Legionella pneumophila replication in macrophages inhibited by selective immunomodulatory effects on cytokine formation by epigallocatechin gallate, a major form of tea catechins

Epigallocatechin gallate (EGCg) is a major form of tea catechin and has a variety of biological activities, including antitumor as well as antimicrobial activity against some pathogens. Although the biological activities of EGCg have been extensively studied, its immunological effects are not well known. In the present study, the ability of EGCg to modulate macrophage immune functions in an in vitro Legionella pneumophila infection model of macrophages was examined. The study showed that EGCg inhibited the growth of L. pneumophila in macrophages at a concentration as low as 0.5 microg/ml without any direct antibacterial effect on the organisms. The EGCg selectively upregulated the production of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) and downregulated IL-10 production of macrophages induced by L. pneumophila infection in a dose-dependent manner, but did not alter IL-6 production even at a high dose. The upregulation of the levels of macrophage gamma interferon (IFN-gamma) mRNA by EGCg was also demonstrated. Treatment of macrophage cultures with anti-TNF-alpha and anti-IFN-gamma monoclonal antibodies markedly abolished the anti-L. pneumophila activity of macrophages induced by the EGCg treatment. These results indicate that EGCg selectively alters the immune responses of macrophages to L. pneumophila and leads to an enhanced anti-L. pneumophila activity of macrophages mediated by enhanced production of both TNF-alpha and IFN-gamma. However, the enhancement of in vitro anti-L. pneumophila activity by EGCg may not be directly mediated by IL-10 and IL-12 production modulation. Thus, the results of this study revealed the immunomodulatory effect of EGCg on macrophages, which have a critical role in infections.     

Cyclic AMP inhibition of lipopolysaccharide-induced restriction of Legionella pneumophila growth in macrophage cultures.

The mechanism of the effects of lipopolysaccharide (LPS) on macrophages in terms of replication of intracellular facultative bacteria is unclear. It was found in the present study that the anti-Legionella pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by dibutyryl cyclic AMP (DcAMP). A 24-h pretreatment of murine thioglycolate-elicited macrophages with LPS resulted in an enhanced ability of these cells to inhibit the intracellular growth of L. pneumophila. This anti-L. pneumophila activity of macrophages induced by LPS was inhibited when DcAMP (10(-3) to 10(-5) M) was present during preincubation with LPS. The addition of DcAMP to the cultures was more effective before LPS treatment than after treatment. The effect of DcAMP was dose dependent. The secretion and production of acid phosphatase by LPS-activated macrophages were also inhibited by the addition of DcAMP before LPS treatment. Furthermore, the anti-L. pneumophila activity of macrophages induced by LPS could also be reversed in vitro by treatment with prostaglandin E2, colchicine, isoproterenol, theophylline, or hydrocortisone, all of which are known to increase the intracellular levels of cyclic AMP in various tissues. These observations indicate that the anti-L. pneumophila activity induced by LPS treatment can be modified by mechanisms involving cyclic nucleotide metabolism.     

Effector mechanisms responsible for gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts.

To facilitate identification of the effector mechanism(s) responsible for gamma interferon (IFN-gamma)-mediated host resistance to Legionella pneumophila, a murine model of legionellosis in BALB/c mice with a targeted disruption in the IFN-gamma gene (gamma knockout [GKO] mice) was developed. Immunocompetent BALB/c mice and GKO mice were inoculated intratracheally with virulent L. pneumophila (10(6) bacteria per mouse), and bacterial clearance and the pulmonary inflammatory response were assessed. L. pneumophila did not replicate in, and was rapidly cleared from, the lungs of immunocompetent BALB/c mice, demonstrating that immunocompetent BALB/c mice are resistant to replicative L. pneumophila pulmonary infections. In contrast, similarly infected GKO mice developed persistent, replicative intrapulmonary L. pneumophila infections with extrapulmonary dissemination of the bacteria to the spleen. Histopathologic and flow cytometric analysis of L. pneumophila-infected lung tissue demonstrated that while immunocompetent BALB/c mice develop multifocal pneumonitis which resolves, similarly infected GKO mice develop diffuse pneumonitis with persistent neutrophil recruitment into the lung. Intratracheal administration of exogenous IFN-gamma to L. pneumophila-infected GKO mice facilitated intrapulmonary clearance of the bacteria, confirming the pivotal role of IFN-gamma in innate host defenses to L. pneumophila lung infection in this murine host. The potential role of endogenous reactive nitrogen intermediates, including nitric oxide (NO), in IFN-gamma-mediated resistance to L. pneumophila pulmonary infections in immunocompetent BALB/c mice was subsequently assessed. Macrophage inducible nitric oxide synthetase (an enzyme responsible for the production of NO) was induced in alveolar cells from L. pneumophila-infected immunocompetent BALB/c mice (with maximal expression at 48 h postinfection) but was not induced in similarly infected GKO mice. However, administration of the NO synthetase inhibitor N-monomethyl-L-arginine did not significantly inhibit clearance of L. pneumophila from the lung of immunocompetent BALB/c mice (compared with that in similarly infected mice not administered N-monomethyl-L-arginine). In contrast, we have previously demonstrated that IFN-gamma-induced host resistance to replicative L. pneumophila lung infections in a susceptible murine host (A/J mice) is mediated, in part, by endogenous NO. Taken together, these studies identify a differing role of endogenous NO in IFN-gamma-mediated resistance to L. pneumophila pulmonary infection in susceptible and resistant murine hosts.     

Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice

The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.     

Host-dependent trigger of caspases and apoptosis by Legionella pneumophila

The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.     

Growth of Legionella pneumophila in thioglycolate-elicited peritoneal macrophages from A/J mice.

Legionella pneumophila is a facultative intracellular bacterium which readily grows in cultures of guinea pig and human mononuclear phagocytes. In this report, we demonstrate that the Legionella sp. also grows in thioglycolate-elicited macrophages obtained from A/J mice but not in cells from other mouse strains tested, such as BDF1, DBA/2, C3H/HeN, C57BL/6, and BALB/c. Growth of Listeria monocytogenes and interleukin-1 production in A/J mice were similar to their growth and production in other strains tested, and the growth of Staphylococcus epidermidis was restricted by A/J macrophages. This finding suggests that although A/J macrophages share functional capabilities with cells from other mouse strains, they differ in growth restriction capacity for the Legionella sp. Resident macrophages were less permissive than were thioglycolate-elicited cells in that resident cells from A/J mice failed to support the growth of Legionella pneumophila. Also, resident cells from BDF1 mice rapidly eliminated the bacteria, rather than merely restricting growth. This finding was also observed in in vivo studies in which thioglycolate pretreatment of mice resulted in the enhanced recovery of viable bacteria from the peritoneal cavity of mice infected intraperitoneally. Higher numbers of bacteria were obtained from A/J mice and, in addition, this strain was more susceptible to the lethal effects of Legionella infection. These data suggest that, as with other intracellular bacteria, macrophages may serve a pivotal role in the early stages of Legionella infection and further suggest that the A/J mouse represents a useful animal model for the study of Legionella infection and immunity.     

Roles for tumor necrosis factor alpha and nitric oxide in resistance of rat alveolar macrophages to Legionella pneumophila.

Legionella pneumophila is an intracellular parasite of alveolar macrophages, and recovery from legionellosis is associated with activation of alveolar macrophages to resist intracellular bacterial replication. Gamma interferon (IFN-gamma) is known to activate alveolar macrophages to suppress L. pneumophila, but the role of macrophage-derived cytokines in modulating alveolar macrophage resistance is unknown. To test the hypothesis that macrophage-derived mediators contribute to the resistance of alveolar macrophages to L. pneumophila, we incubated adherent rat alveolar macrophages with Escherichia coli lipopolysaccharide (LPS), recombinant tumor necrosis factor alpha (TNF-alpha), recombinant IFN-gamma, neutralizing anti-TNF-alpha, and/or N(G)-monomethyl-L-arginine (L-NMMA) for 6 h before challenge with L. pneumophila. Monolayers were sonically disrupted and quantitatively cultured on successive days. We also measured bioactive TNF-alpha release by infected macrophages in the presence or absence of IFN-gamma. We found that pretreatment of alveolar macrophages with LPS or, to a lesser degree, TNF-alpha, significantly inhibited intracellular replication of L. pneumophila. Both LPS and TNF-alpha acted synergistically with IFN-gamma at less than the maximally activating concentration to suppress L. pneumophila growth. The independent and coactivating effects of LPS were blocked by anti-TNF-alpha. Killing of L. pneumophila by IFN-gamma at the maximally activating concentration was inhibited by anti-TNF-alpha. The synergistic effects of TNF-alpha. or LPS in combination with IFN-gamma were inhibited by L-NMMA. Infected alveolar macrophages secreted TNF-alpha in proportion to the bacterial inoculum, and secretion of TNF-alpha was potentiated by cocultivation with IFN-gamma. These data indicate that secretion of TNF-alpha is an important autocrine defense mechanism of alveolar macrophages, serving to potentiate the activating effects of IFN-gamma through costimulation of nitric oxide synthesis.     

Epigallocatechin gallate,a potential immunomodulatory agent of tea components,diminishes cigarette smoke condensate-induced suppression of anti-Legionella pneumophila activity and cytokine responses of alveolar macrophages

Even though cigarette smoking has been shown to suppress immune responses in the lungs, little is known about the effect of cigarette smoke components on respiratory infections. In the present study, the effects of cigarette smoke condensate (CSC) on bacterial replication in alveolar macrophages and the immune responses of macrophages to infection were examined. Furthermore, a possible immunotherapeutic effect of epigallocatechin gallate (EGCg), a major form of tea catechins, on the CSC-induced suppression of antimicrobial activity and immune responses of alveolar macrophages was also determined. The treatment of murine alveolar macrophage cell line (MH-S) cells with CSC significantly enhanced the replication of Legionella pneumophila in macrophages and selectively down-regulated the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) induced by bacterial infection. The treatment of macrophages with EGCg not only overcame the CSC-induced suppression of antimicrobial activity but also strengthened the resistance of macrophages to infection. EGCg also markedly up-regulated the CSC-suppressed IL-6 and TNF-alpha production by macrophages in response to infection. The results of exogenous TNF-alpha treatment and neutralization treatment with anti-TNF-alpha and anti-gamma-interferon (IFN-gamma) antibodies and the determination of IFN-gamma mRNA levels indicate that CSC-suppressed macrophages can be activated by EGCg to inhibit L. pneumophila growth by up-regulation of TNF-alpha and IFN-gamma production. Thus, this study revealed that CSC selectively alters the immune responses of macrophages to L. pneumophila infection and leads to an enhancement of bacterial replication in macrophages. In addition, the tea catechin EGCg can diminish such suppressive effects of CSC on alveolar macrophages.     

Genetic susceptibility and caspase activation in mouse and human macrophages are distinct for Legionella longbeachae and L. pneumophila

Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.     

Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J mouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan, and iron(III).

A/J mouse macrophages infected with Legionella pneumophila and treated with gamma interferon (IFN-gamma) in vitro developed potent antimicrobial activity. This antilegionella activity was independent of the macrophage capacity to generate reactive oxygen intermediates, since the oxygen radical scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on the antilegionella activity of IFN-gamma-activated macrophages. Likewise, whereas the ability of IFN-gamma-activated macrophages to synthesize reactive nitrogen intermediates was markedly inhibited by the L-arginine (Arg) analogs, NG-monomethyl-L-arginine and L-aminoguanidine, as well as by incubation in L-Arg-free medium, their ability to inhibit the intracellular growth of L. pneumophila remained intact. The intracellular growth of L. pneumophila in A/J macrophages was inhibited by the iron(III) chelator desferrioxamine and reversed by Fe-transferrin as well as by ferric salts. Additionally, IFN-gamma-activated macrophages incorporated 28% less 59Fe(III) compared with nonactivated cells. Nonetheless, only partial blocking of growth restriction was observed when IFN-gamma-stimulated macrophages were saturated with iron(III). Indole-propionic acid, which appears to inhibit the biosynthesis of L-tryptophan (L-Trp), was an L-Trp-reversible growth inhibitor of L. pneumophila in macrophages, implying that the intracellular replication of this pathogen is also L-Trp dependent. However, an excess of exogenous L-Trp did not reverse the growth inhibition due to IFN-gamma, though a small synergistic effect was observed when the culture medium was supplemented with both iron(III) and L-Trp. We conclude that IFN-gamma-activated macrophages inhibit the intracellular proliferation of L. pneumophila by reactive oxygen intermediate- and reactive nitrogen intermediate-independent mechanisms and just partially by nutritionally dependent mechanisms. We also suggest that additional mechanisms, still unclear, may be involved, since complete reversion was never obtained and since at higher concentrations of IFN-gamma, iron(III) did not induce any significant reversion in the L. pneumophila growth inhibition.     

Macrophages from mice with the restrictive Lgn1 allele exhibit multifactorial resistance to Legionella pneumophila

Although Legionella pneumophila can multiply in diverse cell types from a variety of species, macrophages from most inbred mouse strains are nonpermissive for intracellular replication and allow little or no growth of the bacteria. This phenomenon is likely genetically controlled by the mouse naip5 (birc1e) gene located within the Lgn1 locus. In this study, we have investigated the resistance of C57BL/6J macrophages to L. pneumophila infection by examining the fate of both the bacterium and the infected cells compared to that in macrophages from the permissive A/J strain. Our results indicate that although the trafficking of the L. pneumophila-containing vacuole is partially disrupted in C57BL/6J macrophages, this cannot account for the severity of the defect in intracellular growth observed in this strain. Infected macrophages are lost shortly after infection, and at later times a larger fraction of the C57BL/6J macrophages in which L. pneumophila undergoes replication are apoptotic compared to those derived from A/J mice. Finally, a loss of bacterial counts occurs after the first round of growth. Therefore, the resistance mechanism of C57BL/6J macrophages to L. pneumophila infection appears to be multifactorial, and we discuss how early and late responses result in clearing the infection.     

Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection.

Resistance to infection with Legionella pneumophila is primarily dependent upon cell-mediated immunity rather than humoral immunity. Recent evidence suggests that activation of cell-mediated immunity depends on Th1 cells and activation of humoral immunity depends on Th2 cells. In this report, delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid of marijuana and an immunomodulator, suppressed development of secondary immunity to L. pneumophila, which correlated with a reduction in Th1 activity. BALB/c mice, infected with a primary sublethal dose of L. pneumophila, developed resistance to a larger challenge infection 3 to 4 weeks later. However, intravenous injection of THC (4 mg/kg of body weight) 1 day prior to primary infection resulted in increased mortality after the challenge infection. The level of anti-L. pneumophila antibodies in serum increased in both THC-treated and control mice; however, in the THC group IgG1 antibodies which are stimulated by Th2 cells were elevated while Th1-regulated, IgG2a antibodies were depressed. Furthermore, cultured splenocytes from THC-treated mice had less L. pneumophila-specific lymphoproliferation, indicating a deficiency in cell-mediated immunity. Normal mouse splenocytes treated in vitro with THC and pokeweed mitogen showed suppressed production of gamma interferon, a cytokine associated with Th1 cells, but increased production of interleukin 4, a cytokine produced by Th2 cells. Splenocytes from THC-treated mice, stimulated in vitro with either pokeweed mitogen or anti-CD3 antibodies, also produced less gamma interferon, indicating less Th1 activity in these mice. These results suggest that THC decreases the development of anti-L. pneumophila immunity by causing a change in the balance of Th1 and Th2 activities.     

Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection

The in vivo role of endogenous interleukin-18 (IL-18) in modulating gamma interferon (IFN-gamma)-mediated resolution of replicative Legionella pneumophila lung infection was assessed using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with virulent bacteria (10(6) L. pneumophila organisms per mouse) resulted in induction of IL-18 protein in bronchoalveolar lavage fluid (BALF) and intrapulmonary expression of IL-18 mRNA. Real-time quantitative RT-PCR analysis of infected lung tissue demonstrated that induction of IL-18 in BALF preceded induction of IL-12 and IFN-gamma mRNAs in the lung. Blocking intrapulmonary IL-18 activity by administration of a monoclonal antibody (MAb) to the IL-18 receptor (anti-IL-18R MAb) prior to L. pneumophila infection inhibited induction of intrapulmonary IFN-gamma production but did not significantly alter resolution of replicative L. pneumophila lung infection. In contrast, blocking endogenous IL-12 activity by administration of anti-IL-12 MAb) alone or in combination with anti-IL-18R MAb inhibited induction of intrapulmonary IFN-gamma and resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection. Taken together, these results demonstrate that IL-18 plays a key role in modulating induction of IFN-gamma in the lung in response to L. pneumophila and that together with IL-12, IL-18 regulates intrapulmonary growth of the bacteria.     

Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease.

The effect of inhaled amoebae on the pathogenesis of Legionnaires' disease was investigated in vivo. A/J mice, which are susceptible to replicative Legionella pneumophila infections, were inoculated intratracheally with L. pneumophila (10(6) bacteria per mouse) or were coinoculated with L. pneumophila (10(6) bacteria per mouse) and Hartmannella vermiformis (10(6) amoebae per mouse). The effect of coinoculation with H. vermiformis on bacterial clearance, histopathology, cellular recruitment into the lung, and intrapulmonary levels of cytokines including gamma interferon and tumor necrosis factor alpha was subsequently assessed. Coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of L. pneumophila in A/J mice. Histopathologic and flow cytometric analysis of lung tissue demonstrated that while A/J mice inoculated with L. pneumophila alone develop multifocal pneumonitis which resolves with minimal mortality, mice coinoculated with H. vermiformis develop diffuse pneumonitis which is associated with diminished intrapulmonary recruitment of lymphocytes and mononuclear phagocytic cells and significant mortality. Furthermore, coinoculation of mice with H. vermiformis resulted in a fourfold enhancement in intrapulmonary levels of gamma interferon and tumor necrosis factor alpha compared with mice infected with L. pneumophila alone. The effect of H. vermiformis on intrapulmonary growth of L. pneumophila in a resistant host (i.e., BALB/c mice) was subsequently evaluated. While BALB/c mice do not develop replicative L. pneumophila infections following inoculation with L. pneumophila alone, there was an eightfold increase in intrapulmonary L. pneumophila in BALB/c mice coinoculated with H. vermiformis. These studies, demonstrating that intrapulmonary amoebae potentiate replicative L. pneumophila lung infection in both a susceptible and a resistant host, have significant implications with regard to the potential role of protozoa in the pathogenesis of pulmonary diseases due to inhaled pathogens and in the design of strategies to prevent and/or control legionellosis.     

Murine macrophages differentially produce proinflammatory cytokines after infection with virulent vs. avirulent Legionella pneumophila

Virulent Legionella pneumophila replicate readily in thioglycollate-elicited peritoneal macrophages from genetically permissive A/J mice, but avirulent L. pneumophila do not. The production of cytokines by macrophages infected with L. pneumophila has been studied, but the correlation of bacterial virulence with immune responses of macrophages, such as proinflammatory cytokine production, is not well understood. In this regard, production of the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1alpha, IL-1beta, and IL-6 were examined in macrophage cultures infected in vitro with virulent vs. avirulent L. pneumophila. Infection of macrophages from A/J mice with the virulent L. pneumophila up-regulated mRNA expression for these cytokines, whereas avirulent bacteria resulted in only a slight or no detectable increase in cytokine mRNA. Similarly, virulent L. pneumophila induced the macrophages to produce relatively high levels of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 proteins as measured by enzyme-linked immunosorbent assays, whereas avirulent bacteria induced only low or often undetectable amounts of these cytokines. Thus, these results show the murine macrophages from susceptible A/J mice are readily infected with virulent L. pneumophila in vitro and stimulated to produce the proinflammatory acute-phase cytokines TNF-alpha, IL-1alpha, IL-1beta, and IL-6, but avirulent L. pneumophila did not. Such differences in induction of these proinflammatory cytokines by macrophages in response to virulent vs. avirulent L. pneumophila infections may be an important factor in the pathogenesis induced by these intracellular bacteria.     

A major role of macrophage activation by interferon-gamma during mouse hepatitis virus type 3 infection. I. Genetically dependent resistance

Resistance of mice to mouse hepatitis virus type 3 (MHV3) infection is genetically determined. Normal adult A/J mice are resistant, and BALB/c mice are susceptible. Higher titers of virus and interferon (IFN) in vivo were found in MHV3-infected BALB/c mice compared with A/J mice. In vitro activation of macrophages (M phi) by lipopolysaccharide (LPS) delayed MHV3 replication only in cells that originated from A/J mice, although cell populations from both A/J and BALB/c mice were able to synthesize comparable amounts of IFN-alpha/beta. Using specific antibodies, we have shown that the delayed MHV3 replication in LPS-activated A/J M phi was due, in part, to IFN-alpha/beta. A/J M phi were found to be more sensitive to IFN-gamma than to IFN-alpha/beta, and BALB/c M phi did not develop an antiviral state to either IFN. Cultured spleen cells from A/J mice synthesized more IFN-gamma than BALB/c spleen cells after specific or non-specific stimulation. The results indicate that IFN-activated M phi may play a crucial role in the resistance to MHV3 infection. Since IFN-gamma is produced in large amounts by A/J spleen cells after specific stimulation with MHV3 and is efficient in activating the A/J M phi, a T cell-dependent mechanism is likely to be involved.     

Role of gamma interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model.

Legionella pneumophila has been shown to induce gamma interferon (IFN-gamma) both in vitro and in vivo during experimental infections of mice. With complement-mediated serologic depletion of murine splenocytes, the cellular sources of IFN-gamma following in vitro stimulation with L. pneumophila antigens were Thy-1.2+, Lyt-2-, L3T4-, and asialo-GM1+, which is consistent with the natural killer (NK) cell phenotype. Additionally, Percoll density discontinuous centrifugation demonstrated that maximal production of IFN coincided with high NK activity in fractions which were enriched for large granular lymphocytes. Furthermore, 18- to 24-h incubation of splenocytes with L. pneumophila whole-cell vaccine resulted in augmented NK cytotoxic activity against YAC-1 tumor target cells in a 51Cr release assay. The addition of macrophages to purified large granular lymphocyte populations augmented both IFN-gamma production and NK activity, suggesting that antigen is required for optimal responses. In an experimental infection model using an intratracheal inoculation route, NK activity was enhanced in the spleen, peripheral blood, and lung cells of infected mice, with maximal stimulation in the lung leukocytes at the site of infection. The results of the present study indicate that NK cells respond in vivo and in vitro to stimulation by L. pneumophila by producing IFN-gamma and by increased cytolytic activity.     

A hemidominant Naip5 allele in mouse strain MOLF/Ei-derived macrophages restricts Legionella pneumophila intracellular growth

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10(3)-fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.     

Gamma interferon production, but not perforin-mediated cytolytic activity, of T cells is required for prevention of toxoplasmic encephalitis in BALB/c mice genetically resistant to the disease

We previously showed the requirement of both T cells and gamma interferon (IFN-gamma)-producing non-T cells for the genetic resistance of BALB/c mice to the development of toxoplasmic encephalitis (TE). In order to define the role of IFN-gamma production and the perforin-mediated cytotoxicity of T cells in this resistance, we obtained immune T cells from spleens of infected IFN-gamma knockout (IFN-gamma(-/-)), perforin knockout (PO), and wild-type BALB/c mice and transferred them into infected and sulfadiazine-treated athymic nude mice, which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE and died after discontinuation of sulfadiazine treatment due to the reactivation of infection. Animals that had received immune T cells from either wild-type or PO mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma(-/-) mice developed severe TE and died as early as control nude mice. T cells obtained from the spleens of animals that had received either PO or wild-type T cells produced large amounts of IFN-gamma after stimulation with Toxoplasma gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those in wild-type T-cell recipients. Furthermore, PO mice did not develop TE after infection, and their IFN-gamma production was equivalent to or higher than that of wild-type animals. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for the prevention of TE in genetically resistant BALB/c mice.