Defensins Augment the Proinflammatory Responses of Human Conjunctival Epithelial Cells

Abstract

Purpose:: Human defensins are important components of innate immunity with broad spectrum anti-microbial killing ability. Previously we have reported that both human alpha defensin (HNP1) and beta defensin (HBD2) stimulate the proliferation and certain collagen and MMP gene expression of conjunctival fibroblasts which indicates a possible role in ocular surface wound healing. In this study we explored the effect of HNP1 and HBD2 on the proinflammatory responses of human conjunctival epithelial cells.

Methods:: IOBA-NHC cells were cultured and incubated with recombinant human HNP1 and HBD2 in DMEM/F12 (1:1) medium without supplement. Gene expression, cytokine production, p42/44 MAP kinase and Akt were analyzed by Taqman real time PCR, ELISA and western blot analysis, respectively. Proliferation of NHC cells was quantitated by BrdU incorporation.

Results:: Twenty-four hours after stimulation, HBD2 at the concentrations of 10 and 20 µg/ml but not 5µg/ml stimulated the production of IL-6 up to 3.16 fold and IL-8 up to 14.88 fold in the cell culture supernatant. Insignificant increase of IL-1α secretion was also observed. No changes of TNFα or IL1ß protein were observed under the same conditions. However, HNP1 had no effect on the secretion of the above cytokines at the same concentrations. IL-6 and IL-8 transcripts increased 2-4 fold 4hrs after stimulation and returned to normal levels between 16-24hrs. Unlike conjunctival fibroblasts, neither defensin showed mitogenic effect on NHC cells. Furthermore, NHC cells showed no response to the presence of either HNP1 or HBD2 in the gene expression for MMP1, MMP3, MMP9, collagen Iα and collagen IVα at the concentrations up to 20µg/ml and incubation period of 2-16hrs. Both defensins phosphorylate p42/44 MAP kinase and Akt at 30mins after the incubation; however, HBD2 is a more potent activator of both kinases than HNP1 at the same concentration.

Conclusions:: NHC cells are more responsive to HBD2 than HNP1 at the same concentration. HBD2 stimulated IL-6 and IL-8 production in NHC cells suggested that it can augment the host innate immune responses and provide links to adaptive immunity in addition to the antimicrobial activitites.

Keywords: conjunctiva • inflammation • cytokines/chemokines