Comparative study of respiratory burst induced by phorbol ester and zymosan in human granulocytes

Objectives: We have comparatively evaluated respiratory burst and some steps of its signaling in human granulocytes towards different stimuli. Materials and methods: An alternative method to remove erythrocytes by dextran differential sedimentation was employed. Respiratory burst (RB) was assessed in a flow cytometer by the oxidation of a fluorescent probe (dichlorodihydrofluorescein). Stimuli were phorbol ester (PMA) and zymosan. Results: Granulocytes obtained with dextran sedimentation mounted a normal RB for the two stimuli but cells obtained by erythrocyte lysis were ineffective to respond to zymosan (P < 0.05). EGTA did not affect PMA-induced, but inhibited zymosan-induced RB (P < 0.05). PMA-induced RB was blocked by a protein kinase C inhibitor (P < 0.05), whereas zymosan was not. Microfilament integrity was essential for zymosan but not for PMA, whereas microtubule depolymerization does not seem to be essential for both stimuli. Granulocytes obtained from recently diagnosed leukemia patients responded relatively well to both stimuli but after chemotherapy, the response to zymosan was increased whereas cells were unable to respond to PMA. Conclusion: The use of the proposed method allows the study of both RB and phagocytosis, making it useful to study these granulocyte responses in the clinical and experimental settings.