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Histol Histopathol (2000) 15: 199-205 and 001 : 10.14670/HH-15.199 http://www.hh.um.es Histopathology Cellular and Molecular

Invited Review

Current understanding of type 1 responses during intracellular infections

Z. Xing Department cf Pathology and Molecular Medicine, and Division cf Infectious Diseases, Centre for Gene Therapeutics, McMaster University ,Hamilton, Ontario, Canada

Summary. are important effector cells in macrophages which is in turn dependent upon type 1 cell-mediated against intracelllular infection. , particularly IFNy, released by activated Among cytokines that macrophages are able to release -specific T cells. However, recent evidence from are IL-12 and TNFa. IL-12 is a critical linker between us and others indicates that activated macrophages the innate and adaptive cell-mediated immunity, capable themselves can be a significant source of IFNy in of T h I differentiation and IFNy release by T and NK addition to other type I cytokines, during intracellular cells. IFNy is critically required for the activation of infection. This thus suggests that macrophages may play macrophage bactericidal activities. Recently emerging a much greater role than previously thought in ceII­ evidence suggests that macrophages are able to release mediated immunity. Furthermore, it also suggests that not only IL-12 and TNFa but also IFNy. However, the the critical role by T cells in host defense against mechanisms that control the release of each of these type intracellular infections goes beyond just the provision to I cytokines in macrophages appear different. While macrophages of soluble activation signals such as IFNy, macro phages release TNFa in an indiscriminate and IL- which we know now, can be at least in part fulfilled by 12-independent way, the release of IL-12, particularly rnacrophages themselves (Fenton et aI., 1997; Munder et bioactive IL-12 p70, and IFNy is under tight control. We aI. , 1998; Wang et aI., 1999). Emerging evidence are just beginning to understand what controls the supports a critical role of T cells in host resistance to release of IL-12 p70, a question of fundamental intracellular infection via their cytotoxic activities importance to understanding the mechanisms underlying (Bonecini-Almeida et aI., 1998; Oddo et aI., 1998). We the initiation of cell-mediated immunity. Our recent are just in the beginning to understand the regulatory findings have shed more insights into the regulatory mechanisms of macrophage activation and the mechanisms of macrophage IFNy responses. It has interaction between macrophages and T cells involved in become evident that IL-12 is required not only for T h I anti-intracellular infection immune responses. Further differentiation but also for IFNy responses by both T understanding in this regard will undoubtedly foster the cells and macrophages during intracellular infection. In development of better and medicine for this overview, we have discussed about the current intracellular infectious diseases. understanding of the regulation of macrophage type I cytokine responses during intracellular infection, based Type 1 and type 2 cytokines upon the recent findings from us and others. Cytokines that are involved in adaptive immunity Key words: Macrophages, , Mycobacteria, can be divided into Thl and Th2 categories, depending Cytokines, Intracellular infection on whether they are released primarily by murine T h I or Th2 clones (Mosmann and Sad, 1996). The Thl cytokines typically include IL-2 and IFNy whereas Th2 Introduction cytokines include IL-4 and IL-S. However, it is known that a number of cytokines can be released by both Th I Many microbes are obligate intracellular and Th2 clones and these include IL-3, GM-CSF and of macrophages. It is generally believed that eradication TNFa. Furthermore, there are cytokines such as IL-12 of these pathogens lies in adequate activation of and IL-18 that are not released primarily by either Th clone but are critical to Thl differentiation and/or Thl Offprint requests to: Zhou Xing , MD. Ph.D, Health Sciences Centre, Rm. cytokine production. It is of help to group these immune­ 4H19, Department of Pathology and Molecular Medicine, McMaster modulatory cytokines according to the type of tissue University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, responses they mediate (Xing et aI., 1999). The type I Canada. Fax: 905-522·6750. e·mail: [email protected] cytokines include IL-12, IFNy and TNFa which are 200 Type I cytokine production by macrophages

involved in the development of cell-mediated immunity mycobacterial infection still remains to be completely or type 1 tissue responses characterized by macrophage understood, we and others have recently provided formation and activation. Type 1 tissue experimental evidence to support a critical role of each responses are essential to host defense against of these cytokines in host defense against mycobacterial intracellular infections by , protozoan parasites infection. Mice deficient in IL-12 lacked IFNy and or fungi. The type 2 cytokine category encompasses IL- TNFa responses, granuloma formation and suffered an 4, IL-5, IL-3 and GM-CSF which play an important role increased susceptibility to intracellular infections in the development of type 2 tissue responses. Type 2 established via a systemic or local pulmonary route tissue responses usually involve both humoral and cell (Mattner et aI., 1996; Cooper et aI., 1997; Wakeham et immune components, characterized by increased aI., 1998; Xing et aI., 1998a). Mice deficient in IFNy or antigen-specific IgE/IgG1, , and TNFa suffered a similarly weakened immune protection and T cell activation, and they underlie the pathogenesis from mycobacterial infection (Cooper et aI., 1993; Flynn of allergic diseases and play an important role in host et aI., 1993,1995). Very recently, mice deficient in IL-18 responses to extracellular metazoan parasitic infection. have also been found to have impaired Th1 responses to In other instances, although the characteristic tissue mycobacterial infection (Takeda et aI., 1998). Since responses may not be a feature, type 1 or type 2 unlike IL-12, IL-18 seems to have no direct effect on cytokines constitute part of molecular mechanisms of Th1 differentiation, the mechanisms by which IL-18 host responses. These include organ-specific auto­ contributed to the overall Th1 responses remain fully immune diseases, allograft rejection and recurrent understood. Of particular interest, recent findings from abortion (type 1 responses), and successful pregnancy us (unpublished data) and others (Oxen ius et aI., 1999) (type 2 responses) (Romagnani, 1997). suggest that in sharp contrast to intracellular bacterial infection, IL-12 is not required in a significant manner Type 1 cytokines and mycobacterial infection for type 1 immune responses to local lung or systemic viral infection in vivo. IL-12 is released from activated macrophages or The critical role of these type 1 cytokines in host dendritic cells and drives a unique Th1 phenotypic resistance to intracellular infections by mycobacteria or differentiation and IFNy release from antigen-specific T other intracellular pathogens has been further supported cells. IL-12 also stimulates NK cells to release IFNy. by recent findings in humans. Patients with a genetic Bioactive IL-12 (IL-12 p70) is a heterodimeric cytokine deficiency in the IFNy or IL-12 receptor have increased composed of p35 and p40 subunits, encoded by two susceptibility to atypical mycobacterial infections or separate genes (Trinchieri, 1997; Okamura et aI., 1998). developing systemic tuberculous infection following The p35 subunit is constitutively expressed and remains BCG vaccination (Jouanguy et aI., 1996; Altare et aI., intracellular in a wide range of cell types including 1998a). Likewise, hosts genetically deficient in the gene macrophages while it can be further induced under coding for IL-12 suffer disseminated BCG and inflammatory and immune conditions. In comparison, infection (Altare et aI., 1998b). expression of the p40 subunit is highly regulated and is expressed primarily by macrophages and dendritic cells. Regulation of IL-12 responses in macrophages and Normally, cells express the p40 subunit at a level 10 to dendritic cells during intracellular infection 100 higher than the p35 subunit, thus resulting in a similarly greater secretion of IL-12 p40 monomers IL-12 is released primarily by macrophages and and/or homodimers than IL-12 p70 heterodimers. IL-12 dendritic cells and has been believed to act primarily p40 has been found to be, particularly in rodents, upon T and NK cells. IL-12 thus serves as an important antagonistic to bioactive IL-12 p70 by competing for the linker between the innate and adaptive cell-mediated IL-12 receptor. IFNy has long been considered to be immunity. However, macrophages or dendritic cells primarily a T cell-derived cytokine and is a key constitute the first defense line confronting almost any macrophage activator capable of markedly enhancing invading organisms/ or stimuli and yet, the host MHC molecule expression and bactericidal activities of does not always mount a cell-mediated immune macrophages in addition to its role in Th1 differentiation response. Thus, IL-12 and the nature of antigens likely and activation (Orme et aI., 1993). TNFa is released operate as a crucial check point to determine whether a primarily from stimulated macrophages but can also be type 1 should result. So little is known released from activated T cells. TNFa has broad about exactly how the host makes such a decision at this stimulatory effects on a variety of cell types including point. Conceivably, whether IL-12 is released upon macrophages and endothelial cells. We have found that encounter of macrophages/dendritic cells with organisms all of these type 1 cytokines but not type 2 cytokines IL- or antigens or any other stimuli, which form of IL-12, 4 and GM-CSF are elevated both in the lung and p40 or bioactive p70, is released, how sustained the level peripheral during pulmonary mycobacterial of IL-12 p70 is, and whether there is active antigen infection (Wakeham et al., 1998). presentation by these cells, all playa part in host's While the relative importance of type 1 cytokines decision-making (Fig. 1). Therefore, it is of particular IL-12, IFNy and TNFa in host defense against importance to understand the regulation of I L-12, 201 Type I cytokine production by macrophages

particularly IL-12 p70 in macrophages and dendritic comparison, dead , latex beads and cells. amastigotes cannot effectively induce IL-12 release The majority of studies have been carried out in (Skeen et aI., 1996; Weinheber et aI., 1998). These vitro or in vivo by examining either gene expression or findings convey a clear message that unlike other total IL-12 protein (p40 and p70) or only p40 protein such as TNFa, IL-1 and IL-6, IL-12 release release by cells, and little is known about the relative is under tight control in macrophages/ (Fig. level of IL-12 p40 and bioactive IL-12 p70. IL-12 is 1). It is worth noting that other macrophage agonists elevated at the tissue sites or by peripheral blood have not been tested for comparison and whether IL-12 monocytes in patients with or p70 is released under all of these conditions is still (Zhang et aI. , 1994). We and others have observed unclear. In this regard, a very recent study carried out by markedly released or expressed IL-12 during pulmonary using human monocytes and alveolar macrophages has or systemic mycobacterial infection in mice (Cooper et demonstrated that while LPS up-regulates expression of ai., 1997; Wakeham et ai., 1998; Xing et ai., 1998a). We both IL-12 p35 and p40 genes, it induces the release of have further demonstrated that lung macrophages are a only IL-12 p40 but not IL-12 p70 and, IL-lO release significant source of IL-12 during pulmonary myco­ stimulated by LPS is found accountable for such an bacterial infection and these macrophages release further inhibition of IL-12 p70 release (Isler et ai., 1999). These increased amounts of IL-12 upon stimulation by LPS or important findings suggest that whether endogenous IL- mycobacterial antigens PPD (Wang et ai. , 1999). Most, 10 release is induced is one mechanism of inhibitory but not all, of intracellular pathogens including both nature that controls the form of IL-12 released by macro­ bacteria and or microbial products have been phages. We are currently investigating the regulation of found capable of releasing IL-12 from macrophages in IL-12 release in models of LPS- or Gram-negative vitro. Live tuberculosis or BCG, live bacterium-induced acute lung . Listeria, LPS and peptidoglycans from certain types of The positive signals, in addition to microbial agents, bacteria are inducers of total IL-12 release from that are required for active IL-12 release by macro­ macrophages (Flesch et aI., 1995; Skeen et aI. , 1996; phages, have remained to be completely understood. It Ladel et aI. , 1997; Lawrence and Nauciel, 1998). In has been believed that IL-12 is the first cytokine released from macrophages which ignites the downstream events of cascade including IFNy responses in cell-mediated immunity. However, recently emerging experimental Non-immtme stressors Mycobacteria evidence has challenged this notion and suggested far Acute bacterial infection Other intracellular pathogens more complicated regulatory mechanisms involved in ! ! the up-regulation of IL-12. It has been noticed that the presence of NK cells or activated T cells facilitates IL- ~. . -.' .. 12 release or gene expression in human macrophages/ monocytes infected with BCG or Cryptococcus / 1°\ / neoformans (Matsumoto et ai., 1997; Retini et aI., 1999). In addition to potential soluble molecules such as IFNy IL-8 N o IL-12 TNFa IL-12p70 or only that may have played a role, the interaction between lL- 12p40 CD40 on macrophages and CD40 ligand on T cells was ! found to play a role (Retini et ai., 1999). The role of

'K Tbl IFNy in IL-12 induction in macrophages has been N'. ' ,CDSj. CD4 speculated for quite some time. While the data are still ;­ ! ! , conflicting, IFNy has been shown to have either potent Tissue IFNy effects (Matsumoto et ai., 1997; Jiang and Dhib­ Fig. 1. Schematic presentation of the mechanisms that determine when Jalbut, 1998) or stimulatory effects on IL-12 release a type 1 cell -mediated immune response will result. Upon encounter from macrophages (Isler et ai., 1999; Retini et ai., 1999). with non -immune stressors or bacteria that normally cause an acute Priming by rIFNy was found to be required for BCG- or inflammatory response, macrophages do not release bioactive IL-12 p70 . Released such as IL-8 and pro­ LPS-induced IL-12 p40 release by -marrow-derived inflammatory cy10kine TNFo will orchestrate the development of tissue macrophages (Flesch et aI., 1995). Furthermore, in mice neutrophilia and macrophage/neutrophil activation. Such a tissue deficient in receptors for either IFNy or TNFa, IL-12 response is transient. only involving the innate immune components. p40 release was found dependent upon the function of However, upon encounter with intracellular pathogens and perhaps either IFNy or TNFa (Flesch et aI., 1995). Although all when co-stimulated by IFNy that is released from NK cells via an IL·12- of these studies have restrictions in various ways, they independent manner, macrophages release bioactive IL-12 p70. On one hand. this macrophage-derived I L-12 drives Th 1 differentiation and IFN y collectively suggest a role of IFNy in active IL-12 release from Th1 cells and on the other hand. it stimulates IFNy release release. Since IFNy release by T or NK cells is believed from macrophages that have been infected with intracellular pathogens. largely dependent up the action of IL-12 and/or IL-18, This macrophage IFNy response is tightly controlled by both IL-12 and the intriguing question is whether there is early IFNy intracellular infection and may represent the most effective auto­ release independently of IL-12 or IL-18. Much less is stimulatory mechanism to activate macrophages. During these responses, TNFo is released in an IL-12-independent manner and its known about the regulation of IL-18 during intracellular level may be in check by both IL-10 and IL-12. infection and it is known that IL-18 can be released, 202 Type I cytokine production by macrophages

compared to IL-12, by a much wider spectrum of cell of active IFNy release until recently. Indeed, macro­ types including tissue structural cells. It is possible that phages do not release IFNy under conventional IL-18 is regulated in different ways from IL-12 and it stimulatory conditions that result in the release of a wide accounts for such an early IL-12-independent IFNy array of other cytokines (Fultz et aI., 1993; Marzio et aI., release from NK cells. Apparently, the full under­ 1994; Fenton et aI., 1997). However, there has been standing of these processes entails vigorous studies evidence that human alveolar macrophages express IFNy ahead. Of particular interest and importance is the recent mRNA during pulmonary tuberculosis (Robinson et aI., finding that it is dendritic cells but not macrophages that 1994). Perhaps, the first authentic evidence that rapidly produce IL-12 in the early stage of Toxoplasma macrophages are able to release lFNy protein has been gondii infection (Sher and Reis e Sousa, 1998) and this provided by Puddu et al. (1997). They found that murine early IL-12 production is IFNy-independent. This peritoneal macrophages cultured in sustained presence of finding suggests an alternative mechanism for recombinant IL-12 could release IFNy whereas freshly macrophage IL-12 release, which is that ­ isolated cells released little in response to IL-12 derived IL-12 serves as an ignition switch for type 1 stimulation. This finding suggests that macrophage IFNy cytokine responses in macrophages by stimulating NK response requires multiple signals. Subsequent to this cells to release IFNy which will in turn trigger massive finding, Fenton et al. (1997) reported that human IL-12 release from macrophages. alveolar macrophages could release IFNy in response to Much less is known about how IL-12 p70 release is Mycobacterium tuberculosis infection but not LPS. regulated in macrophages or dendritic cells (DC) during Recently, by using murine cell-derived intracellular infection. Recent evidence suggests that macrophages, Munder et al. (1998) have reported that examination of only IL-12 p35 and p70 mRNA IL-12 or IL-18 alone only induces IFNy mRNA expression can be misleading (Isler et aI., 1999). Based expression but not protein release whereas stimulation upon limited information available to date, it appears with both IL-12 and IL-18 can induce the release of that much less IL-12 p70 is released than IL-12 p40 in IFNy protein. While these studies have established that both in vitro and in vivo systems. A recent study by macrophages are able to produce IFNy under certain using human macrophages has provided evidence to conditions, they involved artificial in vitro manipulations suggest that IFNy and/or activated are and the precise regulatory mechanisms governing IFNy required for enhanced mycobactericidal activities and release in macrophages still remain to be fully the release of IL-12 p70 but not p40 (Bonecini-Almeida understood. et aI., 1998). A different study has compared the release of IL-12 p70 by human bone marrow-derived macro­ Role of IL-12 and intracellular pathogens phages, peripheral blood monocytes and ­ derived dendritic cells (Smith et aI., 1998). The overall We have been interested in dissecting the regulatory message from this study is that LPS stimulation alone role of macrophages in cell-mediated immunity to releases lots of IL-12 p40, particularly from dendritic pulmonary mycobacterial infection. Following the cells but no or little IL-12 p70 unless IFNy is present. A demonstration that the lack of IL-12 led to a lack of tight control of IL-12 p70 release by both macrophages IFNy and severely impaired TN Fa responses in the lung and dendritic cells was further demonstrated in a murine and by T cells during pulmonary mycobacterial infection in vitro system. While both dendritic cells and (Wakeham et aI., 1998), we set out to examine the macrophages readily release IL-12 p40 upon stimulation contribution of pulmonary macrophages to type 1 by Leishmania amastigotes, LPS+IFNy or cytokine responses with the expectation that macro­ LPS+IFNy+amastigotes, they release IL-12 p70 only phages would be a primary source of IL-12 and TNFa when stimulated with amastigotes+IFNy (DC) or but not IFNy. We found, however, that lung macro­ LPS+IFNy (macrophages), regardless of significant phages freshly isolated from infected immune-competent TNFa released under all of these stimulatory conditions. mice spontaneously released not only IL-12 and TNFa Of importance, IFNy alone has no effect on IL-12 p70 but also IFNy (Wang et aI., 1999). The release of these release by dendritic cells. Even lesser information is type 1 cytokines was remarkably further enhanced by available regarding the level of IL-12 p70 protein in exposure to mycobacterial antigens PPD or LPS. To our vivo. In a model of Listeria or Brucella infection, IL-12 surprise, while lung macrophages from infected IL-12-/­ p70 protein is detected for a brief time post-infection in mice released no IFNy at all, they released, serum or homogenates. This IL-12 p70 release spontaneously or upon stimulation, high levels of TNFu. appears TNFa-dependent but IFNy-independent (Zhan These findings represent the first in vivo evidence that and Cheers, 1998). Afld dead bacteria are ineffective in macrophages are able to release IFNy and that IL-12 is inducing IL-12 p70 release. required, directly or indirectly, for macrophage IFNy but not TNFu release, during pulmonary mycobacterial Regulation of lFNy responses in macrophages during infection. These findings also help interpret our in vivo intracellular infection observations that IFNy was almost completely absent in the lung whereas TNFa response was severely, but not It has been believed that macrophages are incapable completely, inhibited over a course of 71 days 203 Type I cytokine production by macrophages

(Wakeham et aI., 1998). IL-12 is apparently required for induced by IL-12+LPS (Wang et aI., 1999). We found IFNy release from both T cells and macrophages the lack that ovalbumin did activate macrophages as demon­ of which resulted in impaired macrophage accumulation. strated by increased TNFa release. These findings However, a minimum number of macrophages suggest that in addition to IL-12, the second signal must accumulating in the lung that we observed likely be microbial in nature, live or dead intracellular remained capable of releasing TN Fa since TNFa release pathogens or intracellular -derived antigens. by macrophages is not dependent upon IL-12 (Wang et aI. , 1999). Role of IFNy, IL-18 and TNFa The limitation to our in vivo/ex vivo observations is that they would not allow us to dissect the factors that IFNy has been shown to be able to induce its own were directly required for macrophage IFNy responses release from macrophages (Marzio et aI. , 1994). We since macrophages in the lung of IL-12-/- mice were therefore investigated whether exogenously added IFNy, infected already by mycobacteria and lacked the in doses similar to IL-12 that we used, could replace IL- stimulation not only by IL-12 but also by other cytokines 12 to induce endogenous IFNy release in the presence of including IFNy. In order to investigate the regulatory live BCG. Without BCG, IFNy, particularly in moderate mechanisms, we undertook an in vitro approach by using doses, induces low levels of its own secretion. With freshly isolated lung macrophages from non-infected BCG, IFNy induced only a small increase in the naive IL-12-/ - mice. We found that IL-I2-/ ­ secretion of endogenous IFNy, thus suggesting that IFNy macrophages released no IFNy but lots of TNFa in cannot replace the role of IL-12 (Wang et aI., 1999). response to LPS stimulation or M. bovis BCG infection IL-I8 is also called IFNy-inducing factor. Not only (Wang et aI. , 1999). IL-12 alone, even when used at high can macrophages release this cytokine but they respond doses, was only a weaker stimulator of IFNy, which to this cytokine (Munder et aI., 1998; Okamura et aI., supports the findings by Munder et a1. (1998). However, 1998). At this point, although we have not been able to large quantities of IFNy were released by these measure the level of IL-18 release by stimulated lung macrophages when co-stimulated with LPS+IL-I2 or macrophages, it is likely that this cytokine was released. BCG+IL-12. The latter released the greatest amount of If this was the case, our observation that without IL-12, IFNy from macrophages. These findings indicate that BCG infection alone cannot release IFNy already both IL-12 and microbial agents are critically required indicates that IL-I8 cannot compensate for the function for active release of IFNy, but not TNFa, and suggest of IL-12. However, we could not rule out that that IFNy, but not TNFa, response in macrophages is endogenous IL-18 may have potentiated the effect of IL- under tight control. Furthermore, these findings provide 12 on macrophage IFNy release, as it does on NK and T the explanation regarding why macrophages, a cell type cells (Trinchieri, 1997). We thus investigated the role of of the first defense line, are built up with armamentaria endogenous IL-18 by using an anti-IL-I8 monoclonal to respond to all kinds of encounters and yet, the host . We found that endogenous IL-18, if any, does not always mount a type 1 immune response. It is played a marginal role in IFNy responses in IL-12/BCG­ plausible to speculate that the key is the nature of stimulated macrophages, thus establishing a unique role stimulus which in turn determines the form of IL-12, p40 of IL-12 in macrophage type 1 cytokine responses or p70 or both, released by macrophages. Once sufficient (Wang et aI., 1999) (Fig. 1). IL-12 p70 is released, this IL-12 p70 will not only Since we found that TN Fa was released upon activate T cells but also, together with intracellular stimulation by any macrophage agonists, independent of pathogens, macrophages, to release IFNy (Fig. 1). IL-12, we examined whether this cytokine may have Apparently, the question still remains regarding whether potentiated the effect of IL-12 on macrophage IFNy the second signal that acts together with IL-I2, has to be release by using a TNFa neutralizing polyclonal an intracellular pathogen in vivo since in our in vitro antibody. Similar to the results with anti-IL-I8 system, LPS+IL-12 also released IFNy. It is plausible to , endogenous TNFa played a marginal, if any, speculate that since LPS alone does not seem able to role (unpublished data). stimulate IL-I2 p70 release from macrophages (Smith et aI., 1998), it is unlikely that macrophages will be turned Regulation of TNFa responses in macrophages on to release IFNy during endotoxic although durIng intracellular infection they produce many other pro-inflammatory cytokines (Xing et aI., 1994, 1998b) (Fig. 1). We have provided unequivocal evidence that TNFa Since the above experimental design does not allow release represents a non-specific response of macro­ to address whether dead intracellular organisms or phages to almost any stimuli including non-microbial various types of antigens, in the presence of IL-12, can antigen (ovalbumin), microbial products (LPS) or also stimulate IFNy release from macrophages, we tested antigens (PPO) or live or dead mycobacteria (BCG) in the effect of heat-inactivated M. bovis BCG, myco­ an IL-12-independent manner (Wang et aI., 1999). These bacterial antigens PPD or ovalbumin. Of interest, in the findings fit well the role of this cytokine in host presence of exogenous IL-12, ovalbumin failed to induce responses to "stress" or "alarm" signals (Xing et aI. , any IFNy release whereas heat-inactivated BCG or PPO 1999). This non-selective nature of macrophage TNFa induced moderate release of IFNy, similar to that responses contrasts the tight control of macrophage IFNy 204 Type I cytokine production by macrophages

responses which require con-current stimulation by IL- F., Drysdale P., Jouanguy E., Doffinger R., Bernaudin F., Jeppsson 12 and intracellular pathogens/antigens (Wang et aI., 0 ., Gollob JA, Meinl E. , Segal AW., Fisher A., Kumararat ne D. and 1999) (Fig. 1). Casanova J.L. (1998a). Impairment of mycobacterial immunity in Since IL-12, IFNy and TNFa as well as myco­ human IL-12 receptor deficiency. Science 280, 1432-1435. bacteria are all present in the lung during mycobacterial Altare F. , Lammas D. , Revy P. , Jouanguy E., Doffinger R., Lamhamedi infection, we have further investigated the role of type 1 S. , Drysdale P., Scheel-Toellner D., Girdlestone J. , Darbyshire P., cytokines IL-12 and IFNy in macrophage TNFa Wadhwa M., Dockrell H., Salmon M., Fischer A., Durandy A., res ponses (unpublished data). Contrast to our Casanova J-L. and Kumararatne D,S, (1998b). Inherited IL-1 2 expectation, IL-12 alone had little effect on TNFa deficiency in a child with BCG and Salmonella enteritidis release from IL-12-/- lung macrophages whereas IFNy disseminated infection. J. Clin . Invest. 102, 2035-2040. released TNFa in a dose-dependent fashion . These Bonecini-Almeida M.G., Chitale S, Boutsikakis I. , Geng J., Doo H., He findings suggest that IL-12 has a specialized effect on S. and Ho J. L. (1998). Induction of in vitro human macrophage anti­ macrophages and is involved primarily in macrophage Mycobacterium tuberculosis activity: requirement for IFN -gamma IFNy responses whereas IFNy has a global macrophage and primed lymphocytes. J. Immunol. 160,4490-4499. activation effect. Thus, th at lung macrophages from Cooper A.M ., Dalton O.K., Stewart T.A. , Griffin J.P., Russell D.G. and infected IL-12-/- mice lacked surface activation markers Orme I.M . (1993) . Disseminated tuberculosis in y gene­ MHC II and CDllb (Wang et aI., 1999), was likely a disrupted mice. J. Exp. Med. 178, 2243-2247. result of the lack of IFNy but not IL-12. Cooper AM., Magram J., Ferrante J. and Orme I.M. (1997) . Not only is IL-12 alone unable to directly induce 12 (IL-12) is crucial to the development of protective immunity in TNFa release from macrophages, but we have recently mice intravenously infected with Mycobacterium tuberculosis . J. Exp. found that it inhibits TNFa release by macrophages Med . 186, 39-45. when co-stimulated with both IL-12 and live BCG Fenton M.J. , Vermeulen M.W., Kim S., Burdick M., Strieter R,M. and mycobacteria but not LPS. These findings suggest that Kornfeld H. (1997) . Induction of gamma interferon production in while stimulating IFNy release from macrophages during human alveolar macrophages by Mycobacterium tuberculosis. intracellular infection, IL-12 operates to put the level of Infect. Immun. 65, 5149-5156. TNFa responses by macro phages under check. This may Flesch I.EA, Hess J.H., Huang S., Aguet M. , Rothe J., Bleuthmann H, reflect an active mechanism by which the host attempts and Kaufmann S.H.E. (1995). Early interleukin-12 production by to minimize the toxic effect of TNFa since this cytokine macrophages in response to mycobacterial infection depends on tends to be over-produced. interferon-gamma and tumor factor-alpha. J. Exp, Med. 181 , 1615-1622, Concluding remarks Flynn J.L., Chan J. , Triebold K.J., Dalton D.K. , Stewart TA and Bloom B.A. (1993). An essential role for in resistance to IL-12 plays a unique role in host defense against Mycobacterium tuberculosis infection. J. Exp. Med. 178,2249-2254. intracellular infections. Its function cannot be Flynn J.L., Goldstein M.M. , Triebold K.J., Pfeffer K. , Lowenstein C.J., compensated for by other IFNy-inducing factors such as Schreiber R., Mak T.w. and Bloom B.R . (1995) . TNF alpha is IL-18 during pulmonary mycobacterial infection. IL-12 required in the protective immune response against Mycobacterium is a critical type 1 cytokine, critically required not only tuberculosis in mice. Immunity 2, 561- 572. for Th1 differentiation but also for IFNy responses in Fultz M.J., Barber SA, Dieffenbach C.W. and Vogel S.N. (1993) . both T cells and macrophages. While it by itself does not Induction of IFN gamma in macrophages by LPS. Inter. Immunol. 5, induce TNFa release from macrophages, it may serve to 1383-1392. modulate the level of TNFa release during intracellular Isler P., de Rochemonteix B.G ., Songeon F., Boehringer N. and Nicod infection. Further studies are warranted to investigate the L.P . (1999) . IL-12 production by human alveolar macrophages is regulation of IL-12 p70 release by macrophages and controlled by the autocrine production of IL-10. Am , J, Respir. Cell dendritic celis, the mechanisms by which IL-12 controls Mol. 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