Vol. 300, Issue 3, 868-875, March 2002

Leukotriene B₄ Production in Human Mononuclear Phagocytes Is Modulated by Interleukin-4-Induced 15-Lipoxygenase

Mirella Profita, Angelo Sala , Liboria Siena, Peter M. Henson, Robert C. Murphy, Alessandra Paternò, Anna Bonanno, Loredana Riccobono, Angela Mirabella, Giovanni Bonsignore and Antonio M. Vignola

Istituto di Fisiopatologia Respiratoria, Consiglio Nazionale delle Ricerche, Palermo, Italy (M.P., L.S., A.P., A.B., L.R., G.B.); Dipartimento di Scienze Famacologiche, Università di Milano, Milan, Italy (A.S.); National Jewish Medical and Research Center, Denver, Colorado (A.S., P.M.H., R.C.M.); and Istituto di Medicina Generale e Pneumologie, Università di Palermo, Palermo, Italy (A.M., A.M.V.).

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 µM) or opsonized zymosan. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], LTB₄, and arachidonic acid (AA) release were measured by high-performance liquid chromotography/radioimmunoassay, liquid chromotography/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.