Distribution of IFN-γ, IL-4 and TNF-α protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas

In order to examine the immune response at the site of pathology in tuberculosis, we analysed cytokines present in lung granulomas, their associations with each other and with caseous necrosis as well as the phenotype of the cellular infiltrate. Paraffin-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-yγ TNF-α and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-α, IFN-γ and IL-4 protein. Furthermore, CD68-positive myeloid cells expressed IL-12p40 mRNA, as expected, but a subset of CD3-positive lymphocytes also expressed this mRNA. These lymphocytes producing IL-12p40 also stained positive for CD8 but not CD4. A total of 141 granulomas were scored for the presence or absence of cytokine or necrosis and two major associations were identified. The first association was between IFN-γ and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-γ. The second association was between TNF-α and caseous necrosis, where all necrotic granulomas were TNF-α positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular infiltrate and cytokine expression is observed between adjacent granulomas in the same patient.