Regulation of Leukotriene-Biosynthetic Enzymes During Differentiation of Myelocytic HL-60 Cells to Eosinophilic or Neutrophilic Cells

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Abstract
Leukotrienes (LTs) are potent mediators of bronchial inflammation and are predominantly produced by myeloid cells. As myelocytic cells differentiate towards either eosinophils or neutrophils, the profile of leukotrienes they produce upon stimulation diverges. Eosinophils produce mainly cysteinyl leukotrienes whereas neutrophils predominantly synthesize 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB4). The mechanism by which this change in leukotriene composition occurs is unknown. In this study, we investigated the control of leukotriene biosynthetic enzymes during myeloid cell differentiation. Western-blot analyses of myelocytic leukemia cell lines, HL-60#7 and HL-60, differentiated towards eosinophilic or neutrophilic cell types, respectively, demonstrated that as myelocytic cells differentiate towards eosinophils or neutrophils, the protein levels of cytosolic phospholipase A2 (cPLA2) remain constant, whereas 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) levels are simultaneously elevated. As myelocytic cells become more eosinophil-like, 5(S)-hydroxy-6(R)-S -glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4) synthase activity and expression of both the protein and messenger RNA in the cells are dramatically increased (approximately 75-fold), while the LTC4 synthase level and activity in neutrophil-like cells remain constant at very low levels. In contrast, in neutrophilic cells, the amount of 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA4) hydrolase was elevated approximately 100-fold greater than the increase in LTA4 hydrolase from eosinophilic cells. These results indicate that as a myeloid cell differentiates towards a granulocyte, similar mechanisms of regulation may be applied to the leukotriene biosynthetic pathway up to the point at which the pathway diverges. At the stage in the leukotriene biosynthetic pathway where LTA4 may be converted to either LTC4 or to LTB4, specific regulators of transcription may become activated as a myelocytic cell differentiates, thereby causing increased LTA4 hydrolase or LTC4 synthase expression.