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Jing Li, Hong Yuan Zhu, Roger W. Beuerman; Stimulation of Specific Cytokines in Human Conjunctival Epithelial Cells by Defensins HNP1, HBD2, and HBD3. Invest. Ophthalmol. Vis. Sci. 2009;50(2):644-653. doi: 10.1167/iovs.08-1838.
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purpose. To investigate the effect of human defensins HNP1, HBD2, and HBD3 on human conjunctival epithelial cell cytokine secretion.
methods. HNP1, HBD2, and HBD3 were used to test cytotoxicity (1–50 μg/mL) and to stimulate (1–20 μg/mL) primary cultured and immortalized human conjunctival epithelial (IOBA-NHC) cells. Cytokine concentrations in the culture medium were measured by cytokine array and a multiplexed microbead analysis. Protein kinase activation was determined by Western blot analysis after defensin stimulation and with specific inhibitors.
results. HBD3, but not HNP1 or HBD2, killed more than 50% of IOBA-NHC cells at concentrations greater than 12.5 μg/mL. Only IL-6, IL-8, and RANTES were detected in the culture medium in the absence of defensins. All three cytokines increased in the presence of HNP1, HBD2, and HBD3 at concentrations of 5 to 20 μg/mL and between 2 and 8 hours and further accumulated at 24 hours Stimulation with HBD2 and HBD3 increased the secretion of IL-2 and MIP-1β in IOBA-NHC cells but only of MIP-1β in primary cultured cells. Activation of p42/44 mitogen-activated protein (MAP) kinase, Akt, and STAT3 was observed in primary and IOBA-NHC cells after defensin stimulation. Cytokine secretion was significantly decreased by the inhibition of p42/44 MAPK in IOBA-NHC cells.
conclusions. HNP and HBD selectively increase the secretion of specific proinflammatory cytokines in conjunctival epithelial cells in a time- and concentration-dependent manner, suggesting a supporting role to the innate immune system of the ocular surface.
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