Primitive Myeloid Cells Express High Levels of Phospholipase A2 Activity in the Absence of Leukotriene Release: Selective Regulation by Stem Cell Factor Involving the MAP Kinase Pathway

Abstract

The activation of phospholipase A2 (PLA2) with release of eicosanoids and prostanoids in mature myeloid cells and the augmentation (priming) of this activity by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) are central to the inflammatory process. Yet, there are few data concerning PLA2 activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA2activity of mobilized human CD34 antigen-positive (CD34+) stem cells by quantitation of the extracellular release of3H-arachidonate. The PLA2 activity of CD34+ cells stimulated with calcium ionophore (A23187) was of similar magnitude to that of mature neutrophils and monocytes. Preincubation of CD34+ cells with stem cell factor (SCF) before A23187-stimulation resulted in primed PLA2 activity, whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor  had no significant effect. When CD34+ cells were induced to differentiate, PLA2 activity remained responsive to SCF for several days, but after 8 days, at which stage morphological and functional evidence of maturation was occurring, priming of PLA2 by SCF could no longer be elicited, whereas responses to GM-CSF and IL-3 had developed. The further metabolism of arachidonic acid to eicosanoids by CD34+ cells was not detected by either thin-layer chromatography, enzyme immunoassay, or differential spectroscopy. SCF stimulated the rapid but transient activation of ERK2 (p42 MAP kinase) in CD34+ cells, and we used the MAP kinase kinase inhibitor, PD 098059, which at 30 μmol/L blocks ERK2 activation in CD34+ cells, to investigate whether SCF-mediated priming of arachidonate release was mediated by this kinase. PD 098059 only partially inhibited A23187-stimulated PLA2 activity primed by SCF, suggesting the involvement of ERK2 and possibly a further signal transduction pathway. Methyl arachidonyl fluorophosphonate (5 μmol/L), a dual inhibitor of i and cPLA2 isoforms, completely inhibited arachidonate release without affecting ERK2 activation, demonstrating the lack of cellular toxicity. These data provide the first evidence that primitive myeloid cells have the capacity to release arachidonate, which is regulated by an early acting hematopoietic growth factor important for the growth and survival of these cells.