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HUVEC exposed to IL-1 alpha, TNF alpha or LPS showed a time dependent increase in E-selectin expression which was maximal at between 4-6h after stimulation. Stimulation of HUVEC with IL-1 alpha, TNF alpha or LPS for between 2 and 6h followed by removal or neutralisation of IL-1 alpha, TNF alpha or LPS and incubation in new media up to 6h resulted in identical levels of E-selectin expression at 6h, as cells which had been continuously stimulated for 6h with IL-1 alpha, TNF alpha or LPS. These studies demonstrated that HUVEC were committed to the induction of E-selectin following a 2 hr incubation with either IL-1 alpha, TNF alpha or LPS. The protein tyrosine kinase (PTK) inhibitors ST271, ST638 or genistein (0-100M) were ineffective in reducing cytokine or LPS stimulated E-selectin expression during a 2h cytokine or LPS stimulation of cells, in which inhibitors were either coincubated with cytokine/LPS or previously preincubated with the PTK inhibitors. However when PTK inhibitors were present during both agonist activation (2h) and subsequent expression of E-selectin after removal of agonist (4h) the PTK inhibitors resulted in a dose dependent reduction in both IL-1 alpha and LPS stimulated E-selectin expression (IC50 = 50M). Moreover when PTK inhibitors were only incubated with cells for the 4h after cytokine or LPS activation of cells, PTK inhibitors resulted in a more effective dose dependent reduction in IL-1 alpha or LPS stimulated E-selectin expression (IC50 = 10M). Determination of total and surface expressed E-selectin showed that the reduction in E-selectin expression represented a reduction in E-selectin protein and analysis of E-selectin mRNA by RT-PCR demonstrated that inhibition of E-selectin protein synthesis reflected reduced E-selectin mRNA. Other cytokine or LPS signalling pathways such as the activation of MAP-kinase (ERK-2) was unaffected by pre and coincubation with the PTK inhibitors. These studies suggest that HUVEC can become committed to the induction of E-selectin after removal of the stimulus and that protein tyrosine kinase inhibitors do not effect initial (5-30 min) cytokine or LPS signals which result in E-selectin expression but can inhibit the expression of cytokine/LPS induced E-selectin expression at a step distal to MAP-kinase activation.

Using a fixed cell ELISA system E-selectin expression and adhesion of U937 cells were measured in both endotoxin (LPS: serotype 0111:B4) tregted and untreated cultures of human umbilical vein endothelial cells (HUVEC). Basal levels of E-selectin were unde-tectable whereas significant induction of both E-selectin and U937 cell adhesion were observed after exposure of HUVEC to LPS for 6h. The effect of LPS was serum dependent and unaffected by coincubation with neutralising antibodies to either IL-1α, IL-1β or TNFα either alone or in combination, even though each antibody was capable of neutralising the effect of exogenously added cytokine. In addition IgG fractions of neutralising anti-cytokine antibodies were purified, concentrated and microinjected into the cytosol of adherent HUVEC prior to treatment with 1 μg/ml LPS for 6 hrs. Immunofluorescence staining showed that cells microinjected with antibodies to IL-1α, IL-1β and TNFα either alone or in combination were positive for LPS-stimulated E-selectin, d...