Role of Cytokines in Tuberculosis

Immunobiol., vol. 189, pp. 316-339 (1993)

©

1993 by Gustav Fischer Verlag, Stuttgart

Department of Immunology, University of Ulm, Ulm, Germany

Role of Cytokines in Tuberculosis INGE E. A. FLESCH and STEFAN H. E. KAUFMANN

Abstract Mycobacterium tuberculosis and Mycobacterium bovis are facultative intracellular pathogens which preferentially utilize the macrophage as their host cell. Acquired resistance against mycobacteria depends on T cells which activate antimicrobial macrophage functions via the release of cytokines. The data summarized below suggest an important role for interferon-y (IFN-y) as well as the B cell-stimulatory factors interleukin-4 (IL-4) and IL-6 in the induction of tuberculostatic macrophage functions. Growth inhibition of mycobacteria by cytokine-stimulated macrophages is mediated by reactive nitrogen intermediates (RNI) derived from L-arginine. Tumor necrosis factor-a (TNF-a) and IL10 act as autocrine regulators in the induction of the enzyme NO-synthase. Both cytokines are produced by macrophages stimulated with IFN-y and infected with M. bovis. While TNF-a mediates activation of the NO-synthase and production of RNI, IL-I0 suppresses this enzyme activity. The outcome of mycobacterial infection is probably regulated by a complex network between stimulatory and inhibitory cytokines.

Introduction In 1882 ROBERT KOCH identified the tubercle bacillus as the etiologic agent of an infectious disease which was at that time the most frequent cause of death in adults living in the major cities in Europe. Even today, in the era of chemotherapy, more people die from Mycobacterium tuberculosis infection than from any other pathogen. It is assumed that 60 million people suffer from active tuberculosis, and that about 1/3 of the total world population is infected with M. tuberculosis. Although most cases occur in developing countries, incidences have substantially increased in industrialized states Abbreviations: BMM = bone marrow-derived macrophages; GM-CSF = granulocyte/ macrophage colony-stimulating factor; IFN-y = interferon-y; IL = interleukin; mAb = monoclonal antibody; MHC = major histocompatibility complex; MP = mononuclear phagocyte; NK cells = natural killer cells; r = recombinant; RNI = reactive nitrogen intermediates; ROI = reactive oxygen intermediates; TH cells = T helper cells; TGF-~ = transforming growth factor-~; TNF = tumor necrosis factor

Cytokines in tuberculosis . 317

since 1985. The occurrence of multidrug resistant strains and the expanding AIDS threat make the problem even worse. Besides M. tuberculosis the socalled atypical mycobacteria of the Mycobacterium avium/ Mycobacterium intracellulare (MAI)-complex are frequently responsible for secondary infections in AIDS patients. M. tuberculosis/Mycobacterium bovis are facultative intracellular pathogens which preferentially utilize the mononuclear phagocyte (MP) as their habitat and only rarely, if at all, inhabit nonprofessional phagocytes. In contrast to M. tuberculosis/ M. bovis, other intracellular pathogens frequently inhabit both nonprofessional and professional phagocytes. MP are potent effector cells of the host defense system which are able to phagocytose and kill invading microorganisms. Yet, besides tubercle bacilli, many pathogens have developed mechanisms to escape from the antimicrobial potential of macrophages (1, 2). For example, Listeria monocytogenes escapes from the phagosome into the cytoplasm. Salmonella typhi and Brucella abortus interfere with the production of reactive oxygen intermediates (ROI). Toxoplasma gondii inhibits phagosome-lysosome fusion and persists in the phagosome. Salmonella typhimurium and Mycobacterium leprae are resistant against lysosomal enzymes (1, 2). ARMSTRONG & HART (3) investigated the growth of M. tuberculosis H37Rv in murine peritoneal macrophages by electron microscopy. They

Figure 1. Thin-section through a macrophage which has phagocytosed M. tuberculosis (electron microscopy by J. GOLECKI and S. H. E. KAUFMANN).

318 . 1. E. A.

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KAUFMANN

observed bacterial replication within phagosomes and inhibition of phagolysosome fusion. Killed M. tuberculosis or bacteria opsonized with antibodies did not interfere with phagolysosome fusion. MYRVIK et al. (4) provided evidence, that some strains of M. tuberculosis evade from the phagosome into the cytoplasm. Yet other studies suggest that M. tuberculosis remains inside the phagosome. Figure 1 illustrates uptake and intracellular location of M. tuberculosis in murine macrophages. Although the resting MP provides the major habitat for tubercle bacilli, it can differentiate into an essential effector cell of host defense following appropriate activation. The term «activated macrophage» was introduced by MACKANESS (5) who observed increased resistance towards L. monocytogenes in mice chronically infected with M. bovis BCG. The microbicidal effector function was attributed to MP and their activation to lymphocytes. Later LANE & UNANUE (6) and NORTH (7) found that macrophage activation was a function of specific T lymphocytes (reviewed in 8). The interaction between lymphocytes and macrophages was found to be mediated by soluble factors, now called cytokines (9, 10). Meanwhile, the immunological basis of acquired resistance against intracellular bacteria is better - though not fully - understood (8, 11, 12). Microbial antigens are processed by macrophages and presented in the context of gene products of the major histocompatibility complex (MHC) which stimulate antigen-specific T lymphocytes. The activated T cells differentiate and produce cytokines which ultimately induce macrophage activation. Although this interplay is generally accepted as a major factor in the acquisition of host defense, additional mechanisms such as target cell lysis by cytolytic T cells may be required for optimal protection to occur (13).

The T cell system The production of cytokines is the main function of T helper cells (TH cells). These T H cells typically bear the CD4-molecule on their surface and recognize antigenic peptide in association with MHC class II molecules. According to their cytokine secretion pattern, T H cells segregate into T HI cells which produce interleukin-2 (IL-2) and interferon-y (IFN-y) and T HZ cells which secrete IL-4, IL-5, IL-6 and IL-I0 (14, 15). IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are produced by either subset. Besides CD4 T cells, mycobacteria reactive CD8 T cells and y/o T cells have also been shown to produce certain cytokines (IFN-y, TNF-~), both in the murine and in the human system (16-18). Immunity of mice to Leishmania major is regulated by a THl/THZ cell interplay. In resistant strains of mice, the THl cell response dominates and production of IFN-y leads to resolution of infection. In susceptible mouse strains, the induction of T H2 cells results in exacerbation of the disease. In the leishmania system production of IFN -y promotes T HI cell development

Cytokines in tuberculosis . 319 Table 1. Cytokines in antimycobacterial resistance. Cytokine

Major source

Likely mode( s) of action

IL-t

MP

Attraction of phagocytes

IL-4

T cell

Macrophage activation Control of phagocyte influx

IL-6

MP, T cell

Macrophage activation

IFN-y

NK, T cell

Macrophage activation

TNF-a

MP

Macrophage activation Granuloma formation

Chemokines

MP, endothelial cell

Attraction of phagocytes

IL-tO

T cell, MP

Inhibition of macrophage functions

TGF-~

MP

Inhibition of macrophage functions

GM-CSF

T cell, endothelial cell, fibroblast, MP

Phagocyte differentiation Macrophage activation

and production of IL-4 favors T H2 cell differentiation (19). The murine response to M. tuberculosis/ M. bovis is characterized by strong IL-2 and IFN -y production (20, 21). In the case of mycobacterial infections T H2 cells have been found to develop only in lepromatous leprosy (22). In the following, the relevance of macrophage- and T cell-derived cytokines in the resolution of mycobacterial infections will be discussed. Table 1 summarizes the cytokine contribution to antimycobacterial immunity.

Cytokines involved in the defense against mycobacteria Interferon-y IFN-y which is produced by CD4 T cells, CDS T cells, y/o T cells and natural killer cells (NK cells) is probably the major macrophage-activating factor (23). It primes macrophages for tumor cell lysis, release of ROI and of reactive nitrogen intermediates (RNI). It induces secretion of arachidonic acid metabolites and stimulates expression of MHC class II antigens. IFN-y plays a central role in resistance against intracellular pathogens including T. gondii, L. donovani, Chlamydia psittaci, Rickettsia prowazeki and L. monocytogenes (24-2S). ROOK et al. (29) showed that recombinant (r) IFN-y enhances the ability of murine peritoneal macrophages to inhibit intracellular growth of M. tuberculosis in vitro. Murine bone marrow-derived macrophages (BMM) restrict growth of M. bovis and M. tuberculosis H37Rv after activation with rIFN-y, although, growth of M. tuberculosis Middelburg is not affected

5.34

anti-IFN-y

0.24

t::,

4.39

4.17

liver

0.22

t::,

3.4

2.9

lung

C57Bl/6 mice loglo colony-forming units

0.5

t::,

5.4

5.28

spleen

0.12

t::,

4.7

4.44

liver

0.26

t::,

3.64

2.8

lung

BALB/c mice loglo colony-forming units

0.84

t::,

C57Bl/6 and BALB/c mice were infected intravenously with 4 x 10 5 of live M. bovis organisms (day 0). The anti-IFN-y mAb R46A2, 0.5 mg per mouse, was administered intraperitoneally in days -2, -1, 6 and 12. Mice were sacrificed on day 20 and numbers of colony-forming units were determined in spleen, liver and lung. Data represent mean values from groups of 5 mice.

5.1

spleen

Nil

Treatment

Table 2. Effect of anti-IFN-y-mAb on the growth of M. bovis in spleen, liver and lung of C57BI/6 and BALB/c mice.

<.;J

~

'"3:

c

~

t'1

;r:

Y'

::; 0-

.,

'"() :t

m

'TI r

?>

trJ

:--<

N C>

Cytokines in tuberculosis· 321

(30). These data show that rlFN-y can activate antimycobacterial functions in certain macrophage populations in vitro. Different strains of M. tuberculosis, however, vary in their susceptibility towards IFN-y-activated BMM. Continuous infusion of rIFN-y into BALB/c mice leads to increased resistance against a lethal dose of M. tuberculosis H37Rv, and to decreased growth of bacteria in spleen, liver and lung. Conversely, depletion of IFN-y by monoclonal antibodies (mAb) enhances the susceptibility of mice to a lethal dose of M. tuberculosis (31). BANERJEE et al. (32) reported that treatment with rIFN-y enhances resistance to infection with BCG in euthymic BALB/c mice, but fails to restrict growth of BCG in athymic mice. In our hands, growth of M. bovis was enhanced significantly in the lungs of BALB/c mice after treatment with a neutralizing anti-IFN-y mAb (Table 2). In four independent experiments only marginal enhancement of growth was observed in liver and spleen of anti-IFN-y-treated mice (FLESCH & KAUFMANN, unpublished results). In contrast to DENIS (31), mAb were not administered by continuous infusion, but by weekly intraperitoneal injections. Perhaps the concentration and/or the half-life of circulating mAb was too low to affect growth of mycobacteria in liver and spleen. In any case, these studies suggest a major role of IFN-y in protection against tuberculosis in the primary organ of disease manifestation, the lung. Neutralization of IFN-y by administration of mAb does not ensure complete removal of the molecule from the bloodstream. The creation of mutant mice with an inactivated IFN-y gene by homologous recombination (IFN-y knock-out mice) has made it possible to reevaluate the role of IFNy in the immune response. Infection of IFN-y knock-out mice with a sublethal dose M. bovis leads to increased mortality and to an increase of bacterial numbers in lung, liver and spleen (33). Tumor necrosis factor

TNF-a is a major mediator of inflammation and septic shock, and is secreted by MP after stimulation with M. tuberculosis, mycobacterial protein or lipoarabinomannan (34-36). TNF has also been detected in pleural fluids of patients with tuberculous pleuritis (37). In our hands, TNF-a alone had no effect on the antimycobacterial effector functions of murine BMM in vitro, however, it synergized with rlFN-y in the induction of macrophage tuberculostatic activity (38). A synergism between IFN-y and TNF-a was also found in the defense of Schistosoma mansoni and L. major by macrophages (39, 40). Involvement of TNF in tuberculoid granuloma formation was found by KINDLER et al. (41), who observed local TNF production in M. bovisinduced liver granulomas of mice. Administration of anti-TNF antibodies neutralized TNF and concomitantly suppressed granuloma development and bacteriostatic functions in the lesions. According to DENIS (31), infu-

322 . I. E. A.

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KAUFMANN

sion of rTNF-a increases resistance of BALB/c mice to M. tuberculosis and antibodies against TNF-a enhances susceptibility to experimental tuberculosis. In contrast, application of anti-TNF-a antiserum or of a rTNF-a receptor protein to M. bovis-infected C57B1I6 mice, has no effect on the multiplication of bacteria in liver and spleen (FLESCH & KAUFMANN, unpublished results). TNF possesses fibrinogenic activity suggesting that it is involved in encapsulation of tuberculous granulomas (42). Encapsulation would contribute to bacterial containment and, hence, be of beneficial value. On the other hand, high TNF levels released into the circulation could contribute to the cachexia characteristic for the advanced stages of tuberculosis known as consumption (11). Interleukin-4 IL-4 (B cell stimulatory factor-l) was originally defined as a T cell-derived growth factor which induces entry of resting B cells into cell cycle S phase after stimulation with anti-immunglobulin antibodies (43). Meanwhile, it has become evident that IL-4 is also a macrophage-activating factor. Mouse peritoneal macrophages express IL-4 receptors, and IL-4 alone is able to activate proteose peptone-elicited peritoneal macrophages to enhanced tumor cytotoxicity and increased Ia antigen expression (44). Concomitant with increased Ia antigen expression, IL-4 enhances the antigen-presenting ability in mouse BMM (45). STUART et al. (46) showed that IL-4 is capable of inducing both class I and class II antigen expression. IL-4 induces monocyte infiltration in vivo (47) and multinucleated giant cell formation in vitro (48). Multinucleated giant cells are formed by the fusion of macrophages and are prominent in granulomatous lesions in tuberculosis where they are thought to sequester persisting bacteria. In murine BMM rIL-4 induces tuberculostasis, but it is only active when given after infection (38). On the other hand, recent experiments with the listeriosis system indicate a disease exacerbating role of IL-4 in vivo (49). Interleukin-6 IL-6 is identical to B cell-stimulatory factor 2, IFN-~2, 26-kD protein and hybridoma plasmacytoma growth factor. It was originally shown to stimulate B cells at late developmental stages. IL-6 activates B cell functions without causing growth, and is thereby a maturation rather than a growth factor. It is now evident that IL-6 is a pleiotropic mediator which is produced by a variety of cells including T lymphocytes, fibroblasts treated with IL-l and TNF, endothelial cells and monocytes/macrophages. Besides its effects on B cell maturation, IL-6 stimulates T cell growth and differentiation, is involved in acute-phase response of hepatocytes, and activates hemopoietic stem cells (50-52). It has been shown that body fluids of patients with local acute bacterial infections contain elevated levels of IL-6, indicating that this cytokine plays a role in the host response to infectious

Cytokines in tuberculosis . 323 agents (53). Blood MP from subjects infected with M. tuberculosis produce increased amounts of IL-6 (35). In vitro, IL-6 induces antimycobacterial activities in infected BMM in a way similar to IL-4. Stimulation with IL-6 before infection with M. bovis failed to activate macrophage tuberculostatic activities (54). Other cytokines

Besides IFN-y, TNF, IL-4 and IL-6 the cytokines, IL-l, IL-2, IL-8 and GM-CSF are involved in the immune repsonse against intracellular pathogens. According to ]EEVAN and ASHERsoN (55) in vivo administration of IL-2 significantly reduces bacterial counts in the spleens of M. bovisinfected mice. In humans, depressed IL-2 production has been observed in patients with lepromatous leprosy and tuberculosis (56). IL-8 is secreted by human MP following phagocytosis of M. tuberculosis (57). Since IL-8 is an attractant for neutrophils, it may directly contribute to granuloma formation. IL-8 is a member of the chemokine family, which comprises cytokines of similar molecular weight primarily involved in the control of lymphocyte migration (58). Probably these chemokines play a central role in early attraction of blood phagocytes through the endothelial wall to the site of bacterial growth. Therefore, they probably are important mediators of early defense and of the formation of granulomatous lesions. GM-CSF, alone or in combination with TNF stimulates effector functions of human macrophages against M. avium (59, 60).

Mechanisms involved in mycobacterial growth inhibition After activation, MP combat extra- and intracellular pathogens using a variety of defense mechanisms. Factors of potential relevance to antimycobacterial resistance include: 1. generation of ROI (61, 62), 2. production of RNI (63-65), 3. phagosome acidification and phagolysosome fusion (1, 66), 4. degradation of tryptophan (67, 68), 5. limitation of intracellular iron (69, 70), 6. production of defensins (71). In the following section, we briefly describe these defense mechanisms and discuss their impact on resistance against tuberculosis (Fig. 2). Generation of ROI

Phagocytosis of particles or bacteria activates the enzyme NAD(P)Hoxidase, which is located in the plasma membrane of MP. This enzyme catalyses the production of superoxide anion (02-) from oxygen. From O 2the more toxic radicals hydrogen peroxide (H 2 0 2 ), singulett oxygen (0 2 1)

324 . 1. E. A. FLESCH and S. H. E.

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and hydroxyl anion (OH-) are formed (62). It has been postulated that ROI are responsible for mycobacterial growth inhibition by macrophages (72-74). Recently, however, evidence has been provided that ROI are not or only marginally involved in macrophage tuberculostasis. Phagocytosis of M. bovis by rIFN-y-activated macrophages does not lead to an oxidative burst, and scavengers of ROI fail to reverse antimycobacterial macrophage functions (30, 75). SCHLESINGER et al. (76) showed that mycobacteria are phagocytosed by human MP via receptors for C3 breakdown products, and that this pathway interferes with production of ROI. Furthermore, molecules from mycobacteria including sulfatides, lipoarabinomannan and

Arginine

®~NO Citrulline NADP

Figure 2. Defense mechanisms of activated MP with potential relevance to tuberculosis and evasion strategies of mycobacteria. 1 Normal uptake or phagocytosis via fibronectin receptors. 2 Uptake via C3b-receptors (CRl) interferes with production of R01. 3 Phagocytosis. 4 Phagocytosis via pathway 1 activates NADP(H)-oxidase and production of R01. Phagocytosis via pathway 2 avoids activation of NADP(H)-oxidase. 5 Low molecular weight components of mycobacteria inhibit ROI production (GL = glycolipids, ST = sulfatides, LAM = lipoarabinomannan). 6 Activation of NO-synthase and RNI production. 7 Phagosome acidification. Production of NH4 + by tubercle bacilli interferes with phagosome acidification. 8 Phagolysosomefusion. 9 Low molecular weight components of mycobacteria interfere with phagolysosome fusion. 10 Escape of tubercle bacilli to the cytoplasm is equivocal.

Cytokines in tuberculosis . 325 phenolic glycolipid inhibit production of ROI by macrophages (77-79). These results suggest that ROI are of minor importance for macrophage tuberculostasis. Generation of RNI

RNI are formed from the amino acid L-arginine by the inducible NOsynthase located in the cytoplasm of macrophages. Of the RNI, nitric oxide (NO,) has been identified as the intermediate effector molecule. The shortlived NO· reacts with itself, oxygen and water to generate nitrite (NO z-) and nitrate (N0 3-) (80-83). It has been shown before that T. gondii, L. major, Cryptococcus neoformans and S. mansoni are killed by macrophages via the production of RNI. In vitro, infection of rlFN-y-stimulated BMM with M. bovis induces the production of RNI. Inhibition of RNI production by macrophages, either by the addition of the L-arginine anologue monomethyl-L-arginine or by L-arginine depletion, reverses macrophage tuberculostatic activity (84). Similar in vitro findings were obtained by using peritoneal macrophages and M. tuberculosis (80, 85). BCG-induced peritoneal macrophages from IFN-y knock-out mice only produce low amounts of nitric oxide in response to lipopolysaccharide when compared to cells from wild-type mice (33). Since RNI are also produced by hepatocytes (86), entothelial cells (87) and fibroblasts (88) this might represent a basic mechanism of local resistance against intra- and extracellular pathogens. The role of nitric oxide in the defense of mycobacteria by human macrophages is discussed below. Phagosome acidification and phagolysosome fusion

Newly ingested viable M. tuberculosis and M. avium are found in phagosomes which are not acidic but maintain a pH which favors their multiplication. Ammonia produced by M. tuberculosis neutralizes endosomal pH (89). In contrast, killed mycobacteria are associated with acidic vesicles (90). Both, M. tuberculosis and M. avium are able to suppress phagosomelysosome fusion, and in this way evade the antimycobacterial activity of acid-dependent lysosomal enzymes (3, 91). Mechanisms which induce phagosome acidification and phagolysosome fusion should, therefore, lead to growth inhibition of mycobacteria. The lipophilic tertiary amines chloroquine, tetracaine and tributylamine are known to induce phagolysosome fusion in vitro (92). Infection of unstimulated macrophages with M. bovis in the presence of chloroquine, tetracaine or tributylamine induces significant growth inhibition of mycobacteria (75). These results show that agents which reverse the inhibition of phagolysosome fusion by mycobacteria lead to control of infection. Clearance of M. leprae also seems to be related to the induction of phagolysosome fusion. SIBLEY et al. (93) examined the intracellular fate of M. leprae organisms in normal and activated mouse peritoneal mac-

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rophages. Activation of infected macrophages with spleen cell cytokines or rIFN-y enhanced phagolysosome fusion and induced fragmentation of bacilli. Degradation of tryptophan

Human fibroblasts treated with IFN-y can suppress growth of the obligate intracellular pathogen T. gondii through the degradation of tryptophan. IFN-y induces the synthesis of the enzyme indoleamine 2,3-dioxygenase which degrades tryptophan to kynurenine, so that the intracellular parasites are probably starved of this essential amino acid (67). The IFN-y-induced growth inhibition of Chlamydia trachomatis by HEp-2 cells is also due to tryptophan degradation (68). Thus far the role of tryptophan degradation in the defense against mycobacteria by IFN-y-activated murine or human macrophages, has not been investigated. Limitation of intracellular iron

Growth of a number of bacterial pathogens, including L. monocytogenes, Legionella pneumophila and the malaria parasite, has been shown to depend on iron. The major sources of iron are iron-saturated transferrin which is phagocytosed via transferrin receptors and intracellular ferritin. Human monocytes inhibit intracellular growth of L. pneumophila by limitation of intracellular iron after activation with IFN-y. Limitation of iron is achieved by downregulation of transferrin receptors, decrease of the intracellular concentration of ferritin and iron binding by intraphagosomal lactoferrin (70). ALFORD et al. (69) have shown that mouse macrophages need iron to support listeriocidal mechanisms. Higher concentrations of iron, however, favor intracellular growth of listeriae. So far, the role of iron limitation in the resistance against tuberculosis has not been investigated. It is without doubt, however, that iron is a requirement for mycobacterial growth, and that mycobactins facilitate iron uptake by mycobacteria (94). Production of defensins

Defensins are basic polypeptides with microbicidal activities which occur in neutrophils and alveolar macrophages (71). Purified defensins are active against S. typhimurium and L. monocytogenes. Recently, it has been shown that resistance to defensins is encoded by a pag gene product (95). Although the activity of defensins against mycobacteria has not been investigated thus far a major role seems unlikely.

Cytokines in tuberculosis . 327

Regulation of IFN-y-stimulated tuberculostasis by endogenous TNF and IL-10 In infections with L. major, T. gondii, L. monocytogenes and s. mansoni, TNF-a has been identified as an endogenous effector molecule in the induction of L-arginine-dependent effector mechanisms (96-98). Recently, it has been shown that endogenous TNF-a is also involved in the defense against mycobacteria. BMM stimulated with rlFN-y for 24h express TNFa mRNA. Biologically active TNF-a is detectable in culture supernatants of BMM stimulated with rlFN-y and infected with M. bovis. To investigate the function of endogenous TNF-a in macrophage tuberculostasis neutralization of endogenous TNF-a with antibodies was employed. Stimulation of BMM with rlFN-y in the presence of anti-TNF-a antiserum had no effect on macrophage tuberculostatic activity. Addition of the antiserum during the infection of rlFN -y-stimulated BMM with M. bovis, led to significant inhibition of nitrite production and reversed intracellular growth inhibition of M. bovis (99). For the induction of macrophage tuberculostatic activity we propose the following scheme: Resident macrophage + IFN -y + M. bovis Endogenous TNF-a

1 Activated macrophage T uberculostasis IL-I0 is another cytokine which has been detected in culture supernatants of BMM stimulated with rlFN-y and infected with M. bovis (99). IL-I0, originally termed cytokine synthesis inhibitory factor, is produced by TH2 cells, B cells, mast cells and macrophages (100-102). It downregulates certain macrophage functions via the inhibition of cytokine production by T HI cells, including the production of IL-l, IL-6 and TNF, Ia antigen expression and respiratory burst activity (102, 103). In a recent study IL-I0 has been shown to inhibit the activation of macrophages for cytotoxicity against S. mansoni by blocking endogenous TNF-a production and RNI release (104). To investigate the role of IL-I0 in macrophage tuberculostatic activity, BMM were incubated with rlL-10 prior to stimulation with rlFN-y. Low doses of rlL-10 impaired the ability of BMM to produce TNF-a and nitrite after infection with M. bovis. Concomitantly, growth inhibition of M. bovis was found to be suppressed. Importantly, IL-IO had to be present at least

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KAUFMANN

2 h prior to stimulation with rIFN-y; addition of rIL-I0 after activation with rlFN -y did not suppress macrophage tuberculostatic activity as effectively as production of TNF-a and of RNI. Thus, the window for macrophage downregulation by IL-IO appears to be narrow with respect to tlmmg. These results are summarized in Figure 3. Stimulation with rIFN-y and infection with M. bovis induce the production of endogenous TNF-a as well as IL-I0. TNF-a induces a signal transduction pathway which leads to the activation of the enzyme NO-synthase and to the production of RNI. On the other hand, IL-IO impairs macrophage activation by IFN -y and, therefore, suppresses activation of NO-synthase and tuberculostatic macrophage activity. The outcome of mycobacterial infection seems to be regulated by a sensitive interplay between both cytokines (99).

The tuberculostatic activity of human monocytes and macrophages In the human system attempts to stimulate tuberculostasis have led to conflicting results. DOUVAS et al. (105) observed enhanced replication of M. tuberculosis in human macrophages after stimulation with IFN-y. ROOK et al. (106) reported that IFN -y-stimulated monocytes cause variable effects in MP from different donors ranging from significant enhancement to

IFN-y IFN-y

T~F -(t

~ ~ m RNA NO·

IL-l0m RNA \ \

L-Arginine

'-/

'---~

NO-Synthase

-- --

--

./

Figure 3. The interplay of endogenous TNF-a and IL-IO in the defense of mycobacteria. TNF-a and IL-IO are both produced by macrophages stimulated with rIFN-y and infected with M. bovis. TNF-a stimulates NO-synthase and production of RNL IL-IO inhibits activation of macrophages by IFN-y.

Cytokines in tuberculosis . 329

significant inhibition of growth of M. tuberculosis, which is in line with our own results. Monocytes from one of twelve donors inhibited growth of M. bovis after activation with rIFN-y. The monocytes of another donor enhanced growth of M. bovis after treatment with rIFN-y. Replication of M. bovis was not affected in the monocytes of the remaining ten donors (FLESCH & KAUFMANN, unpublished results). According to DENIS (107), growth of M. avium was reduced after stimulation of monocytes with IFNy in the presence of indomethacin, an inhibitor of prostaglandin synthesis. The combination of TNF and GM -CSF also induced growth inhibition of M. avium in human macrophages (59). ZERLAUTH et al. (108) showed that human monocytes stimulated with lipopolysaccharide or TNF-a inhibited growth of M. aviumlintracellulare. In a recent paper, BERMUDEZ (109) has shown that infection of human monocyte-derived macrophages with M. avium induces secretion of TGF-~, a potent immunoregulatory molecule. Treatment of macrophages with anti-TGF-~ antibodies before infection with M. avium induced intracellular killing of M. avium in response to IFN-y. Thus, the unresponsiveness of human monocytes and macrophages may be related to the production of TGF-~, which in turn inhibits macrophage activation by cytokines. Another immunomodulator which is thought to be involved in tuberculostasis is 1,25-dihydroxyvitamin D3, the biologically active form of vitamin D3 (110, 111). Since human MP possess la-hydroxylase activity, IFN-y activation of MP at the site of mycobacterial growth could result in the formation of 1,25-dihydroxyvitamin D3 from the circulating precursor 25-hydroxyvitamin D3 which then activates macrophage antimycobacterial functions. In vitro, the monocytes from some but not all donors responded to 1,25-dihydroxyvitamin D3 with growth inhibition of M. bovis (FLESCH & KAUFMANN, unpublished results). It is likely that 1,25-dihydroxyvitamin D3 acts as a costimulator of tuberculostasis in vivo, but that other molecules, such as TNF, are also involved (110). According to DENIS (112), the combination of 1,25-dihydroxyvitamin D3, IFN-y and TNF induced significant growth inhibition of M. tuberculosis H37Rv by human monocytes. Until now, the involvement of RNI in the antimicrobial activity of human monocytes and macrophages remains equivocal. Several authors reported that human monocytes and macrophages are unable to produce RNI (113, 114). Others provided circumstantial evidence for RNI production by human MP (59, 115-116). NG-monomethyl-L-arginine or arginase, two inhibitors of RNI production which reversed activation of murine macrophages to kill intracellular L. monocytogenes, had no effect on the Listeria killing by human macrophages (109a). Only DENIS (59) claimed to have demonstrated involvement of RNI in TNF-a and GM-CSF induced growth restriction of M. avium by human macrophages.

330 . 1. E. A. FLESCH and S. H. E.

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Table 3. Growth inhibition of M. bovis, M avium and M. intracellulare by BMM activated with rIFN-y. Strain

Culture conditions

3H-uracil uptake (cpm)

Growth inhibition (%)

rIFN-y

34,540 10,920

68

rIFN-y

6,000 4,900

18

rIFN-y

6,000 6,300

0

M. bovis M. avium M. intracellulare

BMM (5 x 104/assay) were stimulated with rIFN-y (2,500 U/ml). Untreated BMM were used as a control. After 24h cells were infected with viable mycobacteria (1 x 106 /assay). Intracellular survival of bacteria was determined after 4 days by 3H-uracil incorporation. Percent growth inhibition=(1- 3H-uracil uptake after culture with rIFN-y-activated BMMPH-uracil uptake after culture with nonactivated BMM) x 100.

Defense to atypical mycobacteria The atypical mycobacteria M. aviumlintracellulare (MAl -complex) are widespread throughout nature and the risk of contact is extraordinarily high. In the normal host, cellular immune mechanisms are able to eradicate invading atypical mycobacteria. In the immunocompromised host, however, infections disseminate and granulomas are not formed (117, 118). In vitro, M. avium and M. intracellulare proved to be resistant to murine macrophages stimulated with rIFN-y. As shown in Table 3, growth of M. bovis was inhibited by 68 % whereas growth of M. avium was suppressed by 18 % and multiplication of M. intracellulare was not affected at all. Possible mechanisms of resistance of MAl towards activated macrophages include (i) resistance against acid pH, (ii) resistance against RNI and (iii) inhibition of RNI production. M. avium is more resistant to acid pH than M. bovis and M. tuberculosis H37Rv. Incubation of mycobacteria at pH 5.5 reduces growth of M. bovis by 80 % and of M. tuberculosis H37Rv by 70 % compared to controls cultured at neutral pH. In contrast, growth of M. avium is not suppressed at pH 5.5. RNI can be generated in vitro by incubation of NaNO z at pH 6.5 (119). As shown in Table 4, M. avium is resistant to low concentrations of RNI. At higher concentrations growth of M. avium is suppressed to the same extent as growth of M. bovis and M. tuberculosis. Production of RNI by activated macrophages is not suppressed by M. avium (data not shown). In summary, atypical mycobacteria are more resistant to acid pH and low concentrations of RNI than M. bovis and M. tuberculosis, and this resistance may contribute to their survival in IFN-y-stimulated macrophages.

M. tuberculosis 1,700 2,200 1,100 70

M. avium

42,000 56,200 41,100 1,700

M. bovis

3,160 2,850 2,370 150

3H-uracil uptake (cpm)

10 25 95

M. bovis

2 96

M. avium

Growth inhibition (%)

35 96

M. tuberculosis

Mycobacteria (5 x lOs/assay) were incubated in medium at pH 6.5 together with NaN0 2 • After 3 days growth of bacteria was assessed by 3H-uracil uptake. Percent growth inhibition was calculated as indicated in Table 3.

0.5 5 125

NaN0 2 (ftg/ml)

Table 4. Effect of RNI on the growth of M. bovis, M. avium and M. tuberculosis H37Rv.

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Concluding remarks This treatise has attempted to discuss our current knowledge about the role of cytokines in the immune defense against tuberculosis and atypical mycobacteriosis. Emphasis was put on the murine system, which clearly demonstrates IFN-y and RNI as the important mediator or effector molecules, respectively. In contrast, the situation in the human system remains elusive. Although the central role of IFN-y is unquestioned, mediators not necessarily belonging to the immune system, but to the endocrine system, also appear essential. Furthermore, in the human system, the ultimate effector mechanism has still to be elucidated. Without doubt, certain human cells, e.g. hepatocytes, produce RNI which are so powerful in the murine system (120). It remains therefore an important goal to assess whether human macrophages fail to produce RNI (i) because of an intrinsic deficiency or (ii) because autocrine inhibitors or toxic molecules are produced during stimulation and infection. Investigations showing production of inhibitory cytokines such as TGF-~ and IL-10 and of toxic molecules which interfere with RNI synthesis would be consistent with the latter notion (99, 109). If this proves true, neutralization of such autocrine inhibitors might open up new avenues of immunotherapy of tuberculosis, which becomes increasingly important through the growing spread of multi-drug resistant tubercle bacilli. Acknowledgements Financial support from the SFB 322 and the Landesschwerpunkt «Chronische Infektionskrankheiten» is gratefully acknowledged. The authors wish to express their thanks to ANKE GRITZAN for her excellent secretarial help.

References 1. MOULDER, J. W. 1986. Comparative biology of intracellular parasitism. Microbiol. Rev. 49: 298-337. 2. SPEERT, D. P. 1992. Macrophages in bacterial infection. In: LEWIS, C. E., and J. MCGEE (eds.): The macrophage. Oxford University Press, pp. 215-263. 3. ARMSTRONG, J. A., and D. HART. 1971. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes.]. Exp. Med. 134: 713-719. 4. MYRVIK, Q. N., E. S. LEAKE, and M. J. WRIGHT. 1984. Disruption of phagosomal membranes of normal alveolar macrophages by the H37 Rv strain of Mycobacterium tuberculosis. Ann. Rev. Respir. Dis. 129: 322-328. 5. MACKANESS, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120: 105-120. 6. LANE, F. c., and E. R. UNANUE. 1972. Requirement of thymus (T) lymphocytes for resistance to listeriosis. J. Exp. Med. 135: 1104-1112. 7. NORTH, R. J. 1973. Importance of thymus-derived lymphocytes in cell-meciated immunity to infection. Cell. Immunol. 7: 166-176.

Cytokines in tuberculosis .

333

8. HAHN, H., and S. H. E. KAUFMANN. 1981. Role of cell-mediated immunity in bacterial infections. Rev. Infect. Dis. 3: 1221-1250. 9. BLOOM, B. R., and B. BENNETT. 1966. Mechanism of reaction in vitro associated with delayed -type hypersensitivity. Science 153:: 80-82. 10. DAVID, J. R. 1966. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc. Natl. Acad. Sci. USA 56: 72-77. 11. KAUFMANN, S. H. E. 1993. Immunity to intracellular bacteria. Ann. Rev. Immunol. 11: 129-163. 12. KAUFMANN, S. H. E. 1993. Immunity to intracellular bacteria. In: PAUL, W. E. (ed.): Fundamental Immunology, 3rd edition. Raven Press, New York, pp. 1251-1286. 13. KAUFMANN, S. H. E. 1988. CD8+ T lymphocytes in intracellular microbial infections. Immunol. Today 9: 168-174. 14. MOSMANN, T R., and K. L. COFFMAN. 1989. TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7: 145-173. 15. MOSMANN, T R., and K. W. MOORE. 1991. The role of IL-1 0 in crossregulation of TH1 and TH2 responses. Immunol. Today 12: A49-A53. 16. DELIBERO, G., I. FLESCH, and S. H. E. KAUFMANN. 1988. Mycobacteria reactive Lyt2+ T cell lines. Eur. J. Immunol. 18: 59-66. 17. FOLLOWS, G. A., M. E. MUNK, A. J. GATRILL, P. CONRADT, and S. H. E. KAUFMANN. 1992. Interferon-y and IL-2 but no detectable interleukin 4 in y/o T cell cultures after activation with bacteria. Infect. Immun. 60: 1229-1231. 18. REES, A. D. M., A. SCOGING, A. MEHLERT, D. B. YOUNG, and J. IVANYI. 1988. Specificity of proliferative response of human CD8 clones to mycobacterial antigens. Eur. J. Immunol. 18: 1881-1887. 19. SHER, A., and R. L. COFFMAN. 1992. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Ann. Rev. Immunol. 10: 385-410. 20. PEDRAZZINI, T, and J. A. LOUIS. 1986. Functional analysis in vitro and in vivo of Mycobacterium bovis strain BCG-specific T cell clones. J. Immunol. 136: 1828-1834. 21. KAUfMANN, S. H. E., and 1. E. A. FLESCH. 1986. Function and antigen recognition pattern of L3T4+ T cell clones from Mycobacterium tuberculosis-immune mice. Infect. Immun. 54: 291-296. 22. BLOOM, B. R., R. L. MODLIN, and P. SALGAME. 1992. Stigma variations: observations on suppressor T cells and leprosy. Ann. Rev. Immunol. 10: 453-488. 23. ADAMS, D.O., and T A. HAMILTON. 1984. The cell biology of macrophage activation. Ann. Rev. Immunol. 2: 283-318. 24. MCCABE, R. E., B. J. LUFT, and J. S. REMINGTON. 1984. Effect of murine interferonyon murine toxoplasmosis. J. Infect. Dis. 150: 961-962. 25. NATHAN, C. F., H. W. MURRAY, M. E. WIEBE, and B. Y. RUBIN. 1993. Identification of interferon-y as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158: 670-689. 26. ROTHERMEL, C. D., B. Y. RUBIN, and H. W. MURRAY. 1983. Interferon-y is the factor in lymphokines that activates human macrophages to inhibit intracellular Chlamydia psittaci replication. J. Immunol. 131: 2542-2548. 27. TURCO, J., and H. H. WINKLER. 1983. Comparison of the properties of antirickettsial activity and interferon in mouse lymphokines. Infect. Immun. 42: 27-34. 28. BUCHMEIER, N. A., and R. D. SCHREIBER. 1985. Requirement of endogenous interferon-y-production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. USA 82: 7404-7408. 29. ROOK, G. A. W., ]. STEELE, M. R. AINSWORTH, and B. R. CHAMPION. 1986. Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis:

334 . 1. E. A. FLESCH and S. H. E. KAUFMANN comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages. Immunol. 59: 333-338. 30. FLESCH, K., and S. H. E. KAUFMANN. 1987. Mycobacterial growth inhibition by interferon-y-activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138: 4408-4413. 31. DENIS, M. 1991. Involvement of cytokines in determining resistance and acquired immunity in murine tuberculosis. J. Leuk. BioI. 50: 495-501. 32. BANERJEE, D. K., A. K. SHARP, and D. B. LOWRIE. 1986. The effect of gammainterferon during Mycobacterium bovis (BCG) infection in athymic and euthymic mice. Microb. Pathogen. 1: 221-224. 33. DALTON, K. D., S. PITTS-MEEK, S. KESHAV, I. S. FIGARI, A. BRADLEY, and T. A. STEWART. 1993. Multiple defects of immune cell function in mice with disrupted interferon-y genes. Science 259: 1739-1742. 34. WALLIS, R. S., M. AMIR-TAHMASSEB, and J. J. ELLNER. 1990. Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte Western blot. Proc. Nat!. Acad. Sci. USA 87: 3348-3352. 35. OGAWA, T., H. UCHIDA, Y. KUSUMOTO, Y. MORI, Y. YAMAMURA, and S. HAMADA. 1991. Increase in tumor necrosis factor-a and interleukin-6-secreting cells in peripheral blood mononuclear cells from subjects infected with Mycobacterium tuberculosis. Infect. Immun. 59: 3021-3025. 36. MORENO, C. J., J. TAVERNE, A. MEHLERT, C. A. W. BATE, R. J. BREALEY, A. MEAGER, G. A. W. ROOK, andJ. H. L. PLAYFAIR. 1989. Lipoarabinomannan from Mycobacterium tuberculosis induces the production of tumor necrosis factor from human and murine macrophages. Clin. Exp. Immunol. 76: 240-245. 37. BARNES, P. F., S.-J. FONG, P. BRENNAN, P. E. TWOMEY, A. MAZUMDER, and R. L. MODLIN. 1990. Local production of tumor necrosis factor and IFN-y in tuberculous pleuritis. J. Immunol. 145: 149-154. 38. FLESCH, I. E. A., and S. H. E. KAUFMANN. 1990. Activation of tuberculostatic macrophage functions by interferon-y, interleukin-4 and tumor necrosis factor. Infect. Immun. 58: 2675-2677. 39. ESPARZA, 1., D. MANNEL, A. RUPPEL, W. FALK, and P. H. KRAMMER. 1987. Interferon-y and lymphotoxin or tumor necrosis factor-a act synergistically to induce macrophage killing of tumor cells and schistosomula of Schistosoma mansoni. J. Exp. Med. 166: 589-594. 40. LIEw, F. Y., Y. LI, and S. MILLOT. 1990. TNF-a synergizes with IFN-y in mediating killing of Leishmania major through the induction of nitric oxide. J. Immunol. 145: 4306-4310. 41. KINDLER, V., A.-P. SAPPINO, G. E. GRAY, P.-F. PIGNET, and P. VASSALLI. 1989. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56: 731-740. 42. VASSALLI, P. 1992. The pathophysiology of tumor necrosis factors. Ann. Rev. Immunol. 10: 411-452. 43. PAUL, W. E., and J. OHARA. 1987. B-cell stimulatory factor-1/interleukin 4. Ann. Rev. Immunol. 5: 429-459. 44. CRAWFORD, R. M., D. S. FINBLOOM, J. OHARA, W. E. PAUL, and M. S. MELTZER. 1987. B cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J. Immunol. 139: 135-141. 45. ZLOTNIK, A., M. FISCHER, N. ROEHM, and D. ZIPORI. 1987. Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages. J. Immunol. 138: 4275-4279.

Cytokines in tuberculosis .

335

46. STUART, P. M., A. ZLOTNIK, and]. G. WOODWARD. 1988. Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4 (B cell stimulatory factor 1). J. Immuno!. 140: 1542-1547. 47. TEPPER, R. 1., P. K. PATTENGALE, and P. LEDER. 1989. Murine interleukin-4 displays potent anti-tumor activity in vivo. Cell 57: 503-512. 48. Mc INNES, A., and D. M. RENNICK. 1988. Interleukin 4 induces cultured monocytes/macrophages to form giant multinucleated cells. J. Exp. Med. 167: 598-611. 49. HAAK-FRENDSCHO, M., J. F. BROWN, Y. IIAZAWA, R. D. WAGNER, and C. J. CZUPRYNSKI. 1992. Administration of anti- IL-4 monoclonal antibody 11B11 increases the resistance of mice to Listeria monocytogenes infection. J. Immuno!. 148: 3978-3985. 50. KISHIMOTO, T., and T. HIRANO. 1988. Molecular regulation of B lymphocyte response. Ann. Rev. Immuno!. 6: 485-512. 51. SNICK, J. VAN. 1990. Interleukin-6. An overview. Ann. Rev. Immuno!. 8: 253-278. 52.0'GARRA, A., S. UMLAND, T. DEFRANCE, and J. CHRISTIANSEN. 1988. «B-cell factors» are pleiotropic. Immuno!. Today 9: 45-54. 53. HELFGOTT, D. c., S. B. TATTER, U. SANTHANAM, R. H. CLARICK, N. BHARDWAJ, L. T. MAY, and P. B. SEHGAL. 1989. Multiple forms of IFN-~2/IL-6 in serum and body fluids during acute bacterial infection. J. Immuno!. 142: 948-953. 54. FLESCH, 1. E. A., and S. H. E. KAUFMANN. 1990. Stimulation of antibacterial macrophage activities by B cell stimulatory factor 2/interleukin-6. Infect. Immun. 58: 269-271. 55. JEEVAN, A., and G. L. ASHERSON. 1988. Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice. Infect. Immun. 56: 660-664. 56. KAUFMANN, S. H. E. 1993. Cell-mediated immunity. In: HASTINGS, R. C. (ed.): Leprosy, 2nd edition. Edinburgh, in press. 57. FRIEDLAND, J. S., D. G. REMICK, R. SHATTOCK, and G. E. GRIFFIN. 1992. Secretion of interleukin-8 following phagocytosis of Mycobacterium tuberculosis by human monocyte cell lines. Eur. J. Immuno!. 22: 1373-1378. 58. OPPENHEIM, J. J., C. o. C. ZACHARIAE, N. MUKAIDA, and K. MATSUSHIMA. 1991. Properties of the novel proinflammatory supergene "intercrine» cytokine family. Ann. Rev. Immuno!. 9: 617-648. 59. DENIS, M. 1991. Tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: Killing effector mechanism depends on the generation of reactive nitrogen intermediates. J. Leuk. Bio!. 49: 380-387. 60. BERMUDEZ, L. E., and L. S. YOUNG. 1990. Killing of Mycobacterium avium: insights provided by the use of recombinant cytokines. Res. Microbio!. 141: 241-245. 61. ANDREW, P. W., P. S. JACKETT, and D. B. LOWRIE. 1985. Killing and degradation of microorganisms by macrophages. In: DEAN, R. T., and W. JESSOP (eds.): Mononuclear Phagocytes: Physiology and Pathology. Elsevier Biomedical Press, Amsterdam, pp. 311-335. 62. BABIOR, B. M. 1984. Oxidants from phagocytes: agents of defense and destruction. Blood 64: 959-966. 63. HIBBS,JR,J. B., R. R. TAINTOR, Z. VAVRIN, andE. M. RACHLIN. 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 1987: 87-94. 64. LIEw, F. Y., and F. E. G. Cox. 1990. Non-specific defence mechanism: the role of nitric oxide. Immuno!. Today 12: A17-A21. 65. MARLETTA, M. A. 1989. Nitric oxide: biosynthesis and biological significance. TIES 14: 488-492.

336 . I. E. A. FLESCH and S. H. E. KAUFMANN 66. HORWITZ, M. A. 1988. Intracellular parasitism. Curr. Opin. Immuno!. 1: 41-46. 67. PFEFFERKORN, E. R. 1984. Interferon-y blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc. Nat!. Acad. Sci. USA 81: 908-912. 68. SHEMER, Y., R. KOL, and I. SAROV. 1987. Tryptophan reversal of recombinant human gamma-interferon inhibition of Chlamydia trachomatis growth. Curr. Microbio!. 16: 9-13. 69. ALFORD, C. E., T. E. KINGJR, and P. A. CAMPBELL. 1991. The role of transferrin, transferrin receptors, and iron in macrophage listericidal activity. J. Exp. Med. 174: 459-466. 70. BYRD, T. F., and M. A. HORWITZ. 1991. Chloroquine inhibits the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin. Invest. 88: 351-357. 71. LEHRER, R. I, T. GANZ, and M. E. SELSTED. 1991. Defensins: endogenous antibiotic peptides of animal cells. Cell 64: 229-230. 72. JACKETT, P. S., V. R. ABER, D. A. MITCHISON, and D. B. LOWRIE. 1981. The contribution of hydrogen peroxide resistance to virulence of Mycobacterium tuberculosis during the first six days after intravenous infection of normal and BCGvaccinated guinea pigs. Br. J. Exp. Patho!' 62: 34-40. 73. JACKETT, P. S., P. W. ANDREW, V. R. ABER, and D. B. LOWRIE. 1983. Guinea pig alveolar macrophages probably kill M. tuberculosis H37Rv and H37Ra in vivo by producing hydrogen peroxide. Adv. Exp. BioI. 162: 99-104. 74. WALKER, L., and D. B. LOWRIE. 1981. Killing of Mycobacterium microti by immunologically activated macrophages. Nature 293: 69-70. 75. FLESCH, I., and S. H. E. KAUFMANN. 1988. Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by interferon-y activated bone marrow macrophages. Infect. Immun. 56: 1464-1469. 76. SCHLESINGER, L. S., C. G. BELLINGER-KAWAHARA, N. R. PAYNE, and M. A. HORWITZ. 1990. Phagocytosis of Mycobacterium tuberculosis is mediated by human complement receptors and complement component C3. J. Immuno!. 144: 2771-2780. 77. ZHANG, L., M. B. GOREN, T. J. HOLZER, and B. R. ANDERSEN. 1988. Effect of Mycobacterium tuberculosis-derived sulfolipid I on human phagocytic cells. Infect. Immun. 56: 2876-2883. 78. PABST, M. J., J. M. GROSS, J. P. BROZNA, and M. B. GOREN. 1988. Inhibition of macrophage priming by sulfatide from Mycobacterium tuberculosis. J. Immunol. 140: 634-640. 79. SIBLEY, L. D., S. W. HUNTER, P. J. BRENNAN, and J. L. KRAHENBUHL. 1988. Mycobacteriallipoarabinomannan inhibits y-interferon-mediated activation of macrophages. Infect. Immun. 56: 1232-1236. 80. CHAN, J., Y. XING, R. S. MAGLIOZZO, and B. R. BLOOM. 1992. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J. Exp. Med. 175: 1111-1122. 81. MARLETTA, M. A., P. S. Yocm, R.IYENGAR, C. D. LEAF, andJ. S. WISHNOK. 1988. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochem. 27: 8706-8711. 82. DING, A. H., C. F. NATHAN, and D. J. STUEHR. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J. Immunol. 141: 2407-2412. 83. GREEN, L. c., D. A. WAGNER, J. GLOGOWSKI, P. L. SKIPPER, Y. S. WISHNOK, and S. R. TANNENBAUM. 1982. Analysis of nitrate, nitrite, and eSN) nitrate in biological fluids. Anal. Biochem. 126: 131-138.

Cytokines in tuberculosis .

337

84. FLESCH, I. E. A., and S. H. E. KAUFMANN. 1991. Mechanisms involved in mycobacterial growth inhibition by y-interferon activated bone marrow macrophages: role of reactive nitrogen intermediates. Infect. Immun. 59: 3213-3218. 85. DENIS, M. 1991. Interferon-y treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell. Immunol. 132: 150-157. 86. CURRAN, R. D., T. R. BILLIAR, D. J. STUEHR, K. HOFMANN, and R. L. SIMMONS. 1989. Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells. J. Exp. Med. 170: 1769. 87. PALMER, R. M. J., D. S. ASHTON, and S. MONCADA. 1988. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664-666. 88. AMBER, 1. J., J. B. HIBBS JR, R. R. TAINTOR, and Z. V AVRIN. 1988. Cytokines induce an L-arginine-dependent effector system in nonmacrophage cells. J. Leuk. BioI. 44: 58-65. 89. D'ARCY HART, P., and M. R. YOUNG. 1991. Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosomeendosome fusion and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J. Exp. Med. 174: 881-889. 90. CROWLE, A. J., R. DAHL, E. Ross, and M. H. MAY. 1991. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect. Immun. 59: 1823-1831. 91. FREHEL, c., J. CHASTELLIER, T. LANG, and N. RASTOGI. 1986. Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium. Infect. Immun. 52: 252-262. 92. D'ARCY HART, P., and M. R. YOUNG. 1978. Manipulation of the phagosomelysosome fusion response in cultured macrophages. Enhancement of fusion by chloroquine and other amines. Exp. Cell. Res. 114: 486-490. 93. SIBLEY, L. D., S. G. FRANZBLAU, and J. L. KRAHENBUHL. 1987. Intracellular fate of Mycobacterium leprae in normal and activated mouse macrophages. Infect. Immun. 55: 680-685. 94. RATLEDGE, c., andJ. STANFORD (eds.). 1982. The biology of the mycobacteria, Vol. 1. Academic Press, pp. 242-248. 95. MILLER, S. 1., A. M. KUKRAL, and J. J. MEKALANOS. 1989. A two-component regulatory system (pho P, pho Q) controls Salmonella typhimurium virulence. Proc. Nat!. Acad. Sci. USA 86: 5054-5058. 96. GREEN, S. J., R. M. CRAWFORD, J. T. HOCKMEYER, M. S. MELTZER, and C. NACY. 1990. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-y-stimulated macrophages by induction of tumor necrosis factor-a. J. Immunol. 145: 4290-4297. 97. LANGERMANS, J. A. M., M. E. B. VAN DER HULST, P. H. NIBBERING, P. S. HIEMSTRA, L. FRANSEN, and R. VAN FURTH. 1992. IFN-y-induced L-argininedependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-a. J. Immunol. 148: 568-574. 98. NAKANE, A., T. MINAGAWA, M. KOHANAWA, Y. CHEN, H. SATO, M. MORIYAMA, and N. TSURNOKA. 1989. Interactions between endogenous y-interferon and tumor necrosis factor in host resistance against primary and secondary Listeria monocytogenes infections. Infect. Immun. 57: 3331-3337. 99. FLESCH, 1. E. A., J. H. HESS, and S. H. E. KAUFMANN. 1993. Growth inhibition of Mycobacterium bovis by interferon-y stimulated macrophages: regulation by endogenous tumor necrosis factor and interleukin 10 (submitted). 100. O'GARRA, A., G. STAPLETON, V. DHAR, M. PEARCE,J. SCHUMACHER, H. RUGO, D. BAREIS, A. STALL, J. CuPP, and K. MOORE. 1990. Production of cytokines by mouse

338 . 1.

E. A. FLESCH and S. H. E. KAUFMANN

B cells: B lymphomas and normal B cells produce interleukin 10. Int. Immunol. 2: 821-832. 101. VIEIRA, P., R. DE WAAL MALEFYT, M.-N. DANG, K. E. JOHNSON, R. KASTELEIN, D. F. FIORENTINO, J. E. DE VRIES, M.-G. RONCAROLO, T. R. MOSMANN, and K. W. MOORE. 1991. Isolation and expression of human cytokine synthesis inhibitory factor (CSIF/IL-I0) cDNA clones: homology to Epstein-Barr virus open reading brame BCRF1. Proc. Nat!. Acad. Sci. USA 88: 1172-1176. 102. WAAL MALEFYT, R. DE, J. ABRAMS, B. BENNETT, C. G. FIGDOR, and J. E. DE VRIES. 1991. Interleukin 10 (IL-I0) inhibitis cytokine synthesis by human monocytes: an autoregulatory role of IL-I0 produced by monocytes. J. Exp. Med. 174: 1209-1220. 103. BOGDAN, c., Y. VODOVOTZ, and C. NATHAN. 1991. Macrophage deactivation by interleukin 10. J. Exp. Med. 174: 1549-1555. 104. OSWALD, P. 1., T. A. WYNN, A. SHER, and S. L. JAMES. 1992. Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor-a required as a costimulatory factor for interferon y-induced activation. Proc. Nat!. Acad. Sci. USA 89: 8676-8680. 105. DOUVAS, G. S., D. L. LOOKER, A. E. VATTER, and A. J. CROWLE. 1985. Gammainterferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect. Immun. 50: 1-8. 106. ROOK, G. A. W., J. STEELE, L. FRAHER, S. BARKER, R. KARMALI, J. O'RIORDAN, and J. STANFORD. 1986. Vitamin D3, y-interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunol. 57: 159-163. 107. DENIS, M. 1991. Growth of Mycobacterium avium in human monocytes: identification of cytokines which reduce and enhance intracellular microbial growth. Eur. J. Immunol. 21: 391-395. 108. ZERLAUTH, G., M. M. EIBL, and J. W. MANNHALTER. 1991. Induction of antimycobacterial and anti-listerial activity of human monocytes requires different activation signals. Clin. Exp. Immunol. 85: 90-97. 109. BERMUDEZ, L. E. 1993. Production of transforming growth factor-~ by Mycobacterium avium-infected human macrophages is associated with unresponsiveness to IFN-y. J. Immunol. 150: 1838-1845. 109a. BERMUDEZ, L. E. 1993. Differential mechanisms of intracellular killing of Mycobacterium avium and Listeria monocytogenes by activated human and murine macrophages. Clin. Exp. Immunol. 91: 277-281. 11 O. ROOK, G. A. W., J. TAVERNE, C. LEVETON, and J. STEELE. 1987. The role of gamma interferon, vitamin D3 metabolites and tumor necrosis factor in the pathogenesis of tuberculosis. Immunology 62: 229-234. 111. CROWLE, A. H., E. J. Ross, and M. H. MAY. 1987. Inhibition by 1,25 (OHhvitamin D3 of the multiplication of virulent tubercle bacilli in cultured human macrophages. Infect. Immun. 55: 2945-2950. 112. DENIS, M. 1991. Killing of Mycobacterium tuberculosis within human monocytes: activation by cytokines and calcitriol. Clin. Exp. Immunol. 84: 200-206. 113. MURRAY, H. W., and R. F. TEITELBAUM. 1992. L-arginine-dependent reactive nitrogen intermediates and the antimicrobial effect of activated human mononuclear phagocytes. J. Infect. Dis. 165: 513-517. 114. PADGETT, E. L., and S. B. BRUETT. 1992. Evaluation of nitrite production by human monocyte-derived macrophages. Biochem. Biophys. Res. Commun. 186: 775-781. 115. SHERMAN, M. P., M. L. LORO, V. Z. WONG, and D. P. TASHKIN. 1991. Cytokineand Pneumocystis carinii-induced L-arginine oxidation by murine and human pulmonary alveolar macrophages. J. Protozool. 38: 234S-236S. 116. MUNOZ-FERNANDEZ, M. A., M. A. FERNANDEZ, and M. FRESNO. 1992. Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-a

Cytokines in tuberculosis .

339

and IFN-y through a nitric oxide-dependent mechanism. Immunol. Letters. 33: 35--40. 117. ANON. 1986. Diagnosis and management of mycobacterial infection and disease in persons with human T Iymhotropic virus type IIIIlymphadenopathy-associated virus infection. MMWR 35: 448--451. 118. Centers for Disease Control. 1987. Diagnosis and management of mycobacterial infection and disease in persons with human immunodeficiency virus infection. Ann. Intern. Med. 106: 254-256. 119. STUEHR, D. J., and C. F. NATHAN. 1989. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169: 1543-1555. 120. NUSSLER, A. K., M. DI SILVIO, T. R. BILLIAR, R. A. HOFFMANN, D. A. GELLER, R. SELBY, J. MADARIAGA, and R. L. SIMMONS. 1992. Stimulation of nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J. Exp. Med. 176: 261-264.

Dr. INGE E. A. FLESCH, Department of Immunology, University of Ulm, AlbertEinstein-Allee 11, 89081 Ulm, Germany