NEUTRALIZATION OF TNF BY THE ANTIBODY CA2 REVEALS DIFFERENTIAL REGULATION OF ADHESION MOLECULE EXPRESSION ON TNF-ACTIVATED ENDOTHELIAL-CELLS

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studi ed, but the reversibility of their expression has not been well charac terized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell ad hesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM -1) on cultured human umbilical vein endothelial cells (HUVECs), Addit ion of cA2 following TNF stimulation of HUVECs enhanced the rate of E- selectin and VCAM-1 down-regulation from the cell surface and also red uced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule an d not microfilament dependent, Neutralization of TNF only slightly red uced ICAM-1 cell surface levels following initial TNF stimulation, sug gesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-s electin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell s urface expression were similarly partially inhibited, however, E-selec tin levels were unaffected, presumably due to the dual, opposing effec t of inhibiting protein expression and inhibiting internalization. Mic rofilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E -selectin or ICAM-1. These data support the notion that E-selectin, VC AM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of t heir ability to reduce the levels of pre-existing adhesion proteins.