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PubMed1995

[Expression and role of complement regulatory proteins on human gametes and pre-implantation embryos].

Human gametes and pre-implantation embryos express selectively several complement regulatory proteins. Membrane cofactor protein (MCP, CD46) and decay accelerating factor (DAF, CD55) are regulators for C3 convertases and protectin (CD59) is an inhibitor of the membrane attack complex. These three proteins were identified on human sperm and found to be functional. CD55 and CD59 were both expressed by the plasmic membrane of unfertilized oocytes and pre-implantation embryos. CD46 was not present on unfertilized oocytes but appeared at the 6/8 cell-stage embryo when human gene expression first occurs. Complement receptor 1 (CR1, CD35) and MHC class I antigens were not found on oocytes neither on embryos. Such a selective expression of complement regulatory proteins associated with the lack of MHC class I antigens may represent an immune protective mechanism by which human gametes and pre-implantation embryos escape from complement-mediated damage during their travel through the female genital tract. Indeed uterine, tubal and follicular fluids contain all the components of the complement cascade, including classical and alternative pathways. Nevertheless participation of CD46 and CD59 in cell to cell interaction during fertilization and/or implantation cannot be excluded. CD59 is an adhesive molecule involved in the rosette phenomena and CD46 has been described as the human receptor for measles virus, which binds through a fusion protein. Monoclonal antibodies raised against these two proteins (CD46 and CD59) are able to inhibit heterospecific fertilization between zona-free hamster oocytes and human spermatozoa suggesting the role of these proteins during fertilization.

Animals↗
PubMed1982

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells.

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.

Animals↗
PubMed1982

Partial H (beta 1H) deficiency and glomerulonephritis in two families.

H (beta 1H) controls the C3b amplification loop by its ability to displace Bb from the alternative pathway convertase, C3b,Bb, and acts as a cofactor with I (C3b inactivator) to produce inactive C3b. Serum C3 levels are dependent to a large extent on the levels of H and I. Partial H deficiency was found in two families. The index case in Family 1 had vasculitis, thrombocytopenia, proteinuria, and depressed serum H and C3 levels. The index case in Family 2 had depressed serum H and B (Factor B) levels and IgA nephropathy which progressed to renal failure. His sister also had IgA nephropathy and depressed serum H and C3 levels. The depressed serum C3 level, B level, and H level could be responsible for the development of the immune diseases found in some members of these families.

Adolescent↗
PubMed2004

The Kaposi's sarcoma-associated herpesvirus complement control protein mimics human molecular mechanisms for inhibition of the complement system.

Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.

Amino Acid Motifs↗
PubMed1987

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition.

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.

Antibodies↗
PubMed1974

Properdin factor D: characterization of its active site and isolation of the precursor form.

The activity of properdin factor D was measured by the generation of the hemolytically active cellular intermediate, EAC43B(D), bearing the C3b-dependent alternate pathway C3 convertase. Treatment of factor D with DFP prevented formation of EAC43B(D); thus, a serine esterase is essential for the generation of the alternate pathway C3 convertase, a situation analogous to the role of C1 in the formation of the classical C3 convertase, C42. The definition of factor D as a serine esterase prompted a search for its proenzyme form, and resulted in the chromatographic isolation from plasma of a single peak of trypsin-inducible factor D activity, distinct from activated factor D. Analytical gel filtration indicated an apparent mol wt of 25,000. This protein from which trypsin elaborated factor D activity, as assessed by the formation of EAC43B(D), the generation of the CoVF-dependent C3 convertase, and the cleavage of factor B in the presence of C3b, was designated "precursor factor D." The DFP resistance of precursor factor D, and the susceptibility of its trypsin-activated form to inactivation by DFP is analogous to the behavior of other plasma serine esterases, including C1.

Animals↗
PubMed1976

Reversible activation of proactivator (factor B) of the alternative pathway without cleavage of the molecule.

The serum of a patient (M.C.) with chronic glomerulonephritis and renal deposits of IgA, C3, and properdin converted C3 on overnight exposure of 0 degrees C. The cold reaction was dependent on immunoglobulin, initiating factor, Factors B and D, and magnesium but not on properdin. Factor B, the C3-cleaving enzyme in this reaction, was used in zymogen form. After participation in this rection, Factor B zymogen in M.C. serum could be fully activated by cobra venom factor (CVF) at 37 degrees C. That activation without fragmentation was not due to an abnormal form of Factor B was shown by its typical cleavage on incubation of MC. serum with CVF or C3b or after depletion of C3b inactivator. The evidence indicates that in the cold reaction only the initial C3 convertase of the alternative parhway is formed and that this enzyme is responsible for the observed C3 consumption.

Chronic Disease↗
PubMed1987

C3 nephritic factor determination. A comparison between two methods.

C3 nephritic factor (NEF), an IgG autoantibody to the alternative pathway C3 convertase, is usually measured by crossed immunoelectrophoresis (CI) but recently a reliable haemolytic assay (HA) was described by Rother (1982). This method is more specific than CI because it is negative in sera with immune complexes, SLE and sera incubated with IgG aggregates. The haemolytic assay is sensitive enough to detect NEF antibody in serum from patients with only slightly low C3 levels and NEF negatives by CI. The haemolytic assay is easy to perform and reproducible, the interassay coefficient of variation being 10.7% compared to 64% in the CI. The intra-assay coefficient of variation in CI was 28% compared to 5.5% in the haemolytic assay. The haemolytic method enabled us to study the kinetic effects of NEF on C3b.Bb bound to sheep erythrocytes, and the lysis mediated by ShE.C3b.Bb.NEF complex. Also the C and NEF binding to sheep erythrocytes was studied.

Animals↗
PubMed1983

Synthesis of complement by macrophages and modulation of their functions through complement activation.

During the last decade considerable progress has been made to characterize intimate functional links between macrophages, a major cellular component of immunoinflammatory responses, and the complement system representing the major humoral mediator of inflammation. Macrophages of various species and tissue sites have been shown to synthesize and release most of the complement components providing these cells with their own "pericellular" complement system. Circumstantial evidence for the assembly of both classical and alternative pathway convertases has been adduced. An intricate network of feedback loops involving endogenous and extrinsic factors operates to adjust complement production to acute requirements, for example augmenting production in the face of accelerated turnover at sites of inflammation, and returning it to baseline levels once the inflammatory stimulus has subsided, in order to maintain a fine-tuned balance. The molecular mechanisms underlying regulation of complement synthesis by macrophages are beginning to be elucidated by use of gene technology. On the other hand, complement activation products exert a number of effects on macrophages via specific surface receptors causing internalization of offending agents, microbes, and immune complexes, promotion of intracellular killing, controlling migration behavior, inducing release of potent biologic substances such as lysosomal enzymes, arachidonic acid metabolites, and interleukin 1. In these interactions, two important humoral mediator systems of inflammation, the complement system and the arachidonic acid cascade, are functionally linked at the level of the macrophage. Stimulation of the release of immunomodulating compounds from macrophages invoke a role for complement in immune regulation. This multifaceted interplay is of particular importance considering the mobility of macrophages that allows them to gain almost unrestricted access to sites of ongoing immunoinflammatory responses. The time seems to have come to abandon the petrified thinking in socalled systems as, for instance, humoral versus cellular, specific versus unspecific, and to proceed to interlocking functions guided by physiology proper.

Anaphylatoxins↗
PubMed1983

The human C3b receptor.

The cellular receptor for the C3b fragment of the third component of complement is a 205,000 molecular weight glycoprotein expressed by erythrocytes, polymorphonuclear leukocytes, monocytes, B lymphocytes, a subset of T lymphocytes and glomerular podocytes. The receptor molecule is a potent inhibitor of complement activation by both the alternative and classical pathways. It serves as a cofactor in the proteolytic degradation of C3b bound to immune complexes. On neutrophils and monocytes, the receptor enhances immunoglobulin-dependent phagocytosis of opsonized particles and triggers internalization of soluble ligands bearing C3b. The number of C3b receptor molecules expressed on erythrocytes is genetically determined and was found to be low in patients with systemic lupus erythematosus: these abnormalities when associated with a low number of receptors in the kidney of patients with non-systemic lupus erythematosus nephritis may predispose to immune complex diseases.

Animals↗
PubMed1976

C3 requirements for formation of alternative pathway C5 convertase.

Although alternative pathway C3 and C5 convertases both have active proteolytic sites dependent on the same protein, Bb, the quantitative requirements for the expression of these activities are sufficiently different to permit their delineation in terms of B input an cell-bound C3b. That the labile component of each active site is Bb was established by their parallel decay rates, regeneration of the original specificities with B in the presence of D, and stabilization of each convertase by C3NeF. The evidence that the spatial relationships of Bb and C3b on the cell surface for C3 and C5 convertase activities are distinct is based not only upon the decay and regeneration of each original convertase but more so upon their interconversion. C3 convertase is converted to C5 convertase by interaction with additional C3 whereas C5 convertase reverts to a C3 convertase by treatment with C3 INA. The capacity of C3 INA treatment to abolish C5 convertase sites without affecting C3 convertase sites indicates the existence of two functional species of C3b, one of which is protected in the C3bBb complex whereas the other is exposed.

Cell Membrane↗
PubMed1976

C3 nephritic factor (C3NeF): stabilization of fluid phase and cell-bound alternative pathway convertase.

C3 nephritic factor (C3NeF) defined by the capacity of nephritic serum and its fractions to initiate loss of the B antigen of C3 in normal serum was purified from the serum of three different donors and shown to function by stabilization of membrane-bound and fluid phase alternative pathway C3 convertase. C3NeF converts cell-bound C3B sites in a dose-related manner to CEB(NeF) sites, which exhibit an approximate 10-fold increase in half-life. The linear relationship between the C3NeF input and the residual hemolytic sites on EAC43B present after incubation for 20 min at 30 degrees C, during which labile C3B sites have decayed, indicates that the number of residual C3B sites is directly related to the dose of C3NeF. The capacity of C3NeF to stabilize the C3B convertase in a temperature- and dose-dependent manner, which is independent of binding or consumption of C3NeF, in a fluid phase reaction mixture of 125I-B, 131I-C3 and D permits isolation of a 10S complex containing radiolabeled C3 and B and exhibiting C3, convertase activity on an exogenous C3 source. Thus, the stabilizing effect of C3NeF is not limited to membrane-bound C3B but is also sufficient to permit recovery of a fluid phase C3 convertase formed during the interaction of C3, B, and D.

Binding Sites↗
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↗
PubMed1988

Naturally occurring anti-band 3 antibodies and complement in phagocytosis of oxidatively-stressed and in clearance of senescent red cells.

Treatment of human red blood cells with diamide and opsonization with whole serum enhanced their phagocytosis by mononuclear phagocytes. Opsonization of diamide-treated red cells with whole serum containing 20-100 times the physiologic concentration of naturally occurring anti-band 3 antibodies further increased the extent of phagocytosis. Enhanced phagocytosis was due to an anti-band 3 mediated binding of C3b to red cells via the alternative pathway. Red cell-bound anti-band 3 was slightly elevated on diamide-treated cells and elicited a C3 binding that exceeded the amount of bound antibody by two orders of magnitude. Pretreatment of red cells with a monoclonal anti-CR1 did not significantly inhibit opsonization and phagocytosis if cells were opsonized at elevated anti-band 3 concentrations. On the other hand, phagocytosis of mildly oxidized (20 microM diamide) red cells was completely inhibited by blocking CR1 if cells were opsonized with serum containing physiologic concentrations of anti-band 3. The results suggest that two types of opsonization mediate in vitro phagocytosis: one operating at physiologic anti-band 3 concentrations with mildly oxidized red cells (IC-like mechanism) and one that operates with either heavily oxidized (greater than 200 microM diamide) red cells at physiologic anti-band 3 concentrations, or with mildly oxidized cells opsonized at elevated concentration of anti-band 3. The latter mechanism is relevant in vivo. It is most likely that it starts by Fab-dependent binding of anti-band 3 to diamide-induced band 3 protein oligomers. Complement activation may occur by assembly of an alternative convertase on C3b covalently bound to red cell-associated anti-band 3. This mechanism is also likely to mediate clearance of senescent red cells, as it was primarily from senescent red cells that we could isolate complexes containing IgG covalently bound to C3b.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2001

The ancestral complement system in sea urchins.

The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat pathogens with complement leading to phagocytosis and removal of foreign cells, a system that would be significantly more effective than an opsonin that binds upon contact as a result of simple diffusion. An understanding of the immune response of the sea urchin, an animal that is a good estimator of what the ancestral deuterostome immune system was like, will aid us in understanding how adaptive immunity might have been selected for during the early evolution of the vertebrates and how it might have been integrated into the pre-existing innate immune system that was already in place in those animals.

Amino Acid Sequence↗
PubMed1993

Facilitation of complement-dependent killing of the Lyme disease spirochete, Borrelia burgdorferi, by specific immunoglobulin G Fab antibody fragments.

In the absence of specific antibody, Borrelia burgdorferi is resistant to the bactericidal action of complement, despite the capacity of the spirochete to activate complement. Complement-mediated killing of B. burgdorferi requires the presence of antiborrelial immunoglobulin G (IgG). The effect of bactericidal IgG takes place after formation of the C5 convertase. Therefore, we examined the ability of Fab fragments from bactericidal IgG to mediate killing of B. burgdorferi by complement. The complement-activating domain of IgG, the Fc fragment, was not required for killing of borreliae, as monovalent Fab fragments prepared from immune IgG were also able to mediate killing. However, the killing efficiency of the Fab fragments was less than that of intact IgG, suggesting that the bactericidal activity of IgG is enhanced by divalency. IgG Fab-mediated killing occurred without increased complement activation or C3 fluid-phase consumption. Cell killing proceeded via the classical complement pathway, as no killing of Fab fragment-sensitized cells was observed in human serum deficient in C2. These results demonstrate directly that the bactericidal effect of anti-B. burgdorferi IgG is independent of the complement-activating properties of the antibody.

Animals↗
PubMed1984

Freeze-thaw activation of the complement attack phase: II. Comparison of convertase generated C--56 with C--56 generated by freezing and thawing.

The activation of the C-attack phase does not necessarily involve the components of the C5 convertases. C--56 hemolytic activity was generated from the same source of C7 depleted serum by the alternative pathway convertase or by freezing and thawing resp. In contrast to activation by the convertase, biological activities of C5a (chemotaxis, serotonin release) were not detected following activation by freezing. The yields of C--56 hemolytic activities were similar and the properties of the activated products were identical. No difference was found in the molecular weight, in the hydrophobicity or with respect to charge. The two activities were in the absence of C7 stable at 37 degrees C and decayed rapidly in the presence of C7. It is proposed that a conformational change in the tertiary structure of the molecule(s) is the critical event in the formation of an active C--56 complex. In this light the cleavage of C5a from the native molecule by the convertase appears as a side reaction, not by itself essential for activation.

Complement Activating Enzymes↗
PubMed1988

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae.

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.

Blood Bactericidal Activity↗