Effect of Hyperosmolality on β-Defensin Gene Expression by Human Corneal Epithelial Cells

Abstract

Purpose-As human β-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-1β (IL-1β)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1β 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-1β (culture supernatants and cell lysates) and pro-IL-1β (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-1β to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1β 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. Keywords hyperosmolality; human β-defensins; corneal epithelium; cytokines Hyperosmolality at the ocular surface is a characteristic of diseases such as dry eye and diabetes mellitus. The normal osmolality of the tear fluid is approximately 300 mOsm/kg, 1 whereas the suggested "gold standard" diagnostic of dry eye is 312 mOsm/kg or greater. 2 It has been proposed that tear fluid hyperosmolality occurs because of either normal tear evaporation associated with reduced tear flow or excessive tear evaporation with a normal tear flow. 3 The hyperosmolar tear film is thought to affect ocular surface epithelial function and differentiation in dry eye disease, with osmolalities as high as 417 mOsm/kg being reported. 1,4,5 Also, Aragona et al 6 showed that type 1 diabetic patients had a significantly higher mean tear osmolality (332.2 ± 18.3 mOsm/L), poor tear stability, altered tear function, and symptoms of ocular irritation akin to patients with dry eye than normal patients. Hyperosmolality in diabetes mellitus is probably attributable to increased levels of tear glucose seen in patients with both types 1 and 2. 7

Dry eye modulates the expression of toll-like receptors on the ocular surface

“…Topical application of hyperosmolar solutions in the range 800-900 mOsm/kg has been found to induce ocular surface burning sensations similar to those reported for dry eye (Liu et al, 2009) leading to the suggestion that similar transient spikes in osmolarity may be responsible for some dry eye symptoms, although such spikes are currently difficult to detect. In the present study, HOS was induced by increasing the osmolarity of the culture media by 70-170 mOsm/kg, which is large compared to the increase in tear film osmolarity found in the DES subjects in this study, however this is consistent with the increase in the tear film osmolarity described in subjects with severe DES (Farris, 1994), as well as several in vitro (Li et al, 2006; Narayanan et al, 2006a) and in vivo (Luo et al, 2005) studies.…”

The Core Mechanism of Dry Eye Disease Is Inflammation

“…However, in the presence of IL-1β, induction of hBD-2 and IL-6 expression was reduced as compared with IL-1β alone, suggesting that hyperosmolar shock may affect ocular surface inflammation via altering functions of key inflammatory cytokines. 33 …”

Dry Eye Disease and Microbial Keratitis: Is There a Connection?

“…This change may not be physiologically relevant in dry eye, as hBD-2 may lose some of its antimicrobial effectiveness on ocular surface of dry eye patients due to hyperosmolar stress and potential interactions with mucins in the tear film. 192,195 …”