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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↗
PubMed1988

Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways.

The serum complement system is a major mediator of inflammation reactions. Two of the complement proteins, the third (C3) and fifth (C5) components, are precursors of potent phlogistic molecules, C3a and C5a. C5a has potent chemotactic activity and plays an active role in pulmonary inflammation. We present evidence suggesting that several complement proteins, including C5, are synthesized locally in the lung in alveolar type II epithelial cells. Lung tissue from normal mice synthesized and secreted C5 protein similar to the C5 protein in mouse serum, whereas lung tissue from C5-deficient mice did not. Lung tissues from both normal and C5-deficient mice synthesized C3. Rat lung tissue synthesized and secreted C5, as well as C2, C4, C3, and factor B. Cultures of type II cells (95% type II cells, 5% macrophages) regularly synthesized all these proteins. In contrast, cultures of macrophages alone synthesized large amounts of C2 and factor B, and in some experiments C3 and C4, but never C5. The C5 synthesized by the rat cells was slightly larger than serum C5 (200 kD compared with 180 kD) and was not processed to the two-chain molecule seen in serum. Rat lung tissue and purified type II cells contained C5 mRNA with the same molecular mass as the C5 mRNA in rat liver and in mouse lung and liver. Human type II cells also synthesized C5, as well as C2, C4, C3, and factor B. Human pulmonary macrophages synthesized only C2, factor B, and, in some experiments, C3. Synthesis of complement proteins in cells that line the alveolar wall may provide a local source of these proteins for inflammatory responses in the lung. Local synthesis of complement proteins could be regulated independently of the synthesis in the liver.

Animals↗
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↗
PubMed1995

Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3.

A human myeloid cell subline, P39+, is found to be a target for human complement (C) via the alternative pathway and to allow the deposition of multiple C3 fragments on its membranes, though expressing the complement regulatory proteins decay-accelerating factor and membrane cofactor protein. The parent cell line, P39-, which is phenotypically similar to the P39+ subline, does not allow the deposition of homologous C3 fragments. In this study, we established a monoclonal antibody, M161 Ab, which reacted with P39+ but not P39- cells. This Ab recognized a 43-kD protein in P39+ cell lysate transblotted onto nitrocellulose. Using this Ab as a probe, we purified the 43-kD protein, namely, M161 antigen (Ag). M161 Ag had a basic isoelectric point (pI), 9.3-9.4 by chromatofocusing, and was precipitated as an insoluble material at the pI point. The purified M161 Ag was a single-chain protein and did not possess N- or O-linked carbohydrates. When the purified M161 Ag was transblotted onto nitrocellulose and incubated with Mg(2+)-EGTA serum, human C3 fragments were efficiently deposited on M161 Ag. The major species of the deposited C3 fragments was C3b. Furthermore, the C3 fragments bound to the M161 Ag were detached by 1 M hydroxylamine, suggesting that a covalent ester linkage sustains M161 Ag-C3b interaction. NH2-terminal amino acid analysis revealed that M161Ag is a novel membrane protein. Hence, it appeared that M161 Ag is a potent activator of human alternative complement pathway on human cells that activates homologous C3 and allows the deposition of C3b on itself. Thus, under some conditions, homeostasis of complement is maintained even on human cells, not only by the complement regulatory proteins, but also by membrane C3-activating molecules on which C3b is deposited.

Antibodies, Monoclonal↗
PubMed1973

Interactions of the classical and alternate complement pathway with endotoxin lipopolysaccharide. Effect on platelets and blood coagulation.

The contributions of the classical and alternate pathways of complement activation to the biological effects of endotoxin have been examined in the guinea pig, with particular reference to thrombocytopenia, leukopenia, and the development of the hypercoagulable state. Injection of endotoxin into normal guinea pigs led to a 95% fall in the level of circulating platelets within 15 min as well as a fall in circulating granulocytes. C4-deficient guinea pigs, known to have a complete block in the activity of the classical complement pathway, but with the alternate pathway intact, sustained no fall in platelets. The development of granulocytopenia proceeded normally. Endotoxin did activate the alternate complement pathway in C4D guinea pigs, as evidenced by the fall in C3-9 titers. With restoration of serum C4 levels, endotoxin-induced thrombocytopenia was observed in C4D animals. Thus, function of the classical complement pathway was an absolute requirement for the development of thrombocytopenia. Experiments performed in cobra venom factor (CVF)-treated normal guinea pigs, with normal levels of C1, C4, and C2, but with less than 1% of serum C3-9 demonstrated the importance of the late components in the development of thrombocytopenia but not leukopenia.C4-deficient guinea pigs had normal clotting times demonstrating that C4 was not required for normal clotting. In addition, development of the hypercoagulable state, evidenced by a marked shortening of the clotting time, was not observed on injection of endotoxin into C4D animals. Therefore, development of the hypercoagulable state paralleled the development of thrombocytopenia and required function of the classical complement pathway. Again, the importance of the late components of complement was emphasized by the failure of CVF-treated normal animals to develop hypercoagulability. These results demonstrate that endotoxin is capable of activating both the classical and alternate complement pathways in guinea pigs but that function of the classical pathway is an absolute requirement for the development of thrombocytopenia and the hypercoagulable state.

Animals↗
PubMed1996

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site, is a selective inhibitor of the alternative complement pathway.

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b alpha chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.

Binding, Competitive↗
PubMed1984

Activity of classical and alternative pathways of complement in preterm and small for gestational age infants.

Complement activity was compared in 50 low birth weight infants divided into appropriate and small for gestational age groups; the influence of birth weight and gestational age on complement development was also investigated. CH50 and kinetics (tH50) of both classical and alternative pathway activity of complement, C3, and Factor B levels were significantly higher in small for gestational age infants (classical pathway CH50, 630 HU/ml +/- 184 SD; CP tH50, 77 min +/- 47; aternative pathway CH50, 44.8 HU/ml +/- 11.3; AP tH50, 56 min +/- 43; C3, 73.98 mg/dl +/- 12.68; and Factor B, 13.17 mg/dl +/- 3.67) than in weight-matched appropriate for gestational age infants (CP CH50, 523 HU/ml +/- 152; CP tH50, 105 min +/- 49; AP CH50, 38.8 HU/ml +/- 13; AP tH50, 90 min +/- 53; C3, 58.14 mg/dl +/- 9.43; and Factor B, 9.32 mg/dl +/- 1.73). Complement values were lower in low birth weight infants than in adult controls (P less than 0.001 in all cases). All complement parameters were mainly correlated with gestational age; CH50 values of the classical and alternative pathways were also highly correlated with each other (r = 0.64; P less than 0.001). Low birth weight infants, especially preterm infants, have an important defect of complement activity. Complement factors increase gradually during gestation and intrauterine growth retardation does not affect complement development. Classical and alternative complement pathway activities have a similar development pattern.

Complement Activation↗
PubMed1988

Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Blotting, Northern↗
PubMed1983

Effects of immunological adjuvants on the mouse complement system. I. The inability of the polyanion heparin to act as an adjuvant is paralleled by inefficient alternative complement pathway inhibition.

Interference with hemolytic complement activity by polyanionic substances was studied in relation to the ability of these compounds to act as an adjuvant for a dead listeria vaccine. Heparin appeared a poor inhibitor of the mouse alternative pathway not only in contrast to its effects on the mouse classical and the human classical and alternative pathways, but also when compared to two polyanions with known adjuvant activity: dextran sulfate and suramin. For the three polyanions mentioned a correlation between adjuvant activity and mouse alternative pathway inhibition was observed. These findings suggest a possible causal relationship between interference with alternative complement pathway activation and adjuvant activity.

Adjuvants, Immunologic↗
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↗