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PubMed1982

Perioperative changes in complement associated with cardiopulmonary bypass.

The total haemolytic complement (CH50), the complement components C3 and C4, the complement breakdown product C3d, alternative pathway activation and transferrin, were measured before, during and after cardiopulmonary bypass. As expected, CH50 decreased after heparinization, remained low during bypass and decreased further up to 8 h after bypass. C3 and C4 decreased significantly during bypass, continued to decrease for a further 8 h after bypass (by 35% and 40% respectively) and thereafter increased gradually up to 48 h. Although the depletions observed were suggestive of complement activation, there were no demonstrable increases in C3d, and in all patients the concentration of C3d remained within the normal range. Hence it was concluded that complement depletions of this magnitude were unlikely to result from complement activation. Non-specific changes in protein concentrations during bypass, as a result of dilution, redistribution or other unidentified factors, are more probable causes of the observed reductions. The acute phase response to surgery may be a factor in the subsequent increase in C3 and C4 which is seen 24 h after bypass. As transferrin concentrations in the plasma are known to decrease during this response the observed decrease in transferrin concentration would support this view.

Adult↗

PubMed2001

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND: Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS: An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS: E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION: Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Animals↗

PubMed1992

Functionally active complement is present in human ovarian follicular fluid and can be activated by seminal plasma.

Human ovarian preovulatory follicular fluids (FF) from 10 women were analysed for their complement contents. Functionally active complement was detected in all the fluids studied in amounts similar to those present in normal human serum. Pooled FF was challenged by seminal plasma in order to determine whether seminal plasma could activate FF complement, the pattern of such an activation and the possible consequences on the reproductive function. FF complement activation occurred during the incubation with seminal plasma with features including alternative pathway activation, factor B and C3 conversion and reduction in total haemolytic complement, as well as an inhibition by seminal plasma of the FF complement response to a new activating challenge. Possible consequences for fertilization, implantation of a fertilized ovum and local defence mechanisms against viruses and bacteria are discussed.

Complement Activation↗

PubMed1998

Complement regulation.

Because of its strong potential for generating inflammation and causing tissue destruction the complement system has to be kept strictly under control. Cells of the host need special protection against the cytolytic complement system. This paper will describe how inappropriate activation of complement in the fluid phase is prevented and how viable human blood cells defend themselves against being destroyed and cleared away by the complement system. Since disturbances in complement regulation occasionally result in disease a brief reference will be made to two of the syndromes caused by complement regulator deficiency, hereditary angioedema (HAE) and paroxysmal nocturnal hemoglobinuria (PNH).

Angioedema↗

PubMed1992

Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes.

Complement C1↗

PubMed1975

Consumption of classical complement components by heart subcellular membranes in vitro and in patients after acute myocardial infarction.

Experiments were conducted to characterize the antibody-independent activation of complement in human serum by isolated human heart mitochondrial membranes in vitro and to determine whether similar patterns of complement consumption occurred in patients after acute myocardial infarction. Direct evidence for the interaction of C1 and heart mitochondrial membranes was obtained by mitochondria-C1 binding and elution experiments. Exposure of normal human sera to isolated human heart mitochondria at 37 degrees C resulted in the consumption of C1, C4, C2, and C3 without significant consumption of the terminal components of the complement system (C6 through C9). The consumption occurred in the absence of detectable anti-heart mitochondria autoantibody, was demonstrated to be calcium dependent, and was inhibited by either 0.01 M EDTA or ethylene glycol bis(bets-aminoethyl ether) N,N,N',N',-tetraacetic acid (EDTA). Although specific absorption of C1q from human sera inhibited the mitochondria-dependent activation of C4, C3 donsumption was not affected. These data indicate that the consumption of C4 and C2 likely occurred due to the mitochondrial membrane-mediated activation of C1, but that the consumption of the C3 did not necessarily involve either the classical or alternative complement pathways. After the in vitro characterization of the mitochondria-dependent activation of the complement system, additional studies were performed to determine whether similar consumption occurred in patients after acute myocaridal infarction. During a 72-h period after hospital admission significant decreases in C1, C4, and C3 occurred in six patients with recent chest pain but no evidence of acute myocardial infarction. These studies suggest that myocardial cell necrosis results in the release of subcellular membrane constituents capable of activating the complement system in the absence of detectable anti-heart autoantibodies; such activation may be responsible in part for the development of acute inflammation and evolution of the infarct size following coronary artery occulusion.

Autoantibodies↗

PubMed1983

Interaction of desialated guinea pig erythrocytes with the classical and alternative pathways of guinea pig complement in vivo and in vitro.

We examined the fate of desialated autologous erythrocytes injected intravenously into guinea pigs (GP). Desialated GP erythrocytes (E) were lysed directly or cleared by the reticuloendothelial system in normal GP (NIH-GP) and cleared by the reticuloendothelial system in GP genetically deficient in the classical complement pathway component C4 (C4D-GP), which activate complement only via the alternative pathway. Desialated E were also cleared in cobra venom factor-treated GP (CVF-GP), which had less than 1% of normal C3 levels, but were not cleared at all in C4D-CVF-GP. Preinjection of asialoorosomucoid (ASOR) and ovalbumin (OVA) had no effect on the rate of E clearance. These in vivo studies indicated that complement activation is essential for clearance of desialated E and that clearance is unaffected by blockade of galactose or mannose receptors. Inhibition of complement-mediated clearance required blockade of both classical and alternative complement pathways. In vitro studies showed that lysis of desialated E could occur in NIH-GP serum (GPS) but not in C4D-GPS. Surprisingly, CVF-GPS also caused lysis of desialated E. Lysis was dependent on both natural antibody to desialated E and classical pathway activation; natural antibody was of both the IgG and IgM classes. C3 uptake studies demonstrated that almost 10 times as many C3 molecules/E were deposited by NIH-GPS as by C4D-GPS or CVF-GPS onto desialated E. Approximately equal numbers of C3 molecules were deposited by CVF-GPS, which did lyse desialated E, and by C4D-GPS, which did not. We suggest that the molecular mechanism of in vivo clearance and in vitro lysis of desialated E by CVF-GP is via classical pathway deposition of C3b into sites on the erythrocyte surface protected from inactivation by H (beta 1H) and I (C4b/3b inactivator). Deposition of C3b into these sites by alternative pathway activation is sufficient to cause clearance but not lysis of desialated E. CVF-GPS may not represent an adequate reagent for testing the complement dependence of various biologic phenomena, particularly if the question involves surfaces that can provide protected sites for C3b molecules.

Animals↗

PubMed1991

Activation of complement by Treponema denticola.

Oral spirochetes have been shown to be associated with periodontal diseases and are present in increased numbers in lesions of greater severity. In this study, the interaction of Treponema denticola with human complement, a major antibacterial defense system, was examined. For each of two strains of T. denticola, it was found that both the classical and alternative pathways of human complement were activated in human serum upon incubation at 37 degrees C. C3 fragments were deposited on the surface of this organism following complement activation; the fragments bound included both of the major C3-derived opsonic fragments C3b and iC3b. Under incubation conditions identical to those carried out for complement activation in serum, T. denticola failed to degrade purified, hemolytically-active C3, although it readily degraded inactivated C3. Thus, despite the documented proteolytic activity of this organism, complement activation and deposition of complement-derived opsonins may be important defense mechanisms in the control of infections with T. denticola.

Complement C3↗

PubMed2003

Expression and characterization of the C345C/NTR domains of complement components C3 and C5.

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.

Amino Acid Motifs↗

PubMed2002

Complement's participation in acquired immunity.

The preliminary evidence for the involvement of complement in promoting primary humoral responses dates back over a quarter of a century. However, it is only in the course of the past decade or so that the detailed mechanisms underlying complement's influence have been characterized in depth. It is now clear that complement serves as a regulator of several B cell functions, including specific antibody production, antigen uptake, processing and presentation, and shaping of the B cell repertoire. Of key importance, in this respect, is the role played by the B cell-signaling triad consisting of the B cell receptor for antigen (BCR), a complex composed of the iC3b/C3d fragment-binding complement type 2 receptor (CR2, CD21) and its signaling element CD19 and the IgG-binding receptor FcgammaRIIb (CD32). The positive or negative outcome of signaling through this triad is determined by the context in which antigen is seen, be it alone or in association with natural or induced antibodies and/or C3-complement fragments. The aim of this review is to describe the present status of our understanding of complement's participation in acquired immunity and the regulation of autoimmune responses.

Animals↗

PubMed1985

Evidence for complement activation by protamine-heparin interaction after cardiopulmonary bypass.

Complement activation by the alternate pathway has been implicated in the pathophysiology of cardiopulmonary bypass (CPB), and laboratory studies suggest that the complement cascade may be activated by the protamine-heparin complex. To determine if the administration of protamine to patients receiving heparin activates complement, we studied 100 patients undergoing CPB by assaying levels of C3a and C4a (classic pathway) at regular intervals before and after protamine administration. In group I (90 patients), protamine was given at the usual interval (median 5 minutes) after CPB. In group II (10 patients), protamine was withheld until skin closure (median 45 minutes) after CPB. Results demonstrated that C4a was not activated during CPB in either group. After CPB, the C4a level in group I was 459 ng/dl and increased to 1047 ng/dl 10 minutes after protamine administration (p less than 0.001). In group II, the C4a level was 484 ng/dl at the end of CPB and 354 ng/dl 15 minutes later, which corresponds to the value immediately after protamine administration in group I. The delayed administration of protamine in group II caused a significant increase in C4a at the time of skin closure (1090 ng/dl; p less than 0.001). Corresponding results from C3a analysis before and after protamine administration confirmed the activation of complement cascade. Our study provides the first clinical evidence that the protamine-heparin complex activates complement via the classic (C4a) pathway. The hemodynamic effects of protamine after CPB may be related to complement activation.

Aged↗

PubMed1983

Complement polymorphism, the major histocompatibility complex and associated diseases: a speculation.

Genes in the major histocompatibility complex code for three major groups of glycoproteins, now referred to as classes I, II, and III. Susceptibility to some autoimmune diseases and to systemic lupus erythematosus is associated with the presence of particular haplotypes of genes in these three classes. An attempt has been made to correlate these finding on the basis of the observation that different polymorphic forms of complement component C4 show varying efficiencies of complement activation. It is suggested that susceptibility to these diseases will be related to the varying efficiency of complement cell lysis and of immune aggregate dissolution by complement. This in turn will depend on the strength of interaction of the different polymorphic forms of C4 with other proteins (some also polymorphic) in the scheme of activation and inactivation of complement. Such an arrangement would lead to preferential association of certain alleles of C2, C4 and factor B and possibly also of class I and II antigens as potential targets of complement reaction.

Animals↗

PubMed1983

[A mechanism of human complement activation by immunostimulators from the bacterial cell wall].

Effects on the human complement system of the cell wall preparations: lipopolysaccharides and polysaccharides of E. coli, Salmonella typhi (pyrogenal, salmosan), Bact. prodigiosum (prodigiosan) and peptidoglycan of Lactobacillus bulgaricus (blastolysin) have been studied. Lipopolysaccharide of E. coli, pyrogenal and salmosan were found to bind efficiently the first complement factor C1q. The constants for inhibition of the C1q binding to antibodies by the mentioned preparations were determined, and their ability to initiate the classical pathway of complement activation (consumption of C4, C2 and C3 factors) was assayed. These preparations only slightly affect the alternative pathway. Prodigiosan, not influencing the classical pathway, initiates the alternative one. Its binding constant with the activated Bb factor was determined. This constant reflects the Bb association with C3b and is (2,4 +/- 0,1) x 10(7) mole-1 l. Blastolysin is effective both in the C1q binding and initiation of the alternative pathway of complement activation. Immunostimulating activity of the bacterial cell wall preparations seems to involve activation of the complement system and the release of the fragments from the complement factors which are mediators of the immune response.

Anti-Bacterial Agents↗

PubMed1998

Therapeutic inhibition of the complement system.

The use of powerful methodologies in molecular biology, biochemistry, and physiology in the last 2 decades had led to impressive progress in our understanding of the mechanisms of complement activation and its role as either a protective or a pathogenic factor in human disease. With respect to disease pathogenesis, the complexity of the complement cascade provides opportunities for several different therapeutic targets within the complement pathways. More than a century after complement was first described, we are about to witness in the near future the availability of a variety of complement inhibitors for specific therapies. Progress in the area of xenotransplantation has been substantial, but formidable obstacles remain to selective inhibition of the factors that block successful clinical xenotransplantation. Bispecific antibodies, designed to enhance rather than inhibit existing complement pathways, hold strong promise for the clearance of viral and bacterial pathogens from the circulation.

Animals↗

PubMed1985

Complementation analysis of locus for hypoxanthine guanine phosphoribosyltransferase in Chinese hamster cells.

The study deals with intragenic complementation between clones of Chinese hamster cells carrying mutations in the HPRT gene. All clones were of independent origin, selected in media containing one of three purine bases: 8-azaguanine (8 AG), 6-mercaptopurine (6MP), or 6-thioguanine (6TG). Some of the clones were spontaneous, others were induced by various mutagens. To make the study less time-consuming, an experimental set-up was proposed for simultaneous complementation testing of up to 10 clones. As a result, about 400 combinations of clones have been analyzed. Twelve pairs of complementating mutants have been identified in HAT medium. A linear complementation map has been constructed for the HPRT locus, showing five complementation groups. The changes in kinetic and other characteristics observed for mutant HPRT show that all the mutants studied carry structural gene mutations. Analysis of the biochemical characteristics of HPRT has revealed considerable differences between mutant enzymes in clones belonging to different complementation groups (three groups were examined). At the same time, the four mutant clones of complementation group II show similar HPRT characteristics, suggesting a relative similarity of their structural variants of the enzyme. The hybrid nature of HPRT in clones resulting from the fusion of mutant cells confirms the intragenic nature of complementation.

Animals↗

PubMed2003

Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.

Cell Line↗

PubMed1996

Apobec-1 interacts with a 65-kDa complementing protein to edit apolipoprotein-B mRNA in vitro.

The editing of apolipoprotein-B (apoB) mRNA involves the deamination of cytidine at nucleotide 6666 to uridine. The catalytic subunit of the editing enzyme, apobec-1, is a cytidine deaminase that requires other unidentified proteins to edit apoB mRNA in vitro. We partially purified an activity from baboon kidney that functionally complements apobec-1. The complementing activity was protease-sensitive and micrococcal nuclease-resistant, had a native molecular mass of 65 +/- 10 kDa on size exclusion chromatography, and sedimented at 4.5 S in glycerol gradients. Purified recombinant His6-tagged apobec-1 immobilized on beads depleted >90% of the complementing activity from partially purified extracts. These beads edited apoB mRNA in vitro in the absence of exogenous apobec-1 or complementing activity. A functional holoenzyme containing apobec-1 and the complementing activity was eluted from the apobec-1-affinity resin using 0.5 M imidazole, whereas buffer containing 0.4 M KCl eluted only the complementing activity. The carboxyl-terminal 59 amino acids of apobec-1 were not required for interaction with the complementing activity in vitro. Our results demonstrate that the complementing protein interacts directly with apobec-1 in the absence of apoB mRNA.

APOBEC-1 Deaminase↗

PubMed2006

Complement activation in the Parkinson's disease substantia nigra: an immunocytochemical study.

BACKGROUND: Inflammatory processes are increased in the Parkinson's disease (PD) brain. The long-term use of nonsteroidal anti-inflammatory drugs has been associated, in retrospective studies, with decreased risk for PD, suggesting that inflammation may contribute to development of this disorder. The objective of this study was to determine the extent of complement activation, a major inflammatory mechanism, in PD. METHODS: Substantia nigra specimens from young normal subjects (n = 11-13), aged normal subjects (n = 24-28), and subjects with PD (n = 19-20), Alzheimer's disease (AD; n = 12-13), and dementia with Lewy bodies (DLB; n = 9) were stained for iC3b and C9, representing early- and late-stage complement activation, respectively. Numbers of iC3b+, C9+, and total melanized neurons in each section were counted in a blinded fashion. Nonparametric analyses were used to evaluate differences between groups and to evaluate correlations between complement staining, numbers of melanized neurons, and the duration of PD. RESULTS: Lewy bodies in both PD and DLB specimens stained for iC3b and C9. Staining was also prominent on melanized neurons. The percentage of iC3b+ neurons was significantly increased in PD vs. aged normal and AD specimens, and in young normal vs. aged normal specimens. C9 immunoreactivity was significantly increased in PD vs. AD specimens, but unlike iC3b, the increased C9 staining in PD and young normal specimens did not achieve statistical significance vs. aged normal specimens. iC3b and C9 staining in PD specimens was not correlated with the numbers of remaining melanized neurons, nor with the duration of PD. CONCLUSION: Complement activation occurs on Lewy bodies and melanized neurons in the PD substantia nigra. Early complement activation (iC3b) is increased on melanized neurons in PD vs. aged normal specimens, and late-stage complement activation (C9) also tends to increase. This latter finding suggests that complement activation may contribute to loss of dopaminergic neurons in some individuals with PD. Complement activation on melanized neurons appears to decrease with normal aging, suggesting a possible neuroprotective role for this process in the normal substantia nigra.

Journal Article↗