EXPERIMENTAL-MODEL FOR THE STUDY BY CHEMILUMINESCENCE OF THE ACTIVATION OF ISOLATED EQUINE LEUKOCYTES

The activation of human polymorphonuclear leucocytes (the respiratory burst) can be studied by measuring their chemiluminescent response. Th is technique was adapted to equine leucocytes to investigate the effec ts of cell number, activator concentration, enhancers of chemiluminesc ence, pH, temperature and inhibitors. Leucocytes were isolated from ci trated blood from healthy horses and chemiluminescence was measured wi th a Bio-Orbit luminometer sensitive to 900 nm light. The optimal cell density for the maximal chemiluminescent response ranged from 10(6) t o 10(7) leucocytes 600 mu l(-1). Chemiluminescence increased as a func tion of temperature, and the concentrations of luminol, lucigenin and phorbol myristate acetate (PMA), and was pH related (optimal pH value = 8.0 for lucigenin and 8.5 for luminol). The inhibition of chemilumin escence by 5 x 10(-5)M azide was 88 per cent for luminol and 37 per ce nt for lucigenin. Superoxide dismutase (100 IU) totally inhibited the chemiluminescence response. Approximately 30 per cent variability in c hemiluminescence was observed under the same assay conditions, dependi ng on the origin of the leucocytes. Based on these results, the condit ions selected for the measurement of equine leucocyte chemiluminescenc e were: 10(6) to 10(7) leucocytes 600 mu l(-1) 1 x 10(-6)M PMA, 1 mM l uminol or 0.4 mM lucigenin, physiological pH (7.4) and physiological t emperature (37.8 degrees C). These conditions were similar to those us ed for measuring the chemiluminescent response of human leucocytes.