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PubMed1993

Activation of the complement system by Francisella tularensis lipopolysaccharide.

We have shown by combining lipopolysaccharide (LPS) extracted and purified from Francisella tularensis live vaccine strain (LVS) with normal complement and back titrating with sensitised sheep red blood cells that the LPS activates complement. Deionising the LPS and converting it into the single salt forms of pyridine, ethanolamine and triethylamine altered the ability to activate complement according to the apparent molecular weight due to aggregation. Francisella tularensis LPS activated complement deficient in a component of the alternative pathway (factor B) but failed to activate complement deficient in a component of the classical pathway (C1q). In addition normal complement suspended in ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) which inactivates the classic pathway was not activated by LPS, and we concluded that the LPS activates complement predominantly via the classical pathway. LPS bound to specific monoclonal antibodies activated complement more than LPS alone. An anti-core monoclonal antibody was approximately tenfold more potent when bound to LPS then an anti-O side chain monoclonal antibody in activating complement.

Antibodies, Bacterial↗
PubMed1997

Detection and quantification of the control proteins of the alternative pathway of complement in 3T3-L1 adipocytes.

The complement peptide C3a desarg is identical to acylation-stimulating protein (ASP), a human plasma protein that potently stimulates adipocyte triacylglycerol synthesis and glucose transport. Both human and murine adipocytes express mRNA and/or protein for the complement components C3 and factors B and D (adipsin) required to generate ASP. However, the regulatory mechanisms controlling this process are unknown. We have established a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique to demonstrate the presence in mouse 3T3-L1 adipocytes of mRNA for all components of the alternative pathway, including the control proteins factors I and H, CR1 and properdin. On differentiation, mRNA for C3 (fivefold) and factor D (> 50-fold) increased, whereas stimulation with tumour necrosis factor (TNF)-alpha and interleukin (IL) 1 beta led to eightfold increases in factor B mRNA. Metabolic labelling followed by immunoprecipitation showed that factor B protein is normally present in small quantities, and is greatly increased by cytokine stimulation. The larger quantities of C3 and H proteins present were little affected, whereas levels of C3a increased on cytokine stimulation. These results suggest that the rate-limiting step in the cytokine-induced production of ASP in adipocytes is factor B synthesis.

3T3 Cells↗
PubMed1984

Effects of various treatments of bovine complement on its lytic efficacy measured by two different tests.

Bovine complement was treated with various agents known to activate or inactivate one or more of the cascade components. The treated complement was then assessed for remaining hemolytic activity by a tube titration test and a radial hemolysis method. Divalent cation chelators (EDTA and EGTA); immune complexes prepared with serum and IgM, IgG1, IgG2, and IgA isotypes; smooth and rough lipopolysaccharides and lipid A; hydrazine; zymosan; cobra venom factor and brown recluse spider venom caused depletion of complement as determined in the tube titration test. Immune complexes (prepared with serum); hydrazine; cobra venom factor; EDTA and smooth lipopolysaccharide caused loss of hemolytic activity in the radial hemolysis test. This evidence suggests that the radial hemolysis test assessed complement consumed by the alternate pathway, while the tube titration method measured classical pathway consumption.

Animals↗
PubMed1987

Alternative pathway of complement activation by stimulated T lymphocytes. I. Binding of C3 fragments.

Human blood lymphocytes cultured for 3 days with concanavalin A (Con A), phytohemagglutinin or pokeweed mitogen, in mixed lymphocyte culture with added interleukin 2 and stimulated by a lymphoblastoid cell line were found to activate and bind C3 molecules when exposed to human serum. The split products of C3 were detected in the supernatants and on the surface of the activated cells. The surface-attached C3 fragment on the Con A blast was identified as C3b by immune adherence i.e. binding of CR1 carrying human erythrocytes. In the Con A-stimulated population the majority of cells that activated and bound C3 were CD3 and Fc gamma receptor (CD16)-positive but complement receptor-negative blasts. In this cell subset both CD4 and CD8-positive cells were detected but their frequency suggested that a proportion of them carried both markers.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Complement activation in septic shock patients.

To evaluate the status of the complement system and to determine the effects of corticosteroids on complement component levels in septic shock, C3, C4, and Factor B were measured in 42 patients with severe late septic shock. Serum levels of C4 and Factor B correlated with C3 levels (r = 0.48 and 0.64, respectively; p less than .01) in patients in shock for more than 4 h, but only Factor B correlated with C3 (r = 0.85; p less than .01) in patients in shock for 4 h or less. C3 and Factor B levels were significantly (p less than .05) lower in patients who died (12,174 +/- 1,524 CH50 U/ml and 14 +/- 1 mg/dl, respectively) than in patients who survived (18,418 +/- 2,833 CH50 U/ml and 21 +/- 2 mg/dl, respectively). Corticosteroids did not alter complement component levels. The alternative pathway appears to be activated early in septic shock, whereas the classical pathway is activated later. C3 and Factor B levels may predict survival of patients in septic shock. In this study, corticosteroids did not change the complement component levels of patients in late severe septic shock.

Complement Activation↗
PubMed1991

Alternative complement pathway-mediated myeloid cell cytotoxicity: repertoire of membrane factors participating in regulation of C3 deposition and cytolysis.

Most human nucleated cells and cell lines possess C3 step regulators, decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg(2+)-EGTA-human serum treatment did not induce C3 deposition on most nucleated cells. Furthermore, less than 20% lysis occurred even after the block of all the three factors. In contrast, three myeloid cell lines, U-937, HL-60 and p39, were found to exhibit unusual C3 deposition or cytolysis. U-937 possessed DAF and MCP but lacked p18, and about 50% was lysed by treatment with anti-DAF and anti-MCP followed by Mg(2+)-EGTA-serum, which caused C3, C5 and C8 deposition. Anti-DAF evoked similar but less complement (C) deposition and cytolysis while anti-MCP alone did not, although it enhanced the anti-DAF-mediated C deposition and cytolysis. Thus, once the C3 step is overcome, U-937 is attacked by the late components leading to cytolysis because of the absence of p18. On the other hand, HL60 allowed the deposition of C3 by blocking of either DAF or MCP followed by the Mg(2+)-EGTA-serum treatment. C5, C8 and C9 were subsequently deposited but resulted in no lysis. Lysis of 60% was attained by the additional blocking of p18. Thus, HL60 is poorly protected by C3 and C9 step regulation. Strikingly, extensive C3 deposition occurs on p39 without any antibody treatment, suggestive of the presence of unique alternative pathway activators. However, little cytolysis was induced on p39 even by blocking of all three inhibitors with antibodies. These results suggest that in activation of the alternative pathway on myeloid cells, C3 step is controlled by the inhibitors and alternative pathway activators, and C-mediated cytolysis is blocked by p18 and additional regulatory mechanisms or factors which assist in protection of nucleated host cells from MAC attack. Susceptibility to homologous C of these cell lines, therefore, reflects relatively low potency of C regulation on their membrane.

Antigens, CD↗
PubMed1983

A simplified quantification method of complex-release activity using peroxidase as immune complex antigen.

The complement-mediated solubilization of precipitable immune complexes (complex-release activity) in serum specimens was determined by a simplified method using peroxidase as an immune complex antigen. The results correlated well with the hemolytic activity via the classical complement pathway and that via the alternative complement pathway. This simplified method proved to be reliable and useful.

Antigen-Antibody Complex↗
PubMedDate not supplied

A complement-sensitive mutant of Pseudomonas aeruginosa.

The role of complement in the bactericidal activity of human serum against a mutant strain of Pseudomonas aeruginosa used as a model was demonstrated. The involvement of complement in the bacterial destruction of P. aeruginosa, and the contribution of the alternative and classical pathways of the complement system were directly evidenced by using sera from complement-deficient patients.

Animals↗
PubMed1994

A role for natural antibody in the pathogenesis of leprosy: antibody in nonimmune serum mediates C3 fixation to the Mycobacterium leprae surface and hence phagocytosis by human mononuclear phagocytes.

We have previously determined that complement receptors on human mononuclear phagocytes and complement component C3 in nonimmune serum mediate phagocytosis of the intracellular bacterial pathogen Mycobacterium leprae, the agent of leprosy. We have also determined that C3 fixes selectively to the major surface glycolipid of M. leprae, phenolic glycolipid 1 (PGL-1). In this study, we have explored the role of natural antibody in nonimmune serum in C3 fixation and C1q binding to M. leprae and PGL-1. At serum concentrations within the range at which phagocytosis of M. leprae is maximal, C3 fixation was mediated by both the classical and the alternative complement pathways. At the low end of this serum concentration range (2.5%), C3 fixation was mediated predominantly by the classical pathway. Consistent with a role for both pathways, C3 fixation to M. leprae was enhanced by the addition of either pure C1q to C1q-depleted serum or pure factor B to factor B-depleted serum. C3 fixation to M. leprae was strictly antibody dependent regardless of the serum concentration used. C3 fixation to M. leprae occurred in nonimmune serum but not in agammaglobulinemic serum unless heat-inactivated nonimmune serum or small amounts of pure immunoglobulin G (IgG) or IgM were added. C3 fixation by both the alternative and the classical complement pathways was mediated by antibody, and the antigen-binding portion of the antibody molecule was required. C3, IgG, IgM, and C1q were readily detected on the surface of M. leprae. Consistent with the previously demonstrated exclusive role of the classical complement pathway in C3 fixation to PGL-1, C1q bound to PGL-1 in a dose-dependent fashion; C1q binding was evident in > 1.25% nonimmune serum. C1q binding to PGL-1 was strictly antibody dependent. When PGL-1 was incubated with pure C1q, little or no C1q bound to PGL-1 unless heat-inactivated nonimmune serum or pure IgG or IgM was added. When PGL-1 was incubated in nonimmune serum, C3 bound directly to PGL-1 and not to anti-PGL-1 antibody, since the amount of C3 bound to PGL-1 was not reduced by acid elution of the antibody. However, the amount of C3 bound to PGL-1 was markedly reduced by hydroxylamine treatment, providing evidence for C3 fixation via a covalent ester bond. Nonimmune serum contained antibody to all four major M. leprae surface carbohydrates. Relative to PGL-1, nonimmune serum contained more antibody to the other surface carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Bacterial↗
PubMed2001

Mast cells mediate complement activation after acid aspiration.

A significant role for the alternative complement pathway in acid aspiration has been demonstrated by the observation that C3 but not C4 genetic knockout mice are protected from permeability edema. Using mast cell-deficient mice (W/Wv), we tested the hypothesis that mast cells mediate complement activation after acid aspiration. Tracheostomy tubes were placed in anesthetized mice and 2 mL/kg 0.1 N HCL was instilled in the trachea. After 4 h, extravasation of 125I-albumin was used to calculate lung vascular permeability. The serum alternative complement pathway hemolytic activity was examined, and lung immunohistochemistry was performed. Lung permeability in W/Wv mice was 62% less than that of mast cell sufficient (+/+) animals and similar to +/+ mice treated with the chymase inhibitor chymostatin (65% decrease). Treatment of +/+ mice with D-PRO2,D-TRP(7,9)-Substance P, an antagonist to the neuropeptide substance P, reduced injury by 66%. Serum complement hemolytic activity was intact in injured w/wv mice and +/+ animals treated with chymostatin or dpdt-sp, but was decreased to 65% in the injured untreated +/+ group. Alveolar C3 deposition was intense in injured untreated +/+ mice but absent in the other groups. We interpret these data to indicate that mast cells mediate complement activation, via chymase degranulation, after acid aspiration. This mast cell activity likely is regulated by the release of substance P.

Animals↗
PubMed1981

The role of complement and IgG on zymosan opsonization.

Zymosan particles require opsonization for optimal interaction with granulocytes and activation of their respiratory burst. In the present study we evaluated the serum factors necessary for zymosan opsonization. First, zymosan was treated with either normal serum or hypogammaglobulinemic serum (HGS), which is deficient in immunoglobulins (Ig) but has a normal concentration of complement components. Using granulocyte chemiluminescence to assay opsonization, the activity of particles treated with HGS was 66 +/- 1.9% (mean +/- SEM) of that with normal serum (p less than 0.001), suggesting a role of Ig. HGS opsonic activity was restored to normal when the particles were also treated with IgG; however, neither heat-inactivated normal serum (56 degrees C, 30 min) nor pure human IgG alone had opsonic activity. The roles of the classical (CCP) and alternative (ACP) pathways of the complement system were also investigated. ACP activity seemed essential, since inactivation of the ACP (50 degrees C, 30 min) eliminated the activity of normal serum. However, when the ACP was intact, CCP action appeared to participate in opsonization, since selective CCP inactivation with EGTA and MgCl2 reduced the opsonic activity of normal serum by 26 +/- 3.3% (p less than 0.005). Thus, it is concluded that the ACP, CCP, and IgG all participate in zymosan opsonization.

Agammaglobulinemia↗
PubMed1984

Activation of vertebrate complement by Helix pomatia haemolymph.

Haemolymph plasma from the pulmonate snail Helix pomatia contains a constituent, not yet identified, which causes activation of vertebrate complement via the alternative complement pathway in fluid phase. The activation of vertebrate complement by snail plasma is closely analogous to the activation caused by cobra venom factor (CVF), the snake's C3b, with one notable exception; the snail factor requires vertebrate C3 for the formation of C3 convertase which cobra venom factor does not. Our results do not allow any definite conclusion on the exact mechanism but we favour the idea that the haemolymph contains a complement-like protein which functions as an opsonin in the snail, and which can interact with vertebrate alternative complement pathway components.

Animals↗
PubMed1987

Hypocomplementemic glomerulonephritis in an infant and mother. Evidence for an abnormal form of C3.

A mother developed hematuria during the fourth month of pregnancy, and her nursing infant son from this otherwise uncomplicated pregnancy developed hematuria at 3.5 months of age. Both had a mild glomerulonephritis characterized by mesangial prominence, focal thickening and mottling of the glomerular basement membrane and electron-dense deposits, predominantly in the intramembranous and subendothelial positions. Immunofluorescence studies revealed striking accumulations of C3 and other complement components associated with alternative complement pathway activation within glomeruli, and the presence of small or equivocal amounts of immunoglobulin. C1q, C4 and factor B were not detectable. The glomerular lesion was accompanied by hypocomplementemia. Sera of both mother and infant displayed half normal levels of C3 and factor B, increased levels of C4, and normal levels of 12 other complement proteins. High normal or slight elevation in nephritic factor-like activity was observed in serial serum samples. Studies suggested that this mother and son represent the second kindred having an abnormal form of C3 which produces an alternative complement pathway C3 convertase, C3b, Bb, resistant to control by factor H. No additional affected family members were identified. The course of the nephritis over 7 years without drug therapy has been mild with resolving hematuria and no abnormal proteinuria or decrease in creatinine clearance.

Adult↗
PubMed1985

Increased local complement levels upon intraperitoneal injection of mice with Listeria monocytogenes and regulation by polyanions.

Alternative complement pathway activity in mouse peritoneal fluid was determined by a sensitive microtiter assay with rabbit erythrocytes as target cells and cobra venom factor as the inducer of bystander hemolysis. The intra- and interstrain variations in five mouse strains were 30% and, maximally, 64%, respectively. No activity was detected in genetically C5-deficient mice. In C5-sufficient mice, intraperitoneal injection of heat-inactivated Listeria monocytogenes resulted in a 250% increase in local complement activity within 30 min. This effect was reversed by simultaneous injection of dextran sulfate but not heparin. These results are discussed in relation to the ability of listeriae to activate the mouse alternative complement pathway in vitro and the effectiveness of dextran sulfate and the failure of heparin in functioning as an adjuvant in the induction of a protective response in mice by a heat-inactivated listeria vaccine.

Animals↗
PubMed1999

Heparin-coated cardiopulmonary bypass equipment. II. Mechanisms for reduced complement activation in vivo.

OBJECTIVE: Our objective was to study mechanisms for reduced complement activation by heparin coating of cardiopulmonary bypass equipment in clinical heart surgery. METHODS: Adults undergoing elective coronary artery bypass grafting were randomized to cardiopulmonary bypass with Duraflo II heparin-coated (n = 15) or uncoated (n = 14) sets (Duraflo coating surface; Baxter International, Inc, Deerfield, Ill). Blood samples were analyzed with the use of enzyme immunoassays for C1rs-C1 inhibitor complexes and the activation products Bb, C4bc, C3bc, C5a-desArg, and the terminal complement complex. Data were compared by repeated-measures analysis of variance. RESULTS: C1 was activated during bypass, and increases in C1rs-C1 inhibitor complexes were larger with heparin coating (P =.03). C4bc increased after administration of protamine, without intergroup differences (P =.69). Bb (P =.22) and C5a-desArg (P =.13) tended to increase less with heparin coating. Formation of C3bc (P =.03) and the terminal complement complex (P <.01) was significantly reduced with heparin coating. C5a-desArg increased 2-fold during bypass, whereas the terminal complement complex increased 10- to 20-fold. Maximal terminal complement complex concentrations were significantly correlated to maximal Bb and C3bc (R = 0.6, P <.001), but not to C1rs-C1 inhibitor complexes or C4bc (R < 0.05, P >.8). CONCLUSIONS: C1 activation during bypass was increased by heparin coating, but further classical pathway activation was held in check until administration of protamine. Heparin coating significantly inhibited C3bc and terminal complement complex formation. Terminal complement complex concentrations were related to alternative pathway activation and may be useful for evaluation of differences in bypass circuitry. Increases and intergroup differences in terminal complement complex concentrations were much larger than those in C5a-desArg.

Anticoagulants↗
PubMed2004

Variable degree of alternative complement pathway-mediated hemolysis in Indian visceral leishmaniasis induced by differential expression of 9-O-acetylated sialoglycans.

BACKGROUND: Increased expression of linkage-specific 9-O-acetylated sialoglycans (9-O-AcSGs) has been demonstrated on erythrocytes from patients with visceral leishmaniasis (VL) by use of Achatinin-H. We assessed the capacity of this glycotope to influence hemolysis via activation of the alternative complement pathway in patients with VL, compared with that in healthy control subjects. METHODS: The differential expression of 9-O-AcSGs on surfaces of erythrocytes was measured, 9-O-AcSGs were affinity purified, and the molecular determinants were identified by Western blotting. The degree of alternative complement pathway-mediated hemolysis was compared with expression of 9-O-AcSGs on erythrocytes. RESULTS: Enhanced expression of linkage-specific 9-O-AcSGs was demonstrated on erythrocytes from patients with active VL. Six distinct molecular determinants present only on diseased erythrocytes were affinity purified and were absent after elimination of parasite burden. A correlation (r2=0.9) was observed between the presence of 9-O-AcSGs and the degree of alternative complement pathway-mediated hemolysis. CONCLUSION: The 9-O-AcSGs expressed on erythrocytes from patients with VL are potent complement activators, causing enhanced hemolysis via activation of the alternative complement pathway, and may account for the anemia that is a common manifestation of VL.

Adolescent↗
PubMed1985

Generation of leukotrienes by human monocytes upon stimulation of their beta-glucan receptor during phagocytosis.

Human monocytes possess a receptor for ingestion of particulate activators of the human alternative complement pathway that functions in the absence of plasma proteins and is distinct from the receptors for Fc-IgG and the major cleavage fragment of the third component of complement (C3b). Incubation of monolayers of monocytes with 1.1 X 10(6) to 2.2 X 10(7) glucan particles per ml initiated a phagocytic response comparable to that obtained with zymosan particles, of which beta-glucan is a constituent along with mannan. Maximal quantities of 4.93 +/- 3.43 ng of leukotriene B4 (LTB4) and 0.43 +/- 0.23 ng of leukotriene C4 (LTC4) (mean +/- SD, n = 3) were released by 10(6) monocytes stimulated with 1.1 X 10(7) glucan particles per ml. Preincubation of monocytes with 50 micrograms of soluble beta-glucan per ml reduced subsequent monocyte ingestion of 5 X 10(6) zymosan particles per ml and 2.2 X 10(6) glucan particles per ml by 52% and 55%, respectively, and diminished release of LTB4 by monocytes stimulated with 2 X 10(8) zymosan particles per ml and 8.6 X 10(6) glucan particles per ml by 73% and 61%, respectively. Preincubation with 1 mg of soluble mannan per ml had little effect on monocyte phagocytosis or LTB4 generation in response to either zymosan or glucan particles, and neither soluble beta-glucan nor mannan stimulated generation of LTB4 or LTC4. The effect of pretreatment of monocytes with soluble beta-glucan was time dependent, with the maximal effect being evident within 20 min of pretreatment, and was specific for zymosan or glucan particles in that the LTB4 and LTC4 release induced by 2.5 microM calcium ionophore A23187 was unaffected. That both phagocytosis and leukotriene generation are inhibited by soluble beta-glucan but not by mannan at a rate compatible with the phagocytic process of monocyte monolayers indicates ligand specificity for a beta-glucan receptor. As the beta-glucan receptor recognizes particulate activators of the alternative complement pathway, the nonimmune response to a single stimulus induces complement activation, phagocytosis, and leukotriene generation.

Complement Pathway, Alternative↗