American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1640-1646, (2002)
© 2002 American Thoracic Society
Dendritic Cell Involvement in Pulmonary Granuloma Formation Elicited by Bacillus Calmette-Guérin in Rats
Tomoyoshi Tsuchiya,
Kingo Chida,
Takafumi Suda,
Eveline E. Schneeberger and
Hirotoshi Nakamura
Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan; and Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Correspondence and requests for reprints should be addressed to Takafumi Suda, M.D., Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, Shizuoka, 431-3192 Japan. E-mail: suda{at}hama-med.ac.jp
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ABSTRACT
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Dendritic cells (DCs) are the most potent antigen-presenting cells that play a central role in initiating the primary immune response. However, their role in granulomatous inflammation has not been well studied. The aim of the present study was to elucidate the role of DCs in granuloma formation. Using a rat model of bacillus Calmette-Guérin (BCG)-elicited pulmonary granulomas, we investigated the distribution of DCs in the granulomas by immunohistochemistry with a rat-DC–specific monoclonal antibody, OX62. We found numerous large, pleiomorphic OX62+ cells accumulating at the borders of the pulmonary granulomas. The OX62+ cells isolated from the granulomatous lung showed intense surface expression of major histocompatibility complex class II, B7–1, and B7–2, and a lack of T cell– and monocyte/macrophage-specific markers. Their ultrastructural morphology was characteristic of DCs. Functionally, they had potent capacity to stimulate allogeneic T cells as well as purified protein derivative-specific syngeneic T cells in the absence of exogenous peptides. Based on these findings, the OX62+ cells infiltrating the granulomas were considered to be DCs expressing BCG-derived peptides. These results indicate that DCs contribute to pulmonary granuloma formation elicited by BCG by means of their potent antigen-presenting function, providing a novel insight into DC function in T cell–mediated granulomatous immune responses.
Key Words: dendritic cells • granuloma • BCG
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INTRODUCTION
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Dendritic cells (DCs) are the most potent antigen-presenting cells and they play a central role in initiating the primary immune response (1, 2). DCs act as sentinels, being widely distributed in virtually all organs (3). They are strategically positioned to take up antigens, after which they migrate to lymphoid organs, where they present antigens to naive T cells, leading to the initiation of T cell immunity (1–4). In addition to their T cell–stimulatory properties, recent studies have revealed that DCs can directly stimulate antibody production by B cells and induce B cell proliferation (5, 6). DCs also govern immunoglobulin class-switching, such as immunoglobulin A2 expression (7), indicating that DCs regulate the humoral immune response, in part, via a direct interaction with B cells (8). In contrast, DCs have also been implicated in the induction of tolerance (9–11) and have been shown to induce antigen-specific unresponsiveness in certain experimental models (12, 13). These cumulative data indicate that DCs have various roles in regulating immunity in vivo. Although DCs appear to participate in a large variety of immune responses, their role in initiating granulomatous inflammation has not been clearly determined.
Granuloma formation is a chronic inflammatory response to a variety of persistent pathogens and foreign particles as well as to certain etiologically unknown factors (14). Granuloma formation, the hallmark of a large number of human diseases, including tuberculosis, leprosy, schistosomiasis, beryliosis, and sarcoidosis, can be broadly classified into a hypersensitive type (immunologic) and a foreign body type (nonimmunologic) (15, 16). Histologically, the foreign-body granuloma is composed predominantly of macrophages and their derivatives, such as epithelioid and giant cells. In contrast, the hypersensitive-type granuloma is more complex and contains lymphocytes in addition to macrophages and their derivatives. Currently, the hypersensivity-type granuloma is believed to be induced by the interaction of activated T cells and monocytes/macrophages in an antigen-specific manner (16, 17). However, the precise mechanisms involved in its development are still not clearly understood. In light of the potent antigen-presenting function of DCs, we postulated that they may directly participate in granuloma formation.
The present study was conducted to elucidate the role of DCs in hypersensitivity-type granuloma formation. For this purpose, using a rat model of bacillus Calmette-Guérin (BCG)-elicited pulmonary granulomas, we examined the distribution of DCs in the granulomas by immunohistochemistry with a rat-DC–specific mAb, OX62. In addition, DCs were isolated from the granulomatous lung, and their phenotype, ultrastructure, and antigen-presenting function were determined. We found that a number of OX-62+ cells accumulated at the margins of the pulmonary granulomas. The OX62+ cells isolated from the granulomatous lung showed intense surface expression of major histocompatibility complex (MHC) class II, B7–1, B7–2 costimulatory molecules, a lack of other cell specific markers, and ultrastructural morphology characteristic of DCs. Functionally, they had a potent mitogenic capacity in an allogeneic mixed lymphocyte reaction, and effectively stimulated purified protein derivative (PPD)-specific syngeneic T cells in the absence of exogenous peptides. Based on these results, the OX62+ cells infiltrating the granulomas were considered to be DCs expressing BCG-derived peptides, suggesting that DCs participate in pulmonary granuloma formation elicited by BCG.
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METHODS
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Animals
Eight-week-old male Lewis (RT1Al) and male DA (RT1Ab) rats (200–250 g) were purchased from Charles River Laboratories (Yokohama, Japan).
Antibodies
The following mAbs were used: OX6 (anti-rat MHC class II, Serotec, Oxford, UK), OX8 (anti-rat CD8; Harlan Sera-Lab, Loughborough, UK), OX52 (anti-rat pan-T cell; Harlan Sera-Lab), OX62 (anti-rat DC; Serotec), V65 (anti-rat   -T cell; Pharmingen, San Diego, CA), ED1 (anti-rat dendritic cell/macrophage/monocyte; Serotec Ltd.), and ED2 (anti-rat macrophage; Serotec Ltd.). Phycoerythrin (PE)-conjugated OX6, fluorescein isothiocyanate (FITC)-conjugated 3H5 (anti-rat CD80, B7–1), and 24F (anti-rat CD86, B7–2) were purchased from Pharmingen. FITC-conjugated W3/25 (anti-rat CD4) and R7.3 (anti-rat TCR  /ß) were obtained from Serotec.
Induction of Pulmonary Granulomas
Bacillus Calmette-Guérin (BCG)-elicited pulmonary granulomas were induced using a modification of a method previously described (18). Briefly, Lewis rats were sensitized by intravenous injection of 0.1 mg of heat-killed BCG strain of Mycobacterium bovis (gift from Dr. Q.N. Myrvik). Three weeks later, sensitized rats were challenged by intravenous administration of 0.5 mg of heat-killed BCG.
Immunohistochemistry
As we described previously (19), frozen sections of lungs were prepared. The sections were fixed in acetone for 10 minutes at room temperature. Nonspecific staining was blocked with horse serum. Slides were then incubated with OX62 mAb for 1 hour at room temperature and then treated with 0.3% hydrogen peroxide for 20 minutes at room temperature. After incubation with biotinylated horse anti-mouse immunoglobulin antibody (Vector Laboratories) for 20 minutes at room temperature, the slides were incubated with streptavidin-biotin-peroxidase complex (Vector Laboratories) for 15 minutes at room temperature. Reaction product was developed with 3-amino-9 ethylcarbazole and the sections were counterstained with hematoxylin.
For double immunolabeling, acetone-fixed frozen sections were first reacted with OX62 mAb as described in the previous paragraph. Reaction product was generated using 3-amino-9 ethylcarbazole. The sections were then incubated with OX6, OX52, OX8, V65, ED1, or ED2 mAb, followed by biotinylated horse anti-mouse immunoglobulin antibody, and the antigen was visualized with an avidin-biotinylated alkaline phosphatase complex using a chromogen kit (Vector Laboratories).
Isolation OX62+ DCs from Granulomatous Lung
To obtain low-density, single cells from the lung parenchyma, we performed collagenase digestion and density gradient centrifugation, as we reported previously (19). The low-density cells were then sorted using a magnetic cell separator (Vario MACS, Miltenyi Biotec, Auburn, CA). Briefly, the cells were reacted with OX62 mAb for 5 minutes at 4°C, followed by incubation with rat–anti-mouse immunoglobulin G1-conjugated magnetic beads (Miltenyi Biotec Inc.) for 15 minutes at 4°C. After washing with separation buffer containing 0.2 mM ethylenediaminetetraacetate and 2% bovine serum albumin, magnetic bead-bound cells were magnetically sorted in the magnetic cell separator.
Flow Cytometry
After blocking with 10% normal goat serum, the cells were first stained with OX62 mAb for 30 min at 4°C, followed by incubation with FITC-conjugated goat–anti-mouse immunoglobulin G for 30 min at 4°C. The cells were stained with PE-conjugated OX6, B7–1, B7–2, ED1, ED2, or V65, mAbs for 30 min at 4°C. They were fixed in 2% paraformaldehyde/phosphate-buffered saline, and analyzed with an EPICS profile (Beckman Coulter Inc., Fullerton, CA).
Allogeneic Mixed Lymphocyte Reaction
Splenic T cells from DA (RT1Ab) rats were purified by passage through an affinity column (Cedarlane Laboratories, Hornby, Canada). Residual contaminating antigen-presenting cells were removed by panning using OX6-coated plates. The final cell population was greater than 95% OX52-positive as judged by flow cytometric analysis. Varying numbers of irradiated (2,500 rads) DCs (1 x 103– 3 x 104 cells/well) were then placed in 96-well flat-bottom tissue culture plates (Corning, Acton, MA) alone or with allogeneic T cells (1 x 105 cells/well) obtained from DA rats. On Day 5, cells were pulsed with [3H]thymidine (1 µCi/well; specific activity of 50–75 Ci/mmol; Amersham Japan, Tokyo, Japan) for 16 hours. The cultures were then harvested with a cell harvester and the incorporated radioactivity was quantified in a liquid scintillation counter (LSC-3100; Aloka, Tokyo, Japan).
Generation of PPD- and Hen Egg Lysozyme-specific T Cells
Lewis rats were immunized with an emulsion of 100 µL of complete Freund's adjuvant alone or 100 µg of hen egg lysozyme (HEL) (Sigma Chemical, St. Louis, MO) in 100 µL of complete Freund's adjuvant in each footpad. Popliteal lymph nodes were harvested 14 days after immunization. The lymph node cells were incubated with 20 µg/ml of PPD (Japan BCG Laboratory, Tokyo, Japan) or 100 µg/ml of HEL. Interleukin-2 (100 U/ml; Peprotech EC, London, UK) was added to the medium at Day 5. Every 3–4 weeks, the cultures were restimulated in the presence of 20 µg/ml of PPD or 100 µg /ml of HEL and irradiated (2,500 rads) syngeneic spleen cells.
Antigen Presenting Assay
Varying numbers of irradiated DCs (1 x 103 to 1 x 104 cells/well) were plated in 96-well flat-bottom tissue culture plates. PPD- or HEL-specific T cells (5 x 104 cells/well) were added to each well without exogenous PPD or HEL. After 72 hours incubation, wells were pulsed with [3H]thymidine (1 µCi/well; specific activity of 50–75 Ci/mmol; Amersham Japan, Tokyo, Japan) for 6 hours. The cultures were then harvested with a cell harvester and the incorporated radioactivity was measured.
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RESULTS
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BCG-induced Pulmonary Granuloma Formation
Administration of BCG induced the formation of numerous granulomas in the lungs of Lewis rats. The granulomas were conspicuous by Day 3, and increased in size over time, reaching maximal size on Day 14, and then gradually becoming smaller (Figure 1) . The lesions were composed predominantly of centrally packed epithelioid-like cells and surrounding small and large mononuclear cells. Although the granulomas were still obvious between Days 21 and 28, the aggregates of epithelioid-like cells became loose and scattered.
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Figure 1. Histologic appearance of sequential pulmonary granulomas elicited by BCG. A–E are representative lesions 3, 7, 14, 21, and 28 days after challenge, respectively. Original magnification: x400.
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Identification of OX62+ DCs in Pulmonary Granulomas
As shown in Figure 2
, 14 days after challenge a number of large, pleiomorphic OX62+ cells were seen surrounding epithelioid-like cells in pulmonary granulomas, whereas normal lung had a few OX62+ cells (data not shown). The majority of OX62+ cells had abundant cytoplasm and pleiomorphic morphology, whereas the remaining OX62+ cells were small and round, resembling lymphocytes. Because a small proportion of T cells also react with OX62 mAb (20), we further analyzed the OX62+ cells using dual immunostaining with other cell-specific markers. Figures 3A–3C
show that the large, pleiomorphic OX62+ cells were negative for OX52 (anti-rat pan-T cell mAb), OX8 (anti-rat cytotoxic T cell), and V65 mAb (anti-rat T cell mAb), whereas some of the small, round OX62+ cells reacted with these T cell–specific mAbs. All of the large, pleiomorphic OX62+ cells exhibited intense staining for OX6 mAb (anti-rat MHC class II mAb) (data not shown). No reactivity with ED2 mAb, specific for rat macrophages, was observed in these cells (Figure 3D), whereas approximately 20% of OX62+ cells reacted with ED1 mAb, recognizing a lysosomal antigen present in rat monocyte, macrophages, and a subset of DC (Figure 3E). Based on these data, showing that the large, pleiomorphic OX62+ cells accumulating at the margins of the granulomas had no reactivity with T cell- or macrophage-specific mAbs, they were considered to be phenotypically DCs. Moreover, a close association between the infiltrating DCs and T cells at the margins of the granulomas was noted (Figure 3A and 3B).
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Figure 2. Immunohistochemic staining of pulmonary granulomas with OX62 mAb against rat DCs. Frozen sections of the lung on Day 14 after challenge were immunostained with OX62 mAb. Numerous large, pleiomorphic OX62-positive cells are present at the borders of pulmonary granulomas. Original magnification: x200.
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Kinetics of the Number of OX62+ DCs in Pulmonary Granulomas
Figure 4
shows pulmonary granulomas at various times after BCG challenge, immunostained with OX62 mAb. To chronologically evaluate the numeric density of OX62+ DCs in the granulomas, a morphometric analysis was performed. The number of OX62+ DCs per granuloma increased over time, becoming maximal (118 cells/granuloma) on Day 14, and decreasing thereafter (Figure 5)
. The kinetics of the number of DCs in the granulomas closely corresponded to alterations in the granuloma size (data not shown).
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Figure 4. Immunohistochemical staining with OX62 mAb of pulmonary granulomas at various times after BCG-challenge. A–E are representative lesions at 3, 7, 14, 21, and 28 days after challenge, respectively. Original magnification: x400.
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Figure 5. Kinetics of the number of OX62-positive DCs in pulmonary granulomas. The numbers of OX62-positive DCs per granuloma were counted in a minimum of 20 granulomas. The OX62-positive DCs were defined on the basis of their large, pleiomorphic morphology, intense reactivity with OX62 mAb, and lack of staining for OX52, OX8, V65, and ED2 mAbs. The data points represent the mean and SEM of six animals for each time point.
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Yield of OX62+ DCs from Granulomatous Lung
To further examine the phenotype, ultrastructural morphology, and function of OX62+ DCs in the pulmonary granulomas, we isolated these DCs from the granulomatous lung. Because both the granuloma size and the number of OX62+ DCs were maximal on day 14, we isolated OX62+ DCs at 14 days after BCG challenge. The average cell yield from the enzymatic digestion of the granulomatous lung on Day 14 was 211 ± 17 x 106 cells/rat. After density separation on bovine serum albumin, the average number of cells in the low-density fraction retrieved was 42 ± 3.1 x 106 cells/rat. A final cell yield of 0.80 ± 0.09 x 106 cells/rat was obtained by immuno-magnetic cell sorting with OX62 mAb. From normal rat lungs, the same isolation procedures resulted in a yield of negligible numbers of cells (less than 1 x 102 cells/rat).
Phenotypic and Morphologic Analysis of Isolated DCs
In the low-density fraction after bovine serum albumin separation, only approximately 18% of cells were double-stained with OX6 and OX62 mAbs, and were thus phenotypically considered DCs. Subsequent immunomagnetic separation yielded a final population that contained more than 98% OX6+/OX62+ cells (Figure 6A)
. More than 95% of this population strongly expressed the costimulatory molecules B7–1 and B7–2 (Figure 6B and 6C). The isolated OX62+ cells did not react with the ED2 or V65 mAbs (Figure 6D and 6E). Comparable to the results of dual immunostaining, approximately 20% of the isolated OX62+ cells were positive for ED1 mAb (Figure 6F). The purity of the final cell population obtained from the granulomatous lung was greater than 95%, as assessed by the flow cytometric analysis that showed that the population was OX62+, MHC class II+, B7–1+, and B7–2+, and lacked staining for other cell-specific markers. An electron micrograph of a typical, freshly isolated DCs (Figure 7)
shows veil-like cell projections, a lobulated nucleus, and few cytoplasmic lysosomes. No Birbeck granules were observed in any of the isolated DCs examined.
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Figure 7. Electron micrograph of purified OX62-positive cells from pulmonary granulomas. The purified cells show villous cell processes, a lobulated nucleus, and few cytoplasmic lysosomes. No cytoplasmic Birbeck granules were found in their cytoplasmic. Original magnification: x8,000.
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Capacity of Isolated DCs to Stimulate Allogeneic T Cells
DCs are unique among antigen-presenting cells in their ability to stimulate naive T cells, as measured by an allogeneic mixed lymphocyte reaction. The ability of the OX62+ DCs isolated from the granulomatous lung to stimulate naive T cells was tested in an allogeneic mixed lymphocyte reaction. The OX62+ DCs strongly induced the proliferation of allogeneic T cells (Figure 8)
, even with as few as 1,000 DCs per well.
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Figure 8. Capacity of purified OX62-positive cells from pulmonary granulomas to stimulate allogeneic T cells in a primary allogeneic mixed lymphocyte reaction. OX62-positive cells were purified from pulmonary granulomas and splenic T cells from DA rats were isolated as described in METHODS. Counts of the purified OX62-positive cells alone and T cells alone were under 500 counts per minute (cpm). The figure shows the mean cpm and SEM of triplicate cultures in one representative experiment out of five.
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Capacity of Isolated DCs to Stimulate PPD-specific T Cells
To determine whether the OX62+ DCs isolated from the granulomatous lung had BCG-derived peptides bound to surface MHC class II molecules, we examined the capacity of the DCs to stimulate syngeneic PPD-specific T cells in the absence of exogenously added antigens. PPD- and HEL-specific T cell blasts we generated were more than 95% OX52-, W3/25-, and R7.3-positive as judged by flow cytometric analysis. The OX62+ DCs elicited the strong proliferative response in PPD-specific T cells in the absence of added PPD, but failed to stimulate HEL-specific T cells (Figure 9) , indicating that the DCs in the granulomatous lung bear BCG-derived peptides associated with their MHC class II molecules.
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Figure 9. Capacity of purified OX62-positive cells from pulmonary granulomas to stimulate PPD- and HEL-specific T cells in the absence of exogenous PPD. OX62-positive cells were purified from pulmonary granulomas and syngeneic PPD- and HEL-specific T cells were generated as described in METHODS. The figure shows the mean cpm and SEM of triplicate cultures in one representative experiment out of more than three. PPD and HEL indicate the counts of PPD-specific T cells + OX62-positive DCs and HEL-specific T cells + OX62-positive DCs, respectively. As a positive control, syngeneic spleen cells (1 x 105 cells/well) irradiated at 2,500 rads were cocultured with PPD- or HEL-specific T cells (5 x 104 cells/well) in the presence of PPD (20 µg/ml) or HEL (20 µg/ml). Counts of syngeneic spleen cells + PPD-specific T cells in the presence of PPD (20 µg/ml) and syngeneic spleen cells + HEL-specific T cells in the presence of HEL (20 µg/ml) were 93,200 ± 7,600 and 76,400 ± 5,300, respectively. Counts of the purified OX62-positive cells alone, PPD- and HEL-specific T cells alone, and syngeneic spleen cells alone were under 1,000 cpm.
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DISCUSSION
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Using a model of synchronized formation of BCG-elicited granuloma, which is a well-defined, delayed type, hypersensitivity T cell–mediated response, we investigated the involvement of DCs in granuloma formation in situ. The isolation of DCs from the pulmonary granulomas enabled us to examine the morphologic, phenotypic, and functional properties of these DCs. Our results clearly demonstrate that these cells have the characteristic phenotype and function of DCs and indicate that DCs play an important role in the T cell–mediated immune reaction occurring in granulomatous inflammation.
To explore the hypothesis that DCs participate in granuloma formation, we first examined the distribution of DCs in the pulmonary granulomas by immunohistochemistry with an anti-rat DC mAb (OX62). Strikingly, numerous large, pleiomorphic OX62+ cells were present at the margins of the granulomas (Figure 2). Although the OX62 mAb was originally reported as a rat-DC–specific marker, it also reacts with a subset of T cells (20, 21). Recently, Brenan and Rees cloned the rat integrin subunit recognized by OX62 mAb (22), and showed that this integrin subunit is expressed on dendritic epidermal T cells in the skin, intraepithelial T cells in the small intestine, as well as DC-lineage cells. Thus, to eliminate the possibility that the large, pleiomorphic OX62+ cell population in the pulmonary granulomas contains T cells and other types of cells, we analyzed them by dual labeling immunohistochemistry of the granulomas, and showed that they had no reactivity for pan-T cell, T cell, or macrophage-specific markers. Next, we isolated these OX62+ cells from the granulomatous lung to further characterize them. The combination of bovine serum albumin density gradient centrifugation and immuno-magnetic cell sorting resulted in the isolation of highly purified OX62+ cells from the rats challenged by BCG. In contrast, only negligible numbers of OX62+ cells were obtained from normal rats. Consistent with this finding, very few OX62+ DCs are found in the peripheral lungs of the normal rats using immunohistochemistry, their location being confined to the connective tissues around vessels and small airways, and in bronchus-associated lymphoid tissues (our unpublished data). Although it is possible that trace numbers of resident OX62+ DCs contaminated the purified population, most of the OX62+ DCs isolated from the granulomatous lung are likely to be DCs associated with the granulomas in situ. These purified OX62+ cells also revealed intense expression of MHC class II and costimulatory molecules, which are known to be strongly expressed by DCs (23, 24). Morphologically, they had dendritic cytoplasmic processes and eccentric, lobulated nuclei which are characteristic of DCs. Functionally, the purified OX62+ cells possessed potent accessory cell capacity in an allogeneic mixed lymphocyte reactions, even when present in small numbers, which is a distinctive feature of DCs (25). Collectively, these phenotypic, morphologic, and functional data indicate that the OX62+ cells infiltrating the pulmonary granulomas are DCs.
To further clarify the functional role of DCs in granuloma formation, we examined the capacity of granuloma-associated DCs to stimulate PPD-specific T cells. We found that DCs isolated from granulomatous lung could effectively induce the proliferation of PPD-specific T cells in the absence of exogenous peptide, but failed to stimulate HEL-specific T cells. This suggests that these DCs bear BCG-derived peptides on their surface. It is of interest that the distribution of DCs within the pulmonary granulomas was exclusively confined to the borders of the granulomas. Previous histologic studies of BCG-induced granuloma models showed that in mature granulomas, a halo of lymphocytes surrounded the centrally packed epithelioid and giant cells (26). Consistent with this observation, we found that the granuloma-associated T cells were present at the borders of the granulomas, where DCs were primarily localized. The interaction between antigen-presenting cells, such as monocytes and macrophages, and T cells has been shown to be essential for initiating and maintaining hypersensitive-type granulomas (14, 15). Considering the close association of the T cells and DCs in the granulomas and the potent capacity of these DCs to proliferate PPD-specific T cells, we suggest that DCs infiltrating the granulomas may directly stimulate T cells in an antigen-specific manner, resulting in the augmentation of granuloma formation.
Previous immunohistochemical studies suggested the possibility that DCs were present in infectious granulomas (27, 28). However, these studies used relatively nonspecific antibodies for DCs, such as B7–2 and S-100, which did not allow definitive identification of DCs, and the workers did not perform functional analyses of these cells. Recently, Yoneyama and coworkers demonstrated the recruitment of F4/80- B220- CD11c+ DC precursors from the circulation into Propionibacterium acnes–induced granulomas in mouse liver. They prepared low-density, DC-enriched cells from the granulomatous liver, and examined their phenotype and capacity to stimulate allogeneic T cells. However, the preparation procedure was lengthy, including overnight culture in the presence of granulocyte/macrophage-colony stimulating factor (GM-CSF), which may have induced phenotypic and functional maturation of harvested DCs. Thus, it is likely that their phenotypic and functional analysis did not actually reflect the characteristics of DCs associated with granuloma in situ. Further, Yoneyama and colleagues provided no data to indicate that granuloma-associated DCs bore P. acnes–derived peptides. In the present study, our isolation procedure of DCs without lengthy culture enabled us to precisely characterize the phenotype and function of granuloma-associated DCs. In addition, we showed that granuloma-associated DCs expressed BCG-derived peptide, suggesting the direct involvement of DCs in BCG-elicited granuloma formation by presenting its derived peptides to infiltrating T cells.
In conclusion, our findings indicate that DCs participate in pulmonary granuloma formation elicited by BCG in rats, possibly through their potent antigen-presenting function. The results of the present study clearly show that DCs play a pivotal role in T cell mediated granulomatous immune responses. These results shed light on a novel role of DCs in T cell–mediated immune responses, and provide important knowledge that contributes to the further understanding of the cellular components elaborating granulomatous inflammation.
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Acknowledgments
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Supported by a grant-in-aid for scientific research (11,670,572 and 13,670,595) (T.S.) from Japan Society for the Promotion of Science and by NIH grant HL36781 (E.E.S.).
Received in original form October 23, 2001;
accepted in final form March 5, 2002
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