Induction of cyclooxygenase-2 in human synovial cells by β2-microglobulin

Induction of cyclooxygenase-2 in human synovial cells by β2-microglobulin.BackgroundProstaglandins (PGs) are important mediators of inflammation in arthritis. We evaluated the role of the cyclooxygenase-2 (COX-2) enzyme, which regulates PG biosynthesis, in osteoarthropathy associated with hemodialysis-associated amyloidosis (HAA) by characterizing COX-2 expression in β2-microglobulin–treated human synovial cells.MethodsWe examined the effects of β2-microglobulin (β2m), a major constituent protein of amyloid fibrils in HAA, on the COX-2 protein and mRNA expression in human synovial cells using Western blot and reverse transcriptase-polymerase chain reaction.Resultsβ2m selectively increased the biosynthesis of COX-2 protein and induction of COX-2 mRNA in a dose-dependent manner. Immunoabsorption of β2m–containing media by anti-β2m–specific antibody abrogated β2m–mediated COX-2 expression on synovial cells. On the other hand, dexamethasone markedly suppressed the induction of COX-2 protein and mRNA in β2m–stimulated synovial cells.ConclusionsOur results suggest that induction of COX-2 expression by β2m may be an important component of the inflammatory process in hemodialysis-associated osteoarthropathy

Immunolocalization of Adhesion Molecules in Rheumatoid and Osteoarthritic Synovial Tissues

To elucidate the potential role of adhesion molecules in the pathogenesis of rheumatoid arthritis (RA), we stained specimens of synovial tissue from patients with RA and osteoarthritis (OA) with monoclonal antibodies against adhesion molecules using an immunohistochemical method. Positive staining with anti-ICAM-1 monoclonal antibody was detected on the synovial lining cells, the sublining cells and the capillary endothelial cells in the synovium from patients with RA, and, to a lesser degree, in that from patients with OA. The capillary endothelial cells from patients with RA intensively expressed both ELAM-1 and VLA-5α molecules, in contrast to that from OA patients. The intensity of both ICAM-1 and ELAM-1 on the capillary endothelial cells in RA synovium was comparable to disease activity and to the degree of synovial proliferation. A high density of expression of LFA-1α , VLA-4α and VLA-5α was observed on the mononuclear cells that infiltrated the RA synovium, especially in the lesions with aggregated mononuclear cells. The findings clearly demonstrated an up-regulation of the expression of adhesion molecules on synovial cells, capillary endothelial cells and infiltrated mononuclear cells in the synovial tissues of patients with RA. This enhanced expression of adhesion molecules may play an important role in the migration of mononuclear cells into the synovial tissues and thus perpetuate the inflammatory response in these tissues

ウシα1-3 galactosyltransferaseのCOS-7細胞への導入

αGal抗原は異種抗原として知られ,ヒトおよび旧世界サル以外の生物に広汎に存在し,異種移植における超急性拒絶反応の原因抗原と考えられている.このαGal抗原はα1-3 galactosyltransferase (α1-3GT)によって合成される.異種移植の分野では,α1-3GTを様々な方法で修飾することにより,移植可能な動物の作製が試みられている.本実験では,黒毛和牛からα1-3GTを単離し,これをαGal陰性のCOS-7細胞に導入し,我々のクローニングしたウシα1-3GTが機能するかどうかについて検討した.黒毛和牛末梢血から,RT-PCR法を用いてα1-3GTcDNAをクローニングした.これを,ネオマイシン耐性遺伝子を含むベクターに挿入し,αGal抗原発現ベクターを作製した(N3GA). N3GAをリポフェクション法により,αGal抗原陰性であるCOS-7細胞に導入した.N3GA導入翌日にIB4レクチンを用いた蛍光抗体染色を行った.N3GAを導入したCOS-7細胞(COS(+))では,αGal抗原陽性所見を認めた.また,N3GA導入2週間後に,同じくIB4レクチンを用いて行ったflow cytometryでもCOS(+)ではαGal抗原陽性所見を認めた.以上より,我々が黒毛和牛からクローニングしたα1-3GTは酵素機能を有しており,異種細胞においても機能することが示唆された.The polysaccharide antigen, called aGal epitope is ubiquitously found in all animals except for humans and old world monkeys. An αGal epitope is a main xenoantigen which hampers the success of the clinical xenotransplantation. The αGal epitope is synthesized by an enzyme, α1-3 galactosyltransferase (α1-3GT). Thus, disruption of the α1-3GT gene in large animals is one strategy for the successful xenotransplantation. In the present study, we isolated α1-3GT cDNA of Japanese Black Cattle (JBC), and transfected it into an α1-3GT negative COS-7 cell line to demonstrate that the cDNA was functional. The α1-3GT cDNA of JBC was amplified by a polymerase chain reaction. The α1-3GT cDNA was cloned to an expression vectors, named N3GA and transfected into α1-3GT negative COS-7 cells. Cell surface expression of αGal epitope was examined by immunofluorescent staining and flow cytometry with IB4 lectin. Transfected COS-7 cells were positive for the immunoreactive IB4 lectin examined at 24 hours after the transfection. Then, transfected COS-7 cells were selected with neomycin for 10 days. Flow cytometry of COS-7 cells performed 4 weeks after the transfection demonstrated positive log shift for αGal epitope. In conclusion, we isolated α1-3GT cDNA of JBC and obtained stable cell line expressing the gene

ウシα1-3 galactosyltransferaseのCOS-7細胞への導入

αGal抗原は異種抗原として知られ,ヒトおよび旧世界サル以外の生物に広汎に存在し,異種移植における超急性拒絶反応の原因抗原と考えられている.このαGal抗原はα1-3 galactosyltransferase (α1-3GT)によって合成される.異種移植の分野では,α1-3GTを様々な方法で修飾することにより,移植可能な動物の作製が試みられている.本実験では,黒毛和牛からα1-3GTを単離し,これをαGal陰性のCOS-7細胞に導入し,我々のクローニングしたウシα1-3GTが機能するかどうかについて検討した.黒毛和牛末梢血から,RT-PCR法を用いてα1-3GTcDNAをクローニングした.これを,ネオマイシン耐性遺伝子を含むベクターに挿入し,αGal抗原発現ベクターを作製した(N3GA). N3GAをリポフェクション法により,αGal抗原陰性であるCOS-7細胞に導入した.N3GA導入翌日にIB4レクチンを用いた蛍光抗体染色を行った.N3GAを導入したCOS-7細胞(COS(+))では,αGal抗原陽性所見を認めた.また,N3GA導入2週間後に,同じくIB4レクチンを用いて行ったflow cytometryでもCOS(+)ではαGal抗原陽性所見を認めた.以上より,我々が黒毛和牛からクローニングしたα1-3GTは酵素機能を有しており,異種細胞においても機能することが示唆された.The polysaccharide antigen, called aGal epitope is ubiquitously found in all animals except for humans and old world monkeys. An αGal epitope is a main xenoantigen which hampers the success of the clinical xenotransplantation. The αGal epitope is synthesized by an enzyme, α1-3 galactosyltransferase (α1-3GT). Thus, disruption of the α1-3GT gene in large animals is one strategy for the successful xenotransplantation. In the present study, we isolated α1-3GT cDNA of Japanese Black Cattle (JBC), and transfected it into an α1-3GT negative COS-7 cell line to demonstrate that the cDNA was functional. The α1-3GT cDNA of JBC was amplified by a polymerase chain reaction. The α1-3GT cDNA was cloned to an expression vectors, named N3GA and transfected into α1-3GT negative COS-7 cells. Cell surface expression of αGal epitope was examined by immunofluorescent staining and flow cytometry with IB4 lectin. Transfected COS-7 cells were positive for the immunoreactive IB4 lectin examined at 24 hours after the transfection. Then, transfected COS-7 cells were selected with neomycin for 10 days. Flow cytometry of COS-7 cells performed 4 weeks after the transfection demonstrated positive log shift for αGal epitope. In conclusion, we isolated α1-3GT cDNA of JBC and obtained stable cell line expressing the gene

Characterization of CD133+ parenchymal cells in the liver: Histology and culture

AIM: To reveal the characteristics of CD133+ cells in the liver