Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: Comparison of an immortalized cell line to primary cells, and their response to retinoic acid

Abstract

PURPOSE. The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS. Primary cells, maintained in serum free medium, were exposed to 10 -6 M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (a 3, a 6, a V, ß 1, ß 2, ß 3 and ß 4) , laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS. Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins a 6 (CD49f) and a V (CD51) were expressed by primary and immortalized cells. Expression of integrin a 6 was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin a V expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin a 6 expression by primary cells. Both cell types also expressed integrins ß 1, ß 2 and ß 3, but ß 4 was detected only in immortalized cells. Lacrimal acinar cells do not express integrin a 3. CONCLUSIONS. Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin a V, but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.