title: Leukotriene B4 production in human mononuclear phagocytes is modulated by interleukin-4-induced 15-lipoxygenase.
abstract: The aim of this study was to evaluate the consequences of interleukin (IL)-4-induced 15-lipoxygenase (15-LO) expression on leukotriene B4 (LTB4) synthesis in human monocytes. Human monocytes incubated for 24, 48, and 72 h with IL-4 (10 ng/ml) were stimulated with Ca2+-ionophore A23187 (calcimycin; 5 microM) or opsonized zymosan. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], LTB4, and arachidonic acid (AA) release were measured by high-performance liquid chromatography/radioimmunoassay, liquid chromatography/tandem mass spectrometry (LC/MS/MS), or gas chromatography/mass spectrometry. 15-LO activity was evaluated in AA-treated monocytes. 15-LO, 5-lipoxygenase (5-LO) and 5-LO activating protein (FLAP) expression were analyzed by reverse transcription-polymerase chain reaction. Neutrophil chemotactic activity was evaluated using a microtaxis chamber assay. A23187-induced synthesis of 15(S)-HETE was significantly increased after treatment with IL-4 (10 ng/ml) for 48 and 72 h (p < 0.001). Concomitant decrease of LTB4 release was observed after 72 h of incubation with IL-4 (p < 0.001). LC/MS/MS analysis confirmed the production of 15(S)-HETE and the significant inhibition of LTB4 synthesis in IL-4-treated monocyte after challenge with opsonized zymosan. IL-4 treatment induced 15-LO enzymatic activity as well as 15-LO mRNA, but did not affect either 5-LO or FLAP mRNA expression in monocytes. Supernatant from IL-4-treated monocytes showed significantly lower neutrophil chemotactic activity than controls. 15(S)-HETE significantly inhibited LTB4 production induced by A23187-stimulated human monocytes without affecting AA release. IL-4-induced expression of 15-LO in monocytes caused a significant reduction of LTB4 production. Whereas this effect did not reflect changes in 5-LO and FLAP mRNA expression, synthetic 15(S)-HETE was able to significantly inhibit the synthesis of LTB4, without affecting AA release.
authors.affiliation: Istituto di Fisiopatologia Respiratoria, Consiglio Nazionale delle Ricerche, Palermo, Italy.
authors.forename: Mirella
authors.initials: M
authors.lastname: Profita
authors.forename: Angelo
authors.initials: A
authors.lastname: Sala
authors.forename: Liboria
authors.initials: L
authors.lastname: Siena
authors.forename: Peter M
authors.initials: PM
authors.lastname: Henson
authors.forename: Robert C
authors.initials: RC
authors.lastname: Murphy
authors.forename: Alessandra
authors.initials: A
authors.lastname: Paternò
authors.forename: Anna
authors.initials: A
authors.lastname: Bonanno
authors.forename: Loredana
authors.initials: L
authors.lastname: Riccobono
authors.forename: Angela
authors.initials: A
authors.lastname: Mirabella
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bonsignore
authors.forename: Antonio M
authors.initials: AM
authors.lastname: Vignola
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001093:Arachidonate 15-Lipoxygenase / Q000096:biosynthesis*; D016718:Arachidonic Acid / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis / Q000378:metabolism; D066298:In Vitro Techniques; D015847:Interleukin-4 / Q000494:pharmacology*; D007975:Leukotriene B4 / Q000096:biosynthesis*; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D007975:Leukotriene B4; D015847:Interleukin-4; D016718:Arachidonic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001093:Arachidonate 15-Lipoxygenase