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Expression of an alternative spliced IL4 mRNA was found in in vitro activated T cells. In this study we show that the expression of IL4 mRNA, as well as the expression of this alternatively spliced form of IL4 mRNA, is not restricted to these cells. We analyzed different human lymphoid tissues and cell lines of different origin and found that the alternatively spliced IL4 transcripts are also expressed in human lymphoid tissues, in purified B cells, in the various B cell-derived cell lines, and even in nonlymphoid cell lines. Stimulation with the phorbol ester PMA enhanced expression of both transcripts in all cells studied. The two IL4 transcripts were cloned and sequenced from the B cell line Namalwa. In the alternatively spliced form, the same exon 2 is deleted as has been observed in in vitro activated T cells. In principle, such alternatively spliced mRNA may give rise to a truncated IL4 protein, as the deletion does not result in a frameshift. We tested supernatants of activated PBMC, cell lines, and cell extracts for the presence of IL4 protein. We found IL4 protein expression in activated PBMC, but not in any of the stimulated cell lines or in the purified B cells. Using a modified in situ hybridization method with Dig-labeled PCR products, however, these cells did express IL4 mRNA. This shows that transcription of both IL4 forms is not restricted to T cells and can be induced in other cell types as well. Using these non-T cells, no protein has been found in the supernatant, however. It is possible that transcription of the IL4 gene is not necessarily followed by translation and that translation into the IL4 protein requires an additional signal.

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 peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human 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 shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. 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 follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.