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PubMed1991

Complement consumption in a patient with necrobiotic xanthogranuloma and paraproteinemia.

Necrobiotic xanthogranuloma and paraproteinemia have been reported to be associated with acquired deficiency of the early components of the classic pathway of complement. We describe a patient in whom activation of complement was suggested by a high level of C3d, an accelerated catabolism of C1q, and a rapid consumption of C4 after an intravenous infusion of fresh-frozen plasma. This infusion was followed by an episode of pulmonary edema. Patients with complement deficiency or depletion should be monitored closely while receiving plasma, to avoid this potential complication.

Complement C1q↗
PubMed2001

Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity.

Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galalpha1 3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galalpha1 3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galalpha1 3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galalpha1 3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galalpha1 3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.

Animals↗
PubMed2002

Structural and functional studies of complement inhibitor C4b-binding protein.

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of the complement system. This large plasma glycoprotein consists of seven identical alpha-chains and a unique beta-chain held together by disulphide bridges. Both types of subunits are composed almost exclusively of complement control protein domains (CCPs). Using homology-based computer modelling and mutagenesis of recombinant proteins we have localized binding sites for several ligands of C4BP: complement factor C4b, heparin and vitamin K-dependent anticoagulant protein S (PS). We found that C4b requires CCP1-3 of the alpha-chain for binding. The interaction is ionic in nature and mediated by a cluster of positively charged amino acids present on the interface between CCP1 and CCP2 of the alpha-chain. Loss of C4b-binding resulted in a loss of all inhibitory functions of C4BP within the classical pathway of complement. Binding of heparin required CCPs 1-3 of the alpha-chain, with CCP2 being the most important, as well as the cluster of positively charged amino acids involved in binding of C4b. The interaction between C4BP and PS is of very high affinity and conveyed by a cluster of surface exposed hydrophobic amino acids localized on CCP1 of the beta-chain. Furthermore, C4BP is captured on the surface of several pathogens, which may contribute to their serum resistance and pathogenicity. We have localized interaction of C4BP with Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes and Escherichia coli to various regions of the alpha-chain.

Animals↗
PubMed1995

Antibody dependent, complement mediated liver uptake of liposomes containing GM1.

PURPOSE: We have previously reported that GM1 exhibits an opposite effect on regulating liposome circulation time in mice and rats (Liu et al. Pharm. Res. Vol. 12:508-512 (1995)). Inclusion of GM1 into liposomes significantly prolongs liposome circulation time in mice, while it dramatically decrease the blood half life and increases liver uptake of liposomes in rats. The purpose of this study was to elucidate the mechanism that underlies this phenomenon. METHODS: Single-pass liver perfusion in vitro and complement mediated liposome lysis assay was used. RESULTS: Serum appeared to play an important role in determining the liver uptake of GM1 liposomes. Specifically, rat serum enhanced the uptake of GM1-containing liposomes by the perfused liver. Such activity was also found in human and bovine serum, but not in mouse serum. Taking human serum as an example, we demonstrated that such serum activity can be blocked by EDTA and EGTA/Mg2+. Antibodies against human IgM and the third component of complement system (C3) also inhibited serum activity. CONCLUSIONS: The presence of naturally occurring anti-GM1 antibodies in rats, through the activation of the classic pathway of complement system, is likely the cause of rapid blood clearance of GM1-liposomes. The third component of complement is likely to serve as the opsonin that is directly involved in mediating liposome clearance.

Animals↗
PubMed1997

Complement activation by HIV-1-infected cells: the role of transmembrane glycoprotein gp41.

To characterize the mechanisms of complement activation by human immunodeficiency virus type 1 (HIV-1)-infected cells, Cl-4 cells stably expressing the envelope glycoproteins of HIV-1 and the parent African green monkey cell line CV-1 were tested for C1q binding and complement activation. While the parent cell line CV-1 only showed a weak spontaneous activation of the alternative pathway, Cl-4 cells additionally triggered the classical pathway of complement activation independent of anti-HIV antibodies by direct C1q binding. Earlier studies had shown different complement activating potential of cells infected with various HIV isolates. Recombinant soluble CD4-induced shedding of gp120 from the surface of HIV-1-infected cells converted a weak activator isolate (MVP-899) into a strong complement activator. The increase in complement activation was paralleled by the concomitant unmasking of a previously hidden gp41 epitope comprising the major complement-activating domain of gp41 (aa. 601-613). Our results strongly suggest that the transmembrane protein gp41 induces the activation of complement on the surface of infected cells as has been described previously for purified HIV-1 virions. Furthermore, we present evidence that the different potential of HIV isolates to activate the complement system on the cell surface is caused by different degrees of spontaneous gp120 shedding by various HIV isolates.

Animals↗
PubMed2004

A discrepancy between liquid phase and gel phase assays for evaluation of total complement activity and some possible explanations.

This study was conducted to investigate the frequency and origin of discrepant assay results between two haemolytic assays which both measure activity of the classical pathway of complement (CH50) by haemolysis of sheep red blood cells (SRBCs). One is conducted in gel phase using undiluted sera and the other in liquid phase with sera in 1/100 dilution. The majority of discrepant readings are observed as low or absent haemolysis in the gel phase, with values within or above the normal range in the liquid phase. The incidence of discrepant assay readings was evaluated in 300 samples. Furthermore, 28 samples showing the most discrepant readings were investigated further for disturbing factors. Factors evaluated in the test sera were mannose-binding lectin, C-reactive protein (CRP), immune complexes, antibodies to SRBCs, rheumatoid factor and immunoglobulin A (IgA) and IgG anti-C1q antibodies. The results showed that discrepant readings are present in 10% of the 300 samples and false low gel assay readings account for 6.3%. The majority (68%) of the discrepant samples contained a heat-stable-inhibiting factor, and the main mediators found were elevated levels of IgA anti-C1q antibodies and antibodies to SRBCs. This could indicate a clinically relevant factor in the test sera but can also result from the difference in assay design.

Antigen-Antibody Complex↗
PubMed2004

[C4d: a pathogenetically relevant marker in kidney transplantation].

Antibody-mediated allograft rejection should be discriminated from cell-mediated rejection processes since these two distinct pathogenic mechanisms are likely to require different therapeutic approaches. The complement split product C4 d (although biologically inactive by itself) turned out to be a reliable diagnostic marker of antibody-induced complement activation in kidney allografts. In contrast to other organs like heart and bowel, there is no indication that ischemia/reperfusion injury might induce the classical pathway of complement activation in the kidney. Endothelial C4 d deposits in peritubular capillaries of transplanted kidneys are therefore robust indicators of antibody-mediated alloreactivity with potentially unfavourable outcome. The high efficiency of therapeutic strategies aimed at removing alloantibodies from the recipient's circulation (i.e. immunoadsorption and plasmapheresis) in cases of C4d positive rejection further underscores the important pathogenic role of antibodies in renal allograft rejection and the diagnostic relevance of endothelial C4 d deposits. C4 d deposits can also occur de novo in late allograft biopsies. Their association with basement membrane injury in peritubular capillaries and glomeruli, which are the hallmarks of chronic rejection, indicates a contribution of humoral immune reactions to chronic rejection. Due to the clinical relevance of humoral rejection and due to the fact that the latest update of the Banff classification introduced the pathogenically based subdivision of acute rejection into a cell-mediated and an antibody-mediated variant every renal allograft biopsy should be stained for C4 d. It is important to be aware that humoral and cellular rejection often occur simultaneously and that C4d deposits alone are not an unequivocal proof of humoral rejection that immediately requires treatment.

Basement Membrane↗
PubMed1989

Effect of complement binding on a solid-phase immunometric TSH assay.

The binding of serum thyrotropin (TSH) to plastic beads coated with a monoclonal antibody to human TSH was inhibited unless EDTA was present during the incubation. The inhibitory factor in serum was heat labile, and its effect could be abrogated by the addition of human albumin-anti-albumin immune complexes. Subsequently it was shown that the antibody-coated beads were able to bind the first component of complement, C1q, and that this binding was inhibited by addition of albumin-anti-albumin complexes. The results show that a surface coated with a monoclonal murine antibody is able to bind complement, and that binding of complement may interfere in solid-phase immunometric assays.

Antibodies, Monoclonal↗
PubMed1993

Binding ability of complement receptor CR1 to C3 bound on the surface of M+ group A streptococci.

A previous study demonstrated that although M+ bacteria bound C3, mainly C3b and iC3b, via the classical pathway of complement activation, they were not phagocytosed by polymorphonuclear leucocytes (PMN). To elucidate this mechanism, we attempted to distinguish between the possibilities that M+ bacteria are effectively adhering to PMN but are not being endocytosed, or that the C3 deposited on M+ bacteria is not able to interact with the complement receptors on PMN. In the present study, we studied the interaction of C3-coated M+ bacteria with complement receptor CR1, which was isolated from the stroma of human erythrocytes. We show that the isolated complement receptor CR1 can associate with C3-coated M+ bacteria as well as with C3-coated M- bacteria, and the C3 deposited on M+ bacteria is cleaved and releases a C3 fragment in the presence of factor I and liquid-phase CR1. These results suggest that the C3 bound on the surface of M+ bacteria is able to promote adherence to the complement receptor CR1 on PMN. We also studied the distribution of C3 deposited on M+ bacteria in normal human serum (NHS) or normal human plasma (NHP). By immunofluorescence, we show that the C3 bound to M+ bacteria in NHS was deposited uniformly over the surface of the bacteria. On the other hand, the C3 bound to M+ bacteria in NHP was deposited at both ends between adjacent daughter cocci. The results suggest that an additional factor contained in NHP is related to the enhancement of anti-phagocytic activity of M+ bacteria.

Antibodies↗
PubMed1992

Localization of sites through which C-reactive protein binds and activates complement to residues 14-26 and 76-92 of the human C1q A chain.

Studies were initiated to localize the C-reactive protein (CRP) binding site on the collagen-like region (CLR) of C1q. CRP bound preferentially to the A chain of reduced C1q, in contrast to aggregated immunoglobulin G (Agg-IgG), which reacted preferentially with the C chain. A group of C1q A chain peptides, including peptides identical to residues 81-97, 76-92, and 14-26, respectively, were synthesized from predicted binding regions. Peptide 76-92 contained two proximal lysine groups, and peptide 14-26 contained four proximal arginine groups. CRP-trimers and CRP-ligand complexes did not bind to immobilized peptide 81-97, but bound avidly to immobilized peptides 76-92 and 14-26. Agg-IgG did not bind to any of the peptides. Peptide 76-92 partially, and peptide 14-26 completely, inhibited binding of CRP to intact C1q. Peptide 14-26 also blocked C consumption initiated by CRP, but not by IgG. Replacement of the two prolines with alanines, or scrambling the order of the amino acids, resulted in loss of ability of peptide 14-26 to inhibit C1q binding and C activation by CRP, indicating a sequence specificity, and not a charge specificity alone, as the basis for the inhibitory activity of the peptide. Similar investigations with scrambled peptides showed a sequence specificity for the effects of peptide 76-92 as well. DNA and heparin inhibited binding of CRP trimers to intact C1q, as well as to each peptide 14-26 and 76-92, suggesting involvement of these regions in C1q-CLR binding reactions generally. Collectively, these data identify two cationic regions within residues 14-26 and 76-92 of the C1q A chain CLR as sites through which CRP binds and activates the classical C pathway, and suggest that these residues represent significant regions for C1q CLR binding reactions generally. To our knowledge, this represents the first delineation of sites on C1q through which binding and activation of the classical C pathway can occur.

Amino Acid Sequence↗
PubMed1994

Beta-sheet secondary structure of the trimeric globular domain of C1q of complement and collagen types VIII and X by Fourier-transform infrared spectroscopy and averaged structure predictions.

C1q plays a key role in the recognition of immune complexes, thereby initiating the classical pathway of complement activation. Although the triple-helix conformation of its N-terminal segment is well established, the secondary structure of the trimeric globular C-terminal domain is as yet unknown. The secondary structures of human C1q and C1q stalks and pepsin-extracted human collagen types I, III and IV (with no significant non-collagen-like structure) were studied by Fourier-transform i.r. spectroscopy in 2H2O buffers. After second-derivative calculation to resolve the fine structure of the broad amide I band, the Fourier-transform i.r. spectrum of C1q showed two major bands, one at 1637 cm-1, which is a characteristic frequency for beta-sheets, and one at 1661 cm-1. Both major bands were also detected for Clq in H2O buffers. Only the second major band was observed at 1655 cm-1 in pepsin-digested C1q which contains primarily the N-terminal triple-helix region. The Fourier-transform i.r. spectra of collagen in 2H2O also showed a major band at 1659 cm-1 (and minor bands at 1632 cm-1 and 1682 cm-1). It is concluded that the C1q globular heads contain primarily beta-sheet structure. The C-terminal domains of C1q show approximately 25% sequence identity with the non-collagen-like C-terminal regions of the short-chain collagen types VIII and X. To complement the Fourier-transform-i.r. spectroscopic data, averaged Robson and Chou-Fasman structure predictions on 15 similar sequences for the globular domains of C1q and collagen types VIII and X were performed. These showed a clear pattern of ten beta-strands interspersed by beta-turns and /or loops. Residues thought to be important for C1q-immune complex interactions with IgG and IgM were predicted to be at a surface-exposed loop. Sequence insertions and deletions, glycosylation sites, the free cysteine residue and RGD recognition sequences were also predicted to be at surface-exposed positions.

Amino Acid Sequence↗
PubMed1996

Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor.

Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.

Animals↗
PubMed2005

Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease that is restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind the classical pathway of complement down-regulatory protein C4b-binding protein (C4bp) to evade killing by human complement. Strains of N. gonorrhoeae that resisted killing by human serum complement were killed by serum from rodent, lagomorph, and primate species, which cannot be readily infected experimentally with this organism and whose C4bp molecules did not bind to N. gonorrhoeae. In contrast, we found that Yersinia pestis, an organism that can infect virtually all mammals, bound species-specific C4bp and uniformly resisted serum complement-mediated killing by these species. Serum resistance of gonococci was restored in these sera by human C4bp. An exception was serotype Por1B-bearing gonococcal strains that previously had been used successfully in a chimpanzee model of gonorrhea that simulates human disease. Por1B gonococci bound chimpanzee C4bp and resisted killing by chimpanzee serum, providing insight into the host restriction of gonorrhea and addressing why Por1B strains, but not Por1A strains, have been successful in experimental chimpanzee infection. Our findings may lead to the development of better animal models for gonorrhea and may also have implications in the choice of complement sources to evaluate neisserial vaccine candidates.

Amino Acid Sequence↗
PubMed1991

Proposed structure of the F' allotype of human CR1. Loss of a C3b binding site may be associated with altered function.

Human CR1 is composed of tandem long homologous repeating (LHR) segments that encode separate binding sites for C3b or C4b. Homologous recombination with unequal crossover has been proposed as the genetic mechanism that gave rise to the CR1 alleles that differed in their total numbers of LHR. The F allotype has four LHR, named LHR-A, -B, -C, -D, 5' to 3'. The site in LHR-A preferentially binds C4b and those in LHR-B and -C prefer C3b. A previous study revealed the presence of a fifth LHR with sequences similar to LHR-B and a third C3b binding site in the S allotype of higher m.w. In the present study, an 18-kb EcoRV fragment that was associated with the expression of the lower m.w. F' allotype hybridized with a unique pattern of cDNA and intron probes specific for LHR-C. Deletion of LHR-B and one C3b binding site was proposed as the mechanism for the appearance of this F'-specific fragment. Functional differences among the CR1 variants were sought by comparative analyses of soluble rCR1 having one, two or three C3b binding sites. Although these three variants did not exhibit any significant differences in their capacities to act as cofactors for the cleavage of monomeric C3b, their relative affinities for dimeric ligand varied more than 100-fold. Furthermore, the variant with only one C3b binding site was at least 10-fold less effective in the inhibition of the alternative pathway C3 and C5 convertases. These observations suggested that the F' allotype may be impaired in its capacity to bind opsonized immune complexes, to inhibit the formation of the alternative pathway C3 and C5 convertases, and perhaps to mediate other CR1-dependent cellular responses.

Alleles↗
PubMed2001

Beta-amyloid fibrils activate the C1 complex of complement under physiological conditions: evidence for a binding site for A beta on the C1q globular regions.

Previous studies based on the use of serum as a source of C have shown that fibrils of beta-amyloid peptides that accumulate in the brain of patients with Alzheimer's disease have the ability to bind C1q and activate the classical C pathway. The objective of the present work was to test the ability of fibrils of peptide Abeta1-42 to trigger direct activation of the C1 complex and to carry out further investigations on the site(s) of C1q involved in the interaction with Abeta1-42. Using C1 reconstituted from purified C1q, C1r, and C1s, it was shown that Abeta1-42 fibrils trigger direct C1 activation both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions consistent with the physiological context in serum. The truncated peptide Abeta12-42 and the double mutant (D7N, E11Q) of Abeta1-42 did not yield C1 activation, providing further evidence that the C1 binding site of beta-amyloid fibrils is located in the acidic N-terminal 1-11 region of the Abeta1-42 peptide. Binding studies performed using a solid phase assay provided strong evidence that C1q interacts with Abeta1-42 fibrils through its C-terminal globular regions. In contrast to previous studies based on a different experimental design, no significant involvement of the C1q collagen-like domain was detected. These findings were confirmed by additional experiments based on C1 activation and C4 consumption assays. These observations provide direct evidence of the ability of beta-amyloid fibrils to trigger activation of the classical C pathway and further support the hypothesis that C activation may be a component of the pathogenesis of Alzheimer's disease.

Amino Acid Sequence↗
PubMed1985

Complement and cancer: activation of the alternative pathway as a theoretical base for immunotherapy.

Activation of the APC is pointed out as the common factor in all sufficiently studied cancer treatments employing nonspecific, active immunotherapy. This chapter outlines the molecular biology of both APC and classical pathway of complement, summarizes the alternative pathway's biologic activities especially in relation to the C3/C5 convertase C3b,Bb, and its implications in the mechanism of host defense against malignancies, particularly relating to the activated macrophage. The many involvements of the APC in the various agents used for nonspecific active immunotherapy are reviewed, and possible clinical implications outlined. It is concluded that activation of the APC can be proposed as the specific theoretical basis so far lacking for this treatment modality and that it is accordingly feasible to attempt to monitor clinical application of this principle by fine-tuning of APC activation in cases of human cancer.

Adjuvants, Immunologic↗