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Results for “Complement C3b Inactivator Proteins”

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PubMed2006

Complement activation is involved in biological responses to leukocyte adsorptive apheresis.

We examined the effects of complement activation on the biological responses of cellulose acetate (CA) beads. Peripheral blood containing the complement activation inhibitor nafamostat mesilate (NM) or heparin was incubated with CA beads in vitro. Thereafter, the fraction of adsorbed granulocytes as well as the generation of complement activation fragments (C3a and C5a) and interleukin 1 receptor antagonist (IL-1ra) were measured. Granulocyte adsorption, complement activation, and IL-1ra release were significantly inhibited in the presence of NM. Adsorption was significantly increased onto CA beads pretreated with plasma containing heparin even in the presence of NM and adding C3a or C5a enhanced IL-1ra release. These results suggested that bound complement fragment (e.g., C3b) on CA beads plays a central role in granulocyte adsorption to CA beads and that C3a and C5a augment the release of anti-inflammatory substances. We therefore conclude that complement activation is involved in these biological responses of leukocyte apheresis.

Adsorption↗

PubMed1977

Enhancement of C56-initiated lysis by cell-bound C3 fragments: evidence for a mechanism independent of the prior binding of C56 to C3b.

Cell-bound C3b can reversibly bind C56, the activated complex of the fifth (C5) and sixth (C6) components of complement, and in this way potentiate C56-initiated lysis by favoring the formation of C567 at the cell surface. We report here another way in which cell-bound C3 fragments can enhance C56-initiated lysis, which involves C567 generated in the fluid phase rather than at the cell surface. Evidence for the involvement of fluid phase C567 was obtained by use of dextran sulfate, which is known to inhibit the hemolysis of E mediated by fluid phase C567. Dextran sulfate strongly inhibited the formation of C567 sites on cells bearing C4b and C3b (EAC4b3b) as well as on unmodified E when C56 and C7 were added simultaneously to the cells. By contrast, dextran sulfate had virtually no effect on the reaction sequence involving the prior binding of C56 to C3b and subsequent formation of C567 at the cell surface. Treatment of EAC4b3b with either anti-C3 Fab' fragments or the C3b inactivator reduced but did not eliminate the enhancement of hemolysis, raising the possibilities that a C3 fragment(s) other than C3b also can enhance C56-initiated lysis and/or that the enhancement is indirect without a requirement for an interaction between C567 and the cell-bound C3 fragment itself.

Complement C3↗

PubMed1996

Neutrophil lactoferrin release induced by IgA immune complexes can be mediated either by Fc alpha receptors or by complement receptors through different pathways.

Our previous results showed that neutrophil secondary granule release, indicated by release of lactoferrin, was a slow process when induced by IgA immune complexes (IC) formed in heat-inactivated serum, but became very fast if IgA IC were formed in normal human serum. This phenomenon did not apply to the IC of other Ab isotypes. In this paper, we demonstrate that the fast lactoferrin release is caused by complement, mainly due to the deposition of C3b and iC3b on IgA IC. Either CR1 or CR3 can mediate the response and both receptors have to be blocked to prevent it. Complement also influences FcalphaR-mediated lactoferrin release, in that this is enhanced by the anaphylatoxin peptides, C5a and C5a(desArg). Divalent cations are required for FcalphaR and CR3- but not for CR1-mediated lactoferrin release. Genistein, a protein tyrosine kinase inhibitor, totally inhibits FcalphaR-mediated response, but has little effect on CR1-mediated response. Therefore, it is clear that different pathways of intracellular signaling are utilized. In addition, stimulation through FcalphaR promotes the receptor up-regulation, which is abolished by the presence of EDTA or genistein.

Antigen-Antibody Complex↗

PubMed1993

Opsonic effect of the alternative complement pathway on channel catfish peripheral blood phagocytes.

This study determined the effect of the alternative complement pathway (ACP) on neutrophil and monocyte phagocytes from peripheral blood of channel catfish, Ictalurus punctatus. Fluorescent-labeled latex microspheres, Edwardsiella ictaluri and Escherichia coli were used to quantify phagocytic attachment and ingestion. Activation of the ACP enhanced the attachment of bacteria and microspheres to neutrophils and monocytes. Activation of the ACP by serum opsonization of Escherichia coli increased its ingestion by neutrophils in comparison with Edwardsiella ictaluri and microspheres. Inactivation of the ACP and C3b diminished attachment by neutrophils and monocytes, and ingestion by neutrophils of microspheres and bacteria. Ingestion by monocytes was not affected by activation of the ACP. In the present study, we found that the ACP functions in opsonophagocytosis in catfish and that ingestion by neutrophils was especially enhanced.

Animals↗

PubMed2006

Opsonic requirements for the respiratory burst of neutrophils against Giardia lamblia trophozoites.

BACKGROUND: Giardia lamblia is an important cause of parasitic diarrheal disease worldwide. Occasionally, polymorphonuclear neutrophils (PMNs) may participate as effector cells against Giardia lamblia. The present study was performed in order to examine the role of specific antibody and complement components in promoting the respiratory burst (RB) of PMNs against Giardia lamblia. METHODS: PMNs from human adult volunteers were incubated with Giardia trophozoites in the presence of non-immune (NS) or hyperimmune (HS) serum (anti-Giardia titer, >1:1024). Adherence was scored visually on coverslide after staining with Giemsa. The ability of Giardia to trigger the oxidative response of PMNs was measured by the anion superoxide (O2(-)) production using a cytochrome C reduction method and by the luminol amplified chemiluminescence (CL) assay. RESULTS: Incubation with NS or HS increased Giardia adherence to PMNs from 6.9 +/- 3.2% (basal adherence of Giardia incubated in buffer) to 39 +/- 18.6% (p <0.01) and 76 +/- 19.5% (p <0.001), respectively. In absence of serum, Giardia failed to trigger an oxidative response of PMNs. Opsonization with NS or HS increased the PMN O2(-) production from 3.9 +/- 0.92 nmol/2.5 x 10(6) PMNs/10 min to 9.04 +/- 1.68 (p <0.05) and 17.9 +/- 1.32 (p <0.001), respectively. A similar enhancement of the CL response was also observed. The inactivation of complement activity by heat as well as the elimination of specific antibodies by absorption produced a significant abrogation of the oxidative response but in the case of HS heat inactivation alone did not abolish the response. Similar findings (variable abrogation of the oxidative PMN response) were observed when PMNs were incubated with monoclonal antibodies directed against complement C3, C3b or the low-affinity Fc receptors (CR1, CR3 or FcRlo). CONCLUSIONS: These results show that complement components and specific antibodies influence in the Giardia-PMN interaction. Although components of complement can contribute to the RB of PMNs, specific antibodies are critical for an optimal oxidative PMN response.

Animals↗

PubMed1999

Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases.

C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.

Adenoviridae↗

PubMed1976

Formation in the presence of C3 nephritic factor (C3NeF) of an alternative pathway C3 convertase containing uncleaved B.

C3 nephritic factor (C3NeF) interacts with native C3 and B in the absence of D to generate a C3 convertase containing an uncleaved form of B. Dose response studies with C3NeF and B, respectively, revealed incremental C3 inactivation without loss of B. These findings are in agreement with the previous isolation from such reaction mixtures of a 10S complex containing haemolytically inactive C3 and active B and manifesting C3 convertase activity. Functional contamination of C3 with C3b was negated by demonstrating that pretreatment of C3 with C3bINA had no effect on its subsequent interaction with B and C3NeF to generate C3 convertase activity, while pretreatment of C3b eliminated its effective interaction with B and C3NeF. Relatively higher concentrations of C3bINA present during interaction of C3, B and C3NeF suppressed C3 inactivation, indicating its dependence on amplification by utilization of the initial C3b generated. Trace quantities of D were not found by functional analyses of C3, B and C3NeF and pretreatment of these proteins with a concentration of DFP sufficient to suppress D activity had no effect on their effective interaction. The introduction of D to mixtures of C3NeF, B, and C3 resulted in B clevage and more efficient expression of C3 convertase function as defined by a reduced requirement for C3NeF.

Complement C3↗

PubMed1979

Further evidence for the antibody nature of C3 nephritic factor (C3NeF).

C3 nephritic factor (C3NeF), found in the sera of some patients with membranoproliferative glomerulonephritis, has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. Purified C3NeF binds to the amplification convertase of complement, C3b,Bb, and thereby prevents decay of its C3-cleaving potential. The capability of C3NeF to bind to C3b,Bb was used as a means for purifying C3NeF to homogeneity. The investigation described in this report suggests that binding of C3NeF to C3b,Bb occurs via the Fab portion of the molecule. Pepsin treatment of eight C3NeF preparations resulted in an average loss of 76% of C3NeF functional activity. Papain treatment induced a loss of approximately 90%. The decrease in functional activity could be attributed to the accelerated rate of dissociation of 125I-F(ab')2 and 125I-Fab fragments from stabilized cell-bound C3b,Bb. The dissociation rate of 125I-F(ab')2 from C3b,Bb was comparable with the decay of the functional activity of C3b,Bb stabilized by F(ab')2 or Fab fragments of C3NeF. Although these results suggest that the stabilizing activity of C3NeF is mediated by the Fab portion of the molecule, it was found that the Fc portion also contributes to its functional activity.

Antibodies↗

PubMed1973

Human lymphocytes bear membrane receptors for C3b and C3d.

Human peripheral blood lymphocytes have membrane receptors for EAC43b (sheep erythrocytes sensitized with antibody and complement) and also for EAC43d, obtained by treating EAC43b with C3b inactivator. Human granulocytes bind only EAC43b, C3 fragments obtained by limited trypsin digestion of purified human C3 display both C3b and C3d sites, since they inhibit rosette formation of lymphocytes with EAC43b and EAC43d. These findings raise the possibility that C3b and C3d receptor sites may be selectively distributed among normal subpopulations of B lymphocytes as well as among leukemic leukocytes.

Animals↗

PubMed1993

The possible role of membrane complement regulators in vasculitis.

Potentially, damage to endothelial cells of small vessel walls may be induced by the deposition of immune complexes and subsequent complement activation. The degree of deposition of C3b onto the endothelial cell surface under normal conditions is regulated by a number of membrane-bound regulators. Under inflammatory conditions various cytokines could potentially deregulate the expression and function of the membrane-bound complement inhibitors. Such a process could potentially increase the susceptibility of the endothelial cell layer to complement-mediated attack and provoke a vascular lesion.

Animals↗

PubMed1975

The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation.

Minor elevation of the concentration of the C3b inactivator (KAF) in whole serum (by 15-25%) markedly inhibits the capacity of the serum to support complement activation by inulin, aggregated IgG and by low concentrations of CVF. However, there was no effect when large concentrations of CVF were used nor was the spontaneous ageing of C3 slowed by increased KAF concentrations. These findings show that the activity of the C3b feedback cycle can be reduced by raising the KAF concentration above physiological levels. This finding may provide a mechanism for damping down complement activation locally in vivo. It seems that the spontaneous ageing of C3 in vitro does not involve a KAF-inhibitable step and therefore cannot be involved to explain the 'C3 tickover' in vitro.

Animals↗

PubMed1982

Activation of the alternative pathway of human complement by the extracellular slime glycolipoprotein of Pseudomonas aeruginosa.

The capability of the extracellular slime glycolipoprotein (GLP) of Pseudomonas aeruginosa to activate human complement was investigated. When slime GLP was added to type AB human serum, C3 and factor B were converted to their respective major cleavage fragments, C3b and Bb. This activation also occurred when slime GLP was incubated with serum-ethylene glycol bis(trichloroacetate)-Mg++, a result which indicates that the alternative complement pathway is involved. Additional support for the hypothesis of alternative pathway activation was provided by the fact that when serum-ethylene glycol bis(trichloroacetate)-Mg++ was preheated to inactivate factor B, slime GLP did not induce conversion of C3. The activation of the alternative pathway of human complement by slime GLP may represent an early nonimmune defense against P. aeruginosa infection.

Animals↗

PubMed1991

Glycoprotein C of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) is a receptor for the complement component C3b. We have previously isolated HSV-1 gC- strains (TN1, TN2 and TN3) from a patient with recurrent keratitis at three different times. These are very rare isolates because gC was thought to be essential for the virus in vivo. To determine whether gC modifies the interaction of complement with cell-free virus or virus-infected cells, we constructed gC+ recombinant viruses in which the intact gC gene of strain KOS was inserted into the TN1 virus genome. TN1 virus was inactivated by complement and TN1 virus-infected cells were lysed by complement; however, gC+ recombinant viruses became resistant to these effects of complement. These results suggest a role for gC in protection of both the virion envelope and the infected cell surface against damage by complement. TN1 virus was inactivated by complement from rats (Wistar, WKA, F344 and SD), guinea-pigs (Hartley) and humans, but not by complement from mice (C3H, DDD and BALB/c), which indicates that mice seem to be inappropriate as an experimental model for the study of HSV infection in which complement factors need to be considered.

Animals↗

PubMed1994

Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri.

Herpesvirus saimiri (HVS) is a lymphotropic herpesvirus that induces T-cell transformation in vitro and causes lymphomas and leukemias in New World primates other than its natural host, the squirrel monkey. Nucleotide sequence analysis of the HVS genome revealed two open reading frames with significant homology to genes for human complement regulatory molecules. One of these genes encodes a predicted protein (designated HVSCD59) with 48% amino acid sequence identity to the human terminal complement regulatory protein CD59 (HuCD59). The CD59 homolog from squirrel monkey (SMCD59) was cloned, and the corresponding amino acid sequence showed 69% identity with HVSCD59. BALB/3T3 cells stably expressing HVSCD59, SMCD59, or HuCD59 were equally protected from complement-mediated lysis by human serum. However, only HVSCD59-expressing cells were effectively protected from complement-mediated lysis when challenged with rat serum, suggesting that HVSCD59 was less species restrictive. The complement regulatory activity of HVSCD59 and SMCD59 occurred after C3b deposition, indicating terminal complement inhibition. Treatment of BALB/3T3 stable transfectants with phosphatidylinositol-specific phospholipase C prior to complement attack decreased the complement regulatory function of HVSCD59, suggesting cell surface attachment via a glycosyl-phosphatidylinositol anchor. Cells expressing HVSCD59 effectively inhibited complement-mediated lysis by squirrel monkey serum in comparison with SMCD59-expressing cells. Finally HVSCD59-specific transcripts were detected in owl monkey cells permissive for lytic HVS replication but not in T cells transformed by HVS, which failed to produce virions. These data are the first to demonstrate a functional, virally encoded terminal complement inhibitor and suggest that HVSCD59 represents a humoral immune evasion mechanism supporting the lytic life cycle of HVS.

3T3 Cells↗

PubMed1998

Expression of factor I-resistant mutants of the human complement component C3 in heterologous systems.

Complement plays a major role in hyperacute rejection of xenografts. In order to overcome this, we are developing, by minimal mutagenesis, a modified C3 molecule that, like cobra venom factor (CVF), escapes normal complement regulatory processes and inhibits complement-mediated responses by systemic depletion of C3. Unlike CVF, this protein should have little or no immunogenicity and be suitable for repeat administrations. As an initial step in this process, we have modified human C3 to make it resistant to inactivation by factor I. The factor I resistant C3 is capable of forming an active C3 convertase. Preincubation with normal human serum abrogated subsequent complement-mediated cytolysis by both the classical and alternative pathways, while wild-type (wt) C3 was inactive. The modified human C3 also blocked complement activity of guinea-pig serum. For economical and rapid production, we have developed expression of recombinant C3 wt and mutant proteins in the Baculovirus system. Large quantities are also being produced from stably transfected CHO cell lines. In addition, we have developed a fast C3 purification method by engineering a 6XHIS tag into the C3a portion of the molecule, thereby avoiding the need for subsequent separation of the tag from active C3b molecules.

Animals↗

PubMed1997

Response of SCC-12F, a human squamous cell carcinoma cell line, to complement attack.

We studied the response of a human squamous cell carcinoma cell line, SCC-12F, to human complement attack and found that the cells were completely resistant to complement lysis. In the absence of lysis, there was significant C3 deposition and C5b-9 deposition on the cells. Removal of the lipid-linked complement regulatory proteins CD59 and decay-accelerating factor (DAF) by treatment of the cells with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in increased C3b and C5b-9 deposition on the cells and a slight increase in cell death. Treatment of the cells with complement caused them to release membrane vesicles containing the terminal complement proteins. In addition, complement induced SCC-12F to produce significant amounts of prostaglandin F2alpha (PGF2alpha). We conclude that CD59 and DAF are important in the resistance of SCC-12F to complement and that these cells produce membrane vesicles and PGF2alpha in response to complement attack. These responses, in the absence of cell death, may be important in the pathogenesis of inflammatory skin disease in which complement is deposited.

Animals↗

PubMed1980

Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.

A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.

Animals↗

PubMed1977

Correlations between serum factor B and C3b inactivator levels in normal subjects and in patients with infections, nephrosis and hypocomplementaemic glomerulonephritis.

In normal subjects, factor B and C3b inactivator (KAF) levels were linearly related (r = 0-71); factor B levels in subjects with low normal levels of KAF were significantly lower than in those with high normal levels of KAF. This relationship is postulated to be the result of modulation by the level of KAF of the availability of nascent, spontaneously formed, C3b, in turn responsible for a constant low-grade activation of the C3b feedback which determines in part the level of factor B. The correlation did not obtain in patients with infections; levels of KAF and, to a greater extent, factor B were elevated. In patients with glomerulonephritis and hypocomplementaemic because of in vivo classical pathway activation, KAF and factor B levels were also poorly correlated, presumably because many factors were influencing the levels of these proteins transcending the modulating effect of the concentration of KAF. On the other hand, in patients with nephrosis and with MPGN Type II, KAF and factor B levels were low but were directly correlated. In nephrosis the correlation may be the combined result of an equal rate of catabolism of these proteins and of modulation of factor B levels by the level of KAF as in normal subjects. In MPGN Type II, the correlation may reflect the fact, noted in in vitro studies, that with alternative pathway activation, the level of KAF influences the extent of factor B and C3 conversion to a greater extent than when complement is activated by the classical pathway.

Analysis of Variance↗