Induction of endothelial cell decay-accelerating factor by vascular endothelial growth factor: a mechanism for cytoprotection against complement-mediated injury during inflammatory angiogenesis

OBJECTIVE: Decay-accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up-regulated by tumor necrosis factor alpha and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis. METHODS: Human umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement-regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF. RESULTS: Incubation of large- or small-vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48-72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady-state messenger RNA levels and de novo protein synthesis. Although VEGF-induced EC proliferation was inhibited by both p38 and p42/44 mitogen-activated protein kinase (MAPK) antagonists, DAF up-regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF-stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement-mediated EC lysis. These protective effects were abolished by anti-DAF monoclonal antibody 1H4. CONCLUSION: This study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up-regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement-mediated injury during angiogenesis in inflammatory rheumatic diseases.     

Induction of decay-accelerating factor by cytokines or the membrane-attack complex protects vascular endothelial cells against complement deposition.

Vascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.     

Induction of endothelial cell decay‐accelerating factor by vascular endothelial growth factor: A mechanism for cytoprotection against complement‐mediated injury during inflammatory angiogenesis

Objective

Decay‐accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up‐regulated by tumor necrosis factor α and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis.

Methods

Human umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement‐regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF.

Results

Incubation of large‐ or small‐vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48–72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady‐state messenger RNA levels and de novo protein synthesis. Although VEGF‐induced EC proliferation was inhibited by both p38 and p42/44 mitogen‐activated protein kinase (MAPK) antagonists, DAF up‐regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF‐stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement‐mediated EC lysis. These protective effects were abolished by anti‐DAF monoclonal antibody 1H4.

Conclusion

This study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up‐regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement‐mediated injury during angiogenesis in inflammatory rheumatic diseases.

     

Statin-induced expression of decay-accelerating factor protects vascular endothelium against complement-mediated injury

Complement-mediated vascular injury is important in the pathophysiology of atherosclerosis and myocardial infarction. Because recent evidence shows that statins have beneficial effects on endothelial cell (EC) function independent of lipid lowering, we explored the hypothesis that statins modulate vascular EC resistance to complement through the upregulation of complement-inhibitory proteins. Human umbilical vein and aortic ECs were treated with atorvastatin or simvastatin, and decay-accelerating factor (DAF), membrane cofactor protein, and CD59 expression was measured by flow cytometry. A dose-dependent increase in DAF expression of up to 4-fold was seen 24 to 48 hours after treatment. Statin-induced upregulation of DAF required increased steady-state mRNA and de novo protein synthesis. L-Mevalonate and geranylgeranyl pyrophosphate reversed the effect, confirming the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition and suggesting that constitutive DAF expression is negatively regulated by geranylgeranylation. Neither farnesyl pyrophosphate nor squalene inhibited statin-induced DAF expression, suggesting that the effect is independent of cholesterol lowering. Statin-induced DAF upregulation was mediated by the activation of protein kinase Calpha and inhibition of RhoA and was independent of phosphatidylinositol-3 kinase and NO activity. The increased DAF expression was functionally effective, resulting in significant reduction of C3 deposition and complement-mediated lysis of antibody-coated ECs. These observations provide evidence for a novel cytoprotective action of statins on vascular endothelium that is independent of the effect on lipids and results in enhanced protection against complement-mediated injury. Modulation of complement regulatory protein expression may contribute to the early beneficial effects of statins in reducing the morbidity and mortality associated with atherosclerosis.     

Effect of IL-4 on altered expression of complement activation regulators in rat pancreatic cells during severe acute pancreatitis

AIM: To investigate the effect of IL-4 on the altered expression of complement activation regulators in pancreas and pancreatic necrosis during experimental severe acute pancreatitis (SAP). METHODS: SAP model of rats was established by retrograde injection of 5% sodium taurocholate (1 mL/kg) into the pancreatic duct. We immunohistochemically assayed the expression of three complement activation regulators: decay accelerating factor (DAF; CD55), 20 ku homologous restriction factor (HRF20; CD59) and membrane cofactor protein (MCP; CD46), in the pancreatic acinar cells of rats at 0, 3, 6, 12, and 24 h after the induction of SAP model. Meanwhile the levels of amylase and lipase were determined, and morphological examination was performed. Then, 61 rats were randomly divided into three groups. Group A (n = 21) received no treatment after the SAP model was established; group B (n = 20) was given IL-4 (8 μg/animal) intraperitoneally 0,5 h before the SAP model was established; group C (n = 20) was given IL-4 (8 μg/animal) intraperitoneally 0.5 h after the SAP model was established. Plasma amylase and lipase, extent of pancreatic necrosis and expression of complement activation regulators were investigated 6 h after the induction of SAP model. RESULTS: Three complement activation regulators were all expressed in pancreatic acinar cells. MCP was not found on the basolateral surface as reported. Contrary to the gradually increasing plasma level of amylase and lipase, expression of complement activation regulators decreased after SAP model was set up. At the same time, the severity of pancreatic necrosis was enhanced. A strong negative correlation was found between the ex- pression of MCP, DAF, CD59 in pancreatic acinar cells and the severity of pancreatic necrosis (r=-0.748, -0.827, -0.723; P＜0.01). In the second series of experiments, no matter when the treatment of IL-4 was given (before or after the induction of SAP model), the serum level of amylase or lipase was decreased and the extent of pancreatic necrosis was ameliorated significantly. Compared to SAP control group, the expression of DAF and CD59 in pancreas was reinforced when IL-4 was given before the induction of SAP model (P＜0.01, P＜0.05), but the expression of MCP was not influenced (P＞0.05). The expression of DAF was enhanced, when IL-4 was given after the induction of SAP model (P＜0.05), but the expression of CD59 and MCP did not change (P＞0.05). CONCLUSION: Complement activation regulators may participate in the pathogenesis of pancreatic inflammation. Downregulation of complement activation regulators expression may be one of the causes of pancreatic necrosis. IL-4 treatment may control SAP aggravation by enhancing expression of DAF and CD59 in pancreas and decreasing pancreatic necrosis. Moreover, DAF and CD59 may play an important role in the regulation of complement activation regulators during SAP.     

EXPRESSION OF COMPLEMENT REGULATORY MOLECULES AND OTHER SURFACE MARKERS ON NEUTROPHILS FROM SYNOVIAL FLUID AND BLOOD OF PATIENTS WITH RHEUMATOID ARTHRITIS

In an effort to elucidate the activation status of neutrophils(PMN) in inflammatory joint disease the expression of relevantcell surface proteins was examined using immunofluorescenceand flow cytometry. Paired samples of SF and peripheral bloodwere obtained from 18 patients with RA and PMN purified usingmethods designed to minimize activation in vitro. We then usedflow cytometry to measure expression of the four membrane complementregulatory molecules, decay accelerating factor (DAF; CD55),complement receptor 1 (CR1; CD35), membrane cofactor protein(MCP; CD46) and CD59; two adhesion molecules of the integrinfamily LFA1 (     

Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack

Decay-accelerating factor (DAF) and CD59 are 2 glycosylphosphatidylinositol-anchored membrane proteins that inhibit complement activation at the C3 and C5b-9 step, respectively. CD59 is considered critical for protecting erythrocytes from spontaneous complement attack, as deficiency of CD59 or CD59/DAF, but not of DAF alone, on human erythrocytes renders them sensitive to complement lysis in paroxysmal nocturnal hemoglobinuria syndrome. To evaluate the relative roles of CD59 and DAF in vivo, we have generated and studied a CD59 knockout and a CD59/DAF double-knockout mouse. CD59-deficient and CD59/DAF-double-deficient mouse erythrocytes were highly sensitive to antibody-induced complement lysis in vitro, yet neither CD59 knockout nor CD59/DAF double-knockout mouse developed spontaneous hemolytic anemia. Consistent with the latter observation, erythrocytes from the 2 strains of mutant mice were shown to have a normal lifespan in vivo. In contrast, mouse erythrocytes deficient in complement receptor 1 (CR1)-related gene y (Crry), a membrane C3 inhibitor with DAF and membrane cofactor protein activities, were rapidly eliminated from the circulation by a complement-dependent mechanism. Compared with DAF-deficient erythrocytes, Crry-deficient erythrocytes incurred higher levels of spontaneous C3 deposition in vivo. These findings demonstrate that CD59 and DAF are not indispensable on murine erythrocytes. Rather, effective C3 regulation on the cell surface, provided by Crry rather than DAF, is necessary for mouse erythrocytes to resist spontaneous complement attack. Our results raise the possibility that proper control of C3 activation may also be critical on human erythrocytes, where CR1 but not DAF could be the principal regulator of spontaneous C3 activation.     

Expression of cell membrane complement regulatory glycoproteins along the normal and diseased human gastrointestinal tract

Background/Aims—Uncontrolled complementactivation may be of immunopathological importance in inflammatorydiseases of the gastrointestinal tract. Expression of membrane boundfactors that regulate complement activation was therefore studied in situ.
Methods—Frozen tissue specimens were obtained frompatients with Helicobacter pylori gastritis, coeliacdisease, Crohn's disease, or ulcerative colitis, and fromhistologically normal controls. Sections were examined byimmunofluorescence with monoclonal antibodies to protectin (CD59),decay accelerating factor (DAF), and membrane cofactor protein (MCP).
Results—Protectin and MCP were widely expressed innormal and diseased mucosae. MCP was generally observed basolaterallyon all epithelial cells, whereas apical protectin expression was moreintense on the epithelium of normal colonic mucosa than in the normalduodenum (p = 0.001). Epithelial DAF and to some extent protectin wereupregulated in gastritis, coeliac disease, and inflammatory boweldisease. Areas of the stomach with intestinal metaplasia expressed DAF,unlike the adjacent gastric epithelium. Parietal cells of the gastricbody expressed neither protectin nor DAF.
Conclusion—Epithelial complement inhibitorymolecules were expressed differently at various normal gastrointestinalsites and also in association with mucosal disease, suggesting variable protective potential. Such molecules could play a role in the development of gastric atrophy by protecting areas of intestinal metaplasia. Conversely, parietal cells appeared to be potentially vulnerable targets for complement attack.

Keywords:Helicobacter pylori; coeliac disease; Crohn's disease; ulcerative colitis; immunofluorescence; complementregulatory proteins

     

Decay-accelerating factor in the periovulatory rat ovary

One of the most prominent inflammatory reactions is the activation of the complement system. The activated complement system does not distinguish between pathogens and the host cell. In order to prevent autologous complement-mediated attack, host cells express a variety of both membrane-bound and fluid-phase complement regulatory proteins which control activity of the complement cascade by acting on convertase enzymes or the membrane-attack complex. Although the process of ovulation is facilitated by the inflammatory reaction, this reaction has the potential to cause serious damage to growing follicles, ovulated follicles, and other important ovarian tissues. This study was undertaken to characterize the expression and regulation of decay-accelerating factor (DAF), a complement regulator, as a potential mediator of ovarian tissue protection from ovulatory inflammation. DNA microarray and Northern blot analyses showed that an ovulatory gonadotropin stimulus dramatically yet transiently induced DAF mRNA expression in the immature rat ovary. Northern blot and PCR analyses revealed that of the three known DAF isoforms, glycosylphosphatidylinositol (GPI)-, soluble-, and transmembrane-(TM) DAF, GPI-DAF was the predominant form. In situ hybridization localized GPI-DAF mRNA expression in the theca-interstitial cells of the periovulatory ovary. Neither the anti-progestin RU486 nor the cyclooxygenase inhibitor indomethacin significantly inhibited human chorionic gonadotropin (hCG)-induced GPI-DAF mRNA expression in vivo. In vitro theca cell culture studies indicated that hCG induces GPI-DAF mRNA expression through the protein kinase A pathway. This study suggests that gonadotropin-induced GPI-DAF may be involved in the protection of ovarian tissues from the potential attack by the complement system activated by the inflammatory response associated with ovulation.     

Expression of decay-accelerating factor on synovial lining cells in inflammatory and degenerative arthritides

Summary The decay-accelerating factor (DAF) is a complement regulatory cell surface protein that protects cells from complement-mediated lysis. We analysed synovial tissue biopsies from patients with chronic arthritides for the presence of DAF using immunohistochemistry. DAF was expressed in the synovial lining cell layer both in rheumatoid arthritis (RA) and in osteoarthritis (OA). DAF was also on vascular endothelial cells of synovial tissue. A significant correlation was found between the expression of DAF and of HLA-DR in the lining layer, suggesting that DAF may be induced during a local inflammatory response. In addition, C5b-9 terminal complement complexes were found in several DAF-positive cases, suggesting that complement activation might, in itself, induce DAF expression. We propose that the occurrence of DAF may represent a physiological mechanism for local complement regulation in synovial tissue.     

Expression of complement components and inhibitors on platelet microparticles

Platelet microparticles (PMP) are released from activated platelets and play an important role in hemostasis, thrombosis and inflammation. Since platelets were recently found to demonstrate an intrinsic capacity for activating both classical and alternative pathways of the complement system, the present study extended these observations to PMP. PMP were generated by treating platelets with 10 microM A23187 (37 degrees C, 5 min). PMP were identified by flow cytometry, based on size, Annexin V binding, and expression of P-selectin and GPIIb (CD41). PMP expressed gC1qR/p33, a multifunctional cellular protein that was recently described to activate the classical complement cascade. PMP also expressed the classical pathway and contact system regulator, C1 inhibitor (C1-INH), as well as CD55 and CD59. Despite C1-INH expression, PMP supported classical pathway C4 activation in the presence of purified C1 and C4. Moreover, statistically significant deposition of C3b and C5b-9 was detected on PMP exposed to plasma, concurrently with expression of CD55 and CD59. These data provide the first evidence for the ability of PMP to support in situ complement activation. Complement activation contributes to a variety of vascular and inflammatory disease states including atherosclerosis and ischemia/reperfusion injury.     

Expression of complement components and inhibitors on platelet microparticles

Platelet microparticles (PMP) are released from activated platelets and play an important role in hemostasis, thrombosis and inflammation. Since platelets were recently found to demonstrate an intrinsic capacity for activating both classical and alternative pathways of the complement system, the present study extended these observations to PMP. PMP were generated by treating platelets with 10 µM A23187 (37°C, 5 min). PMP were identified by flow cytometry, based on size, Annexin V binding, and expression of P-selectin and GPIIb (CD41). PMP expressed gC1qR/p33, a multifunctional cellular protein that was recently described to activate the classical complement cascade. PMP also expressed the classical pathway and contact system regulator, C1 inhibitor (C1-INH), as well as CD55 and CD59. Despite C1-INH expression, PMP supported classical pathway C4 activation in the presence of purified C1 and C4. Moreover, statistically significant deposition of C3b and C5b-9 was detected on PMP exposed to plasma, concurrently with expression of CD55 and CD59. These data provide the first evidence for the ability of PMP to support in situ complement activation. Complement activation contributes to a variety of vascular and inflammatory disease states including atherosclerosis and ischemia/reperfusion injury.     

Helicobacter pylori eradication decreases the expression of glycosylphosphatidylinositol-anchored complement regulators, decay-accelerating factor and homologous restriction factor 20, in human gastric epithelium

BACKGROUND: It has previously been reported that there is a strong correlation between the expression of glycosylphosphatidylinositol (GPI)-anchored complement membrane inhibitor in gastric epithelium and the severity of inflammation of gastric mucosa. To investigate the regulation of complement activity in gastric epithelium during Helicobacter pylori (H. pylori)-associated gastritis, the expression of GPI-anchored complement membrane inhibitors, decay-accelerating factor (DAF) and 20-kDa homologous restriction factor 20 (HRF20), and membrane cofactor protein (MCP), which is a transmembrane protein, were evaluated after removal of the H. pylori stimulus. Furthermore, the expression of the complement fragment, C3c, was also investigated. METHODS: Forty-six patients with epigastric symptoms and endoscopically confirmed peptic ulcer or gastritis who had H. pylori infection of the gastric mucosa were enrolled in the present study. Biopsy specimens were obtained from the gastric antrum and corpus 1 month before and after eradication. Helicobacter pylori infection was determined by the rapid urease test, histology, and culture before eradication, and by histology, culture, and urea breath test after eradication. Gastric biopsy specimens obtained before and after eradication were evaluated for infiltration by neutrophils and mononuclear cells. The expression of complement membrane inhibitors, DAF, HRF20, and MCP and that of the main complement fragment, C3c, was immunohistochemically evaluated. RESULTS: One month after the eradication of H. pylori, the infiltration by neutrophils and mononuclear cells in the gastric mucosa decreased significantly (P < 0.0001) as compared with that before eradication. The expression of DAF, HRF20, and C3c on gastric mucosal epithelium also significantly decreased in both the antrum and the corpus (P < 0.05) 1 month after eradication. However, no change was observed in the expression of MCP. CONCLUSIONS: The decrease in the expression of GPI-anchored complement regulator and the complement after removal of a chronic microbial stimulus suggests that the gastric epithelium appears to undergo an aggressive stress of complement during H. pylori infection. Conclusively, DAF and HRF20 may play an important protective role against complement-mediated damage induced by chronic microbial stimuli in such a pathological condition.     

The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system. (Blood. 2001;98:1047-1054)     

Endothelial function and hemostasis

The vascular endothelium influences not only the three classically interacting components of hemostasis: the vessel, the blood platelets and the clotting and fibrinolytic systems of plasma, but also the natural sequelae: inflammation and tissue repair. Two principal modes of endothelial behaviour may be differentiated, best defined as an anti- and a prothrombotic state. Under physiological conditions endothelium mediates vascular dilatation (formation of NO, PGI2, adenosine, hyperpolarizing factor), prevents platelet adhesion and activation (production of adenosine, NO and PGI2, removal of ADP), blocks thrombin formation (tissue factor pathway inhibitor, activation of protein C via thrombomodulin, activation of antithrombin III) and mitigates fibrin deposition (t- and scuplasminogen activator production). Adhesion and transmigration of inflammatory leukocytes are attenuated, e.g. by NO and IL-10, and oxygen radicals are efficiently scavenged (urate, NO, glutathione, SOD). When the endothelium is physically disrupted or functionally perturbed by postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension, then completely opposing actions pertain. This prothrombotic, proinflammatory state is characterised by vaso-constriction, platelet and leukocyte activation and adhesion (externalization, expression and upregulation of von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, TNF alpha, etc.), promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall (expression of tissue factor, PAI-1, phosphatidyl serine, etc.) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelet CD40-ligand to endothelial, monocyte and B-cell CD40. Since thrombin formation and inflammatory stimulation set the stage for later tissue repair, complete abolition of such endothelial responses cannot be the goal of clinical interventions aimed at limiting procoagulatory, prothrombotic actions of a dysfunctional vascular endothelium.     

Expression of complement regulatory proteins in diffuse proliferative glomerulonephritis

This study assessed the expression of complement receptor 1 (CR1), decay accelerating factor (DAF) and membrane inhibitor of reactive lysis (CD59) on the erythrocytes and glomerulus of diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE) patients using flow cytometry and immunofluorescence techniques to elucidate their role in the pathogenesis of DPGN. Expression of CR1 on the erythrocytes and glomerulus of DPGN patients was reduced compared with expression in normal subjects. However, expression of DAF and CD59 was increased on both erythrocytes and glomerulus of DPGN patients, suggesting the generation of a protective response against complement-mediated injury.     

Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice

BACKGROUND AND AIMS: The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS: Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS: Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS: Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.     

Congenital Defects in the Expression of the Glycosylphosphatidylinositol-Anchored Complement Regulatory Proteins CD59 and Decay-Accelerating Factor

CD59 and decay-accelerating factor (DAF) are glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins critical for regulating complement activation on the host cell surface. Defective expressions of CD59 or DAF caused by mutations in the genes coding for these proteins or genes involved in the biosynthesis of GPI, such as PIGA, PIT, and PIGM, are associated with various clinical symptoms that are mediated by dysregulated activation of complement, especially the C5 component. Eculizumab, an anti-C5 antibody, is effective in relieving the symptoms seen in patients with complement dysregulation.     

Expression of complement regulatory proteins CR1, DAF, MCP and CD59 in haematological malignancies

Host cells are protected from the lytic effect of the complement system by complement regulatory proteins. This study was designed to investigate the expression of complement regulatory proteins on leukemic blasts which may be susceptible to the lytic effects of the complement system in the circulation. The surface expressions of complement regulatory proteins, complement receptor 1 (CR1, CD35), decay accelerating factor (DAF, CD55), and homologous restriction factor 20 (HRF20, CD59), on peripheral blood and bone marrow blasts were evaluated by using flow cytometry in 16 acute myeloblastic leukemia (AML), 16 acute lymphoblastic leukemia (ALL), 4 chronic lymphocytic leukemia (CLL), 3 chronic myelocytic leukemia (CML) patients and control granulocytes and lymphocytes obtained from 15 healthy volunteers. mRNA expression was investigated by Northern blot analysis. mRNA abundances were calculated after normalization according to 28s rRNA. Surface expressions of CRI and DAF were marginally (p = 0.08 and p = 0.08, respectively) lower in AML, and DAF expression was significantly lower (p=0.0008) in ALL patients in comparison to their normal counterparts. Except from a slight increase that is detected for CD59 in CML patients (p=0.06), there was no significant difference between the surface expressions of CD59 in any of the groups studied. Densitometric analysis of autoradiographs obtained from Northern blots revealed that in AML patients, CR1 mRNA expression were 5.5-fold lower than controls (p=0.06), while DAF mRNA expression was significantly higher (p=0.0046). Furthermore, the mRNA expression of CRI in ALL patients was found significantly lower than in the control group (p = 0.0419). None of the values obtained from the other groups were significantly different from each other. These results suggest that leukemic blasts are protected from the lytic attack of the complement system at all levels, since all of the complement membrane regulatory proteins were expressed in all leukemia types (although at lower amounts in some cases), and it is also possible to use CRI and DAF as differentiation markers in acute leukemias.     

Levels of complement regulatory proteins, CD35 (CR1), CD46 (MCP) and CD55 (DAF) in human haematological malignancies

Levels of the membrane complement regulatory proteins, C3b/C4b receptor (CR1, CD35), membrane cofactor protein (MCP, CD46), and decay-accelerating factor (DAF, CD55), expressed on cells from patients with haematological malignancies and normal subjects were assessed by flowcytometry using the respective monoclonal antibodies (mAbs). All myeloid and most lymphoid leukaemia samples tested were CR1-negative: two of the 42 leukaemia samples expressed minute amounts of CR1. Lack of CR1 in leukaemia cells was confirmed with two mAbs raised against CR1, 31R, and 243R, which recognized different epitopes and induced different degrees of CR1-mediated fluorescent shift on flow-cytometry in granulocytes and erythrocytes. MCP was increased in most chronic myelogenous leukaemia (CML) and chronic lymphocytic leukaemia (CLL), and was also increased in majority of acute nonlymphocytic leukaemia (ANLL), acute lymphocytic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL). Levels of DAF were also high in CML and CLL, and were variable in other types of leukaemia: some were DAF-negative while others expressed extremely high levels of DAF. In CML patients, the high level of MCP and the lack of CR1 were normalized after medical treatment. These results are in agreement with the data obtained with human leukaemia cell lines, and support the hypothesis that CR1 is essentially a differentiated cell antigen and that a high level of MCP reflects some malignant transformation or an immature stage in blood cells.