Induction of E-Selectin by Endotoxin is Direct and not Mediated Through Intracellular or Extracellular Release of Secondary Cytokines

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...