We have used RNA-RNA in situ hybridization to detect the expression of several Mycobacterium tuberculosis genes in tuberculous granulomas in lung tissue sections from tuberculosis patients. The M. tuberculosis genes chosen fall into two classes. Four genes (icl, narX, and Rv2557 and Rv2558) have been implicated in the persistence of the bacterium in the host, and two genes (iniB and kasA) are upregulated in response to isoniazid exposure. Both necrotic and nonnecrotic granulomas were identified in all of the patients. Necrotic granulomas were divided into three zones: an outer lymphocyte cuff containing lymphocytes and macrophages, a transition zone consisting of necrotic material interspersed with macrophages, and a central acellular necrotic region. Transcripts of all of the genes studied were found in nonnecrotic granulomas and in the lymphocyte cuff of necrotic granulomas. Mycobacterial gene expression was associated with CD68-positive myeloid cells. Rv2557 and/or its homologue Rv2558, kasA, and iniB were expressed within the transition zone of necrotic granulomas, whereas icl and narX transcripts were absent from this area. There was no evidence of transcription of any of the genes examined in the central necrotic region, although mycobacterial DNA was present. The differential expression of genes within granulomas demonstrates that M. tuberculosis exists in a variety of metabolic states and may be indicative of the response to different microenvironments. These observations confirm that genes identified in models of persistence or in response to drug treatment in vitro are expressed in the human host.

Mycobacterium tuberculosis is a facultative intracellular pathogen which can survive and replicate within the host macrophage. The majority (about 90%) of individuals are relatively resistant to M. tuberculosis and mount an effective cell-mediated immune response, which results in the development of delayed-type hypersensitivity reactions in the skin but no manifestations of clinical disease. In susceptible individuals, however, an inadequate immune response allows the clinical disease of tuberculosis (TB) to develop. In the majority of patients who develop TB, the symptoms are restricted to the lung (pulmonary TB); however, some patients develop extrapulmonary TB, where the mycobacteria escape from the lungs and colonize other organs.The host immune response plays a crucial role in determining which outcome results from the encounter between the host and the pathogen. There have been many studies of cellmediated immunity in patients with TB or other granulomatous diseases and in subjects exposed to M. tuberculosis but exhibiting no symptoms. Studies of the peripheral circulation and the site of pathology, e.g., the tuberculous effusion, bronchoalveolar lavage fluid, and lymph node biopsies, have been carried out (3,12,17). There is general consensus that CD4 ϩ

SUMMARYIn 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 in®ltrate. Paraf®n-embedded tissue from the lungs of seven patients with pulmonary tuberculosis was analysed by immunohistochemistry and in situ hybridization to detect interferon-c (IFN-c), tumour necrosis factor-a (TNF-a) and interleukin-4 (IL-4) proteins and IL-12p40 mRNA. All seven patients had granulomas staining positive for IFN-c, TNF-a and IL-12p40, but only four stained positive for IL-4. Cells with the morphology of lymphocytes, macrophages and giant cells expressed TNF-a, IFN-c 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 identi®ed. The ®rst association was between IFN-c and IL-12, with 76% of granulomas staining positive for both cytokines. Unexpectedly, those granulomas positive for IL-4 were always positive for IFN-c. The second association was between TNF-a and caseous necrosis, where all necrotic granulomas were TNF-a positive. This association was modulated by IL-4. Therefore, heterogeneity of cellular in®ltrate and cytokine expression is observed between adjacent granulomas in the same patient.

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.