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Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
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TNF-alpha and IL-1 upregulate membrane-bound and soluble E-selectin through a common pathway

C W Wyble1, K L Hynes, J Kuchibhotla

  • 1Department of Surgery, University of Chicago, MC 5029, 5841 S. Maryland Ave., Chicago, Illinois 60637, USA.

The Journal of Surgical Research
|February 7, 1998
PubMed
Abstract

Insights

Soluble E-selectin, released from endothelial cells, can bind neutrophils in the bloodstream, reducing inflammation. This study shows shedding of E-selectin receptors, not new synthesis, is the source of soluble E-selectin.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • E-selectin mediates neutrophil (PMN) capture in microcirculation, initiating inflammation.
  • Soluble E-selectin (sE-selectin) can bind circulating PMN, reducing tissue adhesion.

Purpose of the Study:

  • Characterize molecular response to cytokines (TNF-alpha, IL-1).
  • Investigate the balance between cell surface-bound and soluble E-selectin.
  • Determine the source of soluble E-selectin.

Main Methods:

  • Cultured human umbilical veins treated with TNF-alpha or IL-1.
  • Analyzed E-selectin mRNA (Northern blot), cell surface expression (flow cytometry), and sE-selectin release (ELISA).
  • Investigated transcriptional regulation using Raf kinase dominant-negative transfection.

Main Results:

  • Cytokines induced E-selectin mRNA and cell surface expression, peaking at 6h.
  • Soluble E-selectin levels increased later, starting at 12h and continuing to 24h.
  • Raf kinase inhibition reduced both surface and soluble E-selectin expression.

Conclusions:

  • Late increases in sE-selectin accompanied by decreases in cell surface E-selectin indicate shedding of receptors.
  • The primary source of circulating E-selectin is shed receptors, not new synthesis.
  • Enhancing E-selectin shedding may offer therapeutic benefits by reducing PMN adhesion.

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