title: Linoleic acid induces interleukin-8 production by Crohn's human intestinal smooth muscle cells via arachidonic acid metabolites.
abstract: Previously we reported that linoleic acid (LA), but not oleic acid, caused a marked increase in the secretion of IL-8 by Crohn's human intestinal smooth muscle (HISM) cells. Antioxidants inhibited this response, implicating a role for oxidative stress and NF-kappaB, a transcription factor for IL-8 that is activated by oxidative stress. In this study, we examined two mechanisms whereby LA, the dietary precursor for arachidonic acid (AA), could increase the production of IL-8 via activation of AA pathways: 1) by generation of reactive oxygen species by the AA-pathway enzymes to activate NF-kappaB or 2) by AA metabolites. Normal and Crohn's HISM cells were exposed to LA, oxidizing solution (Ox), or oxidizing solution enriched with LA (OxLA). Exposure of cells to Ox or OxLA induced oxidative stress as determined by thiobarbituric acid reactive substances. In normal cells, Ox but not LA activated NF-kappaB as determined by transfection experiments and Western blot. In Crohn's cells, NF-kappaB was spontaneously activated and was not further activated by Ox or LA. In contrast, TNF-alpha markedly increased activation of NF-kappaB in both normal and Crohn's cells. These results indicated that LA did not increase IL-8 by activating NF-kappaB, so we evaluated the second mechanism of an effect of AA metabolites. In normal cells, OxLA, but not LA, markedly stimulated IL-8, whereas in Crohn's cells, both OxLA and LA stimulated IL-8. OxLA, also stimulated production of AA metabolites leukotriene B(4) (LTB(4)), PGE(2), and thromboxane B(2) (TXB(2)) by normal and Crohn's cells. To determine whether AA metabolites mediated the IL-8 response, cells were treated with OxLA plus indomethacin (Indo), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor. Both Indo and NDGA blocked the IL-8 response to OxLA. To determine more specifically a role for AA metabolites, AA was used. Similar to OxLA, OxAA stimulated production of IL-8 and AA metabolites. Pinane thromboxane, a selective thromboxane synthase inhibitor and receptor blocker, inhibited OxAA stimulation of TXB(2) and IL-8 in a dose-response manner. MK886, a selective 5-lipoxygenase inhibitor, inhibited OxAA stimulation of LTB(4) and IL-8 also in a dose-response manner. Analysis of specific gene products by RT-PCR demonstrated that HISM cells expressed receptors for both thromboxane and LTB(4). We conclude that AA metabolites mediated the IL-8 response to LA in HISM cells. Both cyclooxygenase and lipoxygenase pathways were involved. LA did not increase IL-8 by activating NF-kappaB, but NF-kappaB appeared to be involved, because LA increased IL-8 only in situations where NF-kappaB was activated, either spontaneously in Crohn's cells or by Ox in normal cells. We speculate that AA metabolites increased IL-8 production by enhancing NF-kappaB-dependent transcription of IL-8.
authors.affiliation: Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298-0034, USA. swwalsh@vcu.edu
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Alzoghaibi
authors.forename: Scott W
authors.initials: SW
authors.lastname: Walsh
authors.forename: Amy
authors.initials: A
authors.lastname: Willey
authors.forename: Dorne R
authors.initials: DR
authors.lastname: Yager
authors.forename: Alpha A
authors.initials: AA
authors.lastname: Fowler
authors.forename: Martin F
authors.initials: MF
authors.lastname: Graham
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D003424:Crohn Disease / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D016209:Interleukin-8 / Q000096:biosynthesis*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000187:drug effects; D007975:Leukotriene B4 / Q000096:biosynthesis; D019787:Linoleic Acid / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D017482:Receptors, Thromboxane / Q000378:metabolism
chemical_list: D016861:Cyclooxygenase Inhibitors; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D017482:Receptors, Thromboxane; D007975:Leukotriene B4; D016718:Arachidonic Acid; D019787:Linoleic Acid; D008156:Luciferases; D007213:Indomethacin