Factors Associated with Nitric Oxide-mediated {beta}2 integrin Inhibition of Neutrophils [Cell Biology]

June 1st, 2015 by Bhopale, V. M., Yang, M., Yu, K., Thom, S. R.

This investigation explored the mechanism for inhibition of β2 integrin adhesion molecules when neutrophils are exposed to nitric oxide (NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA and cells from knock out mice. Optimal inhibition occurs with exposures to a .NO flux of ~ 28 nmol/min for 2 minute or more, which sets up an auto-catalytic cascade triggered by activating type-2 nitric oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400W), or with cells from mice lacking NOS-2 or the gp91phox component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein crosslinking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator stimulated phosphoprotein, focal adhesion kinase and protein disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The auto-catalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high .NO flux (~ 112 nmol/min) that inactivates NOX.