Characterization of equine E‐selectin

Abstract

SUMMARYExpression of E-selectin on activated endothelium is a critical initial step that leads to extravasation of leucocytes during in¯ammation, yet E-selectin is largely uncharacterized in several animal species including the horse. We have sequenced and compared E-selectin genes derived from activated cultures of puri®ed equine (horse), cervid (black-tailed deer) and ovine (sheep) pulmonary artery endothelial cells (ECs). Phylogenetic and amino acid sequence comparisons indicate that bovine, cervid and ovine E-selectin are similar, whereas human and equine E-selectin are more closely related to each other than to the ruminant molecules. Human E-and P-selectin-speci®c monoclonal antibodies that also recognize equine E-selectin were identi®ed and used to characterize its expression. Expression of E-selectin was more readily induced by lipopolysaccharide treatment in equine ECs than in human ECs and supported adhesion and activation of neutrophils, consistent with the extreme sensitivity of horses to endotoxaemia and septic shock.

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells

“…f The purified EECs were further characterized by immunofluorescence staining with rabbit antiserum to von Willebrand factor (factor VIII); e by the identification of Weibel-Palade bodies, which are highly characteristic of endothelial cells; and by lipopolysaccharide-induced E-selectin (CD65E) expression, as described. 18 The EECs were maintained in endothelial maintenance medium (Dulbecco' s modified essential medium with sodium pyruvate, g heat-inactivated 10% fetal bovine serum, h antimicrobials, i nonessential amino acids, i and L-glutamine i ). All experiments were performed on EEC cultures between passages 5 and 15.…”

Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells

“…Equine pulmonary artery endothelial cells (EECs [23]) were maintained and propagated in Dulbecco's modified essential medium (DMEM, Mediatech, Herndon, VA) supplemented with sodium pyruvate, 10 % fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 100 U of penicillin and 10 lg of streptomycin per ml and 200 mM L-glutamine (Life Technologies, Grand Island, NY) [23,40]. RK-13 (ATCC CCL-37) and BHK-21 (ATCC CCL-10) cells were cultured and maintained in Eagle's minimum essential medium (EMEM) (Mediatech, Herndon, VA) supplemented with 10 % fetal calf serum (FCS; Hyclone, Logan, UT), 100 U of penicillin, 100 lg of streptomycin, and 1 lg of amphotericin B per ml, and 0.06 % sodium bicarbonate at 37°C.…”

“…RK-13 (ATCC CCL-37) and BHK-21 (ATCC CCL-10) cells were cultured and maintained in Eagle's minimum essential medium (EMEM) (Mediatech, Herndon, VA) supplemented with 10 % fetal calf serum (FCS; Hyclone, Logan, UT), 100 U of penicillin, 100 lg of streptomycin, and 1 lg of amphotericin B per ml, and 0.06 % sodium bicarbonate at 37°C. The experimentally derived highly virulent Bucyrus strain (VBS) of EAV (ATCC VR-796) [38] was propagated in EECs to prepare a high-titered working stock as described previously [23,40]. Briefly, EECs infected with the VBS strain of EAV were frozen at -80°C when 90-100 % cytopathic effect (CPE) was observed.…”

Experiences with infectious cDNA clones of equine arteritis virus: Lessons learned and insights gained

“…In horses, EAV replicates in endothelial cells, blood mononuclear cells, selected epithelial cells, and myocytes (Balasuriya and Snijder, 2008;Del Piero, 2000;MacLachlan et al, 1996). Similarly, EAV replicates in a variety of primary cell cultures including equine pulmonary artery endothelial (Hedges et al, 2001), horse kidney, rabbit kidney, and hamster kidney cells, and a number of continuous cell lines including baby hamster kidney (BHK-21) (Hyllseth, 1969;Maess et al, 1970), rabbit kidney-13 (RK-13), African green monkey kidney (VERO) (Konishi et al, 1975;Radwan and Burger, 1973), rhesus monkey kidney (LLC-MK2), MARC-145, and hamster lung (HmLu) (Konishi et al, 1975) cells. In distinct contrast, PRRSV replicates in only a limited number of cell types that include primary porcine alveolar macrophages (PAM) and the African green monkey cell line, MA-104, or its derivative, CL2621, and MARC-145 (Van Breedam et al, 2010).…”