Phosphorylation of cytosolic phospholipase A2 by IL-3 is associated with increased free arachidonic acid generation and leukotriene C4 release in human basophils

• Published in: Journal of allergy and clinical immunology 1998-09, Vol.102 (3), p.512-520
• Main Authors: Miura, Katsushi, Hubbard, Walter C., MacGlashan, Donald W.
• Format: Article
• Language: English
• Subjects: arachidonic acid; Arachidonic Acid - biosynthesis; Arachidonic Acid - metabolism; Basophils - drug effects; Basophils - enzymology; Basophils - physiology; Biological and medical sciences; Cells, Cultured; Complement C5a - pharmacology; Cytosol - enzymology; cytosolic phospholipase A2; Enzyme Activation; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Human basophils; Humans; IL-3; Immunobiology; Interleukin-3 - pharmacology; Ionomycin - pharmacology; Ionophores - pharmacology; leukotriene C4; Leukotriene C4 - metabolism; Myeloid cells: ontogeny, maturation, markers, receptors; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Phospholipases A - metabolism; Phospholipases A2; Phosphorylation - drug effects; Polynuclears; Protein Kinase C - antagonists & inhibitors; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0091-6749; 1097-6825
• DOI: 10.1016/S0091-6749(98)70142-3

Background: Human basophils secrete leukotriene C4 (LTC4) in response to various stimuli, and a short treatment with IL-3 enhances LTC4 release, although IL-3 alone does not induce LTC4 release. However, the mechanism of this priming effect of IL-3 for LTC4 generation remains unknown in human basophils. Objective: This study was designed to explore the mechanisms by which short treatments with IL-3 enhance stimulated secretion of LTC4, with a focus on the activation of cytosolic phospholipase A2 (cPLA2). Methods: The phosphorylation state of cPLA2 in human basophils was examined by its shift in electrophoretic mobility as detected by Western blotting. Free arachidonic acid (AA) and LTC4 were measured by gas chromatography–negative ion chemical ionization mass spectrometry and LTC4-specific RIA, respectively. Result: Human basophils expressed cPLA2. IL-3, as well as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, caused a shift in the electrophoretic mobility of cPLA2, which indicated phosphorylation of cPLA2 and therefore its activation. Ionomycin at a concentration of 0.1 μg/mL was used to induce a modest elevation of cytosolic calcium response ([Ca 2+] I), no apparent cPLA2 phosphorylation, and little free AA and LTC4 generation. Pretreatment with IL-3 (1 to 10 ng/mL) markedly enhanced ionomycin (0.1 μg/mL)-mediated AA and LTC4 generation. The concentration dependence of cPLA2 phosphorylation by IL-3 and its effects on free AA and LTC4 generation were similar. The selective PKC inhibitors, bis-indolylmaleimide II and Ro-31-8220 inhibited the phorbol 12-myristate 13-acetate–mediated cPLA2 electrophoretic mobility shift, but not the IL-3–mediated shift, suggesting that the IL-3 effect is PKC independent. Both the anaphylatoxin split product of the C component C5 (C5a) and f-Met-Leu-Phe induced PKC-independent cPLA2 phosphorylation with a similar time course most notable for the absence of observable changes in cPLA2 phosphorylation before 30 seconds. These results suggested an explanation for the absence of free AA generation by C5a. When [Ca 2+] I was elevated in response to C5a, there was no phosphorylation of cPLA2, and by the time cPLA2 became phosphorylated, [Ca 2+] I had returned to resting levels. Treatment with IL-3 preconditioned the cPLA2 by causing its phosphorylation so that the transient [Ca 2+] I response, which followed stimulation by C5a, could induce the generation of free AA and LTC4. Conclusion: Taken together, thes