Activation of Human Microvascular Endothelial Cells with TNF-Alpha and Hypoxia/Reoxygenation Enhances NK-cell Adhesion, but not NK-Cytotoxicity

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Abstract

Background.

Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-α and hypoxia/reoxygenation in an in vitro model of I/R.

Methods.

MVEC were stimulated in vitro for 8 h with TNF-α, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using 51Cr-release assays.

Results.

Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-α but unaffected by hypoxia/reoxygenation in the absence of TNF-α. ICAM-1 expression was further increased by a combination of TNF-α and hypoxia/reoxygenation, whereas TNF-α-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-α and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-α and slighty reduced by hypoxia/reoxygenation.

Conclusions.

Endothelial activation with TNF-α, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

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