Measurement of the respiratory burst in human monocytes and polymorphonuclear leukocytes by nitro blue tetrazolium reduction and chemiluminescence

Abstract

We compared measurements of chemiluminescence (CL) assessing the rate of production of oxygen intermediates at a given instance, and of nitro blue tetrazolium (NBT) reduction detecting total superoxide produced during the assay period, for the assessment of the respiratory burst of both human monocytes (Mø) and polymorphonuclear leukocytes (PMN). In the CL experiments, opsonized and non-opsonized zymosan was used to stimulate peripheral blood Mø and PMN. Opsonized zymosan yielded an earlier and 2-fold higher peak response than non-opsonized zymosan. The stimulatory action of zymosan in CL varied with the time of incubation and depended on the concentration of cells and zymosan. No light generation was observed in the absence of viable cells. In contrast to NBT reduction, incubation of PMN with dextran sulfate did not result in increased light generation but rather in a quenching of the response in CL. Opsonized zymosan also yielded a higher level on NBT reduction than non-opsonized zymosan. Comparing NBT reduction with CL in 21 healthy individuals, with 2 × 10 5 Mø and 2.5 × 10 5 PMN for NBT and 5 × 10 5 PMN for CL tests, we found that NBT brings about spontaneous oxidative metabolism, possibly reflecting the intracellular compartment, the chemiluminescent response of resting cells being only marginal. The data suggest higher sensitivity of the CL method. These results provide useful comparative data for 2-established methods used to document the respiratory burst in phagocytic cells.