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Although allograft rejection, the major complication of human organ transplantation, has been extensively studied, little is known about the exact cellular localization of the cytokine expression inside the graft during rejection. Therefore, we used in situ hybridization and immunohistochemistry to study local cytokine mRNA and protein expression in human heart allografts, in relation to the phenotypical characteristics of the cellular infiltrate. Clear expression of mRNA for interleukin (IL)-6, IL-8, IL-9, and IL-10 and weak expression for IL-2, IL-4, IL-5, and tumor necrosis factor (TNF)-alpha was detected in biopsies exhibiting high rejection grades (grade 3A/B). Also at lower grades of rejection, mRNA for IL-6 and IL-9 was present. Some mRNA for IL-1 beta, TNF-beta, and interferon (IFN)-gamma was detected in only a few biopsies. Using immunohistochemistry, IL-2, IL-3, and IL-10 protein was detected in biopsies with high rejection grades, whereas few cells expressed IL-6, IL-8, and IFN-gamma. In biopsies with lower grades of rejection, a weaker expression of these cytokines was observed. IL-4 was hardly detected in any of the biopsies. The level of IL-12 expression was equal in all biopsies. Although mRNA expression of several cytokines was expressed at a low level compared with the protein level of those cytokines, there was a good correlation between localization of cytokine mRNA and protein. Expression of IL-2, IL-4, IL-5, TNF-alpha, and IFN-gamma was mainly detected in lymphocytes. IL-3, IL-6, IL-10, and IL-12 were not detected or not only detected in lymphocytes but also in other stromal elements (eg, macrophages). Macrophage production of IL-3 and IL-12 was confirmed by immunofluorescent double labeling with CD68. We conclude that cardiac allograft rejection is not simply regulated by T helper cell cytokine production, but other intragraft elements contribute considerably to this process.

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.