C3-depleted Serum Research Articles

Inhibitory effect of free sialic acid on complement activation and its significance in hypocomplementemic glomerulonephritis

The role of free sialic acid on complement activation was investigated. The serum levels of free sialic acid and total sialic acid were measured by previously described methods in 16 patients with acute post-infectious glomerulonephritis (AGN), 27 patients with systemic lupus erythematosus (SLE), 15 patients with persistent hypocomplementemic membranoproliferative glomerulonephritis (MPGN), and 13 healthy controls. A statistical study demonstrated an increased level of free sialic acid in patients with AGN and SLE in which the hypocomplementemia improved throughout the course and a decreased level of free sialic acid in patients with MPGN and SLE in which hypocomplementemia continued throughout the course. The levels of total sialic acid were significantly increased in patients with AGN and SLE and were significantly decreased in patients with MPGN. There was no correlation between the levels of free sialic acid and total sialic acid in patients with AGN, in whom the levels of both total and free sialic acids were increased. To examine the effect of free sialic acid on the complement cascade, lipopolysaccharide (LPS) was incubated with normal human serum (NHS) in the various concentrations of N-acetyl neuraminic acid (NANA), a member of the sialic acid group. The incubation mixtures were examined by enzyme immunoassay using monoclonal anti-iC3b antibody or anti-Bb antibody. Native C3 or Factor B in NHS broke down less following the addition of NANA. To elucidate the role of NANA on the hemolytic function of C3, a rabbit erythrocyte (Ra E) hemolytic assay was carried out. Ra E lysed completely in the presence of R3 with native C3. However, hemolysis occurred to a lesser degree in C3-depleted serum (R3) or R3 with NANA-treated C3. To investigate the influence of NANA on complement components, the levels of complement components were measured in the incubation mixture with various doses of NANA and NHS. The levels of C3 and C5 were significantly decreased after the addition of NANA, even though the levels of Factor H and Factor I were not markedly changed. These data indicate that NANA exerts an influence on the complement components even though it has no effect on the regulatory proteins of complement. Our in vitro findings, together with the in vivo data, suggest that free sialic acid might have an inhibitory effect on the activation of C3 and the following complement cascade, and might also have been responsible for the improvement of hypocomplementemia.

Human immunodeficiency virus type 1 gp41 binds to Candida albicans via complement C3-like regions.

Oral candidiasis in human immunodeficiency virus type 1 (HIV-1)-infected persons is believed to be caused by the acquired T lymphocyte immunodeficiency. The direct interaction of C. albicans and HIV-1 in vitro was investigated. Twice as many yeasts adhered to cells transfected with the HIV-1 env gene as they did to controls. HIV-1 rsgp160 and rsgp41 but not rsgp120 were found to bind to Candida albicans via two C3-like regions within gp41. Normal human serum, but not C3-depleted serum, was able to inhibit rsgp41 binding to C. albicans. Vice versa, rsgp160 and rsgp41 were able to block rosetting of C. albicans with iC3b-coated sheep erythrocytes. Binding to C. albicans, and its inhibition by rsgp41 or rsgp160, was confirmed for the whole virus. Therefore, oral candidiasis in HIV-1-infected subjects may be augmented or may even be initiated by direct interaction between C. albicans and HIV-1 or HIV-1-infected cells.

Effects of complement activation on the expression of CD59 by human mesangial cells.

Human mesangial cells (HMC) were analyzed for their expression of 20-kDa homologous restriction factor (CD59), a glycophospholipid-anchored regulatory protein of the complement cascade. Flow cytometric analysis showed that CD59 was expressed on the HMC membrane and, following the activation of the terminal pathway complement components, CD59 expression on the HMC membrane increased. Northern blot analysis showed that CD59 mRNA levels increased by complement activation. Of interest, CD59 mRNA levels increased by soluble complement activation product, zymosan-activated C8-depleted serum, but not zymosan-activated C5-depleted serum. This effect of soluble complement activation product was due to the presence of the action of C5a. Thus, recombinant C5a increased CD59 mRNA levels. The capacity of CD59 to reinsert into rat E and inhibit C-mediated lysis was inhibited by mAb against CD59 (1F5). Metabolic labeling using [35S]cysteine showed that the molecular mass of CD59 in HMC was 20 kDa. In conclusion, CD59 is present on HMC and the expression of CD59 is controlled by at least two steps of complement activation pathway, C5 and C8.

Complement C3 participation in monocyte adhesion to different surfaces.

As part of an ongoing investigation into the role of the monocyte/macrophage in biocompatibility, a major goal is to identify the adhesion mechanisms that initiate and promote the observed in vivo morphologic progression of monocyte-to-macrophage-to-foreign body giant cell on biomaterials. We have exploited differently modified polystyrenes, specific component-depleted sera, and monoclonal antibodies (mAbs) to leukocyte integrins to ask what adhesion mechanisms mediate human blood monocyte adhesion to different surfaces in vitro. Preliminary findings are that monocyte interactions with fluorinated, siliconized, nitrogenated, and oxygenated surfaces are reduced by 50-100% when complement component C3-depleted serum is used for adsorption; reductions vary with material surface properties. Adhesion is restored on all surfaces when C3-depleted serum is replenished with purified C3. Monocyte adhesion to serum-adsorbed surfaces is inhibited by mAbs to the leukocyte integrin beta subunit, CD18 (mAbs 60.3 and MHM23), and partially inhibited by a mAb to the alpha subunit, CD11b (mAb 60.1), suggesting adhesive interactions between adsorbed C3bi (the hemolytically inactive form of the C3b fragment) and the leukocyte integrin CD11b/CD18. However, adsorbed fibrinogen reduces the effectiveness of these mAbs, indicating that alternative adhesion mechanisms may operate depending on the propensities of critical adhesion-mediating components to be adsorbed onto different surfaces.

Serum bactericidal activity and induction of chemiluminescence of polymorphonuclear leukocytes: complement activation pathway requirements in defense against Neisseria meningitidis.

Serum bactericidal activity and chemiluminescence (CL) responses of polymorphonuclear leukocytes (PMNL) to pathogenic Neisseria meningitidis serogroups B and W-135 and to nonpathogenic serogroup 29E were examined with pooled normal human serum depleted of the complement proteins C1q, factor D, properdin and C5. Purified C1q, factor D, properdin and C5 were added alone or in combination. For investigation of serogroup W-135 meningococci, a C1q, factor D and properdin-depleted postvaccination serum with high concentrations of anticapsular antibodies was also used. Serogroup B and W-135 cultured to log phase were resistant to the bactericidal activity of pooled normal human serum but were efficiently killed through the classical pathway alone when the bacteria were cultured to stationary phase. Nonpathogenic serogroup 29E meningococci in log or stationary growth phases were efficiently killed in serum, predominantly through the classical pathway. Serogroup W-135 meningococci grown to log phase were resistant to classical pathway-mediated bactericidal activity in postvaccination serum but were killed on addition of alternative pathway proteins. Stationary phase serogroup W-135 meningococci were killed through both pathways in the postvaccination serum. In the pooled normal human serum CL responses of PMNL were consistently more pronounced with fully reconstituted C1q, factor D, properdin, C5-depleted serum than with serum reconstituted with C1q, factor D and properdin suggesting contribution of actions related to terminal components. In the absence of C1q, serogroup W-135 meningococci in postvaccination serum induced a significant but delayed alternative pathway-mediated CL response. CL responses induced by serum-opsonized meningococci, in contrast to serum bactericidal activity, were not influenced by culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of complement activation on hemodialysis membranes by immobilized heparin.

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18).

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.

Neutrophil adhesion to xenogeneic endothelium via iC3b.

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.

Activation of complement by hemodialysis membranes: Polyacrylonitrile binds more C3a than cuprophan

Conventionally, complement activation by hemodialysis membranes has been determined by measuring fluid phase C3a. Based on such measurements, polyacrylonitrile (PAN) membranes have been classified as weak activators compared to cuprophan. Previous studies have demonstrated, however, that PAN adsorb fluid phase C3a. Based on that observation, we hypothesized that complement activation by PAN might be artifactually underestimated if relatively large amounts of C3a remained membrane bound. In the present study, a method that allows the simultaneous quantification of both fluid phase and membrane bound C3a was used to assess complement activation by PAN and cuprophan. Pieces of membrane were incubated with C3-depleted serum that had been repleted with radiolabeled C3. Subsequently, the supernates and membranes were subjected to SDS-PAGE, and complement activation was quantified by determining the radioactivity of the C3a bands in the gel. The results showed that while the serum exposed to cuprophan membranes contained almost five times more C3a than that exposed to PAN, approximately 80 times more C3a was bound to the PAN membranes. Consequently, the total amount of C3a generated in the presence of PAN was higher than that generated in the presence of cuprophan. We conclude that assessment of complement activation by hemodialysis membranes using fluid phase C3a measurements alone may be misleading.

Generation of C5-Dependent Bioactivity by Tissue-Bound Anti-BMZ Autoantibodies

We previously reported that complement-binding antibasement membrane zone (BMZ) autoantibodies can mediate complement-dependent directed migration and adherence of leukocytes to the BMZ in cryostat skin sections and that there is heterogeneity in the ability of anti-BMZ autoantibodies to mediate that response. Those observations suggested that directed migration and adherence of leukocytes to the BMZ might be dependent on the amount of complement-activating autoantibody deposited at the BMZ and the extent to which those antibodies could activate complement and generate C5-derived peptides (C5a, C5a des arg). In this study, we have examined the role of autoantibody concentration and C5 in mediating the adherence response. When cryostat skin sections were pretreated with anti-BMZ autoantibodies and subsequently incubated with neutrophils suspended in fresh serum, neutrophils adhered to the BMZ. Adherence was anti-BMZ autoantibody specific and proportional to anti-BMZ autoantibody concentration. To determine the role of C5 in mediating adherence, neutrophils were suspended in increasing concentrations of: 1) fresh serum, 2) heat-inactivated serum, 3) serum pretreated with antihuman C5, 4) serum pretreated with antihuman IgG, 5) C5-depleted serum, 6) purified C5, and 7) C5-depleted serum reconstituted with increasing concentrations of purified C5. The suspensions were then incubated with autoantibody-treated skin sections. The results showed a dose-dependent requirement for fresh serum and for C5-depleted serum reconstituted with increasing doses of C5. Adherence could be detected with C5 concentrations less than 200 ng/ml, which correspond to a C5a/C5a des arg concentration of 10(-8)-10(-9) molar. These results suggest that complement-dependent neutrophil adherence is a highly sensitive method for detecting and quantitating the ability of tissue-deposited anti-BMZ autoantibodies to activate complement and generate C5-derived bioactive peptides, for estimating the amount of C-activating anti-BMZ autoantibody deposited at the BMZ in vivo, and for evaluating the potential role of C-activating anti-BMZ autoantibodies in the pathogenesis of lesions.

The selective localization of B lymphocytes in the spleen and the role of complement receptors.

The role of complement receptors on the localization of T and B cells in the spleen of mice was studied using short-term homing experiments in cobra venom factor (CoF)-treated animals. The localization ratio of B and T cells in the spleen of CoF-treated mice decreased significantly compared to control recipients. No changes could be found in the relative distribution of resident T and B cells in the spleen or other lymphoid organs of CoF-treated animals and when their spleen or lymph node cells were transferred, the localization pattern was normal. When cells were incubated in serum prior to transfer a disturbed localization ratio in the spleen of untreated recipients was observed. This was due to a blockade of complement receptors as determined by the inability of the incubated cells to form EAC rosettes. No blockade of EAC rosettes and no changes in localization ratios upon transfer could be observed when the cells were incubated in functionally C3-depleted serum. The results suggest a role for the complement-receptor on B cells in the initial localization in the spleen, whereas no influence upon the selective localization in high endothelial venules-bearing organs was found.

Complement-dependent cellular cytotoxicity due to alternative pathway C3 activation by the target cell membrane

The cytotoxic activity of human blood lymphocytes toward Raji cells was strongly elevated when human serum (HS) was included in the cytotoxicity assay. This phenomenon also occurred when the effector cells were activated by interferon (IFN). Hypogammaglobulinemic serum (HyS) and heat-inactivated serum could also augment cytotoxicity, but C3-depleted serum was inefficient. IFN treatment of Raji cells decreased their sensitivity to lysis and this effect was counteracted by addition of HS to the system. It is likely that C3 activation by, and deposition on, Raji cells when used as targets for cytotoxicity facilitate their recognition and lysis by lymphocytes. These events may represent one mechanism operating in the natural killing phenomenon.