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