TNF-ALPHA AND IL-1 UP-REGULATE MEMBRANE-BOUND AND SOLUBLE E-SELECTIN THROUGH A COMMON PATHWAY

Background: Endothelial cell adhesion molecules such as E-selectin pro mote the capture of neutrophils (PMN) in the microcirculation and init iate the inflammatory response. In contrast, when ''shed'' into the mi crocirculation, soluble E-selectin can bind PMN in the blood stream, r educing the number available for adhesion to injured tissue. These exp eriments were designed to better characterize the molecular response t o cytokines and the balance between cell surface (bound) and soluble ( unbound) E-selectin. Methods: Cultured human umbilical veins, exposed to human recombinant TNF-alpha or IL-1 (10 pg/ml), were analyzed for E -selectin mRNA induction (Northern blot), E-selectin cell surface expr ession (flow cytometry), and sE-selectin release (ELISA). Transcriptio nal regulation was analyzed via Raf kinase dominant negative gene tran sfection. Results: E-selectin mRNA expression was markedly increased a t 2 h and sustained through 8 h. No further induction was noted at 12 h. Upregulation of cell surface E-selectin was noted (mean fluorescenc e) as early as 2 h for TNF-alpha (baseline, 12.28 +/- 1.32; TNF-alpha, 23.03 +/- 1.81) or 4 h for IL-1 (baseline, 12.28 +/- 1.32; IL-1, 70.0 0 +/- 3.04) with maximum expression at 6 h (TNF-alpha, 118.8 +/- 15; I L-1, 94.11 +/- 9.34). Expression returned to baseline levels by 24 h, Soluble E-selectin (ng/ml) assays demonstrated later increases beginni ng at 12 h (TNF-alpha, 0.313 +/- 0.077; IL-1, 0.159 +/- 0.075) and con tinuing through 24 h (TNF-alpha, 0.340 +/- 0.062; IL-1, 0.157 +/- 0.03 0). Transfection of endothelial cells with Raf kinase 301 dominant neg ative gene resulted in proportionate decreases in the peak expression in both surface (bound) E-selectin (TNF-alpha, 51.7%; IL-I, 29.6%) and sE-selectin (TNF-alpha, 49.2%; IL-1, 34.5%). Conclusion: The temporal sequence of late decreases in cell surface E-selectin accompanied by increases in soluble E-selectin indicates that the source of E-selecti n in the microcirculation is shed receptors rather than synthesis of a different type of receptor. Enhancement of such ''shedding'' may decr ease PMN adhesion to injured tissue and have therapeutic potential. (C ) 1997 Academic Press.