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PubMed1993

Complement deposits in epidermal cells after ultraviolet B exposure.

Ultraviolet B (UVB) radiation is known to induce formation of sunburn cells (SBC) in the epidermis. Since it was unknown whether this process might be accompanied by complement (C) activation, we analyzed C-deposition in skin biopsies taken before and 24 h and 48 h after UVB exposure (fourfold minimal erythema dose or fourfold minimal phototoxic dose) from 14 patients (5 receiving potentially photosensitizing drugs and 9 without such medication) by immunohistology. Local C-activation was visualized by direct or indirect immunofluorescence staining with polyclonal antibodies against C3b, C3d, C5, C9 and monoclonal antibodies to C3b, C3d, C9 and neoantigens on the terminal complement complex (TCC). Neutrophils were identified immunohistologically by antibodies to polymorphonuclear elastase. All but one specimen taken before UVB irradiation were completely negative; biopsies obtained 24 h after UVB revealed complement C3b- and/or C3d-deposits within scattered cells of the epidermis, with wide individual variations in the number of C3-positive cells. C3b and TCC were found predominantly in the cytoplasma; C3d deposits were more often accentuated at the cell surface of C-positive cells. A significantly higher number of keratinocytes was C3d-positive 48 h after UVB exposure than in specimens taken 24 h after UVB. Such a reactivity pattern might indicate a rapid decay of intracellular C3b to C3d. Patients medicated with potentially photosensitizing drugs developed significantly higher numbers of strongly C3-positive cells than those without such medication. Infiltration with elastase-positive cells, presumably representing neutrophils, was observed in the upper third of the dermis and the epidermis in all but one biopsy (n = 9) after UVB.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

Amino Acid Sequence↗
PubMed1983

Precipitability and composition of HBsAg-anti-HBs immune complexes formed in the presence of complement. A model of circulating immune complex analysis.

Immune complexes (IC) of partially purified HBsAg and human anti-HBs were prepared at different antigen/antibody ratios in the presence of complement in normal human serum (NHS), and under conditions not allowing complement activation in buffers or in NHS containing 10 mM EDTA (NHS-EDTA). Commercial preparations of the radiolabelled antigen and antibody were used. IC formed in NHS were not significantly precipitated even after incubation for 24 h at 4 degrees C, whereas a typical precipitation curve was observed with complexes formed in the absence of complement. Thus, complement activation was found to markedly and permanently inhibit precipitability of HBsAg-anti-HBs immune complexes (HBsAg-IC). HBsAg-IC were precipitated from sera with 3.5% polyethylene glycol (PEG), boiled in sodium dodecyl sulphate (SDS)-urea buffer, and analysed by SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE). With complexes formed in the presence of complement, about one-sixth of the antibody activity was found in high molecular weight fractions corresponding in size to IgG oligomers. By contrast, with complexes formed without complement, no significant amount of antibody was found in these fractions. With blotting technique and radiolabelled anti-human-C3 antibody, it was demonstrated that anti-HBs was covalently bound to C3b fragments in IC formed in the presence of complement and was in the high molecular weight fractions.

Animals↗
PubMed1999

Mechanism of complement-dependent haemolysis via the lectin pathway: role of the complement regulatory proteins.

Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.

Animals↗
PubMed1987

Isolation of bovine complement factor H.

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.

Animals↗
PubMed1983

An inherited defect in the C3 convertase, C3b,Bb, associated with glomerulonephritis.

The control of the amplification C3 convertase, C3b,Bb, of the serum complement system has been found to be defective in five members of a family spanning three generations. One of the five has membranoproliferative glomerulonephritis (MPGN) type III and another has mild idiopathic rapidly progressive glomerulonephritis. The defect is manifested by low serum concentrations of C3 and usually factor B with normal levels of the proteins which control the convertase, H and I. C3 nephritic factor (C3NeF) was not demonstrable. Enhanced C3 conversion was produced by the incubation of their serum at 37 degrees C for 30 min. This conversion was further accelerated by incubation after increasing the serum magnesium concentration by increments ranging from 0.25 to 1.9 mM. Incremental additions of H to serum depleted of H indicated that the amplification convertase of affected family members required more H for its inhibition than did that of normal subjects. This requirement was reduced by the addition of purified normal C3 but not by the addition of purified C3 of the propositus. It is postulated that affected family members are heterozygous for a gene producing an abnormal C3 which, as a constituent of the amplification convertase, C3b,Bb, confers resistance to H. Investigation of this apparently nephritogenic defect may provide insight into the pathogenesis of these glomerulonephritides.

Adult↗
PubMedDate not supplied

The role of antibody and complement in the reticuloendothelial clearance of pneumococci from the bloodstream.

An experimental model of pneumococcal bacteremia in guinea pigs has been developed. By use of this model, complement has been shown to play a critical role in clearance of Streptococcus pneumoniae from the bloodstream and in survival of guinea pigs after iv challenge with type 7 S. pneumoniae. In nonimmune animals, complement activation occurs primarily via the alternative pathway. However, anticapsular antibodies increase the rate of clearance of pneumococci primarily via activation of the classical complement pathway. Detailed studies of the reticuloendothelial localization of cleared radiolabeled pneumococci showed that clearance took place primarily in liver and spleen and that anticapsular antibody increased hepatic and decreased splenic sequestration. This effect could be blocked by depleting complement with cobra venom factor. Comparison of nonimmune animals injected with unencapsulated pneumococci or encapsulated types 7 or 12 pneumococci showed that the virulence of these organisms for guinea pigs correlated with the extent of splenic sequestration. Sensitization of encapsulated pneumococci with anticapsular antibodies led to an antibody dose-dependent increase in the rate of bloodstream clearance. Sensitization of encapsulated pneumococci with anticell wall antibodies had no effect on clearance rates despite the ability of these antibodies to bind to the bacteria and to activate and fix complement to the organisms. In vitro studies showed that C3b deposited by these opsonically ineffective antibodies interacted poorly with C3b receptors. Electron microscopic studies showed that C3b deposited by anticapsular antibodies bound to the pneumococcal capsule while C3b deposited by anti-cell wall antibodies did not. Thus, the localization of C3b deposition on the pneumococcus markedly affects its opsonic potential.

Animals↗
PubMed1987

Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes exhibit abnormalities in decay accelerating factor (DAF), acetylcholinesterase, and resistance to autologous C5b-9 attack. To investigate the nature of the lesion underlying PNH cells, we examined the relationship of these abnormalities to one another. Analyses of DAF in acetylcholinesterase-negative erythrocytes revealed that these two abnormalities involve functionally independent molecules, coincide precisely in the same cell populations, and are similarly expressed in PNH II and more complement-sensitive PNH III erythrocytes. The DAF and acetylcholinesterase deficiencies contrast with the C3b/C4b receptor (CR1) deficit, which is less profound and similarly distributed in complement-insensitive cell populations. Hemolytic studies showed that defective resistance to autologous C5b-9 attack is mediated by another mechanism. Whereas reconstitution of PNH II erythrocytes with DAF completely corrected their complement sensitivity, DAF reconstitution of PNH III erythrocytes restored their ability to circumvent C3b uptake but had no effect on their heightened susceptibility to reactive lysis. Assays of complement-insensitive (PNH I) erythrocytes surviving after reactive lysis disclosed partial DAF and acetylcholinesterase deficits. These findings indicate that the PNH lesion involves multiple membrane components and that PNH I erythrocytes are also abnormal.

Acetylcholinesterase↗
PubMed2003

Immune evasion by herpes simplex virus type 1, strategies for virus survival.

Many viruses capable of persistent or recurrent infections have evolved strategies to evade host immunity. Viral evasion molecules target components of innate and acquired immunity, including complement proteins, natural killer cells, MHC Class I or Class II molecules and antibody. Our work focuses on HSV-1 glycoproteins gC and gE that impair antibody and complement responses. gC inhibits complement activation by binding C3b and blocking activities mediated by this pivotal complement protein, while gE binds the IgG Fc domain, blocking Fc-mediated activities, including complement activation and antibody-dependent cellular cytotoxicity. HSV-1 mutant viruses that lack the ability to bind C3b, IgG Fc, or both are much less virulent than wild-type virus in a murine model. These HSV-1 immunoevasins help explain the virus' ability to produce recurrent infections despite intact immunity. Strategies to prevent immune evasion may be required to develop successful HSV vaccines.

Animals↗
PubMed1981

The separation of functionally distinct forms of the third component of human complement (C3).

Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.

Chromatography, Affinity↗
PubMed2005

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD).

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.

Antigens↗
PubMed1988

Identification of the C3b receptor-binding domain in third component of complement.

We report here that complement receptor type one (CR1) binds to a region of C3b that is contained within the NH2 terminus of the alpha' chain. In an enzyme-linked immunosorbent assay, CR1 bound to C3b, iC3b, and C3c but not to C3d, and this binding was inhibited by soluble C3b and C3c. Further attempts to generate a small C3 fragment capable of binding CR1 were unsuccessful. However, elastase degradation of C3 generated four species of C3c (C3c I-IV), two of which bound CR1. NH2-terminal sequence analysis and sodium dodecyl sulfate-gel electrophoresis of the C3cs indicated that the beta chains and the 40,000-dalton COOH-terminal alpha' chain fragments were identical; the NH2-terminal alpha' chain fragments of C3c I-IV varied from 21,000 to 27,000 daltons and accounted for the differential binding to CR1. C3c-I and II, which do not bind CR1, were missing 8 and 9 residues from the NH2 terminus of the alpha' chain when compared with the intact alpha' chain of C3b. C3c-III and IV, which bind CR1, had NH2 termini identical to the intact NH2-terminal alpha' chain of C3b. Using iodinated concanavalin A and endoglycosidase H, we showed that the NH2-terminal alpha' chains of C3c-I and III were glycosylated, while C3c-II and IV were not. Therefore, these data indicated that the amino terminus of the NH2-terminal alpha' chain fragment of C3c was responsible for binding CR1 while the COOH terminus of this fragment was not involved since the presence or absence of this region in C3c did not affect CR1 binding to C3c. Subsequently, two peptides were synthesized from the NH2-terminal alpha' chain fragment of C3c: X42, 42 residues in length from the NH2 terminus and C30, 30 residues in length from the COOH terminus. X42 inhibited binding of CR1 to C3b, and this effect was also observed with antipeptide antibodies against the X42 peptide. The C30 and other C3-derived peptides and antipeptide antibodies had no effect on the binding of CR1 to C3b.

Amino Acid Sequence↗
PubMed1993

Differences in the activity of the alternative pathway of complement in BALB/c and C57Bl/6 mice.

The deposition of C3b on the surface of zymosan was assessed in inbred strains of mice. Mg(2+)-EGTA plasma from C57Bl/6 and BALB/c mice was incubated with zymosan and deposited C3b was eluted by methylamine. Eluted C3b was detected and quantified by Western blot and an enzyme-linked immunosorbent assay. 3- to 4-fold more C3b was deposited on zymosan incubated with C57Bl/6 plasma than on zymosan incubated with BALB/c plasma (p < 0.01). Data on the kinetics of deposition of C3b suggest a faster and greater activity of the alternative pathway of complement (ACP) in C57Bl/6 than in BALB/c. Higher C3b deposition in C57Bl/6 plasma occurs in the presence of higher plasma factor H concentration (1.3-fold of BALB/c concentration; p < 0.01) and is accompanied by higher factor H deposition on zymosan (3- to 10-fold of BALB/c concentration; p < 0.01). Demonstrable differences in the activity of the proteins of the ACP may provide a biological basis for the genetic variation in innate resistance to infectious organisms observed in inbred strains of mice.

Animals↗
PubMed1989

Preparation of monoclonal antibodies to C3b by immunization with C3b(i)-sepharose.

We have prepared and characterized four monoclonal antibodies (MAbs) to human C3b of high specificity and affinity. Our procedure did not require a purified source of C3b for immunization. Instead, C3b and C3bi were deposited on Sepharose 4B via the alternative pathway of complement activation in normal human serum, and this C3b(i)-Sepharose served as the immunogen. C3b(i)-Sepharose was also prepared from a number of primate and non-primate sources, and this allowed us to demonstrate that the anti-human C3b MAbs cross-reacted with primate-derived C3b, but not with C3b from non-primates. The procedures we have developed may be useful in the further investigation of species-specific C3 fragment-binding proteins from both primate and non-primate sources.

Animals↗
PubMed2004

Inhibition of complement activation by a secreted Staphylococcus aureus protein.

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

Bacterial Proteins↗
PubMed1983

The C3/C5 convertase of the alternative pathway of complement: stabilization and restriction of control by lanthanide ions.

The alternative pathway C3 convertase (C3b,Bb) is a Mg-dependent, labile enzyme with a t/2 of 3 min at 37 degrees C and of 14 min at 24 degrees C (at half physiological ionic strength). To stabilize the enzyme, metal ions of the lanthanide series were tested. Formation and decay of the enzyme as well as binding of radiolabeled Factor B, Factor H or properdin to C3b were measured using C3b-bearing sheep erythrocytes (EC3b). Binding of Factor B to EC3b in presence of 40 microM gadolinium (Gd) was two to three times greater than in presence of 1 mM Mg. Binding of Factor H and of properdin to EC3b was partially inhibited by Gd. Although it enhanced Factor B uptake by EC3b, Gd was unable to substitute for Mg in enzyme formation by Factor D and completely inhibited (at 10 microM) Mg-dependent enzyme activation. However, the preformed enzyme was not inhibited by Gd. Instead, exposure of EC3b,Bb to 40-100 microM Gd increased the t/2 at 37 degrees C from 3 min to 12-28 min, and at 24 degrees C from 14 min to 32 min. The slow decay of the enzyme correlated with slow release of Bb. Similar enzyme stabilization was observed using terbium, ytterbium, dysprosium and lanthanum. The Gd stabilized enzyme was also less susceptible to control by Factor H and properdin than the unstabilized enzyme. Furthermore, Gd protected surface bound-C3b from being cleaved by Factor I. The Gd effects were instantaneously reversed upon addition of 10 mM EDTA. Thus, Gd is able to stabilize preformed C3b,Bb and to render the enzyme refractory to control by Factors H and I.

Cations, Divalent↗
PubMedDate not supplied

Molecular understanding of cellular adhesion on artificial surfaces.

This study was conducted to clarify cellular adhesion mechanisms of blood cells (platelets [PLT] and white blood cells [WBC]) and vascular endothelial cells at the molecular level. This study indicated that the adhesion of three cellular systems to proteins such as fibronectin and fibrinogen proceeds via the RGD (Arg-Gly-Asp) ligand-receptor interaction, in which the RGD tripeptidyl sequence is the minimal amino acid sequence common to adhesive proteins. This was evident from the dose-dependent inhibitory effect of RGD-containing peptide on cellular adhesion. Additional supporting evidence was the presence of PLT and WBC receptors, which molecularly recognize RGD, verified by fluorescein-labelled RGD-containing peptide. The adhesion of vascular endothelial cells was also predominantly controlled by the ligand-receptor mechanism, and participation of complement activation on WBC adhesion was demonstrated as well. The adhesion of WBCs on surface hydroxyl group-bearing polymers proceeded via the CR3 receptor-C3b ligand interaction, in which activated complement factor C3b is chemically fixed upon complement activation. Thus, the molecular understanding of cellular adhesion mechanisms provide the basis of biocompatibility for implantation and extracorporeal circulation, as well as molecular design of artificial and bioartificial organs.

Animals↗