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PubMedDate not supplied

Underlying complement deficiency in patients with disseminated gonococcal infection.

The complement system was evaluated in 22 individuals with disseminated gonococcal infection. Three of the 22 patients exhibited a total serum complement activity that was greater than 2 SD below the normal mean. Of these three, one had a complete deficiency of C1r, a second patient had pre-existing systemic lupus erythematosus with low levels of C4, and the third had a C8 concentration that was 60% of normal. We conclude that the prevalence of inherited or acquired complement deficiency among patients with disseminated gonococcal infection exceeds that among the general population and is an important host factor predisposing to systemic infection with Neisseria gonorrhoeae.

Adolescent↗
PubMed1993

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines.

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1 beta or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5-107.6 ng/10(6) cells/24h) in different MC lines. IL-1 beta up-regulated the synthesis by 2.4-4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1.76 ng/h/10(6) cells under IL-1 stimulation versus basal rate of 0.37 ng/h/10(6) cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.

Biotechnology↗
PubMed1987

Formation of the membrane attack complex of complement (MAC) on erythrocytes from monocyte-produced terminal complement components.

By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.

Animals↗
PubMed1981

The membrane attack mechanism of complement: photolabeling reveals insertion of terminal proteins into target membrane.

We have utilized a membrane-restricted, photoactivable glycolipid probe to investigate the protein-lipid interactions involved in complement (C) mediated lysis of a target membrane. The purified C proteins C5b-6, C7, C8, and C9 were added to artificial membrane vesicles containing the 14C-labeled photoreactive probe anchored in the outer monolayer of the membrane, and 6-carboxyfluorescein trapped in the lumen as an indicator for effective lysis. Irradiation of the membrane samples at different stages of functional complex assembly resulted in labeling of each of the 5 terminal C proteins, indicating that all 5 proteins become inserted into the hydrophobic milieu of the membrane during some stage of complex assembly. However, at the final stage of complex assembly, only C9 appeared to be labeled. Because we can demonstrate that the photoreactive probe has no strong affinity for C9 over the other terminal components (C5b-C8), the extensive change in labeling specificity during assembly is evidence for substantial changes in protein-lipid and possibly protein-protein interactions during formation of the C lesion.

Cell Membrane↗
PubMed1989

Inherited deficiencies of the late-acting complement components other than C9 found among healthy blood donors.

Among sera from 145,640 healthy blood donors in Osaka, 16 were found to have abnormalities in late-acting complement components other than C9. It was found that of these 16 sera, 2 were deficient in C5, 4 in C6, 6 in C7 and 4 in C8 alpha-gamma-subunit. The incidence of deficiency of each component among the Osaka blood donors was calculated as follows: C5 deficiency, 0.0014%; C6 deficiency, 0.0027%; C7 deficiency, 0.0041%; C8 alpha-gamma-subunit deficiency, 0.0027%. We confirmed that 13 donors were healthy and 12 had no past history related to a complement component deficiency. From these results, not only C9 deficiency but also deficiencies of the other late-acting complement components were found among the healthy blood donors, but no early-acting component deficiencies were noted.

Blood Donors↗
PubMedDate not supplied

The role of complement in anti-bacterial defence.

The complement system consists of several proteins present in human serum interacting among themselves and with the other compounds of the immune system in the host defence process. In particular, late complement component (C5, C6, C7, and C8) deficiencies (LCCD) are closely associated with Neisseria, mainly meningitidis, infections. The aim of our study was to verify this association in an Italian population by analyzing the complement profile in survivors of meningococcal meningitis. Ten out of the 59 (17%) subjects studied had homozygous LCCD (6 C8, 3 C7 and 1 C6). The meningococcal C strain was the most widely diffused (68%) and had infected all homozygous LCCD subjects. In addition meningococcal serogroup C seemed to be the least immunogenic when compared to serogroups A and B. These data confirm the close association between homozygous LCCD and meningococcal infections from common serogroups (A, B and C) in the Italian population. Anti-meningococcal vaccination is usually recommended for LCCD subjects because it increases, both quantitatively and qualitatively, the antibody component of anti-meningococcal immune defence. We therefore analyzed the levels of anti-polysaccharide (PS) A and PSC antibodies in the members of 4 families including normal subjects and subjects with homozygous and heterozygous C7, C8 or factor H defects, before and after vaccination with only PSA+C. Surprisingly, we found the highest levels of antibodies before vaccination in homozygous subjects, followed by heterozygous and normal controls, whereas, after vaccination, homozygous subjects showed the lowest increase of specific antibodies, indicating their relative incapacity to respond to meningococcal PS alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1989

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.

Cell Fractionation↗
PubMed1984

Membrane attack by complement.

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.

Animals↗
PubMed1987

Paroxysmal nocturnal hemoglobinuria type III. Lack of an erythrocyte membrane protein restricting the lysis by C5b-9.

The complement-mediated lysis is inefficient when complement and target cells are homologous with regard to the species. In erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), the species restriction is lost: PNH-erythrocytes (PNH-E) are susceptible to lysis by human complement. In human erythrocytes (huE) the species restriction is ascribed to an integral membrane protein, designated C8-binding protein (C8bp). In the present study, we tested membranes of PNH-E type III for the presence of C8bp. A protein with C8-binding capacity could not be detected. C8bp, which was isolated from the membrane of huE, inhibited the lysis of PNH-E by C5b-9 as well as the C9 polymerization. Thus, addition of C8bp restored the species restriction in PNH-E. In conclusion, we propose that lack of C8bp might represent the defect in PNH-E type III membranes, which is responsible for their enhanced lytic susceptibility towards lysis by the late complement components.

Complement C8↗
PubMed2000

Free radicals upregulate complement expression in rabbit isolated heart.

Both free radicals and complement activation can injure tissue. Our study determined whether free radicals alter complement production by the myocardium. Isolated hearts from New Zealand White rabbits were perfused on a Langendorff apparatus and exposed to xanthine (X; 100 microM) plus xanthine oxidase (XO; 8 mU/ml) (X/XO). The free radical-generating system significantly (P < 0.05) increased C1q and also increased C1r, C3, C8, and C9 transcription compared with controls. Immunohistological examination revealed augmented membrane attack complex deposition on X/XO-treated tissue. X/XO-treated hearts also exhibited significant (P < 0.05) increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in left-ventricular developed pressure. N-(2-mercaptopropionyl)-glycine (3 mM), in conjunction with the superoxide dismutase mimetic SC-52608 (100 microM), significantly (P < 0.05) reduced the upregulation of C1q, C1r, C3, C8, and C9 mRNA expression elicited by X/XO. The antioxidants also ameliorated the deterioration in function caused by X/XO. Local complement activation may represent a mechanism by which free radicals mediate tissue injury.

Animals↗
PubMed1978

Electron spin resonance studies on interaction of complement proteins with erythrocyte membranes.

Sheep erythrocytes have been spin labeled with 5-, 12-, and 16-nitroxystearic acid in order to investigate complement-induced changes in the physical state of the lipid bilayer. Formation of osmotic lesions in the membrane causes an increase in the fluidity of the membrane which overcomes the decrease in membrane fluidity caused by the interaction of the complement proteins. A decrease in membrane fluidity is observed only when complement-lysed membranes are resealed or when complement proteins react with isosmolar ghosts that do not undergo osmotic lysis. The decrease in bulk fluidity of the membrane is first observed when C8 binds to the membranes bearing C5b67 and is enhanced upon the subsequent binding of C9. The decrease in membrane fluidity shown by the electron spin resonance spectra of spin-labeled fatty acids suggests that certain of the complement proteins penetrate the membrane and interact with hydrophobic regions of the lipid bilayer.

Animals↗
PubMed1990

[Paroxysmal nocturnal hemoglobinuria].

Paroxysmal nocturnal hemoglobinuria, first described in the late 19th century, is an acquired disorder characterized by hemoglobinemia and hemoglobinuria. The major clinical manifestation of PNH is chronic intravascular hemolysis of various severity. Patients-mostly young adults - may also present with episodes of abdominal or back pain. Common cause of death is thrombosis especially of the hepatic veins. Granulocytopenia and thrombocytopenia may be the initial manifestation of PNH, indicating that the disorder is a primary bone-marrow disease, affecting not only the erythrocytes but also other peripheral blood cells and the haematopoietic stem cell. The course of the disease is variable. Partial complete recovery was described, but also fatal thrombosis. The major phenotypic expression of PNH is an increased susceptibility of the erythrocytes to the lytic action of complement in vitro. The enhanced complement susceptibility is most probably due to membrane defects: two membrane proteins regulating the complement cascade in PNH cells were missing, the decay-accelerating factor, DAF, inhibiting the activation of the lytic complement complex and the C8 binding protein, C8bp, which interferes with the lytic process. Aside from the lack of the complement regulators also other membrane defects have been described (e.g. of acetylcholinesterase or alkaline phosphatase). The proteins as well as DAF and C8bp are linked to the cell membrane via a phosphatidylinositol (PI) anchor, leading to the speculation that the disease results from a deficiency in the post-translational PI anchoring mechanism. The diagnosis of PNH is based on the Hamtest, but will be extended to the quantitation of the above described membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement System Proteins↗
PubMed1995

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Antigen-Antibody Complex↗
PubMed1988

Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane.

Recently, a protein isolated from the membrane of human E, the so-called C8 binding protein (C8bp), has been described. C8bp is characterized as a 65-kDa protein that binds to C8 and inhibits the C5b-9-mediated lysis in a homologous system. In the present study, membranes of peripheral blood cells were tested for the presence of C8bp by SDS-PAGE and immunoblotting. In all cells a protein band reacting with anti-C8bp was seen, the Mr, however, was only about 50 kDa. To further analyze the 50-kDa protein, we isolated the protein by phenol-water extraction and isoelectric focusing from papain-treated platelets. The isolated protein behaved similar to the E-derived C8bp: it inhibited the lysis of model target cells by C5b-9. To examine the function of C8bp in platelets, we tested platelets from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). These platelets were deficient in C8bp, being in accordance with their higher lytic susceptibility in vitro. In response to sublytic C5b-9 doses, the PNH platelets released considerably more serotonin and thromboxane B2 than normal platelets. By addition of purified C8bp, the thromboxane B2 release was suppressed, indicating that C8bp not only restricts the lytic complement attack, but also regulates the C5b-9-mediated stimulation of target cells. Thus, lack of C8bp might not only result in enhanced hemolysis, but also in enhanced stimulation of platelets, which in turn might contribute to the thrombotic complications seen in some PNH-type III patients.

Blood Platelets↗
PubMed1975

Complement fixation by rheumatoid factor.

The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum.

Animals↗
PubMed1988

Complement fixation by pemphigus antibody. V. Assembly of the membrane attack complex on cultured human keratinocytes.

Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.

Autoantigens↗