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

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 al6 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 In human corneal epithelial cells (HCECs) in culture, chronic (up to 48 hours) hypertonic (350–600 mOsm/kg) stress was found to decrease proliferation rates and reduced the regulatory volume response (a mechanism by which cells recover their original volume in a hypertonic environment) by altering Na-K-2Cl cotransporter expression.8 Rabbit corneal epithelial cells exposed to a hyperosmolar media (330–407 mOsm/kg) showed decreased intercellular connections, increased desquamation, and loss of microplicae.9 Furthermore, HCECs cultured in high-osmolality media (412–512 mOsm/kg) secreted greater amounts of proinflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α.10 A recent study showed that the hyperosmolality (350–500 mOsm/kg)-induced production of the matrix metalloproteinases (MMP)-1, -3, and -9 was partially mediated by activation of stress-activated protein kinase pathways (c-Jun N-terminal kinase pathway).11 These findings are consistent with observations in other tissues that hyperosmolality can cause a variety of effects on cellular function. For example, hyperosmolality reduces proliferation rates and induces apoptosis in murine renal medullary cells,12 increases hepatocyte growth factor secretion in human mesangial cells,13 increases platelet-derived growth factor secretion in human umbilical vein endothelial cells,14 activates various serine/threonine kinases,15 and alters calcium mobilization patterns in rat vascular smooth muscle cells.16 Recent studies have shown that the ocular surface epithelia produce antimicrobial peptides called β-defensins.17,18 Human β-defensin-1 (hBD-1) and hBD-3 are constitutively expressed by the corneal and conjunctival epithelia,18–20 whereas the expression of hBD-2 is inducible by proinflammatory cytokines such as IL-1α, IL-1β, TNF-α, bacterial by-products such as lipopolysaccharide and heat-killed Pseudomonas aeruginosa, and injury.19–22 We have also observed that hBD-2 is expressed by conjunctival epithelial cells from patients with moderate dry eye but not in cells from normal subjects.20 Because it has been reported that proinflammatory cytokines are secreted in greater amounts by HCECs in hyperosmolar media,10 we reasoned that a differential expression of β-defensins by HCECs may be observed when the cells are in a hyperosmolar environment. Such a response may underlie our observation of conjunctival hBD-2 expression in dry eye patients but not normal subjects. Altered β-defensin expression associated with hyperosmolality may be important in conditions such as dry eye. Although it is known that the antimicrobial activity of some defensins is compromised by high salt concentrations,23 which is in keeping with the observation that the compromised ocular surface of patients with dry eye caused by Sjogren syndrome is at risk for microbial infection.24 However, in addition to their antimicrobial effects, defensins are known to modulate the behavior of mammalian cells; thus, even if hyperosmolality-induced defensin expression cannot contribute to antimicrobial protection, the defensins may have other effects on ocular surface epithelial cells such as promoting migration and proliferation.25 Therefore, the goal of this study was to investigate if differential expression of β-defensins by HCECs in culture was observed under hyperosmolar growth conditions.