Secretion and effects of defensins on human conjunctival epithelial cells and fibroblasts

Abstract

Abstract: : Purpose: Defensins are a family of peptides, found in the tears, and with known antimicrobial functions. However, there is little information regarding the response of ocular surface cells to defensins. In this study we have examined the effects of defensins on human conjunctival epithelial cells and fibroblasts. Methods: Human conjunctival epithelium and fibroblasts were isolated from cadaveric conjunctival tissues by dispase I digestion. Fibroblasts grew out from the stroma tissue after 7 days of culture in DMEM with 10% FBS. Conjunctival epithelial cells were cultured in KGM supplemented with BPE and 10 ng/ml EGF (Cambrex, USA). P2–P4 cells were used throughout the study. Cell proliferation (BrdU, Amersham) and migration (Chemicon, USA) were determined using human neutrophil peptide 1 (HNP1, Sigma) and ß–defensin2 (ßD2, Chemicon). MAP kinase and Akt participation was analyzed by Western blot analysis (Cell Signaling Technology, USA). Results:Conjunctival epithelial cells, but not fibroblasts, were found to up–regulate only ßD1, 2 and 3 following stimulation with 20 ng/ml lipopolysaccride or wounding as detected by RT–PCR analysis. The existence of defensin proteins was confirmed by mass spectrum analysis of culture medium. In response to ßD2 and HNP1 (5µM), conjunctival epithelial cells showed increased secretion of IL8 and TIMP2 in the culture medium, and in total cell lysates MIF, TGF beta2, TIMP1,2 and FGF4 levels were increased compared to samples obtained from cells in KGM medium (supplemented with EGF and BPE). HNP1 (5µM) also stimulated the proliferation of conjunctival fibroblasts when compared to 10% FBS (3.27+/–0.977 vs 6.86+/–1.77 fold for FBS, n=8, p=0.032), but epithelial cells were not responsive. Similar results were obtained by using 5µM ßD2. Both HNP1 and ßD2 stimulated the migration of fibroblasts but not epithelial cells. The magnitude of which was similar to that of 10% FBS (fluorescent intensity 1.50x10⁵ vs 1.55x10⁵ units for FBS, n=2, assayed in 6 replicated wells). Fibroblast proliferation correlated with the activation of p42/44 MAPK and Akt. However, neither p38 or c–Jun kinase activities were changed upon defensin stimulation. Conclusions:We conclude that under stress human conjunctival epithelial cells express and secrete beta defensins. Defensins affect conjunctival fibroblasts by stimulating the proliferation and migration of these cells. Defensins might play an important role in conjunctival wound healing and defense mechanisms

Keywords: conjunctiva • wound healing