The Principal Eosinophil Peroxidase Oxidant, HOSCN, Is a Uniquely Potent Phagocyte Oxidant Inducer of E-Selectin, ICAM-1 and VCAM-1 Expression on Endothelial Cells and Promotes Leukocyte-Endothelial Cell Adhesion. A Potential Mechanism Regulating Leukocyte Trafficking in Eosinophilic Inflammatory States.

We have shown that SCN- is the principal substrate for EPO in vivo and that its product, HOSCN, a weak sulfhydryl-reactive oxidant, is a singularly effective oxidant inducer of tissue factor activity in human umbilical vein endothelial cells (HUVEC) through activation of the NF-κB transcription factor. Because their genes contain upstream NF-κB binding sites, we hypothesized that HOSCN would induce expression of the cell adhesion molecules E-selectin, ICAM-1 and VCAM-1 and thereby enhance leukocyte-endothelium adhesion. HUVEC monolayers were exposed to various phagocyte-derived oxidants or 10 μg/ml LPS in M199 medium with 10% FCS and assayed by western blotting and flow cytometry. We find that 150 μM HOSCN induces VCAM-1 and ICAM-1 expression starting at 2 h that peaks stably at up to 10-fold from 4–12 h while upregulation of E-selectin is first detectable by 2h, peaks up to 10-fold at 4 hours, then rapidly diminishes to baseline. This induction is dose-dependent and does not occur in the presence of the other phagocyte oxidants HOCl, HOBr and H2O2. HOSCN inducton of adhesion molecule expression is transcriptionally mediated as documented by semi-quantitative RT-PCR analysis. Moreover, HOSCN, but not HOCl, HOBr or H2O2 strongly activates the NF-κB p65/p50 heterodimer assayed by EMSA and cytoplasmic IκB-α degradation assayed by western blotting. To test the functional significance and specificity of these findings we performed neutrophil/HUVEC static adhesion assays with blocking monoclonal antibodies. 150 μM HOSCN induces a 6-fold increase in neutrophil adhesion that is totally blocked by 10 μg/ml of andrographolide, a specific inhibitor of the NF-κB pathway. Adhesion peaks at 4 h then wanes, consistent with the kinetics of E-selectin expression. Neutrophil/HUVEC adhesion was decreased 30–40% by blocking antibodies to either E-selectin or ICAM-1 but not VCAM-1. In an in vivo model of neutrophil rolling and adhesion, mice were intraperitoneally injected with reagent HOSCN (10 nmol/g) or buffer control 2 h before the cremaster muscle was externalized and leukocyte/endothelial interactions in post-capillary venules were quantitated by intravital microscope using a CCD video camera. Peritoneal cavities were lavaged and leukocytes enumerated and identified. HOSCN treatment induces a 4.6-fold (n = 8, p = 0.006) increase in total endothelium-adherent leukocytes but no significant change in leukocyte rolling flux. HOSCN also stimulates a 20-fold increase in peritoneal neutrophils. These findings show that HOSCN is a uniquely potent oxidant inducer of HUVEC E-selectin, ICAM-1 and VCAM-1 expression and promotes neutrophil-HUVEC adhesion. We propose that HOSCN generated by eosinophils adhering to and infiltrating endothelium may stimulate the recruitment and activation of neutrophils, eosinophils and other leukocytes to sites of eosinophilic inflammation and thereby participate in the pathogenesis of allergic diseases and the hypereosinophilic syndrome. The recent unexpected finding that neutrophil myeloperoxidase generates equimolar amounts of HOSCN and HOCl as its principal physiologic products raises the possiblility that neutrophils also utilize HOSCN as an inflammatory amplification mechanism.