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PubMed1985

The cobra complement system: I. The alternative pathway of activation.

The complement system of the cobra snake is of particular interest because cobra venom contains cobra venom factor (CVF), a protein that is related to C3 and forms a stable C3 convertase with mammalian Factor B. We investigated the alternative pathway of cobra complement. Cobra plasma lysed erythrocytes in presence of Mg-EGTA of some mammalian species, but not cobra erythrocytes. The hemolytic activity was inhibited by EDTA and destroyed by heating. Preincubation of cobra plasma with alternative pathway activators (zymosan, inulin, lipopolysaccharide), with small nucleophiles (methylamine, hydrazine), or with chaotropes (KSCN) abrogated the hemolytic activity. The activity of cobra plasma was not affected by CVF. Cobra plasma also lysed EAC1423 cells in presence of EDTA but not EAC142 cells (prepared with sheep erythrocytes, rabbit antibody, and human complement proteins) indicating the presence of C5 in cobra plasma that is susceptible to activation by the human C5 convertase. These results indicate that the cobra has a complement system with an alternative pathway very similar to mammalian complement.

Animals↗
PubMed1989

C3 binds covalently to the C gamma 3 domain of IgG immune aggregates during complement activation by the alternative pathway.

Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

Antigen-Antibody Complex↗
PubMed1981

Hypocomplementaemia due to a genetic deficiency of beta 1H globulin.

An 8-month-old Asian boy who presented with the haemolytic uraemic syndrome was found to have a low haemolytic complement, and in particular a very low C3 level, with a normal C4 level. These abnormalities persisted after recovery and were not associated with the presence of circulating C3 nephritic factor. A clinically healthy 3-year-old brother was found to have an identical complement profile, which indicated increased alternative pathway activation. Both brothers had normal levels of the C3b inactivator, but very low levels of beta 1H globulin (less than 10% of a reference standard serum). The parents, who were first cousins, had half-normal levels of beta 1H globulin, and low levels were found in other members of the family, indicating that the defect was inherited.

Child, Preschool↗
PubMed2002

Activation of complement pathways in xenotransplantation: an in vitro study.

Pig-to-human xenotransplantation faces the problem of hyperacute graft rejection due to the presence of human naturally occurring antibodies against the disaccharide Galalpha1-3Gal (anti-Gal antibodies) expressed on pig endothelium. Antibody-mediated complement activation is usually referred to as classical pathway activation. In this study we examined if the alternative complement pathway is also directly activated through anti-Gal antibodies or if the classical pathway is indispensable. We therefore developed a hemolysis test with rabbit erythrocytes (E), which have an activating surface for the alternative complement pathway and express abundant amounts of Galalpha1-3Gal, and used this assay in addition to the standard complement tests CH50 and AP50. In this rabbit E CH50 (RECH50) assay we were able to study activation of both major complement pathways simultaneously. FACS analysis was used to trace complement and antibody deposition on rabbit E. Anti-Gal depletion of human serum by immunoabsorption revealed a 65% reduction of rabbit E hemolysis in the RECH50 test (value before absorption: 28 +/- 5.8, after absorption: 9.9 +/- 2.8, P<0.001), but only a 35% reduction of lysis in the AP50 test (AP50 before 11.3 +/- 2.1, after 7.4 +/- 2.0, P<0.002). Repletion with purified anti-Gal fully restored hemolysis in both assays. Serum depleted of Clq showed a reduced lysis of rabbit E as compared to normal human serum; this effect increased with higher serum dilutions. The reciprocal picture, i.e. less effect on hemolysis with increasing dilution, was seen with factor D depleted serum. Comparison of the RECH50 values with the AP50 values revealed an 8.4-fold increase of lysis in the RECH50 test, in which both complement pathways are running. By FACS analysis, complement deposition on rabbit E was determined and components of the classical pathway were found, especially in sera where the alternative pathway was disrupted. We conclude that in our model anti-Gal induce lysis via both classical and alternative complement pathways, but that the alternative pathway activation is of minor importance. In addition, we saw that with higher serum dilutions, the classical pathway (i.e. anti-Gal-mediated lysis) takes a predominant role in lysing the rabbit E. As anti-Gal-mediated activation of the alternative complement cascade seems of minor importance based on our results, and as there are only few surfaces in transplanted organs that would favor the alternative pathway to be executed, the specific inhibition of early steps of the classical pathway appears as a realistic strategy in pig-to-primate xenotransplantation that-to the benefit of the patient-leaves the mainly anti-bacterial defense by the alternative pathway intact.

Animals↗
PubMed2004

Immunoglobulin M-enriched intravenous immunoglobulin inhibits classical pathway complement activation, but not bactericidal activity of human serum.

Acute or even hyperacute humoral graft rejection, mediated by classical pathway complement activation, occurs in allo- and xenotransplantation due to preformed anti-graft antibodies. Intravenous immunoglobulin (IVIg) preparations can prevent complement-mediated tissue injury and delay hyperacute xenograft rejection. It is known that IgM-enriched IVIg (IVIgM) has a higher capacity to block complement than IVIgG. Different IVIgs were therefore tested for specificity of complement inhibition and effect on anti-bacterial activity of human serum. IVIgM-I (Pentaglobin), 12% IgM), IVIgM-II (IgM-fraction of IVIgM-I, 60% IgM), and three different IVIgG (all >95% IgG) were used. The known complement inhibitor dextran sulfate was used as control. Hemolytic assays were performed to analyze pathway-specificity of complement inhibition. Effects of IVIg on complement deposition on pig cells and Escherichia coli were assessed by flow cytometry and cytotoxicity as well as bactericidal assays. Complement inhibition by IVIgM was specific for the classical pathway, with IC50 values of 0.8 mg/ml for IVIgM-II and 1.7 mg/ml for IVIgM-I in the CH50 assay. Only minimal inhibition of the lectin pathway was seen with IVIgM-II (IC50 15.5 mg/ml); no alternative pathway inhibition was observed. IVIgG did not inhibit complement in any hemolytic assay. Classical pathway complement inhibition by IVIgM was confirmed in an in vitro xenotransplantation model with PK15 cells. In contrast, IVIgM did not inhibit (mainly alternative pathway mediated) killing of E. coli by human serum. In conclusion, IgM-enriched IVIg is a specific inhibitor of the classical complement pathway, leaving the alternative pathway intact, which is an important natural anti-bacterial defense, especially for immunosuppressed patients.

Animals↗
PubMed1990

[Fatal pneumococcal meningitis in a 1-year-old child with homozygous C2 deficiency].

The 1-year old girl died of recurrent bacterial meningitis. Streptococcus pneumoniae was isolated from the cerebrospinal fluid. The analysis of the immune system revealed only a defect of the complement system. The following results were obtained: 1. No function of the classical complement pathway. 2. Reduced function of the alternative complement pathway. 3. No functional C2 activity. 4. No C2 protein. The parents had half normal C2 titers. HLA typing was only possible for the parents with the following results: A1, A32(w19), B18, DR2, DRw11(5) (father) and A3, A10, B18, B7, DR2 (mother). These data are compatible with a B18, DR2 haplotype of the child which is found in most cases of homozygous C2 deficiency. Our patient list another example for the high risk of recurrent severe infectious diseases in persons with a total complement defect.

Complement C2↗
PubMed1984

Defective binding of the third component of complement (C3) to Streptococcus pneumoniae in multiple myeloma.

Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Complement C3↗
PubMed2000

Binding of immune complexes to erythrocyte CR1 (CD35): difference in requirement of classical pathway components and indication of alternative pathway-mediated binding in C2-deficiency.

Deficiency of complement components within the classical pathway is associated with increased risk for immune complex disease. However, C2-deficient individuals often have a mild disease and about 50% are healthy. To study the importance of the different components for immune complex clearance, bovine serum albumin (BSA)/anti-BSA complexes were opsonized in human serum and the binding to erythrocyte complement receptor type 1 (CR1, CD35) was measured in vitro. In C2-depleted serum the complexes were opsonized and bound to CR1 but the reaction needed a longer opsonization time than in normal human serum (NHS). In contrast, serum reagent lacking C1q, C4 or C3 did not promote binding in this assay system. We also demonstrated that elevated levels of factor B could restore binding of complexes to erythrocytes in C2-depleted serum via alternative pathway activation. These results indicate that in spite of lack of a complete classical pathway, C2-deficient individuals could retain some immune complex opsonizing activity via the alternative pathway. This finding could contribute to the understanding of differences in association between complement deficiency and immune-complex disease.

Antigen-Antibody Complex↗
PubMed1983

Adherence of murine peripheral blood eosinophils and neutrophils to the different parasitic stages of Nematospiroides dubius.

Eosinophils and neutrophils, purified by density gradient centrifugation from the blood of infected mice resistant to reinfection, were tested for their ability to adhere to the different parasitic larval stages of the murine nematode parasite Nematospiroides dubius. Cells were tested for adherence to larvae which had been sensitised with immune mouse serum (IMS) or normal mouse serum (NMS) in the presence of CA2+ and Mg2+ ions. EDTA, or EGTA. Differences were observed in the degree of cell adherence to the different stages of the parasite. However, the adherence of the two cell types to any given stage of the parasite was similar. Adherence to the sheathed infective third-stage (L3) larvae, 96 h post-infective larvae and to adult worms depended to a large degree on conditions suitable for complement activation (viz. fresh serum and the presence of Ca2+ and Mg2+ ions). Complement was activated both via the alternative pathway by the parasite itself and via the classical pathway by parasite-bound antibodies. In these conditions, cell adherence probably occurred predominantly through the interaction of leucocyte third component of complement (C3) receptors with parasite-bound C3. In contrast, adherence of cells to exsheathed L3 and to the 48 h and 72 h post-infective larval stages appeared to involve antibody/Fc receptor as well as C3/C3 receptor interaction. The data indicate that N. dubius may undergo a series of antigenic changes during its life cycle and that antibodies capable of mediating granulocyte attachment are elicited predominantly against the early tissue developmental forms of the parasite.

Animals↗
PubMed1987

Effect of Haemophilus influenzae type b lipopolysaccharide on complement activation and polymorphonuclear leukocyte function.

Purified lipopolysaccharide (LPS) from Haemophilus influenzae type b (Hib) was examined for its capacity to interact with human hemolytic complement, generate conversion products of C3, C4, and factor B, stimulate C5a activity, and affect human neutrophil chemiluminescence and phagocytosis. Salmonella typhimurium LPS and Salmonella minnesota Rb LPS (R345 mutant) were examined for comparison. Incubation of Hib LPS with human serum deficient in gamma-globulin or with normal human serum containing 10 mM EGTA and 7 mM MgCl2 resulted in some depletion of hemolytic complement and conversion of C3 to degradation products (determined by inhibition of passive hemolysis and electrophoresis/immunofixation, respectively), indicating that complement activation occurred by the alternative pathway. Complement activation by Hib LPS and S. minnesota Rb LPS was similar, but significantly less effective than by S. typhimurium LPS (p less than 0.01). Solubilized Hib lipid A, but not LPS, induced conversion products of C4 in hypogammaglobulinemic serum, indicating activation of the classical pathway. Similar levels of C5a activity were generated by incubation of Hib LPS and S. typhimurium LPS in hypogammaglobulinemic serum, as determined by neutrophil shape change and neutrophil aggregation. Hib LPS directly stimulated neutrophil chemiluminescence, whereas S. typhimurium LPS had little effect. Phagocytosis of radiolabeled, opsonized Hib by neutrophils was diminished by S. minnesota Rb LPS, Hib LPS, or solubilized Hib lipid A (p less than 0.001), but was slightly increased by S. typhimurium LPS. Neither the oligosaccharide of Hib LPS or Hib capsular polysaccharide was capable of interacting with complement or altering neutrophil chemiluminescence or phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Activation↗
PubMed1981

Interactions of killed Listeria monocytogenes with the mouse complement system.

Incubation of mouse serum with Listeria monocytogenes involved activation of the alternative complement pathway, resulting in depletion of both classical and alternative pathway activity. The activation process gave rise to reactive (calcium- and magnesium-independent) lysis of, specifically, rabbit erythrocytes, which become resistant to this form of hemolysis by sensitization with antibodies. The possible implications of these findings for L. monocytogenes as an intracellular parasite and for rabbit erythrocytes as target cells for mouse alternative complement pathway activity are discussed.

Animals↗
PubMed1986

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine.

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.

Animals↗
PubMed1990

Complement-mediated enhancement of HIV-1 infection of the monoblastoid cell line U937.

To assess the role of complement and complement receptors in HIV-1 infection of monocytes and macrophages, we studied the infectivity of HIV-1, isolated from the peripheral blood of a patient with subacute AIDS-related encephalopathy, on the human monoblastoid cell line U937. HIV-1 and HIV-1-infected cells were capable of activating the complement system via the classical and the alternative pathways, respectively. Low concentrations of HIV-1 were able to infect U937 cells more easily in the presence than in the absence of complement. At higher virus concentrations, infectivity was no longer facilitated by the presence of complement. Infection of U937 cells was reduced in the presence of any of the monoclonal antibodies (MAbs), OKT4a (anti-CD4), OKM1 (anti-CR3), or M522 (anti-CR3). A combination of all three of these MAbs reduced the infection by an even greater amount. These data indicate that complement receptors may be a port of entry for complement-coated HIV-1.

AIDS Dementia Complex↗
PubMed1980

Role of Kupffer cells, complement, and specific antibody in the bactericidal activities of perfused livers.

The relative roles of Kupffer cells, complement, and specific antibody in liver antimicrobial activities were investigated by using a rat liver perfusion model. Normal livers trapped an average of 60% of Salmonella typhimurium in a single pass and in the presence of plasma killed more than 60% of these organisms in 30 min. Livers depleted of Kupffer cell function by silica treatment had significantly less bactericidal ability (ca. 15%) in the presence of plasma, showing that viable Kupffer cells are required for optimal antimicrobial activity. To determine the importance of complement in Salmonella killing, plasma complement activity was inhibited by heating at 57 and 50 degrees C, zymosan absorption, chelation with disodium ethylenediaminetetraacetate (EDTA) and depletion of rat C3 by using specific immunoabsorbent. All treatments significantly reduced bactericidal activity in the perfused liver. Chelation of plasma with EDTA had no effect, suggesting that the alternate and not the classical pathway for complement activation was involved. Immune plasma alone was bactericidal. When immune plasma was heated, zymosan absorbed, or chelated with EDTA, bactericidal activity was inhibited in the perfused liver, but bacterial trapping increased. These results suggest that complement is required for bactericidal activity in perfused livers and that specific antibody only enhances bacterial trapping.

Animals↗
PubMed1980

A complement inhibitor produced by Stachybotrys complementi, nov. sp. K-76, a new species of fungi imperfecti.

A complement inhibitor, K-76, was isolated and purified from the culture supernatant of a fungus, Stachybotrys complementi, nov. sp. K-76, isolated from soil of Ishigaki Island, Okinawa. K-76 is a sesquiterpene compound and it can be oxidized to a monocarboxylic derivative (K-76 COOH), the sodium salt of which is very soluble and much less toxic than K-76. K-76 and K-76 COOH both inhibited complement activation by either the classical or alternative pathway. They inhibited generation of the factor chemotactic to human polymorphonuclear leukocytes from human serum by aggregated immunoglobulin. When sensitized erythrocytes were treated with complement in the presence of K-76 COOH, the resulting unlysed cells were found to be in the state of EACl, 4b, 2a, 3b. Thus K-76 COOH is considered to block mainly the C5 intermediate step. K-76 COOH did not inhibit any proteases or esterases tested, except when tested at high concentration.

Chemotactic Factors↗
PubMed2005

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases.

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Bacterial Proteins↗
PubMed2003

Clionasterol: a potent inhibitor of complement component C1.

Clionasterol (1a), clionasterol monoacetate (1b) and 5alpha,8alpha-epidioxy-24alpha-ethylcholest-6-en-3-ol (2), isolated from the marine sponge Xestospongia exigua, and beta-sitosterol (3) were tested for their influence on the classical (CP) and alternative (AP) pathways of activation of the human complement system in vitro. All the sterols inhibited the CP in a dose-dependent manner but no detectable effect was observed in the AP even at concentrations of 400 microM. Clionasterol was found to be a potent inhibitor of CP (IC50 = 4.1 microM) being ten-fold more active than beta-sitosterol. The presence of the epidioxy group on C-5 and C-8 of compound 2 caused a pronounced decrease of the inhibitory effect. Mechanistic studies on the anticomplementary effect of clionasterol revealed that it interferes with the complement component C1.

Animals↗