Submitted on April 25, 2003
Accepted on November 19, 2003
Linoleic Acid Induces Interleukin-8 Production by Crohn's Human Intestinal Smooth Muscle Cells via Arachidonic Acid Metabolites
Mohammad A. Alzzoghaibi1, Scott W. Walsh2*, Amy Willey3, Dorne R. Yager4, Alpha A. Fowler, III5, and Martin F. Graham6
1 Department of Physiology, Virginia Commonwealth University, Richmond, VA, USA
2 Department of Physiology, Virginia Commonwealth University, Richmond, VA, USA; Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, USA
3 Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, USA
4 Department of Surgery, Virginia Commonwealth University, Richmond, VA, USA
5 Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA
6 Department of Pediatrics, Virginia Commonwealth University, Richmond, VA, USA
* To whom correspondence should be addressed. E-mail: swwalsh{at}hsc.vcu.edu.
Previously we reported that linoleic acid (LA), but not oleic acid, caused a marked increase in
the secretion of interleukin-8 (IL-8) by Crohn's human intestinal smooth muscle (HISM) cells.
Antioxidants inhibited this response implicating a role for oxidative stress and nuclear factor -
kappa B (NF-
B), 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-B; 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 TBARS. In normal cells, Ox, but not LA, activated NF-B as
determined by transfection experiments and Western blot. In Crohn's cells, NF-B was
spontaneously activated and was not further activated by Ox or LA. In contrast, TNF
markedly
increased activation of NF-B in both normal and Crohn's cells. These results indicated that LA
did not increase IL-8 by activating NF-B, 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 B4 (LTB4), prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) by
normal and Crohn's cells. To determine if 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 TXB2 and IL-8 in a dose response manner. MK886, a selective 5-lipoxygenase
inhibitor, inhibited OxAA stimulation of LTB4 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 LTB4. Conclusion: 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-B, but NF-B appeared to be involved because LA
increased IL-8 only in situations where NF-B 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-B dependent transcription of IL-8.
