Effect of the protein phosphatase inhibitors, okadaic acid and calyculin A, on dexamethasone-mediated inhibition of neutrophil apoptosis.

The onset of constitutive cell death by apoptosis in human neutrophils can be delayed by treatment with glucocorticosteroids such as dexamethasone [ 1-31. The mechanisms underlying this steroid-mediated prolongation of neutrophil survival are however unknown. Okadaic acid and calyculin A, specific and potent inhibitors of protein phosphatases 1 and 2A, have been shown to influence the rate of apoptosis in different cell types [4, 51. Moreover, protein dephosphorylation has been implicated as an essential step for glucocorticoid-mediated apoptosis in T-cell hybridomas [6]. In this study, okadaic acid and calyculin A were used to investigate whether phosphorylation/dephosphorylation plays a role in the intracellular signalling pathways controlling dexamethasone-mediated inhibition of neutrophil apoptosis. Human neutrophils were isolated from the peripheral blood of healthy volunteers, as described previously [7]. Harvested neutrophils were resuspended in Iscove's DMEM supplemented with 5% autologous serum and 50 U/ml penicillin and streptomycin, and finally cultured at 37 OC in a humidified 5% C02-rich atmosphere. Neutrophils (3 x 10s) were incubated in the presence of various concentrations of okadaic acid (0.5 nM 500 nM) and calyculin A (1 nM 100 nM) both alone and in combination with dexamethasone (1 pM), in a final volume of 150 pl in flat-bottomed 96 well flexiplates. After 20h in culture, neutrophils were harvested for assessment of morphology, viability (trypan blue exclusion) and recovery. Cytocentrifuge preparations of duplicate cell samples in each experiment were prepared, fixed in methanol, and stained with Diff-Quick. Slides were examined microscopically to determine the number of cells with apoptotic morphology in five fields of view (at least 500 cells were counted) on each preparation. Results were expressed as mean f SEM for each preparation and analysed by analysis of variance followed by Student-Newman-Keuls post test. When p<0.05 (*), the results were considered to be significant. Figure 1 demonstrates that okadaic acid (100 nM) had no