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Results for “Complement Pathway, Alternative”

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PubMed1989

Complement-fixing properties of human IgA antibodies. Alternative pathway complement activation by plastic-bound, but not specific antigen-bound, IgA.

The complement-fixing properties of human IgA antibodies bound to specific antigen, or coated directly on plastic surfaces, were examined in comparison with those of IgG antibodies. Use was made of antigen-binding (anti-staphylococcal alpha-toxin) IgA and IgG monoclonal antibodies and normal polyclonal IgA and IgG, purified greater than 99.9% by avoidance of denaturing processes. Complement-fixation ELISA was used, with a high density of biotin-conjugated staphylococcal alpha-toxin bound to avidin-coated plates for the efficient capture of antibodies, and conditions were adjusted for the assessment of classical and alternative pathways of complement activation. Although IgA coated directly on plastic surfaces activated the alternative complement pathway in a dose-dependent manner, IgA antibodies bound to antigen failed to fix complement by either classical or alternative pathways. In contrast, IgG antibodies, either bound to antigen or coated directly on plastic, activated complement mainly by the classical pathway. It was concluded that the complexation of IgA antibodies with antigen is insufficient to elicit complement activation: rather a degree of denaturation seems to play a part in the expression of alternative complement pathway-activating properties by IgA.

Complement Activation↗

PubMed1992

Human keratinocytes and A-431 cells synthesize and secrete factor B, the major zymogen protease of the alternative complement pathway.

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein. Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis. Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B. Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media. Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes). Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.

Animals↗

PubMedDate not supplied

Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

Acetylation↗

PubMed1979

The quantitation of alternative pathway complement function by timed lysis assay.

A simple timed lysis assay is described for quantifying haemolytic complement activity in human serum. Classical pathway complement function was determined by measuring the time taken to lyse 50% of a standard suspension of antibody-coated sheep erythrocytes; the time required for 50% lysis of a standard rabbit erythrocyte suspension was similarly used to evaluate alternative pathway function. Because target erythrocytes prepared on different days gave slightly different 50% lysis times, it was necessary first to construct a series of calibration curves for converting 50% lysis times into CH50 U/ml. For this purpose, a range of dilutions of the standard human serum, of known haemolytic activity, was tested against erythrocytes prepared on 10 separate occasions. The standard serum was subsequently included with each batch of unknown sera and used to select the appropriate calibration curve for direct conversion of the 50% lysis time into CH50 U/ml. Eleven samples of normal human serum were tested by both the timed lysis assay and by the dilution methods of Mayer (1971) (classical) and Platts-Mills and Ishizaka (1974) (alternative pathway). Comparable results were obtained in all cases.

Animals↗

PubMed1990

A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b.

In this work we studied the role of the classical pathway complement component C4b in the activation of the alternative pathway. It was found that nascent C3b attaches with high efficiency to C4b and that C3b in C4bC3b complexes is protected from inactivation by factors H and I. Activation of C3 by factors B and D in the presence of Mg2+ ions and excess C4b led to 35% incorporation of nascent C3b into C4bC3b complexes in the fluid phase. In comparison, when human IgG was tested as an acceptor under similar conditions, only 12% of generated C3b was incorporated into IgGC3b complexes. The half-life time of dissociation of C3b from purified C4bC3b complexes was approximately 2.3 h at 37 degrees C. C4b in these complexes protected C3b from inactivation as effectively as any known alternative pathway activator. Thus, C3b bound to C4b was tenfold more stable than free C3b or C3b bound to a nonactivating surface. In comparison, the protection provided by attachment to human IgG was only 67% of that of C4b. The results provide an explanation for observations of alternative pathway recruitment following classical pathway activation and for the stability of the classical pathway C5 convertase on surfaces which do not provide protection for C3b from factors H and I.

Complement C3-C5 Convertases↗

PubMed1992

Activation of the alternative complement pathway accompanies disease flares in systemic lupus erythematosus during pregnancy.

OBJECTIVE: To assess the activity of systemic lupus erythematosus (SLE) during pregnancy and to distinguish it from preeclampsia. METHODS: We prospectively measured the complement activation products Ba, Bb, SC5b-9, and C4d, as well as the conventional complement determinants C3, C4, and CH50, during pregnancy in 14 patients with SLE and 10 women with preeclampsia. RESULTS: Four of the 14 SLE patients were considered to have disease flares, 3 occurring in the second trimester and 1 postpartum. In these patients, significant abnormalities of Ba, Bb, SC5b-9, and CH50 were noted. In contrast, measures of C4d did not distinguish between pregnant patients who had flares and those whose SLE remained stable. Although decreased values of C3 were rarely seen in the patients with stable disease, normal values of C3 during lupus pregnancy were not reliably associated with stable disease. Three of 10 non-SLE patients with preeclampsia had elevated levels of Ba; however, in each case, the CH50 level was close to or within the normal range. This was in sharp contrast to the findings observed in the 4 patients with active SLE, in whom high levels of plasma Ba were always associated with low CH50 values. Moreover, the ratio of CH50 to Ba was significantly lower in the patients with lupus flares than in the non-SLE patients with preeclampsia. CONCLUSION: While a decline in the CH50 level alone could otherwise be attributed to decreased synthesis of complement components, these data demonstrate that ongoing activation of the alternative complement pathway can accompany disease flares in pregnant women with SLE.

Complement Activation↗

PubMedDate not supplied

Alternative complement pathway in hypocomplementemic/normal C1s-C1 inhibitor complex patients with SLE.

To test whether alternative complement pathway activation explains normal C1s-C1 inhibitor complex in hypocomplementemic (low CH50cl) patients with systemic lupus erythematosus, we examined alternative pathway hemolytic complement (CH50alt) factor B, and Ba fragment in hypocomplementemic sera with normal and with elevated C1s-C1 inhibitor complex. Sera with and without high C1s-C1 inhibitor complex were similar in CH50cl, C3, and C4. There was little evidence for important alternative complement pathway activation in either group, but patients with classical pathway activation (elevated C1s-C1 inhibitor complex) had slightly lower CH50alt and slightly higher factor B and Ba compared to patients with normal C1s-C1 inhibitor complex. Pregnant patients did not differ from non-pregnant patients. Alternative complement pathway activation does not account for hypocomplementemia in this group of patients.

Complement Activation↗

PubMed1991

Plasma levels of proteins of the alternative complement pathway in inbred mice that differ in resistance to Trypanosoma congolense infections.

Inbred BALB/c, A/J, and C57B1/6J mice were infected with Trypanosoma congolense (Trans Mara strain), clone TC13, and monitored for parasitemia, survival times, and plasma levels of complement components C3, C5, factor B, and factor H. Parasitemia was highest in BALB/c, intermediate in A/J, and lowest in C57Bl/6J mice. The mean survival times were 11.5 +/- 0.9, 23.8 +/- 2.3, and 119 +/- 26 days for BALB/c, A/J, and C57Bl/6J mice, respectively. Preinfection levels of factor H were significantly correlated with survival times (r = 0.7722, P less than 0.001). Marked differences were observed between the plasma levels of C3, factor B, and factor H in the 3 mouse strains following infection. Complement C5 levels showed the fewest changes. In the initial postinfection period, BALB/c mice had highest increases in the levels of the 4 complement proteins but also had the greatest declines toward the end of the infection. Factor H levels showed a biphasic increase in BALB/c and C57Bl/6J, but not in A/J mice, with peaks at days 3 and 9. Complement C3 levels declined in all mice toward the terminal stage of the disease. In the late stages of infection, factor B levels markedly decreased in BALB/c but significantly increased in C57Bl/6J mice. Factor B levels measured at the terminal stages in BALB/c, A/J, and C57Bl/6J were correlated positively with their respective survival times (r = 0.714, P less than 0.01). The results suggest that genetic differences in the alternative complement pathway might affect the resistance to T. congolense infections.

Animals↗

PubMed1976

Activation of the alternative complement pathway in systemic lupus erythematosus.

Serum factors activating the alternative pathway of complement in vitro, independent of classical pathway activation was demonstrated in six of eleven patients with systemic lupus erythematosus (SLE). These serum factors were detected by lysis of gluthathione-sensitized human erythrocytes and by C3 and factor B conversion in the presence of EGTA (10 mM) and MgCl2 (0-3 mM), conditions which blocked activation of the classical pathway but permitted activation of the alternative pathway. In order to determine if in vivo activation of the alternative pathway of complement was present in SLE, highly-purified factor B was labelled with radioactive iodine (125I), and its metabolism studied in the eleven patients with SLE and in twelve control subjects. All six patients with serum factors capable of activating the alternative pathway in vitro, had in vivo evidence of alternative pathway activation as measured by increased fractional catabolic rate (FCR) of factor B. Two patients without demonstrable alternative pathway activating factors in their sera had an elevated FCR of factor B. Six of the patients with increased FCR of factor B had disease limited to skin or joint and one had lupus nephritis which was inactive at the time of study. One of the four patients who were in clinical remission had elevated FCR. This study demonstrates that a significant number of patients with SLE of relatively mild disease activity had evidence of alternative complement pathway activation. This activation did not appear to be limited to patients with lupus nephritis and raises the possibility that it could also be related to some of the extra-renal manifestations of SLE.

Adolescent↗

PubMed1989

Characterization of complement activity in turkeys: evidence for classical and alternative complement pathways.

Complement activity in turkey serum was examined by using inhibitors or activators of mammalian complement. Hemolytic test systems using sheep red blood cells sensitized with specific antibody (SSRBC) and horse red blood cells (HRBC) were developed to measure residual complement activity of turkey sera treated with the various inhibitors or activators. Lysis of SSRBC was blocked by treatment with 6-mM EDTA, 10-mM ethylene glycol-bis-beta aminoethylether N,N,N',N' tetraacetic acid (EGTA), and carrageenan. In contrast, lysis of HRBC was blocked by 6-mM EDTA, but not by 10-mM EGTA or carrageenan. Addition of magnesium to EGTA-chelated serum facilitated the lysis of HRBC but not the lysis of SSRBC. Treatment of serum with zymosan at 1 mg/mL and inulin at 5 mg/mL depleted hemolytic activity against both SSRBC and HRBC, suggesting depletion of components common to both pathways. Differences in the hemolytic activities of sera against SSRBC and HRBC after treatment with the various complement inhibitors and activators demonstrate the presence of two complement pathways in turkeys.

Animals↗

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↗

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↗

PubMed1991

Alternative complement pathway activation by HIV infected cells: C3 fixation does not lead to complement lysis but enhances NK sensitivity.

HIV infected T and monocytic cell lines could activate and fix C3 fragments when incubated in human serum under conditions allowing for activation of the alternative complement pathway. Normal T lymphocytes incubated with HIV could also activate and fix C3. This activity was, at least in part, the property of the virus itself since cell-free HIV could efficiently activate C3. The C3 activating HIV infected cells were resistant to complement-mediated lysis, even after prolonged incubation periods. However, their sensitivity to cell-mediated natural killing increased, presumably due to their interaction with complement receptor bearing NK lymphocytes. The results suggest that the alternative complement pathway may contribute to the depletion of CD4+ T lymphocytes during HIV infection in vivo.

Cell Line↗

PubMed1979

Activation of the alternative complement pathway by Torulopsis glabrata.

Torulopsis glabrata is a yeast-like fungus with increasing importance as an opportunistic human pathogen. The role of serum complement in resistance to this infection was studied by chemotactic and opsonic assays. The results indicate that T. glabrata activates complement via the alternative pathway. This may be an important mechanism in host immunity to this fungus.

Candida↗