INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.2.810–815.2001

Feb. 2001, p. 810–815 Vol. 69, No. 2

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Trehalose 6,69-Dimycolate (Cord Factor) of Mycobacteriumtuberculosis Induces Foreign-Body- and Hypersensitivity-Type

Granulomas in MiceHIROKAZU YAMAGAMI,1,2* TAKAYUKI MATSUMOTO,2 NAGATOSHI FUJIWARA,1

TETSUO ARAKAWA,2 KENJI KANEDA,3 IKUYA YANO,4 AND KAZUO KOBAYASHI1

Departments of Host Defense,1 Gastroenterology,2 and Anatomy,3 Osaka City University Graduate School of Medicine,1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, and Institute of BCG, Kiyose-shi, Tokyo 204-0022,4 Japan

Received 17 July 2000/Returned for modification 14 September 2000/Accepted 8 November 2000

Granulomatous inflammation is characterized morphologically by a compact organized collection of mac-rophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipidcomponents of the Mycobacterium tuberculosis cell wall participate in the pathogenesis of infection. Strains ofM. tuberculosis have cord factor (trehalose 6,6*-dimycolate [TDM]) on their surface. To clarify host responsesto TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology,and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challengedwith TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritantand T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions thanunimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregu-latory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies thatboth nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobac-terial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic moleculeagainst the host and plays an important role in the pathogenesis of tuberculosis.

The pathogenesis of tuberculosis is a function of the patho-gen, Mycobacterium tuberculosis, and of the immune responseof the host to the pathogen (5, 14). Tuberculosis is a chronicinfection with M. tuberculosis complex, including M. tuberculo-sis and Mycobacterium bovis, that is characterized morpholog-ically by granulomatous inflammation, a compact organizedcollection of macrophages and their derivatives, such as epi-thelioid and giant cells, at the site of infection (19). The patho-genicity of M. tuberculosis is related to its ability to escapekilling by macrophages and induce delayed-type hypersensitiv-ity (DTH) (5, 14, 19).

Granulomatous inflammation can be broadly classified aseither a hypersensitivity (immunologic, T-cell-dependent) typeor a foreign-body (nonimmunologic, T-cell-independent) type(19, 20). There is much known, but we still have a long way togo to understand the mechanism of M. tuberculosis pathoge-nicity. Mycobacteria are rich in lipids. Lipid components of theM. tuberculosis cell wall participate in pathogenesis. Cord fac-tor (trehalose 6,69-dimycolate [TDM]), a surface glycolipid,causes M. tuberculosis to grow in serpentine cords in vitro.Virulent strains of M. tuberculosis have TDM on their surface(2), and injection of purified TDM into experimental animalsinduces lesions characterized by chronic granulomatous in-flammation (6, 29).

To clarify host responses to mycobacterial TDM, includingimmunogenicity and pathogenicity, we have analyzed the foot-

pad reactions, histopathology, and cytokine profiles of experi-mental granulomatous lesions in immunized and unimmunizedmice challenged with TDM.

MATERIALS AND METHODS

Animals. Specific-pathogen-free female euthymic and athymic nude nu/nuBALB/c mice at 8 weeks of age were purchased from SLC (Shizuoka, Japan).Experiments were conducted according to the standard guidelines for animalexperiments of Osaka City University Graduate School of Medicine.

Preparation of TDM. M. tuberculosis Aoyama B was grown in Sauton mediumfor 5 weeks at 37°C. Glycolipids were serially extracted with chloroform-meth-anol at 4:1, (vol/vol), 3:1, and 2:1. Each mycolate was purified by developing thelipids on a thin-layer plate of silica gel (Analtech, Inc., Newark, Del.) withchloroform-methanol-acetone-acetic acid (90:10:6:1) and subsequently withchloroform-methanol-water (90:10:1). This procedure was repeated until a singlespot was obtained (25). The preparation contained less than 80 ng of protein/100mg of TDM, as determined by a protein assay kit (Bio-Rad, Hercules, Calif.).

Preparation of w/o/w emulsion. To prepare 100 ml of sample, 100 mg ofpurified TDM was dissolved in 3.2 ml of Freund’s incomplete adjuvant (FIA)(Difco Laboratories, Detroit, Mich.) in a Teflon grinder. After addition of 3.2 mlof 0.1 M phosphate-buffered saline (PBS), a water-in-oil emulsion was made.Then, 93.6 ml of saline containing 0.2% Tween 80 was added at the final con-centration (3.2%) of FIA, and a water-in-oil-in-water (w/o/w) emulsion was madeby mixing (28). As controls, w/o/w micelles without TDM were used.

Immunization. Mice were immunized by subcutaneous injections of 100 mg ofmethylated bovine serum albumin (MBSA) (Sigma Chemical Co., St. Louis,Mo.) emulsified with Freund’s complete adjuvant (FCA) into the inguinal region,the front footpad, and the base of the tail. FCA was prepared by adding heat-killed M. tuberculosis Aoyama B in FIA at a concentration of 2 mg/ml (15).

Footpad assays for DTH. Eight days after immunization, hind footpads werechallenged with 20 ml of TDM (1 mg/ml) in the form of w/o/w emulsion, w/o/wmicelles alone, MBSA (1 mg/ml), or egg albumin (1 mg/ml) (grade V; SigmaChemical Co.). Five mice were used for each group. Triplicate measurements offootpad thickness were performed with an engineer’s micrometer (MitsutoyoCo., Kanagawa, Japan) before and 24 h after the challenge (15). The differencebetween the measurements was calculated and expressed as the mean 6 stan-dard deviation (SD) in millimeters.

* Corresponding author. Mailing address: Department of Host De-fense, Osaka City University Graduate School of Medicine, 1-4-3Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Phone: 81-6-6645-3746. Fax: 81-6-6645-3747. E-mail: yamagami@med.osaka-cu.ac.jp.

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Induction of pulmonary granulomas. Ten days after immunization, mice wereinjected intravenously with either 100 mg of TDM in 100 ml of w/o/w emulsion,100 ml of w/o/w micelles alone, or 100 mg of MBSA in 100 ml of PBS. Unimmu-nized mice were similarly challenged. To exclude the possibility of boosting withrepeated TDM exposures, we used two different sets of mice for DTH footpadassays and induction of lung granulomas throughout the study.

Determination of lung index. To determine the activity of granulomatousinflammation, lung indices were used. Previous reports have indicated that aconsiderable proportion of the increase in lung weight as a consequence ofgranulomatous inflammation is due to an increase in cellularity in the organ (15,30). The index was calculated as follows (6): lung index 5 lung weight (grams) 3100/body weight (grams).

Histology. Lungs were fixed with 10% formalin for 5 days, dehydrated, andembedded in paraffin (15). Sections were stained with hematoxylin and eosin.For immunohistochemical analysis (13), lungs were fixed in a periodate–ly-sine–3% paraformaldehyde solution overnight at 4°C and then frozen in liquidnitrogen. Sections were made using a CM3000 cryostat (Leica InstrumentsGmbH, Nussloch, Germany) and immediately air dried. To block endogenousperoxidase activity, the sections were incubated with 0.3% hydrogen peroxide inmethanol for 20 min at room temperature. After washing with PBS, the sectionswere incubated with normal rat serum for 20 min at room temperature. Subse-quently, sections were incubated with diluted rat anti-mouse CD4 monoclonalantibody (1:300) (RM4-5; Pharmingen, San Diego, Calif.) overnight at 4°C. Afterbeing washed with PBS, they were treated with diluted biotinylated anti-ratimmunoglobulin G antibody (1:400) (Dako, Copenhagen, Denmark) for 60 minat room temperature, followed by incubation with avidin-biotin-peroxidase com-plex (Vectastain kit; Vector Laboratories, Burlingame, Calif.). Reaction prod-ucts were visualized after incubation with 0.025% diaminobenzidine and 0.003%hydrogen peroxide.

Enumeration of infiltrating cells. The number of CD41 cells in immuno-stained sections of the lung was counted 3 days after the challenge with TDM orw/o/w micelles in unimmunized and immunized mice. Three mice were used foreach group, and 10 microscopic fields at a magnification of 3200 were randomlyselected. The average number per microscopic field (0.34 mm2) was then calculated.

Protein expression of chemokines and cytokines in the lung. Aqueous extractsof granulomatous lungs were prepared by a method described previously (15,30). Briefly, lungs were inflated with 1 ml of PBS and homogenized in 1 ml ofsaline by using a Polytron (Brinkmann Instruments, Westbury, N.Y.) for 30 s.Homogenized tissues were then centrifuged in a refrigerated unit at 5,000 3 g for30 min, and the tissue pellet was discarded. Protein concentrations were deter-mined with a protein assay kit (Bio-Rad). Aqueous lung extracts contained 2 to4 mg of protein per ml of PBS (15, 30). The contents of cytokines and chemo-kines were measured by commercially available enzyme immunoassay (EIA) kitsfor murine interleukin-1b (IL-1b), IL-4, IL-12, tumor necrosis factor alpha(TNF-a), gamma interferon (IFN-g), macrophage inflammatory protein-1a(MIP-1a), and monocyte chemotactic protein-1 (MCP-1) (Genzyme, Minneap-olis, Minn.) and expressed as the amount per milligram of protein in the extract.The sensitivity was ,3.0 pg/ml for IL-1b, ,2.0 pg/ml for IL-4, ,2.5 pg/ml forIL-12, ,5.1 pg/ml for TNF-a, ,2.0 pg/ml for IFN-g, ,1.5 pg/ml for MIP-1a, and,2.0 pg/ml for MCP-1, according to the manufacturer’s instructions. The EIAwas conducted in duplicate.

Statistical analyses. Data were analyzed with a Power Macintosh G3 usingStatView 5.0 (SAS Institute Inc., Cary, N.C.) and expressed as the mean 6 SD.Data that appeared to be statistically significant were compared by an analysis ofvariance for comparing the means of multiple groups and were consideredsignificant if P values were less than 0.05.

RESULTSInduction of footpad DTH responses by TDM. We found

delayed-type footpad responses to specific antigens (TDM andMBSA) in immunized euthymic mice (Table 1). Althoughfootpad responses induced by TDM, but not by MBSA, wereseen in unimmunized euthymic mice and in athymic nude miceregardless of immunization, these responses were significantlylower than those in immunized euthymic mice. Regardless ofimmunization, w/o/w vehicles alone induced mild swelling offootpads. An irrelevant antigen, egg albumin, could not elicitthe response. TDM could elicit both antigen-specific and non-specific responses in euthymic mice, although athymic miceshowed only nonspecific inflammatory responses.

Granulomatous inflammation of the lung. It has been dem-onstrated that intravenously injected TDM micelles aretrapped in alveolar vessels and induce granulomatous inflam-mation of the lung (6, 8). A significant increase of lung indiceswas found in immunized euthymic mice 3 to 7 days after thechallenge with TDM compared with unimmunized mice (Fig.1). Regardless of immunization, athymic nude mice showed amoderate increase in the index. The kinetics and intensity weresimilar to those of unimmunized mice. The level showed amarked increase within 3 days, reached the maximum by day 7,and gradually declined thereafter. No significant increase wasfound in euthymic and athymic mice challenged with w/o/walone or MBSA regardless of immunization.

Histopathologic features of the lung. In unimmunized eu-thymic mice challenged with TDM, there was mild, diffuse andinflammatory cell infiltration in alveolar and perivascular areasat early stages (1 to 3 days) (Fig. 2). The infiltrate was com-posed primarily of macrophages, lymphocytes, and scattered

FIG. 1. Lung indices of unimmunized and immunized mice. Theresults are expressed as the mean 6 SD obtained from three to sixmice for each condition. The asterisks indicate a P value of ,0.01,compared to unimmunized mice.

TABLE 1. Footpad responses to antigens in euthymic and athymicmicea

Antigen

Difference in footpad thicknessb

Euthymic mice Athymic mice

Unimmunized Immunized Unimmunized Immunized

TDM 1.28 6 0.32c 1.88 6 0.46c,d 1.10 6 0.23c 1.07 6 0.15c

w/o/w 0.46 6 0.15 0.44 6 0.07 0.29 6 0.06 0.36 6 0.06MBSA 0.05 6 0.02 0.43 6 0.03d 0.08 6 0.10 0.16 6 0.10Egg albumin 0.19 6 0.11 0.07 6 0.07 0.11 6 0.09 0.09 6 0.05

a BALB/c mice were immunized with MBSA emulsified in Freund’s completeadjuvant. Eight days after immunization, mice were challenged with each anti-gen. The thickness of footpads was measured immediately before and 24 h afterantigenic challenge. Differences were calculated.

b Data represent the mean (millimeters) 6 SD from three separate experi-ments for five mice each condition.

c Statistically significant (P , 0.01) compared to mice challenged with w/o/w.d Statistically significant (P , 0.01) compared to athymic mice regardless of

immunization and to unimmunized euthymic mice.

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neutrophils. At 1 and 2 weeks after the challenge, such miceshowed randomly distributed, organized granulomas com-posed of macrophages and lymphocytes. The lesions subsidedthereafter. Unimmunized mice challenged with w/o/w micellesexhibited no significant lesions. By contrast, accelerated andaugmented lesions, including cell infiltration and granulomaformation in the alveolar and perivascular regions, were foundin immunized euthymic mice challenged with TDM but not inthose challenged with w/o/w micelles. The lesions in immu-nized mice were composed of macrophages, lymphocytes, anda few neutrophils. Athymic nude mice challenged with TDM,regardless of prior immunization, developed lesions that weresimilar to those in unimmunized mice.

Immunohistochemical studies demonstrated that CD41

cells (Th cells) were abundant in granulomas and perivascularcell infiltration of unimmunized and immunized mice 3 daysafter TDM challenge, although the number of CD41 cells wassignificantly increased in immunized mice compared to unim-munized mice (Fig. 3). Regardless of preimmunization, thenumber did not differ in mice challenged with w/o/w alone.

Protein expression of chemokines and cytokines. We nextstudied the profile of cytokines and chemokines in the lung(Fig. 4), because a prominent accumulation of mononuclearcells, such as granulomatous inflammation, was seen. A signif-icant amount of CC chemokines, including MCP-1 and MIP-1a, was detected in lung extracts from immunized mice chal-lenged with TDM compared to unimmunized mice. Inimmunized and unimmunized mice, the peak activity waspresent 1 to 3 days after the challenge and declined thereafter.Lung extracts from mice challenged with w/o/w micelles alonecontained a smaller amount of CC chemokines regardless ofpreimmunization. Proinflammatory cytokines such as IL-1band TNF-a were prominent in immunized mice challengedwith TDM. In contrast to the case for immunized mice, thelevel of proinflammatory cytokines in the extracts from unim-munized mice challenged with w/o/w micelles alone was notonly considerably less but also was maintained for a shortertime. A considerable amount of Th1- and IFN-g-inducing im-munoregulatory cytokines, IL-12 and IFN-g, was detected inthe extracts prepared from immunized mice challenged withTDM, but there was much less from immunized mice chal-lenged with w/o/w micelles alone and unimmunized mice chal-lenged with TDM. The peak was reached by day 3, and thelevel declined thereafter. Regardless of prior immunization,IL-4 was not found in the extracts from mice challenged withTDM or w/o/w micelles alone (data not shown). To examinethe effects of the procedure on enzymatic degradation andhalf-lives of cytokines, we measured cytokine levels in theextracts prepared from either immunized or unimmunizedmice in the presence of standard recombinant cytokines, in-cluding chemokines. The results showed that the levels of all

cytokines tested in our study were not affected by proceduresof immunization and extract preparation (data not shown).

DISCUSSION

In the present study, we have demonstrated for the first timethat TDM derived from virulent M. tuberculosis can induceboth foreign-body-type (nonimmune) and hypersensitivity-type(immune) granulomas by acting as a nonspecific irritant and aT-cell-dependent antigen. This result implies that both nonim-mune and immune mechanisms participate in granulomatousinflammation induced by mycobacterial infection.

Granulomatous inflammation is manifested in chronic in-flammatory diseases that often result in tissue destruction andend-stage fibrosis. The common histologic feature of granulo-matous inflammation, i.e., infiltrating mononuclear leukocytesand their derivatives (epithelioid cells and multinucleated gi-ant cells), is observed in a variety of granulomatous diseasescaused by infectious agents (tuberculosis, leprosy, schistosomi-asis, leishmaniasis, and histoplasmosis), non-infectious agents(silicosis and berylliosis), or unknown agents (sarcoidosis,Crohn’s disease, and Wegener’s granulomatosis) (20). The le-sion is usually surrounded by a collar of lymphocytes andoccasionally eosinophils. Granulomatous inflammation can bebroadly classified as either a hypersensitivity (immunologic,T-cell-dependent) type or a foreign-body (nonimmunologic,T-cell-independent) type (19, 20). The classification is basedon the participation of antigen-specific T lymphocytes in thelesions. The granulomas induced by M. bovis bacillus Calmette-Guerin (BCG) in mice were developed regardless of preim-munization with M. tuberculosis or BCG, although augmentedlesions were observed in preimmunized mice (15, 30).

Disease progression to active tuberculosis is dependent onan interplay between bacterial and host factors. Mycobacteriaare rich in lipids. These include mycolic acids, which are long-chain fatty acids (C78 to C90) (2). The pathogenicity of M.tuberculosis is related to its ability to induce cell-mediatedimmunity and DTH. (5). Strains of M. tuberculosis have cordfactor (TDM), a surface glycolipid. A variety of foreign lipidsand glycolipids, including several found in the cell walls andcell membranes of pathogenic mycobacteria, are recognized byCD1-restricted T cells in humans, and the CD1 system pro-vides a novel mechanism for host responses to mycobacterialinfection, such as the development of cell-mediated immunity(21, 27). To confirm the contribution of T cells in TDM-induced hypersensitivity, it is necessary to demonstrate TDM-specific T cells, although in the present study this was not done.This approach will also explore the possibility that the putativeT-cell contribution might arise from contamination withminute amounts of protein in the TDM preparation. The im-mune response to microbial pathogens relies on both innateand adaptive components. The role of CD1, CD14, and Toll-

FIG. 2. Histopathologic features of the lung. In unimmunized euthymic mice challenged with TDM, mild, diffuse and inflammatory cellinfiltration in alveolar and perivascular areas at early stages (1 to 3 days). The infiltrate was composed primarily of macrophages, lymphocytes, andscattered neutrophils. One week after the challenge, such mice showed organized granulomas (arrowheads) composed of macrophages andlymphocytes. The lesions subsided thereafter. Unimmunized mice challenged with w/o/w micelles exhibited no significant lesions. By contrast,accelerated and augmented granulomatous lesions were found in immunized euthymic mice challenged with TDM but not in those challenged withw/o/w micelles. The lesions in immunized mice were composed of macrophages, lymphocytes, and a few neutrophils. In athymic nude mice, slightgranuloma formation was seen at day 7 regardless of preimmunization. Hematoxylin and eosin staining was used. Bar, 200 mm.

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like receptors (1, 26) in host responses to TDM is intriguing,although this is not addressed in our present study. Futurestudies are needed to clarify the issue.

In the present study we have demonstrated that TDM caninduce hypersensitivity granulomas in euthymic mice immu-nized with FCA and also can elicit foreign-body lesions inunimmunized euthymic and athymic nude mice. This conten-tion is supported by the result that footpad DTH responses toTDM were augmented in immunized mice compared to unim-munized euthymic and athymic nude mice. In addition, TDMitself acts as a nonspecific inflammatory stimulus, because sim-ilar and moderate footpad swelling was observed in euthymicand athymic mice. The precise mechanism of the delayed gran-ulomatous response in athymic nude mice remains unknown,however, mature T lymphocytes may be involved in the re-sponse, because athymic mice lack them. Collectively, our re-sults imply that mechanisms of granulomatous inflammation intuberculosis are composed of both foreign-body and hypersen-sitivity types.

Granuloma formation is the expression of a series of com-plex inflammatory events. Evidence suggests that proinflam-matory cytokines play important roles in the initiation andmaintenance of granuloma formation (9, 10, 31). Histopatho-logically, the bulk of both hypersensitive and foreign-bodygranulomas are composed of macrophages and their deriva-tives. In most tissues the presence of inflammatory macro-phages results from the recruitment of peripheral blood mono-cytes. Besides granulomas, our studies showed prominentperivascular infiltration of mononuclear cells around the le-sion. This may be attributed to recruitment of blood and tissuemononuclear cells through local expression of CC chemokinesfor monocytes and lymphocytes, such as MCP-1 and MIP-1a(4, 7). MIP-1a is known to be an efficient chemoattractant forTh1 cells, although MCP-1 exerts chemotactic activity for bothTh1 and Th2 cells (32). This may lead to expression of cell-mediated immunity in immunized mice challenged with TDM,because the expression of MIP-1a (days 1 to 3) persistedlonger than that of MCP-1 (day 1) in the lesion.

The very early expression of CC chemokines was detectedwithin 1 to 3 days after the challenge with TDM. Human bloodmonocytes preferentially produce CC chemokines in response

to M. tuberculosis (11, 12). Both MCP-1 and MIP-1a may bepivotal in the initial recruitment of monocytes to sites of sub-sequent granuloma formation. It has been demonstrated thatproinflammatory cytokines such as IL-1 and TNF-a participatein granulomatous inflammation (9, 10, 19, 20, 31), althoughthey lack direct chemotactic activity for monocytes (22). How-ever, chemokines are inducible by stimulating macrophages/monocytes with IL-1 and TNF-a (23). The cytokine networkmay form a powerful amplification circuit of granulomatousinflammation.

IL-12, a cytokine produced mainly by macrophages in re-sponse to mycobacteria, augments cytotoxicity and cytokineproduction by T cells and NK cells and initiates developmentof CD41 Th1 cells (33). CD41 Th1 and NK cells stimulatedwith IL-12 produce and secrete IFN-g, which activates macro-phages to inhibit or kill intracellular mycobacteria via reactivenitrogen intermediates (3). Thus, macrophages accumulate atthe site of microbial growth and become activated through theCD41 Th1 cell-cytokine-macrophage axis (14). IL-12 inducesthe differentiation of Th1 cells from uncommitted T cells and,consequently, initiates cell-mediated immunity, which plays aprotective role in infections with mycobacteria. This cytokinerepresents an important regulatory bridge between innate re-sistance and adaptive immunity.

TDM can stimulate production of IL-12 from mouse mac-rophages (24), and IL-12 is found in the active lesion elicitedby experimental mycobacterial infection in mice, including that

FIG. 3. Immunocytochemical analyses of CD4-positive cells in thelesion. The number of CD41 cells per unit square (0.34 mm2) in thelungs of unimmunized and immunized mice was calculated 3 days afterthe challenge with either TDM or w/o/w micelles alone. Ten micro-scopic fields were examined at a magnification of 3200. Data representthe mean 6 SD from three mice. The asterisk indicates a P value of,0.05 compared to unimmunized mice.

FIG. 4. Protein expression of chemokines and cytokines in thelung. Results represent the mean 6 SD from three (unimmunized) orsix (immunized) mice challenged with TDM, w/o/w micelles, or MBSA.The asterisks indicate a P value of ,0.05 compared to unimmunizedmice. The EIA was conducted in duplicate.

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with Mycobacterium avium (17, 18) and Mycobacterium leprae(16). In immunized mice challenged with TDM, lesional IL-12and IFN-g reached peak levels concomitantly 3 days afterchallenge, whereas unimmunized mice express less of them.The temporal profiles of IL-12 and IFN-g may indicate theirclose functional relationship. Our data that mice bearing gran-ulomas showed significant expression of IL-12 and IFN-g butnot IL-4 suggest that TDM challenge may favor dominance ofthe Th1 response via through the local cytokine profile. In-deed, such mice did not produce anti-TDM antibodies thatmight reflect a Th2 response (data not shown).

Infection with mycobacteria results in either host defense ordisease expression such as granulomatous inflammation (5, 14,19). Our present study suggests that TDM, a surface glycolipidderived from the cell walls of virulent strains of M. tuberculosis,plays a critical role in the process. Taken together with theprevious reports that mycobacterial TDM can induce apoptosis(6) and angiogenesis (29), this study indicates that TDM is apleiotropic molecule against the host and participates in thepathogenesis of tuberculosis.

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Health andWelfare (Research on Emerging and Re-emerging Infectious Dis-eases, Health Sciences Research Grants) and the U.S.-Japan Cooper-ative Medical Science Program against Tuberculosis and Leprosy.

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