The Major Phagocyte Peroxidase Product HOSCN Stimulates PMN and Eosinophil Adhesion to Endothelium by p38 MAPK-Dependent Enhancement of CD11b/18-ICAM-1 Binding,

Abstract 3205

Hypothiocyanous acid (HOSCN) is the predominant physiologic product of eosinophil peroxidase (EPO) and a major (50%) product of myeloperoxidase (MPO), a product of H2O2 oxidation of the pseudohalide substrate thiocyanate (SCN-). We have previously shown that this sulfhydryl-specific, cell-permeant oxidant, unlike HOCl, can potently induce expression of several NF-kB regulated genes, including the adhesion molecules ICAM-1 and VCAM-1, in human umbilical vein endothelial cells (HUVEC) through a transcriptional mechanism reflecting NF-κB activation. We here tested the hypothesis that HOSCN generated within activated PMN and eosinophils (EO) promotes their adhesion to ICAM-1 and VCAM-1 by enhancing the avidity for their leukocyte integrin counterligands, CD11b/18 (Mac-1) and CD49d/29 (VLA-4), respectively. We isolated human PMN and EO and tested the influence of reagent HOSCN upon their adhesion to purified human ICAM-1 and VCAM-1 immobilized in the bottom of bovine serum albumin (BSA)-blocked of tissue culture plate wells using a method based on crystal violet staining of nuclei and spectrophotometric assay of acetic acid extracts of adherent cells. 3–150 μM HOSCN enhanced adhesion to ICAM-1 of EO 10–18-fold over baseline binding and PMN 2–3-fold. This increase was inhibited >90% by both CBRM1/5, a blocking antibody against CD11b/18 and M1/70, which blocks ICAM-1, showing the specificity of this interaction. Similar concentrations of H2O2 did not significantly influence EO and PMN adhesion. HOSCN did not affect PMN or EO binding to immobilized VCAM-1. Pretreatment of EO with 33–150 μM HOSCN for 15 min prior to washing out HOSCN and layering them over TNF-α-activated HUVEC monolayers for 30 min stimulated a 5–15-fold increase in both EO and PMN adhesion that was > 75% inhibited by blocking antibodies against ICAM-1 and VCAM-1 (EO only) as well as CD11b. Assessing the potential mechanism behind HOSCN enhancing of CD11b/18-dependent EO and PMN adhesion, flow cytometry experiments showed HOSCN did not induce total cell surface expression of EO CD11b/18, but confocal microscopy showed prominent clustering of CD11b 20 min after exposure. Hypothesizing that the “stress” kinase p38 MAPK plays a role in HOSCN promotion of EO adhesion, we probed western blots whole cell lysates of EO exposed 1–10 min to 75 μM HOSCN for activated phospho-p38 MAPK and found a pronounced (10x baseline) but evanescent (1–2 min long) increase starting at 2 min. 1 μM SB203580, a potent and specific p38 MAPK inhibitor, blocked by >80% EO and PMN HOSCN stimulation of adhesion to both immobilized ICAM-1 and activated HUVEC. Collectively, these data show that HOSCN is a uniquely effective phagocyte oxidant inducer of both EO and PMN binding to ICAM-1 by a mechanism depending upon activation of p38 MAPK and enhancement of the CD11b/18-ICAM-1 interaction. This, in turn, suggests that endogenous MPO- and EPO-generated HOSCN may play a hitherto unsuspected physiologic autocrine amplification role in the regulation of adhesion and, hence, inflammation. In view of this, p38 MAPK inhibitors may have promise as therapeutic inhibitors in pathologic human inflammatory states, especially allergic diseases involving EOs.