Regulation of P-selectin expression by inflammatory mediators in canine jugular endothelial cells

Canine endothelial cells express the adhesion molecule P-selectin to mediate the initial attachment of leukocytes to the vessel wall. Although it is known that agents like histamine and thrombin stimulate the surface 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-selectin expression has not been investigated. Therefore, the objective of this study was to analyze the regulation of P-selectin messenger RNA (mRNA) and protein by these cytokines in canine endothelial cells isolated from jugular veins. Analyses of cytoplasmic RNA by Northern blotting showed that stimulation of culture endothelial cells with either LPS (100 ng/ml) or recombinant human TNF-alpha (30 U/ml) for 3 or 6 hours significantly increased (P < 0.05) steady-state levels of mRNA for 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 increase for TNF-alpha at 3 and 6 hours, respectively). P-selectin mRNA had decreased by 48 hours to levels found in unstimulated cells. In contrast, human IL-1 beta had no effect on P-selectin mRNA. Increased levels of mRNA with LPS stimulation were associated with the synthesis of new protein, as demonstrated by the positive staining in LPS-stimulated cells using immunocytochemistry with a monoclonal antibody against canine P-selectin (MD3). These results reveal that important inflammatory mediators and cytokines such as LPS and TNF-alpha induce the synthesis of new P-selectin and suggest that this process could represent a means of sustaining local leukocyte recruitment for several hours during an acute inflammatory reaction.