SummaryTo understand the selective accumulation of memory T helper lymphocytes and of macrophages in delayed-type hypersensitivity (DTH) granulomas, we studied the in situ production of RANTES, a chemokine initially characterized on the basis of its in vitro chemotactic properties for each of these cell populations. RANTES gene expression was studied by in situ hybridization in 15 human lymph nodes presenting typical DTH lesions related to either sarcoidosis or tuberculosis. A positive signal was detected in all cases. Labeling was specific for the DTH lesions, as very few if any positive cells were detected in the normal residual lymphoid tissue surrounding them or in reactive lymph nodes involved in a B lymphocyte response. R.ANTES gene expression was associated with the production of the protein, which was detected by immunochemistry in DTH lymph nodes. The morphological characteristics and distribution of positive cells in in situ hybridization and immunochemical experiments indicated that macrophages and endothelial cells, two cell populations not previously reported to produce RANTES, contributed to its production in DTH reactions. The ability of macrophages and endothelial cells to produce R.ANTES was confirmed by in vitro studies with alveolar macrophages and umbilical vein endothelial cells. In view of the chemotactic properties of RANTES for a limited range of cell populations, these results suggest that RANTES production in DTH granulomas may play a role in the selective accumulation of macrophages and memory T helper lymphocytes characterizing this type of cell-mediated immune reaction, and that macrophages and endothelial cells are involved in this production.

The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients. rIL-2 administration increased the frequency of positive cells in all patients (P < 0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P < 0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56+ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.

Interleukin-6 is a major B lymphocyte growth factor, and may play a role in the proliferation of malignant B lymphocytes. In order to provide arguments supporting such a role, the intratumoral production of IL-6 was studied by in situ hybridization and immunohistochemistry in 53 neoplastic tissues from B cell chronic lymphocytic leukemia or B lymphomas. IL-6-producing cells were detected in all samples but 5. However, the number of IL-6 producing cells was variable amongst the different cases. Increased density of IL-6-producing cells was highly dependent on the presence of malignant immunoblasts within the neoplastic clone. IL-6 was produced in a paracrine way, macrophages and endothelial cells being the main producers of the cytokine while malignant immunoblasts expressed the IL-6 receptor. Taken together, these results suggest that IL-6 may indeed act as a growth factor for malignant cells in some B lymphoproliferations and that this paracrine loop could be the target of new therapeutic approaches.

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