TNF-alpha and IL-1 upregulate membrane-bound and soluble E-selectin through a common pathway.
BACKGROUND
Endothelial cell adhesion molecules such as E-selectin promote the capture of neutrophils (PMN) in the microcirculation and initiate the inflammatory response. In contrast, when "shed" into the microcirculation, soluble E-selectin can bind PMN in the blood stream, reducing the number available for adhesion to injured tissue. These experiments were designed to better characterize the molecular response to 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 expression (flow cytometry), and sE-selectin release (ELISA). Transcriptional regulation was analyzed via Raf kinase dominant negative gene transfection.


RESULTS
E-selectin mRNA expression was markedly increased at 2 h and sustained through 8 h. No further induction was noted at 12 h. Upregulation of cell surface E-selectin was noted (mean fluorescence) 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, 70.00 +/- 3.04) with maximum expression at 6 h (TNF-alpha, 118.8+/-15; IL-1, 94.11 +/- 9. 34). Expression returned to baseline levels by 24 h. Soluble E-selectin (ng/ml) assays demonstrated later increases beginning at 12 h (TNF-alpha, 0.313 +/- 0.077; IL-1, 0.159 +/- 0.075) and continuing through 24 h (TNF-alpha, 0.340 +/- 0.062; IL-1, 0.157 +/- 0.030). Transfection of endothelial cells with Raf kinase 301 dominant negative gene resulted in proportionate decreases in the peak expression in both surface (bound) E-selectin (TNF-alpha, 51. 7%; IL-1, 29.6%) and sE-selectin (TNF-alpha, 49.2%; IL-1, 34.5%).


CONCLUSIONS
The temporal sequence of late decreases in cell surface E-selectin accompanied by increases in soluble E-selectin indicates that the source of E-selectin in the microcirculation is shed receptors rather than synthesis of a different type of receptor. Enhancement of such "shedding" may decrease PMN adhesion to injured tissue and have therapeutic potential.
Wyble
C W
CW
Department of Surgery, University of Chicago, MC 5029, 5841 S. Maryland Ave., Chicago, Illinois 60637, USA.
Hynes
K L
KL
Kuchibhotla
J
J
Marcus
B C
BC
Hallahan
D
D
Gewertz
B L
BL
D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019040:E-Selectin / Q000235:genetics* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D019040:E-Selectin; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019908:Proto-Oncogene Proteins c-raf