Cw. Wyble et al., TNF-ALPHA AND IL-1 UP-REGULATE MEMBRANE-BOUND AND SOLUBLE E-SELECTIN THROUGH A COMMON PATHWAY, The Journal of surgical research, 73(2), 1997, pp. 107-112
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.