April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Human goblet cell function in an in vitro allergic microenvironment
Author Affiliations & Notes
  • Laura Garcia-Posadas
    Ocular Surface Group, IOBA-University of Valladolid, Valladolid, Spain
  • Dayu Li
    Schepens Eye Research Institute/Massachusetts Eye and Ear, Harvard Medical School, Boston, MA
    Department of Ophthalmology, Harvard Medical School, Boston, MA
  • Robin R Hodges
    Schepens Eye Research Institute/Massachusetts Eye and Ear, Harvard Medical School, Boston, MA
    Department of Ophthalmology, Harvard Medical School, Boston, MA
  • Marie A. Shatos
    Schepens Eye Research Institute/Massachusetts Eye and Ear, Harvard Medical School, Boston, MA
    Department of Ophthalmology, Harvard Medical School, Boston, MA
  • Yolanda Diebold
    Ocular Surface Group, IOBA-University of Valladolid, Valladolid, Spain
  • Darlene Dartt
    Schepens Eye Research Institute/Massachusetts Eye and Ear, Harvard Medical School, Boston, MA
    Department of Ophthalmology, Harvard Medical School, Boston, MA
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2759. doi:
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      Laura Garcia-Posadas, Dayu Li, Robin R Hodges, Marie A. Shatos, Yolanda Diebold, Darlene Dartt; Human goblet cell function in an in vitro allergic microenvironment. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2759.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To determine the effect of the Th2-type cytokines IL4, IL5, and IL13 and allergic mediator histamine on cultured human goblet cells (GC), and to compare their effect with our previous results in cultured rat GC.

Methods: Human conjunctival GC were grown from tissue explants and used after passage 1. The expression of goblet (CK7) and stratified squamous (CK4) specific cell markers was analyzed by immunofluorescence, and the lectin HPA was used to identify GC secretory products. The expression of MUC5AC and IL receptor mRNAs was evaluated by RT-PCR. The effect of histamine on [Ca2+]i was studied in cells treated for 15 min or 24 h with the cytokines IL4, IL5 or IL13 (10ng/ml each) or buffer (n = 7). To measure [Ca2+]i, cells were loaded with fura2 and analyzed using the InCyte Im2TM Ratio Imaging System. To evaluate high molecular weight glycoconjugate secretion, cells were serum starved for 24 h. Then, supernatants from untreated and 24 h cytokine-treated cells were analyzed using an enzyme-linked lectin assay (n = 3).

Results: Human cultured cells expressed GC markers (CK7, secretory products identified by HPA, and MUC5AC), but not CK4. Receptors for the analyzed ILs were also expressed by GC. Histamine increased [Ca2+]i to 490.2 ± 189.4 nM, but individual ILs did not significantly increase it. A 15 min treatment with IL-4, IL-5, or IL-13 decreased the effect of histamine on [Ca2+]i to 133.6 ± 40.9 nM (p = 0.01), 114.4 ± 38.9 nM (p = 0.007), and 117.0 ± 36.7 nM (p = 0.007), respectively. However, the histamine effect on [Ca2+]i was not altered when cells were treated with cytokines for 24 h. These results are similar to what we previously found in rat GC. Regarding high molecular weight glycoconjugate secretion, histamine induced a 2.1 ± 0.2 fold increase compared to basal. IL5 and IL13 increased GC secretion to 1.3 ± 0.07 (p = 0.01) and 1.2 ± 0.04 (p = 0.008) fold, respectively. A 24 h pretreatment with IL4 or IL5, but not IL13, blocked histamine-induced glycoconjugate secretion. No significant effect on histamine-induced secretion was found in rat GC.

Conclusions: Cultured human GC respond to an allergic microenvironment, simulated with histamine and/or Th2 cytokines, by increasing [Ca2+]i and secreting their products. Interestingly, Th2 cytokines act by blocking the histamine effect, instead of increasing it as expected. This action could be an attempt by GC to regulate the oversecretion that characterizes allergy.

Keywords: 474 conjunctiva • 557 inflammation • 490 cytokines/chemokines  
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