Cellular Signalling Vol. 3. No. 6. pp. 635-644. 1991. 0898-6568/91 $3.00+.00 Printed in Great Britain. © 1991 Pergamon Press plc PRIMING OF CALCIUM MOBILIZATION IN HUMAN NEUTROPHILS BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR: EVIDENCE FOR AN INVOLVEMENT OF PHOSPHOLIPASE D-DERIVED PHOSPHATIDIC ACID PAUL H. NACCACHE,* BERNARD HAMELIN, MURIELLE GAUDRY and SYLVAIN BOURGOrN Centre de recherche en inflammation, immunologic et rhumatologie, Centre de recherche du CHUL, and Department of Medicine, Faculty of Medicine, Universit6 Laval, 2705 boulevard Laurier, Ste Foy, Qu6bec Canada GIV 4G2 (Received 23 July 1991; and accepted 27 August 1991) Abstract--Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM- CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidie acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors. Key words: Neutrophils, calcium, inositol phosphates, phospholipase D, haemopoiesis, phosphatidic acid, GM-CSF. INTRODUCTION THE HAEMATOPOEITIC growth factor granulo- cyte-macrophage-colony-stimulating factor (GM-CSF) modulates in important ways the *Author to whom correspondence should be addressed at: CHUL, room 9800, 2705 Boulevard Laurier, Ste-Foy, Qu6bec, GIV 4G2, Canada. Tel: (418) 656-4141 x7531. Fax: (418) 654-2765. Abbreviations: GM-CSF--granulocyte-macrophage colony-stimulating factor; fMet-Leu-Pbe---N- formylmethionyl-leucyl-phenylalanine; PIC--phosphatidy- linositol 4,5-bisphosphate-spccific phosphoinositidase; PLD---phospholipas¢ D; Ins(l,4,S)P3--inositol 1,4,5-tris- phosphate; PEt--phosphatidylethanol; PtdOH--phospha- tidie acid. 635 functional responsiveness of mature human neutrophilic polymorphonuclear leukocytes (neutrophils). GM-CSF has thus been shown to alter the locomotory [1-3] and phagocytic [4, 5] behaviour of neutrophils as well as their oxida- tive metabolism [3, 4, 6-8], three important elements of these cells' ability to fulfill their role in the body's first line of defence against patho- genic organisms. Furthermore, the demonstra- tion of the presence of GM-CSF in pathological media including synovial fluids [9] lends strong support to the idea that the in vivo behavior of phagocytes is likely to be modulated by this, as well as other, cytokines.