IN-SITU HYBRIDIZATION FOR CYTOKINE MESSENGER-RNA WITH DIGOXIGENIN-LABELED RIBOPROBES - SENSITIVITY OF DETECTION AND DOUBLE-LABEL APPLICATIONS

A sensitive in situ hybridization procedure using both digoxigenin and S-35-labeled riboprobes is described that allows detection of single T cells expressing cytokine mRNA species in both single and double lab el formats. Modifications to existing procedures have been developed t hat allow in situ hybridization to be performed in either fresh frozen tissue sections or cytocentrifuge preparations of cultured cells. For single label studies, the digoxigenin labeling technique is equivalen t to S-35 labeling for sensitivity of detection and is superior with r espect to precise localization and ease of use. A procedure to detect two cytokine mRNA species in individual cells can be performed using o ne digoxigenin-labeled riboprobe and one S-35-riboprobe, with equivale nt sensitivity between the two labels and no non-specific mixing of th e two signals. Since production of many T cell cytokines are controlle d by transcriptional mechanisms, the use of in situ hybridization will be useful to investigate the biology of T cell activation, patterns o f cytokine phenotype development, and histological localization of cyt okine expressing cells in inflammatory lesions. Initial studies using this method to examine cytokine expression by a panel of T cell clones reveals that individual cytokine genes are not necessarily expressed in coordination in individual cells and relatively few individual cell s in a Th0 clone express Th1-like and Th2-like cytokines simultaneousl y.