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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.

The tissue distribution of human IgG Fc receptors (Fc gamma Rs) classified in three clusters of differentiation (CD16, using 5 antibodies, CD32, using 2 antibodies; and CD64, using 3 antibodies) was evaluated by immunohistochemistry on lymphoid (lymph node, spleen, thymus, tonsil) and non-lymphoid (heart, jejunum, kidney, liver, lung, muscle, stomach, and skin) tissues. Macrophage-like cells, including Kupffer cells, expressed all three classes of Fc gamma R. Part of the cells coexpressed HLA-DR. Interdigitating dendritic cells that were present in high density in interfollicular areas of a lymph node showing dermatopathic lymphadenopathy were immunoreactive for CD32, but not for CD16 or CD64 antibodies. In lymphoid tissue, mantle zones of secondary follicles were labeled by CD32 and some CD16 antibodies. The immunolabelling of mantle zones was not present after washing the sections at low pH, which suggests that the molecules detected were passively absorbed on the cell surface (i.e. soluble Fc gamma R). The immunolabelling of tonsil sections by various CD16 antibodies showed three patterns. The first (anti-Leu-11b) revealed labelling of solitary macrophage-like cells. The second (BW209/2 and 3G8) revealed, in addition, labelling of germinal centres. The third (CLBgran1 and CLBgran11) revealed labelling of solitary cells and follicle mantles. This labelling on tissue sections was also seen in the analysis of follicular dendritic cells isolated from tonsil. The cells were faintly immunoreactive for 3G8, as well as for CD16 mAb CLBgran1, and both CD32 mAbs. In all tissues investigated there was immunoreactivity for Fc gamma Rs in varying intensity on endothelial cells of blood vessels.