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PubMed1994

Interaction between serum amyloid P component and C4b-binding protein associated with inhibition of factor I-mediated C4b degradation.

Serum amyloid P component (SAP) is a member of the pentraxin protein family. Although present in all types of amyloid deposits, it is also a normal constituent of blood and extravascular tissues. In blood, SAP forms a calcium-dependent, noncovalent complex with C4b-binding protein (C4BP). C4BP regulates the classical complement pathway as it binds C4b and functions as cofactor in its degradation by factor I. Although SAP and C4b bind to distinct sites on C4BP, it is not known whether the SAP-C4BP interaction affects the function of C4BP. We report that in a fluid phase system, SAP inhibited degradation of C4(H2O) (which is C4b-like) in a dose-dependent manner. Phosphorylethanolamine was found to alleviate the inhibitory effect of SAP on C4(H2O) degradation. Because this compound is known to inhibit the SAP-C4BP interaction, this indicated direct binding of SAP to C4BP to be required for inhibition of C4(H2O) degradation. Even though C4BP, C4(H2O), and SAP form a multimolecular complex in fluid phase, SAP was found to inhibit binding of C4BP to immobilized C4(H2O). The inhibitory effect was calcium dependent and alleviated by phosphorylethanolamine. Heparin, which is known to inhibit the interaction between SAP and C4BP, was also found to counteract the inhibitory effect of SAP on C4BP binding to C4(H2O). However, the effect of heparin was biphasic because high concentrations of heparin directly inhibited binding of C4(H2O) to C4BP. The inhibition of C4BP function by SAP suggests that SAP may be involved in regulation of the classical complement pathway C3 convertase.

Calcium↗
PubMed1991

Influence of C4 null alleles on C4 activation in systemic lupus erythematosus.

Deficiencies of early components of the classical complement pathway are known to be associated with systemic lupus erythematosus (SLE). C4 null alleles, C4A Q0 and C4B Q0, are prime candidates for the major histocompatibility complex associated factor which determines susceptibility to SLE. There is poor correlation, however, between the presence of low concentrations of C4 and possession of C4 null alleles, and thus the basis of the association between C4A Q0, C4B Q0 and SLE remains obscure. The possibility that activation of C4 may be related to the possession of C4 null alleles was examined. C4 phenotypes were investigated, and C4 concentration and activation were estimated in patients with SLE. C4 activation was determined by measuring the concentration of C4d--a split product of C4. Twenty five of 35 patients had C4 phenotypes which include null alleles. No association between low C4 concentrations and C4 null alleles was found, but a significant association between low C4d concentrations and C4 phenotypes including null alleles, particularly those with C4A Q0, was noted. No correlation between concentrations of C4 and C4d was found. These results show an influence of C4 null alleles on the activation of the C4 molecule, which is independent of the concentration of C4. The possession of silent genes coding for C4 null alleles might predispose to SLE by conditioning poor C4 activation, a critical event for the clearance of immune complexes mediated by the classical complement pathway.

Alleles↗
PubMed1986

Emergence of bactericidal and opsonizing antibody to Vibrio vulnificus following bacterial infection.

Virulent isolates of Vibrio vulnificus resist the bactericidal and opsonizing effects of normal human serum, in contrast to environmental isolates, which are highly serum susceptible. Immune responses to bacteremic V. vulnificus infections in human subjects have not been characterized. Serum from a patient who survived sepsis caused by V. vulnificus had substantial bactericidal and opsonizing immunoglobulin G (IgG) for his own bloodstream isolate. Killing was mediated by the classical complement pathway, whereas opsonization was effected by either the classical or the alternative pathway. IgG that reacted strongly with 55-, 58-, and 68-kilodalton outer membrane proteins was present in the patient's convalescent-phase serum but was absent from normal human serum. These findings suggest that humoral immunity to V. vulnificus, mediated by bactericidal and opsonizing antibody, emerges during infection and may be due, in part, to IgG directed against identifiable outer membrane proteins.

Antibodies, Bacterial↗
PubMed1985

The role of C1, C1-inactivator and C4 in modulating immune precipitation.

To clarify the mechanism of inhibition of immune precipitation by early components of the classical pathway of complement, aggregation of 125I-BSA-rabbit-anti-BSA antibody complexes was performed in the presence of purified C1, C1-inactivator (C1-In) and C4. C1 delayed the rate of immune precipitation in a concentration dependent manner. This phenomenon was not influenced by the presence of 0.3 mM p-nitrophenyl-p-guanidinobenzoate (NPGB) which inhibits C1 activation. The antiaggregational effect of C1 was reversed by 10 mM EDTA and by C1-In at a C1-In/C1 molar ratio of greater than or equal to 4/1. C1-In was not effective when the reaction was performed in the presence of NPGB. Thus, although the inhibitory effect of C1 on immune precipitation was not dependent upon C1 activation, the formation of C1 was required to observe the effect of C1-In. The addition of C4 to C1 did not modify the slow aggregation of complexes, even when a limiting concentration of C1 was used. C1-In and EDTA were both able to cause similar rapid precipitation of complexes prepared in the presence of C1 and C4, demonstrating that C4 did not play a significant role in delaying the precipitation reaction. However, soluble complexes prepared in the presence of C1 and C4 were specifically precipitated by the addition of excess anti-C4 antibody, attesting to the binding of C4 to immune complexes. These observations suggest that the processing of immune complexes in vivo may not be similar in different classical pathway complement deficiency states.

Animals↗
PubMed1994

Complement activation in platelet concentrates is surface-dependent and modulated by the platelets.

Activation of platelets during storage may contribute to the loss of function and viability known as the platelet storage lesion. We investigated the role of complement activation as a possible mediator of the platelet storage lesion. We studied platelet bags stored up to 5 days under standard blood bank conditions and monitored the generation of several complement activation fragments from both the classical and alternative pathways. To assess the role of noncellular artificial surfaces in the activation of complement, parallel bags were prepared containing platelet-poor plasma. The levels of C4d and C3a increased steadily over time in storage, as did the level of the inactivated membrane attack complex SC5b-9. Generation of C4d, C5a, and SC5b-9 was greater in the absence of platelets than when platelets were present in the container. C5a levels in both groups were low and remained so during storage, suggesting that the C5a generated became surface associated. Using flow cytometry we detected C3 and C3a, but not C9, on the platelet surface. The percentage of C3-positive platelets peaked at the third day of storage; by day five platelet-associated C3 had declined. Decay accelerating factor expression on the platelet surface increased with time in storage and in parallel with CD63 expression. Based on the C4d levels, complement activation proceeded via the classical pathway; minimal generation of the alternative pathway activation fragment. Bb was seen in either the presence or absence of platelets. As an indirect measure of the activation of C1, the functional level of C1 esterase inhibitor (C1INH) was determined. C1INH levels declined over time in storage in bags containing only plasma; however, in the presence of platelets, the levels remained constant presumably because of release of C1INH from the alpha-granules of activated platelets.

Blood Platelets↗
PubMed1995

Cell surface molecules related to factor J in human lymphoid cells and cell lines.

Factor J (FJ) is a cationic glycoprotein that is able to inhibit in vitro both the classical and alternative pathways of complement. FJ was purified to homogeneity from human urine by sequential chromatographic steps. To examine the expression of FJ in human cells we obtained mAbs against urine-purified FJ. Preliminary studies by immunocytochemistry revealed that one of the anti-FJ mAbs recognized cell surface components of certain cell lines, such as K562 and U937 cells, so we have focused subsequently on the detection of these homologue membrane-bound FJ Ags (FJ-h Ags) in cell lines of lymphoid (Ramos and Jurkat) and mieloyd (U937 and K562) origin, as well as in peripheral blood cells. The flow cytometry analysis of the examined cell lines revealed partial staining ranging from 10% (U937) to 29% (K562) positive cells. Flow cytometry of peripheral blood cells showed a positive staining in a small but consistent population of lymphocytes (mean = 11%, n = 17) but none at all on monocytes, granulocytes, erythrocytes, or platelets. Double Ab immunostaining of lymphocytes showed that the FJ-h positive population included mainly B lymphocytes (a mean of 63% CD19+ were FJ-h positive). When we analyzed peripheral blood lymphocytes from a patient with chronic lymphocytic leukemia B (95% CD19+/CD5+), the majority of these (55%) bore FJ-h on their surface. Acid strip of these cells did not abrogate the surface staining, which supports the finding that the Ag is tightly bound to the membrane. Immunoprecipitation from U937 cell lysates showed a single 65 kDa band under reducing conditions. FJ-h Ags purified from K562 and U937 cells displayed inhibitory activity in the functional EAC14 assay for the classical complement pathway, as did urine FJ, and they were recognized immunochemically by five different (one polyclonal and four monoclonal) anti-FJ Abs. In conclusion, FJ-homologues are present in the membranes of several human cell lines that show functional and antigenic characteristics similar to soluble urine FJ. They are also found in a small subset of peripheral blood lymphocytes, mainly B cells. The structural relationship between both soluble urine FJ and these membrane-bound FJ-h remains to be established.

Animals↗
PubMed1998

Complement activation following reoxygenation of hypoxic human endothelial cells: role of intracellular reactive oxygen species, NF-kappaB and new protein synthesis.

Complement plays an important role in ischemia-reperfusion injury. We recently demonstrated that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) activated the classical complement pathway and augmented iC3b deposition. In the present study, we investigated the potential role of oxygen-derived free radicals, NF-kappaB and new protein synthesis in this model. HUVECs subjected to 12 or 24 h hypoxic stress (1% O2) and then reoxygenated (0.5, 1, 2 or 3 h; 21% O2) in 30% human serum activated complement and deposited iC3b. Addition of hydrogen peroxide (H2O2; 1-100 micromol/l) to normoxic HUVECs increased iC3b deposition in a concentration-dependent manner. H2O2 (10 micromol/l), a concentration that did not significantly increase iC3b deposition on normoxic HUVECs, augmented iC3b deposition on hypoxic/reoxygenated HUVECs. We observed a significant increase in intracellular H2O2 and hydroxyl radical (OH.) production in hypoxic/reoxygenated HUVECs using dihydrorhodamine 123. Further, treatment of HUVECs with dimethylthiourea (DMTU, 1-100 micromol/l), deferoxamine (DEF, 1-100 micromol/l), or oxypurinol (10 micromol/l), but not superoxide dismutase (SOD, 500 U/ml), catalase (300 U/ml) or iron-loaded DEF, attenuated iC3b deposition following hypoxia/reoxygenation in a concentration-dependent manner. Western analysis demonstrated hypoxia-induced nuclear NF-kappaB translocation that increased with reoxygenation. Inhibition of new protein synthesis (i.e. cycloheximide) or inhibition of NF-kappaB (ALLN or SN-50) also significantly decreased iC3b deposition on hypoxic/reoxygenated HUVECs. We conclude that (1) hypoxic/reoxygenated HUVECs generate H2O2 and OH.; (2) treatment of HUVECs with cell permeable reactive oxygen species inhibitors/scavengers (i.e. DEF, DMTU, oxypurinol) but not large molecular weight inhibitors (i.e. catalase or SOD) significantly reduces iC3b deposition and (3) inhibition of new protein synthesis or NF-kappaB activation attenuates iC3b deposition. These data suggest that iC3b deposition on the vascular endothelium may be regulated by intracellular oxygen-derived free radical-induced activation of NF-kappaB, new protein synthesis and activation of the classical complement pathway during ischemia/reperfusion.

Cell Hypoxia↗
PubMed1979

Cryptic peptidoglycan and the antiphagocytic effect of the Staphylococcus aureus capsule: model for the antiphagocytic effect of bacterial cell surface polymers.

The antiphagocytic effect of the Staphylococcus aureus capsule is known to be related to its ability to interfere with opsonization by normal human serum. In this study, evidence is presented with isolated cell surface components which indicates that the capsule hinders opsonization by masking cell wall peptidoglycan. In contrast to intact, encapsulated S. aureus M cells, peptidoglycan particles isolated from the organism were efficiently opsonized by normal human serum and phagocytized by human polymorphonuclear leukocytes. Cell wall particles retaining capsular material were opsonized less efficiently than peptidoglycan. Studies comparing the opsonic capacities of normal, C2-deficient, and heat-inactivated sera led to the conclusion that both the classical and the alternative complement pathways contribute to the opsonization of peptidoglycan in normal human serum. It appears that the capsule interferes with opsonization via both of these complement pathways. Serum from rabbits immunized with S. aureus M had significant heat-stable opsonic activity for the intact organism and cell walls retaining capsular material, but not for peptidoglycan. A general model is proposed to explain how antiphagocytic cell surface polymers may inhibit bacterial opsonization and thereby impede natural immunity.

Cell Wall↗
PubMed1997

Regulation of Classical Pathway of Complement Activation by Interferons.

Though complement activation in normal physiological conditions is under regulation of special proteins relevant to complement system, there is a number of other humoral factors, which may also act as activators or inhibitors of complement activation due to significant increase in their concentration during inflammation. Administration of interferons in rheumatic patients leads to pronounced changes in immune response including complement system. It is very likely that interferons may be regulators of complement activation. In this connection, influence of recombinant alpha2b- and gamma-interferon on C5-convertase formation was investigated. We have obtained stabilized and bound on sheep erythrocyte membrane C3-convertase with prolonged period of half-inactivation and ability of transformation into C5-convertase in the presence of employed interferons. Addition of interferons to the model system caused inhibition of complement activation and abrogation of complement-induced hemolysis. Inhibition constants were found to be 133000 and 170000 IU/ml for alpha2b- and gamma-interferon, respectively. Despite, these values are higher than natural serum interferon concentrations, it is possible that under inflammation local synthesis of interferons may by performed by attracted leukocytes. In this case it seems difficult to define concentration of interferons as well as revealed by us interferon inhibition of complement activation in situ. This inhibition has not been previously described and represents a new mechanism of interferon action. Binding of activated C3b complement component to interferons is probably of great physiological importance leading to constraint of complement cascade activation as well as to inactivation of excessive interferon quantity.

Journal Article↗
PubMed1992

Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence.

The role of a viral gene product in evasion of the host immune response was investigated. The antibody-dependent complement-enhanced neutralization of vaccinia virus infectivity was prevented by the culture medium from vaccinia virus-infected cells. The vaccinia virus complement-control protein (VCP) was identified as the secreted product of vaccinia virus gene C21L and has homology to a group of eukaryotic genes encoding regulators of complement activation. Thus, the culture medium from cells infected with a C21L deletion mutant was VCP deficient and had little or no effect on antibody-dependent complement-enhanced neutralization. In addition, the anticomplement effect was associated with the C21L-encoded protein partially purified from the medium of cells infected with wild-type virus. Antibody-dependent, complement-enhanced neutralization of vaccinia virus occurred with a complement source that was deficient in the classical pathway complement component C4 and required the alternative pathway complement factor B. Furthermore, the presence of VCP abrogated the complement-enhanced neutralization in C4-deficient serum. Together with previous hemolysis data, the present result suggests that VCP can inhibit both the classical and alternative pathways of complement activation. Skin lesions caused by the C21L deletion mutant were smaller than those caused by wild-type virus, demonstrating an important role for VCP in virulence. The C21L deletion mutant also was attenuated in C4-deficient guinea pigs, consistent with in vitro studies. Vaccinia virus appears to have acquired the ability to regulate the complement cascade for the purpose of evading the host immune response.

Animals↗
PubMed1993

Mouse Crry/p65 is a regulator of the alternative pathway of complement activation.

Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement-mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase-treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay-accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.

Animals↗
PubMed1991

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes.

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.

Animals↗
PubMed1996

Factor J, an inhibitor of the classical and alternative complement pathway, does not inhibit esterolysis by factor D.

Factor J (FJ) is an inhibitor of the classical and alternative complement pathways. On the classical pathway factor J disrupts the C1 component, and on the alternative pathway, factor J disrupts the C3 convertase (C3b,Bb) by a direct interaction of FJ with the components C3b and Bb. The aim of this work was to verify whether FJ could have any effect on factor D proteolytic activity since previous experiments could not rule out an eventual inhibition by factor J on factor D enzymatic activity. For this purpose, the reactivity of serine proteinase factor D was determined by using two peptide thioester substrates, Z-Lys-SBzl.HCl and Z-Lys-Arg-SBzl.2HCl, in the presence and in the absence of factor J. Kinetic studies evidenced that FJ did not affect the enzymatic activity of factor D in any case.

Complement C1 Inactivator Proteins↗
PubMed1988

Opsonins in uterine washings influencing in vitro activity of equine neutrophils.

Uterine washings were found to promote neutrophil mediated killing of Streptococcus zooepidemicus. Depletion of complement and/or specific antibody from the washings significantly reduced bactericidal activity. Phagocytosis of yeast by uterine washings was complement dependent. Inhibition of the classical pathway significantly reduced opsonic activity indicating that, in addition to direct activation via the alternate pathway, antibody may also be involved in yeast phagocytosis.

Animals↗
PubMed1979

Hypocomplementemia associated with naturally occurring lymphosarcoma in pet cats.

Eighty cats were classified by indirect immunofluorescence and histologic diagnosis into four categories: normal, feline leukemia virus (FeLV) infected; normal noninfected; lymphosarcoma-FeLV infected; lymphosarcoma, no FeLV present. All viremic cats with lymphosarcoma were found to be hypocomplementemic and activation of the complement system had occurred via the classical pathway. Sera of cats with lymphosarcoma in the absence of FeLV had varying levels of total hemolytic complement (TCH50) ranging from normal to hypocomplementemic. Approximately 50% of the cats that were viremic but histologically and clinically free of disease had TCH50 levels within normal range, and the remainder exhibited varying degrees of hypocomplementemia.

Animals↗
PubMed1977

Classical and alternative complement pathway activation by pneumococci.

Sixty-two strains of Streptococcus pneumoniae were studied for their abilities to consume selected components of classical and alternative complement pathways in human sera. The classical pathway was blocked by chelating calcium with ethyleneglycol-bios (beta-aminoethyl ether)-N,N-tetraacetic acid and by removing C4. The alternative pathway was blocked by removing factor B. Each strain's activation of the two pathways was compared with its nonimmune reactivity with the Fc region of immunoglobulin G (IgG). Activation of the classical complement pathway appeared to be independent of such Fc reactivity. Highly Fc-reactive strains, however, were shown to activate the alternative pathway more effectively than did less Fc-reactive strains. Since pneumococcal activation of the alternative pathway requires non-immunospecific IgG, these findings suggest that nonimmune binding of IgG on the pneumococcal surface endows it with complement-activating properties.

Complement System Proteins↗
PubMed1987

Evidence for activation of complement in patients with AIDS related complex (ARC) and/or lymphoadenopathy syndrome (LAS).

The complement system was examined in 16 patients with AIDS-related complex (ARC) (n = 5) and/or lymphoadenopathy syndrome (LAS) (n = 11). Of these patients 62.5% showed an impairment of classical and/or alternative pathway activity associated with the presence of cleavage fragments of C3 and/or B and a significant reduction of many complement factors. The data indicate pathological complement activation in these patients through the classical and/or alternative pathway. Complement activation was more severe in patients with ARC than in those with LAS, and greater in drug abusers than homosexuals. The lack of efficient complement in the patients can be considered an 'acquired complement deficiency' with possible importance in the failure to combat the HIV attack.

AIDS-Related Complex↗
PubMed2001

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.

Adjuvants, Immunologic↗