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


CONCLUSIONS
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.
Narayanan
Srihari
S
College of Optometry, University of Houston, Houston, TX 77204-2020, USA.
Manning
Jennifer
J
Proske
Rita
R
McDermott
Alison M
AM
D000368:Aged; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008353:Mannitol / Q000494:pharmacology; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023083:beta-Defensins / Q000235:genetics* / Q000378:metabolism
D016895:Culture Media, Serum-Free; C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D012333:RNA, Messenger; D023083:beta-Defensins; D008353:Mannitol