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Войти в исследования Investigative ophthalmology & visual science · 2012
In vitro interactions between peripheral blood lymphocytes and the Wong-Kilbourne derivative of Chang conjunctival cells.
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Chassignol Alexis, Brasnu Emmanuelle, Baudouin Christophe, Riancho Luisa, Warnet Jean-Michel, Brignole-Baudouin Françoise
Аннотация
PURPOSE: 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.
METHODS: 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-G(1) 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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-G(1) 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.
RESULTS: 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.
CONCLUSIONS: 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.