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Afsun Sahin, Wendy Kam, David A. Sullivan; Induction of Leukotriene B4 by Lipopolysaccharide in Human Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4789.
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Leukotriene B4 (LTB4) is a potent inflammatory mediator. This compound, which is produced by leukocytes and non-ocular epithelial cells in response to various stimuli (e.g. Escherichia coli endotoxin [LPS]), is known to attract and activate T cells and/or neutrophils, as well as to induce the formation of reactive oxygen species and the release of lysosomal enzymes. We hypothesize that LTB4 is also synthesized in, and secreted by, ocular surface and adnexal epithelial cells and acts to promote local inflammation. To begin to test this hypothesis, we analyzed whether LPS can elicit the release of LTB4 by human conjunctival epithelial cells.
Immortalized human conjunctival (from Ilene Gipson), corneal (from Jamie Jester), meibomian gland and breast (MCF7; from Ana Soto and Carlos Sonnenschein) epithelial cells were cultured in keratinocyte serum-free medium until reaching 100% confluence. Cells were then exposed to DMEM/F12 medium with 10% BCS for 2 days, followed by an incubation in serum-free DMEM/F12 for 1 day. After this time period, cells were treated with vehicle or 15 ug/ml LPS (E. Coli, strain 0127:B8) for 6, 24 and 48 hours. Culture media were collected and analyzed for LTB4 levels with an ELISA.
Our results show that LPS, but not vehicle, induces the release of LTB4 from both human conjunctival epithelial and the control MCF7 cells. The nature and magnitude of this effect, which was observed in repeated studies, is cell- and time-dependent. Our preliminary investigations also suggest that LPS may stimulate the production of LTB4 in human corneal and meibomian gland epithelial cells. The action of LPS is dose-dependent. At concentrations of 30, 50, 100 and 200 ug/ml, LPS is extremely toxic and kills conjunctival, corneal, meibomian gland and MCF7 cells.
Our findings support our hypothesis that LTB4 can be produced by ocular surface epithelial cells. It is possible that such LTB4 production, when induced by appropriate stimuli, may play a role in generation of inflammation in ocular surface disease.
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