Nasal-associated lymphoid tissue is not an absolute requirement for the induction of rat tear IgA antibody responses.

UNLABELLED: PURPOSE/AIM OF STUDY: The purpose of this work was to determine whether rat nasal-associated lymphoid tissue is required for the induction of tear IgA responses. MATERIALS AND METHODS: Particulate antigen in the form of DNP-BSA encapsulated in cationic microparticles was applied topically to the eyes (ocular topically) of rats that had the nasolacrimal ducts temporarily plugged with chromic gut suture material. Eye washes and serum were monitored for development of antigen specific IgA and IgG, respectively. To track the particulate uptake, fluorescent latex beads were applied topically to the eyes of plugged and unplugged animals. The nasal-associated lymphoid tissue and the draining lymph nodes were then examined for the presence of the fluorescent beads. RESULTS: It was found that the chromic gut suture was effective in blocking the passage of antigen into the nasopharyngeal cavity for at least 24 hr. Tear antigen-specific IgA levels found in the eyes of plugged animals were not significantly lower from those of unplugged animals. Serum IgG antibody levels were also similar between the two groups. In animals with plugged nasolacrimal ducts, fluorescent beads were found predominately in the superficial cervical lymph nodes, which have been shown to drain the surface of the eye. CONCLUSIONS: These results indicate that particulate antigen can be taken up by the conjunctiva and transported to the draining lymph nodes, showing that antigen does not need to access nasal-associated lymphoid tissue to induce tear IgA antibody responses.

Development of immortalized rat conjunctival epithelial cell lines: an in vitro model to examine transepithelial antigen delivery.

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.