AN IMPROVED, SENSITIVE, NONRADIOACTIVE IN-SITU HYBRIDIZATION METHOD FOR THE DETECTION OF CYTOKINE MESSENGER-RNAS

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated p eripheral blood mononuclear cells (PBMC), lymphoid cell lines and huma n lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was show n on ethidium bromide-stained agarose gels by a higher molecular weigh t of the PCR products with incorporated Dig-dUTPs when compared to con trol PCR products without digoxigenin. Cytospin-centrifuged cells of P HA-stimulated PBMC or lymphoid cell lines and frozen sections of vario us human lymphoid tissues were hybridized with the Dig-labelled cytoki ne probes and the hybridized probes were detected immunohistochemicall y. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the fo llicular lymphoma cell line DoHH2, and in human lymph nodes and tonsil s. The in situ hybridization had a high sensitivity as the results cor related closely with the detection of cytokine mRNA by reverse transcr iptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, aft er activation with the phorbol ester PMA, expression of several cytoki ne mRNAS was enhanced.