Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha.

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) model we assessed whether early IL-12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti-tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.

Activation of natural killer cells by heat-killed Listeria monocytogenes requires additional signals from lymphoid cells.

Regulatory and protective functions have been attributed to murine natural killer (NK) cells in a number of infectious diseases including listeriosis. We have developed an in vitro model to study parameters underlying the activation of naive NK cells using heat-killed Listeria monocytogenes (HKL) as stimulator. Independent from expression of the cell surface marker NK1.1, NK cells lysed YAC-1 cells after in vitro stimulation with HKL or HKL + Interleukin (IL)-2, but not medium or IL-2 alone. In contrast, NK cells from severely immunocompromised SCID or RAG-1-/-mutant mice failed to respond to HKL alone, but required exogenous IL-2. Using single-gene-disruption mutant mice, we show that NK-cell activation can be supported by either T-cell receptor (TCR) alpha beta cells, TCR- gamma delta cells. MHC class I or MHC class II gene products. We conclude from these data that recognition of listerial components alone is insufficient for activation of naive NK cells, and that additional costimulatory signals are necessary. These can be provided by various lymphoid cells and appear to be cytokines.

Role of cytokines in tuberculosis.

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-gamma (IFN-gamma) 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-alpha (TNF-alpha) and IL-10 act as autocrine regulators in the induction of the enzyme NO-synthase. Both cytokines are produced by macrophages stimulated with IFN-gamma and infected with M. bovis. While TNF-alpha mediates activation of the NO-synthase and production of RNI, IL-10 suppresses this enzyme activity. The outcome of mycobacterial infection is probably regulated by a complex network between stimulatory and inhibitory cytokines.

Effect of fetal calf serum on cytokine release by bone marrow-derived macrophages during infection with intracellular bacteria.

Bone marrow-derived macrophages (BMM) comprise a population of quiescent cells which can be activated by defined signals. Here, we directly compare the release of chemokines and monokines by BMM raised either in serum-supplemented or in serum-free medium in response to Listeria monocytogenes EGD or Mycobacterium bovis BCG infection. We focused on this issue because there have been several controversial reports on the production of cytokines by BMM due to different in vitro culture conditions. Culture in serum-supplemented medium primed BMM for release of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-12, but had no effect on macrophage inflammatory protein (MIP)-1alpha and tumor necrosis factor (TNF)-alpha production in response to L. monocytogenes infection. After challenge infection with M. bovis, BMM raised and stimulated in serum-supplemented medium secreted higher levels of MCP-1, MIP-1alpha, IL-6, and TNF-alpha but not of IL-12 as compared to BMM cultured and infected in a serum-free medium. The effects of serum could be partially mimicked by interferon-gamma. Because the serum components responsible for BMM priming are undefined, BMM cultured under serum-free conditions provide an appropriate cell population for defining macrophage activating signals.

Macrophages and hepatocytic cells as chemokine producers in murine listeriosis.

The major target organ of systemic infection with the intracellular bacterium Listeria monocytogenes is the liver, to where inflammatory leukocytes are rapidly recruited. We determined by reverse transcriptase polymerase chain reaction the early chemokine response in the liver after systemic infection of mice with listeriae, and in parallel compared chemokine release from macrophages and hepatocytic cells in vitro. Murine bone marrow-derived macrophages (BMM) grown in fetal calf serum-supplemented medium were used as macrophages and the TIB75 cell line as hepatocytic cells. Within 1-3 hours, gene expression of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha, MIP-2, KC, and interferon-gamma inducible protein-10 (IP-10) was upregulated in liver tissue of infected mice. BMM infected in vitro with L. monocytogenes showed a generalized chemokine response, and readily released MCP-1, MIP-1 alpha, MIP-2, and KC, as measured by enzyme-linked immunosorbent assay. In contrast, the chemokine response of hepatocytic cells was more restricted, and infection induced MCP-1 and KC, but not MIP-2 and MIP-1 alpha. Interferon gamma enhanced chemokine release from hepatocytic cells, but unexpectedly had either no or a negative effect on chemokine secretion by BMM cultured in serum-supplemented medium. Listeriolysin (Hly)-negative avirulent listeriae as well as listeriae killed by heat or gentamycin initiated a similar chemokine response in BMM and hepatocytic cells as did wild-type L. monocytogenes. Stimulation of hepatocytic cells with the monokines, tumor necrosis factor alpha and interleukin (IL-)1 alpha, but not IL-6, augmented liberation of chemokines. Together, our data demonstrate an early hepatic chemokine response to L. monocytogenes in murine listeriosis. Probably, not only macrophages but also parenchymal cells participate in chemokine production.

Activation of tuberculostatic macrophage functions by gamma interferon, interleukin-4, and tumor necrosis factor.

The capacity of the cytokines gamma interferon (IFN-gamma), interleukin-4 (IL-4), and tumor necrosis factor (TNF) to activate tuberculostatic functions in murine bone marrow-derived macrophages (BMM phi) was investigated. In confirmation of earlier findings, IFN-gamma rendered BMM phi capable of inhibiting subsequent infection with Mycobacterium bovis. In contrast, IL-4 and TNF failed to inhibit mycobacterial growth. However, in already infected BMM phi, tuberculostasis was induced by subsequent stimulation with IL-4. Although TNF alone was ineffective, it showed synergy with IFN-gamma in the stimulation of tuberculostasis. Our data suggest that not only IFN-gamma but also IL-4 and TNF participate in the control of mycobacterial growth.

Cytokines in antibacterial resistance: possible applications for immunomodulation.

Acquired resistance to intracellular bacteria is primarily mediated by T cells which--by secreting multiple interleukins (ILs)--activate antimicrobial activities in mononuclear phagocytes. Experimental infection of mice with Listeria monocytogenes and Mycobacterium bovis has helped to clarify the role of ILs in antimicrobial infection. These studies revealed: 1) application of IFN-gamma reduces the number of L. monocytogenes and treatment with anti-IFN-gamma antibodies worsens listeriosis; 2) in vitro, tuberculostatic and listericidal macrophage functions are activated by IFN-gamma, IL-4, and IL-6; 3) tumor necrosis factor (TNF) synergizes with IFN-gamma; 4) treatment with anti-TNF antibodies worsens listeriosis and interferes with M. bovis-induced granuloma formation; 5) concomitant application of mycobacterial products and TNF induces necrotic reactions. In humans at least some effects of IFN-gamma on macrophages may be mediated via 1,25-dihydroxyvitamin D3. These findings illustrate the multifaceted role of ILs in antimicrobial resistance and point to a central role of IFN-gamma.

Role of macrophages and alpha beta T lymphocytes in early interleukin 10 production during Listeria monocytogenes infection.

Immunity to intracellular bacteria including Listeria monocytogenes is determined by Th1 cells and CD8 T cells which produce interferon-gamma. Here we show that high levels of IL-10 are released by splenocytes from mice infected with L. monocytogenes. IL-10 was detected on day 1 after infection, peaked on day 4, and subsequently declined. Cell separation studies and experiments with RAG-1-deficient mice, which do not possess mature B cells or T cells, revealed that the macrophage is the major cellular source of early IL-10 production. Elevated IL-10 production in RAG-1 mutants and TCR beta mutants, but not in TCR delta mutants, is consistent with an inhibition of macrophage IL-10 release by alpha beta T cells. High IL-10 production was also seen after infection with another intracellular bacterium, Mycobacterium bovis. Since IL-10 inhibits Th1 cell responses, certain pathogens might use induction of this cytokine as an evasion mechanism from the protective immune response of the host. However, our findings showing high levels of IL-10 production in infectious models which are dominated by Th1 cell responses suggest that IL-10 alone is insufficient for directing Th0 differentiation into the Th2 cell pathway. These findings therefore challenge the view of IL-10 as a unique and decisive determinator of the Th2 cell pathway.

Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates.

Murine bone marrow-derived macrophages are able to inhibit the growth of Mycobacterium bovis after stimulation with recombinant gamma interferon. This antimycobacterial activity was inhibited by NG-monomethyl-L-arginine, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Furthermore, there was a complete lack of mycobacterial growth inhibition in a medium deficient in L-arginine. Nitrite is generated by gamma interferon-activated bone marrow-derived macrophages after infection with M. bovis, and a correlation between mycobacterial growth inhibition and nitrite production was observed. These results indicate that reactive nitrogen intermediates derived from L-arginine are crucially involved in macrophage antimycobacterial activity.

Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6).

Mononuclear phagocytes provide the major habitat of intracellular bacteria, including Mycobacterium tuberculosis and Mycobacterium bovis. The capacity of B-cell stimulatory factor 2 (interleukin-6 [IL-6]) to activate tuberculostatic functions was investigated by using murine bone marrow-derived macrophages (BMM phi). BMM phi stimulated with recombinant IL-6 and subsequently infected with M. bovis organisms failed to inhibit mycobacterial growth. In contrast, marked tuberculostasis was induced by IL-6 in BMM phi that were already infected with M. bovis, indicating that IL-6 has a macrophage-activating function.

Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages.

Bone marrow-derived murine macrophages are able to inhibit the growth of Mycobacterium bovis and of some strains of M. tuberculosis after stimulation with either recombinant gamma interferon (rIFN-gamma) or lymphokines from antigen-specific T-cell clones. To elucidate the mechanism(s) involved in antimycobacterial activity, macrophages were infected with M. bovis in the presence of agents thought to influence the antimicrobial effects of phagocytes. Scavengers of toxic oxygen metabolites failed to influence the capacity of IFN-gamma-activated bone marrow macrophages to inhibit the growth of M. bovis. Suramin slightly affected mycobacterial growth in IFN-gamma-activated macrophages, and chloroquine markedly induced growth inhibition of M. bovis in unstimulated macrophages. We conclude that growth inhibition of M. bovis by IFN-gamma-activated macrophages is an oxygen-independent process.

The role of T cell--macrophage interactions in tuberculosis.

Acquired resistance against tuberculosis paradigmatically depends on specific T lymphocytes and mononuclear phagocytes. The etiological agent, Mycobacterium tuberculosis is capable of replicating in mononuclear phagocytes which act both as habitat and as effectors of protection. Upon interaction with antigen-specific T lymphocytes infected mononuclear phagocytes acquire tuberculostatic activities. Here, data from experimental tuberculosis studies in mice are summarized which show that: interleukins produced by cloned T cells and recombinant interferon-gamma are capable of activating tuberculostatic capacities in macrophages; both CD4 and CD8 T cells, after adequate stimulation, produce interferon-gamma; CD8 T cells lyse macrophages in an antigen-specific way; not only CD8 but also CD4 T cells possess an antigen-specific cytolytic potential; lysis of infected macrophages results in mycobacterial growth inhibition. Evidence is also presented that tuberculostatic activities of activated macrophages depend on phagosome-lysosome fusion and are independent of reactive oxygen metabolites and that some strains of M. tuberculosis are resistant against interferon-gamma activities macrophages. These findings suggest that both helper and cytolytic T cells participate in the immune response to tuberculosis and that similar T cell mechanisms contribute to resistance as well as pathogenesis. Protection against tuberculosis, therefore, depends on subtle coordination of the immune response.

IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early listeriosis.

IL-4 is a major promotor of Th2 differentiation and an antagonist of IFN-gamma production. Although experimental listeriosis is characterized by a Th1 response, IL-4-producing cells were detected in spleens of mice promptly after Listeria monocytogenes infection. We identified this early IL-4 as inducer of the chemokine, monocyte chemoattractant protein-1 (MCP-1), which mainly attracts monocytes/macrophages, but not neutrophils. MCP-1-secreting cells were demonstrable in spleens of mice infected with L. monocytogenes, and IL-4 neutralization with anti-IL-4 mAb 11B11 markedly diminished frequencies of MCP-1-producing cells. Cell depletion experiments and studies with gene disruption mutant mice lacking distinct T cell subsets and surface MHC molecules point to CD4+ NK1+ T cells as a cellular source of early IL-4. Since monocyte infiltration to infective foci contributes to early control of listeriosis, our results suggest that IL-4-producing CD4+ NK1+ T cells participate in the innate immune response against L. monocytogenes through MCP-1 induction.

Effects of IL-13 on murine listeriosis.

Acquired resistance against Listeria monocytogenes is a typical T helper (Th) 1 dominated immune response, whereas Th2 cytokines are thought to worsen listeriosis. We investigated effects of recombinant IL-13 (rIL-13) on the host response to L. monocytogenes in mice. Although IL-13 has been described as a Th2 cytokine with deactivating anti-inflammatory activities, it was found to enhance antilisterial resistance. In vitro, rIL-13 increased IL-12 p40 and p70 production by bone marrow macrophages infected with L. monocytogenes. In vivo, numbers of viable bacteria in spleens and livers were decreased after treatment of mice with rIL-13. In addition, granuloma formation was impaired and NK cell activity of spleen cells was enhanced. At the onset of infection, frequencies of IL-12-producing cells were increased and numbers of IL-4- and IFN-gamma-secreting cells were diminished in rIL-13-treated mice as compared to controls. In contrast, on day 6 after infection, IL-12, IL-4 and IFN-gamma levels in rIL-13-treated animals were equal to or even higher than those in controls. Although direct activation of host macrophages by IL-13 is possible, we consider it more likely that IL-13 acted indirectly through stimulation of IL-12 production and inhibition of IL-4 release early after infection. In contrast, our data argue against an apparent role of IFN-gamma in IL-13-induced antilisterial resistance.

NADPH diaphorase staining suggests a transient and localized contribution of nitric oxide to host defence against an intracellular pathogen in situ.

Nitric oxide (NO) is formed constitutively in neurons by the constitutive enzyme NO synthase (cNOS) and acts as a neurotransmitter. It has already been shown that cNOS-containing neurons are identical to neurons staining for NADPH diaphorase and vice versa. Effector cells of the immune response produce high NO levels after appropriate stimulation and this NO is formed by inducible NO synthase (iNOS). The NO produced by macrophages is considered an important effector molecule of antimicrobial host defence. We have applied NADPH diaphorase staining for the detection of NO producing cells in situ during infection with an intracellular pathogen. Macrophages which produce NO in vitro are stained for NADPH diaphorase. Expression of iNOS mRNA and macrophage NADPH diaphorase staining was inhibited by iNOS-specific antisense oligonucleotides. These data suggest coincidental similarity between NADPH diaphorase activity and NO production by macrophages. Cells staining for NADPH diaphorase were identified in cryostat frozen sections of livers from mice infected with the intracellular pathogen, Listeria monocytogenes, and co-localized with cells labelled by MAC-1 mAbs. The purple-blue reaction product of NADPH diaphorase staining was visible in discrete granulomatous lesions but was absent from the liver parenchyma. Our results provide direct evidence for localized and transient participation of NO in antimicrobial immunity in the infected organ. This restriction may focus NO production to lesions, leaving unrelated tissue sites unaffected.

Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.