Infection of Mycobacterium bovis bacillus Calmette-Guérin in antibody-mediated gamma delta T-cell-depleted mice.

To evaluate the hypothesis that gamma delta T cells participate in protective immunity against mycobacterial infection, we depleted gamma delta T cells from mice by administration of anti-T-cell receptor (TCR)gamma delta monoclonal antibody (mAb) and analysed protection against Mycobacterium bovis bacillus Calmette-Guérin (BCG). The gamma delta T-cell-depleted mice did not show any exaggerated bacterial multiplication compared with control mice. In contrast, alpha beta T-cell-depleted mice, which were administrated anti-TCR alpha beta mAb before BCG infection, showed a depressed protective immunity. These results suggest that gamma delta T cells are not essential for coping with a primary BCG infection.     

Acquired Resistance but Not Innate Resistance to Mycobacterium bovis Bacillus Calmette-Guérin Is Compromised by Interleukin-12 Ablation

Interleukin-12 (IL-12) is one of the first cytokines produced by macrophages, key mediators of innate resistance, during the host’s immune response to infections. Therefore, in this study we propose that IL-12 has an important role in the early phase of the immune response to Mycobacterium bovis BCG. IL-12 has been shown to enhance the maturation of protective Th1 cells and gamma interferon (IFN-γ) production during mycobacterial infection. Therefore, it may play a crucial role during the immune phase of infection as well. To examine the role of IL-12 in both the innate and the immune phase of infection, we compared BCG-resistant mice, B10.A (Bcg^r), to the susceptible congenic strain B10.A (Bcg^s) following administration of a blocking monoclonal antibody to IL-12 (10F6). Anti-IL-12-treated susceptible animals exhibited a two- to threefold increase in spleen CFU by day 21. In contrast, anti-IL-12 treatment had little or no effect on the response of the genetically resistant animals to infection. The B10.A (Bcg^r) but not the B10.A (Bcg^s) mice had an increase in IFN-γ mRNA relative to baseline levels as early as day 1 of infection irrespective of anti-IL-12 treatment. By day 14, B10.A (Bcg^r) mice showed a decrease in IFN-γ mRNA while the B10.A (Bcg^s) mice showed a significant increase in IFN-γ mRNA levels. Thus, during BCG infection, the B10.A (Bcg^r) mice mount an early IFN-γ response against BCG whereas the B10.A (Bcg^s) mice have a delayed IFN-γ response correlating with their genetic permissiveness expressed as an increased mycobacterial load by day 21. Overall, our data demonstrate that the inherent resistance of B10.A (Bcg^r) mice to mycobacteria does not depend on optimal levels of IL-12 to maintain effective control of the bacteria, whereas IL-12 is important for the susceptible animals’ response to BCG during the peak of infection.     

Urease activity does not contribute dramatically to persistence of Mycobacterium bovis bacillus Calmette-Guérin.

Multiplication of BCGure-, an isogenic urease-negative mutant of Mycobacterium bovis BCG constructed by allelic exchange (J. M. Reyrat, F. X. Berthet, and B. Gicquel, Proc. Natl. Acad. Sci. USA 92:8768-8772, 1995), was examined in human macrophages and mice. Although ureolytic activity was not essential to BCGure-growth, a slight decrease in the multiplication and persistence of the mutated strain compared with wild-type BCG was observed in lungs of infected mice.     

Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guérin.

C57BL/6 and BALB/c mice were vaccinated with either live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin (BCG) organisms, and splenic T cells were used to screen the stimulatory potential of fractionated somatic and secreted mycobacterial proteins by production of gamma interferon (IFN-gamma). Maximum responses were obtained with fractionated secreted proteins of Mycobacterium tuberculosis. There was no single dominant antigen, but five regions of mycobacterial proteins induced high concentrations of IFN-gamma. However, only two of the five regions stimulated T cells from both mouse strains: two were exclusively recognized by T cells from BALB/c mice, and one was exclusively recognized by T cells from C57BL/6 mice. T cells from mice vaccinated with heat-killed M. bovis BCG organisms failed to respond to fractionated secreted proteins but recognized several somatic antigen fractions. As late as 1 year after primary vaccination, memory T cells responded to similar protein regions, and IFN-gamma production was intensified by secondary infection. Our data confirm a central role for secreted proteins in immunity to mycobacteria. Moreover, we demonstrate that a major set of mycobacterium-reactive T cells is stimulated only by vaccination with live but not with heat-killed M. bovis BCG organisms. Because a major impact of genetic host factors on antigen recognition was observed, we favor the use of live carrier organisms which secrete mycobacterial proteins over subunit vaccines as an improved antituberculosis vaccine.     

Adoptive immunity to Mycobacterium bovis strain bacillus Calmette-Guérin by long-term cultured T-cell line in nude mice.

Tuberculin-active peptide-reactive T-cell lines were established from the popliteal lymph node of BALB/cA mice immunized with heat-killed Mycobacterium tuberculosis to investigate the cellular mechanisms in the protective immunity of tuberculosis. These T-cell lines, consisting mainly of L3T4 surface antigen-positive cells, were transferred intravenously into nude mice infected with M. bovis strain bacillus Calmette-Guérin (BCG). Four or 6 weeks after transfer, footpad swelling and hepatic granuloma formation were observed and viable counts in the liver were decreased. Reduction of viable counts in the liver was obviously preceded by the hepatic granuloma formation. An effect of Lyt-2+ T cells which might be included in the inoculum could be ruled out by the experiment using T-cell lines pretreated with anti-Lyt-2 monoclonal antibody and complement. These results indicated that T cells required for protective immunity in these experiments belong to the L3T4-positive TDTH subset. However, their protective activity was inferior to that of freshly prepared immune lymph node T cells. From the observation of migration patterns of 51Cr-labelled T cells in BCG-infected nude mice, a relatively high proportion of freshly isolated T cells, but only a small part of T-cell line, deposited in the spleen of infected nude mice. This difference in migration pattern of freshly isolated and cultured T cells could be one of the reasons for the less in vivo anti-bacterial activity of the latter.     

Systemic and Mucosal Immune Responses after Intranasal Administration of Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Expressing Glutathione S-Transferase from Schistosoma haematobium

A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes. One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers. In this study, Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase from Schistosoma haematobium (Sh28GST). The gene encoding Sh28GST was placed under the control of the mycobacterial hsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker. The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded. Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost. Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum. In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly. Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathione S-transferase from Schistosoma mansoni, although the two antigens have over 90% identity. This difference was not observed after intraperitoneal administration.     

Humoral and Cellular Immune Responses in Mice Immunized with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Producing a Pertussis Toxin-Tetanus Toxin Hybrid Protein

The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60 promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.     

T cell response to purified filtrate antigen 85 from Mycobacterium bovis Bacilli Calmette-Guérin (BCG) in leprosy patients.

Precipitating autoantibodies to the URNP particles were used to select 80 patients, and were further characterized by immunoblotting and quantitative ELISA. These immunochemical results have been related to clinical diagnosis, the frequency of nephritis, and Raynaud's phenomenon. Autoantibodies to the 70-kD polypeptide of the U1RNP particle were present in 16 out of 19 patients with mixed connective tissue disease (MCTD) and in 27 out of 61 patients with systemic lupus erythematosus (SLE). The ratio of anti-U1RNP/Sm by ELISA and the frequency of antibody to 70-kD protein were directly related to the frequency of Raynaud's phenomenon and inversely related to the frequency of nephritis.     

The Association Between Bacille Calmette-Guérin Vaccination and Nontuberculous Mycobacterial Pulmonary Disease

This study aimed to investigate the association between Bacille Calmette-Guérin (BCG) vaccination and nontuberculous mycobacterial pulmonary disease (NTM-PD). Patients in the prospective NTM-PD cohort were matched to healthy controls to measure the association between BCG and NTM-PD development. The clinical course of NTM-PD patients was also evaluated to investigate the association between BCG and NTM-PD progression. BCG scars were not associated with NTM-PD development (adjusted odds ratio [OR], 2.04; 95% confidence interval [CI], 0.96–4.34) or progression (adjusted OR, 1.61; 95% CI, 0.92–2.81). In conclusion, BCG vaccination was not associated with the development or progression of NTM-PD.     

Activation of CD8 T cells with specificity for mycobacterial heat shock protein 60 in Mycobacterium bovis bacillus Calmette-Guérin-vaccinated mice.

Heat shock protein 60 (hsp60)-specific CD8 T cells lysed Mycobacterium bovis BCG-infected macrophages in vitro and adoptively transferred protection against mycobacterial infection. Moreover, CD8 T cells with this hsp60 specificity were activated in vivo by BCG vaccination. Our studies suggest there is participation of hsp60-specific CD8 T cells in BCG-induced immunity.     

Lysis of human macrophages by cytolytic CD4+ T cells fails to affect survival of intracellular Mycobacterium bovis-bacille Calmette-Guérin (BCG).

Human CD4+, mycobacteria-specific, cytolytic T cell clones were used to lyse BCG-infected macrophages, and the effect on the subsequent growth and viability of the organisms was examined. The survival of released bacteria following cell lysis was assessed by both 3H-uridine labelling and colony-forming unit (CFU) estimation. The results indicate that even when effective antigen-specific or lectin-mediated cytolysis of the infected macrophages was achieved, there was no evidence for a direct mycobactericidal effect on the intracellular bacteria. This remained the case even if the period of co-culture of T cells and macrophages was extended up to 48 h. Pretreatment of the macrophages with interferon-gamma (IFN-gamma) was not able to act together with T cell-mediated lysis to produce inhibition of mycobacterial growth.     

Bacillus Calmette-Guérin (BCG) decreases resistance to Listeria monocytogenes infection in mice.

Bacillus Calmette-Guérin (BCG) inoculation has been shown to inhibit certain immune functions. To determine whether this inhibition adversely affects host defences against infection, the effect of BCG on Listeria infection in mice was investigated. Mice were injected intravenously (i.v.) with Listeria monocytogenes and 24-96 hr later were inoculated with 8 x 10(6) BCG. Mice given BCG and Listeria had a greater mortality and higher spleen Listeria counts than mice given Listeria alone. An increased number of bacteria in spleens was noted as early as 24 hr after BCG inoculation. Peritoneal macrophages from mice receiving both organisms had a decreased capacity to kill Listeria in vitro. In addition, BCG inoculation suppressed delayed hypersensitivity responses and in vitro spleen cell proliferative responses to Listeria antigen. Suppression of spleen cell proliferative responses was associated with an adherent, non-T lymphocyte subpopulation. The data indicate that BCG administration decreases resistance to intracellular pathogens by abrogating normal cellular defences.     

Protection against Mycobacterium tuberculosis Infection by CD8+ T Cells Requires the Production of Gamma Interferon

The role of CD8 T cells in controlling Mycobacterium tuberculosis infections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-γ), it was shown that IFN-γ production was essential for CD8 cell mediation of protective immunity against M. tuberculosis.

Cell-mediated immunity is crucial for the control of mycobacterial infections. Athymic mice (4) and mice whose T cells have been depleted (22, 23) are much more susceptible to infection with mycobacteria than euthymic or unmanipulated mice. However, the contributions of the different components of the T-cell response are unclear. CD4 T cells are thought to play a major role in controlling infections with the primary human tubercle bacillus, Mycobacterium tuberculosis; individuals with reduced CD4 counts, from infection with human immunodeficiency virus, for example, are known to be more susceptible to M. tuberculosis infections (12). Activation of CD4 cells by antigen in association with major histocompatibility complex (MHC) class II molecules results in clonal expansion and the production of cytokines, most notably gamma interferon (IFN-γ), which activate macrophages so that they become mycobactericidal. Mice with deletions of the IFN-γ gene are much more susceptible to M. tuberculosis infection than wild-type mice (5, 9). However, in addition to CD4 cells, other components of the cell-mediated response are thought to play roles in controlling infection with M. tuberculosis. For example, CD8 T cells have been shown to be involved (20, 24): β2 microglobulin-deficient knockout mice, which lack an effective CD8 response, show increased susceptibility to M. tuberculosis infection (10). Other cell types, such as T cells bearing the γ/δ T-cell receptor (19) and NK cells (1), are also thought to have roles in protection against intracellular bacteria, while a number of T cells with novel phenotypes and unknown functions have been shown to recognize mycobacterial antigens (2, 28).CD8 T cells are known to contribute to the protective response against M. tuberculosis, but the mechanism(s) by which they exert this protective effect is unknown. CD8 T cells produce a range of cytokines, including IFN-γ (11, 17, 25, 26), but their primary role is thought to be cytotoxic. However, it has recently been shown that mice with a targeted disruption in either the perforin gene or the granzyme gene and mice which are Fas receptor defective are no more susceptible to infection with M. tuberculosis than are wild-type mice (6, 16). Since perforin (13, 18) and Fas-Fas ligand interactions (21, 27, 31) are thought to be the primary mechanisms of cytotoxicity mediated by CD8 T cells, such cells may contribute their antimycobacterial activity through noncytotoxic pathways.In this study, we have used MHC class II-deficient mice and athymic mice to confirm the role of non-CD4 T-cell-mediated mechanisms in protection against M. tuberculosis infection. Using transfer of purified CD4 and CD8 cells into athymic mice, we have demonstrated that these cells contribute equally to protective immunity in this system. However, by using mice with deletions of the IFN-γ gene as T-cell donors, we have shown that production of IFN-γ is required in order for CD8 T cells to exert their antimycobacterial effect.In preliminary experiments, the levels of growth of M. tuberculosis in MHC class II knockout, athymic, and normal mice were compared. MHC class II knockout (Aβ^−/−) mice were obtained as a breeding nucleus (kindly provided by D. Gray, Hammersmith Hospital, London, United Kingdom, with permission from D. Mathis, Institut National de la Santé et de la Recherche Médicale). These mice were bred from heterozygous (Aβ^+/−) parents and genotyped as described previously (7). Heterozygous littermates were used as controls. Stock cultures of M. tuberculosis H₃₇Rv were grown in Dubos 7H9 broth for 14 days, and then they were aliquoted and stored in liquid nitrogen. For infection, aliquots were thawed, diluted in phosphate-buffered saline, and inoculated intraperitoneally into mice. The infection was monitored by removing the lungs and spleens of infected mice at various intervals; the baseline level of infection of each tissue was estimated by harvesting organs from the mice 18 h after infection and determining viable counts. The tissues were weighed and homogenized by shaking with 2-mm-diameter glass beads in chilled saline with a Mini-Bead Beater (Biospec Products, Bartlesville, Okla.), and 10-fold dilutions of the suspension were plated onto Dubos 7H11 agar with Dubos oleic albumic complex supplement (Difco Laboratories, Surrey, United Kingdom). Numbers of CFU were determined after the plates had been incubated at 37°C for approximately 20 days. The results are shown in Fig. ​Fig.1A1A and B. In control mice, there was a transient increase in bacterial counts in the spleen, followed by a steady decline over 60 days and then by a levelling out of the infection at approximately 10⁴ CFU per g of tissue. In MHC class II knockout mice, there was an initial growth of the infection over the first 60 days, followed by a plateau phase during which the infection appeared to be controlled but was significantly more severe than in wild-type mice (Fig. ​(Fig.1A).1A). In lung tissue (Fig. ​(Fig.1B),1B), a similar pattern emerged, except that in the MHC class II knockout mice, control of the infection broke down in some of the mice after about 60 days, when there was a sudden increase in bacterial counts. By day 80, counts had reached approximately 10⁷ CFU per g of tissue, a 10,000-fold increase over the counts seen in wild-type mice. Open in a separate windowFIG. 1Growth of M. tuberculosis in the tissues of MHC class II knockout, control, and athymic mice. (A and B) Growth in spleens and lungs, respectively, of MHC class II knockout mice (•) and their wild-type littermates (▪). (C and D) Growth in spleens and lungs, respectively, of MHC class II knockout (•) and athymic (▴) mice. Data are the geometric means ± the standard errors of the means for three to five mice. An asterisk indicates a significant difference between values for MHC class II knockout and control mice (P < 0.05 by Students’ t test). A double asterisk denotes that at the indicated time, all remaining mice in the group were killed because of the widely disseminated nature of the infection.These results emphasize the importance of the MHC class II-CD4 T-cell pathway in controlling M. tuberculosis infection. However, in spite of the fact that after the first few days of infection there was always a highly significant difference between the level of viable M. tuberculosis organisms in MHC class II knockout mice and the level in control mice, some control of bacterial multiplication did appear to occur in the MHC class II knockout mice. In order to demonstrate that this apparent partial control of the infection in MHC class II knockout mice was mediated by T cells, we compared growth in these mice with growth in athymic mice. Athymic (nude) BALB/c mice were obtained from a breeding colony at the National Institute for Medical Research. Athymic and MHC class II knockout mice were infected intraperitoneally, and the infections were monitored as described above. Whereas the MHC class II knockout mice were again able to control the infection to some degree, growth in athymic mice was unchecked and the mice had to be killed at 40 days because of overwhelming infection (Fig. ​(Fig.1C1C and D).These results confirm the importance of CD4 cells in controlling M. tuberculosis infections but also suggest that a contribution is made by non-CD4-mediated mechanisms. It has previously been shown that depletion of CD8 cell populations in mice with anti-CD8 antibodies (20) or abolition of a CD8 response by disruption of the β2 microglobulin gene (10) renders mice highly susceptible to infection with M. tuberculosis. CD8 T cells have also been implicated in human tuberculosis; CD8⁺ T cells with specificity for mycobacterium-pulsed target cells have been described (14, 32), and an individual with recurrent tuberculosis was found to have a specific reduction in CD8 T cells (3).In order to investigate the contribution of CD8 T cells to the control of M. tuberculosis infections in mice, total spleen cells, CD4 T cells, and CD8 T cells were transferred from control BALB/c mice into infected athymic BALB/c mice. Splenocytes were incubated in hypotonic medium to lyse erythrocytes and washed twice. To obtain highly purified populations of CD4 and CD8 cells, cell suspensions were enriched by negative selection with T-cell-subset columns (R & D Systems Inc., Minneapolis, Minn.) according to the manufacturer’s instructions. The resulting populations were >90% CD4 or CD8 T cells, as determined by flow cytometric analysis. The cells were washed, resuspended in sterile saline, and injected intravenously such that recipient mice received 5 × 10⁶ cells. The mice were then infected with M. tuberculosis, and organs were harvested 21 days later for CFU counts. The results of a typical experiment are shown in Fig. ​Fig.2.2. In athymic mice which had not received any transferred cells, the infection reached approximately 10⁷ CFU per g in the lung (Fig. ​(Fig.2A)2A) and 10⁸ CFU per g in the spleen (Fig. ​(Fig.2B).2B). Transfer of total spleen cells from naive BALB/c mice reduced the number of CFU 100- to 1,000-fold in both tissues. It appeared that CD4 and CD8 T cells contributed approximately equally to the observed protection. Open in a separate windowFIG. 2Infection of athymic mice with M. tuberculosis following transfer of splenocytes from euthymic mice. (A and B) Results for the lungs and spleen, respectively, of mice infected intravenously with approximately 10⁶ CFU 21 days prior to harvest. Transfer of cells was carried out 24 h before infection. Data are the means ± the standard errors of the means for three to five mice. Mice received either no cells, total spleen cells, CD4 cells, or CD8 cells. All three groups of mice which received cells showed significantly reduced CFU counts compared to controls (P < 0.05 by Student’s t test).The mechanism by which CD8 T cells exert this antimycobacterial response is not understood. It has been suggested that the cytotoxicity of mycobacterium-laden target cells could be involved, perhaps through the release of M. tuberculosis bacilli from ineffective macrophages to cells with greater antimycobacterial potential (15). However, perforin or granzyme knockout mice and Fas receptor-defective mice, when infected, did not display any increased susceptibility to infection, compared to wild-type controls (6, 16). Interestingly, both the perforin knockout mice and the Fas receptor-defective mice had elevated levels of cytokines, including IFN-γ, in the absence of infection, and levels in infected mice were similar to those seen in wild-type mice (16). Thus, neither perforin-, granzyme-, nor Fas-mediated cytotoxicity appeared to be involved in the control of these experimental infections (6, 16). Conversely, however, Silva and colleagues (29) produced CD8⁺ T-cell clones which were capable of conferring protection against M. tuberculosis in recipient mice, and the level of protection correlated with the level of cytotoxic activity rather than with the level of IFN-γ secretion.In a recent study of human cytotoxic cells with mycobacterial specificity, it was found that CD4⁻ CD8⁻ T cells lysed macrophages through a Fas-Fas ligand interaction but the lysis was not associated with mycobacterial killing, whereas CD8⁺ T-cells lysed macrophages by a Fas-independent pathway and the lysis resulted in the killing of mycobacteria (30). The human T-cell lines used for these experiments were unusual in that they were CD1 restricted.Since CD8 T cells were clearly able to confer significant levels of protection against M. tuberculosis in our cell transfer model, we next investigated the role of IFN-γ in this protection. Again athymic mice were recipients of either total spleen cells or CD8 cells. This time, however, donor mice were either normal BALB/c mice or IFN-γ knockout mice (8) and recipient mice received 3 × 10⁶ cells. The results (Fig. ​(Fig.3)3) clearly demonstrate the requirement for IFN-γ. Transfer of total spleen cells or CD8 T cells from normal mice gave protection, although the level of protection was slightly lower than that seen in the previous experiment (Fig. ​(Fig.2).2). This was probably because the number of cells transferred was lower (3 × 10⁶ rather than 5 × 10⁶). However, the protection seen in both organs was significant (P < 0.05). Importantly, transfer of cells from IFN-γ knockout mice gave no protection. Open in a separate windowFIG. 3Infection of athymic mice with M. tuberculosis following transfer of splenocytes from control BALB/c and IFN-γ knockout (IFN-γ −VE) BALB/c mice. (A and B) Results for the lungs and spleen, respectively. The experimental design was identical to that for Fig. ​Fig.2.2. Mice received either no cells, total spleen cells from wild-type BALB/c mice, CD8 cells from BALB/c mice, total spleen cells from IFN-γ knockout mice, or CD8 cells from IFN-γ knockout mice. Mice which received cells from control BALB/c mice (total spleen or CD8 cells) showed significantly reduced CFU counts compared to naive athymic mice (P < 0.05); there were no significant differences between values for naive athymic mice and mice which received either total spleen cells or CD8 cells from IFN-γ knockout BALB/c mice.Thus, the results reported in this study confirm the role of CD8 T cells in the control of M. tuberculosis infections in mice. We have also demonstrated that this control requires the ability of the CD8 cells to produce IFN-γ, suggesting that such cells may exert their effects through classical cytokine-mediated macrophage activation rather than through a cytotoxic mechanism. The recent demonstration that human CD1-restricted CD8 T cells were able to kill mycobacteria in vitro through a cytotoxicity-mediated pathway (30) suggests that different subpopulations of CD8 cells may have different effector mechanisms; since no murine equivalent of the CD1-restricted CD8 T cell has been described, this mechanism may be absent in mice. Alternatively, the results reported earlier for murine CD8 T-cell lines (29) or human CD8, CD1-restricted T-cell lines (30) may reflect the activity of primed or memory T cells, whereas the results reported in the present study reflect the activity of unprimed cells. Primed CD8 T cells have been shown to be hyperreactive to antigenic challenge in vitro and may employ different effector mechanisms. That production of IFN-γ by CD8 T cells is required in order to control infection has also been reported for viral infections (11, 26), where cytotoxicity has long been thought to be the major mechanism of CD8-mediated antiviral activity. IFN-γ and other cytokines have been shown to be major components of the mechanism by which hepatitis B virus is controlled in mice by CD8 cells without the killing of hepatocytes (11). The results reported in this study demonstrate that IFN-γ is essential for CD8-mediated protection against M. tuberculosis infection in mice.     

Internalization of Mycobacterium bovis Bacillus Calmette-Guérin into Osteoblast-Like MC3T3-E1 Cells and Bone Resorptive Responses of the Cells against the Infection

Mycobacterium bovis BCG (BCG) is a live vaccine used worldwide against tuberculosis. However, it has unfavourable side effects such as osteitis or osteomyelitis, and these sometimes lead to vertebral caries in some patients as a result of bone resorption. Osteoblasts might play a role in the bone resorption caused by BCG infection, because they are central cells in bone metabolism. Cultured osteoblast-like cell lines (MC3T3-E1) derived from C57BL mice susceptible to BCG infection cells were infected with BCG at several doses. Interestingly, internalization of BCG-enveloped phagosome-like membrane in osteoblast-like cells were observed by transmission electron microscopy (TEM). Owing to infection, the proliferation and alkaline phosphatase activity of the osteoblast-like cells were reduced in a dose-dependent manner. On the other hand, interleukin (IL)-6 production was considerably enhanced by infection. These results suggest that BCG infects osteoblasts, suppressing their proliferation and differentiation and inducing bone resorption, which may be related to osteitis/osteomyelitis and bone caries caused by BCG infection.     

T-helper 1-like subset selection in Mycobacterium bovis bacillus Calmette-Guérin-infected resistant and susceptible mice.

The Bcg gene has been shown to control natural resistance of mice to intravenous infection with low doses of Mycobacterium bovis (bacillus Calmette-Guérin; BCG). In the present study, we evaluated the impact of the Bcg gene on the development of T-cell reactivity during the early stages of infection. Congenic strains of mice, bearing 'r' and 's' alleles of the Bcg gene on B10.A and BALB/c backgrounds, were studied at different time-points for 2 weeks after infection. The in vitro proliferative response of spleen cells, induced by mycobacteria or concanavalin A, was depressed in the Bcgs mice compared to the Bcgr congenic mice 14 days after infection with 10(5) colony-forming units (CFU) of BCG. Polymerase chain reaction (PCR)-based methodology was used to compare the level of lymphokine gene expression in the spleens of infected congenic mice both ex vivo and after in vitro stimulation. In both cases, preferential expression of interferon-gamma (IFN-gamma), lymphotoxin, interleukin-2 (IL-2) and IL-2 receptor genes was observed. The lymphokine gene expression profiles indicated that T lymphocytes activated in the course of the BCG infection preferentially expressed the T-helper 1-specific pattern, irrespective of the allele of the Bcg gene. We showed that this bias in T-cell differentiation could not be attributed to either down-regulation of IL-4 gene expression or modulation of the macrophage co-stimulatory activity by live M. bovis BCG. We conclude that the mechanism of phenotypic expression of the Bcg gene resides in the differential ability of macrophages to be activated by lymphokines produced by protective T cells, rather than in the lack of these lymphokines in susceptible animals.     

Bacillus Calmette-Guérin (BCG) and immunoglobulins synergistically enhance mineral dust-induced production of reactive oxygen metabolites by human monocytes.

The modulating effect of BCG and polyclonal immunoglobulin on mineral dust-induced production of reactive oxygen metabolites (ROM) by human monocytes was studied using luminol-dependent chemiluminescence. BCG and immunoglobulin synergistically amplified the ROM production induced by chrysotile asbestos and quartz particles, and BCG caused a sharper dose response for poly-immunoglobulin added to the mineral dusts. Immunoglobulins did not affect zymosan yeast-induced ROM production, which was enhanced strongly by BCG. As there is evidence that phagocyte-derived ROM are of importance in mineral dust-induced lung injury, we suggest that the observed synergism between host response inflammatory mediators (poly-immunoglobulin) and exogenic irritants (BCG) may contribute to the outcome of exposure of mineral dusts, and thus in part explain the individual variations in susceptibility to mineral dust-induced diseases.     

H-2-linked control of in vitro gamma interferon production in response to a 32-kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Guérin.

A 32-kilodalton protein antigen (P32) was previously purified to homogeneity from culture filtrate of Mycobacterium bovis BCG (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Spleen cells from BCG-sensitized mice produce significant amounts of gamma interferon (IFN-gamma) in response to this P32 protein. The amount of secreted IFN-gamma is influenced by mouse genotype, with C57BL/6 (H-2b), C57BL/10 (H-2b), and 129/Sv (H-2b) mice producing about four times more than BALB/c (H-2d), CBF1 (H-2d/b), and DBA/2 (H-2d) mice do. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments indicates a probable H-2-linked control of this IFN-gamma induction, with H-2b cells producing high titers and H-2d cells producing low titers in response to the P32 antigen.     

Mycobacterium bovis Bacillus Calmette Guérin infection prevents apoptosis of resting human monocytes

Apoptosis plays an essential role in the development and homeostasis of multi-cellular organisms. Some infectious agents interfere with this programmed cell death to their own benefit. Here, we show that infection of resting human monocytes with Mycobacterium bovis Bacillus Calmette Guérin (BCG) increases monocyte viability by preventing them from undergoing apoptosis. Heat-killed BCG also prevented apoptosis, indicating that replication of BCG is not required to prevent cell death. Analysis of BCG-infected monocytes revealed an up-regulation of the A1 mRNA, whereas the bcl-2 mRNA was not up-regulated. Interestingly, preinfection with BCG renders the cells resistant to interleukin (IL)-10-induced apoptosis which may be one of the mechanisms mycobacteria use to modulate immune responses. BCG infection was also accompanied by an impairment of the capacity of monocytes to secrete IL-10 and by an induction of the capacity to secrete tumor necrosis factor-α, two cytokines known to induce and prevent human monocyte apoptosis, respectively. Since it has been reported that apoptosis is involved in killing of intracellular mycobacteria, the prevention of apoptosis may represent a strategy for mycobacterial survival in the infected host.