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Results for “Complement Inactivator Proteins”

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PubMed1992

Regulation of complement activity by vaccinia virus complement-control protein.

A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.

Complement Activation↗

PubMed2005

Intrinsic resistance of hepatocytes to complement-mediated injury.

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.

Animals↗

PubMed1996

C5b-8 step lysis of swine endothelial cells by human complement and functional feature of transfected CD59.

The authors established several swine endothelial cell (SEC) lines expressing human CD59 by transfection of cDNA, and assessed the function of the transfectant molecules in comparison with those of membrane cofactor protein (MCP) and decay-accelerating factor (DAF) in an in vitro hyperacute rejection model of swine to human discordant xenograft. At the usual expression rate, DAF and MCP protected SEC from human complement mediated cell lysis, but CD59 did not block human complement attack on SEC. However, CD59 protects SEC from cell lysis when sufficiently expressed as in human umbilical vein (HUVEC). The authors examined why CD59 needed so many molecules to protect human complement-mediated SEC lysis and found that SEC underwent lysis by human C5b-8. The degree of C5b-8 step lysis of SEC was approximately 70% of the total activation (C5b-9). Additionally, CD59 protected human complement activities less efficiently at the C5b-8 step than at the C9-step. Therefore, to overcome human complement mediated SEC lysis, C8 activity must be inhibited by dense expression of CD59.

Animals↗

PubMed1995

Physiology and pathophysiology of complement: progress and trends.

The complement system comprises a family of at least 20 plasma and membrane proteins that interact in a tightly regulated cascade system to destroy invading bacteria and prevent the deposition of immune complexes in the tissues. This brief review addresses the basic mechanisms of complement activation and control and describes the active fragments produced during complement activation. The biological importance of the complement system is amply illustrated in patients with complement deficiencies, who are susceptible to bacterial infections and immune complex diseases. The involvement of complement in other immunological diseases is an expanding area of clinical research, supported by the development of new assays for the identification of complement activation. This area is discussed here with particular reference to neurological diseases. A promising new prospect involves the use of complement inhibitory molecules in therapy of complement-mediated disease and this exciting area is also discussed. Novel physiological roles of complement also are being revealed and new evidence that complement and complement receptors play an important role in reproduction is summarized. It is hoped that this brief overview will convey some of the enthusiasm currently pervading research in this underappreciated area of immunology.

Animals↗

PubMed2005

Synergy between two active sites of human complement receptor type 1 (CD35) in complement regulation: implications for the structure of the classical pathway C3 convertase and generation of more potent inhibitors.

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.

Binding Sites↗

PubMed1984

Effects of K-76 monocarboxylic acid, an anticomplementary agent, on various in vivo immunological reactions and on experimental glomerulonephritis.

K-76 monocarboxylic acid (K-76 COOH) caused rapid reduction in hemolytic activities of complement and some of its components, especially C5, when injected into guinea pigs or rats. K-76 COOH suppressed Forssman shock in guinea pigs and mice and heterologous passive cutaneous anaphylaxis in guinea pigs. It also reduced the amount of protein excreted in the urine of rats with nephrotoxic nephritis and greatly prolonged the survival of (NZB X NZW) F1 female mice with a spontaneous systemic lupus erythematosus-like disease. The glomeruli of mice treated with K-76 COOH retained almost the normal histological appearance even at 1 year of age. K-76 COOH-treated mice became less sensitive to the infection of Escherichia coli, staphylococcus aureus, or Streptococcus pneumoniae, probably by enhancement of phagocytosis due to inhibition of factor I. K-76 COOH did not have any significant effect on a delayed-type contact skin reaction in mice, or on experimental allergic encephalitis in guinea pigs.

Anaphylaxis↗

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

The change of membrane complement regulatory protein in chorion of early pregnancy.

Research on membrane complement inhibitors (DAF, MCP, and CD59) has led to understanding of the regulation of complement system; however, their precise role and distribution remain speculative. In this study, we used an indirect fluorescent immunostaining to investigate the distribution of complements, MCP, DAF, and CD59, in the villi before the 10th week of pregnancy in 18 women. DAF and MCP were observed at the surface of syncytiotrophoblasts (ST) and extravillous trophoblast (EVT) in all subjects. They were observed in villous cytotrophoblast (VCT) in the subjects before the 8th week of pregnancy but were not observed in any subject after the 9th week. However, CD59 appeared in ST but never in VCT. MCP, DAF, and CD59 were observed in EVT. These findings indicated that these complement inhibitors played an important role in early pregnancy, and that CD59 continued to appear in early pregnancy, whereas the expression of MCP and DAF depended on the stage of pregnancy.

Antigens, CD↗

PubMed1978

Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator.

We recently described the isolation from human serum of a high molecular weight protein with specific binding affinity for fluid-phase activated C4. We show here that the C4-binding protein (C4-Bp) functions as an essential cofactor in the proteolysis of C4b in the presence of C3b-inactivator (C3bINA). C4-bp, together with C3bINA, cleave the alpha'-chain of C4b into three fragments called alpha2, alpha3, and alpha4, with mol wt of 47,000, 25,000, and 17,000 daltons, respectively. The alpha2 fragment was dissociated from C4b without reduction, whereas the alpha3 and alpha4 fragments were disulfide bonded the other chains of C4b. The reaction did not occur when either C4-bp or C3bINA were omitted, nor in the presence of either protein in combination with beta1H. Native C4 was not affected by C3bINA aand C4-bp. C4b was not cleaved when incubated in serum of a patient with genetic deficiency of C3bINA. However, when purified C3bINA was added, the alpha'-chain of C4b was cleaved and fragments with the same molecular weight as alpha2, alpha3, and alpha4 were generated.

Carrier Proteins↗

PubMed1996

Human carcinomas variably express the complement inhibitory proteins CD46 (membrane cofactor protein), CD55 (decay-accelerating factor), and CD59 (protectin).

Normal human tissues express membrane-associated complement inhibitory proteins that protect these tissues from damage by autologous complement. To determine whether neoplasms also express these proteins, we examined the distribution of the complement inhibitors decay-accelerating factor (DAF), CD59 (protectin), and membrane cofactor protein in frozen samples of human breast, colon, kidney, and lung carcinomas and in adjacent non-neoplastic tissues, using immunohistochemistry. All samples were also studied for deposition of C3 fragments and activated C5b-9. Differences between normal tissues and the corresponding neoplasms were often observed, with loss or gain of expression of one or more inhibitors. Ductal carcinomas of the breast showed the most variation in phenotype; some tumors expressed only one inhibitor while others expressed different combinations of two or three inhibitors. Colon carcinomas, by contrast, stained intensely for all inhibitors. Renal cell carcinomas had weak to moderate expression of one to three inhibitors, generally DAF and CD59, whereas non-small cell carcinomas of the lung usually expressed CD59 and membrane cofactor protein with variable DAF immunoreactivity. The two small cell carcinomas of the lung showed little or no staining for any inhibitor. Activated C5b-9 deposition was seen adjacent to tumor nests in a minority of carcinomas and showed no correlation with complement inhibitor expression. C3 fragment deposition was minimal. Our results demonstrate that most carcinomas, with the exception of small cell carcinomas of the lung, do express one or more complement inhibitors at a level likely to inhibit complement-mediated cellular damage. Unexpectedly, large quantities of DAF and CD59 were often observed in tumor stroma, with only limited deposition in normal connective tissue. This suggests that carcinomas may supplement the activity of membrane-associated complement inhibitors by release of soluble forms of DAF and CD59 into the surrounding extracellular matrix.

Antigens, CD↗

PubMed1996

Human skeletal myoblasts spontaneously activate allogeneic complement but are resistant to killing.

The complement (C) system has previously been implicated in several diseases of muscle. We here report that human myoblasts or rhabdomyosarcoma cell lines spontaneously activate C through the classical pathway, causing release of anaphylatoxins and coating of myoblasts with opsonic C fragments but without causing cell killing. Survival of myoblasts is a consequence of the abundant expression of the membrane C regulatory molecules MCP and CD59, and neutralization of CD59 renders cells susceptible to C killing. The decay-accelerating factor was expressed at a very low level. Myoblasts and rhabdomyosarcoma lines also abundantly express the fluid-phase regulators C1-inhibitor, factor H, C4 binding protein, S-protein, and clusterin and secrete a soluble form of CD59. Expression of membrane and fluid-phase regulators is enhanced by either IFN-gamma or TNF-alpha. Although myoblasts resist C killing, spontaneous activation of C on these cells may have important consequences in inflammatory diseases of muscle where the generation of anaphylactic and opsonic fragments will recruit and activate inflammatory cells. C activation on myoblasts may also have consequences for the use of these cells as vehicles for gene delivery. Inhibition of C using soluble complement receptor I (sCR1) efficiently protected myoblasts from C attack in vitro, and this agent, already being tested in therapy of several C-mediated diseases, might be of value in inflammatory muscle disease and in improving the efficiency of gene delivery.

Cell Death↗

PubMed1997

The cowpox virus-encoded homolog of the vaccinia virus complement control protein is an inflammation modulatory protein.

Vaccinia virus complement control protein (VCP) is encoded by vaccinia virus, with its homolog encoded by other pathogenic poxviruses including variola virus. Since rodents are the primary reservoir hosts of cowpox virus (CPV) and since CPV encodes a highly conserved functional homolog of VCP, termed here the inflammation modulatory protein (IMP), the effects of injection of CPV into the footpads of mice was determined in order to study the precise in vivo effects of IMP. Macroscopic examination of the site of injection with a recombinant virus lacking IMP (CPV-IMP) showed greater tissue damage, with more hemorrhage and induration, than sites injected with the wild-type cowpox virus. In addition, the measurement of the specific swelling response carried out for several weeks revealed significantly greater swelling in mice injected with CPV-IMP. Thus, IMP modulates the complement-activated inflammatory response in vivo. Furthermore, the diminished destruction of host tissue observed in the presence of IMP indicates symbiosis in which the virus ensures the preservation of surrounding host tissue, possibly to support the growth of its progeny.

Amino Acid Sequence↗

PubMed1996

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing.

Adult↗

PubMed1989

Heightened complement sensitivity of acquired immunodeficiency syndrome lymphocytes related to diminished expression of decay-accelerating factor.

Although the human immunodeficiency virus can induce cytopathic changes in human lymphocytes in vitro, the mechanism(s) underlying progressive lymphopenia in patients with AIDS and AIDS-related complex has not been elucidated. To investigate this issue, peripheral blood lymphocytes of AIDS and AIDS-related complex patients and healthy control subjects were examined for their ability to resist homologous complement-mediated lysis. Upon sensitization with monoclonal antibodies to major histocompatibility complex class I antigen, as much as 48% lysis of patients' cells was observed in as little as a 1:32 dilution of human serum compared to 18 +/- 8% (mean +/- SD) lysis of controls' cells even in a 1:8 dilution of human serum. To investigate the mechanism of the abnormal complement sensitivity, AIDS and AIDS-related complex cells were analyzed for expression of decay-accelerating factor (DAF), a complement regulatory protein that functions intrinsically in blood cell membranes to prevent complement activation on their surfaces. Flow cytometric assays using anti-DAF monoclonal antibodies demonstrated that patients' lymphocytes and monocytes were DAF-deficient, in contrast to their polymorphonuclear leukocytes, which showed normal DAF levels. Expression of DAF was diminished on CD4+ as well as CD8+ T-lymphocyte subpopulations as opposed to expression of CD3, which was comparable in patients and controls. Incubation of normal lymphocytes with anti-DAF monoclonal antibodies or phosphatidylinositol-specific phospholipase C, an enzyme that cleaves DAF, enhanced lysis. Conversely, reconstitution of patients' cells with exogenous DAF reduced their lysis. The findings of heightened complement sensitivity and DAF deficiency of patients' lymphocytes in vitro suggest the possibility that the DAF deficit may contribute to the progressive lymphopenia of AIDS in vivo.

AIDS-Related Complex↗

PubMed1977

Complement-dependent hemodynamic and hematologic changes in the rabbit.

The intravenous injection of the anticomplementary protein from cobra venom, cobra factor (CoF),2 induces decreases in mean arterial blood pressure and circulating platelets in rabbits. The changes are rapidly reversed. Both changes require the presence of C3 and occur in rabbits genetically deficient in the sixth component of complement. The hypotensive effects of CoF were blocked by the histamine H2-receptor antagonist burimamide. An acute C3-dependent change in blood pressure and circulating platelets also was demonstrated following the intravenous injection of S. marcescens endotoxin. However, abrogation of these acute changes by C3 depletion did not alter the extent of a second, prolonged fall in blood pressure and platelets induced by S. marcescens endotoxin occurring after 60--90 min. C3 depletion also did not alter the lethal effects of the S. marcescens endotoxin.

Animals↗

PubMed2001

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori.

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.

Amino Acid Sequence↗

PubMed1994

Aberrant expression of membrane cofactor protein and decay-accelerating factor in the endothelium of patients with systemic sclerosis. A possible mechanism of vascular damage.

BACKGROUND: One of the main features of systemic sclerosis (SSc) is vascular damage, the mechanism of which is not understood. The aim of this study was to investigate if complement (C) regulatory molecules, membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59, which normally protect endothelial cells from damage by autologous C, are absent or down-regulated in vascular endothelium of patients with SSc. A deficiency or persistent down-regulation of the above molecules is likely to render vascular endothelium of these patients susceptible to damage by physiologically or pathologically activated C. From this point of view, expression of MCP, DAF, and CD59 was tested on endothelium of skin of normal subjects and patients with diffuse and limited forms of SSc. EXPERIMENTAL DESIGN: Punch biopsies of normal skin (N = 11) and lesional and non-lesional skin of patients with diffuse (N = 5) and limited (N = 5) forms of SSc were obtained and serial sections prepared. Immunoperoxidase staining of these sections was carried out using four monoclonal antibodies (MoAbs) directed to different epitopes of DAF, four to different epitopes of MCP, one to CD59 and one to von Willebrand factor. von Willebrand factor served as a marker of endothelial cells. Besides normal skin, lesional skin of systemic lupus erythematosus and several inflammatory and proliferative diseases, described below, served as controls. RESULTS: The endothelium of normal skin strongly expressed all the three proteins. However, the endothelium of lesional and nonlesional skin of all the 10 patients with diffuse or limited forms of the disease tested, expressed either decreased or undetectable amounts of MCP and DAF. CD59 expression was normal in some patients and lower than normal in others. Aberrant expression of MCP, DAF, or CD59 was not found in other control inflammatory, connective tissue and proliferative diseases studied. CONCLUSIONS: In view of the common function of MCP and DAF to protect self cells from autologous C, their decrease or virtual absence from the endothelium of patients with SSc strongly suggests that this deficiency may contribute to vascular damage, resulting in intima proliferation and, finally, fibrosis.

Antibodies, Monoclonal↗

PubMed1997

Resistance of ovarian teratocarcinoma cell spheroids to complement-mediated lysis.

We have shown previously that it is possible to target complement-mediated killing against cultured ovarian tumour cells in vitro. As malignant ovarian cells usually grow in solid nodules in vivo, we have in the present study examined the effectiveness of complement killing against ovarian teratocarcinoma cells (PA-1) growing in three-dimensional tumour microspheroids (TMSs). Our study shows that PA-1 cells growing in TMSs are less susceptible to complement-mediated killing than cells growing in monolayer cultures, even after neutralization of protectin (CD59), the main inhibitor of complement lysis. Cells in suspension and cells growing in TMSs showed a similar expression of membrane co-factor protein (MCP, CD46) and CD59. Decay-accelerating factor (DAF, CD55) was not detected on the surface of cells in suspension, but appeared focally on the outermost cell layers of the TMSs. Complement-activating antibodies bound to all PA-1 cells in suspension but only to the most peripherally located cells in TMSs, even though the target antigens were similarly expressed in the two systems. Antibody-induced complement activation on PA-1 cells in suspension led to C3 and C5b-9 deposition on most cells, while C3 and C5b-9 were only found on the outermost layers of the TMSs. The increased complement resistance of tumour cells growing in three-dimensional spheroids is partly because of an insufficient penetration of antibodies and complement into the TMSs. TMSs are a useful model for the development of more efficient ways to kill malignant cells in micrometastases with monoclonal antibodies and complement.

Antibodies, Monoclonal↗