In Vitro Lymphocyte Adhesive Interactions With Cultured Rat Lacrimal Gland Acinar Epithelial Cells
Immunity at the ocular surface is dependent upon the function of the lacrimal glands and their association with the entire mucosal immune network, a relationship made possible by the circulation of lymphocytes. The localization of lymphocytes within lacrimal gland tissue contributes to the expression of immunoglobulin A (IgA) antibodies in the tears and is regulated by cellular interactions between lacrimal gland acinar epithelial cells and the lymphoid cells. This study sought to 1) characterize the lymphocyte adhesive interactions with cultured lacrimal gland acinar epithelial cells, 2) compare these interactions with those observed for lacrimal gland cryostat sections and primary cultures, and 3) determine whether molecules mediating lymphocyte-lacrimal gland binding could be isolated from cultured lacrimal acinar epithelial cells. Through use of lymphocyte adherence assays it was found that thoracic duct lymphocytes (TDL) bound to lacrimal gland acinar epithelial cells in greater numbers than thymocytes (294 cells/mm2 versus 8 cells/mm2). Adhesion was found to be not only dose dependent, but temperature and time dependent as well. Use of the chemical inhibitors sodium azide, cytochalasin B, and formaldehyde resulted in diminished lymphocyte adherence (12%, 4%, and 1% of control binding, respectively). Furthermore, binding was dependent on the presence of divalent cations: Inhibition by ethylenediaminetetraacetic acid (32% of untreated) was reversed by replacing the lymphocyte suspensions with calcium (106% of control binding) and magnesium (76% of control binding). In addition, binding seemed to be enhanced when calcium and magnesium were replaced simultaneously (167% of contol binding). TDL adherence was inhibited in the presence of fucoidin or phoshomannan polysaccarides (47% and 65%, respectively). Treating the TDL with anti-selectin monoclonal antibodies A.11.5, HRL-2 and HRL-3 gave very low inhibition (7%, 21%, and 29%, respectively). Addition of anti-integrin monoclonal antibodies WT.l and HP2/1 to the TDL resulted in diminished adherence (38% and 52% of control binding, respectively). 1A.29, an anti-ICAM1 antibody, reduced TDL adherence to only 38% of control binding. Other anti-adhesion molecule antibodies, OX7, against Thy-I, and OX49, against CD44, inhibited adherence by 55% and 43% respectively. Finally, greater than 40% inhibition was observed when TDL were assayed in the presence of several concentrated culture supernatants. Collectively, these results indicate that lymphocyte binding to cultured lacrimal gland acinar epithelial cells requires viable lymphocytes with unimpaired surface function and the presence of divalent cations. They further suggest that the adhesion event involves the action ofintegrins, members of the immunoglobulin superfamily, selectins, and other cellular adhesion molecules. Furthermore, these findings suggest that the lacrimal gland epithelial cell cultures will be useful for isolating adhesion molecules involved in lymphocyte localization. Finally, the results of the lymphocyte adherence characterization studies indicate that thoracic duct lymphocyte adherence to cultured lacrimal gland acinar epithelial cells showed good correlation with previously reported adherence to lacrimal gland cryostat sections and primary cultures.With honors.