Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaGroup, IOBA-University of Valladolid, Valladolid, Spain;2 Networking Research Center on Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Valladolid, Spain; 3 Schepens EyeResearch Institute/Massachusetts Eye and Ear, Boston, MA;4 Department of Ophthalmology, Harvard Medical School, Boston,MA.Purpose: To determine if Th1 and Th2 pro-inflammatory cytokinesaffect cultured rat conjunctival goblet cells and to measure theireffects on proliferation, intracellular calcium levels ([Ca 2+ ] i ) and highmolecular weight glycoconjugate secretion that includes MUC5AC.Methods: Rat conjunctival goblet cells were grown from tissueexplants. Passage 1 cells were cultured in supplemented RPMImedium. The expression of goblet (CK-7) and stratified squamous(CK-4) specific cell markers was analyzed by immunofluorescenceand the lectin UEA-1 was used to identify goblet cell secretoryproducts. To evaluate proliferation cells were serum starved for 24 h,treated with the cytokines IFN-γ (3ng/ml), IL-4 (10ng/ml), IL-5(10ng/ml) or IL-13 (10ng/ml) for 24 h and then stimulated with thecholinergic agonist carbachol (Cch) or the allergic mediatorhistamine for 2 h. Proliferation was measured by WST-8 assay (n=3).To measure [Ca 2+ ] i , cells were loaded with fura2 and analyzed usingInCyte Im2TM Ratio Imaging System. The effect of Cch andhistamine on [Ca 2+ ] i was studied in cells treated for 15 min or 24 hwith cytokines or buffer (n=5). High molecular weightglycoconjugate secretion was measured in supernatants fromuntreated and 24 h cytokine-treated cells using an enzyme-linkedlectin assay (n=3).Results: Rat cultured cells expressed goblet cells markers (CK-7 andsecretory products identified by UEA-1), but not stratified epithelialmarker (CK-4). The Th1 cytokine IFN-γ decreased goblet cellproliferation by 0.79 fold, whereas Th2 cytokines IL-4, IL-5 and IL-13, and histamine increased it by 1.94, 2.65, 2.89 and 2.46 foldrespectively. IFN-γ used for 15 min significantly blocked Cchmediatedincrease in [Ca 2+ ] i (p = 0.009), and IL-4 and IL-13 had thesame effect on histamine-mediated [Ca 2+ ] i increase after 15 min preincubation(p=0.006 and p=0.003, respectively). When cells wereincubated with cytokines for 24 h, only IL-13 maintained theblockade (p=0.037). IFN-γ significantly blocked Cch-inducedsecretion (p=0.002), but Th2 cytokines did not have significanteffects on histamine-induced secretion.Conclusions: Th1- (IFN-γ) and Th2- (IL-4, IL-5 and IL-13) derivedcytokines have opposite effects on proliferation and secretion fromcultured rat goblet cells. These findings could explain the differencesin goblet cell function found in inflammatory ocular surface diseases,such as dry eye and allergic conjunctivitis.Commercial Relationships: Laura Garcia-Posadas, None; DayuLi, None; Robin R. Hodges, None; Marie A. Shatos, None;Yolanda Diebold, None; Darlene A. Dartt, NoneSupport: FEDER-CICYT Grant MAT2010-20452-CO3-01 and FPIScholarship Program BES-2011-046381 and EEBB-I-12-05371(Ministry of Science and Innovation, Spain), and Regional JCyLGrant VA132A11-2. NIH EY019470.Purpose: Benzalkonium chloride (BAK) is the most commonlyfound preservative in eye drops, and has been shown to cause ocularsurface inflammation and toxicity. Lacritin is a human tearglycoprotein secreted from the lacrimal glands that has been found tobe cytoprotective. This study was designed to determine if theprosecretory and mitogenic properties of lacritin confer protection toa cultured human corneal epithelial (HCE) cell line, CRL-11515, andprimary HCE cells after exposure to the ocular preservative agentBAK.Methods: Recombinant human lacritin and negative control fragmentC-25 were cloned into intein fusion vectors, expressed in E. coli, andpurified on chitin beads and DEAE Sepharose. Metabolic curveswere established after exposure of subconfluent CRL-11515 cells toBAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II)provided a measure of autophagy in CRL-11515 cells exposed tolacritin and/or BAK.Results: BAK reduced CRL-11515 cellular metabolic activity in atime and dose dependent manner. BAK-induced cellular stress wasevident by elevated autophagy that increased with risingconcentrations of BAK compared to control (P