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PubMed2005

Complement factor H polymorphism in age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. We report a genome-wide screen of 96 cases and 50 controls for polymorphisms associated with AMD. Among 116,204 single-nucleotide polymorphisms genotyped, an intronic and common variant in the complement factor H gene (CFH) is strongly associated with AMD (nominal P value <10(-7)). In individuals homozygous for the risk allele, the likelihood of AMD is increased by a factor of 7.4 (95% confidence interval 2.9 to 19). Resequencing revealed a polymorphism in linkage disequilibrium with the risk allele representing a tyrosine-histidine change at amino acid 402. This polymorphism is in a region of CFH that binds heparin and C-reactive protein. The CFH gene is located on chromosome 1 in a region repeatedly linked to AMD in family-based studies.

Aged↗
PubMed2002

Expression of complement regulating factors in gastric cancer cells.

AIMS: To investigate the deposition of complement components, C3d and C5b-9, and the expression of complement regulating factors (S protein, membrane cofactor protein (MCP; CD46), protectin (CD59), decay accelerating factor (DAF; CD55), and type 1 complement receptor (CR1; CD35)) in gastric cancers. METHODS: Specimens of gastric cancer were examined by immunohistochemistry and immunoelectron microscopy. RESULTS: Four complement regulating factors (S protein, MCP, protectin, and DAF) were expressed on gastric cancer cells, in ultrastructurally localised areas on the cell membrane. CR1 was not expressed. The staining intensity of DAF in both differentiated and undifferentiated adenocarcinomas was significantly higher than in histologically normal gastric epithelium. Furthermore, the staining intensity of DAF in gastric cancers showing a diffusely infiltrating growth pattern was higher than in gastric cancers showing an expanding growth pattern. CONCLUSIONS: These data indicate that DAF may play a role in cancer cell infiltration and resistance in tumour cells.

Adenocarcinoma↗
PubMed2005

Activation of the extracellular signal-regulated kinase by complement C5b-9.

Extracellular signals may be transmitted to nuclear or cytoplasmic effectors via the mitogen-activated protein kinases. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of phospholipases and protein kinases. This study addresses the complement-mediated activation of the extracellular signal-regulated kinase (ERK). C5b-9 induced ERK threonine202/tyrosine204 phosphorylation (which correlates with activation) in GEC in culture and PHN in vivo. Expression of a dominant-inhibitory mutant of Ras reduced complement-mediated activation of ERK, but activation was not affected significantly by downregulation of protein kinase C. Complement-induced ERK activation resulted in phosphorylation of cytosolic phospholipase A2 and was, in part, responsible for phosphorylation of mitogen-activated protein kinase-associated protein kinase-2, but did not induce phosphorylation of the transcription factor, Elk-1. Activation of ERK was attenuated by drugs that disassemble the actin cytoskeleton (cytochalasin D, latrunculin B), and these compounds interfered with the activation of ERK by mitogen-activated protein kinase kinase (MEK). Overexpression of a constitutively active RhoA as well as inhibition of Rho-associated kinase blocked complement-mediated ERK activation. Complement cytotoxicity was enhanced after disassembly of the actin cytoskeleton but was unaffected after inhibition of complement-induced ERK activation. However, complement cytotoxicity was enhanced in GEC that stably express constitutively active MEK. Thus complement-induced ERK activation depends on cytoskeletal remodelling and affects the regulation of distinct downstream substrates, while chronic, constitutive ERK activation exacerbates complement-mediated GEC injury.

Actins↗
PubMed2003

p38 mitogen-activated protein kinase protects glomerular epithelial cells from complement-mediated cell injury.

In the passive Heymann nephritis (PHN) model of rat membranous nephropathy, complement C5b-9 causes sublytic injury of glomerular epithelial cells (GEC). We previously showed that sublytic concentration of C5b-9 triggers a variety of biological events in GEC. In the current study, we demonstrate that complement activates p38 MAPK in GEC and address the role of p38 in complement-mediated cell injury. When cultured rat GEC were stimulated with complement, p38 kinase activity and phosphorylation were increased by approximately 2.4-fold, compared with control. Treatment with p38 inhibitors significantly augmented complement-mediated cytotoxicity. In contrast, when the constitutively active mutant of transforming growth factor-beta-activated kinase 1 (TAK1), a kinase upstream of p38, was expressed in GEC in an inducible manner, cytotoxicity was significantly reduced, compared with uninduced cells. p38 inhibitors abolished the protective effect of TAK1 expression. By analogy to cultured cells, p38 activity was also increased in glomeruli from rats with PHN and treatment with the p38 inhibitor FR-167653 increased proteinuria. Complement induced phosphorylation of MAPK-associated protein kinase-2 (MAPKAPK-2), a kinase downstream of p38 in GEC. Heat shock protein (HSP27) is a cytoskeleton-interacting substrate of MAPKAPK-2. Overexpression of the wild-type HSP27, but not a non-phosphorylatable mutant, markedly reduced complement-mediated GEC injury. In summary, complement activates p38 MAPK in GEC in vitro and in glomeruli from rats with PHN. The activation of p38 MAPK appears to be cytoprotective for GEC against complement-mediated GEC injury. Phosphorylation of HSP27 may mediate this cytoprotection.

Animals↗
PubMed2005

Experimental membranous nephropathy redux.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Active and passive Heymann nephritis (HN) in rats are valuable experimental models because their features so closely resemble human MN. In HN, subepithelial immune deposits form in situ as a result of circulating antibodies. Complement activation leads to assembly of C5b-9 on glomerular epithelial cell (GEC) plasma membranes and is essential for sublethal GEC injury and the onset of proteinuria. This review revisits HN and focuses on areas of substantial progress in recent years. The response of the GEC to sublethal C5b-9 attack is not simply due to disruption of the plasma membrane but is due to the activation of specific signaling pathways. These include activation of protein kinases, phospholipases, cyclooxygenases, transcription factors, growth factors, NADPH oxidase, stress proteins, proteinases, and others. Ultimately, these signals impact on cell metabolic pathways and the structure/function of lipids and key proteins in the cytoskeleton and slit-diaphragm. Some signals affect GEC adversely. Thus C5b-9 induces partial dissolution of the actin cytoskeleton. There is a decline in nephrin expression, reduction in F-actin-bound nephrin, and loss of slit-diaphragm integrity. Other signals, such as endoplasmic reticulum stress, may limit complement-induced injury, or promote recovery. The extent of complement activation and GEC injury is dependent, in part, on complement-regulatory proteins, which act at early or late steps within the complement cascade. Identification of key steps in complement activation, the cellular signaling pathways, and the targets will facilitate therapeutic intervention in reversing GEC injury in human MN.

Animals↗
PubMed1993

Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.

Animals↗
PubMed1995

Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.

Animals↗
PubMed2001

Complement C5b-9 induces cyclooxygenase-2 gene transcription in glomerular epithelial cells.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. Rat GEC in culture express cyclooxygenase (COX)-1 constitutively, whereas COX-2 expression is induced by C5b-9. Both isoforms contribute to complement-induced prostaglandin generation. The present study addresses mechanisms of complement-induced COX-2 expression in GEC. Downregulation of protein kinase C (PKC) blunted complement-induced upregulation of COX-2 mRNA. Complement and phorbol 12-myristate 13-acetate (PMA) both stimulated COX-2 promoter activity. C5b-9 activated c-Jun NH(2)-terminal kinase (JNK), and inhibition of JNK activity by transfection of a kinase-inactive JNK1 partially inhibited complement-induced (but not PMA-induced) COX-2 promoter activation. Conversely, a constitutively active mitogen-activated protein or extracellular signal-regulated kinase kinase kinase (MEKK)-1, a kinase upstream of JNK, increased COX-2 promoter activity. MEKK-induced COX-2 promoter activation was not affected by downregulation of PKC and was augmented by PMA. Thus, in GEC, PKC and JNK pathways contribute independently to complement-induced COX-2 expression. Nuclear factor-kappaB was also activated by complement in GEC but did not contribute to complement-induced COX-2 upregulation.

Animals↗
PubMed1998

Antiganglioside GM1 antibodies and their complement activating capacity in central and peripheral nervous system disorders and in controls.

So far, the pathogenic significance and use for diagnosis of antiganglioside GM1 antibodies (anti-GM1) are unclear. We therefore compared serum IgM and IgG antimonosialo ganglioside GM1 levels of 33 patients with presumed immune-mediated neuropathies, 100 patients with various other central or peripheral neurological disorders, and 110 controls by ELISA. We also measured the complement-activating capacity of anti-GM1 by C5b-9-GM1-ELISA to evaluate its value to distinguish between pathogenic and nonpathogenic autoantibodies. Low levels of anti-GM1 were observed in all disease categories and in controls (healthy blood donors). Twenty-four of the controls including the 10 with the highest serum IgM or IgG anti-GM1 were examined for neurological disorders in a double-blind checkup study. In the patients, elevated IgM anti-GM1 levels were predominantly found in those with neuropathies (NP), but barely in patients with central nervous system disease (CNSD). We found elevated IgG anti-GM1 levels predominantly in patients with NP of inflammatory origin (multifocal motor neuropathy, chronic inflammatory demyelinating polyneuropathy or Guillain-Barré syndrome), rarely in patients with NP of noninflammatory origin or CNSD, but not in the control disease group myasthenia gravis (MG). Median levels of IgM-, IgG-, (IgM+IgG)-, and C5b-9-binding anti-GM1 were significantly higher in patients with inflammatory NP as compared to the controls (p < 0.025). In addition, median levels of IgG- and (IgM+IgG)-anti-GM1 were significantly higher in inflammatory NP versus CNSD. Elevated complement-binding activity was associated with low or elevated IgM and/or IgG anti-GM1. Nevertheless, there was a significant correlation between anti-GM1 level (IgM+IgG) and the respective complement-activating capacity (r = 0.758; n = 243). Estimation of anti-GM1 and their respective complement-activating capacity may be helpful in the diagnosis of inflammatory neuropathies. However, neither an elevated anti-GM1 level nor an increased C5b-9 binding seems specific for a given disease category (e.g. peripheral nerve disease) nor a disease process (e.g. demyelination or inflammation).

Adolescent↗
PubMed1991

Induction of mediator release from human glomerular mesangial cells by the terminal complement components C5b-9.

Exposure of cultured human glomerular mesangial cells (GMC) to normal human serum and an activator of the complement system results in rapid uptake of the terminal complement proteins C5b-9 by the cells. This 'innocent bystander' complement attack, however, does not result in cell killing, but in the stimulation of the GMC to release prostaglandin E (PGE), interleukin 1 (Il-1) and tumor necrosis factor (TNF). Endogenously synthesized Il-1 in turn activates PGE release, indicating that the C5b-9 attack initiates an autocrine feedback stimulation. Together with the fact that C5b-9 is found in many forms of glomerulonephritis, the data point to a role of the terminal complement proteins in the initiation and perpetuation of an inflammatory response.

Complement Membrane Attack Complex↗
PubMed1991

In vitro formation of complement activation products by lipopolysaccharide chemotypes of Salmonella minnesota.

We have applied immunoassays for complement activation products C4d, fragment Bb and the protein S-C5b-9 neoantigen (S-MAC) to assess activation of classical, alternative and terminal pathways, respectively, by lipopolysaccharides (LPS) from the smooth strain (SS) of Salmonella minnesota and the shallow rough (core) mutants R60, R345, R5 and R7. Incubations of sera (n = 6) with LPS generated small and insignificant quantities of Bb and S-MAC in the case of Rb, Rc and Rd chemotypes and slightly greater quantities with Ra. SS-LPS brought about significant (p = 0.01) increases in the formation of both Bb and S-MAC. No significant changes were observed in the concentration of C4d. Polymyxin B enhanced Bb and S-MAC production by SS-LPS, optimally at the lowest concentration of polymyxin B studied, 10 ng/ml. These data confirm and extend observations about complement activation by LPS and suggest that immunoassay may be useful in studying mechanisms of complement activation.

Adult↗
PubMed2003

Association between complement factor H and proteoglycans in early human coronary atherosclerotic lesions: implications for local regulation of complement activation.

OBJECTIVE: Complement activation has been suggested to play a role in atherogenesis. To study the regulation of complement activation in human coronary atherosclerotic lesions, we examined the spatial relationships between the major complement inhibitor, factor H, and the complement activation products C3d and C5b-9. METHODS AND RESULTS: In early lesions (American Heart Association types II and III), factor H was immunohistochemically found in the superficial proteoglycan-rich layer in association with numerous macrophages and C3d, whereas C5b-9 was found deeper in the intima, where factor H was virtually absent. In vitro experiments involving surface plasmon resonance and affinity chromatography analyses demonstrated that isolated human arterial proteoglycans bind factor H, and functional complement assays showed that glycosaminoglycans inhibit the complement activation induced by modified low density lipoprotein or by a foreign surface. CONCLUSIONS: The present observations raise the possibility that proteoglycans, because of their ability to bind the major complement inhibitor factor H, may inhibit complement activation in the superficial layer of the arterial intima. In contrast, deeper in the intima, where factor H and proteoglycans are absent, complement may be activated and proceed to C5b-9. Thus, the superficial and the deep layers of the human coronary artery appear to differ in their ability to regulate complement activation.

Adult↗
PubMed1999

Complement and atherogenesis: binding of CRP to degraded, nonoxidized LDL enhances complement activation.

Complement activation occurs in temporal correlation with the subendothelial deposition of LDL during early atherogenesis, and complement also plays a pathogenetic role in promoting lesion progression. Two lesion components have been identified that may be responsible for complement activation. First, enzymatic degradation of LDL generates a derivative that can spontaneously activate complement, and enzymatically degraded LDL (E-LDL) has been detected in the lesions. Second, C-reactive protein (CRP) colocalizes with complement C5b-9, as evidenced by immunohistological studies of early atherosclerotic lesions, so the possibility exists that this acute phase protein also fulfills a complement-activating function. Here, we report that addition of LDL and CRP to human serum did not result in significant C3 turnover. Addition of E-LDL provoked complement activation, which was markedly enhanced by CRP. Binding of CRP to E-LDL was demonstrated by sucrose flotation experiments. Binding was Ca(2+)-dependent and inhibitable by phosphorylcholine, and the complement-activating property of E-LDL was destroyed by treatment with phospholipase C. These results indicated that CRP binds to phosphorylcholine groups that become exposed in enzymatically degraded LDL particles. Immunohistological studies complemented these findings in showing that CRP colocalizes with E-LDL in early human atherosclerotic lesions. Thus enzymatic, nonoxidative modification of tissue-deposited LDL can be expected to confer CRP-binding capacity onto the molecule. The ensuing enhancement of complement activation may be relevant to the development and progression of the atherosclerotic lesion.

C-Reactive Protein↗
PubMedDate not supplied

Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that causes substantial clinical morbidity. Activation of complement during CPB contributes significantly to this inflammatory process. We examined the capability of a novel therapeutic complement inhibitor to prevent pathological complement activation and tissue injury in patients undergoing CPB. METHODS AND RESULTS: A humanized, recombinant, single-chain antibody specific for human C5, h5G1.1-scFv, was intravenously administered in 1 of 4 doses ranging from 0.2 to 2.0 mg/kg before CPB. h5G1.1-scFv was found to be safe and well tolerated. Pharmacokinetic analysis revealed a sustained half-life from 7.0 to 14.5 hours. Pharmacodynamic analysis demonstrated significant dose-dependent inhibition of complement hemolytic activity for up to 14 hours at 2 mg/kg. The generation of proinflammatory complement byproducts (sC5b-9) was effectively inhibited in a dose-dependent fashion. Leukocyte activation, as measured by surface expression of CD11b, was reduced (P<0.05) in patients who received 1 and 2 mg/kg. There was a 40% reduction in myocardial injury (creatine kinase-MB release, P=0.05) in patients who received 2 mg/kg. Sequential Mini-Mental State Examinations (MMSE) demonstrated an 80% reduction in new cognitive deficits (P<0.05) in patients treated with 2 mg/kg. Finally, there was a 1-U reduction in postoperative blood loss (P<0. 05) in patients who received 1 or 2 mg/kg. CONCLUSIONS: A single-chain antibody specific for human C5 is a safe and effective inhibitor of pathological complement activation in patients undergoing CPB. In addition to significantly reducing sC5b-9 formation and leukocyte CD11b expression, C5 inhibition significantly attenuates postoperative myocardial injury, cognitive deficits, and blood loss. These data suggest that C5 inhibition may represent a novel therapeutic strategy for preventing complement-mediated inflammation and tissue injury.

Antibodies, Monoclonal↗