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A. Berra, M. Berra; Hyperosmolarity Induce Nuclear Translocation of NF–kB in Human Conjunctival Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4402.
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Purpose: To investigate whether: 1) a normal human conjunctival epithelial cell line (IOBA–NHC) with hyperosmotic stress induce NF–kB nuclear translocation and 2) conjunctival epithelial cells of Sjogren (SS) and non SS dry eye patients has nuclear NF–kB translocation Methods: IOBA–NHC cells and hyperosmotic treatment. Cells were grown on cover slips for 3 days, washed with PBS and switched to a serum–free medium for 24 hours before treatment. Cells were cultured for an additional 1 hour or 24 hours in an equal volume of serum–free media with two different osmolar conditions: a) normal osmolarity (303 mOsM) or b) hyperosmolarity (500 mOsM). The conditioned media were then collected and centrifuged, and the supernatants were stored at –80°C. Patients and specimens. Dry eye patients (n=42) were diagnosed by Schirmer test, tear break–up time, Rose Bengal staining, tear osmolarity, and conjunctival impression cytology (ic) Studied serum autoantibodies were: anti/Ro, anti/La, RF and antinuclear antibodies. SS patients fulfilled three or more Fox’s criteria and were positive for anti/Ro and anti/La antibodies. Patients were divided in two groups (G): GI: 26 dry eye SS patients and GII: 16 postmenopausal dry eye without SS patients. Controls (GIII): 20 normal subjects. Immunochemistry: IOBA–NHC cells or ic specimens were fixed, washed with PBS, incubated overnight with anti NF–kB p65 (Santa Cruz). Secondary antibodies used were FITC–conjugated(Santa Cruz) for IOBA–NHC cells or biotinilated conjugated (Santa Cruz) for ic specimens. ELISA: IL–1, IL–6 and TNF–alpha (Pharmingen) were determined in supernatants and tears Results: IOBA–NHC cells with 303 mOsM medium conditions showed NF–kB in cytoplasm without nuclear translocation. In sharp contrast, these cells showed a weak or a strong nuclear translocation after 1 or 24 hour of 500 mOsM medium conditions. All GI showed more that 450 mOsM in tears and evidence of nuclear translocation by ic, furthermore, 12/16 of GII had more that 380 mOsM in tears and 10/12 of them showed nuclear translocation. Supernatants of 1 and 24 hours with hyperosmolar conditions and tear of GI or GII showed high levels of IL–1, IL–6 and TNF–alpha Conclusions: These data demonstrate (in vitro) and suggest (in vivo) that a hyperosmotic stress were linked to the nuclear NF–kB translocation in human conjunctival epithelial cells with pro–inflammatory cytokines production. The nuclear NF–kB translocation could be involucrate in the initiation of the inflammatory process of the dry eye.
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