Expression of human beta-defensins in conjunctival epithelium: relevance to dry eye disease

Abstract

Purpose: The goals of this study were to investigate whether beta-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human beta-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells.

Methods: RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3. Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa. RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively.

Results: hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects' samples, whereas hBD-1 and -3 were detected in all subjects tested. RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1beta, IL-1beta and tumor necrosis factor-alpha (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3). hBD-2 expression was upregulated from 4 hours of treatment with IL-1beta (at 10 ng/mL; (n = 2-3) and at a concentration of 0.1 ng/mL IL-1beta (24-hour treatment; n = 2-3). Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data.

Conclusions: hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye. Because cytokines such as IL-1beta and TNF-alpha induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate dry eye is mediated by proinflammatory cytokine activity.

Figure 1

hBD expression in human subjects. Duplex RT-PCR was performed for GAPDH+hBD-1 (A) or GAPDH+hBD-2 (B) on RNA extracted from conjunctival impression cytology specimens. One-step RT-PCR was performed to detect GAPDH and hBD-3 mRNA (C). M, size marker; N, normal subject; D, subject with dry eye. Data are from two representative subjects in each group.

Figure 2

hBD expression by human conjunctival epithelial cells in culture. RT-PCR was performed on samples collected from Chang and NHC cells and primary cultured conjunctival cells that were treated with serum-free growth media alone. Products of the constitutively expressed GAPDH gene are shown as well as the 3-hBD. The first two lanes are representative results (one of three experiments) from the cell lines and the last two lanes represent results from primary cultured cells from two of three donors tested.

Figure 3

hBD mRNA and protein expression by primary cultured conjunctival epithelial cells treated with cytokines or bacteria. Primary conjunctival cells (n = 3) were treated with cytokines or PA for 24 hours. (A) RT-PCR results for GAPDH and hBD-1, -2, and 3, and immunoblot analysis to detect hBD-2 protein secretion. (B) Comparison of the effect of TNF-α on hBD-2 mRNA expression by different human conjunctival cells in culture. M, size marker.

Figure 4

The effect of various concentrations of IL-1β on hBD-2 expression in primary cultured conjunctival epithelial cells. (A) RT-PCR products for GAPDH and hBD-2 from primary conjunctival cells (n = 1) treated 24 hours with various concentrations of the proinflammatory cytokine IL-1β. M, size marker; C, media-treated control cells. (B) The changes in hBD-2 protein secretion by primary cultured conjunctival cells in this experiment. Similar results were obtained with the cell lines (n = 2).

Figure 5

The effect of various periods of incubation with IL-1β on hBD-2 expression by primary conjunctival epithelial cells. (A) RT-PCR products for GAPDH and hBD-2 from primary conjunctival cells (n = 1) treated with IL-1β for various lengths of incubation at a concentration of 10 ng/mL. M, size marker; C, media-treated controls. (B) The changes in hBD-2 protein secretion in this experiment. Similar results were obtained with the cell lines (n = 2).