Highly vacuolated or foamy macrophages are a distinct characteristic of granulomas in the lungs of animals infected with Mycobacterium tuberculosis. To date these have usually been considered to represent activated macrophages derived from monocytes entering the lesions from the blood. However, we demonstrate in this study that foamy macrophages express high levels of DEC-205, a marker characteristic of dendritic cells (DCs). In addition to high expression of the DEC-205 marker, these cells were characterized as CD11b+CD11chighMHC class IIhigh, and CD40high, which are additional markers typically expressed by DCs. Up-regulation of CD40 was seen only during the early chronic stage of the lung disease, and both the expression of CD40 and MHC class II markers were down-regulated as the disease progressed into the late chronic phase. Foamy cells positive for the DEC-205 marker also expressed high levels of TNFR-associated factor-1 (TRAF-1), TRAF-2, and TRAF-3, markers associated with resistance to apoptosis. These data indicate that in addition to the central role of DCs in initiating the acquired immune response against M. tuberculosis infection, they also participate in the granulomatous response.

Macrophages play a central effector role in the immune response to infection with Mycobacterium tuberculosis. Once infected by the bacterium, the macrophages presents Ag on both class I and class II MHC molecules to T cells, which, in turn, secrete IFN-γ, resulting in activation of the macrophage to kill the intracellular organism (1, 2, 3, 4, 5).

It is now becoming clear that specialized dendritic cells (DCs)3 are also involved in an effector role against M. tuberculosis infection (6, 7, 8, 9, 10, 11, 12, 13, 14) and are central to the generation of acquired immunity after carriage of Ags to draining lymph nodes, where recognition by T cells can be maximized (12, 15). DCs also harbor intact live M. tuberculosis, but the degree to which these bacilli can be killed by the cell is very low (10). Analysis of DC populations within the lungs of mice infected by aerosol exposure to low doses of M. tuberculosis reveals a process of DC maturation that can be monitored by flow cytometry (9).

The granulomatous response is the hallmark of a chronic stage of infection caused by M. tuberculosis representing a desperate attempt by the host immune system to contain multiplication and further dissemination of the bacteria to other cells and organs. In human tuberculosis, there is evidence that smaller solid granulomas (<3 mm in size) and increased macrophage renewal are indicative of an immune response capable of controlling M. tuberculosis growth. In contrast, larger caseous granulomas (>5 mm in size) and decreased cellular renewal indicate less effective containment of bacterial growth, leading to dissemination of the infection (16, 17).

Highly vacuolated or foamy macrophages are a distinct characteristic of granulomas in the lungs of animals infected with M. tuberculosis. One primary aspect of foamy cells within granulomas is the tendency of some of them to form layers of highly vacuolated cells, regarded as highly vacuolated macrophages and even “angry” macrophages in some of the early literature (16, 18). However, the origin and function of these cells during the granulomatous responses remain unknown.

Programmed cell death, or apoptosis, is an essential mechanism for the maintenance of cell homeostasis, effective control of inflammation, cell renewal, and elimination of microbial infections (19, 20, 21, 22). In some instances, apoptosis can be blocked by TNFR-associated factors (TRAFs), which initiate signaling pathways that promote cell survival or longevity. Within the TRAF family, TRAF-1 is a strong antiapoptotic protein that associates with TRAF-2 (23, 24, 25) and interacts with intracellular domains of cell surface receptors, such as TNFR II (26). TRAF-3 is a cytoplasmic factor that can interact with other TNF family receptors, such as CD40 and, upon receptor oligomerization with TRAF-2, redistributes to the cell surface, activating downstream signaling events that inhibit apoptosis (26). Apoptosis has previously been described in macrophages infected with M. tuberculosis (27, 28, 29, 30, 31); however, the expression of markers from the TRAF family during mycobacterial infections has only been reported during Mycobacterium avium paratuberculosis infections (32).

In the present study we used immunohistochemistry and flow cytometric analysis to further characterize DC populations in M. tuberculosis-infected lungs and to define their location in the developing granuloma. The DC marker DEC-205 (8, 33) was observed to be highly expressed on cells making up the foamy macrophage layers, but minimally expressed on cells in the surrounding parenchyma. In addition to high expression of the DEC-205 marker, these cells were characterized as CD11b+ CD11chighMHC class IIhigh and CD40high, which are additional markers typically expressed by DCs. These cells also showed an increase in the expression of TRAF molecules, which signal resistance to apoptosis. These data indicate that in addition to the central role of DCs in initiating the acquired immune response against M. tuberculosis infection, they also participate in the granulomatous response.

Specific pathogen-free female C57BL/6 mice, 6–8 wk old, were purchased from The Jackson Laboratory. Mice were maintained in the biosafety level 3 biohazard facility at Colorado State University and were given sterile water, mouse chow, bedding, and enrichment for the duration of the experiments. The specific pathogen-free nature of the mouse colonies was demonstrated by testing sentinel animals. All experimental protocols were approved by the Animal Care and Use Committee of Colorado State University.

Mice were challenged by low-dose aerosol exposure to M. tuberculosis strain Erdman using a Glas-Col aerosol generator calibrated to deliver 50–100 bacteria into the lungs. Bacterial counts in the lungs (n = 5) at each time point of the study were determined by plating serial dilutions of homogenates of lungs on nutrient 7H11 agar and counting CFU after a 3-wk incubation at 37°C. Bacterial growth in the lungs was similar to previous reports (34) (data not shown). Lungs and lung draining lymph nodes (DLNs) from other mice (n = 5) in the same groups were harvested for immunohistochemistry, histological analysis, and lung cell suspensions on days 35, 90, and 400 after challenge. The results shown in this study are representative of two experiments.

Lungs were infused with 30% OCT (Tissue-Tek) in PBS through the trachea. Once removed from the pulmonary cavity, whole lungs were embedded in OCT, frozen in a bath of liquid nitrogen for a few seconds, and then stored at −80°C. Serial sections, 5- to 8-μm thick, from each lung were cut on a cryostat (Leica; CM 1850) using the Instrumedics tape transfer system, fixed in cold acetone, and air dried. The sections were washed, and unspecific Ab binding was blocked with 3% BSA-PBS solution. Thereafter, the sections were incubated overnight at 4°C with one of the following purified primary Abs: isotype control rat IgG2a or anti-DEC-205 (clone NDCL 145 IgG2a; Serotec) or rabbit anti-mouse TRAF-1, TRAF-2, anti-TRAF-3, or rabbit IgG (Santa Cruz Biotechnology). All sections were washed three times in PBS and incubated with the secondary detection Ab F(ab′)2 anti-rat or anti-rabbit Ig conjugated to HRP (BioSource International). Finally, the reaction was developed using aminoethylcarbazole (BioGenex) as substrate. The sections were counterstained with Meyer’s hematoxylin and thereafter mounted with crystal/mount (Biomedia).

When lung tissue was analyzed by confocal microscopy, the sections were incubated simultaneously with DEC-205 or any of the three TRAF family Abs. The secondary Abs were substituted by anti-rat or anti-rabbit Ig conjugated to Alexa 488 or Alexa 546 (Molecule Probes). The nuclei in cells of these sections were counterstained using TO-PRO-3 dye (Molecular Probes) and examined using an inverted microscope (Olympus; F-IX70) with a FVX-IHRT Flouview confocal scanning system laser scanning microscope (Olympus) equipped with three laser beams (argon 488, HeNe 546, and HeNe 633) that allow excitation of Alexa 488, Alexa 546, and TO-PRO-3 dye.

The presence of apoptotic cells in lung tissue was analyzed by the TUNEL assay with the MEBSTAIN apoptosis kit (Medical & Biological Laboratories). The protocol provided by the manufacturer was modified to be used for immunohistochemistry. In brief, serial sections, 5- to 8-μm thick, from each lung were cut as indicated above and fixed for 15 min with a solution containing 4% paraformaldehyde. Thereafter, the sections were washed twice with PBS, and 50 μl of TdT buffer II were added and incubated for 10 min at room temperature. The TdT reaction was performed in the presence of biotin-dUTP for 1 h at 37°C. Then, the sections were immersed in TB solution for 15 min. After washing three times with distilled water, the sections were incubated with blocking solution for 10 min at room temperature. The blocking solution was removed, and 200 μl of streptavidin/HRP (Serotec) was incubated for 1 h at 37°C. After three washes with PBS, the reaction was developed using aminoethylcarbazole (BioGenex) as substrate. The sections were counterstained and mounted as described above.

The accessory lung lobe from each mouse was fixed with 10% Formalin in PBS. Sections from these tissues were stained using H&E.

Single-cell suspensions were prepared as described previously (9, 35). Briefly, the lungs were perfused with a solution containing PBS and heparin (50 U/ml; Sigma-Aldrich) through the pulmonary artery, aseptically removed from the pulmonary cavity, placed in medium, and dissected. The dissected lung tissue was incubated with complete DMEM containing collagenase XI (0.7 mg/ml; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 mg/ml; Sigma-Aldrich) for 30 min at 37°C. The digested lungs were further disrupted by gently pushing the tissue through a cell strainer (BD Biosciences). RBC were lysed with ACK buffer, washed, and resuspended in complete DMEM. Total cell numbers per lung were determined using a hemocytometer.

Lung DLNs from either naive or infected mice were also collected. All DLNs from each mouse were pulled together and disrupted by gently pushing the tissue through a cell strainer (BD Biosciences). The cell suspension obtained was resuspended in medium.

Cell suspension from each mouse was incubated with mAbs labeled with FITC, PE, PerCP, or allophycocyanin at 4°C for 30 min in the dark. After washing the cells with deficient RPMI 1640 (Irvine Scientific) containing 0.1% sodium azide (Sigma-Aldrich) the cells were incubated with mAbs. mAbs against CD11c (clone HL3 and hamster IgG1), CD11b (Mac-1, clone M1/70, and rat IgG2a), CD40 (clone 3-23 and rat IgG2a), I-A/I-E MHC class II (clone 2G9 and rat IgG2a) markers, rat IgG2a, rat IgG2b, rat IgG1, mouse IgG2a, and hamster IgG were used in this study. These mAbs were purchased from BD Pharmingen, Serotec, or eBioscience as direct conjugates to FITC, PE, PerCP, PerCP-Cy5.5, or allophycocyanin. Data acquisition and analysis were performed using a FACSCalibur (BD Biosciences) and CellQuest software (BD Biosciences), respectively. Compensation of the spectral overlap for each fluorochrome was performed using CD11b Ag from cells gated in the forward scatter (FSC)mid/high/side scatter (SSC)mid/high region. Macrophages and DCs were gated according to size and granularity, as defined in the FSC and SSC plots. All analyses were performed with an acquisition of at least 500,000 total events and a minimum of 10,000 CD11c+ events.

Cells were first stained for cell surface markers as indicated above; thereafter, the same cell suspensions were prepared for intracellular staining. Staining for markers of the TRAF family and DEC-205 was performed using intracellular staining of cells. Cell membranes were permeabilized according to the kit instructions (Fix/Perm kit; BD Pharmingen). Abs against TRAF-1 and -2 (Santa Cruz Biotechnology) were incubated with the appropriate surface-stained cells for 30 min, and the cells were washed twice and resuspended in deficient RPMI 1640 before analysis.

Data are presented using the mean values from five mice per group and from values from replicate samples and duplicate or triplicate assays. A parametric method, Student’s t test, was used to assess statistical significance between groups of data. Correlations between sets of data were calculated using the Spearman rank correlation.

DEC-205 is an endocytosis receptor highly expressed on lymphoid tissue DCs (36, 37, 38). We used the mAb NLDC-145 from Serotec characterized against the DEC-205 marker to further identify murine lung DC populations. Surprisingly, immunohistochemical staining against the DEC-205 marker of lung sections from M. tuberculosis-infected mice showed that classically defined foamy macrophages (Fig. 1,A) were recognized by the NDLC-145 Ab (Fig. 1, B and C). Alveolar macrophages or other subpopulations of DCs exterior to the granuloma, which are known to be located in lung parenchyma, were rarely positive for the DEC-205 marker (Fig. 1, D and E). Very few cells in lung sections from naive mice were positive for DEC-205 (Fig. 1 F).

FIGURE 1.

Foamy macrophages in the lung granuloma stain positively for DEC-205. Sections from either paraffin-embedded (A) or OCT-embedded (B–F) lungs from mice challenged 90 days previously with M. tuberculosis were stained with H&E (A) or Abs against the DC marker DEC-205 (B–F). A, Typical granuloma formation consisting of characteristic foamy macrophages surrounding a lymphocytic core structure. B and C, High number of foamy cells staining positively for the DEC-205 marker in the granuloma (red color). D, DEC-205-positive cells were concentrated in the granuloma (g) and were rarely found outside the granuloma (arrow). E, There were few DEC-205-positive cells exterior to the granuloma in the surrounding lung parenchyma (arrow). F, There were few DEC-205 cells in lungs of naive mouse (arrow). Total magnification: A, B, D, and E, ×10; C and F, ×20.

FIGURE 1.

Foamy macrophages in the lung granuloma stain positively for DEC-205. Sections from either paraffin-embedded (A) or OCT-embedded (B–F) lungs from mice challenged 90 days previously with M. tuberculosis were stained with H&E (A) or Abs against the DC marker DEC-205 (B–F). A, Typical granuloma formation consisting of characteristic foamy macrophages surrounding a lymphocytic core structure. B and C, High number of foamy cells staining positively for the DEC-205 marker in the granuloma (red color). D, DEC-205-positive cells were concentrated in the granuloma (g) and were rarely found outside the granuloma (arrow). E, There were few DEC-205-positive cells exterior to the granuloma in the surrounding lung parenchyma (arrow). F, There were few DEC-205 cells in lungs of naive mouse (arrow). Total magnification: A, B, D, and E, ×10; C and F, ×20.

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The comparative analysis of alveolar macrophages (R5) and DCs (R6) populations from the lungs of naive and M. tuberculosis-infected mice was performed as previously reported (9). As shown in Fig. 2 A, macrophages and DCs were gated in R1 as FSC/SSCmid/high. Secondary gating of cells in R1 demonstrated that DEC-205-positive cells from lung cell suspensions were also characterized as CD11b+/CD11chigh cells (R6).

FIGURE 2.

DCs in the lungs of infected mice are DEC 205high/CD11bhigh/CD11chigh. A, Flow cytometric analysis of lung cell suspensions in representative samples from naive cells (left) and cells 35 days after infection (right). First, the macrophage and DC populations in these samples were gated on FSC/SSCmid/high (R1). Cells in R1 were further gated as DEC-205-positive cells and on the CD11c vs CD11b dot plots. The CD11b/CD11c dot plot was gated into various regions, including alveolar macrophages (CD11bneg/CD11chigh; R5) and DCs (R6). Cells in other regions are small macrophages or monocytes CD11b+/CD11cneg (lower middle region) and neutrophils CD11b+/high/CD11cneg (lower right region). The MFC and percentage of positive cells for the DEC-205 marker in cells in R5 and R6 from naive mice (□) or infected mice (▪) are shown in B and C. D, Colocalization of cells demonstrating foamy cell morphology when stained by H&E (left panel) or for DEC-205 marker (right panel). Total magnification: F, ×100. E, Percentage of cells in R5 and R6 from naive mice (□) or M. tuberculosis-infected mice 35, 90, or 400 days after challenge (▪).

FIGURE 2.

DCs in the lungs of infected mice are DEC 205high/CD11bhigh/CD11chigh. A, Flow cytometric analysis of lung cell suspensions in representative samples from naive cells (left) and cells 35 days after infection (right). First, the macrophage and DC populations in these samples were gated on FSC/SSCmid/high (R1). Cells in R1 were further gated as DEC-205-positive cells and on the CD11c vs CD11b dot plots. The CD11b/CD11c dot plot was gated into various regions, including alveolar macrophages (CD11bneg/CD11chigh; R5) and DCs (R6). Cells in other regions are small macrophages or monocytes CD11b+/CD11cneg (lower middle region) and neutrophils CD11b+/high/CD11cneg (lower right region). The MFC and percentage of positive cells for the DEC-205 marker in cells in R5 and R6 from naive mice (□) or infected mice (▪) are shown in B and C. D, Colocalization of cells demonstrating foamy cell morphology when stained by H&E (left panel) or for DEC-205 marker (right panel). Total magnification: F, ×100. E, Percentage of cells in R5 and R6 from naive mice (□) or M. tuberculosis-infected mice 35, 90, or 400 days after challenge (▪).

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We next determined the levels of expression and the percentage of cells expressing the DEC-205 marker in regions R5 and R6 in the lungs of naive and infected mice (Fig. 2, B and C). As shown in Fig. 2,B, alveolar macrophages (R5) and DCs (R6) from the lungs of naive mice expressed low levels of the DEC-205 marker (mean fluorescence channel (MFC), 20 and 45, respectively). However, mice infected 35 days previously with M. tuberculosis showed a significant increase (p = 0.02) in the MFC for DEC-205 on DCs gated in R6. Interestingly, and in agreement with the immunohistological analysis shown in Fig. 1,E, alveolar macrophage cells (R5) did not show a significant increase in expression of the DEC-205 marker after infection (p = 0.10; Fig. 2,B). The percentage of DEC-205-positive cells in R6 also increased significantly in lung cell suspensions after infection (DEC-205-positive cells in naive and infected lungs were 25 and 96%, respectively; Fig. 2,C). Fig. 2 D shows that DEC-205-positive cells in the granuloma have a foamy appearance, similar to cells stained by H&E. Combining the results from the histological and flow cytometric analyses indicated that the foamy cells in the lungs of infected mice were CD11b+/CD11chigh/DEC-205high (R6).

As shown in Fig. 2 E, the total percentage of DCs in the lungs after M. tuberculosis infection gradually increased from day 35 until day 90 and thereafter remained constant, whereas the percentages of alveolar macrophages in the same lung cell suspensions were significantly decreased (p = 0.04) as the infection progressed.

Lung cells were further analyzed for the expression of surface MHC class II and CD40 molecules, which are quickly up-regulated by DCs after stimulation and activation (39). Fig. 3,A shows constitutive levels (naive mice) and changes in MHC class II and CD40 molecule expression on alveolar macrophages (R5; CD11bneg/CD11chigh) and DCs (R6; CD11b+/CD11chigh/DEC-205high) during the early stage of chronic infection with M. tuberculosis. The expression of MHC class II and CD40 molecules on alveolar macrophages gated within region R5 and DEC-205-positive cells gated in R6 from samples collected from naive and day 35 M. tuberculosis-infected mice was measured by MFC values for each molecule (Fig. 3 A). The MFCs of MHC class II and CD40 molecules in cells from R6 from naive mice were 4 and 3 times higher, respectively, than those in cells from R5 from the same mice. These results indicate that the constitutive levels of expression of MHC class II and CD40 molecules on the DEC-205-positive cells (R6) were higher than those on alveolar macrophages (R5).

FIGURE 3.

Expression of MHC class II and CD40 molecules on alveolar macrophages and DCs in lungs during chronic infection. A, The MFC of MHC class II and CD40 molecule expression on cells in the R5 region and DEC-205-positive cells in R6 from samples collected from mice (n = 5) before infection (□) or 35 days after aerosol infection (▪). ∗, p < 0.05 (by Student’s t test). B, The MFC ratios for MHC class II or CD40 molecules in cells gated in R5 (▨) or DEC-205-positive cells in R6 (□) 35, 90, and 400 days after challenge. C, Foamy appearance of cells staining positively for MHC class II (left) or CD40 (right) markers in lung sections obtained on day 35 after challenge with M. tuberculosis. Total magnification: C, ×100. D, The mean percentage of naive (○) and M. tuberculosis-infected (•) cells in R5 and DEC-205-positive cells in R6 expressing MHC class II and CD40 molecules at 35, 90, and 400 days after challenge.

FIGURE 3.

Expression of MHC class II and CD40 molecules on alveolar macrophages and DCs in lungs during chronic infection. A, The MFC of MHC class II and CD40 molecule expression on cells in the R5 region and DEC-205-positive cells in R6 from samples collected from mice (n = 5) before infection (□) or 35 days after aerosol infection (▪). ∗, p < 0.05 (by Student’s t test). B, The MFC ratios for MHC class II or CD40 molecules in cells gated in R5 (▨) or DEC-205-positive cells in R6 (□) 35, 90, and 400 days after challenge. C, Foamy appearance of cells staining positively for MHC class II (left) or CD40 (right) markers in lung sections obtained on day 35 after challenge with M. tuberculosis. Total magnification: C, ×100. D, The mean percentage of naive (○) and M. tuberculosis-infected (•) cells in R5 and DEC-205-positive cells in R6 expressing MHC class II and CD40 molecules at 35, 90, and 400 days after challenge.

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These parameters changed as the chronic stage ensued (Fig. 3,B). At the early stage of the chronic infection (35 days after aerosol), the relative MFC values for MHC class II molecules on alveolar macrophages were higher than those on foamy DCs (2.9 and 1.2 times, respectively), but these decreased at later times over the course of infection for both cell populations. On the contrary, during the early chronic stage of infection, the expression of CD40 molecules was up-regulated (3.4 and 3.5 times, respectively) in both cell populations, but again this progressively decreased over the later stages of the infection (90 and 400 days). Immunohistochemical staining of lung sections showed that cells staining positively for DEC-205 and MHC class II or CD40 markers within the granuloma environment had a foamy appearance (Fig. 3 C).

Fig. 3 D shows the percentages of cells expressing CD40 and MHC class II molecules on alveolar macrophages and DCs during the early and late stages of the infection. The percentages of alveolar macrophages and DCs expressing MHC class II or CD40 molecules increased in the early chronic stage of infection, but decreased progressively over the course of infection. These results indicated that MHC class II or CD40 molecules were up-regulated on alveolar macrophages during the early chronic stage of infection. However, DCs expressing the DEC-205 marker were unable to up-regulate MHC class II during either the early or late stage of chronic infection. Together, these results show that during early and late stages of infection with M. tuberculosis, DCs have inadequate activation of molecules required to function as efficient APCs.

TRAFs are known to block apoptotic signals by activating signaling pathways that promote cellular survival and longevity (25). Immunohistochemical staining for TRAF-1, TRAF-2, and TRAF-3 on lung sections from mice infected 35, 90, and 400 days previously with M. tuberculosis showed preferential expression of TRAF-1, TRAF-2, and TRAF-3 on foamy cells in the lung lesions (Fig. 4). Positive staining for TRAF molecules was observed on foamy cell layers also staining positively for the DEC-205 marker (Fig. 4,A), and this was seen throughout the chronic stage of infection (data not shown). Analysis by confocal microscopy verified that the expression of the DEC-205 marker on foamy cells colocalized with the expression of TRAF markers (Fig. 4,B). Furthermore, we evaluated apoptosis in sections from the lungs of infected mice by immunohistochemical staining for DNA fragmentation using the MEBSTAIN TUNEL apoptosis kit. As shown in Fig. 4,C, lung sections from mice challenged with M. tuberculosis 90 days previously showed positive staining for apoptosis on cells located primarily outside the granuloma (Fig. 4 C).

FIGURE 4.

Expression of TRAF markers on DEC-205-positive cells from lungs during the chronic stage of pulmonary infection with M. tuberculosis. Top panels show representative photomicrographs illustrating colocalization of staining for DEC 205, TRAF-1, TRAF-2, or TRAF-3 in lung tissue sections from mice 35 days after challenge. Middle panels show confocal microscopic analysis demonstrating the coexpression of DEC-205 (red) and TRAF-1 (green) and DEC-205 plus TRAF (yellow) in foamy cells of lung sections obtained 35 days after challenge. Bottom panels correspond to TUNEL assay on lung sections from infected mice showing cells staining positively for apoptosis. Only a few cells were positive for apoptosis and were located outside the granuloma (g; left panel). Inside the granuloma, no cells stained positively for apoptosis (middle panel), and cells were stained positively for apoptosis in the area exterior to the granuloma (right panel). Total magnification: A and B, ×20; C, left panel, ×10; C, middle and right panels, ×40.

FIGURE 4.

Expression of TRAF markers on DEC-205-positive cells from lungs during the chronic stage of pulmonary infection with M. tuberculosis. Top panels show representative photomicrographs illustrating colocalization of staining for DEC 205, TRAF-1, TRAF-2, or TRAF-3 in lung tissue sections from mice 35 days after challenge. Middle panels show confocal microscopic analysis demonstrating the coexpression of DEC-205 (red) and TRAF-1 (green) and DEC-205 plus TRAF (yellow) in foamy cells of lung sections obtained 35 days after challenge. Bottom panels correspond to TUNEL assay on lung sections from infected mice showing cells staining positively for apoptosis. Only a few cells were positive for apoptosis and were located outside the granuloma (g; left panel). Inside the granuloma, no cells stained positively for apoptosis (middle panel), and cells were stained positively for apoptosis in the area exterior to the granuloma (right panel). Total magnification: A and B, ×20; C, left panel, ×10; C, middle and right panels, ×40.

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The expression of TRAF markers in DEC-205-positive cells was also demonstrated by flow cytometric analysis (Fig. 5, A and C). Intracellular staining for TRAF-1 or TRAF-2 showed coexpression of DEC-205 and TRAF markers in cells located in the R6 region (Fig. 5,A). Most of DEC-205-positive cells in R6 (70–90%) expressed TRAF-1 or TRAF-2, whereas only 20% of alveolar macrophages expressed these molecules. Colocalization by immunohistochemistry of DEC-205-positive cells expressing TRAF-1 also had a foamy appearance, as shown in Fig. 5,B. In addition, the increase in the percentage of DCs correlated with the increase in the percentage of cells expressing TRAF-1 (r = 0.948) and TRAF-2 (r = 0.715; Fig. 5 C) over the course of the infection.

FIGURE 5.

Expression of TRAF markers on DEC-205high cells. A, Representative dot plots showing expression of DEC-205 and TRAF-1 or TRAF-2 marker expression on cells from regions R5 and R6. Colocalization by immunohistochemistry of DEC205-positive cells (left panel) expressing TRAF-1 marker (middle panel) in foamy cells (right panel) is shown in B. The mean percentage of cells in R5 or DEC-205high cells in R6 expressing TRAF-1 or TRAF-2 markers (C) (right panel). Total magnification: B, left and middle panels, ×40; B, right panel, ×100.

FIGURE 5.

Expression of TRAF markers on DEC-205high cells. A, Representative dot plots showing expression of DEC-205 and TRAF-1 or TRAF-2 marker expression on cells from regions R5 and R6. Colocalization by immunohistochemistry of DEC205-positive cells (left panel) expressing TRAF-1 marker (middle panel) in foamy cells (right panel) is shown in B. The mean percentage of cells in R5 or DEC-205high cells in R6 expressing TRAF-1 or TRAF-2 markers (C) (right panel). Total magnification: B, left and middle panels, ×40; B, right panel, ×100.

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Finally, we determined whether the expression of TRAF molecules was a unique characteristic of cells expressing the DEC-205 marker in lungs after infection, or whether cells expressing DEC-205 from other sources also expressed TRAF markers. Because the DEC-205 marker is mainly expressed on DCs from lymphoid tissue (36), we looked at the expression of these markers in the DLNs. The expression of TRAF-1 and TRAF-2 on the DEC-205-positive cells obtained from the DLNs and lungs from mice challenged with M. tuberculosis 35 days previously are shown in Fig. 6,A. Naive mice had low expression of TRAF-1 and TRAF-2, but this was higher on DCs (R6) than on macrophages (R5). After infection, the expression of TRAF markers on DCs from the lungs was higher than that in similar cells from the DLNs. Finally, immunohistochemical staining of sections of DLNs from infected mice showed expression of the DEC-205 marker on clusters of cells interspersed within lymphocytes (Fig. 6,B). Interestingly, smaller numbers of cells in such clusters were positive for the TRAF-1 marker, and after infection, some cells in the clusters showed a foamy morphology (H&E stain; Fig. 6 B).

FIGURE 6.

DEC-205-positive cells from DLN had low levels of expression of TRAF markers. A, MFC values for TRAF-1 or TRAF-2 on cells in R5 or R6 obtained from lungs (▨) or DLNs (□) of naive or infected mice. B, Representative photographs of DLN tissue sections from mice 35 days after challenge. H&E staining shows clusters of cells, some of which have a foamy morphology (top right). Similar clusters of cells appeared strongly stained for DEC-205 (middle) and had low levels of staining for TRAF-1 (bottom). Total magnification: left panel, ×10; right panel, ×40.

FIGURE 6.

DEC-205-positive cells from DLN had low levels of expression of TRAF markers. A, MFC values for TRAF-1 or TRAF-2 on cells in R5 or R6 obtained from lungs (▨) or DLNs (□) of naive or infected mice. B, Representative photographs of DLN tissue sections from mice 35 days after challenge. H&E staining shows clusters of cells, some of which have a foamy morphology (top right). Similar clusters of cells appeared strongly stained for DEC-205 (middle) and had low levels of staining for TRAF-1 (bottom). Total magnification: left panel, ×10; right panel, ×40.

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The central finding in this study is that foamy macrophages, a distinct component of the granulomatous process and often regarded as a cell type in a state of degradation in such lesions, strongly expressed the DC marker DEC-205. This observation thus provides an interesting new twist to the growing information that DCs play a central role in the host response to M. tuberculosis infection (7, 10, 12, 40, 41) and supports a previous report showing that DCs are important in the regulation of granulomatous responses in the lung (42).

It was also observed that DEC-205-positive cells in the lungs expressed high levels of CD11c, MHC class II, and CD40 molecules, which are characteristic markers of DCs. However, as the chronic disease process continued, these cells lost expression of the two latter molecules and instead strongly expressed antiapoptotic markers of the TRAF family. Additionally, we found that apoptosis was mainly present in cells outside the granuloma. Together, these observations support the hypothesis that the role of DCs in the lungs in response to M. tuberculosis infection is not simply limited to carriage of Ag into nearby lymphoid tissues. It appears that in addition to this important event, these cells expressing markers characteristic of DCs have an active role in the granulomatous response. DCs appear during this process as layers of cells interspersed with aggregates of lymphocytes, the majority of which are T (CD4) and B cells (34).

It is known that M. tuberculosis infects macrophages and that these cells are capable of eliminating the bacteria very efficiently after activation by IFN-γ (3, 10, 43). Although M. tuberculosis can also infect DCs, it is now clear that viable bacteria remain for long periods of time within these cells (8). It has also been demonstrated that DCs have poor mechanisms to eliminate M. tuberculosis (10, 41) or other intracellular bacteria, such as L. monocytogenes (44). It has also been suggested that DCs offer niches for long-term survival of intracellular bacteria (10, 41, 44). Thus, accumulation of DCs at the granuloma site during pulmonary infection with M. tuberculosis may provide niches where the bacteria can survive.

In contrast, the large recruitment of DCs into the granuloma may represent a directly protective event on the part of the host. DCs ingest M. tuberculosis (6, 7, 8, 10, 41), and the process of vacuolation, which is not understood or well defined other than by microscopy, disrupts or slows bacterial cell division by interfering with the phagosomal niche (41, 45, 46). In this study we also concluded that expression of MHC class II Ag is down-regulated on foamy DCs during the chronic stage of infection. Our data support previous reports indicating that M. tuberculosis-infected cells become defective in Ag processing due to down-regulation of cell surface expression of MHC class II Ags and that bacterial Ags are responsible for such down-regulation (9, 47, 48, 49, 50). By losing expression of MHC class II molecules, the cell loses its ability to signal to T cells that it has been infected, a phenomenon that would dampen a chronic inflammatory response, thereby potentially reducing lung pathology. Moreover, by expressing TRAF molecules, the cell changes to a long-lived state by avoiding any possibility of apoptosis, preventing cell death and subsequent dissemination of bacteria to other organs. Finally, the foamy DC layers become enmeshed in a fine lattice of fibrosis (51), further encapsulating the site of infection and protecting the host from bacterial dissemination.

Why DCs positive for the DEC-205 marker accumulate within the granuloma is still unknown. There could be several reasons, including increased differentiation of DCs precursors arriving in the lungs (9), recruitment of DCs to the inflammatory site as observed previously (52, 53, 54, 55), and continued production of chemokines that recruit DCs during inflammatory conditions. The expression of chemokines during the granulomatous response in tuberculosis has been studied (56, 57) to some extent, but not in the context of DC recruitment. Indeed, it is well established that certain cytokines and chemokines control DC migration patterns (58, 59), but this is still unknown for tuberculosis.

The expression of TRAF-1, -2, and -3 markers is associated with increased cell survival (60). In our study only the DCs and, to a much lower extent, macrophages expressed these markers within the granuloma. The environment within the granuloma may promote expression of markers of the TRAF family, because the expression of these markers on DCs in the lymphoid tissue or in other cells outside the granulomatous region was very low. The mechanism used by the bacteria or immune system to increase the expression of markers of the TRAF family in these cells remains to be studied. The lifespan of DCs is only a few days (60), and increasing the expression of antiapoptotic molecules on DCs ensures its long-term survival and protection from apoptosis. In contrast, apoptosis is known as a mechanism that regulates homeostasis of cells in tissues, participates in cell renewal, and eliminates infected cells (19, 20). All these events are required for effective elimination of bacteria and resolution of a granulomatous/inflammatory response during an infection. However, although apoptosis has been observed in mononuclear phagocytic cells after M. tuberculosis infection (27, 28, 29, 30, 31, 61, 62) and in macrophages and T cells from tuberculous lesions (63), our study demonstrated that apoptosis was present primarily in cells exterior to the granuloma. Therefore, we argue against the idea that apoptosis is an effective mechanism within the granuloma environment. Conversely, we have shown that within the granuloma environment, antiapoptotic mechanisms are more predominant than apoptosis, and this may explain the long-term survival and accumulation of cells at the site of infection.

In conclusion, our results demonstrated that the environment of the granuloma during chronic infection with M. tuberculosis resulted in weak and inadequate activation of DCs, low cellular renewal, and increased longevity of DCs at the site of the infection. As a final point, current studies investigating the role of DCs in M. tuberculosis infection have been directed at early events when Ag-presenting properties are paramount (6, 14, 64, 65). The results of this study indicate a more extensive role for DCs in the pathogenesis of tuberculosis in the mouse model that extends well into the chronic and granulomatous stage of the disease process. The development of new strategies of therapeutic value that are aimed at fast DC turnover at the site of tuberculosis infection as well as strong activation of these cells for maximal Ag presentation (15) should also consider that DCs play an important role in the granuloma environment.

We thank Karen Marietta for technical assistance, and Dr. J. Taylor (Colorado State University, Fort Collins, CO) for reviewing the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI44072.

3

Abbreviations used in this paper: DC, dendritic cell; DLN, draining lymph node; FSC, forward scatter; MFC, mean fluorescence channel; SSC, side scatter; TRAF, TNFR-associated factor.

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