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PubMed1995

Calcium-loaded erythrocytes have a defect in complement regulation distinct from that resulting from exposure to 2-aminoethylisothiouronium bromide.

Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET-treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs.

Adult↗
PubMed1989

Terminal complement complexes and C1/C1 inhibitor complexes in autoimmune thyroid disease.

The potential role of complement activation and the membrane attack complex in the pathogenesis of Graves' disease and Hashimoto's thyroiditis has been investigated by measuring serum concentrations of the C1r-C1s-C1 inhibitor complex (C1/C1-inh) and the terminal complement complex (TCC), and by studying the binding to thyroid tissue of monoclonal and polyclonal antibodies against TCC neoantigens. Serum C1/C1-inh and TCC concentrations were significantly increased in 29 patients with untreated Graves' disease compared with 47 healthy subjects (P less than 0.001 for both), and decreased significantly after carbimazole treatment in 18 of these patients for whom post-treatment samples were available (P less than 0.01 and P less than 0.02, respectively). The serum TCC concentration, but not that of C1/C1-inh, was also significantly increased in 15 patients with Hashimoto's thyroiditis compared with the 47 healthy subjects (P less than 0.001). TCCs were identified by immunohistochemical staining around the thyroid follicles in thyroidectomy specimens from patients with Graves' disease (six out of six) and Hashimoto's thyroiditis (two out of two); normal thyroid tissue from two subjects showed no staining. These results suggest a role for complement, in particular the membrane attack complex in the pathogenesis of autoimmune thyroid disease.

Adolescent↗
PubMed2002

Complement activation and formation of the membrane attack complex on serogroup B Neisseria meningitidis in the presence or absence of serum bactericidal activity.

Encapsulated meningococci are complement sensitive only in the presence of bactericidal antibodies by yet-unexplored mechanisms. The objective of this study was to investigate the involvement of major bacterial surface constituents on complement activation and membrane attack complex (MAC) formation on serogroup B meningococci in the presence or absence of antibody-dependent serum bactericidal activity (SBA). The strains used were the encapsulated H44/76, five of its variants differing in capsulation and expression of the class 1 porin (PorA), and its lipopolysaccharide (LPS)-deficient isogenic mutant (LPS(-)) pLAK33. Two normal sera, one with high SBA (SBA(+)) and one with no bactericidal activity (SBA(-)) against H44/76 as well as an a-gamma-globulinemic serum were used for sensibilization of the bacteria. C3b and iC3b deposition on H44/76, its unencapsulated variant v24, and pLAK33 was similar in SBA(+) and SBA(-) serum, and no difference was present between the strains. MAC deposition on H44/76 was higher in SBA(+) serum than in SBA(-) serum and the a-gamma-globulinemic serum. The amounts of C3b on H44/76, v24, and pLAK33 in the a-gamma-globulinemic serum were also not different, indicating immunoglobulin G (IgG)- and LPS-independent complement activation. H44/76 PorA(+) and its PorA(-) variant and the v24 PorA(+) and its PorA(-) variant incubated in SBA(-) serum induced comparable amounts of MAC, despite their different serum sensitivities. Complement formation on the surface of the bacteria occurred almost exclusively via the classical pathway, but the considerable amounts of Bb measured in the serum indicated alternative pathway activation in the fluid phase. We conclude that complement deposition on meningococci is, for the most part, independent of classical pathway IgG and is not influenced by the presence of PorA or LPS on the meningococcal surface. Addition of an anti-PorA chimeric antibody to the nonbactericidal normal serum, while promoting a dose-related bacterial lysis, did not influence the amounts of C3b, iC3b, and MAC formed on the bacterial surface. These findings support the hypothesis that proper MAC insertion rather than the quantity of MAC formed on the bacterial surface is of importance for efficient lysis of meningococci.

Antibodies, Bacterial↗
PubMed2001

Complement (C5b-9) induces DNA synthesis in rat mesangial cells in vitro.

BACKGROUND: The membrane attack complex C5b-9 causes injury in many forms of immune-mediated glomerular diseases characterized by mesangial cell (MC) proliferation and inhibiting C5b-9 decreases MC proliferation in vivo. Membrane insertion of sublytic quantities of the membrane attack complex of complement (C5b-9) is a potent stimulus for cell activation and the production of a variety of cytokines, growth factors, oxidants, matrix components, and other nephritogenic molecules. In vivo, a common response of MC to C5b-9--mediated injury is cell proliferation, an event closely linked to matrix expansion and sclerosis. In this study, we tested the hypothesis that C5b-9 might also serve as a mitogenic stimulus for MCs. METHODS: Rat MCs in vitro were exposed anti-Thy1 antibody and 2% normal PVG serum (a complement source) to induce sublytic C5b-9 attack and DNA synthesis and cell number were measured. Control MCs were exposed to antibody and C6-deficient PVG serum. RESULTS: Sublytic C5b-9--induced injury to MCs is sufficient to induce DNA synthesis. Furthermore, C5b-9 augmented DNA synthesis induced by platelet-derived growth factor (PDGF) and 5% fetal calf serum. C5b-9--induced DNA synthesis was reduced by inhibiting reactive oxygen species (ROS) with superoxide dismutase and catalase, but not by neutralizing the mitogenic growth factors PDGF and basic fibroblast growth factor (bFGF). CONCLUSIONS: This study demonstrates that C5b-9 may directly increase DNA synthesis in cultured MCs, which are mediated in part by the release of ROS, and that C5b-9 also augments DNA synthesis induced in MCs by other known mitogens.

Animals↗
PubMed1991

Alternative complement pathway-mediated myeloid cell cytotoxicity: repertoire of membrane factors participating in regulation of C3 deposition and cytolysis.

Most human nucleated cells and cell lines possess C3 step regulators, decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg(2+)-EGTA-human serum treatment did not induce C3 deposition on most nucleated cells. Furthermore, less than 20% lysis occurred even after the block of all the three factors. In contrast, three myeloid cell lines, U-937, HL-60 and p39, were found to exhibit unusual C3 deposition or cytolysis. U-937 possessed DAF and MCP but lacked p18, and about 50% was lysed by treatment with anti-DAF and anti-MCP followed by Mg(2+)-EGTA-serum, which caused C3, C5 and C8 deposition. Anti-DAF evoked similar but less complement (C) deposition and cytolysis while anti-MCP alone did not, although it enhanced the anti-DAF-mediated C deposition and cytolysis. Thus, once the C3 step is overcome, U-937 is attacked by the late components leading to cytolysis because of the absence of p18. On the other hand, HL60 allowed the deposition of C3 by blocking of either DAF or MCP followed by the Mg(2+)-EGTA-serum treatment. C5, C8 and C9 were subsequently deposited but resulted in no lysis. Lysis of 60% was attained by the additional blocking of p18. Thus, HL60 is poorly protected by C3 and C9 step regulation. Strikingly, extensive C3 deposition occurs on p39 without any antibody treatment, suggestive of the presence of unique alternative pathway activators. However, little cytolysis was induced on p39 even by blocking of all three inhibitors with antibodies. These results suggest that in activation of the alternative pathway on myeloid cells, C3 step is controlled by the inhibitors and alternative pathway activators, and C-mediated cytolysis is blocked by p18 and additional regulatory mechanisms or factors which assist in protection of nucleated host cells from MAC attack. Susceptibility to homologous C of these cell lines, therefore, reflects relatively low potency of C regulation on their membrane.

Antigens, CD↗
PubMed2002

Protection of hDAF-transgenic porcine endothelial cells against activation by human complement: role of the membrane attack complex.

Xenograft rejection in the discordant pig-to-primate model is dependent on binding of natural antibodies to gal-alpha [1-3]-gal epitopes on the porcine endothelial cell (EC). This leads to complement activation and deposition of activation products onto the membrane and results in perturbation of EC function and thrombus formation. Here we investigated the ability of human complement activation products to directly induce activation of porcine EC, with subsequent upregulation of adhesion and pro-coagulant molecules. Porcine aortic EC were isolated from wild-type and hDAF-transgenic pigs and incubated with human serum, either in the presence or absence of the soluble complement inhibitor TP10 (sCR1). Recombinant C5a, C1q-IgG immune complexes, C6-deficient human serum and serum containing anti-C9 Ab were used to identify EC activating complement products. Heat-inactivated human serum was used as a negative control. Cells were stained with antibodies against human C3, the MAC or with antibodies cross-reactive for porcine E-Selectin, VCAM-1 or Tissue Factor, and analyzed by flow cytometry. We found upregulation of E-Selectin and Tissue Factor on wild-type EC after incubation with human serum. This effect coincided with the deposition of C3 and MAC on the membrane of these cells. The addition of TP10 inhibited EC activation by up to 95%. In contrast, greatly reduced C3 and MAC deposition was detected on hDAF transgenic cells, and no complement-mediated EC activation was seen. Experiments with C6-deficient serum and incubation with anti-C9 Ab indicate a major role of the MAC in serum-mediated EC activation, whereas neither C5a nor C1q-IgG caused activation of EC. These data provide further explanation of the protective role of human DAF in the pig-to-primate xenotransplantation model.

Animals↗
PubMed1985

Transmembrane channel-formation by five complement proteins.

Five serum proteins act in concert to form the membrane attack complex (MAC) of complement. The precursor proteins, C5, C6, C7, C8 and C9, are hydrophilic glycoproteins with molecular weights ranging from 70,000 to 180,000. When C5 is cleaved by the serine protease C5 convertase, nascent C5b is produced which forms together with C6 a soluble and stable bimolecular complex (C5b,6). Upon binding of C5b,6 to C7 a trimolecular complex (C5b-7) is formed, which expresses a metastable membrane binding site. Membrane-bound C5b-7 constitutes the receptor for C8 and the tetramolecular C5b-8 complex binds and polymerizes C9. During the assembly process the proteins undergo hydrophilic-amphiphilic transition and the end product consists of C5b-8 (Mr approx. 550,000) and of tubular poly C9 (Mr approx. 1,100,000). The functional channel size varies but its maximal diameter is approximately 10 nm. C9 polymerization appears to involve initial reversible associations of several C9 molecules, which leads to temperature dependent, constrained unfolding. Unfolded C9 monomers then associate laterally with each other and polymerization terminates with closure of the circular structure, which consists of 12-18 C9 monomers. Amino acid composition and sequence indicate that the N-terminal half of the single chain C9 molecule is hydrophilic and the C-terminal half rather hydrophobic. Phospholipid binding and insertion into membranes are functions of the C-terminal portion of the molecule.

Amino Acid Sequence↗
PubMed1994

Neuroimmune mechanisms in Alzheimer disease pathogenesis.

Lesions in Alzheimer disease are characterized by the assembly of a variety of cells and proteins associated with the immune system. Activated microglia express high levels of major histocompatibility complex glycoproteins and receptors for complement. Small numbers of T lymphocytes infiltrate tissue. Proteins of the classical complement pathway are closely connected with beta-amyloid deposits. beta-Amyloid protein binds C1q in vitro and activates the pathway. The membrane attack complex of complement, as well as proteins that defend against that complex, colocalize with dystrophic neurites. These data imply that an autodestructive process is occurring in Alzheimer disease, that overactive microglia might be responsible, and that antiinflammatory drugs might be an effective form of therapy.

Aged↗
PubMed1989

Activation of the classical complement pathway in brain tissue of Alzheimer patients.

Positive immunohistochemical staining of Alzheimer brain tissue was obtained with antibodies to proteins associated with classical, but not the alternative, complement pathway. Clq, C3d, C4d are fractions of complement proteins that bind to tissue when the classical complement pathway is activated. Antibodies to these fractions stained senile plaques, dystrophic neurites and some neurofibrillary tangles. C5b-9 is the membrane attack complex which promotes cell lysis when assembled on the plasma membrane. An antibody to a neoantigenic site on this complex stained dystrophic neurites and many neurofibrillary tangles, but not extracellular amyloid. Properdin and fraction Bb of factor B, two proteins that bind to tissue when the alternative complement pathway is activated, were not detected immunohistochemically.

Aged↗
PubMed1993

Activated complement directly modifies the performance of isolated heart muscle cells from guinea pig and rat.

The complement system has been implicated in the pathogenesis of cardiovascular disorders including ischemia and atherosclerosis. Selective deposition of C5b-9, the membrane attack complex of complement, has been histochemically documented in human myocardium early after reperfusion of ischemic areas and in infarcted zones. However, functional sequelae of the C5b-9 complex binding to myocardial cells have not been identified. Insertion of C5b-9 complexes into the membrane of other cell types can generate transient changes in membrane permeability in the absence of cell lysis. We demonstrate in beating isolated adult guinea pig and rat cardiac myocytes that human derived C5b-9 can transiently augment in a dose-dependent manner both basal cytosolic calcium concentration and calcium transients, resulting in a temporary increase in contractility. If similar changes occur in human heart cells in vivo, they could significantly affect myocardial performance and contribute to functional abnormalities seen in ischemia and other pathological conditions associated with complement activation.

Animals↗
PubMed2006

Deposition of complement proteins on cells treated by photodynamic therapy in vitro.

Activation of the complement system has emerged as a critical event in the response of tumors to photodynamic therapy (PDT) due to its involvement in the vascular effects as well as in the inflammatory and immune reactions observed with this treatment modality. However, the exact mechanism of PDT-induced complement activation has not been precisely characterized. The present study examines the potential of PDT at the cellular level to directly activate the complement system. Mouse tumor SCCVII cells treated by Photofrin-based PDT and post-incubated in the presence of homologous (mouse) serum were analyzed by flow cytometry for binding of complement proteins on their surface. The results show that PDT induced the fixation of complement C3 protein, probably in the form of its activated fragments, and of the terminal membrane attacks complex of complement on the treated SCCVII cells. Deposition of C3/C3 fragments on human umbilical vein endothelial cells (HUVEC) treated by Photofrin-PDT and post-incubated in the presence of human serum was also detected. Complement fixation was preferentially elevated on SCCVII cell undergoing PDT-induced apoptosis. The induction of surface expression of heat shock protein 70 (HSP70) on PDT-treated cells also triggered complement deposition, since the presence of anti-HSP70 antibodies during the post-PDT incubation blocked the anchoring of C3/C3 fragments to SCCVII cells. The fact that PDT-treated cells are recognized by the complement system as their target adds an important element for understanding the mechanism of tumor response to PDT.

Animals↗
PubMed1989

Complement-mediated inhibition of function in complement-resistant Escherichia coli.

C-mediated inhibition of function in C-sensitive strains of Escherichia coli involves the assembly of the membrane attack complex (MAC) on the outer membrane with subsequent inhibition of inner membrane function. The inhibition of inner membrane function is critical for effective cell killing as damage to the outer membrane alone is insufficient to kill a cell in the absence of serum lysozyme. Studies on the measurement of oxygen consumption for cells under complement attack showed that C-sensitive cells were inhibited by assembly of the MAC, and that this represents damage to some component of the respiring inner membrane. Mechanisms of cellular resistance to C attack could include 1) inhibition of the assembly of the MAC, 2) inhibition of effective activation of the assembled MAC, or 3) reversal of the inhibitory effects of the MAC. Demonstration of a transient C-mediated inhibition of inner membrane function in C-resistant cells implies that the latter case should be considered as one possible component of cellular resistance to C attack.

Bacteriolysis↗
PubMed2002

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.

Animals↗
PubMed1988

Studies on serum resistance in Escherichia coli.

Serum-sensitive mutants and their serum-resistant smooth parental E. coli strains (Wf8, Wf26, and WF 52) have been investigated in respect to their binding of different complement components. These pairs consisting of a wild-type and its mutants represent a better model for the investigation of the mechanism of serum resistance than the comparison of unrelated strains. Both strains of a pair bind equivalent amounts of C3. In binding assays using radiolabeled terminal components C6, C7, C8, and C9, the serum-sensitive strains do bind more late acting components than their resistant parental strains. An active membrane attack complex stably bound to the cell surface was found on the mutants, whereas with wild-type bacteria a complex could be isolated from the supernatant which is composed of the late acting complement components and S-protein. This complex is released from the surface of the wild-type bacteria without participation of C9.

Blood Bactericidal Activity↗
PubMed2005

Further characterization of reproductive abnormalities in mCd59b knockout mice: a potential new function of mCd59 in male reproduction.

CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59b-/-) results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities in mCd59b-/- mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were similar in mCd59b-/- and mCd59b+/+. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the abnormal reproductive phenotype of mCd59b-/-, although it was efficient in rescuing their hemolytic anemia. These results indicate that the male reproductive abnormalities in mCd59b-/- are complement-independent, and that mCd59 may have a novel function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex formation.

Anemia, Hemolytic↗
PubMed1986

Antigenic crossreactivity of the alpha subunit of complement component C8 with the cysteine-rich domain shared by complement component C9 and low density lipoprotein receptor.

Complement component C9 contains two distinct cysteine-rich domains exhibiting high sequence resemblance to a domain present in the low density lipoprotein (LDL) receptor and epidermal growth factor precursor, respectively. Antibodies were raised against a peptide corresponding to the most conserved region of the LDL receptor/C9-homology segment. The antibodies were shown by immunoblotting to bind specifically to C9 but also to crossreact with C8 alpha, the alpha subunit of complement component C8. Moreover, a monoclonal antibody to a neoantigen present in polymerized C9 bound to an epitope exposed on C8 within the C5b-8 complex but buried in monomeric C8, suggesting that C8 and C9 undergo similar conformational changes during membrane-attack-complex assembly. Isolated C8 alpha-gamma exhibited the propensity to polymerize in the presence of Zn2+ and urea, as already demonstrated for C9. These data indicate that C8 alpha is closely related, both structurally and functionally, to C9.

Complement C8↗
PubMed1997

Complement activation after ischemia-reperfusion in human liver allografts: incidence and pathophysiological relevance.

BACKGROUND & AIMS: Little is known about the occurrence and possible consequences of local complement activation in human liver transplantation. The aim of this study was to search for signs of complement activation in human liver allografts and evaluate their relationship to cell injury and leukocyte sequestration. METHODS: In 33 postreperfusion biopsy specimens, 9 preoperative specimens, and 10 intraoperative specimens, the cytolytic membrane attack complex of complement was localized, expression of complement inhibitors was evaluated, and intragraft accumulation of leukocytes and platelets was quantitated. RESULTS: In control samples and preoperative biopsy specimens, the membrane attack complex was detected only in extracellular deposits, associated with its soluble inhibitors clusterin and vitronectin. Comparable observations were performed in 14 postoperative specimens. In the remaining 19 postoperative specimens, membrane attack complex decorated a variable proportion of hepatocytes. The extent of membrane attack complex deposition correlated with the increase in postoperative aspartate aminotransferase levels in serum (P = 0.002) and the decrease in postoperative factor V levels in serum (P = 0.002). It also correlated with the number of leukocytes and platelets accumulating within the graft (P < or = 0.001). CONCLUSIONS: Local complement activation is frequent during human liver transplantation and probably contributes to cell injury and leukocyte sequestration in the allograft.

Adult↗
PubMed1994

Expression of the complement regulatory protein CD59 on human spermatozoa: characterization and role in gametic interaction.

Protectin (CD59) is a complement regulatory protein which blocks the membrane attack complex during complement activation. CD59 was identified on the human sperm surface by means of H19, an IgG1 anti-protectin mouse monoclonal antibody. Using indirect immunofluorescence, flow cytometry and immunoperoxidase, CD59 was found to be present on the whole plasma membrane including the head and tail of fresh ejaculated, capacitated and acrosome-reacted spermatozoa. Immunoperoxidase staining of normal testicular sections indicated that this protein was already present on intraluminal germ cells. Analysis of this sperm protein by gel electrophoresis and immunoblotting revealed that its molecular weight of 20 kDa was comparable to that of CD59 expressed on peripheral blood cells (erythrocytes, lymphocytes) and that it was bound to the membrane through a glycophospholipid tail which could be released after treatment with phosphatidylinositol-specific phospholipase C. Associated to membrane cofactor protein (CD46) and decay accelerating factor (CD55) located in the acrosomal membranes, CD59 may participate to the protection of male gametes against complement-mediated damage as they travel through the female genital tract. Moreover CD59, known as an adhesion molecule involved in lymphocyte rosettes, may also participate in cell to cell adhesion during gametic interaction since H19 inhibited sperm binding and reduced the penetration rate and index during the hamster egg penetration test.

Animals↗