title: Characterization of bovine immortalized luteal endothelial cells: action of cytokines on production and content of arachidonic acid metabolites.
abstract: BACKGROUND
  The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.
  
  
  METHODS
  The primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated.
  
  
  RESULTS
  We received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release.
  
  
  CONCLUSIONS
  TNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.
authors.affiliation: Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland. a.korzekwa@pan.olsztyn.pl
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Korzekwa
authors.forename: Gabriel
authors.initials: G
authors.lastname: Bodek
authors.forename: Joanna
authors.initials: J
authors.lastname: Bukowska
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Blitek
authors.forename: Dariusz J
authors.initials: DJ
authors.lastname: Skarzynski
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000502:physiology*; D016207:Cytokines / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000096:biosynthesis; D004851:Epoxide Hydrolases / Q000096:biosynthesis; D005260:Female; D005982:Glutathione Transferase / Q000096:biosynthesis; D006911:Hydroxyprostaglandin Dehydrogenases; D007371:Interferon-gamma / Q000494:pharmacology*; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis; D008184:Luteal Cells / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016207:Cytokines; D019332:Endothelin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D007371:Interferon-gamma; D006911:Hydroxyprostaglandin Dehydrogenases; C031051:prostaglandin-F synthase; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases; C041116:leukotriene-C4 synthase; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; C043134:leukotriene A4 hydrolase