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Abstract
Rainbow trout macrophages maintained in short term culture when incubated with either calcium ionophore, A23187, or opsonized zymosan synthesize a range of lipoxygenase products including lipoxins and leukotrienes. These cells are unusual in that they generate more lipoxin than leukotriene following such challenge. The main lipoxin synthesized was lipoxin (LX) A4. This compound was identified by cochromatography with authentic standard during reversephase high performance liquid chromatography, by ultra violet spectral analysis, radiolabeling following incorporation of [14C]arachidonic acid substrate into macrophage phospholipids, and gas chromatography electron impact mass spectrometry of the methyl ester, trimethylsilyl ether derivative. Other 4-series lipoxins synthesized by trout macrophages were identified as 11-trans-LXA4, 7-cis-11-trans-LXA4, and 6(S)-LXA4. These cells also produced 5-series lipoxins tentatively identified as LXA5, 11-trans-LXA5 and possibly 6(S)-LXA5. No LXB4 or LXB5 was, however, detected. The dynamics of leukotriene and lipoxin release were also determined. Lipoxin generation was slower than leukotriene generation the latter reaching a maximum after 30 min of exposure to ionophore (5 microM, 18 degrees C) compared with 45 min for the former.
Highlights
These cells produced 5-series lipoxins tentatively [19] generate them from endogenous substrates, as can identified as LXAS,ll-trans-LXASand possibly 6(S)- porcine leukocytes when incubated with a snakevenom phos
This paper reports on the full identification of lipoxins and other lipoxygenase products synthesized by rainbow trout, Oncorhynchus mykiss, macrophages in response to both cal
PGB, (100 ng) was added as Identification of Lipoxins-Rainbow trout macrophages synthesized a range of oxygenated fatty acid metabolites in
Summary
Any nonadherent cells remaining were removed by vigorous washing Biological Challenge-Zymosan was opsonized for 60 min a t 18"C with Ca/Mg-containing HBSS Theremaining HBSS were incubated with opsonized zymosan (0.25, 1.0, 2.5, 5.0, or adherent cells (195% macrophages) were covered with 5 ml of fresh 10 mg.ml") for 2 h a t 18"C followed by eicosanoid extraction and. Ionophore Challenge-Macrophage cultures (-1 X lo cells/flask) lenged with E. coli Olll.B4 LPS (0.1, 1.0, and 10 pg.rn1-I) for 2 h at were incubated with 5 p~ calcium ionophore, A23187, for 5-60 min 18 "C. at 18 "C, the medium removed, and centrifuged to pellet any cells (10,000 X g, 5 min, room temperature).
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