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Alexis Chassignol, Emmanuelle Brasnu, Christophe Baudouin, Luisa Riancho, Jean-Michel Warnet, Françoise Brignole-Baudouin; In Vitro Interactions between Peripheral Blood Lymphocytes and the Wong-Kilbourne Derivative of Chang Conjunctival Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1492-1498. doi: 10.1167/iovs.11-7708.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the interactions between conjunctival cells and peripheral blood lymphocytes (PBLs) in vitro and to analyze the role of benzalkonium chloride (BAC)-induced apoptosis in this model.
Wong-Kilbourne derivative (WKD) cells were cocultured in cell-contact cultures or on cell inserts for 1 to 7 days with PBLs, activated or not with phorbol 12-myristate 13-acetate (PMA). Morphologic analyses of cell interactions were performed using membrane stainings (green PKH67 for WKD cells and red PKH26 for PBL), F-actin immunostaining, and scanning electron microscopy. Sub-G1 peak, CD95/Fas, and HLA-DR expression were assessed by flow cytometry (FCM). Specific interactions through the E-cadherin–CD103 complex were studied with FCM and standard immunofluorescence. Five different concentrations of BAC were tested in microplate cytofluorometry assays, to evaluate cytotoxic effects on cell viability and apoptosis.
WKD/PBL coculture allowed obvious cell interactions, as shown through plasma membrane exchanges. Direct-contact coculture potentiated the BAC cytotoxic effects and increased HLA-DR and CD95/Fas expression on WKD cells. Trichostatin A-pretreated WKD/PBL coculture induced a slight increase in CD103 expression on PBLs. Moreover, the presence of PBLs during the recovery period after WKD cell BAC stimulation reduced WKD cell apoptosis.
These results suggest that the in vitro interaction of PBLs with WKD cells participates in BAC-induced epithelial toxicity regulation, probably through cell membrane contacts.
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