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PubMed1986

A model system for the study of the assembly and regulation of human complement C3 convertase (classical pathway).

The formation of classical C3 convertase of complement and its regulation by C4b-binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b-like C4) and iodine-treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b-like C4)-C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P-glycerol/P-choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane-associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle-associated C3 convertase.

Buffers↗
PubMed1987

Effect of differences in antibody and complement requirements on phagocytic uptake and intracellular killing of "c" protein-positive and -negative strains of type II group B streptococci.

The influence of antibody and complement on the polymorphonuclear leukocytic uptake and killing of type II group B streptococci (GBS) was examined with 11 adult sera and three type II strains possessing the trypsin-resistant and trypsin-sensitive components (II/TR+TS) of the "c" (formerly Ibc) protein or two type II strains lacking both components (II/no c) of the c protein. All tested sera mediated a greater than 1 log10 reduction in colony-forming units (CFUR) of a type II/no c strain, even in the absence of measurable type-specific antibody (less than 1.08 micrograms/ml), but only 5 of 11 mediated a greater than 1 log10 CFUR of any type II/TR/TS strain, even in the presence of moderate levels of type-specific antibody. The classical pathway of complement activation appeared to be more important than the alternative pathway, and even absorbed or immunoglobulin G (IgG)-depleted serum (IgG, 10 mg/dl) mediated a greater than 1 log10 CFUR without magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (magnesium EGTA) chelation. Chelation with magnesium EGTA reduced the CFUR in 4 of 11 test sera and greatly reduced the CFUR in absorbed or IgG-depleted sera for type II/no c strains. Despite variation in the phagocytic killing of two representative strains of type II GBS, both strains were well phagocytized, as measured by radiolabeled bacterial uptake or electron microscopy. This study suggested that poorly killed type II/TR+TS GBS were easily phagocytized but apparently resisted intracellular killing.

Antibodies, Bacterial↗
PubMed1986

Immunoregulatory disorders associated with hereditary angioedema. II. Serologic and cellular abnormalities.

Hereditary angioedema is defined biochemically by a deficiency in the functional activity of the inhibitor of Cl, Cl esterase inhibitor (Cl INH). Deficiency of this regulator of the early classic pathway of complement results in chronic activation of this cascade with a resultant deficiency of C4 and C2. Ninety-seven patients with either complicated (associated with autoimmune disorders) or uncomplicated hereditary angioedema were evaluated for laboratory evidence of immunoregulatory defects. Specific cellular and humoral abnormalities were found and included increased mean total lymphocyte counts, increased mean Leu 4+ (total) and Leu 3+ (helper) T cells, an increased mean Leu 3/Leu 2 (helper/suppressor T cell) ratio, polyclonal B cell activation, and evidence of circulating immune complexes. C4 functional titers were negatively correlated with percent Leu 3+ cells and absolute Leu 3+ cell numbers. We failed to detect any evidence of immune deficiency in this population, and yet a statistically significant number of patients demonstrated elevated levels of antibodies to Epstein-Barr virus antigens when patients were compared to a control group. Thus, early classic complement pathway activation and/or partial complement component deficiency may effect T cell subpopulations and B cell activation. However, additional predisposing factors (e.g., genetic or viral) appear necessary for the development of a particular autoimmune disease in hypocomplementemic patients.

Adolescent↗
PubMed1986

Hemolytic activity of leukemic sera: the role of complement and sucrose.

In sera of patients with acute myeloblastic leukemia (AML), hemolytic activity can be demonstrated in vitro in the presence of sucrose. To investigate the nature and the mode of action of this hemolytic activity, serum samples from 24 patients with AML were studied by incubation of normal human erythrocytes together with patient serum in the presence of sucrose at low ionic strength (inverse sucrose hemolysis test, ISHT). Fifty-five percent of the serum samples collected during the active stage of the disease gave a hemolysis rate of greater than 4%, whereas in remission only 15% of the samples lysed erythrocytes. Substitution of raffinose, lactose, or polyethylene glycol 400 for sucrose resulted in an almost complete failure of hemolysis under standard conditions, indicating a minor role of the low ionic strength in the ISHT. Heat inactivation, preincubation with inulin, and addition of EDTA, Mg2+-EGTA, or heparin completely abolished hemolytic activities of AML sera when the incubation was carried out for 30 minutes (standard conditions of the ISHT). A prolongation of the incubation time resulted in delayed hemolysis only with the Mg2+-EGTA-treated AML sera. The kinetics of this hemolysis by Mg2+-EGTA-treated AML sera were similar to those of normal human serum in the presence or absence of Mg2+-EGTA. Hemolysis was also obtained by performing the ISHT with normal sera and erythrocytes preincubated with AML sera. These observations suggest a mediation of membrane modification of normal human erythrocytes by AML sera in the presence of sucrose, resulting in an activation of the classical pathway of complement.

Adult↗
PubMed1985

Defective complement activity in chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) are at an increased risk for infections with bacteria which require complement for osponization. We explored the possibility that patients with CLL have a defect in binding the potent opsonin C3b to bacteria. Bacteria selected for these experiments included Streptococcus pneumoniae type 3, which binds C3 by activating the classical complement pathway (CCP), type 25, which can bind normal amounts of C3b by the alternative complement pathway (ACP), type 14, which can activate both the CCP and ACP, and Staphylococcus aureus and Escherichia coli, both of which activate the CCP. Bacteria were treated with normal serum or serum from 15 patients with CLL, and the bound C3b was quantified spectrophotofluorometrically. Despite normal serum concentrations of C3, C4, Factor B, C-reactive protein, and total hemolytic complement activity, all 15 CLL sera bound reduced amounts of C3b to at least one bacterial species; 9 to S pneumoniae type 3, 8 to types 14 and 25, 11 to S aureus, and 13 to E coli. Mixing normal serum with CLL serum restored C3b binding to all bacteria, suggesting a deficiency rather than an inhibitor of activity. Serum from ten hypogammaglobulinemic CLL patients bound less C3b (62.7 +/- 5% of normal) (means +/- SEM) than those with normal immunoglobulin levels (81.9 +/- 5%) (p less than .005). Nevertheless, the addition of specific antibacterial antibodies to CLL serum did not enhance C3b binding to any of the bacteria. Serum from patients with a history of a bacterial infection bound less C3b (62.3 +/- 5%) than those without a history of infections (76.1 +/- 6%) (p less than .05). Thus, there is a defect in either the activation or activity of C3 in CLL serum which may contribute to the increased incidence of infections in these patients.

Blood Bactericidal Activity↗
PubMedDate not supplied

Immunomodulatory activity of an aqueous extract of Azadirachta indica stem bark.

The interference of an aqueous extract of the stem bark of Azadirachta indica with different parts of the human immune system was investigated. The extract showed strong anticomplementary effects which were dose-and time-dependent and most pronounced in the classical complement pathway assay. Moreover, a dose-dependent decrease in the chemiluminescence of polymorphonuclear leukocytes was observed and a dose-dependent increase in the production of migration inhibition factor by lymphocytes.

Adjuvants, Immunologic↗
PubMed1985

Requirement for calcium ions in antibody-dependent complement-mediated cytolysis of Toxoplasma gondii.

The mechanism of antibody-dependent complement-mediated cytolysis of Toxoplasma organisms was studied by using a selective chelating agent for Ca++, ethyleneglycol-bis (beta-aminoethyl ether)-N,N-tetraacetic acid (EGTA). Toxoplasma organisms were lysed by treatment with human serum containing anti-Toxoplasma antibodies and with fresh human-plasma at 37 degrees C for 1 h. The reaction was prevented by heat treatment of the fresh plasma at 56 degrees C for 30 min, confirming that the cytolysis of Toxoplasma organisms had been mediated by an activation of complement. The lytic reaction was completely inhibited by an addition of EGTA at final concentrations of more than 2.5 mM. Addition of Ca++ cleared the inhibitory effect of EGTA on the reaction. These results demonstrate that the antibody-dependent complement-mediated cytolysis of Toxoplasma organisms requires the presence of Ca++, indicating that the reaction is mediated by an activation of the classical complement pathway.

Animals↗
PubMed1986

Susceptibility of phenotypic variants of Haemophilus influenzae type b to serum bactericidal activity: relation to surface lipopolysaccharide.

After three serial passages of Haemophilus influenzae type b strain Fuju in rats, we recovered a variant, which differed in colonial morphology, serum sensitivity, and lipopolysaccharide configuration from the parent strain. The parent organism (Fuju) appeared iridescent and transparent on Levinthal agar, and the rat-passaged variant (rat3 Fuju) appeared iridescent and opaque. The transparent, parent strain Fuju was sensitive to the complement-mediated bactericidal activity of normal rat serum, and the opaque, rat-passaged strain rat3 Fuju was serum resistant. Serum killing of the serum-sensitive strain appeared to be mediated by the classic complement pathway. Both serum-resistant and serum-sensitive strains were killed equally well by immune rat and human serum. These two strains did not differ in the amount of capsular polysaccharide that they elaborated nor in their major outer membrane protein patterns on SDS-PAGE. Lipopolysaccharide isolated from these two strains demonstrated different electrophoretic mobility patterns. Furthermore, the organisms showed different reactivities with two monoclonal antibodies directed against determinants of Hib lipopolysaccharide. Thus, the difference in susceptibility to complement-mediated bactericidal activity of normal rat serum displayed by these two strains is associated with their phenotypes, appears to be unrelated to differences in major outer membrane proteins or in the amounts of capsular polysaccharide elaborated, and is associated with differences in surface lipopolysaccharides.

Animals↗
PubMed1987

Bactericidal activity for Neisseria meningitidis in properdin-deficient sera.

We investigated serum bactericidal reactions against Neisseria meningitidis (serogroups A, B, C, D, Y, W-135, 29E, X, and Z) in the sera of two healthy adults with properdin deficiency. Bactericidal reactions mediated via the classic complement pathway (unchelated system) were not impaired in properdin-deficient serum. The properdin-deficient sera supported alternative pathway-mediated killing (Mg++EGTA-chelated system) of some, but not all, of the strains investigated. Vaccination of the properdin-deficient individuals with serogroup A and C polysaccharide clearly increased the concentrations of antibody to meningococci. At least some of the antibodies induced by vaccination supported the bactericidal activity of properdin-deficient serum. Some antibodies to meningococci, probably of the IgM class, promoted alternative pathway-mediated bactericidal reactions in the absence of properdin. By contrast, presensitizing meningococci with IgG enhanced the alternative pathway-mediated reactions, but this was strictly a properdin-dependent effect.

Antibodies, Bacterial↗
PubMed1986

Determination of anaphylatoxin concentrations in suction blisters in patients with psoriasis.

Concentrations of C3a and C4a anaphylatoxins in suction blister fluids were determined by radioimmunoassay in patients with psoriasis and normal controls. Comparison of anaphylatoxin levels between serum samples and blister contents in the same subjects revealed that the levels of both C3a and C4a anaphylatoxins were significantly higher in the former than the latter even in those raised on normal skin, suggesting that the classic complement pathway is activated during suction procedure. Therefore we cannot regard suction blister fluid to be simply representative of undisturbed interstitial tissue fluid as far as the complement system is concerned. There was no difference in anaphylatoxin levels between those from uninvolved skin of psoriatic patients and those from normal controls. However, significantly high anaphylatoxin levels were noted in fluids of suction blisters raised on lesional skin as compared with those produced on uninvolved skin in psoriatic patients.

Adolescent↗
PubMed1986

Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura.

Many chronic ITP patients have increased amounts of platelet-associated IgG, C3, C4 and C9, suggesting in vivo complement activation. In this study, we assessed the ability of various antiplatelet antibodies (APA) to activate and deposit complement proteins and to cause platelet lysis in vitro. Platelet sensitization with rabbit APA, anti-P1A1 antibody (one patient), anti-HLA antibody (two patients) and ITP autoantibodies (four patients) resulted in the deposition of C4 and C3 onto platelets in an amount proportional to the quantity of antibody-containing sera used to sensitize the platelets. Although C9 deposition onto platelets could not be quantitatively demonstrated on platelets sensitized with ITP serum, platelet lysis (51Cr release) was noted after incubation with each of three ITP sera and complement. When compared, ITP autoantibodies, anti-HLA and anti-P1A1 antibodies activated complement to a similar degree. We conclude that some autoantibodies in chronic ITP activate the classical complement pathway. The demonstration of in vitro platelet lysis by autoantibodies and complement suggests that in vivo platelet lysis may occur in some chronic ITP patients.

Autoantibodies↗
PubMed1979

Chemotaxigenesis by cell surface components of Staphylococcus aureus.

In an attempt to delineate the staphylococcal cell surface components of importance in chemotaxigenesis, we incubated intact Staphylococcus aureus H, crude cell walls, purified cell walls, peptidoglycan, teichoic acid, and cell membranes with human sera. The results reported indicate that both crude cell walls and purified cell walls, as well as peptidoglycan, were potent chemotaxigens. These particles led to the generation in normal human serum of a factor that was chemotactic for human polymorphonuclear leukocytes. Cell wall peptidoglycan and teichoic acid both appeared to play a role in chemotaxigenesis. Kinetic studies employing C2-deficient serum and immunoglobulin-deficient serum revealed that optimal chemotaxigenesis required the presence of an intact classical complement pathway, as well as antibody. Granulocyte aggregometry studies showed that significant levels of C5a were generated in normal serum and that this activated complement component appears to be a major chemotactic factor produced in serum upon interaction with staphylococcal cell wall components.

Cell Membrane↗
PubMed1992

Classical complement activation induced by pregnancy: implications for management of connective tissue diseases.

AIMS: To determine the effect of pregnancy on C4d concentrations and to assess whether C4d remains a useful disease activity marker in the management of connective tissue diseases during pregnancy. METHODS: Plasma C3, C4, and C4d concentrations were measured in 83 women at various stages of normal pregnancy and compared with those in 80 non-pregnant controls. RESULTS: C3 concentrations in the pregnant women were significantly raised (p = 0.0001) and the C4 concentrations were reduced (p = 0.0007), and accompanied by a significant increase in C4d (p = 0.0001). The C4d:C4 ratio was higher in the pregnant women (p = 0.0001). CONCLUSIONS: Pregnancy induces activation of the classical complement pathway. C4d concentrations cannot be used to monitor disease activity in patients with connective tissue diseases during pregnancy.

Adolescent↗
PubMed1989

Molecular basis of complement activation in ischemic myocardium: identification of specific molecules of mitochondrial origin that bind human C1q and fix complement.

Mitochondria may be a source of molecules that activate complement during ischemic injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. To identify specific molecules that activate the classical complement pathway, detergent lysates of canine cardiac mitochondria were fractionated by polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose paper (NCP). The NCP replicas of the gels were incubated with isolated C1q and fresh sera as a source of complement, washed briefly, and overlaid with sensitized sheep erythrocytes (RBC) in agarose. A cluster of four to six molecules between 45 and 53 kDa as well as four others, 34, 30, 26, and 23 kDa, consumed complement thereby preventing complement-mediated lysis of sensitized sheep RBC in the agarose overlay. Additional molecules reactive with C1 were identified by their ability to bind isolated human C1q and to serve as assembly sites for later acting complement components. Sites of localization of complement were demonstrated by incubating NCP replicas of fractionated mitochondria with antisera specific for C1q, C3, C5, and C9, followed by peroxidase-conjugated anti-immunoglobulin and substrate. A total of 12 C1q binding molecules ranging in size from 67 kDa to 23 kDa, which can fix later acting complement components, were identified. At least two of these reacted with antisera prepared against canine cardiac lymph collected in the first 3-4 hours after a 45-minute coronary artery occlusion. These studies present direct evidence that specific molecules, released from subcellular fractions of myocardial cells rich in mitochondria, can activate the complement cascade.

Animals↗
PubMed1986

Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor-deficient patients.

The first component of complement (C1) inhibitor plays a critical role in the regulation of the classical complement pathway and the contact system, and the deficiency of C1 inhibitor protein or function is associated with recurrent angioedema. In this study we evaluated the size of the C1 inhibitor antigens present in the plasmas of C1 inhibitor-deficient patients. We found that the C1 inhibitor in the plasmas existed in three forms: high molecular weight forms in complex with proteases, native 110-kD C1 inhibitor, and a modified inactive 94-kD form. The proportion of the total C1 inhibitor in the 94-kD form was 28% in nine hereditary angioedema patients, 92% in five acquired C1 inhibitor-deficiency patients, and 1.2% in five normal controls. In vitro activation of normal plasma with kaolin, but not heat-aggregated gamma-globulin generated 94-kD C1 inhibitor from 110-kD C1 inhibitor. Neither kaolin activation nor heat-aggregated gamma-globulin activation generated 94-kD C1 inhibitor in Hageman factor-deficient plasma. These results suggest that 94-kD C1 inhibitor is generated in vitro by activation of the contact system. The in vivo mechanism of 94-kD C1 inhibitor generation in C1 inhibitor-deficient patients is not known.

Angioedema↗
PubMed1992

Induction of contact sensitivity by cell-associated immunocomplexes requires activation of the early complement components.

Lymph node cells collected from CBA/J mice 4 days after painting the skin with picryl chloride behave like antigen presenting cells and induce contact sensitivity when injected into naive recipient mice. The immunizing capacity of these '4-day' cells is due to T cells which carry on their membrane hapten-IgM immunocomplexes. Incubation of the cells with complement from mouse strains that express high C4 serum levels (C4H), abolishes their immunizing capacity. This effect is related to the activation of the early components of the classical complement pathway, as supported by experiments using C3 and C4-depleted or C5 and C6-genetically deficient mouse sera. The detection of different amounts of C3b and C4b on the surface of 4-day T cells after incubation with C4L and C4H sera supports the possibility that membrane bound activated complement components could modify the immunizing capacity of these cells. Results herein reported suggest that membrane-bound C3b and C4b are not per se inhibitory but interfere with the residual complement activating capacity of 4-day T cells. The role of complement activation by 4-day T cells is pivotal as complement depletion of recipient mice by cobra venom factor (CVF) inhibits the immunizing capacity of untreated 4-day T cells, while 4-day T cells treated with complement in vitro and injected together with C4a anaphylatoxin are able to immunize recipient mice.

Animals↗
PubMed1990

[Complement activation by killed polyvalent bacterial vaccine on the pleurodesis].

Broncasma Berna (B.B.) prepared from killed polyvalent bacterial vaccine is used as a pleural irritant for pleurodesis on patients with spontaneous pneumothorax. The effects of B.B. on the complement system in vivo after injections into pleural spaces were investigated. Five patients with spontaneous pneumothorax underwent intrapleural injections of B.B. Increased body temperatures with positive C-reactive proteins and increased polymorphonuclear leukocyte counts in peripheral blood were observed in all patients. Serum levels of complement components (Clq, Cls, C4, C3 and B), hemolytic activity of complement (CH50) and plasma levels of C3a were gradually elevated after intrapleural injections of B.B. Analysis of pleural fluids from 4 patients showed that levels of C3a and C5a were increased compared with plasma levels, and an accumulation of polymorphonuclear leukocytes were observed. These findings suggest that intrapleural injections of B.B. activate the complement system and cause inflammatory reactions accompanied by an increase of C3a and C5a and an accumulation of polymorphonuclear leukocytes. Such inflammatory reactions might inversely induce the synthesis of complement components and promote the migration of polymorphonuclear leukocytes from bone marrow. The deposit of fibrin during these inflammatory processes might contribute to the development of pleurodesis. These inflammatory reactions might be mediated through the activation of the classical complement pathway by B.B.

Adult↗