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J. Li, J.–B. Shen, D. Tan, R.W. Beuerman; Effect of HNP1 and HBD2 on Cytokine Gene Expression in Primary Cultured Human Conjunctival Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4377.
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© ARVO (1962-2015); The Authors (2016-present)
Human α defensins are released by neutrophils into the tears and ß defensins are expressed by conjunctival and corneal epithelial cells. The expression of human ß defensin 2 (HBD2) is regulated by pro–inflammatory cytokines such as IL–1 and TNFα. However, little is known about the effect of defensins on cytokine gene expression. This study was designed to understand the effects of human α defensin 1 (HNP1) and HBD2 on cytokine gene expression in primary cultured human conjunctival epithelial cells.
Normal human bulbar conjunctival epithelial cells (CjEC) were isolated from cadaver tissue and cultured in serum free keratinocyte culture medium (SFM) supplemented with insulin and bovine pituitary extraction (BPE). P2 CjEC cells were incubated with 10µg/ml of HNP1 and HBD2 for 2, 4, 6, 16 and 24 hrs and RNA extracted for real time PCR analysis. The expression of the genes for IL–1, IL–6, IL–8, TNFα, TLR2 and TLR4 was analyzed. At 3 and 5 hrs after HNP1 and HBD2 stimulation CjEC cells were harvested and NFΚB activity measured by ELISA.
At two hours HNP1 stimulation at 10µg/ml increased the transcription of IL–1α, IL–1ß, IL–6, IL–8 and TNFα 2–4 fold. However, the increased transcription of these genes was not sustained and at 6 hrs the transcription level of these genes returned to normal except for TNFα, which decreased to normal after 8 hrs. For all genes analyzed HBD2 at 10µg/ml was less stimulatory than HNP1. Neither defensin affected TLR2 and TLR4 gene expression. However, both HNP1 and HBD2 increased the transcriptional activity of the p50 and p65 subunits of NFΚB at 3 and 5 hrs after stimulation.
HNP1 is secreted by neutrophils, which appears on the ocular surface as part of the inflammation/wound response. The rapid increase in cytokine gene expression induced by HNP1 suggests that it is involved in host cell immune response via a receptor–mediated mechanism; however, despite the activation of NFΚB, HBD2 did not change cytokine gene expression, suggesting functional differences between α and ß defensins in their role in innate immunity. Moreover, these defensins do not lead to changes of message for TLR 2 and 4.
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