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PubMed1980

Evaluation of a hemolytic assay of the alternative complement pathway in human serum.

Alternative pathway activity of human serum was titrated by use of unsensitized rabbit erythrocytes (RE). Under the conditions of the assay, the von Krogh equation could be used to relate the proportion of RE lysed to the level of alternative pathway activity. The use of a 50% hemolytic endpoint provided maximum sensitivity in the assay. The 50% hemolytic endpoint could be calculated from a single measurement in the region of 20% to 80% lysis or RE. Factor B was required for lysis of RE in the test, but neither C2 nor C8 was limiting under the conditions of the assay. Alternative pathway activities of three sera with abnormal IgG levels were in the normal range, but normal serum absorbed with RE at 0 C before testing had diminished lytic activity with the test. Lysis of RE in acute-phase sera of 16 patients who had bacteremic pneumococcal pneumonia was significantly below normal (P < 0.01). Results with Re lysis in these patients correlated well with levels of Factor B that were measured immunochemically and with consumption of whole complement by zymosan.

Animals↗
PubMedDate not supplied

Alternative pathway complement activation in rheumatoid arthritis.

Serum and synovial fluid (SF) levels of the complement components C3, C5, factor B (B), properdin (P), beta 1H and C3b inactivator (C3bINA), and EDTA-plasma and SF concentrations of C3d and Ba were measured in 40 rheumatoid arthritis (RA) and 5 patients with osteoarthritis. Decreased SF concentration of B and P and increased levels of Ba showed that increased alternative pathway turnover occurred in RA. Reduced SF C3bINA concentrations occurred, but levels of beta 1H were not reduced. The results showed that alternative pathway turnover was dependent upon C3 turnover, but failed to support the notion that levels of C3bINA or beta 1H control alternative pathway turnover in RA.

Arthritis, Rheumatoid↗
PubMed1983

A study of optimal reaction conditions for an assay of the human alternative complement pathway.

The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37 degrees C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37 degrees C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 +/- SD for 45 normal adults was 25.3 +/- 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.

Adult↗
PubMed1981

Alternate complement pathway activation by recluse spider venom.

Venom from the poisonous brown recluse spider (Loxosceles reclusa) catalyses the lysis of dithiothreitol-treated human erythrocytes when incubated with serum complement but not in heat inactivated serum, a characteristic of complement system activation via the alternate pathway. This activity of the venom was shown to reside in a Sephadex G-75 fraction of the venom which also contains the dermonecrotoxin. Isoelectric focusing of this fraction identified the complement inactivating molecules(s) in the region near pH=6. Analysis of complement after interaction with venom indicated a loss of haemolytic C3. Immunoelectrophoretic development of venom-complement mixtures with anti C3 proactivator revealed the appearance of the C3 activator. These data indicate that recluse spider venom activates the alternate complement pathway.

Animals↗
PubMed2006

Retrovirus-mediated over-expression of decay-accelerating factor rescues Crry-deficient erythrocytes from acute alternative pathway complement attack.

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry(-/-)) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 +/- 127 DAF molecules/cell (DAF(high)) compared with 249 +/- 94 DAF molecules/cell (DAF(low)) and 774 +/- 135 Crry molecules/cell on control mouse erythrocytes. DAF(high)-Crry(-/-) erythrocytes were significantly more resistant than either DAF(low)-Crry(-/-), DAF(-/-) -Crry(+/+) or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry(-/-) erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAF(high)-Crry(-/-) erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

Animals↗
PubMedDate not supplied

[Role of the classical and alternative complement pathways in chemotaxis of human C2 deficiency (author's transl)].

The roles of the classical and alternative complement pathways in the formation of chemotactic factors were comparatively evaluated in a normal serum and a serum from a child with congenital C2 deficiency. Neutrophil chemotactic index was determined by a direct microscopic observation using serum activated by an immune complex for the classical pathway, and by zymosan A or liposaccharide of Salmonella enteritidis for the alternative pathway. Chemotactic activity was observed only when the C2-deficient serum was activited by the alternative pathway in the presence of zymosan A. But, in comparison to normal serum, the chemotactic activity of C2-deficient serum was impaired by dilution or short incubation time. Addition of purified C2 resulted in the normalisation of chemotactic activity induced by activation of classical and alternative pathways. These results suggest that integrity of the classical pathway of the complement is necessary for the optimal formation of the chemotactic mediators induced by the alternative complement pathway. The implications of the alternative complement pathway alteration with regard to susceptibility to bacterial infections, are discussed.

Antigen-Antibody Complex↗
PubMed1979

Deficient alternative complement pathway activity in newborn sera.

Neonatal susceptibility to overwhelming bacterial infection is commonly attributed to a relative deficiency in serum opsonic activity. However, few studies have compared the functional capacity of the classical complement pathway with that of the alternative complement pathway in the neonate. The opsonic activity of nine maternal infant serum pairs were studied by determining percent uptake of radiolabeled Escherichia coli. Seven mother-infant paired sera were studied using E. coli strains known to be opsonized via the alternative complement pathway: the mean percent uptake of E. coli opsonized in neonatal sera was 16.8%; of those opsonized in maternal sera, 54%; and of those opsonized in control sera, 45% (P less than 0.005). Two E. coli strains requiring the classical complement pathway for opsonization were phagocytized equally well in maternal and infant sera of seven mother-infant pairs. Determination of anti-O hemagglutination inhibition (HI) antibody titers in six maternal sera for one classical complement pathway activating and one alternative complement pathway strain showed no correlation between percent phagocytosis and HI antibody titer. These data would suggest that serum levels of classical pathway components are probably adequate for opsonization of E. coli via the classical pathway, but that low alternative complement pathway activity in neonatal sera may contribute to the newborn's increased susceptibility to bacterial sepsis.

Complement Activation↗
PubMed1979

Activation of the alternative pathway of complement by antiserum to factor B.

Monospecific rabbit and goat antisera to human complement proteins and human immunoglobulins were tested for their ability to activate the alternative complement pathway. This activation was detected by two methods where classical pathway activation was blocked with EGTA and alternative pathway activation was promoted with added magnesium ions. These two methods consisted of lysis of GSHE and conversion of factor B into split products. C1q-depleted serum was used in a third assay system. Only antiserum to human factor B was able to activate the alternative pathway in the various systems used. None of the other anticomplement sera showed such activity. When antiserum to factor B was fractionated by ammonium sulfate and column chromatography, activation of the alternative pathway was found in the IgG fraction, and this activity was completely removed by absorption with purified factor B but not with other purified complement components.

Animals↗
PubMed1990

Expression of complement alternative pathway proteins by endothelial cells. Differential regulation by interleukin 1 and glucocorticoids.

We have studied the secretion of proteins of the alternative pathway of complement C3, factor B and factor H by human umbilical vein endothelial cells (HUVEC). Results showed that factor H and factor B are quantitatively secreted in abundance whereas C3 could only be detected when the cells are maintained in culture during long periods of time. Interferon-gamma stimulated factor H, factor B and, to a lesser extent, C3 secretions. Interleukin (IL) 1 had a differential effect on spontaneous C3, factor B and factor H secretions. In the presence of IL 1, there was a significant secretion of C3 occurring within a short period of culture. IL 1 also stimulated factor B secretion. There was a synergistic stimulating effect between IL 1 and interferon-gamma to bring C3 and factor B productions by HUVEC to very high levels. In contrast, factor H secretion was consistently inhibited by IL 1. Local increase in C3 and factor B secretions by endothelial cells in the presence of IL 1 may have important implications in the inflammatory reaction. In striking contrast, the glucocorticoid dexamethasone (DXM) had modulatory effects which are consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, decreased C3 and factor B secretions and increased factor H secretion. Local modulation of complement protein secretion by DXM appears to be a new mechanism by which this glucocorticoid may control inflammation.

Complement C3b Inactivator Proteins↗
PubMed1982

Reduced alternative complement pathway control protein levels in anorexia nervosa: response to parenteral alimentation.

Serum levels of 16 proteins, including 11 component and control proteins of the complement system were determined before and after nutritional repletion in five female patients with severe malnutrition secondary to anorexia nervosa. Before parenteral alimentation significantly decreased serum levels were found for IgG, IgM, transferrin, Clq, C2, C3, factor B, beta lH, C3b inactivator, properdin, and C4 binding protein. A significant increase in posttreatment serum levels compared with pretreatment levels were found for transferrin, C3, factor B, beta lH, and C3b inactivator. Of the proteins measured, the C3b amplification loop control and component proteins, beta lH, C3b inactivator, C3, and factor B rose to the normal range in response to therapy most rapidly. In the absence of an acute phase reaction, these proteins appear to be particularly good indices of malnutrition and its response to therapy.

Adolescent↗
PubMed1978

[Activity of the alternative complement pathway by various axenic strains of Entamoeba histolytica].

The activation of the alternative pathway of complement by axenic cultures of E. histolytica is reported. The activation is carried out in different proportions by several axenic strains, i.e., HK9:NIH, HM1:IMSS, HM2:IMSS and HM3:IMSS; the end result consists of a destruction of the parasite as well as consumption of the serum complement. The participation of the alternative pathway was studied in the presence of EGTA and Mg++, zymosan, heat-inactivation at 50 degree C for 20 min and immunoelectrophoretic studied with anti-factor B.

Animals↗
PubMed1979

Classical and alternate complement pathway activities in paired dairy cow--newborn calf sera.

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.

Animals↗
PubMed1990

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.

Amino Acids↗
PubMed1985

Activation of the alternative complement pathway by pneumococcal lipoteichoic acid.

Cell wall teichoic acids of some gram-positive bacteria are potent activators of the alternative pathway of complement. It is unclear, however, whether the other form of teichoic acid, cell membrane lipoteichoic acid (LTA), can also activate the alternative pathway. In the present study, radiolabelled pneumococcal LTA was found to bind spontaneously to sheep erythrocytes in a temperature- and time-dependent fashion. In addition, the presence of pneumococcal LTA on the erythrocyte surface was verified by the fact that they could be agglutinated by a myeloma protein (TEPC-15) specific for choline, a constituent of pneumococcal LTA. Pneumococcal LTA when fixed to the surface of erythrocytes was able to activate the alternative pathway of complement in both guinea pig serum deficient in the fourth component of complement and human serum deficient in the second component of complement, resulting in lysis of the sensitized erythrocytes. The sensitizing principle of the LTA preparation was removed before erythrocyte sensitization by immunoabsorption, using the choline-specific TEPC-15 myeloma protein. These data demonstrate that purified pneumococcal LTA will bind to sheep erythrocytes and endow them with the ability to activate the alternative pathway.

Animals↗
PubMed2007

Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.

Clinical and experimental data indicate that anti-neutrophil cytoplasmic autoantibodies (ANCAs) cause glomerulonephritis and vasculitis. Here we report the first evidence that complement is an important mediator of ANCA disease. Transfer of anti-myeloperoxidase (MPO) IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice caused crescentic glomerulonephritis that could be completely blocked by complement depletion. The role of specific complement activation pathways was investigated using mice with knockout of the common pathway component C5, classic and lectin binding pathway component C4, and alternative pathway component factor B. After injection of anti-MPO IgG, C4-/- mice developed disease comparable with wild-type disease; however, C5-/- and factor B-/- mice developed no disease. To substantiate a role for complement in human ANCA disease, IgG was isolated from patients with myeloperoxidase ANCA (MPO-ANCA) or proteinase 3 ANCA (PR3-ANCA) and from controls. Incubation of MPO-ANCA or PR3-ANCA IgG with human neutrophils caused release of factors that activated complement. IgG from healthy controls did not produce this effect. The findings suggest that stimulation of neutrophils by ANCA causes release of factors that activate complement via the alternative pathway, thus initiating an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease.

Animals↗
PubMed1993

Activation of the human complement alternative pathway by Listeria monocytogenes: evidence for direct binding and proteolysis of the C3 component on bacteria.

The capacity of the intracellular pathogen Listeria monocytogenes to activate the alternative pathway of human complement was examined. Incubation of L. monocytogenes with human serum in optimal conditions (20% Mg2+EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]-chelated serum) consumed (31.3 +/- 3.9)% of C3 hemolytic activity and led to similar amounts of C3 deposition among the 27 strains tested, except for a rough mutant and the penicillin-induced L forms of strain EGD, which bound reduced amounts of C3. The same results were obtained with strains belonging to related species (L. innocua, L. seeligeri, L. welshimeri, and L. ivanovii). Direct evidence is provided that L. monocytogenes induces the deposition of C3b and its cleavage products iC3b and C3d through ester and amide linkages, as demonstrated by the analysis of the released products of radiolabelled purified C3 after treatment with hydroxylamine. Our results clearly demonstrate that L. monocytogenes activates the alternative pathway of human complement, suggesting that bacteria in the blood or in tissues of infected patients are opsonized and targeted to C3 receptor-bearing cells such as macrophages.

Cell Membrane↗