells. C3b was present on stored CPD cells only. The indirect labeling technique showed a significant increase in C3d and C4d on all cells following incubation i- compatible FNS, whereas bound C3b was significantly increased only with stored CPD cells. There was no increase in bound C4b following serum incubation. The average number of C3d molecules per cell on normal EDTA cells was 557 and average Ko was 3.6 x 10(7) l/mol.

Animals↗
PubMed1981

Human membrane-bound C3 receptors. II. Physiochemical properties of the C3b and C3d receptors isolated from tonsil cells by immunoprecipitation.

The aim of the present study was to define the physicochemical structure of C3b and C3d receptors of lymphoid cells. C3b and C3d receptors were isolated from KBr lysates of the 20,000 g fraction of human tonsil homogenates by immunoprecipitation with an anti-C3 receptor serum (AC3RS). Sodium dodecyl sulphate (SDS) gel filtration and polyacrylamide gel electrophoresis (PAGE) of unreduced immunoprecipitates revealed a highly predominant component with an apparent molecular weight (mol. wt.) greater than 1 x 10(6) and a small component with a mol. wt. of 80,000. After reduction, the SDS-PAGE profile was made up of a constant major 38,000 mol. wt. component and a inconstant smaller 18,000 mol. wt. component. The 38,000 (and also the 18,000) component could be isolated only from C3 receptor-active lysates, and not from C3 receptor-negative lysates. Taken together, the results of this study suggest that the active C3 receptor molecule of tonsil cells is a lipoprotein complex with a mol. wt. greater than 1 x 10(6); its protein moiety consists predominantly of disulphide-bridged polypeptide chains with a mol. wt. of 38,000; C3b and C3d receptors are composed of equal-sized polypeptide chains, but the specific binding sites for C3b and C3d are located on different molecules.

Chromatography, Gel↗
PubMed1994

CR2 is the primary acceptor site for C3 during alternative pathway activation of complement on human peripheral B lymphocytes.

Human cells infected with certain viruses acquire the ability to activate the alternative pathway (AP) of complement. Complement receptor 2 on EBV-infected lymphoblastoid cell lines has been reported to act as the covalent binding site for C3b during AP activation. Using flow cytometry, we investigated the ability of normal human peripheral blood leukocytes to activate the AP in homologous serum. Deposition of C3 fragments was determined as a measurement of complement activation on each of the subpopulations of the blood cells. Incubating human peripheral blood leukocytes with homologous or autologous serum resulted in C3 deposition on B cells and, to a lesser extent, on monocytes and polymorphonuclear leukocytes. Complement activation in the presence of Mg2+ ions and EGTA revealed major involvement of the AP in the case of B cells, and to a lesser extent for other leukocyte populations examined. Preincubation of the leukocytes with polyclonal anti-complement receptor 2 Ab markedly decreased the C3 fragment deposition, as a result of in vitro AP activation, on B cells, indicating that on normal human B cells this receptor may be involved in AP activation. Freshly isolated, normal human B cells also bear low but significant amounts of C3d,g fragments on their membranes, indicating that this AP activation also occurs in vivo. AP activation was partially decreased in the presence of autologous erythrocytes (RBC) suggesting that complement regulatory proteins on RBC play a role in limiting the AP activation in vivo.

Antigens, CD↗
PubMed1980

Component concentrations and activation of the complement system in neonatal illness: a preliminary study of necrotizing enterocolitis.

Determinations of C3, C4, and C5 concentrations by radial immunodiffusion, and assays for the activation products of C3, C3c and C3d by counterimmunoelectrophoresis, were performed on 80 infants. Seven nonbacteremic preterm infants with necrotizing enterocolitis (NEC) or probable NEC (PNEC) were found at the time of diagnosis to have a significantly lower mean concentration of C3 (P less than 0.05, 1-tailed) without C3 activation when compared to other noninfected preterm infants. Ten full-term and 63 preterm infants were studied prospectively during the first days of life, and were then followed for the postnatal development of localized or systemic infection. Assays for the detection of C3 activation products were negative in all these infants. Four preterm infants who developed PNEC after 5 or more days without clinical illness had low original concentrations of complement components. The pathogenesis of NEC may not involve primarily complement activation, and susceptibility to this condition may be related to pre-existing deficiencies in complement component concentrations relative to gestational age, or to defective activation of C3 in the presence of certain bacterial species and strains.

Complement Activation↗
PubMed1993

Complement activation during tryptophan immunoadsorption treatment.

Antibodies against human lymphocyte antigens (HLA) are frequently seen among patients undergoing repeated renal transplantations. Graft survival can be improved by eliminating these antibodies by plasmapheresis before transplantation. In this study, we have tried a new extracorporeal procedure to remove the anti-HLA antibodies. An immunoadsorption column (IM-TR) with a matrix of polyvinyl alcohol (PVA) gel conjugated with a hydrophobic amino acid tryptophan was utilized. Previous results have shown that repeated IM-TR treatments are at least equally effective as plasmapheresis in reducing levels of specific immunoglobulins in treated patients. In this study, 7 HLA-immunized patients were treated before renal transplantation. Each patient was subjected to a total of 12 treatment sessions divided into 3 sessions per week. After each treatment session, the reduction of the immunoglobulins was less than what has been reported for plasmapheresis. This suggests that mechanisms other than immunoglobulin depletion are involved in the reduction of the total immunoglobulin levels. The IM-TR treatment resulted in a strong complement activation triggered by the alternative pathway. Since the adsorbed plasma was returned to the patient, exceedingly high levels of the activation fragment C3d (C3dg) were found in plasma during and after the treatment. We conclude that the extensive generation of C3dg may be one of the factors that plays a role in the reduction of the antibody levels since the C3dg fragment has been shown to down-regulate the immune response.

Adult↗
PubMed1975

Evidence for macrophage C3d-receptor active in phagocytosis.

Evidence is presented that guinea pig peritoneal macrophages possess membrane receptors able to recognize the C3b inactivator-cleaved third component of complement. This conclusion is based on the finding that rosette formation and phagocytosis by macrophages of EA-IgM-C1423 were equal regardless of whether EA-IgM-C1423 had been pretreated with C3b inactivator or not.

Animals↗
PubMed1992

Expression and localization of proteins of the complement system in human skin.

The complement system participates in the immune recognition of foreign antigens, many of which may penetrate the skin by physical injury or transcutaneous adsorption. In this study, we examined the presence of complement components and complement regulatory proteins in the human skin and cultured human keratinocytes. Immunofluorescence studies showed C3, Factor B, decay accelerating factor, the C3b receptor (CR1), and C3d receptor (CR2), distributed among cells of the epidermis as well as on cultured keratinocytes. Immunoblot analysis of keratinocytes supernatants showed the presence of C3 with a molecular weight of approximately 180 kD. The decay accelerating factor was localized as previously reported on elastic fibers; additionally it was observed in the basement membrane zone. In situ hybridization studies suggest the expression of CR1 and CR2 mRNA in human epidermis. These results show the presence in the human epidermis of complement components that are capable of generating the initial C3 convertase of the alternative pathway. The presence of complement regulatory proteins could endow keratinocytes with immune functions such as the regulation of complement activation and endocytosis of C3 opsonized particles.

Antigens, CD↗
PubMed2000

Immunomodulatory functions of murine CR1/2.

C3-fragments generated upon complement activation play an important role in the formation and regulation of immune responses. Receptors interacting with various activation fragments of this versatile complement component are expressed on a wide variety of cell types, such as lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes, consequently C3-products may influence several biological functions at different sites of the body, where complement activation takes place. In the last decade, genes, protein structure and functions played by murine complement receptors CR1 and CR2 (mCR1/2) have been deciphered. In this review, we wish to relate these properties, and fit it into the context of events following in vivo complement activation. We separately address the roles played by murine mCR1/2 as BCR coreceptor and as BCR independent structure, and propose a mchanism for the utilization of antigen-C3d conjugates bound on B cells. Finally, we raise some of the questions that remain to be elucidated in order to get a more precise picture of the functions of mCR1/2.

Animals↗
PubMed1982

Structure-function relations in the third component of human complement (C3)-I. Hydrophobic site.

Charge shift electrophoresis and crossed hydrophobic interaction immuno-electrophoresis were used to demonstrate the presence of hydrophobic sites in the human C3 molecule. C3b and C3d were true amphiphilic proteins that could bind to hydrophobic surfaces. To the contrary, native C3, that presented the characteristics of amphiphilic proteins upon charge shift electrophoresis, did not bind to hydrophobic surfaces. These results suggested that the hydrophobic sites were located in the internal part of the C3 molecule and they were exposed in the external part when C3 was activated. The action of chaotropes on C3 was studied in detail and showed that the hydrophobic sites protected the thiolester bond (present in the labile site) from hydrolysis by water and thereby preserved the biological properties of native C3.

Binding Sites↗
PubMed2003

Flow cytometry based detection of HLA alloantibody mediated classical complement activation.

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.

Case-Control Studies↗
PubMed1979

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.

Animals↗
PubMed1990

Complement fragments C3b and iC3b coupled to latex induce a respiratory burst in human neutrophils.

The complement fragments C3b and iC3b were purified from human serum by affinity chromatography with Sepharose-coupled monoclonal antibody against the C3d region of C3. The resulting preparations were more than 95% pure and contained less than 0.1% native IgG. Purified C3b and iC3b were coupled to latex beads (0.8 micron diameter) by means of F(ab')2 fragments of monoclonal antibodies against the beta chain or the C3d region of C3, thus orienting the C3b and the iC3b on the latex with the C3b- and iC3b-specific regions outwards. These particles were found to activate the respiratory burst of freshly isolated human neutrophils to 20-30% of the maximal capacity. Latex particles randomly coated with C3b or iC3b were about 3 times less stimulatory. C3b, iC3b and IgG coupled to latex in an oriented fashion were about equally effective in stimulating the respiratory burst. Neutrophils from a patient with a total deficiency of CR3 responded normally to C3b-coated latex but did not respond to iC3b-coated latex. A monoclonal antibody against the alpha chain of CR3 inhibited the activation by iC3b-coated latex and a polyclonal antibody against CR1 partially inhibited the activation by C3b-coated latex. We found an additive effect between IgG-coated latex and C3b-coated latex, regardless of the presence of IgG and C3b on the same particle or on different particles. Thus, binding of ligands to either CR1 or CR3 per se is sufficient to induce an activating signal to the NADPH oxidase in human neutrophils.

Antigens, CD↗
PubMedDate not supplied

Alternative pathway complement activation in rheumatoid arthritis.

Serum and synovial fluid (SF) levels of the complement components C3, C5, factor B (B), properdin (P), beta 1H and C3b inactivator (C3bINA), and EDTA-plasma and SF concentrations of C3d and Ba were measured in 40 rheumatoid arthritis (RA) and 5 patients with osteoarthritis. Decreased SF concentration of B and P and increased levels of Ba showed that increased alternative pathway turnover occurred in RA. Reduced SF C3bINA concentrations occurred, but levels of beta 1H were not reduced. The results showed that alternative pathway turnover was dependent upon C3 turnover, but failed to support the notion that levels of C3bINA or beta 1H control alternative pathway turnover in RA.

Arthritis, Rheumatoid↗
PubMed1989

Complement C3 proteins in psoriasis.

A new polymorphism of the complement factor C3 in human plasma was demonstrated by isotachophoresis in agarose gels followed by immunodetection with rabbit anti-human C3c and C3d immunoglobulins. Four bands were detected in the immunoprint of freshly drawn EDTA-plasma, which were C3s1, C3s2, C3f1 and C3f2. At least four additional C3 components in Mg2+ -zymosan activated plasma were present, which were C3b1 to C3b4. The different forms of C3 in frozen and thawed heparin-plasma from 20 patients with psoriasis and 20 healthy individuals were studied from the immunoprint. The total content of C3 components was 29% greater in the patients with psoriasis than controls. The major difference was in the C3b components which were increased by 46%. In psoriatic patients, the two slow C3 components C3s1 and C3s2 were increased by 24 and 56% respectively, when compared with controls. The two fast C3 components C3f1 and C3f2 were decreased to 29 and 37%. The results suggest a direct involvement of the complement factor C3 in psoriasis.

Adult↗
PubMed1986

Hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins reacting optimally at 22 degrees C.

A 65-year-old white man had severe hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins showing optimum reactivity at 22 degrees C. The IgG agglutinins were detected by manual indirect antiglobulin test (IAT) using anti-IgG, and had a titer of 1 at 37 degrees C, 128 at 22 degrees C and 16 at 4 degrees C against adult type O red blood cells (RBC). The corresponding titers with cord RBC were 1 (37 degrees C), 64 (22 degrees C) and 8 (4 degrees C). Proteolytic enzyme and neuraminidase treatment of RBC did not decrease these titers. No known specificity could be assigned to these agglutinins. The isolated agglutinins (recovered by cold adsorption, warm elution) were shown by immunoelectrophoresis to be IgG lambda antibodies. They did not bind complement in vitro, consistent with the finding that the patient had negative manual direct antiglobulin test (DAT) by anti-C3d. It could be shown only by automated IAT that patient's serum also contained IgM cold agglutinins which also reacted best at 22 degrees C and appeared to be of lambda light-chain type. The patient responded to corticosteroid therapy and remains well without treatment 14 months after the hemolytic episode. The presence of IgG cold agglutinins may be predictive of a favorable response to corticosteroid therapy.

Aged↗
PubMed1984

Inhibition of immune precipitation by complement.

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.

Animals↗
PubMed2005

Complement dependent trapping of infectious HIV in human lymphoid tissues.

OBJECTIVES: HIV-1 bound extracellularly to follicular dendritic cells (FDC) in germinal centers (GC) of lymphoid tissues (LT) represents the largest viral reservoir in HIV-infected individuals; however there is no direct evidence as to whether HIV trapped in human GC remains infectious. In the GC, complement receptors and Fc gamma receptors have been suggested to participate in trapping of HIV; therefore, the relative contribution of complement- and Fc gamma receptors in binding HIV on LT was investigated and the infectivity of this virus was tested. DESIGN: As it is difficult to isolate FDC without contaminations of productively infected cells, HIV was detached from LT of HIV positive individuals using antibodies blocking complement- and Fc gamma receptors. Isolated virus was tested in an infectivity assay. METHODS: HIV detached from LT was quantified by HIV p24 ELISA, PCR and in an in vitro infectivity assay. Presence and accessibility of viral envelope proteins, complement factors and immunoglobulins on the surface of detached viral particles were evaluated through viral capture by respective antibodies. RESULTS: Although both C3d-fragments and IgG molecules were identified on the surface of HIV detached from LT, trapping of HIV was mediated solely by CR2-C3d interactions, whereas contribution of Fc gamma receptors was not detectable. Infectivity assays with HIV stripped from LT of HIV positive individuals revealed that in four out of ten patients HIV particles were infectious. CONCLUSIONS: These findings indicate that in the GC infectious virus is trapped on CR2-expressing FDC (or B cells). Reduction of this pool of HIV could be a therapeutic goal.

Cells, Cultured↗
PubMed2000

A possible role of CD46 for the protection in vivo of human renal tumor cells from complement-mediated damage.

It is still unclear which membrane-bound regulatory proteins (mCRP) are important in vivo to protect tumor cells from complement-mediated damage. To address this question, the expression levels of CD46, CD55, and CD59 were measured semi-quantitatively in situ on renal cell carcinomas and compared with the expression level and cellular distribution of these mCRP in proximal tubuli within each patient (n = 31). It was also determined whether the expression of mCRP on tumor cells is associated with deposition of C3d and C5b-9. CD46 expression was decreased on tumor cells; in contrast, CD55 was expressed on tumor cells (12 out of 31 samples), while it was not detected on proximal tubular epithelial cells (PTEC). Also, expression of CD59 on tumor cells was increased as compared with its expression on PTEC. Furthermore, the localization on the cell surface of mCRP as observed on PTEC was altered on tumor cells. Because expression of mCRP may limit a complement-mediated anti-tumor response, we determined whether complement deposition was associated with the expression level of CD46, CD55, and CD59. The presence of C3d on tumor cells was associated with a low expression level of CD46 (p < 0.02). The expression level of CD46 was also associated with a low tumor stage (p < 0.04). The results suggest that in vivo CD46 plays a role in the protection of human renal tumor cells from complement-mediated injury.

Antigens, CD↗