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RL Redfern, Y Pei, AM McDermott; Cytokine Stimulated Expression of Human beta-Defensin-2 by Corneal Epithelial Cells is Mediated via Activation of NFkB . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1639.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previously (IOVS 2001, 42, #199, s35) we have shown that cytokines such as interleukin (IL) -1 stimulate the expression of the antimicrobial peptide human ß-defensin 2 (hBD-2) by human corneal epithelial cells (HCEC). Here we have further investigated the characteristics of the effect and studied the intracellular pathways involved. Methods: SV40 transformed HCEC were treated with 10ng/ml IL-1ß for 30 min to 36 hrs or with 0.01-100ng/ml IL-1ß for 24 hrs. Cells treated with serum-free culture media acted as controls. At the end of the incubation the culture media was collected and the cells harvested. Immunoblotting was performed to detect hBD-2 secreted by the cells in to the culture media. RNA was extracted from the cells and RT-PCR was performed to detect hBD-2 mRNA expression. To study the pathways involved various inhibitors (50uM genistein, 5uM SB203580, 1uM dexamethasone, 100uM PDTC) were added 30 mins before 10ng/ml IL-1ß. At the end of the 3 hr. incubation cells were harvested for RT-PCR. Results: Untreated control HECE did not express hBD-2. IL-1ß stimulated expression of hBD-2 mRNA in HCEC could be detected at 3 hrs and every time point thereafter (n=2). Secreted hBD-2 protein was detectable by 6-12 hrs and was maximal at 24 hrs (n=2). The effect of IL-1ß was concentration dependent with the maximal effect being seen at 10ng/ml (n=2). Of the inhibitors tested PDTC (NFkB inhibitor) completely blocked IL-1ß stimulated hBD-2 mRNA expression (n=4). Dexamethasone (anti-inflammatory glucocorticoid) attenuated the action of IL-1ß whereas genistein (tyrosine kinase inhibitor) or SB203580 (p38MAPK inhibitor) had little or no effect (n=2-3). Conclusion: These studies show that IL-1ß stimulates hBD-2 mRNA expression in HCEC in a time and concentration dependent manner by a pathway involving NFkB and that mRNA upregulation is rapidly followed by protein secretion. Such a pathway likely operates in vivo to help protect the cornea from infection following epithelial injury.
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