Relative chlorinating, nitrating, and oxidizing capabilities of neutrophils determined with phagocytosable probes

The capabilities of stimulated neutrophils to initiate intraphagosomal and extracellular chlorination, nitration, and other oxidative reactions has been evaluated using a fluorescent particle and soluble phenolic compounds as target molecules. Neutrophils activated by the soluble stimulus, phorbol myristate acetate, both chlorinated fluorescein that was covalently attached to polyacrylamide microspheres and initiated tyrosine dimerization. When nitrite ion was present at millimolar concentration levels in the medium, nitration of the phenolic rings also occurred; the relative extent of nitration increased as the nitrite concentration was increased. Myeloperoxidase (MPO) also catalyzed nitration and chlorination of fluorescein and the fluorescein-conjugated particles in cell-free solutions; the relative nitration yields increased with increasing [NO2-]/[Cl-] ratios. Nitration did not involve intermediary formation of nitrating agents derived from reaction between MPO-generated HOCl and NO2- because this reaction also occurred in chloride-free media and direct addition of HOCl to solutions containing NO2- and fluorescein gave only chlorinated products. In marked contrast to these extracellular reactions, intraphagosomal nitration of the fluorescein-conjugated particles could not be detected (even at [NO2-] as high as 0.1 M), whereas chlorination of the probe was extensive. These data indicate that intraphagosomal aromatic nitration in neutrophils is negligible, although extracellular nitration of phenolic compounds by secreted MPO could occur at physiological concentration levels of NO2-.