The Immunopathogenesis of Granulomatous Inflammation Induced byMycobacterium tuberculosis

METHODS: A Companion to Methods in Enzymology 9, 204–214 (1996)

Article No. 0027

The Immunopathogenesis of GranulomatousInflammation Induced byMycobacterium tuberculosis1

Kazuo Kobayashi* and Takeshi Yoshida†*First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai,Shinagawa-ku, Tokyo 142, Japan; and †Tokyo Institute for Immunopharmacology,3-41-8 Takada, Toshima-ku, Tokyo 171, Japan

Tuberculosis kills more adults each year than any ment and immunotherapy. From this point of view,we will review methodologic and immunopathogeneticother infectious disease. Mycobacterium tuberculosis

infects about one-third of the world’s population. Over aspects of animal models of granulomatous inflamma-tion induced by infection with tubercle bacilli.8 million new cases and nearly 3 million deaths occur

each year (1). Tuberculosis is a chronic infection withtubercle bacilli that is characterized morphologicallyby granulomatous inflammation, a compact organized GRANULOMA-INDUCING ACTIVITYcollection of macrophages, and their derivatives such OF M. tuberculosisas epithelioid and giant cells at the site of infection.We still do not understand the exact mechanism of

Although the M. tuberculosis complex encompassesgranulomatous inflammation in tuberculosis. Granulo-M. tuberculosis, M. bovis, including bacillus Calmette-matous inflammation in general is manifested inGuerin (BCG), M. africanum, and M. microti, two spe-chronic inflammatory diseases that often result in tis-cies of mycobacteria cause tuberculosis in humans, M.sue destruction and end-stage fibrosis. The commontuberculosis and M. bovis, which have often been em-histologic feature of granulomatous inflammation, in-ployed in experimental animal models of tuberculosis.filtrating mononuclear leukocytes and their derivativesTubercle bacilli are grown in aerated liquid media(epithelioid cells and multinucleated giant cells), is ob-(Sauton, Dubos, Proskauer and Beck, and Middlebrookserved in a variety of granulomatous diseases caused7H9 broth) containing bovine serum albumin and de-by infectious agents (tuberculosis, leprosy, schistosomi-tergent, Tween 80. Single bacterial suspensions areasis, leishmaniasis, histoplasmosis), noninfectiousmade by vigorously shaking 7-day-old cultures, centri-agents (silicosis, berylliosis), or unknown agents (sar-fuging at 200g to settle clumps, passing the supernatescoidosis, Crohn disease, Wegener granulomatosis). Thethrough a 1.2-mm membrane filter, and resuspendinglesion is usually surrounded by a collar of lymphocytesthe filtrates in normal saline. This procedure providesand occasionally eosinophils. Granulomatous inflam-more than 95% single or double bacterial cells. Heat-mations can be broadly classified as a hypersensitivitykilled bacteria are homogenized in a glass tube fitted(immunologic, T cell-dependent) type and a foreignwith a Teflon pestle to break up bacterial rafts. Thebody (nonimmunologic, T cell-independent) type (2).final preparations are vortex agitated for 3 min andThe classification is based on the degree of antigen-sonicated for 10–30 s to disrupt clumps before use.specific T-lymphocyte involvement in the lesions. Ex-To induce granulomatous inflammation, doses of 100–8

perimental animal models have been used to investi-CFU live or 101–4 mg dead tubercle bacilli are used togate a series of fundamental questions about the patho-inoculate animals. Strains of M. bovis are more patho-genesis of tuberculosis. Knowledge of the pathogenesisgenic for experimental animals than other species ofwould allow us to determine the appropriate strategiesmycobacteria. The virulence of tubercle bacilli plays afor tuberculosis control such as new vaccine develop-key role in the pathogenesis. It is a complex phenotypedependent not only on the genetic and immunologic1 This work was supported by grants from the Ministry of Educa- makeup of the host. M. tuberculosis has no known exo-tion, Science and Culture of Japan, the Showa Medical Foundation,toxins, endotoxins, or histolytic enzymes. Instead, itsTokyo, Japan, and the Mochida Memorial Foundation for Medical

and Pharmaceutical Research, Tokyo, Japan. pathogenicity is related to its ability to escape killing

204 1046-2023/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

/ 6706x$252s 04-19-96 05:36:01 metha AP: Methods

205TUBERCULOUS GRANULOMAS

by macrophages and induce cell-mediated immunity bacilli. There is considerable variation among animalsin the number of bacilli within lesions, degree of im-(3). Generally, granuloma-inducing agents persist in

the tissue because they are insoluble or poorly degrad- mune responses, and amounts of caseation, fibrosis,cavity formation, and calcification. Rabbits (3) andable. The ability of M. tuberculosis cell wall components

to elicit granulomatous inflammation in the absence of guinea pigs (9) show cavitary lesions and caseous ne-crosis with disseminated disease similar to human dis-host immune responses has been reported. Virulent

strains of M. tuberculosis have cord factor, a surface ease, whereas mice produce delayed-type hypersensi-tivity (DTH) and granulomas but a lesser degree ofglycolipid, whereas avirulent strains do not. The injec-

tion of purified cord factor into animals induces granu- other pathology (10). On the other hand, the disadvan-tage of experimental tuberculosis using rabbits andlomas (4). Its granulomatogenicity is usually correlated

with the amount of the glycolipid trehalose 6,6*-dimy- guinea pigs is the lack of recombinant cytokines, mono-clonal antibodies, and inbred strains of animals,colate (TDM) in the organism (5). Wax D represents a

heterogeneous group of peptidoglycolipids composed of whereas many immunological reagents and inbredstrains are available for the mouse models. There arethe cell wall. They have unique adjuvant activity in

that they not only enhance antibody production but multiple routes (aerogenic including aerosols and in-tratracheal, intravenous, subcutaneous, intraperito-also induce cell-mediated immune responses. The

smallest common denominator of adjuvant activity of neal, or intramuscular) of inoculation with tubercle ba-cilli, but the most commonly used are the intravenouswax D is muramyl dipeptide (MDP), N-acetyl-mura-

myl-L-alanyl-D-isoglutamine (6). Both TDM and MDP and aerogenic routes. Oral infection is far less efficientin transmission of tuberculosis, because mycobacterialcan induce foreign body-type granulomatous lesions

without participation of T lymphocytes in euthymic and viability is reduced by gastric acidity. The challengeby the aerogenic route using an aerosol chamber orathymic nude animals (7).

Purified protein derivative (PPD) of M. tuberculosis intratracheal instillation mimics human tuberculosis,because M. tuberculosis is transmitted by inhalation ofcan induce hypersensitivity-type granulomatous in-

flammation by injecting PPD-coupled indigestible mi- droplets coughed or sneezed into the air by patientsand the disease is most commonly seen in lungs. Re-croparticles into animals (8). Strains of M. bovis are

less aerotolerant than M. tuberculosis, but they are gardless of preimmunization, animals develop granulo-matous lesions 2–3 weeks after the challenge andmore pathogenic for experimental animals. In human

the port of entry for M. bovis is usually the gastrointes- reached peak intensity 4–6 weeks later, and the lesionsdecline gradually thereafter, although accelerated andtinal tract. Extrapulmonary lesions are prevalent, es-

pecially in the cervical and mesenteric lymph nodes, stronger responses are usually observed in preimmu-nized animals (11). These are summarized in Table 1.bones, and joints. When inhaled, M. bovis can also

cause pulmonary tuberculosis indistinguishable fromthat caused by M. tuberculosis. Rabbits

Several rabbit closed colonies showing difference inresistance or susceptibility to tuberculosis have been

ANIMALS established (3, 12). When infected with the virulentstrain of M. bovis (BCG), the resistant rabbit familiesdeveloped cavitary tuberculosis resembling that foundDifferent animal species vary in their susceptibilities

to infection by the different types of virulent tubercle in immunocompetent adult humans. The susceptible

TABLE 1

Experimental Infection with Tubercle Bacilli in Animal Models

Rabbits Guinea pigs Mice

Susceptibility to bacilli Bovine ú human Bovine Å human Bovine ú humanInoculation doses (CFU) 103–7 100–7 104–8

Bacilli in lesions // / ///Granuloma formation /// /// //(weeks after infection) (§2–3) (§2–3) (§2–3)Epithelioid and giant cells /// /// /Caseous necrosis /// /// {DTH // /// //

Note. Summarized from the literature. Susceptible strains of animals are used. {, usually absent; //, intermediate; ///, high. CFU,colony forming units; DTH, delayed-type hypersensitivity.


206 KOBAYASHI AND YOSHIDA

ones developed hematogenously spread tuberculosis re- dominant gene on mouse chromosome 1, called Bcg,Lsh, or Ity. The Bcg gene affects the capacity of macro-sembling that found in infants and immunocompro-

mised individuals. Genetic analysis of these rabbits has phages to destroy ingested intracellular parasites inearly phases of infection. In inbred mouse strains, in-not been reported (3). Most of the commercially avail-

able rabbits are of intermediate resistance to tuberculo- fection with mycobacteria is biphasic, with an earlynonimmune phase (0–2 weeks) characterized by rapidsis. Therefore, many will develop cavities when infected

with virulent tubercle bacilli, but some will die early proliferation of the bacteria in reticuloendothelial or-gans of susceptible strains and absence of bacterialfrom hematogenous spread of disease. Since the rabbits

are not inbred, considerable variation in resistance to growth in resistant strains. The late phase (3–6 weeks)is associated with specific immune response, leadingtubercle bacilli is to be expected. Human strains of tu-

bercle bacilli (M. tuberculosis) are less virulent for rab- either to complete clearance of bacterial load or to per-sistent infection in the reticuloendothelial organs of thebits than the bovine strains (M. bovis).susceptible strains. While the late phase of infection is

Guinea Pigs under the control of genes linked to the major histocom-Studies of the comparative biology of the guinea pig patibility complex (16), the difference in innate suscep-

have revealed a number of remarkable similarities be- tibility detected in the early phase is controlled by thetween this species and humans. Many of those funda- expression of the Bcg gene, which is present in twomental similarities bear directly or indirectly on the allelic forms in inbred strains, Bcgr (resistant, autoso-relevance of the guinea pig as a model of human tuber- mal dominant) and Bcgs (susceptible, recessive). Theculosis. In guinea pigs, the susceptibility to human Bcg locus is identical to two other genes, Ity and Lsh,strains of tubercle bacilli is almost equivalent to bovine which control natural resistance to infection with Sal-strains. Guinea pigs can be infected with small num- monella typhimurium and Leishmania donovani (17).bers (1–2 CFU) of viable M. tuberculosis (9), and they The Bcg gene is phenotypically expressed as an intrin-produce cavitary lesions and caseous necrosis similar sic function of macrophages. The cloning of the cDNAto human disease (13). Much of the work with this for the Bcg gene, designated Nramp (natural resis-model has employed outbred guinea pigs, although in- tance-associated macrophage protein), has beenbred guinea pigs could have significant advantages to achieved (18). Structural homology of Nramp proteinanalyze the genetic factors involved in the pathogene- to a eukaryotic nitrate transporter has been demon-sis. However, direct comparison of the response of out- strated (10). The Bcg gene regulates the developmentbred Hartley, inbred Strain 2, and inbred Strain 13 of granulomas induced by BCG (19, 20). The biologicguinea pigs to low-dose pulmonary infection reveals no features of the Bcg gene are summarized in Table 2.significant differences among them in bacillary loads In addition, granuloma formation in tuberculosis canin lung or spleen 4 to 6 weeks postinfection. Prior vacci- be studied in immunodeficient mice including aged,nation with BCG protected all three strains equally athymic (T cell-deficient), SCID (T and B cell-deficient),well. Parameters of T-lymphocyte reactivity to myco- beige (NK cell-deficient), and gene-disrupted micebacterial antigens in vivo and in vitro are essentially (CD4, CD8, major histocompatibility complex, inter-the same in all three strains (14). feron-g, and interferon-g receptor). Furthermore, ex-

MiceTABLE 2Mice are generally resistant to low-dose inocula of

M. tuberculosis. The mouse model, however, is useful The Bcg Genefor genetic analysis of host susceptibility to the infec-

1. A single dominant gene located on chromosome 1 of the murinetion and also for immunological analysis with a largegenomenumber of currently available immunological reagents.

2. Two allelic forms and phenotypic expression in inbred mouseThese include monoclonal antibodies to cytokines and strainsthe surface phenotypes of cell populations and recombi- Bcgr (resistant, dominant): AKR, A/J, CBA/J, C3H/HeJ,

DBA/2nant murine cytokines. In addition, generation of mu-Bcgs (susceptible, recessive): BALB/c, C57BL/6, C57BL/10,tant mice through targeted gene disruption by homolo-

DBA/1gous gene recombination has markedly improved our3. Controls granulomas and early resistance to infection with

understanding of the immunopathogenetic mecha- intracellular pathogens including Mycobacteriumnisms. The ability of a host to resist infection with a tuberculosis, Mycobacterium bovis, Mycobacterium avium,

Mycobacterium lepraemurium, Salmonella typhimurium,wide range of microbial pathogens is strongly influ-Leishmania donovanienced by genetic factors (15). The genetic regulation of

4. Regulates host tissue responses to the pathogensexperimental mycobacterial infection in mouse models Bcgr (resistant, dominant): low producers of granulomasis apparent. Thus, natural resistance of infection with Bcgs (susceptible, recessive): high producers of granulomasintracellular pathogens is controlled by an autosomal



periments using monoclonal antibodies to deplete spe- About 75% stained positively for nonspecific esterase.Some lymphocytes (5–10%), neutrophils, mast cells,cific targets (cells, cytokines, and cytokine receptors)

can delineate their role in the pathogenesis of disease and fibroblasts are also found in the enzymatically dis-persed granuloma cells (29, 31).as well as granulomatous inflammation.

Other Animal Models for Experimental Tuberculosis Aqueous Extracts of Infected TissuesExperimental tuberculosis using rats, hamsters, and Aqueous extracts of the granuloma-bearing organs

monkeys has been reported (21, 22). The information are prepared to measure the biologically active sub-obtained from these studies is rather sketchy and their stances in the infected site (8, 19). The solid organsbiological significance remains uncertain until further such as livers and spleens are placed in a Waringstudies of those species. blender containing culture medium or phosphate-buf-

fered saline and ground for 30 s at a low speed. Lungsshould be filled with fluid before being placed in theblender. Tissue was centrifuged at 200g for 5 min andASSESSMENT OF GRANULOMATOUSresuspended in medium. The granuloma suspension isLESIONS then homogenized using a polytron (Brinkmann In-struments, Westbury, NY) for 30 s. During these proce-

The quantitative assay methods of the intensity of dures, tissues are kept on ice. The homogenized tissuesgranulomatous inflammation have been described pre- are then centrifuged at 2000g for 30 min. Samples areviously. Briefly, the infected tissues are fixed in 10% sterilized by filtration through a millipore membrane.phosphate-buffered formalin and 4-mm paraffin sec-

Isolation of Total Cellular RNAtions are stained with hematoxylin and eosin. Acid-fast bacilli are stained by the Ziehl–Neelsen technique. To analyze gene expression, total cellular RNA fromTypical means to evaluate granulomatous responses granuloma cells or tissues is isolated (32). Either gran-have included morphometric area/volume measure- uloma cells or tissue homogenates are lysed by a solu-ments from histopathology by measuring the diameter tion containing 25 mM Tris, pH 8.0, 4.2 M guanidineor radius of lesions (8, 23, 24), arbitrary scoring of isothiocyanate, 0.5% N-lauroylsarcosyl, and 0.1 M 2-changes in histopathology (25, 26), wet-weight organ mercaptoethanol. After homogenization, the above sus-measurements (20, 27), measuring the content of mac- pension is added to a solution containing an equal vol-rophage-derived lysosomal enzymes, N-acetyl-b-D-glu- ume of 100 mM Tris, pH 8.0, 10 mM EDTA, and 1.0%cosaminidase, and lysozyme in the extract of infected sodium dodecyl sulfate. The mixture is then extractedorgans (28, 29), and enumerating the number of cells with chloroform–phenol and chloroform–isoamyl alco-obtained from bronchopulmonary lavage fluid (30). hol. The RNA is alcohol-precipitated and the pellet is

dissolved in diethyl pyrocarbonate-treated H2O. Iso-lated RNA is used to measure gene expression of cellsand tissues using reverse transcriptase–polymerasePREPARATION OF GRANULOMA CELLS,chain reaction and Northern blot analysis.AQUEOUS EXTRACTS, AND RNA

Isolation and Dispersion of Granuloma CellsEFFECTORS IN GRANULOMATOUSTo obtain dispersed granuloma cells, lesions are ho-INFLAMMATIONmogenized for 20 s at a low speed in a Waring blender.

Granulomas are collected over a No. 50 steel mesh.Granulomas are pelleted in 50-ml plastic tubes (Falcon) The development of cell-mediated immunity, rather

than humoral immunity, plays a key role in the out-and resuspended in 3 vol of digestion medium con-sisting of phosphate-buffered saline containing 10 mM comes of infection with M. tuberculosis. Persons with

defective cell-mediated immunity, such as those withHepes, 1000 U/ml collagenase from Clostridium histo-lyticum, and 20 mg/ml DNase I. The mixture is vigor- human immunodeficiency virus (HIV) infection and

chronic renal failure, are at markedly increased riskously agitated for 30 min at 377C. The resulting suspen-sion is strained through a No. 100 steel mesh. After for tuberculosis. In contrast, persons with defective hu-

moral immunity, such as hypogammaglobulinemia andcentrifugation, the dispersed cells are resuspended andstrained through a 50-mm nylon sieve for removal of multiple myeloma, show no increased predisposition to

tuberculosis (34). Experimental evidence indicates thatclumps and debris. This procedure routinely providessingle cell suspensions with 80–90% viability as as- antimycobacterial immune defenses are mediated pri-

marily by T lymphocytes and macrophages, and adop-sessed by trypan blue dye exclusion. By Wright’s stain-ing, over 80% of the cells are typical macrophages. tive transfer of resistance against tuberculosis in ani-



mal models is mediated by T cells (35). There are three (IFN-g), which activates macrophages to inhibit or killintracellular mycobacteria via reactive nitrogen inter-important aspects to be considered in the pathogenesis

of tuberculosis: (i) the virulence of the organism, (ii) the mediates, including NO, NO2, and HNO3 (42). Addi-tionally, activated macrophages produce 1,25-dihy-relationship of hypersensitivity to immunity against

infection, and (iii) the pathogenesis of tissue destruc- droxyvitamin D3, which can inhibit intracellularmycobacterial growth (43, 44). Furthermore, vitamintion and caseous necrosis. Protection of the infected

host is achieved initially by a local tissue reaction with D3 is important in transforming macrophages into epi-thelioid cells and multinucleated giant cells (45). Micro-granulomatous inflammation, which is the orches-

trated expression of mononuclear cells and secreted cy- bial proteins are processed and presented to T cells,which are in turn activated. Thus, macrophages accu-tokines in response to M. tuberculosis. In the process,

cell-mediated immune responses to tubercle bacilli play mulate at the site of microbial growth and become acti-vated through the CD4/ Th1 cell–cytokine–macro-a key role, although the response is a two-edged sword

that may contribute to both clearance of infecting phage axis (3, 46).The bulk of experimental and clinical data favor aagents and tissue damage.

dominant but not exclusive role for CD4/ T cells inCells Involved in the Pathogenesis immune defenses against tuberculosis. Mice depleted

of CD4/ T cells prior to the infection are unable toMycobacteria are endowed with the capacity to sur-vive and replicate inside mononuclear phagocytes such control mycobacterial growth (47, 48). The depletion of

CD4/ T lymphocytes due to HIV infection strikinglyas macrophages. Mononuclear phagocytes are potenteffector cells that are able to engulf and kill bacteria increases the patients’ susceptibility to tuberculosis.

HIV-infected patients with decreased levels of CD4/ T(3). Mycobacteria attach to macrophages through fibro-nectin (36) and vitronectin receptors (37). Mycobacteria cells (£200 cells/ml) develop severe type tuberculosis

(49). In good accord with this observation, MHC classsecrete fibronectin-binding molecules that promotemacrophage phagocytosis by fibronectin receptors (36). II gene knockout mice usually die of the infection (10).

Cytotoxic CD8/ T lymphocytes are also crucial in pro-In addition, complement components activated on thesurface of mycobacteria opsonize the organism and fa- tection against tubercle bacilli because mice with the

disrupted gene for b2-microglobulin (lack of functionalcilitate its uptake via the macrophage complement re-ceptor CR3 without triggering the respiratory burst CD8/ T cells) exhibit severe manifestations of tubercu-

losis (50). Lysis of incapacitated macrophages andnecessary to kill the organisms (38). Phagosomal up-take of bacteria is usually followed by their fusion with other host cells by CD8/ cytotoxic T lymphocytes in-

duces the release and subsequent uptake of mycobact-lysosomes and degradation of the bacteria. However,mycobacteria can inhibit phagolysomal fusion, possibly eria by more efficient phagocytes, thus contributing to

host protection (51). At the same time, the lysis of hostthrough the action of surface sulfatides (4). Althoughopsonization of mycobacteria with serum antibody par- cells promotes microbial dissemination and causes tis-

sue injury such as caseating granulomas with necrotictially reverses the inhibition of fusion, mycobacteriacontinue to replicate within mononuclear phagocytes. center and cavity formation. Direct toxicity of certain

mycobacterial components to macrophages may alsoMacrophages are heterogeneous with respect to theirantimycobacterial capacity, depending on their tissue contribute to the necrosis (3). It is known that BCG

infection can lead to an increase in gd T cells in thelocalization and activation by endogenous and exoge-nous stimuli. Naive macrophages are unable to kill my- lesion (52). The role of gd T cells in tuberculosis re-

mains unknown, although they could certainly producecobacteria, which multiply, lyse host cells, infect othermacrophages, and sometimes disseminate through the IFN-g, because TCRb gene knockout mice could cope

with primary BCG infection (10).blood. Natural killer (NK) cells may be pivotal in anti-mycobacterial defense mechanisms at the early phase

Participation of Cytokinesof infection (39). After a few weeks, however, T cell-mediated immunity develops, which becomes apparent It is well known that monocytes/macrophages regu-

late various cellular functions by virtue of synthesizingfrom a positive skin reaction to PPD and which involvesthe interaction of mycobacteria-activated T cells with unique cytokines that initiate and control inflamma-

tory and immune reactions in both local and distantlymacrophages.It is now recognized that interleukin 12 (IL-12), a located cell populations. Inhaled M. tuberculosis organ-

isms are initially engulfed by alveolar macrophagescytokine produced mainly by macrophages in responseto various stimuli, including M. tuberculosis (40), aug- that can produce a plethora of cytokines, including IL-

1 (19, 20), IL-6 (53), IL-8 (54), IL-10 (55), IL-12 (40),ments cytotoxicity and cytokine production by T cellsand NK cells and initiates development of CD4/ type migration inhibition factor (MIF) (13, 19), TNF-a (56),

monocyte chemoattractant protein-1 (MCP-1) (54),1 T-helper (Th1) cells (41). CD4/ Th1 and NK cellsstimulated with IL-12 produce/secrete interferon-g macrophage inflammatory protein-1a (MIP-1a) (57),



and granulocyte-macrophage colony-stimulating factor immune responses. Th2 cells produce IL-4, IL-5, IL-6,and IL-10, support B-cell growth and differentiation,(GM-CSF) (58). Functionally, these cytokines have

been classified largely into two groups: the chemoki- and augment humoral immune responses. In thecourse of an immune response to a specific pathogen,netic cytokine group, which includes chemokines (IL-

8, MCP-1, and MIP-1a) and MIF, and the immunoregu- Th1 and Th2 cells exert cross-regulation that favorspredominance of one subpopulation (63). In addition tolatory cytokine group, which includes IL-1, IL-6, IL-10,

IL-12, TNF-a, and GM-CSF. IL-8, MCP-1, and MIP- macrophage-derived TNF-a, IFN-g produced by CD4/

Th1 lymphocytes and NK cells plays a protective role1a are chemokines that facilitate the recruitment ofinflammatory cells, including neutrophils, monocytes, via production of reactive nitrogen intermediates and

1,25-dihydroxyvitamin D from activated macrophages,and lymphocytes, to the site of infection (59). MIF pro-duced by both macrophages (60) and T lymphocytes which exert antimycobacterial activity (64, 65). IFN-g

inhibits intracellular growth of M. tuberculosis in mu-(61) inhibits mobility of infiltrated macrophages. MCP-1, MIP-1a, MIF, TNF-a, and GM-CSF also activate rine macrophages. Mice lacking the gene for either

IFN-g or IFN-g receptor suffer from severe experimen-macrophages and may augment mycobactericidal ac-tivity. tal infection with BCG or M. tuberculosis. These mice

are extremely susceptible to mycobacterial infectionIL-12 has been identified as the most potent inducerof IFN-g production by Th1 and NK cells and also stim- and eventually die from disseminated tuberculosis. In

organs of these knockout mice, increased numbers ofulates the production of GM-CSF and TNF-a (41).IL-12 induces the differentiation of Th1 cells from un- tubercle bacilli and necrotic granulomas develop and

eventually lead to caseation and extensive tissue de-committed T cells and, consequently, initiates cell-mediated immunity, which plays a protective role in struction (66–69). The necrotic lesions seen in the

gene-disrupted mice are uncommon in normal lit-infections with intracellular pathogens such as myco-bacteria. This cytokine represents an important regula- termate mice (10). Macrophages from IFN-g or IFN-g

receptor-deficient mice cannot produce reactive nitro-tory bridge between innate resistance and adaptive im-munity. It has been reported that IL-12 mRNA and gen intermediates, which are considered essential for

antimycobacterial protection in mice. Thus, the criticalprotein were present at the site of human disease inpleural tuberculosis, and bioactive IL-12 was produced role of IFN-g as a macrophage activating factor in my-

cobacterial infection is evident in murine models of tu-by pleural fluid mononuclear cells in response to M.tuberculosis (40). By using a mouse model of dissemin- berculosis.ated M. avium infection, we have recently demon-strated that treatment with recombinant murine IL-

TABLE 312 reduces significantly the number of viable bacteriain infected mice. IL-12 itself, however, could not di- Cytokines Involved in Granulomatous Inflammationrectly inhibit the mycobacterial growth in vitro. Histo-

Produced bypathologically, IL-12 augmented granulomatous re-sponses. Nevertheless, IL-12 treatment shows minor

Cytokine Mf Th1 Th2 Granuloma-inducing activitytoxicities. Thus, IL-12 treatment protects mice from M.avium infection. IL-12 may prove of therapeutic value IL-1 / 0 0 /by promoting cell-mediated immunity to the infection IL-8 / 0 0 0

IL-12 / 0 0 /(Kobayashi et al., 1995. Antimicrob. Agents Chemother.MCP-1 / 0 0 /39, in press). Collectively, these results imply that IL-MIP-1a / 0 0 /12 contributes to the antimycobacterial immune re-MIF / / 0 /

sponse by enhancing production of IFN-g that activates TGF-b / 0 0 0macrophages to inhibit mycobacterial growth, facilitat- TNF-a / / / /

IL-2 0 / 0 0ing development of Th1 cells and augmenting cytotoxic-IFN-g 0 / 0 0ity of T cells and NK cells. Although the precise mecha-TNF-b 0 / 0 0nism of IL-12-mediated antimycobacterial defense inIL-4 0 0 / 0

the host has not been defined, induction of Th1 and NK IL-5 0 0 / 0cells and macrophage activating cytokines, including IL-6 / 0 / 0

IL-10 / 0 / 0IFN-g, TNF-a, and GM-CSF, is thought to orchestrateGM-CSF / / / ?antimycobacterial defense mediated by IL-12.

As shown in Table 3, CD4/ T cells comprise two func-Note. Mf, macrophage; Th, T helper; IL, interleukin; MCP, mono-tionally distinct subpopulations that differ in patterns

cyte chemoattractant protein; MIP, macrophage inflammatory pro-of cytokine production (62). Th1 cells produce IFN-g, tein; MIF, migration inhibition factor; TGF, transforming growthIL-2, and lymphotoxin (TNF-b), enhance microbicidal factor; TNF, tumor necrosis factor; IFN, interferon; GM-CSF, granu-

locyte-macrophage colony-stimulating factor.activity of macrophages, and augment cell-mediated



The cytokine network in control of intracellular my- granulomatous type of reaction occurs coincidentallywith the development of DTH after 2 or 3 weeks (3). IL-cobacterial growth represents a complex one. Th2 cell

responses may be described as cross-regulatory in that 1b and TNF-a are produced locally within tuberculousgranulomas in relation to disease activity (19, 20, 74)they inhibit the development of cell-mediated immu-

nity induced by Th1 cells. Models of in vitro mycobacte- and are sufficient to induce formation of granulomaswhen mice are injected intratracheally with inert agar-rial infection of macrophages suggest that some of the

Th2 cell-derived cytokines such as IL-10 promote intra- ose beads coupled to macrophage-derived cytokinessuch as recombinant IL-1b and TNF-a (75, 76). Addi-cellular mycobacterial growth (70). In murine models

of tuberculosis, an initial ‘‘protective’’ Th1 response is tionally, foreign body pulmonary granulomas are aug-mented by TNF-a (77). Neutralizing anti-TNF-a anti-followed by a Th2 response, which may serve to limit

the inflammatory response and minimize tissue injury body administered to mice inhibits the formation ofbactericidal granulomas following BCG challenge andat the site of infection (71). In addition, macrophages

play a regulatory role in tuberculosis. To reduce exces- results in progressive, lethal BCG infection (74). Theseobservations closely link granuloma formation, macro-sive inflammation and tissue damage, immunosup-

pressive cytokines such as IL-10 and TGF-b, both of phage-derived cytokines, and immunity in animal mod-els of mycobacterial infection (78). Although the precisewhich are produced by macrophages at the site of infec-

tion (55, 72), may downregulate the immune response mechanism remains unknown, IL-12, a macrophage-derived cytokine, may be involved either directly orand limit the extent of tissue injury. On the other hand,

excessive production of immunosuppressive cytokines indirectly in granuloma formation, because the admin-istration of IL-12 into mice infected with M. aviummay result in failure to control infection.results in augmented granulomatous responses (Ko-

Immunopathology of Granuloma Formation bayashi et al., 1995. Antimicrob. Agents Chemother. 39,in press).Granulomatous inflammation is manifested in

chronic inflammatory diseases that often result in tis- Direct chemotactic activity of IL-1 (79) and TNF-a(80) for macrophages has not been confirmed, althoughsue destruction and end-stage fibrosis. Granulomatous

inflammation can be broadly classified as hypersensi- these cells are the major component of granulomatouslesions. In contrast, chemokines including IL-8, MCP-tivity (immunologic, T cell-dependent) type and a for-

eign body (nonimmunologic, T cell-independent) type 1, and MIP-1a are directly chemotactic for inflamma-tory cells (59, 79). However, these chemokines are in-(2). The BCG-induced granulomas in mice were devel-

oped regardless of preimmunization with M. tuberculo- ducible by stimulating macrophages/monocytes withIL-1 and TNF-a (59). The accumulation and activationsis or BCG, although augmented responses were ob-

served in preimmunized mice (19, 20). MDP, a major of inflammatory cells in the granulomatous lesions maybe due to direct actions of chemokines induced by IL-cell wall component of M. tuberculosis, possesses im-

munostimulatory activity and can induce granuloma- 1 and/or TNF-a. Thus, MCP-1 was found in the site ofglucan-induced granulomas (81) and MIP-1a was de-tous lesions without participation of T lymphocytes (7).

Mice depleted of T lymphocytes produce small but still tected in the granulomatous lesions induced by Schisto-soma mansoni eggs. The administration of anti-MIP-significant degree of granulomas by infection with

BCG, but fail to develop antibacterial immunity (73). 1a antibodies inhibited the early development of thelesions (82). The locally produced MIF activates re-These data support the contention that granulomatous

inflammation in tuberculosis is composed of both hy- cruited macrophages and inhibits their mobility in thelesions (8, 13, 19, 61). In addition, adhesion moleculespersensitivity and foreign body types.

Induction of granuloma formation by tubercle bacilli may be crucial in granulomatous inflammation, be-cause recruited monocytes/macrophages are tightlyappears to a critical event in antimycobacterial defense

of the host, in which cytokines, particularly macro- bound in the lesion. Indeed, intercellular adhesion mol-ecule-1 and its ligand are detected in the lesion causedphage products, play a key role as mentioned above

(summarized in Table 3). The initial inflammatory re- by mycobacteria (83), schistosoma (84), and foreignbodies (85). Adhesion molecules are inducible by stimu-sponse of tuberculosis (within a few days) is the same

as that seen in any bacterial pneumonia and consists lation with proinflammatory macrophage-derived cyto-kines including IL-1 and TNF-a (86, 87). Thus, proin-mainly of fibrin, edema, and polymorphonuclear leuko-

cytes (19). At the early stage, IL-8, a potent chemokine flammatory cytokines and chemokines form a complexnetwork of cellular interactions in granulomatous in-for polymorphonuclear leukocytes, is produced tran-

siently by macrophages in response to M. tuberculosis flammation. Such complex interactions are shown inFig. 1.(54). The extent of this primary exudative response var-

ies with the number and virulence of the bacilli in- In contrast, the role of T cell-derived cytokines ingranuloma formation is less certain. Necrotic granulo-haled, the native resistance of the host, and the effec-

tiveness of the immune response. The change to a mas and widespread tissue destruction develop even in



IFN-g (a T cell-derived cytokine) or IFN-g receptor antagonist protein produced by macrophages regulatesschistosome egg granuloma formation and the regionalgene knockout mice infected with M. tuberculosis or

BCG (67, 68, 88). Mycobacteria are not contained lymph node response (94). The role of endogenous adre-nal corticosteroids in the disappearance of tuberculouswithin the lesions and are broadly disseminated. Mac-

rophages of the knockout mice cannot produce reactive granulomas is uncertain, although administration ofexogenous dexamethasone inhibits the development ofnitrogen intermediates. These mice finally die of myco-

bacterial infection, and IFN-g therapy can prolong sur- foreign body granulomas in mice (95) by its activity todownregulate production of granulomatogenic cyto-vival. Exogenous administration of IFN-g can suppress

granuloma formation in chronic granulomatous disease kines including IL-1 (96), TNF-a (97), and MCP-1 (59).Immunoendocrine feedback regulation may be involved(89, 90) and leprosy (91). Furthermore, we have demon-

strated that administration of IFN-g-coupled beads in the regression, because locally produced IL-1 andTNF-a, which are abundant in granulomatous lesions,cannot induce murine granulomas in vivo (76) and in

vitro (26). These results imply that IFN-g plays a criti- may enter circulation and then stimulate the hypothal-amus–pituitary–adrenal axis (98). Moreover, exoge-cal role in host resistance against the infection but not

in granuloma formation. T cell-derived cytokines, such nous steroids have been used successfully in treatingsarcoidosis, Crohn disease, and Wegener granulomato-as IL-2 and IL-4, are not detected in granulomatous

lesions induced by BCG (19, 20), antigen-coupled beads sis. Prostaglandin E2 also exerts suppressive activityin granuloma formation induced by foreign bodies (25)(hypersensitivity type) (8), or dextran beads (foreign

body type) (20, 75). Furthermore, the administration and S. mansoni eggs (99). This may be due to its inhibi-tory activity against the production and/or activity ofof beads coupled to IL-2 could not induce murine granu-

lomas (26, 75, 76). IL-1 (100) and TNF-a (101). Since macrophage-derivedcytokines can induce prostaglandin E synthesis fromThe process of resolution of granulomatous inflam-

mation after the elimination of pathogens is virtually macrophages/monocytes (102, 103), prostaglandin Emay act as an endogenous autoregulator of monokineunexplored. Some cytokines, especially IL-4 (55), IL-10

(92), and TGF-b (93), downregulate monocyte function production (104), thus suppressing granulomatous in-flammation.and may act at this stage. Endogenous IL-1 receptor

Expression of DTH and AnergyT cell-mediated immunity and/or DTH are important

in hypersensitivity granuloma formation as describedabove. The granulomatous reaction at the site of infec-tion occurs coincidentally with the development of DTHdemonstrated by positive skin reaction to PPD after 2or 3 weeks of mycobacterial infection (3). Up to 25% ofpatients with pulmonary tuberculosis have a negativetuberculin skin test (anergy) on initial evaluation (105).This percentage is increased in those with dissemin-ated or miliary tuberculosis (106). Up to 60% of pa-tients demonstrate reduced responses to PPD in vitroin terms of T cell blastogenesis, production of IL-2 andIFN-g, and surface expression of IL-2 receptors (107,108). Anergy is defined as the absence of an appropriateDTH reaction and may be either nonspecific, with lackof T cell responses to many types of antigens, or spe-cific, with lack of T cell responses to a particular anti-gen (109). Anergy is determined by absence of in vivo(DTH skin test) and in vitro (lymphocyte proliferationand IL-2 production) reactivity to antigens againstwhich the patient is sensitized. Antigen-nonspecific an-ergy occurs in tuberculosis (105, 106), sarcoidosis (110),and lymphoproliferative disease (111). Using experi-mental mouse models, we have demonstrated that an-

FIG. 1. Cellular and molecular mechanisms of granulomatous in- ergy is found in the active stage of granulomatous in-flammation induced by Mycobacterium tuberculosis. Th, T helper; flammation induced by the intratracheal challengeIL, interleukin; MCP, monocyte chemoattractant protein; MIP, mac-

with dextran beads (foreign body type) (24), antigen-rophage inflammatory protein; MIF, migration inhibition factor;TNF, tumor necrosis factor; IFN, interferon. coupled beads (hypersensitivity type) (112), and BCG



(19). Anergic animals show the diminution of both in stimuli to synthesize and secrete a diverse set of ef-fector molecules, many of which serve as mediators tovivo (footpad responses) and in vitro (lymphoprolifera-

tion and IL-2 production) manifestations of cell-medi- initiate and regulate inflammatory as well as immunereactions in either local or distant sites. Granuloma-ated immunity while active lesions contain a large

amount of granulomatogenic cytokines such as IL-1 tous inflammation is a phenotypic expression of thehost defense against M. tuberculosis infection. Al-and MIF. High granuloma producers, namely the mice

bearing the Bcgs gene, but not the mice bearing the though we now have a good grasp of the cells and mole-cules involved in both natural resistance and T cell-Bcgr gene, exhibited the suppression of cell-mediated

immunity, and the kinetics of anergy correlated with mediated immune responses that participate in thereduction of bacterial growth and in granuloma forma-local production of IL-1 and MIF (19). It has been dem-

onstrated that anergic patients with active sarcoidosis tion, more information is needed to understand the pre-cise mechanism for pathogenesis and protection from(110) and lymphoproliferative diseases (111) have high

levels of serum MIF activity. Based upon these obser- tuberculosis. Experimental animal models describedhere may still be useful for essential future studies onvations, antigen-nonspecific anergy may be explained

by (i) compartmentalization of effector cells in the le- tuberculosis. Hopefully, such studies may lead to thedevelopment of a new vaccine and/or immunotherapysion, (ii) loss of chemotactic gradient by the excess

amount of circulating chemokinetic cytokines, and/or for tuberculosis.(iii) cytokine-dependent downregulation (stimulationwith the hypothalamus–pituitary–adrenal axis andinduction of prostaglandin E).

REFERENCESProtection versus Pathogenesis

1. Kochi, A. (1991) Tubercle 72, 1–6.The cell-mediated immune response to tubercle ba-2. Kunkel, S. L., Chensue, S. W., Strieter, R. M., Lynch, J. P., andcilli is a double-edged sword that may contribute to

Remick, D. G. (1989) Am. J. Respir. Cell Mol. Biol. 1, 439–448.both protection from infection and tissue damage3. Dannenberg, A. M., Jr. (1991) Immunol. Today 12, 228–233.(pathogenesis) of the host (3). Indeed, the most destruc-4. Britton, W. J., Roche, P. W., and Winter, N. (1994) Trendstive tissue lesions, pulmonary cavities, occur only in

Microbiol. 2, 284–288.PPD-positive individuals. Cytokines produced by T5. Wayne, L. G. (1982) Am. Rev. Respir. Dis. 125(Part 2), 31–41.cells are involved in activating macrophages to kill or6. Zhang, Y. H., Doerfler, M., Lee, T. C., Guillemin, B., and Rom,inhibit the growth of M. tuberculosis. Tubercle bacilli

W. N. (1993) J. Clin. Invest. 91, 2076–2083.are resistant to generally cytocidal reactive oxygen in-7. Tanaka, A., Emori, K., Nagao, S., Kushima, K., Kohashi, O.,termediates, such as hydrogen peroxide and hydroxyl

Saito, M., and Kataoka, T. (1982) Am. J. Pathol. 106, 165–170.radical, while they are susceptible to reactive nitrogen

8. Kobayashi, K., Allred, C., Cohen, S., and Yoshida, T. (1985) J.intermediates, particularly nitric oxide produced by ac- Immunol. 134, 358–364.tivated macrophages (42). The cytokines required to 9. Smith, D. W., and Wiegeshaus, E. H. (1989) Rev. Infect. Dis.activate macrophages to produce reactive nitrogen in- 11(Suppl. 2), S385–S393.termediates are IFN-g and TNF-a (113). For example, 10. Ehlers, S., Mielke, M. E. A., and Hahn, H. (1994) Immunol.TNF-a has been shown to be critically important for Today 15, 1–4.protection from infection and prevention from dissemi- 11. Collins, F. M., Morrison, N. E., and Montalbine, V. (1978) Infect.

Immun. 20, 430–437.nation (74). However, TNF-a and other products of acti-12. Dannenberg, A. M., Jr. (1982) Am. Rev. Respir. Dis. 125(Partvated macrophages, including radicals, proteases, and

2), 25–29.cytokines, are also known to be toxic molecules induc-13. Masih, N., Majeska, J., and Yoshida, T. (1979) Am. J. Pathol.ing tissue damages (114). Therefore, it is still a funda-

95, 391–406.mental question whether one could dissect protective14. Cohen, M. K., Bartow, R. A., Mintzer, C. L., and McMurray,immunity from hypersensitivity (33).

D. N. (1987) Infect. Immun. 55, 314–319.

15. Skamene, E. (1989) Rev. Infect. Dis. 11(Suppl. 2), S394–S399.

16. Brett, S., Orrell, J. M., Swanson Beck, J., and Ivanyi, J. (1992)Immunology 76, 129–132.CONCLUDING REMARKS

17. Skamene, E., and Pietrangeli, C. E. (1991) Curr. Opin. Immu-nol. 3, 511–517.

Both host- and pathogen-specific factors influence18. Vidal, S. M., Malo, D., Vogan, K., Skamene, E., and Gros, P.

disease outcome and progression. The host response (1993) Cell 73, 469–485.to M. tuberculosis infection is multifaceted, with the 19. Kobayashi, K., Allred, C., Castriotta, R., and Yoshida, T. (1985)recruitment and activation of inflammatory cell popu- Am. J. Pathol. 119, 223–235.lations being of central importance. The macrophage is 20. Sato, I. Y., Kobayashi, K., Kasama, T., Kaga, S., Kasahara, K.,

Kanemitsu, H., Nakatani, K., Takahashi, T., Nakamura, R. M.,a critical cellular participant and is induced by invasive



Skamene, E., and Yoshida, T. (1990) Infect. Immun. 58, 1210– 49. Barnes, P. F., Bloch, A. B., Davidson, P. T., and Snider, D. E.,Jr. (1991) N. Engl. J. Med. 324, 1644–1650.1216.

50. Flynn, J. L., Goldstein, M. M., Triebold, K. J., Koller, B., and21. Ribi, E., Anacker, R. L., Barelay, W. R., Harris, S. C., Leif,Bloom, B. R. (1992) Proc. Natl. Acad. Sci. USA 89, 12013–W. R., and Simons, J. (1971) J. Infect. Dis. 123, 527–538.12017.22. Thoen, C. O., Karlson, A. G., and Himes, E. M. (1981) Rev.

51. DeLibero, G., Flesch, I., and Kaufmann, S. H. E. (1988) Eur.Infect. Dis. 3, 960–972.J. Immunol. 18, 59–66.23. Boros, D. L. (1978) Prog. Allergy 24, 184–243.

52. Inoue, T., Yoshikai, Y., Matsuzaki, G., and Nomoto, K. (1991)24. Allred, C., Kobayashi, K., and Yoshida, T. (1985) Am. J. Pathol.J. Immunol. 146, 2754–2762.121, 466–473.

53. Huygen, K., Vandenbussche, P., and Heremans, H. (1991) Cell.25. Sato, I. Y., Kobayashi, K., Yamagata, N., Shikama, Y., Kasama,Immunol. 137, 224–231.T., Kasahara, K., and Takahashi, T. (1991) Immunopharma-

54. Kasahara, K., Tobe, T., Tomita, M., Mukaida, N., Shao-Bu, S.,cology 21, 73–82.Matsushima, K., Yoshida, T., Sugihara, S., and Kobayashi, K.26. Shikama, Y., Kobayashi, K., Kasahara, K., Kaga, S., Hashi-(1994) J. Infect. Dis. 170, 1238–1247.moto, M., Yoneya, I., Hosoda, S., Soejima, K., Ide, H., and Taka-

55. Barnes, P. F., Lu, S., Abrams, J. S., Wang, E., Yamamura, M.,hashi, T. (1989) Am. J. Pathol. 134, 1189–1199.and Modlin, R. L. (1993) Infect. Immun. 61, 3482–3489.27. Pelletier, M., Forget, A., Bourassa, D., Gros, P., and Skamene,

56. Takashima, T., Ueta, C., Tsuyuguchi, I., and Kishimoto, S.E. (1982) J. Immunol. 129, 2179–2185.(1990) Infect. Immun. 58, 3286–3292.28. Metzger, J. M., and Peterson, L. B. (1988) Immunopharma-

57. Appelberg, R. (1992) J. Leukoc. Biol. 52, 303–306.cology 15, 103–116.58. Barnes, P. F., Chatterjee, D., Abrams, J. S., Lu, S., Wang, E.,29. Kaga, S., Kobayashi, K., Yamagata, N., Takeuchi, H. T., Yo-

Yamamura, M., Brennan, P. J., and Modlin, R. L. (1992) J.shida, K., Matsuda, T., Nakatani, K., Kasama, T., Kasahara,Immunol. 149, 541–547.K., and Takahashi, T. (1991) Int. Arch. Allergy Appl. Immunol.

59. Oppenheim, J. J., Zachariae, C. O. C., Mukaida, N., and Matsu-95, 236–243.shima, K. (1991) Annu. Rev. Immunol. 9, 617–648.30. Remick, D. G., Chensue, S. W., Hiserodt, J. C., Higashi, G. I.,

60. Calandra, T., Bernhagen, J., Mitchell, R. A., and Bucala, R.and Kunkel, S. L. (1988) Am. J. Pathol. 131, 298–307.(1994) J. Exp. Med. 179, 1895–1902.31. Chensue, S. W., Otterness, I. G., Higashi, G. I., Forsch, C. S.,

61. Oppenheim, J. J., and Neta, R. (1994) FASEB J. 8, 158–162.and Kunkel, S. L. (1989) J. Immunol. 142, 1281–1286.62. Mosmann, T. R., and Coffman, R. L. (1989) Annu. Rev. Immu-32. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

nol. 7, 145–173.W. J. (1979) Biochemistry 18, 5294–5299.63. Romagnani, S. (1994) Annu. Rev. Immunol. 12, 227–257.33. Bloom, B. R., and Murray, C. J. L. (1992) Science 257, 1055–

1064. 64. Pryke, A. M., Duggan, C., White, C. P., Posen, S., and Mason,R. S. (1990) J. Cell. Physiol. 142, 652–656.34. Ellner, J. J., Hinman, A. R., Dooley, S. W., Fischl, M. A., Sep-

65. Reichel, H., Koeffler, H. P., and Norman, A. W. (1989) N. Engl.kowitz, K. A., Goldberger, M. J., Shinnick, T. M., Iseman,J. Med. 320, 980–991.M. D., and Jacobs, W. R., Jr. (1993) J. Infect. Dis. 168, 537–

551. 66. Huang, S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann,H., Kamijo, R., Vilcek, J., Zinkernagel, R. M., and Aguet, M.35. Orme, I. M., and Collins, F. M. (1983) J. Exp. Med. 158, 74–(1993) Science 259, 1742–1745.83.

67. Flynn, J. L., Chan, J., Triebold, K. J., Dalton, D. K., Stewart,36. Abou-Zeid, C., Ratliff, T. L., Wiker, H. G., Harboe, M., Bened-T. A., and Bloom, B. R. (1993) J. Exp. Med. 178, 2249–2254.sen, J., and Rook, G. A. W. (1988) Infect. Immun. 56, 3046–

3051. 68. Dalton, D. K., Pitts-Meek, S., Keshav, S., Figari, I. S., Bradley,A., and Stewart, T. A. (1993) Science 259, 1739–1742.37. Rao, S. P., Ogata, K., and Catanzaro, A. (1993) Infect. Immun.

61, 663–670. 69. Cooper, A. M., Dalton, D. K., Stewart, T. A., Griffin, J. P., Rus-sell, D. G., and Orme, I. M. (1993) J. Exp. Med. 178, 2243–38. Schlesinger, L. S., Bellinger-Kawahara, C. G., Payne, N. R.,2247.and Horwitz, M. A. (1990) J. Immunol. 144, 2771–2780.

70. Bermudez, L. E., and Champsi, J. (1993) Infect. Immun. 61,39. Gangadharam, P. R., Edwards, C. K., III, Murthy, P. S., and3093–3097.Pratt, P. F. (1983) Am. Rev. Respir. Dis. 127, 648–649.

71. Orme, I. M., Roberts, A. D., Griffin, J. P., and Abrams, J. S.40. Zhang, M., Gately, M. K., Wang, E., Gong, J., Wolf, S. F., Lu,(1993) J. Immunol. 151, 518–525.S., Modlin, R. L., and Barnes, P. F. (1994) J. Clin. Invest. 93,

1733–1739. 72. Maeda, J., Ueki, N., Ohkawa, T., Iwahashi, N., Nakano, T.,Hada, T., and Higashino, K. (1993) Clin. Exp. Immunol. 92,41. Scott, P. (1993) Science 260, 496–497.32–38.42. Chan, J., Xing, Y., Magliozzo, R. S., and Bloom, B. R. (1992) J.

73. Collins, F. M., Congdon, C. C., and Morrison, N. E. (1975) Infect.Exp. Med. 175, 1111–1122.Immun. 11, 57–64.43. Denis, M. (1991) Clin. Exp. Immunol. 84, 200–206.

74. Kindler, V., Sappino, A.-P., Grau, G. E., Piguet, P.-F., and Vas-44. Rook, G. A. W. (1988) Am. Rev. Respir. Dis. 138, 768–770.salli, P. (1989) Cell 56, 731–740.

45. Abe, E., Miyaura, C., Tanaka, H., Shiina, Y., Kuribayashi, T.,75. Kasahara, K., Kobayashi, K., Shikama, Y., Yoneya, I., Soezima,and Suda, T. (1983) Proc. Natl. Acad. Sci. USA 80, 5583–5587.

K., Ide, H., and Takahashi, T. (1988) Am. J. Pathol. 130, 629–46. Kaufmann, S. H. E. (1993) Annu. Rev. Immunol. 11, 129–163. 638.47. Pedrazzini, T., Hug, K., and Louis, J. A. (1987) J. Immunol. 76. Kasahara, K., Kobayashi, K., Shikama, Y., Yoneya, I., Kaga,

139, 2032–2037. S., Hashimoto, M., Odagiri, T., Soejima, K., Ide, H., Takahashi,T., and Yoshida, T. (1989) Clin. Immunol. Immunopathol. 51,48. Muller, I., Cobbold, S. P., Waldmann, H., and Kaufmann, S. H.

E. (1987) Infect. Immun. 55, 2037–2041. 419–425.



77. Kasama, T., Kobayashi, K., Yamagata, N., Iwabuchi, H., Mat- 94. Chensue, S. W., Bienkowski, M., Eessalu, T. E., Warmington,K. S., Hershey, S. D., Lukacs, N. W., and Kunkel, S. L. (1993)suda, T., Nakatani, K., Kasahara, K., and Takahashi, T. (1992)

Int. Arch. Allergy Immunol. 97, 130–138. J. Immunol. 151, 3654–3662.95. Hashimoto, M., Kobayashi, K., Yamagata, N., Katsura, T., Su-78. Wallis, R. S., and Ellner, J. J. (1994) J. Leukoc. Biol. 55, 676–

gihara, S., Iwabuchi, H., Kasama, T., Kasahara, K., Takahashi,681.T., Takeshita, K., and Otomo, S. (1992) Agents Actions 37, 99–79. Leonard, E. J., and Yoshimura, T. (1990) Immunol. Today 11,106.97–101.

96. Kern, J. A., Lamb, R., Reed, J. C., Daniele, R. P., and Nowell,80. Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue, S. W.,P. C. (1988) J. Clin. Invest. 81, 237–244.Lynch, J. P., Toews, G. B., Westwick, J., and Strieter, R. M.

97. North, R., and Havell, E. A. (1989) J. Exp. Med. 170, 703–710.(1990) J. Clin. Invest. 86, 1945–1953.98. Kroemer, G., Brezinschek, H.-P., Faessler, R., Schauenstein,81. Jones, M. L., and Warren, J. S. (1992) Lab. Invest. 66, 498–

K., and Wick, G. (1988) Immunol. Today 9, 163–165.503.99. Chensue, S. W., Remick, D. G., Higashi, G. I., Boros, D. L., and

82. Lukacs, N. W., Kunkel, S. L., Strieter, R. M., Warmington, K., Kunkel, S. L. (1986) Am. J. Pathol. 125, 28–34.and Chensue, S. W. (1993) J. Exp. Med. 177, 1551–1559.

100. Knudsen, P. J., Dinarello, C. A., and Strom, T. B. (1986) J.83. Sullivan, L., Sano, S., Pirmez, C., Salgame, P., Mueller, C., Immunol. 137, 3189–3194.

Hofman, F., Uyemura, K., Rea, T. H., Bloom, B. R., and Modlin,101. Kunkel, S. L., Spengler, M., May, M. A., Spengler, R., Larrick,R. L. (1991) Infect. Immun. 59, 4154–4160.

J., and Remick, D. (1988) J. Biol. Chem. 263, 5380–5384.84. Lukacs, N. W., Chensue, S. W., Strieter, R. M., Warmington, 102. Nathan, C. F. (1987) J. Clin. Invest. 79, 319–326.

K., and Kunkel, S. L. (1994) J. Immunol. 152, 5883–5889.103. Topley, N., Floege, J., Wessel, K., Hass, R., Radeke, H. H.,

85. Power, C., Kobayashi, K., Nishimura, T., and Yoshida, T. (1994) Kaever, V., and Resch, K. (1989) J. Immunol. 143, 1989–1995.Clin. Immunol. Immunopathol. 73, 321–329.

104. Kunkel, S. L., Chensue, S. W., and Phan, S. H. (1986) J. Immu-86. Bevilacqua, M. P. (1993) Annu. Rev. Immunol. 11, 767–804. nol. 136, 186–192.87. Pilewski, J. M., and Albelda, S. M. (1993) Am. Rev. Respir. Dis. 105. Nash, D. R., and Douglass, J. E. (1980) Chest 77, 32–37.

148, S31–S37. 106. Daniel, T. M., Oxtoby, M. J., Pinto, E., and Moreno, E. (1981)88. Kamijo, R., Le, L. M., Shapiro, D., Havell, E. A., Huang, S., Am. Rev. Respir. Dis. 123, 556–559.

Aguet, M., Bosland, M., and Vilcek, J. (1993) J. Exp. Med. 178, 107. Onwubalili, J. K., Scott, G. M., and Robinson, J. A. (1985) Clin.1435–1440. Exp. Immunol. 59, 405–413.

89. Sechler, J. M. G., Malech, H. L., White, C. J., and Gallin, J. 108. Toossi, Z., Kleinhenz, M. E., and Ellner, J. J. (1986) J. Exp.(1988) Proc. Natl. Acad. Sci. USA 85, 4874–4878. Med. 163, 1162–1172.

109. Kantor, F. S. (1975) N. Engl. J. Med. 292, 629–634.90. Ezekowitz, R. A. B., Dinauer, M. C., Jaffe, H. S., Orkin, S. H.,and Newburger, P. E. (1988) N. Engl. J. Med. 319, 146–151. 110. Yoshida, T., Siltzbach, L. E., Masih, N., and Cohen, S. (1979)

Clin. Immunol. Immunopathol. 13, 39–46.91. Nathan, C. F., Kaplan, G., Levis, W. R., Nusrat, A., Witmer,M. D., Sherwin, S. A., Job, C. K., Horowitz, C. R., Steinman, 111. Cohen, S., Fisher, B., Yoshida, T., and Bettigole, R. E. (1974)R. M., and Cohn, Z. A. (1986) N. Engl. J. Med. 315, 6–15. N. Engl. J. Med. 290, 882–886.

112. Kobayashi, K., Allred, C., and Yoshida, T. (1985) J. Immunol.92. Modlin, R. L., and Nutman, T. B. (1993) Curr. Opin. Immunol.135, 2996–3003.5, 511–517.

113. Denis, M. (1994) J. Leukoc. Biol. 55, 682–684.93. Hirsch, C. S., Yoneda, T., Averill, L., Ellner, J. J., and Toossi,Z. (1994) J. Infect. Dis. 170, 1229–1237. 114. Rook, G. A. W. (1988) Brit. Med. Bull. 44, 611–623.


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The Immunopathogenesis of Granulomatous Inflammation Induced byMycobacterium tuberculosis