Heterogeneity of expression of apical membrane determinants in A6 epithelial cells

Expression of apical membrane antigens by A6 cells is heterogeneous. The A6 cell line is a model system for sodium transport and hormone action in "tight" epithelia. To create probes for the study of sodium transport and aldosterone action, we generated monoclonal antibodies (MoAbs) by immunization of BALB/c mice with intact A6 cells. MoAbs binding to apical membrane determinants were selected by an enzyme-linked immunoadsorbance assay (ELISA) against the apical surface of confluent A6 monolayers. Examination of ELISA-positive MoAbs by indirect immunofluorescence on confluent cells grown on glass cover slips revealed heterogeneity of apical expression: individual cells comprising the monolayer were fluorescently labeled to variable degrees and some cells were unlabeled. Expression of one antigen, recognized by MoAb I4C1, was heterogeneous in cells at low density as well as for confluent cells. Another antibody, 2B8, exhibited heterogeneity of labeling in confluent A6 monolayers, but no labeling of any cells at low density. This heterogeneity of antigen expression suggested that A6 cells are not a single cell type. However, cloned A6 cells established by limiting dilution were also labeled heterogeneously by both antibodies. Thus the heterogeneity observed is not due to genetic inhomogeneity between cells. Expression of the antigens on apical membranes of cells grown on Millipore filters was heterogeneous for the antigen recognized by 2B8, but essentially homogeneous for that recognized by I4C1, suggesting that the degree of differentiation of the cells plays a role in the heterogeneous expression of apical antigens. This heterogeneity of expression of apical antigens adds new complexity to the study of A6 cells and the differentiation of epithelial cells in culture.