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Stimulation of Specific Cytokines in Human Conjunctival Epithelial Cells by Defensins HNP1, HBD2, and HBD3

¹From the Singapore Eye Research Institute, Singapore; and the ²Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

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.