Functional expression of the endogenous Thy‐1 gene and the transfected murine Thy‐1.2 gene in rat basophilic leukemia cells

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Abstract
An interaction of MRC OX7 monoclonal antibody with Thy‐1.1 antigen of rat peritoneal and pleural mast cells has been previously shown to induce rat mast cell activation (L'. Dráberová, Eur. J. Immunol. 1989. 19: 1715). In the present study we analyzed the expression and function of the Thy‐1 antigen in rat basophilic leukemia cells, clone RBL‐2H3. Two RBL‐2H3‐derived cell lines with stable expression of murine Thy‐1.2 antigen, after transfection of a genomic clone of murine Thy‐1.2 or Thy‐1.2 cDNA under the control of simian virus 40 promoter, were also analyzed. Direct radioantibody binding assays, indirect immuno‐fluorescence studies and flow cytometry analyses revealed that both endogenous Thy‐1.1 and transfected murine Thy‐1.2 gene products were expressed on the surface of the cells under study. Analysis of the distribution of the Thy‐1 antigen in situ in cells grown attached to tissue culture vessels located the Thy‐1 predominantly in regions of cell‐cell contacts. Incubation of RBL‐2H3 cells with Thy‐1.1‐specific antibodies, or of the transfected cells with both Thy‐1.1‐ and Thy‐1.2‐specific antibodies, induced a rapid early increase in the concentration of intracellular free calcium ([Ca2+]i) released from internal stores. Sustained increase of [Ca2+]i required the presence of Ca2+ in the extracellular medium. The increase in [Ca2+]i was followed by histamine release from the target cells. The combined data indicate that RBL‐derived cells can be used as a useful model system for analysis of Thy‐1 antigen‐mediated activation of rat mast cells.