Peter F. Barnes

Interleukin 12 at the site of disease in tuberculosis.

Interleukin 12 (IL-12), a heterodimeric cytokine composed of p40 and p35 chains, has potent immunologic effects in vitro. We used tuberculous pleuritis as a model to study the immunoregulatory potential of IL-12 in vivo at the site of human infectious disease. Messenger RNAs for p40 and p35 were detected in pleural fluid from six of six patients by reverse-transcription polymerase chain reaction. By using an ELISA that detected both free p40 and heterodimeric IL-12, we found that mean concentrations were 585 +/- 89 pg/ml in pleural fluid of patients with tuberculous pleuritis, which were significantly higher than those in serum of the same patients (54 +/- 36 pg/ml), or in malignant pleural effusions (123 +/- 35 pg/ml). By using an ELISA specific for heterodimeric IL-12, we found that mean concentrations in pleural fluid of patients with tuberculous pleuritis were 165 +/- 28 pg/ml and undetectable in serum of the same patients, or in malignant pleural effusions. Bioactive IL-12 was detectable in five of five supernatants of pleural fluid cells stimulated with Mycobacterium tuberculosis. Addition of anti-IL-12 antibodies suppressed proliferative responses of pleural fluid cells to M. tuberculosis by 36 +/- 7%. These data indicate that IL-12 may play a role in the human immune response to infectious agents in vivo. We hypothesize that IL-12 contributes to the antimycobacterial immune response by enhancing production of interferon-gamma, facilitating development of Th1 cells and augmenting cytotoxicity of antigen-specific T cells and natural killer cells.

Delayed diagnosis of tuberculosis in patients with human immunodeficiency virus infection

PURPOSE To determine the frequency with which the diagnosis of tuberculosis is delayed in patients with concomitant human immunodeficiency virus (HIV) infection, and to identify reasons for such delays. PATIENTS AND METHODS We reviewed medical records of 52 consecutive HIV-infected patients with culture-proven tuberculosis seen at a 1,900-bed general hospital serving a predominantly indigent population in Los Angeles, where the prevalences of HIV infection and tuberculosis are high. The late-treatment (LT) group consisted of 25 patients in whom tuberculosis was untreated prior to death (n = 6) or treated more than 22 days after presentation (n = 19). The early-treatment (ET) group comprised 27 patients in whom antituberculous therapy was begun less than 16 days after presentation. RESULTS Symptoms, physical and laboratory findings, chest roentgenographic abnormalities suggestive of tuberculosis (hilar adenopathy, pleural effusion, miliary pattern, cavitation, predominant upper lobe infiltrate), and frequencies of concomitant nontuberculous disease were similar in LT and ET groups. Delayed diagnosis of tuberculosis was attributable to errors in management in 21 (84%) of 25 LT group patients. The most common error was failure to obtain at least three sputum samples for acid-fast smear and mycobacterial culture in patients with clinical and chest roentgenographic findings compatible with tuberculosis (15 cases). Acid-fast sputum smears were positive in 25 (61%) of 41 cases of pulmonary tuberculosis. Acid-fast smears of stool were positive in eight (42%) of 19 cases. Blood cultures yielded Mycobacterium tuberculosis in 18 (38%) of 48 cases. CONCLUSIONS Delayed therapy of tuberculosis in HIV-infected patients at our medical center was common and was not due to atypical manifestations of tuberculosis. In most cases, delays could have been avoided if adequate numbers of sputum samples for acid-fast smear and mycobacterial culture had been obtained, and if empiric antituberculous therapy had been given to symptomatic patients in whom chest roentgenographic findings were suggestive of mycobacterial disease.

T cell cytokine responses in persons with tuberculosis and human immunodeficiency virus infection.

Tuberculosis causes more extensive and life-threatening disease in patients with HIV infection than in immunocompetent persons. To investigate the hypothesis that these severe manifestations of tuberculosis may be due to alterations in cytokine production, we evaluated cytokine patterns in HIV-infected tuberculosis patients. Upon stimulation with Mycobacterium tuberculosis in vitro, PBMC from HIV-infected tuberculosis patients had reduced proliferative and type 1 responses, compared with HIV-seronegative tuberculosis patients. The reduction in proliferative responses was independent of the CD4 cell count, but the reduced type 1 response was a direct result of CD4 cell depletion. There was no enhancement of type 2 cytokine production in HIV-infected patients, although production of IL-10 was prominent in all tuberculosis patients. In HIV-infected tuberculosis patients, M. tuberculosis-induced proliferative responses were significantly enhanced by neutralizing antibodies to IL-10 but not by antibodies to IL-4 or by recombinant IL-12. The M. tuberculosis-induced type 1 response was augmented both by antibodies to IL-10 and by recombinant IL-12. Tuberculosis in the context of HIV infection is characterized by diminished type 1 responses, probably induced by immunosuppressive cytokines produced by macrophages/monocytes, rather than by type 2 cells.

Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.

The NKp46 Receptor Contributes to NK Cell Lysis of Mononuclear Phagocytes Infected with an Intracellular Bacterium

We used human tuberculosis as a model to investigate the role of NK cytotoxic mechanisms in the immune response to intracellular infection. Freshly isolated NK cells and NK cell lines from healthy donors lysed Mycobacterium tuberculosis-infected monocytes to a greater extent than uninfected monocytes. Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor, but not the NKp44 receptor. Antisera to NKp46 markedly inhibited lysis of infected monocytes. NK cell-mediated lysis was not due to reduced expression of MHC class I molecules on the surface of infected monocytes or to enhanced production of IL-18 or IFN-γ. NK cell lytic activity against M. tuberculosis-infected monocytes and NKp46 mRNA expression were reduced in tuberculosis patients with ineffective immunity to M. tuberculosis compared with findings in healthy donors. These observations suggest that 1) the NKp46 receptor participates in NK cell-mediated lysis of cells infected with an intracellular pathogen, and 2) the reduced functional capacity of NK cells is associated with severe manifestations of infectious disease.

NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-γ+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+IFN-γ+cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-γ, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-γ, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-γ. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-γ+ T cells by producing IFN-γ, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-γ and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.

Clinical Correlates of Interferon γ Production in Patients with Tuberculosis

To determine if the capacity to produce interferon (IFN) γ is related to the clinical manifestations of tuberculosis, we correlated Mycobacterium tuberculosis-induced IFN-γ production by peripheral blood mononuclear cells (PBMCs) with clinical, radiographic, and laboratory variables for 63 human immunodeficiency virus (HIV)-negative patients and 43 HIV-positive patients with tuberculosis. For HIV-negative patients whose chest radiographs showed moderately advanced disease, the mean IFN-γ concentration ± SD was 1,639 ± 388 pg/mL, whereas that for patients with far-advanced disease was 327 ± 100 pg/mL (P =.0001). For HIV-infected patients who had only pleuropulmonary disease, the mean IFN-γ concentration was 1,002 ± 257 pg/mL, whereas that for patients with disease outside the lungs and pleura was 149 ± 55 pg/mL (P =.0004). Multivariate analysis confirmed that the radiographic extent of disease and the site of disease were the only independent predictors of IFN-γ production in HIV-negative and HIV-infected patients (P ≤.001). We conclude that reduced IFN-γ production by PBMCs is a marker of severe tuberculosis in both HIV-negative and HIV-infected patients with tuberculosis.

NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen

We evaluated the capacity of NK cells to influence expansion of CD4+CD25+FoxP3+ regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4+ cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-γ. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.

IL-22 Produced by Human NK Cells Inhibits Growth of Mycobacterium tuberculosis by Enhancing Phagolysosomal Fusion

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3−CD56+ NK cells produced IL-22 when exposed to autologous monocytes and γ-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

Vimentin Expressed on Mycobacterium tuberculosis-Infected Human Monocytes Is Involved in Binding to the NKp46 Receptor

We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.