REGULATION OF P-SELECTIN EXPRESSION BY INFLAMMATORY MEDIATORS IN CANINE JUGULAR ENDOTHELIAL-CELLS

Canine endothelial cells express the adhesion molecule P-selectin to m ediate the initial attachment of leukocytes to the vessel wall. Althou gh it is known that agents like histamine and thrombin stimulate the s urface expression of P-selectin, the effect of inflammatory mediators and cytokines such as lipopolysaccharides (LPS), tumor necrosis factor -alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) on canine P-sel ectin expression has not been investigated. Therefore, the objective o f this study was to analyze the regulation of P-selectin messenger RNA (mRNA) and protein by these cytokines in canine endothelial cells iso lated from jugular veins. Analyses of cytoplasmic RNA by Northern blot ting showed that stimulation of cultured endothelial cells with either LPS (100 ng/ml) or recombinant human TNF-alpha (30 U/ml) for 3 or 6 h ours significantly increased (P < 0.05) steady-state levels of mRNA fo r P-selectin (3.8- +/- 1.0- and 3.0- +/- 0.4-fold increase for LPS at 3 and 6 hours, respectively, and 2.5 +/- 0.8 and 2.7 +/- 0.9-fold incr ease for TNF-alpha at 3 and 6 hours, respectively). P-selectin mRNA ha d decreased by 48 hours to levels found in unstimulated were associate d with the synthesis of new protein, as demonstrated by the positive s taining in LPS-stimulated cells using immunocytochemistry with a monoc lonal antibody against canine P-selectin (MD3). These results reveal t hat important inflammatory mediators and cytokines such as LPS and TNF -alpha induce the synthesis of new P-selectin and suggest that this pr ocess could represent a means of sustaining local leukocyte recruitmen t for several hours during an acute inflammatory reaction.