BCG: Mycobacterium bovis bacillus Calmette- Guérin.

Macrophages and dendritic cells are host cells for pathogenic mycobacteria, which can invade these cells and persist in them for years or even for decades [1-7]. The latent, symptom-free stage of tuberculous infection is characterized by the formation of granulomas, specific aggregates of immune cells containing mycobacteria [1-3,8-12]. Several potential mechanisms by which mycobacteria can survive in granuloma cells and defy the immune system of the host organism are proposed. First, pathogenic mycobacteria prevent the phagosomes containing them from merging with host cells’ lysosomes and cannot thus be destroyed by lysosomal enzymes [9,13-18]. Secondly, in infected cells, mycobacteria can modulate the expression of proinflammatory (TNFα, IFNγ, IL-1) and anti-inflammatory (IL-10) cytokines involved in the formation of cellular responses and a specific immune response of the host organism to infection [5-9,11,12,19-21]. Finally, mycobacteria cause infected cells to die either by apoptosis, which is characterized by loss of cell membrane elements, chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies, or by necrosis, which is characterized by compromised cell membranes and nuclear envelopes, cytoplasmic swelling and cellular breakdown [22-25]. The apoptotic death of infected macrophages has been proposed to be one of the main mechanisms by which the organism controls tuberculous infection through depopulating pathogenic microorganisms and infected cells [4-5,7-9,25]. By contrast, the necrotic death of infected cells leads to the release of living bacteria into the extracellular environment and further spread of infection throughout animal and human organisms [5,24-28]. At present, autophagy as a way by which host cells eliminate intracellular pathogens during latent chronic tuberculous infection and at its reactivation is extensively studied [5,8,9,23,24,29,30].

As is known, apoptosis can be induced by external (cytokines, FasL) or internal (compromised DNA and mitochondrial integrity) stimuli that trigger the receptor-mediated and/or mitochondrial pathways of a complex, many-staged process of cell death, which leads to nuclear fragmentation and the formation of apoptotic bodies to be destroyed by macrophages [23-25,31,32]. The extrinsic pathway of apoptosis is mediated by specific death receptors (Fas/CD95, TNFR1/CD120a, to mention a few), which are in the tumor necrosis factor receptor superfamily [23-25,33]. Following ligand-receptor interactions, TNFα protein induces apoptosis through the formation of non-membrane complexes composed of various proteins and procaspase-8, where the latter undergoes auto-proteolysis and generates the active caspase-8 [23-25,31,34,35]. In lymphoid cells, the initiator caspase-8 directly activates procaspase-3, which leads to the death of lymphocytes [31]. In other cell types, caspase-8 basically acts by cleaving Bid protein, the active form of which triggers the homooligomerization of Вах protein on the mitochondrial outer membrane and the formation of pores [36]. At this point, the mitochondrial pathway of apoptosis is activated [36]. As a result, cytochrome с translocates, through the pores, from the mitochondrial intermembrane space to the cytoplasm and triggers the formation of a procaspase‑9 activating apoptosome. The initiator caspase-9, in turn, activates procaspase-3 [36]. The substrates for the active effector caspase-3 are multiple intracellular proteins, the destruction of which leads to typical morphological and biochemical signs of apoptotic cell death [22-25]. These processes are inhibited by the antiapoptotic proteins Bcl-2 and Bcl-XL, which act to protect mitochondria from the action of the proapoptotic proteins Bax, Bad, Bak, and Bid [22-25]. Following exposure of cells to genotoxic agents, the P53 protein is stabilized in the cytoplasm [37]. Proapoptotic effects of the P53 protein occur both in the nucleus, by activation of genes for the proapoptotic proteins Fas/CD95 and Вах or suppression of the transcription of the gene for the antiapoptotic protein Bcl-2, and in the cytoplasm, by directly interacting with these proteins in the cells [38-40]. Due to this interaction, cytoplasmic Вах protein undergoes conformational changes, is activated and moves to the mitochondria, on the membrane of which the P53 protein inactivates Bcl-2 protein [38-40]. It was demonstrated [41] that, when nuclear DNA is extensively damaged, a P53-independent pathway of apoptosis can be triggered by induction of expression of the TNFα cytokine and its autocrine interaction with cell death receptors. Bacterial invasion is known to induce TNFα expression in granuloma macrophages, dendritic cells and Т lymphocytes [5-12,34,42]. The TNFα cytokine has been proposed to play an important role in the control of tuberculous infection by not only performing proapoptotic functions, but also engaging in leukocyte activation through the CD30 and CD40 receptors on the cell membrane [34,42]. Suppression of the TNFα cytokine or its receptors led to rapid death of mice from tuberculosis following destruction of granuloma organization, the increase of mycobacteria in the organism and immune dysregulation [43,44].

To date, the main results of analysis of relationships between mycobacteria and host cells have been obtained from in vitro studies with infecting human and mouse monocyte-derived macrophages or constant cell lines. It has been found that mycobaterial strains have different effects on cell death induction in vitro [24]. Non-virulent Mycobacterium tuberculosis strain H37Ra and attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) caused apoptosis of infected macrophages and the elimination of bacteria, while virulent mycobacterial strains (M. tuberculosis H37Rv, Erdman and M. bovis) actively reproduced in the cells and acted to prevent their death in vitro [45-48]. It has also been found that only virulent M. tuberculosis H37Rv the Bcl-2 family, after in vitro infection of human THP-1 cells [49]. In parallel, a weaker induction of TNFα expression was observed in human and mouse cells following in vitro infection with virulent than non-virulent strains [50-52]. Following in vivo infection of zebrafish (Danio rerio) juveniles with M. marinum, mycobacteria spread in their organisms due to the death of some granuloma macrophages and the release of mycobacteria into the extracellular environment, where they were later phagocyted by uninfected macrophages [28,53]. While the formation of granulomas did not depend on TNFα protein at the earliest times of infection, the expression of this cytokine became critical for maintaining the integrity of granuloma structures as infection progressed [28]. Lack of TNFα action on cells due to blockade of TNFα receptors caused increased growth of mycobacteria in granuloma macrophages, necrosis of infected cells and the spread of infection across the fish organisms [27]. Similarly, in the liver of mice treated in vivo with antibodies against TNFα protein, a gradual loss of granuloma structure, decreased granuloma size, decreased granuloma macrophage populations, and increased intracellular BCG mycobacteria reproduction were observed [54]. We have previously demonstrated in our model system of monolayer cultures of cells that migrated ex vivo from individual granulomas of mice with latent BCG infection [55] that although mouse granuloma macrophages with an increased production of proinflammatory cytokines, growth factors, and CD receptors for cell activation could not eliminate intracellular BCG mycobacteria entirely, they could still control mycobacterial reproduction in host cells both in vivo and in ex vivo culture, while the mouse bone marrow and peritoneal macrophages that were not activated after BCG infection in vitro had increased mycobacterial loads and death rates [55-57]. Nevertheless, the in vitro, ex vivo and in vivo studies performed to date are not enough to completely understand the mechanisms by which mycobacteria persist in cells for long periods of time, particularly, at the latent stage of tuberculous infection.

In the present work, an ex vivo model of granulomas from mice with latent tuberculous infection was used to study the expression of inducers, inhibitors and markers of apoptotic cell death relative to mycobacterial loads. In mouse granuloma cells with an increased production of the inducer of apoptosis TNFα, proapoptotic proteins Вах and Ваd, death receptor Fas/CD95 and scavenger receptor CD36, neither significant P53 protein stabilization nor caspase-3 activation was observed in the cytoplasm or nuclei of macrophages, irrespective of the presence or absence of BCG mycobacteria in them. The survival receptor CD30 was detected on the cell membranes of only few granuloma macrophages. It was established that most mouse granuloma cells obtained from spleens and lungs at later times of latent tuberculous infection had considerable amounts of the antiapoptotic protein Bcl-2 in the cytoplasm and, probably, mitochondria, irrespective of the presence or absence of BCG mycobacteria in them. In the macrophages from bone marrow cell cultures and peritoneal exudates infected with BCG mycobacteria in vitro, no activation of Bcl-2 production was detected throughout 4-120 h of analysis. It was thus proposed that Bcl- 2 might be one the factors that protected cells from death in granulomatous inflammatory lesions not only in ex vivo culture, but also in mouse organisms at different times of latent tuberculous infection.

Animals

Two-month-old BALB/c male mice were obtained from the Animal Breeding Facility of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk, Russia). Mice were bred and kept under standard vivarium conditions in accordance with all the known guidelines for laboratory animal care. Animal experiments were conducted in accordance with “The Guidelines for Manipulations with Experimental Animals” issued by the Russian Ministry of Health (guideline 755). All experimental procedures were approved by the Local Ethical Committee of the Research Institute of Biochemistry, Novosibirsk, Russia.

Infection of mice

Mice were infected with a vaccine prepared from an attenuated live strain of M. bovis (the Bacillus Calmette-Guérin vaccine, BCG- 1, the Institute of Microbiology and Epidemiology, Moscow, Russia) at a dose of 0.5 mg per mouse, which amounted to 3 106 viable BCG mycobacteria in 0.9% NaCl solution. Twenty four mice were each infected via tail vein injection with 100 μl of the suspension and four mice were each infected intraperitoneally with 200 μl of the suspension.

Isolation and ex vivo culture of mouse granulomas

Mice were anesthetized and sacrificed by cervical dislocation. Isolation of granulomas from the spleens, bone marrow and lungs of mice after BCG infection was performed as previously described [55-57]. In brief, bone marrow was flushed from femurs with RPMI 1640 medium with 50 μg/ml gentamicin (BioloT, St. Petersburg, Russia). The spleens collected from the animals were cut into small pieces in 5 ml of RPMI 1640 medium with 50 μg/ml gentamicin. For homogenization the dissected lungs were further disrupted by gently pushing the tissue through a metal screen with pores of 1 mm diameter. Granulomas were isolated from the organ homogenates by centrifugation at 150 xg at room temperature. The granuloma pellets in the RPMI 1640 growth medium containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 50 μg/ml gentamicin (BioloT, St. Petersburg, Russia) were placed at low-medium density to 24-well tissue culture plates (Orange Scientific, Belgium) with glass coverslips at the bottom and cultured in 0.5 ml medium for several days at +37 °C in an atmosphere containing 5% CO2. Granulomas were isolated from mice 1 and 2 on day 20 following intraperitoneal infection; mice 1 and 2÷5 after one month following intraperitoneal and intravenous infection, respectively; mice 1÷16 and 21÷24 after two months following intravenous infection; and mouse 25 after two months following intraperitoneal infection. The mouse nomenclature used is explained [55-57]. Peritoneal macrophages were isolated from mice after 20 days and mouse 25 after two months following intraperitoneal infection and cultured under the same conditions as the granuloma cells. After isolation of granulomas from the femur bones of mice 1 and 2 after 20 days following BCG infection, the other bone marrow cells from supernatants were cultured under the same conditions as the granuloma cells. The preparations of cultured, as granuloma cells, peritoneal macrophages of normal BALB/c mice without BCG infection were used as the control groups. Isolation of control fibroblasts from fragments of the splenic capsule or lungs from intact BALB/c mice was performed as previously described [55,57].

Cell cultures and infection in vitro

Isolation of peritoneal and bone marrow cells from four intact mice and BCG infection of each cell culture were performed as previously described in detail [57]. Three independent experiments on obtaining and studying cells in the control (uninfected) mouse bone marrow and peritoneal cultures and in cell monolayers after 4-120 h of acute BCG infection in vitro have been conducted.

Cell staining

At days 2-5 of ex vivo culture, granuloma cells on coverslips were fixed with 4% formaldehyde solution in phosphate buffer saline (PBS, pH 7.4) for 10 min at room temperature. The same procedure was applied to cells in bone marrow cell and peritoneal macrophage cultures at hours 4, 24, 48, 72, 96, and 120 following infection with BCG vaccine in vitro and in control cell cultures without infection. The preparations of peritoneal macrophages and spleen and lung fibroblasts from intact BALB/c mice were fixed as granuloma cells and used as the control for the staining of granuloma cells.

The different antibodies and reagents were used for the immunochemical and the immunofluorescence staining: rat monoclonal primary antibodies to mouse TNFα, CD30, and CD11b (BD, USA, 559064, 553824, and eBioscience, USA, 17-0112, respectively) diluted 1:100, 1:25, and 1:250, respectively; mouse monoclonal primary antibodies to mouse Bcl-2, Bad, CD36, and CD95 (BD, USA, 610538, 610391, 552544 and 610197, respectively) diluted 1:200, 1:200, 1:50, and 1:5000, respectively; rabbit polyclonal primary antibodies to mouse Bax and P53 (BD, USA, 554106, and Novocastra, England, NCL-p53-CM5p, respectively) diluted 1:200 and 1:1000, respectively; rabbit monoclonal primary antibodies to mouse active caspase-3 (BD, USA, 559565) diluted 1:200; goat polyclonal FITC-labeled or Alexa 594-conjugated secondary antibodies to rat IgG (Abcam, England, ab6266 and ab150160, respectively) diluted 1:400 and 1:500, respectively; goat polyclonal FITC-labeled or Alexa 555-conjugated secondary antibodies to mouse IgG (Abcam, England, ab7064, and Invitrogen, USA, A21422, respectively) diluted 1:400 and 1:500, respectively; goat polyclonal Alexa 488-conjugated secondary antibodies to rabbit IgG (Abcam, England, ab7064) diluted 1:400; goat polyclonal biotin-conjugated secondary antibodies to mouse, rabbit, and rat IgG (Sigma, USA, B7264, B7389, and BD, USA, 559286, respectively) diluted 1:100, 1:100, and 1:50, respectively; horseradish peroxidase-conjugated streptavidin (Sigma, USA, S5512), diaminobenzidine (Sigma, USA, D3939), MitoTracker Deep Red FM (Invitrogen, USA, M22426), acidotropic dye LysoTracker Red DND-99 (Invitrogen, USA, L7528).

In the experiments using MitoTracker Deep Red FM, the cell preparations were incubated with 500 nM of the dye for 30 min at +37°C in 5% CO₂ before fixation. The cell preparations were fixed as described above, washed with PBS, permeabilized within 2 min in 0.3% Triton-X100 solution, blocked in PBS solution containing 2% BSA, and finally incubated first with mouse monoclonal primary antibodies to mouse Bcl-2, then with FITC-conjugated goat anti-mouse Ig secondary antibodies.

In the experiments using LysoTracker Red DND-99, the cell preparations were incubated with 50 nM of the acidotropic dye for 5 min at +37°C in 5% CO₂ before fixation. The cell preparations were fixed as described above, washed with PBS, blocked in PBS solution containing 2% BSA, and finally incubated first with mouse monoclonal primary antibodies to CD36, then with FITC-conjugated goat antimouse Ig secondary antibodies.

Some of the fixed cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, blocked in PBS solution containing 2% BSA, and finally incubated first with primary specific antibodies to mouse TNFα, Bax, Bad, P53, active caspase-3, and Bcl-2. Specific immunochemical staining of some cell preparations was visualized using secondary goat polyclonal biotin-conjugated antibodies to mouse, rabbit, and rat IgG, then with horseradish peroxidase-conjugated streptavidin and diaminobenzidine solution containing 0.05% H₂O₂. The cells were further counterstained with 1% methyl green. Fluorescent visualization of TNFα and Bcl-2 was enabled using secondary goat polyclonal Alexa 594-conjugated antibodies to rat IgG and FITC-labeled antibodies to mouse IgG, respectively.

Some of the fixed cell preparations were washed with PBS, blocked in PBS solution containing 2% BSA, and incubated first with first with primary specific antibodies to mouse CD30, CD36, and CD95, and then with secondary goat polyclonal biotin-conjugated antibodies to mouse and rat IgG, then with horseradish peroxidase-conjugated streptavidin and diaminobenzidine solution containing 0.05% H₂O₂. The cells were further counterstained with 1% methyl green. Fluorescent visualization of CD95 was enabled using goat polyclonal Alexa 555-conjugated secondary antibodies to mouse IgG.

Some of the fixed cell preparations were washed with PBS, blocked in PBS solution containing 2% BSA, and incubated with rat monoclonal APC-conjugated primary antibodies to mouse CD11b. After staining for CD, the cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, and incubated with rabbit polyclonal primary antibodies to mouse P53, and then with secondary goat polyclonal Alexa 488-conjugated antibodies to rabbit IgG.

Some of the fixed cell preparations were washed with PBS, blocked in PBS solution containing 2% BSA, and incubated first with rat monoclonal primary antibodies to mouse CD30 and mouse monoclonal primary antibodies to mouse CD95, then with secondary goat polyclonal Alexa 594-conjugated antibodies to rat IgG and FITC-labeled antibodies to mouse IgG.

Some of the fixed cell preparations were washed with PBS, blocked in PBS solution containing 2% BSA, and incubated with mouse monoclonal primary antibodies to mouse CD36. After staining for CD, the cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, and incubated with rat monoclonal primary antibodies to mouse TNFα, and then with secondary goat polyclonal Alexa 594-conjugated antibodies to rat IgG and FITC-labeled antibodies to mouse IgG.

Some of the fixed cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, blocked in PBS solution containing 2% BSA, and incubated with rabbit polyclonal primary antibodies to mouse Bax and mouse monoclonal primary antibodies to mouse Bad. Fluorescent visualization of Bax and Bad was enabled using secondary goat polyclonal Alexa 488-conjugated antibodies to rabbit IgG and Alexa 555-conjugated antibodies to mouse IgG, respectively.

Some of the fixed cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, blocked in PBS solution containing 2% BSA, and incubated first with mouse monoclonal primary antibodies to mouse Bcl-2 and rat monoclonal primary antibodies to mouse TNFα, then with secondary goat polyclonal Alexa 555-conjugated antibodies to mouse IgG and FITC-labeled antibodies to rat IgG.

Some of the fixed cell preparations were washed with PBS, treated within 2 min in 0.3% Triton-X100 solution, blocked in PBS solution containing 2% BSA, and incubated with mouse monoclonal primary antibodies to mouse Bcl-2 and rabbit specific antibodies to mouse Bax, P53, or active caspase-3, then with secondary goat polyclonal Alexa 555-conjugated antibodies to mouse IgG and Alexa 488-conjugated antibodies to rabbit IgG.

The cell preparations were incubated with the appropriate antibodies for 60 min at room temperature. Fluorescent staining was analyzed using the VECTASHIELD^® Mounting Medium with DAPI (4΄,6-diamidino-2-phenylindole) (Vector Laboratories, USA, H-1200).

The confocal images of the cells were recorded; the preparations were washed from VECTASHIELD^® Mounting Medium in PBS for 20 min and re-stained for acid-fast mycobacteria using the Ziehl- Neelsen stain. The cells stained by Ziehl-Neelsen method were further counterstained with 1% methylene blue.

Staining of live cells

After 72 h of ex vivo culture, coverslips with granuloma cells from infected mouse 23 or with control peritoneal macrophages were transferred from culture plates to Petri dishes and washed with PBS. Fc receptors on the cells were blocked by incubating the preparations in non-diluted bovine serum (BioloT, St. Petersburg, Russia) for 10 min at +37°C in 5% CO₂. Next, the cell preparations were incubated with mouse monoclonal primary antibodies to mouse Bcl-2 (BD, USA, 610538) diluted 1:50, and, after washing with PBS, with Alexa 555-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen, USA, A1422) diluted 1:400. The cell preparations were incubated with the appropriate antibodies diluted in PBS solution containing 15% fetal bovine serum for 30 min at +37°C in 5% CO₂. Next, after washing with PBS, the cell preparations were incubated with 50 nМ DiOC₆ (3,3’-dihexyloxacarbocyanine iodide, Sigma, USA, D273) in PBS solution for 20 min at +37°C in 5% CO₂. Finally, the cell preparations washed with PBS were fixed with 8% formaldehyde solution in PBS for 5 min at room temperature and immediately analyzed using the VECTASHIELD Mounting Medium with DAPI under a laser scanning confocal microscope.

Microscopy

The cytological preparations were examined at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, SB RAS, using an Axioscop 2 plus microscope (Zeiss) and objectives with various magnifications (Zeiss), and photographed using an AxioCam HRc camera (Zeiss); the images were analyzed using the AxioVision 4.7 microscopy software (Zeiss). Cell preparations were stained with fluorescent dyes and examined under an LSM 510 or an LSM 780 laser scanning confocal microscope (Zeiss) using the LSM Image Browser and ZEN 2010 software (Zeiss). Granuloma cells were counted separately on each coverslip for each mouse in each test. In each experiment with bone marrow and peritoneal cell cultures, more than 1000 cells were analyzed at each time point. More than 1000 cells were analyzed at each experiment with control peritoneal macrophages and fibroblasts.

Statistical analysis

Statistical data processing was performed using MS Excel 2007 (Microsoft). Differences were tested for significance using Student’s t-test.

The inducer of apoptosis TNFα in granuloma cells from mice with latent tuberculous infection and in bone marrow macrophages after BCG infection in vitro

The production of the proinflammatory cytokine TNFα was analyzed in the cells of granulomas from spleens isolated after day 20 (/20 d; this is how long it takes mice to develop adaptive immunity to BCG [54]), one month (1 m) and two months (2 m) following infection denoted as Gran/20 d, Gran/1 m, and Gran/2 m, respectively. Because none of the mice was observed to have any sign of acute tuberculous infection at the time of granuloma isolation, it was concluded that these granulomas were isolated at the latent stage of BCG infection. Monolayer cultures of cells that had migrated from each granuloma were obtained. Any of the monolayer cultures of granuloma cells that may or may not retain cell clusters in the center of granulomatous lesions will be referred to as the “granuloma” throughout. The cellular composition of each granuloma and the mycobacterial loads in granuloma cells from different mice, the expression of various surface markers, and the production of various proinflammatory cytokines and growth factors by mouse granuloma cells were characterized previously [55-57].

Analysis of the production of the proinflammatory cytokine and inducer of apoptosis TNFα using specific antibodies revealed a large number of macrophages and dendritic cells containing this marker in the granulomas of mice at various times of latent tuberculous infection (Figures 1A-1C and 2). The largest number of TNFα-producing macrophages (>70% of the population size) was in the granuloma cells from mice after 20 days following infection with the BCG vaccine in vivo (Figure 2). The cells of granulomas from mice after one month and two months following BCG infection in vivo showed an about twofold decrease in TNFα-containing cells (Figure 2). A small number of control peritoneal macrophages had TNFα protein (Figure 2) and stained for this marker much less stronger than granuloma cells (Figure 1B). In granuloma macrophages, TNFα protein appeared as dots scattered around perinuclear areas and throughout the cytoplasm, irrespective of the presence or absence of BCG mycobacteria (Figures 1A-1C). Cells inside compacted granuloma structures expressed TNFα protein, too (Figure 1B). All granuloma neutrophils had cytoplasmic TNFα protein. In megakaryocytes, TNFα protein was observed in small cytoplasmic areas. Like control lung and spleen fibroblasts, granuloma platelets and fibroblasts did not stain for TNFα protein either (Figures 1A-1C). No TNFα expression was detectable in mouse 25 peritoneal macrophages isolated after two months following intraperitoneal infection with BCG (Figure 2). The number of TNFα-positive cells did not appear different between the peritoneal macrophages isolated from mice after 20 days following intraperitoneal infection with BCG in vivo and from control cultures (Figure 2).

Figure 1: The cells with TNFα protein in (A-C) the fragments of mouse granulomas (Gran) obtained from the spleens of mice after 20 days (20 d) and two months (2 m) following infection with the BCG vaccine in vivo and after ex vivo culture for several hours, and in (D) the bone marrow macrophages following infection with the BCG vaccine in vitro and after culture for several hours. The scale bars are (A, left panel) 50 μm, ((B, right panel) and ((C, left and central right panels), D) 20 μm each, and (other panels) 10 μm each. (A-B) Immunochemical localization of TNFα in granuloma cells and control mouse peritoneal macrophages (Mph) and splenic fibroblasts (Fibr). The brown color indicates the presence of TNFα in these cells. An TNFα-producing macrophages and megakaryocyte are indicated by the black arrows and star, respectively; black snowflake indicates fibroblast, that do not contain the antigen. The pictures of granuloma fragments (A, right-central and right panels) are enlarged images of the areas defined by black boxes in picture (A, left panel). (C-D) Confocal immunofluorescent localization of TNFα (red signal) in the cells. Nuclei stained by DAPI (blue signal). (C) In the left central and right panels: the same fragments as in the other panels re-stained for acid-fast BCG mycobacteria by the Ziehl-Neelsen (ZN) method. A TNFα-producing macrophages with BCG mycobacteria are indicated by the white arrows on the fluorescent images and the black arrows on the ZN images. (D) A TNFα-producing bone marrow cells are indicated by the white arrows.

Figure 2: Macrophages (Mph), dendritic cells (DC), and lymphocytes (Lym) with TNFα, CD30, CD36, and Fas/CD95. The objects examined are control peritoneal macrophages from three intact mice, peritoneal macrophages obtained from two mice on 20 day following intraperitoneal infection with the BCG vaccine (Mph-20 d), peritoneal macrophages obtained from mouse 25 after two months following intraperitoneal infection with the BCG vaccine (Mph-2 m), and granulomas from the spleens of two to four mice after 20 days (Gran/20 d), one month (Gran/1 m) and two months (Gran/2 m) following BCG infection in vivo and ex vivo culture for several hours. Granuloma cells producing various antigens were analyzed in each granuloma separately; the values were averaged all the mice and then combined for granulomas in each group. Data are expressed as the means ± SEM. *P<0.01 (comparisons of the number of TNFα- or CD-positive macrophages in each Gran group and controls, Mph-20 d, Mph-2 m).

Mouse bone marrow cell cultures that were infected with BCG in vitro and had increased mycobacterial loads and death rates in bone marrow macrophages [57] showed a gradual increase in TNFα- producing macrophages (from 3.88 ± 1.36% at hour 4 and 8.32 ± 1.86% at hour 24 h of analysis to 12.12 ± 1.27% at hour 48) (Figure 1D). The number of TNFα-positive macrophages in the control bone marrow cells was unchanged during culture (on average, 4.2 ± 1.47% throughout hours 4-48 of analysis).

All spleen granulomas from all experimental mice studied were found to contain a considerable number of infected and uninfected macrophages that had no morphological signs of apoptosis or necrosis, their cytoplasm staining for TNFα protein, often very intensely.

Expression of the activation receptor CD30, scavenge receptor CD36 and death receptor Fas/CD95 in mouse granuloma cells

The effect of the TNFα protein on cell viability is known to depend on a variety of factors. An important role in setting up the program for TNFα protein is given to cell receptors, including CD30, a member of the survival receptor subgroup of the TNFα receptor superfamily [42]. Staining with specific antibodies revealed CD30 on the membrane of about one-third of macrophages in mouse spleen granulomas isolated at different times of latent infection, but not in the control peritoneal macrophages (Figure 2, Figures 3A and 3B). CD30-positive cells were detected both among macrophages that had migrated from granulomas and among those that resided in compacted granuloma structures (Figure 3A). We note that the intensity of macrophage staining for CD30 varied considerably in the same granulomas and did not depend on the presence or absence of BCG mycobacteria in them: while some cells had bright microdomain staining across the cell membranes, some had a small number of dot-sized domains (Figures 3A and 3B). Some dendritic cells in the granulomas had CD30, too; however, their population was half the size of that of CD30-positive granuloma macrophages (Figure 2). Like control fibroblasts, granuloma fibroblasts did not stain for CD30, nor was this antigen detected in granuloma megakaryocytes or platelets (Figure 3A). The CD30 receptor is known to be an important activation marker of Т and В lymphocytes, and its transient expression in these cells is associated with antiapoptotic effects and suppression of functions of cytotoxic Т lymphocytes [58]. No lymphocytes with the CD30 marker on their surfaces were observed in peritoneal macrophage cultures (both control and those obtained on day 20 following BCG infection in vivo) or in mouse granulomas at any time of latent tuberculous infection (Figures 3A and 3B). Therefore, CD30 activation appears to be more important for macrophages and dendritic cells in mouse granulomas, also probably for regulation of TNFα action in them.

Figure 3: The cells with cell-surface markers (A-B) CD30, (C-D) CD36, and (B, E-F) Fas/CD95 in (A-C, E-F) the fragments of mouse splenic granulomas (Gran) and control mouse peritoneal macrophages (Mph), and (D) peritoneal macrophages obtained from mouse on 20 day following intraperitoneal infection with the BCG vaccine. The scale bars are (C, left panel) 50 μm, ((B, left, left and right central panels) and ((F, left panel)) 20 μm each, and (other panels) 10 μm each. (A, C, E) The brown color indicates the presence of various surface antigens on the cells after immunochemical staining. CD-producing macrophages are indicated by the black arrows; black snowflakes and stars indicate fibroblasts and megakaryocytes, respectively, that do not contain the antigen. (B, D, F) Confocal immunofluorescent localization of CD-markers in the cells. Colocalization of the markers on the confocal images of cells (yellow signal). Nuclei stained by DAPI (blue signal). In the right panels: the same fragments as in the other panels re-stained for acid-fast BCG mycobacteria by the ZN method. (B) The granuloma macrophage with BCG mycobacterium, CD95 (green signal) and CD30 (red signal) is indicated by the white arrows on the fluorescent images and the black arrow on the ZN image. (D) The peritoneal macrophage with BCG mycobacterium, CD36 (green signal) and LysoTracker Red DND-99 dye (red signal) is indicated by the white arrows on the fluorescent images and the black arrow on the ZN image. (F) The granuloma macrophages with BCG mycobacteria reproducing in it and CD95 (red signal) are indicated by the white arrows on the fluorescent image and the black arrows on the ZN image.

In our previous studies [55-57], we observed mouse granuloma macrophages to actively digest and destroy lymphocytes that had morphological signs of apoptotic death. As is known, phagocytosis of cells dead by apoptosis involves the multifunctional scavenger receptor CD36 jointly with the αvβ3 integrin receptor and thrombospondins. It has also been proposed [59] that thrombospondin 1 binding to CD36 can induce protein kinase signaling cascades on the cell membrane, which leads to caspase activation-mediated apoptotic death of these cells. The use of specific antibodies that detect CD36 revealed this marker on the membranes of a large number of macrophages, dendritic cells and lymphocytes in granulomas isolated from the spleens of mice after two months following infection with BCG vaccine in vivo (Figures 2 and 3C). In control peritoneal macrophages, the membrane of only a small number of cells stained for CD36, less strongly at that (Figures 2 and 3C). Curiously, the number of CD36- positive macrophages did not appear to be different between Gran/1 m and control peritoneal macrophages (Figure 2). The number of CD36-positive cells did not appear to be different between peritoneal macrophages isolated from mice on day 20 following intraperitoneal infection with BCG and control peritoneal macrophages and did not depend on the presence of BCG mycobacteria in the cells (Figures 2 and 3D). Like control lung and spleen fibroblasts, granuloma fibroblasts did not have the CD36 receptor (Figure 3C). All granuloma platelets stained for CD36 intensely; however, this receptor was observed to be virtually absent from granuloma megakaryocytes (Figure 3C). As is known, the glycoprotein CD36 is a receptor for the thrombospondin 1 protein, which is involved in the signaling pathways and interactions of megakaryocytes and platelets, and, therefore, is one of the markers of later stages of their differentiation [60]. Overall, the mouse granuloma macrophages with increased CD36 numbers had a propensity not only to digest dead lymphocytes and platelets in the granulomas, but also to be destroyed via caspase-dependent mechanisms of apoptosis during latent tuberculous infection.

The death receptor Fas/CD95, which induces apoptosis when binding to the Fas/CD95 ligand, was found on the membranes of a large number of granuloma cells from the spleens of all the mice assayed (Figure 2, and Figures 3E and 3F). Most macrophages (whether infected or not), dendritic cells, lymphocytes and neutrophils in granulomas showed a bright microdomain staining for Fas/CD95, often across cell membranes (Figures 3E and 3F). Some granuloma fibroblasts and megakaryocytes had this receptor, too (Figures 3E and 3F). Especially high amounts of Fas/CD95 were observed in granuloma platelets and erythrocytes (Figures 3E and 3F). Cells aggregated inside dense granuloma structures stained for Fas/CD95 as brightly as cells that had migrated from granulomas (Figure 3E and 3F). In the control peritoneal macrophages, Fas/CD95 was detected on the membrane of only a small number of cells, while most lymphocytes had this marker (Figure 2, Figures 3E and 3F). Control lung and spleen fibroblast cultures did not stain for Fas/CD95. We note that the number of Fas/ CD95-positive macrophages in bone marrow cell cultures infected in vitro did not change throughout 4-48 h of analysis and was 4.77 ± 1.25% on average, which was as many as there was in the control bone marrow cell cultures (data not shown). Thus, most mouse granuloma cells had increased Fas/CD95 and, therefore, were prepared for induction of apoptosis by one of receptor-mediated mechanisms.

The Вах, Ваd and P53 proteins, markers of apoptotic cell death, in mouse granuloma cells

To observe relevant proteins, we used antibodies that detect their regular forms, not conformationally changed or phosphorylated. We found the presence of Вах and Ваd proteins in large amounts in all the cells of all granulomas studied from the spleens of mice after one month (3 mice) and two months (9 mice) following infection with the BCG vaccine in vivo (Figures 4A-4D). All control peritoneal macrophages, as well as all control lung and spleen fibroblasts, had these antigens; however, they stained much less strongly than granuloma cells (Figures 4A-4D). Staining for Вах protein was observed both across the cytoplasm of granuloma macrophages and dendritic cells and in small roundish entities of varying size scattered peripherally to the nucleus, where Вах protein showed colocalization with Ваd protein (Figure 4D). As is known, Вах and Ваd are constitutive proteins in mammalian cells and are present in the cytoplasm at all times [31]. When the cell is exposed to an apoptosis stimulus, Вах protein undergoes conformational changes, moves from the cytoplasm to the mitochondrial outer membrane, interacts with dephosphorylated Ваd protein and thus abrogates the antiapoptotic functions of the Bcl-2 and/or Bcl-X_L proteins on the mitochondrial membranes [31]. It has been proposed [36,61] that the balance between Вах, Ваd, Bcl-2 and/or Bcl-X_L proteins in the complex on the mitochondrial outer membrane is critical for what follows. Will cell death be prevented? Or will the apoptotic program (mitochondrial membrane permeabilization, the release of apoptogenic factors from the intermembrane space and, eventually, activation of the executioner caspase-3) continue? Thus, the colocalization of the Вах and Ваd proteins is indicative of their joint proapoptotic activity in the granuloma cells of mice with latent tuberculous infection.

Figure 4: The cells with (A, C) Bax, (B-C) Bad, and (D-E) P53 proteins in the fragments of mouse splenic granulomas and control mouse peritoneal macrophages (Mph) and splenic fibroblasts (Fibr). The scale bars are (B, left panel) 50 μm, ((A, right panel) and ((D, left and right central panels)) 20 μm each, and (other panels) 10 μm each. (A-B, D) The brown color indicates the presence of various antigens in the cells after immunochemical staining. (A-B) Black snowflakes indicate fibroblasts with the antigens. Other stained cells are macrophages, dendritic cells, and lymphocytes. The pictures of granuloma fragments (B, left and right central panels) are enlarged images of the areas defined by black boxes in picture (B, left panel). (D) A P53-positive macrophages are indicated by the black arrows. (C, E) Confocal immunofluorescent localization of the antigens in the cells. Colocalization of the markers on the confocal images of cells (yellow signal). Nuclei stained by DAPI (blue signal). In the right panels: the same fragments as in the other panels re-stained for acid-fast BCG mycobacteria by the ZN method. (C) The granuloma macrophages and dendritic cells without BCG mycobacteria, but with Bax (green signal) and Bad (red signal) proteins. (E) The granuloma macrophages with BCG mycobacteria and CD11b (red signal), but without P53 protein (green signal) are indicated by the white arrows on the fluorescent images and the black arrows on the ZN image.

As is known [38-40], the P53 protein is continuously synthesized in mammalian cells, but is a short-lived protein and degrades rapidly within the complex with the Mdm2 protein, which functions as the ubiquitin ligase. In the cells damaged by genotoxic agents, this complex dissociates, P53 protein is stabilized and escapes proteasome-dependent degradation. As a result, P53 protein can accumulate in the cytoplasm and nuclei and launch the program of a stress-dependent mitochondrial pathway of apoptotic cell death [38,40]. The use of P53-specific antibodies did not reveal this antigen in the cytoplasm or nuclei of macrophages and dendritic cells, irrespective of the presence or absence of BCG mycobacteria in them, from the spleen granulomas of mice after one month and two months following infection with the BCG vaccine in vivo (Figures 4D, 4E and 5). Only a small number of control peritoneal macrophage cultures exhibited a background level of immunofluorescent staining for P53 protein, while most control and granuloma lymphocytes had this marker (Figures 4D and 5). P53 protein was not detected in granuloma fibroblasts or control lung and spleen fibroblasts. All granuloma platelet stained for P53 protein (Figure 4D). Thus, we have no evidence for whatever significant stabilization or accumulation of the P53 protein in the cytoplasm and nuclei of granuloma macrophages, dendritic cells and fibroblasts. Lack of P53 stabilization in granuloma cells, together with the unchanged morphology of their nuclei without evidence of chromatin condensation and nucleolar segregation, indicated that DNA in granuloma cells was undamaged and, consequently, the activators of this process were deficient. As is known [31,36], one of the main inducers of chromosome degradation, Apoptosis Inducing Factor (AIF), a flavoprotein with DNase functions, comes to the cell nuclei from the intermembrane space, where it normally occurs, only after pores have formed on the membrane of these organelles. It is therefore likely that the integrity of the mitochondrial outer membrane is preserved in mouse granuloma macrophages and dendritic cells, irrespective of the presence or absence of BCG mycobacteria in them.

Lack of activation of the executioner caspase-3 in mouse granuloma cells

As is known [62], caspase-3 is permanently present in cells as a non-active proenzyme; however, after its activation in the apoptosome, due to the release of apoptogenic factors from mitochondria, it becomes “executioner” and cleaves more than one hundred structural, signaling and regulatory proteins, making apoptosis inevitable. Consequently, caspase-3 activation is one of the main markers of apoptotic cell death. Cells were analyzed using antibodies that detect the active executioner form of caspase‑3. Caspase-3 activation was detected in the cytoplasm of few macrophages, dendritic cell, fibroblasts and megakaryocytes of spleen granulomas obtained from mice after one month and two months following BCG infection in vivo (Figures 5 and 6). Cells aggregated within compacted granuloma structures did not have this marker (Figure 6). No caspase-3 activation was observed in control macrophage and fibroblast cultures (Figure 6). The presence of this marker in some granuloma lymphocytes, in most granuloma platelets and erythrocytes, as well as in the lymphocytes of control peritoneal macrophage cultures, could be accounted for not only by the apoptotic death of the cells, but also by their differentiation. As is known [63], caspase-3 activation is required for the formation of platelets and erythrocytes, and lymphocyte proliferation. On the whole, lack of caspase-3 activation in the mouse granuloma macrophages, dendritic cells and fibroblasts prevented their caspase-3-dependent apoptosis and was supposed to preserve the integrity of mitochondrial membranes in these cells.

Figure 5: Macrophages (Mph), dendritic cells (DC), and lymphocytes (Lym) with P53, active caspase-3, and Bcl-2 proteins. The objects examined are control peritoneal macrophages from three intact mice (control), bone marrow and peritoneal macrophages obtained from two mice on 20 day following intraperitoneal infection with the BCG vaccine (BM-Mph-20 d and Per- Mph-20 d, respectively), peritoneal macrophages obtained from mouse 25 after two months following intraperitoneal infection with the BCG vaccine (Per-Mph-2 m), and granulomas from the bone marrow (BM/), lungs (L/), and spleens (S/) of two to five mice after 20 days (Gran/20 d), one month (Gran/1 m) and two months (Gran/2 m) following BCG infection in vivo and ex vivo culture for several hours. Gran/2 m granulomas of 12 mice were analyzed for Bcl-2. Granuloma cells producing various antigens were analyzed in each granuloma separately; the values were averaged all the mice and then combined for granulomas in each group. Data are expressed as the means ± SEM. *P<0.001 (comparisons of the number of Bcl-2-positive macrophages in Gran/1 m or Gran/2 m and control or Per-Mph-2 m); ^&P<0.01 (comparisons of the number of Bcl-2-positive macrophages in GranS/1 m or GranS/2 m and GranS/20 d); ^#P<0.05 (comparison of the number of Bcl-2-positive macrophages in GranBM/20 d and BM-Mph-20 d).

Figure 6: Immunochemical localization of the activation of caspase-3 in the cells of mouse splenic granuloma and control mouse peritoneal macrophages (Mph). The scale bars are (left upper panel) 100 μm and (other panels) 10 μm each. The pictures of granuloma fragments are enlarged images of the areas defined by black boxes in (a, left upper panel).The brown color indicates the presence of the antigen in these cells. Macrophages with active caspase-3 in them are indicated by the black arrows. The black stars and snowflakes indicate the megakaryocytes and fibroblasts, respectively that do not contain the antigen.

Thus, in granuloma cells with increased levels of the inducer of apoptosis TNFα, proapoptotic proteins Вах and Ваd, death receptor Fas/ CD95 and scavenge receptor CD36, we did not observe P53 stabilization or caspase-3 activation in macrophages and dendritic cells at the latent stage of BCG infection irrespective of the presence or absence of BCG mycobacteria in them. It is possible that the observed events of prevention of apoptotic death were most probably confined to times before mitochondrial and post-mitochondrial factors and effectors of apoptosis could spring to action and make changes irreversible.

The antiapoptotic protein Bcl-2 in granuloma cells from mice with latent tuberculous infection and in bone marrow and peritoneal macrophages after BCG infection in vitro

The Bcl‑2 protein is one of the main factors that maintain due mitochondrial membrane permeability and protect cells from apoptosis [31,36]. Following immunohistochemical procedures, the cytoplasm of most macrophages, dendritic cells and megakaryocytes in lung and spleen granulomas obtained from mice after one month and two months following BCG infection in vivo for 2-5 days in ex vivo culture exhibited intense staining for Bcl-2 protein (Figures 5, 7A and 7B). However, much fewer macrophages of spleen and bone marrow granulomas had increased Bcl-2 protein after 20 days following BCG infection in vivo than at later times of latent tuberculous infection (Figures 5 and 7C). Nevertheless, there were more Bcl-2-positive macrophages in bone marrow granulomas than in the surrounding stroma (Figure 5). Control peritoneal macrophages as well as control lung and spleen fibroblasts did not stain for this marker (Figures 5, 7A and 7B). The number of lymphocytes with cytoplasmic Bcl-2 protein varied strongly between granulomas (Figures 5, 7A and 7B). No Bcl-2-positive lymphocytes were detected in control peritoneal macrophage cultures (Figures 5 and 7A). In the macrophages of bone marrow and peritoneal cell cultures infected with BCG in vitro, where increased mycobacterial loads and death of infected cells were observed throughout hours 72-120 of analysis [57], no induction of Bcl-2 synthesis was detected throughout hours 4-120 of analysis (Table 1 and Figure 7D). The number of Bcl-2- positive macrophages did not appear different between these cultures and control bone marrow and peritoneal macrophages at the same times of analysis (data not shown).

Figure 7: Antiapoptotic Bcl-2 protein in (A-C) the cells in the fragments of mouse splenic granulomas, control mouse peritoneal macrophages (Mph), splenic fibroblasts (Fibr), and (D) the bone marrow and peritoneal macrophages following infection with the BCG vaccine in vitro and after culture for several hours. The scale bars are (A, left and right upper panels) 50 μm each, (A, left and right down panels) 20 μm each, and (other panels) 10 μm each. (A) The brown color indicates the presence of Bcl-2 protein in the cells after immunochemical staining. The pictures of granuloma fragments (A, left and right central top panels) are enlarged images of the areas defined by black boxes in picture (A, left top panel). (B-D) Confocal immunofluorescent localization of Bcl-2 protein (green signal) and MitoTracker Deep Red FM (red signal) in the cells. Colocalization of the markers on the confocal images of cells (yellow signal). Nuclei stained by DAPI (blue signal). (B) In the left panel: confocal image with phase contrast. (B-C) In the right panels: the same fragments as in the other panels re-stained for acid-fast BCG mycobacteria by the ZN method. A Bcl-2-producing macrophages with BCG mycobacteria are indicated by the white arrows on the fluorescent images and the black arrows on the ZN images.

Table 1: Antiapoptotic protein Bcl-2 in mouse bone marrow and peritoneal macrophages infected with the BCG vaccine in vitro (BCG) or without infection (control) and after culture for as long as indicated.

Immunofluorescent visualization of the Bcl-2 antigen revealed not only a random pattern of staining of the cytoplasm of granuloma macrophages and fibroblasts, but also an intense staining of small roundish entities of varying size. These Bcl-2-positive entities where scattered throughout the cells and compacted granuloma structures as well as in the monolayer cultures of cells that had migrated from lung, spleen and bone marrow granulomas obtained from mice with latent tuberculous infection (Figures 7B,7C, and 8A-8D). Some Bcl- 2-positive granuloma macrophages had acid-fast BCG mycobacteria in them, some did not (Figures 7B,7C, and 8A-8D). Some standalone entities that stained for Bcl-2 protein showed colocalization with the mitochondrial dye MitoTracker Deep Red FM in granuloma macrophages (Figure 7C). Bcl-2 protein was observed to partly colocalize with the proinflammatory cytokine TNFα (Figure 8A), the proapoptotic proteins Вах (Figure 8B), P53 (Figure 8C) and active caspase-3 (Figure 8D) in small roundish entities of varying size in mouse granuloma cells. Some granuloma macrophages and dendritic cells with large amounts of Bcl‑2 protein had BCG mycobacteria in them, some did not (Figures 8C and 8D). Granuloma macrophages, whether with or without acid-fast BCG mycobacteria in them, largely exhibited a background level of immunofluorescent staining for P53 protein and active caspase-3 (Figures 8C and 8D). Thus, granulomas obtained from mice with latent tuberculous infection, especially at later times of its development, characteristically had increased the antiapoptotic protein Bcl-2 in different compartments of granuloma cells, including, probably, mitochondria, irrespective of the presence or absence of BCG mycobacteria in them.

Figure 8: Representative confocal fluorescent images of macrophages stained by the Bcl-2-specific antibodies (red signal) and primary antibodies to (A) TNFα, (B) Bax, (C) P53, and (D) active caspase-3 (green signal) in (B) mouse lung granuloma and its fragment and (A, C-D) the fragments of mouse splenic granulomas. Colocalization of the markers on the confocal images of cells (yellow signal). Nuclei are stained by DAPI (blue signal). The scale bars are 10 μm each. (A) Macrophages and fibroblasts with Bcl-2 protein are indicated by the red and black arrows on the 3D image (right down panel), respectively. (B) The pictures of granuloma fragment are enlarged images of the area defined by black boxes in picture (B, left panel). (C-D) In the right panels: the same fragments as in the other panels re-stained for acid-fast BCG mycobacteria by the ZN method. A Bcl-2-producing macrophages with BCG mycobacteria are indicated by the white arrows on the fluorescent images and the black arrows on the ZN images.

Analysis of cells using the mitochondrial membrane potentialsensitive dye DiOC₆

The fluorescent lipophylic DiOC₆ is widely used on living cells in cytometric studies for analysis of mitochondrial depolarization, as decrease in the fluorescence intensity of this dye is regarded as an indicator of cell death [64]. We had previously observed an intense staining with DiOC₆ of roundish entities of varying size both in granuloma cells from mice with latent tuberculous infection and in control peritoneal macrophages and control lung and spleen fibroblasts (data not shown). In the present work, fluorescent living cell staining revealed the colocalization of the Bcl-2 protein and the DiOC₆ dye on small roundish entities of varying size scattered randomly throughout the cytoplasm of granuloma macrophages from the spleen of mouse 23 following two months after BCG infection in vivo (Figure 9A). It is therefore quite likely that those stained cytoplasmic elements are mitochondria. Confocal microscopy performed immediately after this staining and fixation of granuloma cells revealed a gradual disappearance of the green signal, which was indicative of the presence of DiOC₆ in mitochondria, while the intensity of the red signal, which corresponded to the Bcl-2 protein, did not change. It is likely that we observed DiOC₆ migrating from mitochondria, with increasing mitochondrial depolarization and decreasing mitochondrial membrane potential (ΔΨ_m) in granuloma cell killed by fixation. We also note that even though the living cell staining procedure was quite long, the morphology of granuloma macrophages remained unaffected: no nuclear fragmentation was observed; mitochondria occurring throughout the cells were comparable in size with those previously detected by staining for Bcl-2 protein and MitoTracker Deep Red FM together (Figures 7C and 8A).

Figure 9: Representative confocal fluorescent images of macrophages stained by the mitochondrial DiOC₆ dye (green signal) and Bcl-2-specific antibodies (green signal) in live cells. Nuclei are stained by DAPI (blue signal). The scale bars are 10 μm each. (A) Colocalization of Bcl-2 protein with mitochondria (yellow signal) is observed in macrophages in the fragments of mouse granulomas. (B) Absence of Bcl-2 protein in the cells and lack of its colocalization with DiOC₆-labeled mitochondria (lack of yellow signal) are observed in control peritoneal macrophages. (B, left panel) T1 is the conditional time point, when this macrophage was analyzed. (B, right panel) White arrows indicate a lower level of the green signal of mitochondrial DiOC₆ dye in the macrophage than that on the (B, right central panel) image analyzed 12 min earlier.

Confocal microscopy of the results of two similar experiments with the staining of peritoneal macrophages not infected in vitro revealed immediately proapoptotic changes in cell morphology, lack of staining for Bcl-2 protein and a minimum DiOC₆ fluorescence (Figure 9B). We observed nuclear fragmentation in peritoneal macrophages, typical of apoptotic cell death, and rapidly vanishing dot-sized DiOC₆ signals in perinuclear regions (Figure 9B). We note that control peritoneal macrophages that were not exposed to staining had no morphological signs of cell death in vitro (data not shown). It is possible that the living cell staining procedures, which adversely affect cell viability, were long enough to allow control macrophages to die rapidly, with their nuclei fragmented, spatially oriented mitochondrial network ruined, and organelles translocating to the nuclear region. In contrast to granuloma macrophages, control peritoneal macrophages stained very little for Bcl-2 protein (Figure 9B), and so did other control cultures (Figures 5, 7A and 7B). Thus, control peritoneal macrophages did not have Bcl-2 protein and, therefore, could not prevent the leak of factors and inducers of apoptotic cell death from mitochondria. The observed dotted pattern of cell staining with DiOC₆ (Figure 9B) suggested reduced mitochondrial ΔΨ_m in the control peritoneal macrophages and was yet another hallmark of their apoptotic death.

It was previously established [56] that one-third of macrophages in granulomas from the spleen of mouse 23 after two months following BCG infection in vivo had a considerable number of acid-fast BCG mycobacteria in them, including as growing colonies with the cord morphology (the indicator of mycobacterial virulence). However, mouse 23 granuloma macrophages that stained intensely for the antiapoptotic protein Bcl-2 did not have morphological signs of apoptotic or necrotic death (Figure 9A). Consequently, the Bcl-2 protein, which was observed in large amounts in the cytoplasm and, probably, mitochondria of macrophages in mouse 23 granulomas, can be regarded as one of the factors that protected granuloma cells from death both during long live cell staining procedures in ex vivo culture and, probably, in vivo, in granulomas containing large numbers of BCG-infected cells.

Together, the results obtained suggest that the antiapoptotic protein Bcl-2 promoted the viability of granulomas’ macrophages and other cell types, irrespective of the presence or absence of BCG mycobacteria in them, not only in ex vivo culture or during live cell staining, but also in the animal organisms under a considerable amount of pressure from mycobacterial, proinflammatory and proapoptotic factors in granulomatous inflammatory lesions at different times of latent tuberculous infection in mice.

Apoptotic death of cells infected with mycobacteria is considered to be one of the main mechanisms by which an affected organism can withstand tuberculous infection [4,5,7-9,24,25]. Apoptosis leads not only to disposal of digested apoptotic cells with mycobacteria and their elimination in phagosomes, but also to an efficient processing of mycobacterial antigens used by antigen-presenting cells for the maintenance and enhancement of the immune response to the pathogen [24]. It has been established that in vitro infection of different cell types with non-virulent strains of mycobacteria led these cells to rapid death by TNFα-induced receptor-dependent apoptosis [48,52,65]. However, our previous studies using the ex vivo model of granulomatous inflammatory lesions in mice did not reveal, at any time of latent tuberculous infection, morphological signs of apoptotic or necrotic death in granuloma macrophages or dendritic cells differently loaded with mycobacteria of the attenuated BCG strain [55-57].

Now, analysis of the expression of the markers of the development/ abrogation of apoptosis demonstrated increased levels of the inducer of apoptosis TNFα, proapoptotic proteins Вах and Ваd, death receptor Fas/CD95 and scavenge receptor CD36 in the cells of granulomas obtained from the spleens, lungs and bone marrow of BCG-infected mice at different times of latent tuberculous infection. However, we did not observe any significant stabilization of the P53 protein or activation of the major executioner caspase-3 in the cytoplasm and nuclei of granuloma macrophages, dendritic cells, fibroblasts and megakaryocytes. At the same time, the antiapoptotic protein Bcl‑2 was in large amounts observed in the cytoplasm and mitochondria of granuloma cells, especially at later times of latent tuberculous infection.

As is known, the Bcl-2 protein, which occurs on the mitochondrial outer membrane, interferes with pore formation and the release of proapoptotic cytochrome с and AIF from mitochondrial intermembrane space, and thus prevents activation of caspases, nuclear DNA degradation and apoptosis [36]. Also, Bcl-2 protein is acted on by activated caspase-3 and the Вах and P53 proteins, which abrogates its antiapoptotic functions [31,36,64]. The observed colocalization of the protein Bcl-2 with the proapoptotic proteins Вах and P53 and active caspase-3 on the mitochondria of granuloma cells suggests that they interacted with one another on these organelles in mouse granuloma cells. No colocalization of Bcl-2 protein with Вах protein was observed in the cytoplasm of granuloma cells. This observation is in agreement with studies of Edlich et al. [66,67], who found that non-active Вах protein is continuously recycling in the cells between the cytoplasm and mitochondria, while, on the mitochondrial outer membrane, nonactive Вах protein directly interacts with the antiapoptotic proteins Bcl- 2 and Bcl-xL and then returns to the cytoplasm with their assistance. In the cytoplasm, Вах protein immediately dissociates from the complex assembled on mitochondria. It was demonstrated [66,67] that the balance between mitochondrial and cytosolic Вах protein can additionally be regulated by ВН3-domain-containing proteins, including Bad, which promotes activation of Вах protein in their complex on the mitochondrial outer membrane, by the formation of pores and increase in their permeability, as well as by disabling Вах recycling in the cells. In mouse granuloma cells, we observed the proapoptotic proteins Bad and Вах colocalizing, probably on the mitochondrial outer membrane, and the activation of this complex. However, the accumulation of the potential-sensitive dye DiOC₆ in granuloma macrophage mitochondria suggested that their membranes were intact and that ΔΨ_m was preserved. We proposed that the protection of the mitochondria of granuloma cells from proapoptotic factors depends on Bcl-2 protein, which was found in large amounts in the cytoplasm and mitochondria of granuloma cells. As is known [30,36,61], elevated Bcl-2 protein on the mitochondrial membranes interferes with pore formation due to the action of the proapoptotic proteins Вах and Bad, stabilizes these organelles and abrogates apoptotic dying. For example, overexpression of Bcl-2 protein contained within an expression vector introduced to mouse L929 fibrosarcoma cells protected these cells from death, which was promoted by the inducer of apoptosis TNFα, and the mitochondria of these cells had increased ΔΨ_m [68]. Because ΔΨ_m and the proton gradient on the mitochondrial inner membrane are the main sources of energy for ATP synthesis, reduction in ΔΨ_m, which is normally associated with the opening of non-selective pores, is catastrophic for cellular respiration and energy-related processes in the cell and, eventually, fatal for these cells [69]. Preservation of mitochondrial ΔΨ_m during staining of living granuloma macrophages containing large amounts of the Bcl-2 protein was indicative of its involvement in maintaining the integrity of mitochondrial elements and the protection of granuloma cells from death, because in similar experiments the control macrophages that did not have any Bcl-2 protein in them had considerably reduced ΔΨ_m and exhibited morphological signs of apoptotic death.

In addition to protecting cells from apoptosis, Bcl-2 protein can protect mammalian cells from autophagy by interacting with the ВН3-like domain of the Beclin-1 protein and abrogating its functions in the formation of autophagosomal membranes [70]. It was established [70] that an essential role in preventing autophagy should be given to the Bcl-2 protein that is located on the endoplasmic reticulum, not to that on mitochondrial membranes. In our experiments, not only the mitochondria of granuloma macrophages and dendritic cells stained intensely for Bcl-2 protein, irrespective of the presence or absence of acid-fast BCG mycobacteria in them, but also the cytoplasm near the nucleus, where the endoplasmic reticulum basically occurs. Whether Bcl-2 protein is involved in the regulation of autophagy in the granuloma cells of mice at the latent stage of tuberculous infection remains to be known.

The observed increased Bcl-2 levels in most mouse granuloma cells poses the question about the life cycle of this protein in these cells. The half-life of Bcl-2 protein in cells is 24 h on average [70]. In human follicular lymphoma cells, where the gene encoding this protein was originally identified, the t(14;18) chromosomal rearrangement is often [71]. Following this rearrangement, the Bcl-2 encoding gene is acted on by the activated enhancers of genes for immunoglobulin heavy chains and, consequently, is continuously transcribed, which leads to cellular accumulation of Bcl-2 protein and its mRNA. As a result, lymphocytes lose sensitivity to proapoptotic stimuli, which leads to lymphomas. A mechanism of Bcl-2 accumulation like this is unlikely to operate in mouse granuloma cells, because macrophages and dendritic cells are highly differentiated and do not divide, nor it is likely that this rearrangement may occur in many cells at once. Other mechanisms of cellular Bcl-2 accumulation imply its post-translational modifications, which allow ubiquitination and proteasomal degradation to be escaped. It has been demonstrated [72] that phosphorylation of particular serine and threonine residues protected Bcl-2 protein from degradation and promoted its accumulation in different cell types, while dephosphorylation of these residues by the proinflammatory cytokine TNFα led to the destruction of proteasomal Bcl-2 and apoptosis without any change in Bcl-2 mRNA levels. Furthermore, it has been found [73] that nitrosylation of cysteine residues of Bcl-2 protein by endogenous nitric oxide abrogated its proteasomal degradation and protected cells from apoptotic death. Increased Bcl-2 protein in nucleus-free platelets of the mouse granulomas studied was suggestive of post-translational mechanisms of its stabilization in this cell type. At the same time, increased TNFα production in granuloma macrophages and dendritic cells was indicative of activation of the synthesis and/or maintenance of Bcl-2 mRNA with its continuous de novo production in granuloma cells. It is also known [64] that Bcl-2 protein located in differentMeanwhile, we asked ourselves if increased Bcl-2 protein has relevance to the presence of BCG mycobacteria in the macrophages or it is simply part of the regular proinflammatory response оf the host’s cells to intracellular microbial infection in granulomatous inflammatory lesions. In a study of Mogga et al. [74], it was not before week 16 following infection of mice with the virulent strain H37Rv of M. tuberculosis intraperitoneally that Bcl-2 protein became detectable by immunohistochemistry in lung tissue cells (about 5% of cells studied) that stained for mycobacterial antigens–but not for Вах protein. Natarajan and Sujatha observed a twofold increase in Bcl-2 levels as early as 24 h after in vitro infection of ТНР-1 monocyte-like cells with the virulent strain H37Rv of M. tuberculosis [52]. By contrast, blood mononuclear cells and human ТНР-1 cells infected in vitro with attenuated ВСG mycobacteria or non-virulent M. tuberculosis strain H37Rа had a considerable reduction in Bcl-2 protein after 24 h following infection [52,75]. We did not observe activation of Bcl-2 synthesis at hours 4‑120 of analysis in the macrophages from bone barrow cell cultures and peritoneal exudates infected with BCG mycobacteria in vitro. At the same time, we observed increased mycobacterial loads and death of cells at hours 72-120 of analysis [57]. The granulomas studied had different numbers of infected cells, including macrophages and dendritic cells, from the lowest, as in mouse 25 after two months following BCG infection in vivo, to the highest, as in mice 14, 16, 21, and 23 after two months following BCG infection in vivo; some of the granulomas did not have any infected cells in them at all [56]. However, all granulomas studied from mice after one month and two months following infection with the BCG vaccine in vivo had increased Bcl- 2 virtually in all macrophages and dendritic cells, irrespective of the presence or absence of acid-fast BCG mycobacteria in them. High Bcl-2 levels were also observed in all granuloma fibroblasts, megakaryocytes, platelets and some granuloma lymphocytes. However, peritoneal macrophages isolated in parallel with granuloma cells from mouse 25 two months following intraperitoneal infection did not stain for Bcl- 2 protein. This protein was detected only in a small number of the macrophages and dendritic cells of macrophages obtained from mice after 20 days following infection with the BCG vaccine in vivo. However, the number of Bcl-2-positive macrophages was significantly higher in bone marrow granuloma than in the surrounding stroma. Consequently, high Bcl-2 levels appear to be a feature of granuloma cells, especially as latent tuberculous infection in mice becomes chronic (one month and two months following infection with the BCG vaccine in vivo) in the presence of mycobacterial, proinflammatory and proapoptotic factors in granulomatous inflammatory lesions. Bcl-2 production in mouse granuloma cells appears to be induced and maintained irrespective of the presence or absence of BCG mycobacteria in them. It is possible that the antiapoptotic protein Bcl-2 acts to protect granulomas as a whole and their individual cells from death, including those heavily infected with BCG mycobacteria. As a result, Bcl-2-positive granuloma macrophages with large numbers of BCG mycobacteria in them were viable and did not have a characteristic apoptotic or necrotic morphology, while Bcl- 2-negative macrophages heavily loaded with BCG mycobacteria in the different cultures of mouse cells following BCG infection in vitro had increased death rates [57]. As was proposed [77], the apoptotic death of macrophages infected with mycobacteria in vivo should not necessarily be good for the organism, because the elimination of the cells that have been activated by mycobacteria leads to the weakening of the overall microbicidal response to infection, which, in turn, helps the pathogen survive and evade the host’s immunity. It is possible that it was BCG infection of mice in vivo that triggered not only the formation of granulomatous inflammatory lesions in the organs and tissues, but also the development of protective reactions of cells, including by cellular compartments can be regulated differently. For example, in human peripheral blood Т lymphocytes, mitochondrial Bcl-2 protein had reduced stability–in contrast to Bcl-2 protein on the nuclear envelope [64]. It is also known [76] that the proinflammatory cytokines TNFα and IL-1, an increased production of which was observed in granulomas from BCG-infected mice [55,57], can, through the links with their receptors, activate the transcription factor NF-kB, which induces expression of the bcl-2 gene. Thus, regulation of Bcl-2 levels in different cell types is complex and can be exerted from different layers by a variety of mechanisms. Therefore, further analysis of the ways in which Bcl-2 levels are regulated in the granuloma cells of mice with latent tuberculous infection is required.

Meanwhile, we asked ourselves if increased Bcl-2 protein has relevance to the presence of BCG mycobacteria in the macrophages or it is simply part of the regular proinflammatory response оf the host’s cells to intracellular microbial infection in granulomatous inflammatory lesions. In a study of Mogga et al. [74], it was not before week 16 following infection of mice with the virulent strain H37Rv of M. tuberculosis intraperitoneally that Bcl-2 protein became detectable by immunohistochemistry in lung tissue cells (about 5% of cells studied) that stained for mycobacterial antigens–but not for Вах protein. Natarajan and Sujatha observed a twofold increase in Bcl-2 levels as early as 24 h after in vitro infection of ТНР-1 monocyte-like cells with the virulent strain H37Rv of M. tuberculosis [52]. By contrast, blood mononuclear cells and human ТНР-1 cells infected in vitro with attenuated ВСG mycobacteria or non-virulent M. tuberculosis strain H37Rа had a considerable reduction in Bcl-2 protein after 24 h following infection [52,75]. We did not observe activation of Bcl-2 synthesis at hours 4‑120 of analysis in the macrophages from bone barrow cell cultures and peritoneal exudates infected with BCG mycobacteria in vitro. At the same time, we observed increased mycobacterial loads and death of cells at hours 72-120 of analysis [57]. The granulomas studied had different numbers of infected cells, including macrophages and dendritic cells, from the lowest, as in mouse 25 after two months following BCG infection in vivo, to the highest, as in mice 14, 16, 21, and 23 after two months following BCG infection in vivo; some of the granulomas did not have any infected cells in them at all [56]. However, all granulomas studied from mice after one month and two months following infection with the BCG vaccine in vivo had increased Bcl- 2 virtually in all macrophages and dendritic cells, irrespective of the presence or absence of acid-fast BCG mycobacteria in them. High Bcl-2 levels were also observed in all granuloma fibroblasts, megakaryocytes, platelets and some granuloma lymphocytes. However, peritoneal macrophages isolated in parallel with granuloma cells from mouse 25 two months following intraperitoneal infection did not stain for Bcl- 2 protein. This protein was detected only in a small number of the macrophages and dendritic cells of macrophages obtained from mice after 20 days following infection with the BCG vaccine in vivo. However, the number of Bcl-2-positive macrophages was significantly higher in bone marrow granuloma than in the surrounding stroma. Consequently, high Bcl-2 levels appear to be a feature of granuloma cells, especially as latent tuberculous infection in mice becomes chronic (one month and two months following infection with the BCG vaccine in vivo) in the presence of mycobacterial, proinflammatory and proapoptotic factors in granulomatous inflammatory lesions. Bcl-2 production in mouse granuloma cells appears to be induced and maintained irrespective of the presence or absence of BCG mycobacteria in them. It is possible that the antiapoptotic protein Bcl-2 acts to protect granulomas as a whole and their individual cells from death, including those heavily infected with BCG mycobacteria. As a result, Bcl-2-positive granuloma macrophages with large numbers of BCG mycobacteria in them were viable and did not have a characteristic apoptotic or necrotic morphology, while Bcl- 2-negative macrophages heavily loaded with BCG mycobacteria in the different cultures of mouse cells following BCG infection in vitro had increased death rates [57]. As was proposed [77], the apoptotic death of macrophages infected with mycobacteria in vivo should not necessarily be good for the organism, because the elimination of the cells that have been activated by mycobacteria leads to the weakening of the overall microbicidal response to infection, which, in turn, helps the pathogen survive and evade the host’s immunity. It is possible that it was BCG infection of mice in vivo that triggered not only the formation of granulomatous inflammatory lesions in the organs and tissues, but also the development of protective reactions of cells, including by increasing their levels of the antiapoptotic protein Bcl-2. Whether this event is specific for the granulomas that were induced by mycobacterial infection or it is typical of granulomatous inflammatory lesions of non-infectious origin remains to be known.

Note that the proinflammatory cytokine TNFα, the production of which was induced following exposure to BCG infection both in vivo and in vitro, may have contrasting effects on the infected organism. As is known [2-4,9,19,34], TNFα-induced apoptosis of infected cells considerably reduces the bacterial load. However, overexpression of TNFα protein leads to the development of an uncontrollable inflammatory response in tissues and organs, which can result in the clinical signs of tuberculosis in man [2-4,34]. Therefore, proper regulation of the proinflammatory and costimulating activities of TNFα protein, as well as its proapoptotic effects depending on various factors acting on the cells in granulomatous inflammatory lesions is of major importance. An outstanding role in this choice should be given to granuloma cells’ receptors, which can act pleiotropically. For example, activation of the costimulating molecule CD30, a member of the TNFα receptor superfamily, on the cell membrane, on the one hand, had an antiapoptotic effect on Т and В lymphocytes; while, on the other hand, activated, via its signaling pathways, expression of the gene for the death receptor Fas/CD95 [58]. Overall, it is possible that the involvement of different receptors, which occur on the cell membrane and induce different signaling pathways helps exert a fine-tuned regulation on TNFα activity on granuloma cells and promote either their survival or their death in the granulomatous inflammatory lesions in mice at various times of latent tuberculous infection.

This study of granulomas from mice with latent tuberculous infection using an ex vivo model did not reveal activation of proteins involved in the irreversible development of apoptotic death of granuloma cells irrespective of the presence or absence of BCG mycobacteria in them – not even in the presence of a large number of proapoptotic stimuli and factors. The antiapoptotic protein Bcl-2, found in large amounts in the cytoplasm and, probably, mitochondria of granuloma cells, irrespective of the presence or absence of mycobacteria in them, may have probably been involved in the maintenance of their viability endangered by mycobacterial, proinflammatory and proapoptotic factors in mouse granulomatous inflammatory lesions. Overall, the importance of analysis of intercellular and intracellular relationships between various inducers, effectors and suppressors of cell death, which influence the survival of cell with mycobacteria in the granulomatous inflammatory lesions of animals with latent tuberculous infection, is unquestionable. These interactions can result either in activation of infection into an acute tuberculous process or in a long symptom-free latent stage, which may either end up destroying all mycobacteria or last for even longer and keep mycobacteria persisting in the cells of the host organism.

The author declares that no conflict of interests exists.

The author is thankful to Dr. S.I. Baiborodin and T.E. Aleshina of the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, for technical support; Professor L.E. Panin of the Research Institute of Biochemistry, for support to this work and helpful discussion.