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PubMed1993

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07.

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.

Adenosine Diphosphate↗
PubMed1989

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.

Androgen-Binding Protein↗
PubMed1998

Human heart generates complement proteins that are upregulated and activated after myocardial infarction.

In human heart, we detected mRNAs and proteins for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 with the use of reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical techniques. We found an upregulation of both mRNAs and proteins in areas of recent and old myocardial infarctions. In both situations, the classical complement pathway was activated, with C4d, C3d, and the membrane attack complex (C5b-9) being deposited on damaged cardiac myocytes. These activated complement components were also identified on Western blots of infarcted tissue. Complement mRNAs in infarcted heart tissue were higher than those in liver, and liver complement mRNAs were not upregulated in cases with infarcted hearts. Our results establish that (1) complement proteins are endogenously produced by human heart; (2) the classical complement pathway is fully activated after myocardial infarction; (3) complement activation is directly involved in myocardial damage after ischemic insults; and (4) damage from complement activation may be chronically sustained. These data suggest that inhibition of the complement system should be effective in treating myocardial infarction.

Adult↗
PubMed1996

Complement regulatory molecules on human myelin and glial cells: differential expression affects the deposition of activated complement proteins.

The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four-to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.

Antigens, CD↗
PubMedDate not supplied

Participation of immunoglobulin and the alternative complement pathway in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron.

Studies were conducted to determine the requirements for immunoglobulin and complement in opsonization of Bacteroides fragilis and Bacteroides thetaiotaomicron. The ability of human sera depleted of immunoglobulin or of components of complement to promote the phagocytosis and intracellular killing of the two strains of Bacteroides by human leukocytes was measured in vitro under anaerobic conditions. Neither hypogammaglobulinemic sera nor pooled normal human serum that was heated at 56 C for 30 min supported phagocytosis and killing of the two strains of Bacteroides. Neither sera depleted of terminal complement components by treatment with inulin or cobra venom factor nor human serum deficient in C8 supported phagocytosis of the tested strains. In addition, pooled normal human serum depleted of C3, factor B, or factor D did not support phagocytosis of either strain. Dose-dependent restoration of the opsonic activity of factor B-depleted serum was accomplished by purified human factor B but not by human C2. The results indicate that immunoglobulin and components of the alternative comed in this study.

Animals↗
PubMed1997

The complement membrane attack complex and the bystander effect in cerebral vasospasm.

Activation of complement results in formation of membrane attack complexes (MACs) that can insert themselves either into cells that initiate complement activation or into nearby ("innocent bystander") cells. The MACs form large-conductance, nonspecific ion channels that can cause lytic or sublytic cell damage. The authors used a highly sensitive patch clamp technique to assess the contribution of the bystander effect to the pathophysiology of cerebral vasospasm. They compared the effect of complement activation by autologous aged versus fresh erythrocytes on the membrane conductance of freshly isolated rat cerebral artery smooth-muscle cells. In the presence of autologous serum aged, but not fresh, erythrocytes caused a large increase in membrane conductance, an effect that was prevented by heat-inactivating the serum. Ethyleneglycol tetraacetic acid in the presence of Mg++ attenuated the effect, indicating that complement activation was taking place via the classic pathway. The effect was reproduced by zymosan-activated autologous serum, suggesting that such changes in conductance could result from insertion of MACs secondary to a bystander effect. Both C8- and C9-depleted heterologous sera produced minimal effects that were converted to full effect by addition of the missing complement component. Superoxide dismutase plus catalase did not attenuate the conductance changes produced by autologous serum plus aged erythrocytes. Autologous serum plus aged erythrocyte membrane ghosts that were free of lysate caused a typical increase in conductance. This study demonstrates that complement activation by aged erythrocytes can result in MAC insertion into innocent bystander smooth-muscle cell membranes and that this mechanism, heretofore undescribed, may contribute to development of vasospasm after subarachnoid hemorrhage.

Animals↗
PubMed1990

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

Antibodies, Monoclonal↗
PubMed1991

Collagenase of Clostridium perfringens type A: degradation of human complement component C1q.

The semipurified collagenases from Clostridium perfringens type A strains 2-Cli and ATCC 13124, both characterized by molecular weights of 79.4 kilodaltons, partially degraded purified human complement (C) component C1q. The following purified human serum proteins were refractory: C components C3, C4, C5, C6, C7, C8, and C9; immunoglobulin (Ig)A (from colostrum), IgG, and IgM; alpha 2-macroglobulin, haptoglobin, and C-reactive protein.

Blotting, Western↗
PubMed1988

Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata.

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.

Animals↗
PubMed1979

Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.

The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.

Anthracenes↗
PubMed1994

Sublytic complement attack exposes C-reactive protein binding sites on cell membranes.

C-reactive protein (CRP) is an acute phase serum protein synthesized by the liver. CRP has been localized to acute inflammatory sites and has been postulated to facilitate the removal of damaged cells. CRP binds to a number of ligands that may be present in inflammatory sites, and the extent to which individual ligands are involved in its binding to tissue sites is unknown. Complement activation is important in the tissue damage in many inflammatory conditions causing cell membrane damage and recruitment of inflammatory cells. This paper describes the binding of CRP to complement-damaged cell membranes. Raji cells activate the alternative complement pathway resulting in the deposition of C3b and membrane attack complexes (MAC) on the cell membrane. However, Raji cells are relatively resistant to killing by human complement. Treatment of Raji cells with human serum led to calcium-dependent phosphocholine-inhibitable CRP binding. CRP binding was eliminated by depletion of C3, C5, or C8 and reduced by depletion of C9 from serum. CRP binding preceded cell death and co-localized with MAC on cell membranes. CRP binding to complement-treated liposomes required phosphatidylcholine in addition to the MAC indicating that membrane phospholipids rather than the MAC proteins provide the binding sites for CRP. However, for both liposomes and Raji cells disruption of the lipid bilayer by complement attack was required for CRP binding to occur. These results support the hypothesis that CRP binding at sites of inflammation may be mediated by exposed phospholipids on damaged cell membranes.

Binding Sites↗
PubMed1987

Simultaneous phenotyping of genetic markers for paternity testing.

Time-and cost-saving methods for paternity testing are described. Seventeen genetic systems were divided into six groups: (1) transferrin (Tf), factor B (Bf), and phosphoglucomutase 1 (PGM1); (2) group-specific component (Gc) or alpha 1-antitrypsin (PI) and alpha 2HS-glycoprotein (HSGA); (3) complement components C6 and C7, factor 13B (F13B), and plasminogen (PLG); (4) haptoglobin (Hp), C8 alpha-gamma chain (C81), and factor I (IF); (5) red cell acid phosphatase (ACP), esterase D (ESD), and glutamic-pyruvic transaminase (GPT); and (6) 6-phosphogluconate dehydrogenase (PGD) and glyoxalase I (GLO). Each group of systems was typed simultaneously by electrophoresis or isoelectric focusing (IEF) followed by staining or immunoblotting. These methods are very practical because they afford a considerable saving of time, work and expense, and facilitate semipermanent preservation of electrophoretic patterns.

Blood Grouping and Crossmatching↗
PubMed1981

Isolation and characterization of a low molecular weight complement inhibitor present in normal human serum.

Normal human serum and urine were found to contain a low molecular weight complement inhibitor (LMWI). LMWI was separated from serum by dialysis in membrane tubing or through an Amicon PM10, and then concentrated on an Amicon UM05 membrane. On Bio-Gel P-2 filtration, LMWI was eluted just after the column calibration marker, stachyose hydrate (6666 . 6 daltons), and was estimated to be 500 daltons. Both pathways of complement activation were susceptible to modulation by LMWI. Addition of LMWI reduced the haemolysis of sheep erythrocytes sensitized with antibody, rabbit erythrocytes and guinea-pig erythrocytes bearing human C3 and C4. Formation of EAC142 from EAC14 and guinea-pig C2 was blocked, indicating a failure to generate the classical pathway C3 convertase: however, the lysis of preformed EAC142 was not suppressed. Conversion of factor B and C3 did not occur when LMWI was present during zymosan activation of serum. This indicates that the inhibitor either prevented, or acted at a step prior to, the cleavage of factor B by factor D. LMWI did not prevent formation of erythrocyte C567 intermediates nor their subsequent lysis by C8 and C9. Thus, serum contains a 500-dalton inhibitor which modulates the activities of both complement pathways at an early step in each of the activation sequences. LMWI may serve as a regulator of the inflammatory process by suppressing C3 convertase formation and generation of complement-derived, biologically reactive molecules.

Blood↗
PubMed1987

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

Animals↗
PubMedDate not supplied

Evidence suggesting the occurrence of C3-independent intravascular immune hemolysis. Reactive hemolysis in vivo.

The authors present circumstantial evidence for the involvement of reactive hemolysis, i.e., C3-independent binding of the cytolytic C5b-9 complement complex to bystander red cells (RBC), in a case of intravascular immune hemolysis. Fresh serum obtained from a 6-year-old patient during the hemolytic episode, but not obtained thereafter, induced C5b-9-dependent hemolysis of human RBCs but the indirect C3 antiglobulin test remained negative. Particles (presumably RBC ghosts) isolated from the patient's plasma anticoagulated with EDTA at the peak of hemolysis were coated with C5b-9 complexes, whereas the direct antiglobulin test was strongly positive for IgA, only weakly positive for IgG, and negative for C3. Moreover, neither the autoantibodies isolated by elution (IgG plus IgA), nor free serum autoantibodies (IgA alone) activated complement in vitro. Additionally, serum samples collected later during the 12-month period of observation contained normal levels of C3, C4, C8, and C9, but markedly reduced levels of C7. These serums all produced strong reactive lysis in agarose plates, but not in test tubes. These results appear compatible with the working hypothesis that the intravascular hemolytic episode in this patient might have arisen through a local initiation of complement activation with subsequent C3-independent binding of C5b-9 to and hemolysis of bystander RBCs.

Anemia, Hemolytic, Autoimmune↗
PubMed1999

Complement membrane attack complex and protectin (CD59) in liver allografts during acute rejection.

BACKGROUND/AIMS: The complement system is important in the rejection of xenografts, but very little is known about its activation in the rejection of allografts. Complement lysis is induced by the membrane attack complex (MAC), an aggregate of C5b, C6, C7, C8 and C9 molecules. The main defender against MAC is the CD59 molecule, also called protectin. In this study, the aim was to analyze the possible deposition of MAC and the fate of CD59 on distinct cell populations during liver allograft rejection. METHODS: Liver allografts were monitored by frequent fine-needle aspiration biopsies (FNAB) to demonstrate the immunoactivation of rejection. To examine MAC and CD59 in the FNAB, in relation to the activation markers of rejection, IL2-receptor, MHC class II and ICAM-1 expression, specific monoclonal antibodies and immunoperoxidase staining were used. RESULTS: Ten out of 21 consecutive liver transplants underwent a histologically confirmed episode of reversible acute rejection. In the FNAB, a significant increase of the activation markers IL2-receptor, class II and ICAM-1 correlated with the peak of inflammation during the episode. In association with inflammation, a significant deposition of MAC was recorded in neutrophils and lymphocytes infiltrating the graft and in the parenchymal cells. MAC deposition subsided together with the inflammation. A significant decrease in CD59 expression was seen in neutrophils during rejection, but CD59 expression on other inflammatory cells and hepatic tissue cells varied greatly. CONCLUSIONS: Complement activation was seen in association with acute rejection of liver allografts and it led to MAC assembly on leukocytes and tissue cells. A decrease in CD59 expression was less clear-cut, but it may predispose the cells to complement-mediated elimination.

Acute Disease↗
PubMed1991

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo.

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.

Animals↗