Effect of hyperosmolality on beta-defensin gene expression by human corneal epithelial cells

Abstract

Purpose: As human beta-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs).

Methods: Simian virus 40-transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400-500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1beta (IL-1beta)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1beta function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of active IL-1beta (culture supernatants and cell lysates) and pro-IL-1beta (cell lysates) were detected by enzyme-linked immunosorbent assay.

Results: HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1beta to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1beta was not detected in any cell culture sample.

Conclusion: Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation.

Figure 1

Hyperosmolar media does not alter hBD-1 and hBD-3 expression. SV40-HCECs (n = 4) were cultured for 24 hours in serum-free normal osmolality media or 500 mOsm/kg (NaCl) media. The figure shows RT-PCR products for GAPDH, hBD-1, and hBD-3 from 1 representative experiment. Similar results were obtained with P-HCECs (n = 2; data not shown).

Figure 2

Hyperosmolar medium does not induce hBD-2 expression in HCECs. A, SV40-HCECs (n = 4) were cultured for 24 hours in serum-free normal osmolality media only (N) or with the addition of IL-1β as a positive control (IL-1β) or serum-free hyperosmolar media. B, The cells were exposed to 500 mOsm/kg (NaCl) media for various lengths of time or to IL-1β (positive control) or normal media for 24 hours. The figure shows RT-PCR products for GAPDH and hBD-2. Similar results were obtained when the experiment was repeated with P-HCECs (n = 2; data not shown). M, size marker.

Figure 3

The ability of IL-1β to stimulate hBD-2 and IL-6 expression in HCECs is reduced by 500 mOsm/kg hyperosmolar media. A, SV40-HCECs (n = 4) were cultured for 24 hours in serum-free normal osmolality media (N) or 500 mOsm/kg (NaCl) hyperosmolar media (H). At the end of the incubation, either (1) 10 ng/mL IL-1β was directly added to the culture media of cells in the normal-osmolality media (N+I) and to those in hyperosmolar media (H+I) for 6 hours or (2) the hyperosmolar media was aspirated and substituted with normal-osmolality media containing 10 ng/mL IL-1β for 6 hours (H+N+I). The figure shows representative RT-PCR products for GAPDH and hBD-2 from 1 experiment. M, marker lane. Similar results were obtained with P-HCECs (n = 2, data not shown). B, P-HCECs (n = 2) were cultured for 24 hours in extract-free normal-osmolality media (N) or 500 mOsm/kg (NaCl) hyperosmolar media (H). Experimental conditions were similar to those described for panel A. The figure shows changes in hBD-2 protein secretion by P-HCECs for the various conditions. Similar results were obtained with SV40-HCECs (n = 1, data not shown). C, The experimental conditions were similar to those in panel A. The figure shows RT-PCR products for GAPDH and IL-6. The data are representative of 1 experiment repeated twice with SV40-HCECs. Similar results were obtained with P-HCECs (n = 2, data not shown).