title: Development of immortalized rat conjunctival epithelial cell lines: an in vitro model to examine transepithelial antigen delivery.
abstract: The objective of these studies was to develop conjunctival epithelial cell lines for investigation of antigen translocation across a mucosal barrier. Conjunctival epithelial cells from Fischer 344 rats were immortalized with pSV3(neo) resulting in two cell lines--CJ4.1A and CJ4.3C. Each formed confluent cell layers with epithelial morphology when grown on permeable membrane filters. They expressed the SV40 T antigen, the conjunctiva-specific cytokeratin 4, the goblet cell-specific cytokeratin 7 and were negative for the corneal epithelial cell-specific cytokeratin 12. The cell lines have been in culture for over 60 passages, and the population doubling times were 22+/-7h for CJ4.1A and 23+/-9h for CJ4.3C. When grown on Transwell membranes, each cell line achieved a transepithelial electrical resistance of 600-800 Omega cm2 by 3-4 days and maintained a high resistance for several days. Both cell lines expressed zona occludens-1 at confluence. At 24h following addition of 250 microg of FITC-labeled ovalbumin to the apical chambers, 15+/-6 microg could be detected in the basal chamber of CJ4.1A and 6+/-1 microg in the basal medium of CJ4.3C. In contrast, 82+/-6 microg was detected in the lower chambers of cell-free Transwells. Similarly, Transwells containing confluent CJ4.1A or CJ4.3C cells impeded passage of 0.1 microm diameter polystyrene microspheres (5+/-1% and 4+/-1%, respectively, of the apical input), compared to 26+/-6% of the input microspheres recovered from the basal chambers of cell-free Transwells. Pretreatment with 4mM EGTA for 10 min caused an increase in OVA-FITC translocation across CJ4.3C cells. Incubation in the presence of 4mM EGTA significantly increased OVA-FITC translocation across both cell lines, relative to untreated cell layers. Morphological and functional characterization indicates that these cells provide a useful experimental tool to assess strategies for enhancing transepithelial antigen uptake.
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI 48210, USA. nosulliv@med.wayne.edu
authors.forename: Nancy L
authors.initials: NL
authors.lastname: O'Sullivan
authors.forename: Alfred E
authors.initials: AE
authors.lastname: Baylor
authors.forename: Paul C
authors.initials: PC
authors.lastname: Montgomery
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism*; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D003228:Conjunctiva / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D018613:Microscopy, Confocal; D008858:Microscopy, Phase-Contrast; D008863:Microspheres; D010047:Ovalbumin / Q000493:pharmacokinetics; D051381:Rats; D011916:Rats, Inbred F344; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D000941:Antigens; D005136:Eye Proteins; D007633:Keratins; D010047:Ovalbumin