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Johanna Tukler Henriksson, Xiaobo Zhang, De-Quan Li, Cintia De Paiva, Stephen Pflugfelder; Effects of IL-13 Stimulation on Primary Mouse Conjunctival Epithelial Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2075.
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To investigate the impact of IL-13 stimulation on goblet cell differentiation in primary mouse conjunctival cultures.
Young adult, (6-8 week) female C57BL/6 mice were utilized. Explants were obtained from the forniceal conjunctiva and plated, one explant per well in 48 well plates. First, growth kinetics were evaluated with different media and EGF concentrations. Second, the explants received 200 µl per week of either Keratinocyte media with 3% fetal bovine serum defined (KSFM) or KSFM with added 1ng/ml or 10ng/ml of interleukin-13 (IL-13). Cultures were incubated for 1 or 2 weeks. Subsequently, either cell proliferation was assessed by WST-1 assay or cultures were fixed in methanol and immunostained for MUC5AC and cytokeratin7 (K7), IL- 4 receptor alpha (Rα) or IL-13 receptor alpha1 (Rα1).
On average 50% of explants showed outgrowth using KSFM with 80ng/ml EGF. Both the IL-13 treated and the non-treated cultures showed a statistically significant increase in growth at 2 weeks compared to 1 week (P < 0.05), and the IL-13 1 ng/ml treated cultures had significantly greater outgrowth than control (P < 0.05). 100% of the cultures in all conditions were K7 positive. After 1 week the MUC5AC/K7 ratio was 0.11 in control media versus 0.89 and 0.99 in IL-13 1 ng/ml and IL-13 10 ng/ml treated cultures, respectively (P < 0.05). There was no difference in MUC5AC/K7 ratio at 2 weeks [(0.99 controls, 0.95 IL-13 1 ng/ml and 0.98 IL-13 10 ng/ml (P > 0.05)]. Positive expression for the signal transducing IL- 4 (Rα) and IL-13 (Rα1) receptors was also noted.
This study illustrated successful growth of primary mouse conjunctival cultures with increased proliferation over time. IL-13 cytokine stimulation appears to increase goblet cell differentiation in a dose dependent manner at the early stages of outgrowth. The results from these experiments indicate that conjunctival goblet cell growth and differentiation is regulated by the T helper (Th) 2 cytokine IL-13.
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