Mycobacterium tuberculosis growth at the cavity surface: a microenvironment with failed immunity

Abstract

Protective immunity against pulmonary tuberculosis (TB) is characterized by the formation in the lungs of granulomas consisting of macrophages and activated T cells producing tumor necrosis factor alpha and gamma interferon, both required for the activation of the phagocytes. In 90% of immunocompetent humans, this response controls the infection. To understand why immunity fails in the other 10%, we studied the lungs of six patients who underwent surgery for incurable TB. Histologic examination of different lung lesions revealed heterogeneous morphology and distribution of acid-fast bacilli; only at the surface of cavities, i.e., in granulomas with a patent connection to the airways, were there numerous bacilli. The mutation profile of the isolates suggested that a single founder strain of Mycobacterium tuberculosis may undergo genetic changes during treatment, leading to acquisition of additional drug resistance independently in discrete physical locales. Additional drug resistance was preferentially observed at the cavity surface. Cytokine gene expression revealed that failure to control the bacilli was not associated with a generalized suppression of cellular immunity, since cytokine mRNA was up regulated in all lesions tested. Rather, a selective absence of CD4(+) and CD8(+) T cells was noted at the luminal surface of the cavity, preventing direct T-cell-macrophage interactions at this site, probably allowing luminal phagocytes to remain permissive for bacillary growth. In contrast, in the perinecrotic zone of the granulomas, the two cell types colocalized and bacillary numbers were substantially lower, suggesting that in this microenvironment an efficient bacteriostatic or bactericidal phagocyte population was generated.

FIG. 1.

Radiogram of the lungs of patient 2 before surgery (A) and the resected lung at the time of sample collection (B). The right lung is almost fully destroyed. It contains areas with cavitations, fibrosis, nodules, and granulomas with central necrosis.

FIG. 2.

IS6110 Southern blot hybridization patterns of M. tuberculosis isolates recovered from multiple anatomical sites in the lungs of patients 2 and 3. The six isolates from patient 2 were recovered from sputum (lane 1), right upper lobe open (RUL-O) (lane 2), right lower lobe open (RLL-O) (lane 3), right lower lobe open (lane 4), right middle lobe closed (RML-C) (lane 5), and right lower lobe closed (RLL-C) (lane 6). The seven isolates from patient 3 were recovered from sputum (lane 1), left upper lobe open (LUL-O) (lane 2), left lower lobe open (LLL-O) (lane 3), left lower lobe open (lane 4), left upper lobe closed (LUL-C) (lane 5), left lower lobe closed (LLL-C) (lane 6), and left lower lobe normal (LLL-“Normal”) (lane 7) (Table 2). Lane STD, molecular weight standard.

FIG. 3.

Localization of macrophages and AFB in resected lungs. Macrophages, stained with CD68 (A, C, and E) and AFB stained with carbolfuchsin (B, D, and F) in the left upper-lobe cavity wall of the open lesion of patient 1 are shown. CD68 staining is seen at the cavity (cav) surface (A and C), within the necrotic area (nec) (A and C), and in the granulomatous area below the necrotic area (E). AFB are seen predominantly within macrophages at the cavity surface (D) and not in the Langhans cells or macrophages of the granulomatous tissue (F). The inset in panel D shows AFB in cells at the luminal surface of the right-lower-lobe cavity of patient 2. AFB are also seen in macrophages within the liquefied (liq) material adjacent to the necrotic area (H) of the left-lower-lobe fibrotic nodule undergoing breakdown (liquefaction) in patient 3. Magnifications, ×10 (A and B), ×40 (C, E, F, and G), ×80 (D, inset), and ×200 (D and H).

FIG. 4.

Localization of lymphoid cells in lesions of resected lungs. CD4⁺ (A, B, and C) and CD8⁺ (E, F, and G) T cells are seen in the granulomatous-fibrotic areas of the lung (arrows) but not in the necrotic (nec) zone or at the cavity (cav) surface. CD4⁻ CD8⁻ cytotoxic cells (TIA-1⁺) are seen at the cavity surface but not in the necrotic zone (D) and are less frequent in the granulomatous area (*). These cells contain cytotoxic granules that stain TIA-1⁺ (H, inset). Magnifications, ×4 (A, D, and E), ×40 (B, C, F, G, and H), and ×100 (H, inset).