Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.