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 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.
Footnotes
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Supported by Consiglio Nazionale delle Ricerche Short-Term Mobility Grants (A.S.) and by the National Institutes of Health Grant HL25785 (R.C.M.).
- Abbreviations:
- IL
- interleukin
- 15-LO
- 15-lipoxygenase
- LTB4
- leukotriene B4
- 15(S)-HETE
- hydroxyeicosatetraenoic acid
- AA
- arachidonic acid
- LC/MS/MS
- liquid chromatography/tandem mass spectrometry
- RP
- reversed-phase
- HPLC
- high-performance liquid chromatography
- 5-LO
- 5-lipoxygenase
- FLAP
- 5-LO-activating protein
- RIA
- radioimmunoassay
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- fMLP
- formyl-methionyl-leucyl-phenylalanine
- bp
- base pairs
- A23187
- calcimycin
- LY 223982
- (E)-5-(3-carbobenzoyl)-2-((6-(4-methoxyphenyl)-5-hexenyl)oxy)benzenepropanoic acid
- MK-886
- 3-(1-(4-chlorobenzyl-3-t-butyl-thio-5-isopropylindol-2-yl)-2, 2-dimethylpropanoic acid
- Received June 27, 2001.
- Accepted October 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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