No abstract

Mechanisms of protective immunity to mycobacterial infection in the lung remain poorly defined. In this study, T-cell subset expansion and cytokine expression in bronchoalveolar spaces, lung parenchyma, and mediastinal lymph nodes of mice infected intratracheally with Mycobacterium bovis-Calmette-Guerin bacillus (BCG) were analyzed in parallel with histopathology and bacterial burden. M. bovis-BCG was cleared rapidly from bronchoalveolar spaces without evidence for persistence. In lung parenchyma bacteria grew during the first 4 wk followed by gradual clearance with less than 0.1% of the original inoculum persisting for more than 8 mo. Clearance of M. bovis-BCG from bronchoalveolar lavage was associated with recruitment of both neutrophils and lymphocytes. Lung CD4(+), CD8(+), and gammadelta T-cell receptor-positive T cells expanded maximally by Week 4, and declined by Week 8 to control values despite bacterial persistence. Both CD4(+) and CD8(+) lung T cells produced interferon (IFN)-gamma in response to M. bovis-BCG. Four distinct pathologic states of lung parenchymal infection were noted. Early focal sub-bronchial inflammation with transmigration of cells into airways was followed by diffuse peribronchitis, perivasculitis, and alveolitis with activated macrophages, lymphoblasts, and occasional giant cells. The latter stage corresponded to maximal M. bovis-BCG growth. Resolving infection consisted of small lymphocytes and foamy macrophages, which coincided with decreasing M. bovis-BCG colony-forming units, T-cell infiltration, and IFN-gamma expression. A final quiescent phase consisted of residual lymphoid aggregates and perivasculitis associated with persistent spontaneous IFN-gamma production. Bacterial dissemination to lymph node and spleen occurred by Week 4 and declined in parallel to lung. In contrast to lung, IFN-gamma secretion was detected only late despite early expansion of CD4(+) and CD8(+) T cells. By reverse transcriptase/polymerase chain reaction, IFN-gamma and interleukin (IL)-12 p40 messenger RNA (mRNA) in lung paralleled IFN-gamma protein production. Tumor necrosis factor-alpha, IL-4 and IL-10 mRNA expression was not increased during M. bovis-BCG lung infection. Thus, protective immunity to M. bovis-BCG in the lung evolved differently in air space, lung, and lymph node.

Experimental studies of granuloma formation and antimycobacterial immunity suggest that the lung is uniquely susceptible to Mycobacterium tuberculosis infection. Growth of virulent M. tuberculosis strains and avirulent strains of Mycobacterium bovis BCG is more rapid and destructive in the lung compared to other organs, and far fewer organisms are needed for infection when bacteria are delivered by aerosol compared to intravenous infection (9). Thus, infection of the lung is an important factor affecting the growth and survival of mycobacteria (4). The inhalation of aerosol droplets containing bacilli leads to the deposition of bacilli in both conducting and distal airways where many organisms are removed by mucociliary mechanisms. However, in alveolar spaces resident macrophages phagocytose organisms, resulting in the expression of reactive oxygen and nitrogen radicals and multiple chemokines and cytokines (3,38,39,45,50,52). These innate immune responses do not control the early growth of either virulent M. tuberculosis or avirulent M. bovis BCG in murine lungs. Ultimately, control of both M. tuberculosis and M. bovis BCG infection is dependent on the recruitment and activation of major histocompatibility complex (MHC)-restricted CD4 ϩ and CD8ϩ T cells (6, 9, 11, 21, 33, 52). Whether permissive growth of M. tuberculosis or M. bovis BCG in alveolar spaces is due to an inability of alveolar macrophages to activate T cells, to the capacity of mycobacteria to impair alveolar macrophage function, or to the effects of alveolar protein factors on alveolar macrophage and T-cell function is not known. Recent studies have shown that the 19-kDa lipoprotein which is expressed by both M. bovis BCG and M. tuberculosis can inhibit MHC-II antigen processing and presentation in bone marrow-derived macrophages (31,32,34,47). The inhibition occurs by blocking gamma interferon (IFN-␥) signaling through a Toll-like receptor 2 (TLR-2)-dependent mechanism. The present study was undertaken to determine the ability of alveolar macrophages to serve as antigen-presenting cells (APC) and whether alveolar macrophage function is affected by mycobacterial infection and exposure to mycobacterial lipoproteins. We determined that resident murine alveolar macrophages have the necessary surface molecules to serve as efficient APC for CD4 ϩ T cells, that they can process and present MHC-II-restricted antigens, and that the APC function of alveolar macrophages was inhibited by M. bovis BCG infection and the 19-kDa lipoprotein. Thus, the mycobacterial infection of alveolar macrophages may impede activation of CD4 ϩ T cells and contribute to the permissive pulmonary microenvironment supporting mycobacterial growth and persistence. MATERIALS AND METHODSMice. Specific pathogen-free, female C57BL/6 (H-2 b ) mice were purchased from Charles River Laboratories (North Wilmington, Mass.) and used at between 8 and 12 weeks of age. TLR-2 gene knockout mice were generously provided by O. Takeuchi and S. Akira (Osaka University, Osaka, Japan) and bred onto the...

Although neutrophils have been identified as sources of inflammatory cytokines and chemokines, little is known about their immunologic function during mycobacterial infection in the lungs. In this study, we examined the growth of Mycobacterium bovis BCG in the lungs under experimental conditions that altered neutrophil recruitment to the lungs. Depletion and recruitment of neutrophils was associated with respective increases and decreases in M. bovis BCG growth. Thus, neutrophils may enhance mycobacteriocidal immunity in the lungs

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4؉ T-cell responses. We have previously shown that M. tuberculosis can indirectly inhibit CD4 ؉ T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine if M. tuberculosis could directly inhibit CD4 ؉ T-cell activation. Murine CD4 ؉ T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4 ؉ T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence of M. tuberculosis and its subcellular fractions. CD4؉ T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence of M. tuberculosis. Fractionation identified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannosecapped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosis glycolipids directly inhibited CD4؉ T-cell activation by interfering with proximal T-cell-receptor signaling.Aerosolized Mycobacterium tuberculosis infects alveolar and lung parenchymal macrophages, where it replicates unrestrained in the face of innate responses until T-cell immunity controls its growth. Despite robust activation of innate and adaptive immunity, M. tuberculosis survives and persists as a latent infection (15, 18). CD4 ϩ T cells have a central role in controlling M. tuberculosis during acute and latent infections (53). Animal studies have shown that depletion or absence of CD4 ϩ T cells during primary infection results in unchecked M. tuberculosis growth in the lung and decreased survival (37,38,41). Depletion of CD4 ϩ T cells during latent infection also worsens disease and survival (46). In humans, loss of CD4 ϩ T cells from progressive human immunodeficiency virus infection is directly responsible for the high rates of tuberculosis in human immunodeficiency virus-infected persons (48).Much is known about how M. tuberculosis manipulates macrophages for its survival (19,29,44). However, the way in which M. tuberculosis interferes with adaptive T-cell immunity is not well understood. Our recent studies have demonstrated that M. tuberculosis can modulate CD4 ϩ T-cell function both indirectly and directly. M. tuberculosis, through Toll-like receptor 2 (TLR-2), inhibits gamma-interferon-regulated genes that result in decreased major histocompatibility complex class II (MHC-II) antigen processing by macrophages for effector and memory CD4 ϩ T cells (22,23,40,43). M. tuberculosis can also induce increased adhesion to fibronectin through ␣ 5 ␤ 1 integrin on CD4 ϩ T cells (45)...

Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-alpha, IL-10, and transforming growth factor (TGF)-beta in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-gamma, the effect of IFN-gamma on IL-12 expression was examined. IL-12 mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-alpha protein expression occurred earlier than IL-12 p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-beta did not significantly alter IL-12 induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced increase in IL-12 followed IFN-gamma pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.

Mycobacterium tuberculosis and its antigens are potent inducers of cytokine expression by mononuclear phagocytes. In this study, the ability of live M. tuberculosis to stimulate interleukin-12 (IL-12) expression by human monocytes was examined. Monocytes were purified from peripheral blood mononuclear cells by adherence and either infected with M. tuberculosis or exposed to soluble protein antigens of M. tuberculosis (purified protein derivative [PPD]). Live M. tuberculosis (10 6 to 10 7 CFU/ml) was a potent stimulus for interleukin-12 (IL-12). By using reverse transcription-PCR, p40 mRNA was detected at 3 h, peaked at 6 to 12 h, and decayed to baseline levels at 18 to 24 h following infection. Bioactive IL-12 (p70) was measured by the phytohemagglutinin blast proliferation assay and confirmed the p40 mRNA results. In contrast, soluble PPD at concentrations known to readily induce IL-1 and tumor necrosis factor alpha expression by monocytes (10 to 100 g/ml) was a poor stimulus for IL-12 p40 mRNA expression. The different efficiencies of M. tuberculosis bacilli and PPD for IL-12 expression by monocytes was in part due to a requirement for phagocytosis. Induction of IL-12 in response to M. tuberculosis was reduced by cytochalasin D. Furthermore, phagocytosis of dead M. tuberculosis or inert 2-m-diameter polystyrene beads by monocytes induced IL-12 p40 mRNA. In contrast, 0.5-m-diameter beads, which can enter cells through pinocytosis, did not stimulate IL-12 expression. Functionally, IL-12 readily enhanced PPD-stimulated IFN-␥ production and CD4 ؉ T-cell-mediated cytotoxicity by peripheral blood mononuclear cells from healthy tuberculin-positive donors but induced less enhancement when live M. tuberculosis was the antigen. These results suggest that IL-12 is upregulated as part of the early cytokine response of mononuclear phagocytes to M. tuberculosis and that the cellular events associated with phagocytosis are themselves a potent signal for IL-12 production. IL-12 released by infected macrophages in turn can further upregulate M. tuberculosis-specific CD4 ؉ T-cell effector function. Mycobacterium tuberculosis is an intracellular bacterial pathogen, which readily survives within human mononuclear phagocytes. The majority (Ͼ90%) of healthy individuals infected with M. tuberculosis develop a cellular immune response to this pathogen and successfully control its growth (10). The interaction of T cells and mononuclear phagocytes is critical in this acquired, protective immune response to M. tuberculosis and is associated with a proinflammatory cytokine environment, which includes interleukin-1 (IL-1) IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-␣) and gamma interferon (IFN-␥) (38). Recent studies have demonstrated that upon exposure to M. tuberculosis bacilli or its constituents, monocytes/macrophages can secrete both proinflammatory cytokines such as IL-1, IL-6, and TNF-␣ and inhibitory cytokines such as IL-10 and transforming growth factor ␤ (TGF-␤) (4, 49, 54). Cyto...

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFNgamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosolinfected with GFP-expressing BCG. At 28 d, ~1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b high /CD11c neg-mid lung macrophages (58-68%) or CD11b high /CD11c high DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP− cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.