Effects of Transfected Complement Regulatory Proteins, McP, Daf, and McP/Daf Hybrid, on Complement-Mediated Swine Endothelial Cell Lysis

Miyagawa, Shuji; Shirakura, R; Iwata, Kazunori; Nakata, S; Matsumiya, Goro; Izutani, Hironori; Matsuda, Hikaru; Terado, Atsuko; Matsumoto, Misako; Nagasawa, Shigeharu; Seya, Tsukasa

doi:10.1097/00007890-199410150-00015

Cited by 43 publications

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“…The kinetics of DAF expression were analyzed in greater detail because DAF seems to be the most useful molecule in terms of protecting cells from complement-mediated injury. 16 The temporal changes observed in DAF mRNA and protein expression after culture with CRP were similar to the changes reported after stimulation with vascular endothelial growth factor, 17 tumor necrosis factor-␣, and interferon-␥. 18 Our data suggest that CRP-induced expression of DAF depends on the synthesis of an intermediate transcriptional activator and that the observed increase in DAF expression is functionally relevant, as indicated by the reduction in complement-mediated cell lysis.…”

Section: Discussionsupporting

confidence: 72%

C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells

Szmitko

Weisel

et al. 2004

Circulation

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Background-Because complement-mediated vascular injury participates in atherosclerosis and C-reactive protein (CRP) can activate the complement cascade, we sought to determine whether CRP affects the expression of the protective complement-inhibitory factors on the cell surface of endothelial cells (ECs). Methods and Results-Human coronary artery or human saphenous vein ECs were incubated with CRP (0 to 100 g/mL, 0 to 72 hours), and the expression of the complement-inhibitory proteins decay-accelerating factor (DAF), membrane cofactor protein (CD46), and CD59 were measured by flow cytometry. Incubation with CRP resulted in a significant increase in the expression of all 3 proteins. CRP-induced upregulation of DAF required increased steady-state mRNA and de novo protein synthesis. The increased expression of complement-inhibitory proteins was functionally effective, resulting in significant reduction of complement-mediated lysis of antibody-coated human saphenous vein ECs. Conclusions-These observations provide evidence for a possible protective role for CRP in atherogenesis. Key Words: endothelium Ⅲ atherosclerosis Ⅲ proteins T he inflammatory marker C-reactive protein (CRP) represents one of the strongest independent predictors of vascular death in several settings. 1,2 Originally suggested to play the role of a biomarker, CRP seems to be a mediator of atherosclerosis. 3 CRP elicits a multitude of effects on endothelial biology that favor a proatherosclerotic phenotype, such as decreasing NO release, 4 upregulating adhesion molecules, 5 stimulating vascular smooth muscle cell proliferation and migration, 6 and activating the complement system. 7 The complement system is a complex cascade of enzymes and regulatory proteins that normally participate in host defenses against microorganisms via opsonization, chemoattraction of leukocytes, cell lysis, and cell activation. 8 However, complement activation also has been proposed to participate in both the initiation and progression of atherosclerosis. 9 This is supported by the presence of activated complement components in atherosclerotic plaques, such as the membrane attack complex (MAC, C5b-9), which promotes cellular activation, upregulates adhesion molecules, stimulates chemokine secretion, and can cause cell lysis. 10 To prevent host cell damage, nucleated cells have developed membrane-bound regulators of complement activation. Among these regulatory proteins is decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), which binds to and facilitates the degradation of C3b and C4b, and CD59, which inhibits C5b-9. 11 DAF prevents the formation and accelerates the decay of the C3 and C5 convertases that act early within the complement cascade, 11 functioning to maintain vascular integrity as a key protector against complement-mediated cell lysis. 12 The classical pathway of complement may be activated by CRP bound to enzymatically degraded low-density lipoprotein. 13 The effect of CRP on the expression of complementinhibitory factors is unknow...

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Section: Discussionsupporting

confidence: 72%

C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells

Szmitko

Weisel

et al. 2004

Circulation

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“…showed a substantial inhibitory effect in complement inhibition but none at all in NK cell regulation. In our previous study, we concluded that PEC is lysed by human complement mainly by the classical pathway of complement activation (13). Therefore, there is some possibility that the DAF function in the alternative pathway of complement or the C3b binding site is related to the NK regulatory function or DAF-NK cell interaction.…”

Section: Discussionmentioning

confidence: 96%

“…A pig endothelial cell (PEC) line, MYP30, and a pig fibroblast cell line, D3, provided by the Animal Engineering Research Institute (Ibaraki, Japan), were cultured in DMEM containing 10% FBS with L-glutamine and kanamycin/amphotericin B (13). The human NK like cell line, YT cells, kindly provided by Drs.…”

Section: Cell Culturementioning

confidence: 99%

“…This assay was performed according to a previously described method, using a Kyokuto MTX-LDH kit (Kyokuto Pharmaceutical, Kyokuto, Japan) according to the manufacturer's recommended protocol (13). The transfected cells were plated at a concentration of 2 ϫ 10 4 per well in a flat-bottom gelatin-coated 96-well plate, 1 day before assay.…”

Section: Complement-mediated Cytotoxicity Assaymentioning

confidence: 99%

“…The spontaneous release of LDH activity from the target cells was Ͻ5% of the maximal release of LDH activity, as determined from a complete lysate by sonication. The results are expressed as the percent of specific lysis (13).…”

Section: Complement-mediated Cytotoxicity Assaymentioning

confidence: 99%

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Delta-Short Consensus Repeat 4-Decay Accelerating Factor (DAF: CD55) Inhibits Complement-Mediated Cytolysis but Not NK Cell-Mediated Cytolysis

Miyagawa

Kubo

Matsunami

et al. 2004

The Journal of Immunology

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NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147·F148 to SS and KKK125–127 to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2–4 is required but no relation to its complement regulatory function exists.

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Transgenic pigs expressing human decay‐accelerating factor regulated by porcine MCP gene promoter

Murakami¹,

Nagashima

Takahagi³

et al. 2002

Molecular Reproduction Devel

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Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.

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Effects of Transfected Complement Regulatory Proteins, McP, Daf, and McP/Daf Hybrid, on Complement-Mediated Swine Endothelial Cell Lysis

Cited by 43 publications

References 0 publications

C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells

C-Reactive Protein Upregulates Complement-Inhibitory Factors in Endothelial Cells

Delta-Short Consensus Repeat 4-Decay Accelerating Factor (DAF: CD55) Inhibits Complement-Mediated Cytolysis but Not NK Cell-Mediated Cytolysis

Transgenic pigs expressing human decay‐accelerating factor regulated by porcine MCP gene promoter

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