Primitive myeloid cells express high levels of phospholipase A(2) activity in the absence of leukotriene release: selective regulation by stem cell factor involving the MAP kinase pathway.
The activation of phospholipase A(2) (PLA(2)) 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 PLA(2) activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA(2) activity of mobilized human CD34 antigen-positive (CD34(+)) stem cells by quantitation of the extracellular release of (3)H-arachidonate. The PLA(2) 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 PLA(2) activity, whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor alpha had no significant effect. When CD34(+) cells were induced to differentiate, PLA(2) 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 PLA(2) 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 micromol/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 PLA(2) activity primed by SCF, suggesting the involvement of ERK2 and possibly a further signal transduction pathway. Methyl arachidonyl fluorophosphonate (5 micromol/L), a dual inhibitor of i and cPLA(2) 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.
Roberts
P J
PJ
Department of Haematology, University College London Medical School, London, UK. pamela.roberts@ucl.ac.uk
Mollapour
E
E
Watts
M J
MJ
Linch
D C
DC
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D019891:Leukopoiesis / Q000187:drug effects / Q000502:physiology*; D015289:Leukotrienes / Q000502:physiology*; D010741:Phospholipases A / Q000502:physiology*; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology*
D015289:Leukotrienes; D019089:Stem Cell Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A