A rapid microtiter plate method for the detection of lysozyme release from human neutrophils

An improved method was devised to measure lysozyme secreted from human neutrophils [polymorphonuclear leukocyte (PMN)] using a microtiter plate reader capable of analyzing enzyme kinetics. The assay is an adaptation of the classical photometric method which detects changes in the turbidity of a bacterial suspension, Micrococcus lysodeikticus, caused by the enzymatic activity of lysozyme. A standard curve using chicken egg white lysozyme was generated, and activity was detectable between the range of 1 and 100 ng/ml. Leukotriene B4 (LTB4)-induced lysozyme release from human PMN was comparable in both the standard assay and the microtiter plate adaptation with EC50 values of 6.5 and 7.2 nM, respectively. Other select stimuli and their receptor antagonists were also used to evaluate the method. Dose-response curves for chemotactic hexapeptide (CHP), recombinant human C5a (rhC5a), and platelet-activating factor (PAF) resulted in EC50 values of 0.14, 0.80, and 542.00 nM, respectively. Inhibition of lysozyme release was studied using receptor antagonists N-t-Boc-L-methionyl-L-leucyl-L-phenylalanine (N-t-Boc), LY223982, and protamine, which are putative inhibitors of formyl peptides (i.e., CHP), LTB4, and C5a, respectively. N-t-Boc inhibited CHP-induced (0.2 nM) enzyme release with an IC50 of 2 microM; LY223982 blocked LTB4-induced (20 nM) release resulting in an IC50 of 52 nM; and protamine inhibited rhC5a-induced (1.5 nM) release with an IC50 of 2 microM. Further studies revealed that CHP, LTB4, and rhC5a were selectively inhibited by their respective antagonists, albeit LY223982 and protamine were also weak inhibitors of CHP and LTB4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)