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Modulation of leukotriene release from human polymorphonuclear leucocytes by PMA and arachidonic acid
Abstract:
Stimulation of human neutrophils (PMN) with Ca ionophore A23187, opsonized zymosan and formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) led to a time- and dose-dependent release of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6-trans-LTB4, 12-epi-6-trans LTB4 and LTC4, as detected by reverse-phase HPLC. Preincubation of the PMN suspension in the presence of Ca2+ and Mg2+ with phorbol-12-myristate-13-acetate (PMA) did not release leukotrienes by itself, but modulated the subsequent Ca ionophore-induced leukotriene release. The release of LTC4, 20-OH-LTB4 and 20-COOH-LTB4 was significantly decreased. Lesser effects were observed for the release of LTB4 and the non-enzymatic LTB4 isomers. In contrast, opsonized zymosan and FMLP enhanced the release of LTB4 and LTB4-omega-oxidation products from cells pretreated with PMA. With arachidonic acid as prestimulus, the amounts of the LTB4 isomers (6-trans-LTB4 and 12-epi-6-trans-LTB4) were enhanced significantly on subsequent stimulation with Ca ionophore. Prestimulation of lymphocytes, monocytes and basophilic granulocytes (LMB) with PMA had no significant effects on the ionophore-induced release of LTC4 and LTB4. PMN, but not LMB, suspensions prestimulated with PMA convert exogenously added LTC4 to LTB4 isomers and LTC4 sulphoxide. Our data suggest that preincubation of human granulocytes with PMA modified leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4.
Insights
Stimulating human neutrophils with phorbol-12-myristate-13-acetate (PMA) alters leukotriene release, affecting pathways for leukotriene B4 (LTB4) and leukotriene C4 (LTC4). PMA modulates responses to various stimuli, impacting inflammatory mediator production.
Area of Science:
- Immunology
- Biochemistry
Background:
- Leukotrienes (LTs) are inflammatory mediators derived from arachidonic acid.
- Human neutrophils (PMN) and other leukocytes produce various LTs, including LTB4 and LTC4.
- The phorbol ester phorbol-12-myristate-13-acetate (PMA) is a potent activator of protein kinase C and can modulate cellular responses.
Purpose of the Study:
- To investigate the effect of PMA prestimulation on leukotriene release from human neutrophils.
- To determine how PMA modulates the release of different leukotriene metabolites in response to various stimuli.
- To compare the effects of PMA on leukotriene metabolism in neutrophils versus other leukocytes.
Main Methods:
- Human neutrophils (PMN) and leukocytes (lymphocytes, monocytes, basophils - LMB) were isolated.
- Cells were preincubated with or without PMA.
- Leukotriene release was stimulated using Ca ionophore A23187, opsonized zymosan, or formyl-L-methionyl-L-leucyl-phenylalanine (FMLP).
- Leukotriene metabolites were quantified using reverse-phase High-Performance Liquid Chromatography (HPLC).
Main Results:
- PMN stimulation with A23187, zymosan, or FMLP induced time- and dose-dependent release of LTB4, LTC4, and their metabolites.
- PMA prestimulation of PMN inhibited LTC4, 20-OH-LTB4, and 20-COOH-LTB4 release but enhanced LTB4 and its omega-oxidation products when stimulated with zymosan or FMLP.
- PMA prestimulation did not significantly affect ionophore-induced LTC4 and LTB4 release from LMB, but PMN (not LMB) converted exogenous LTC4 to LTB4 isomers and LTC4 sulphoxide.
Conclusions:
- PMA preincubation modifies leukotriene release in human neutrophils by activating or inhibiting distinct metabolic pathways for LTC4 and LTB4.
- Neutrophils exhibit differential regulation of leukotriene synthesis and metabolism by PMA compared to other leukocytes.
- These findings highlight the complex role of PMA in modulating inflammatory mediator production by immune cells.