Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells

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Abstract
The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1μM), in the presence of calcium. These cells produce 12.94±1.8 ng/106 cells of LTC4 and 3.8±0.4 ng/106 cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1μM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91±0.42 ng/106 cells of LTC4 and 0.41 ng±0.06/106 cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5±0.2 ng of LTC4 and 1.45±0.2 ng of LTB4 per 106 cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.