EFFECTS OF PROTEIN-TYROSINE KINASE INHIBITORS ON CYTOKINE-INDUCED ADHESION MOLECULE EXPRESSION BY HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS

1 Endothelial cells can be stimulated by the pro-inflammatory cytokine s interleukin (IL)-1 alpha and tumour necrosis factor (TNF)alpha to ex press the leukocyte adhesion molecules E-selectin, vascular cell adhes ion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression ar e incompletely understood. We have investigated the role of protein ty rosine kinases (PTK) in adhesion molecule expression by cytokine-activ ated human umbilical vein endothelial cells (HUVEC) using the PTK inhi bitors genistein and herbimycin A, and the protein tyrosine phosphatas e (PTP) inhibitor sodium orthovanadate. 2 Maximal E-selectin expressio n induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml(-1) ) and TNF alpha (100 u ml(-1)) was dose-dependently inhibited by genis tein and herbimycin A. Although similar effects were seen on phorbol 1 2-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibit ors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at conce ntrations which maximally inhibited PMA-induced expression. 3 Genistei n inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expressi on induced by 24 h incubation with either of these cytokines. Herbimyc in A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4 Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependen tly enhanced by sodium orthovanadate. In contrast, vanadate differenti ally affected TNF alpha-induced expression of these molecules with max imal E-selectin and ICAM-1 expression being slightly enhanced and VCAM -1 expression dose-dependently reduced. 5 We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell li ne U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibite d by genistein and herbimycin A but unaffected by daidzein. Adhesion o f U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited b y anti-VCAM-1. 6 Sodium orthovanadate had no effect on TNF alpha-induc ed U937 adhesion but dose-dependently enhanced adhesion to unstimulate d HUVEC. Vanadate-induced adhesion was inhibited by an antibody agains t VCAM-1. 7 These results demonstrate that PTK-mediated phosphorylatio n events are important for the regulation of adhesion molecule express ion by human endothelial cells, and additionally show that PTK inhibit ors differentially affect upregulation of different adhesion molecules , implicating divergent regulatory pathways for cytokine-induced adhes ion molecule expression.