Macrophage permissiveness for Legionella pneumophila growth modulated by iron.

We have investigated the modulation of iron in two populations of macrophages which differ in susceptibility to Legionella pneumophila intracellular proliferation. Previously, we reported that thioglycolate-elicited peritoneal macrophages obtained from the inbred A/J mouse strain readily support the intracellular growth of L. pneumophila, while resident macrophages from the same strain do not. In this study, we show that A/J elicited macrophages exhibit markedly higher expression of transferrin receptor and intracellular iron content than A/J resident macrophages. Furthermore, apotransferrin and desferrioxamine inhibited the intracellular proliferation of L. pneumophila in elicited macrophages, and this suppression was reversed by the additions of Fe-transferrin or ferric nitrilotriacetate. Fe-transferrin and ferric nitrilotriacetate did not further increase the intracellular proliferation of L. pneumophila in thioglycolate-elicited macrophages. However, ferric citrate and ferric nitrilotriacetate stimulated in a dose-dependent manner the growth of L. pneumophila in resident macrophages. Furthermore, equimolar concentrations of desferrioxamine reversed the stimulatory effect of iron in these resident cells. These data provide evidence supporting the hypothesis that differences in susceptibility to L. pneumophila growth between permissive elicited macrophages and nonpermissive resident macrophages from the A/J mouse strain are due to intracellular availability of iron.     

Differential morphologic and metabolic alterations in permissive versus nonpermissive murine macrophages infected with Legionella pneumophila.

Legionella pneumophila infection of macrophages from permissive guinea pigs and from A/J mice compared with infection of cells from nonpermissive BDF1 mice was studied by electron microscopy. The cells from the BDF1 mice were nonpermissive for legionella growth in vitro and showed few if any bacteria in phagosomes by electron microscopic examination. Similar electron micrographic examination of macrophages from A/J mice permissive for legionella growth showed numerous intact intracellular bacteria within 24 to 48 h of culture and the transition of intracellular bacteria from localization in a few large vacuoles early in the course of infection to later localization in areas surrounded and studded by ribosomes. These electron microscopic observations were similar to those seen in the case of guinea pig macrophages infected with legionellae. Biochemical studies of macrophages from permissive versus nonpermissive animals showed little or no differences in respiratory burst and lysosomal enzyme activity for macrophages from all animals tested. However, when zymosan was used as a stimulant, macrophages from the nonpermissive mouse strain produced a larger amount of H2O2 and O2- than did cells from permissive guinea pigs or A/J mice. However, legionella vaccine itself induced no detectable or very little H2O2 and O2- in macrophages tested from any source. These results suggest that permissiveness of A/J mouse macrophages to legionella growth may involve mechanisms similar to those occurring in guinea pig macrophages in terms of morphologic and possibly even biochemical events. The relatively higher production of reactive oxygens by BDF1 mouse macrophages in response to zymosan correlated with nonpermissiveness for legionella growth, although further analysis is necessary to link these observations.     

Spectrum of Legionella species whose intracellular multiplication in murine macrophages is genetically controlled by Lgn1.

We examined the intracellular growth of 20 strains within six species of Legionella in thioglycolate-elicited peritoneal macrophages from A/J and C57BL/6 mice and a congenic strain derived from them (A.B Lgn1). With the exception of Legionella pneumophila Togus-1 and Bloomington-2, the intracellular growth of the 15 L. pneumophila strains tested was controlled by Lgn1. However, the intracellular growth of five Legionella species other than L. pneumophila was not under Lgn1's control.     

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.     

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.     

Legionella pneumophila growth restriction and cytokine production by murine macrophages activated by a novel Pseudomonas lipid A.

Peritoneal exudate macrophages from A/J mice activated by purified lipid A preparations from Pseudomonas vesicularis, which contain 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester as the lipid A backbone, restricted the growth of Legionella pneumophila, an intracellular opportunistic bacteria which readily grows in otherwise permissive macrophages from susceptible A/J mice and induced production of the proinflammatory cytokines interleukin 1 and tumor necrosis factor alpha. Activation of the macrophages was similar to that which occurred after stimulation with more conventional lipid A from other bacteria such as salmonellae. A purified fraction A3 preparation from the Pseudomonas lipid A, which lacked only 1 mol of amide-linked fatty acid, in comparison with another fraction (A2), which contained the fatty acid, also markedly activated the usually permissive macrophages from susceptible A/J mice to resist growth of the legionellae. The fraction A3 also induced both interleukin and tumor necrosis factor alpha. These results show that this novel lipid A from P. vesicularis can activate macrophages to resist infection with an opportunistic bacterium in a manner similar to that induced by conventional enterobacterial lipid A and that the hydrophobic portion of this Pseudomonas molecule may have an important role in activation of macrophages.     

Differential growth of Legionella pneumophila in guinea pig versus mouse macrophage cultures.

Legionella pneumophila is a facultative intracellular bacterium which replicated well in inbred guinea pig strain 2 peritoneal macrophages at a low infectivity ratio. In contrast, the growth of this organism was markedly restricted in mouse (BDF1) peritoneal macrophages, even at a relatively high infectivity ratio. The initial uptake of L. pneumophila organisms by macrophages was similar in both animal species, and both groups of macrophages supported the growth of Listeria monocytogenes. Treatment of L. pneumophila with immune guinea pig serum did not result in restriction of bacterial growth in macrophages, but guinea pig macrophages were readily induced to suppress the growth of L. pneumophila by preincubation with supernatants obtained from mitogen-activated normal guinea pig splenocyte cultures. Thus, lymphokines generated from mitogen-stimulated guinea pig lymphocytes induced a restriction of growth of these organisms similar to that observed naturally with macrophages from mice, which are considered highly resistant to these bacteria. Although guinea pigs are considered highly susceptible to L. pneumophila infections and mice are considered relatively resistant, the mechanism of this difference is not clear. The results of the present study suggest that the restriction of L. pneumophila growth by macrophages relates to host susceptibility to infection and that cell populations permissive for L. pneumophila can be transformed to nonpermissive by products from stimulated lymphocytes but not by opsonization with immune serum.     

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.     

Legionella pneumophila growth restriction in permissive macrophages cocultured with nonpermissive lipopolysaccharide-activated macrophages.

Macrophages can be activated by lipopolysaccharides (LPS) from gram-negative bacteria to evince a number of biological activities, including increased resistance to intracellular infection by opportunistic bacteria. In the present study, intraperitoneal injection of LPS into A/J mice activated peritoneal macrophages so that they resisted subsequent in vitro infection with Legionella pneumophila. Coculture of these macrophages with those from nontreated A/J mice converted the entire population of cells from permissive to nonpermissive. This effect did not appear to be mediated by soluble factors released from the LPS-treated macrophages, since the levels of interleukins-1 and -6 and tumor necrosis factor alpha produced by the macrophages were not found to be markedly elevated at the time when the macrophages from the LPS-treated mice were most effective in converting normal macrophages to nonpermissiveness. Furthermore, macrophages from mice injected intraperitoneally with either interferon or tumor necrosis factor alpha did not evince nonpermissiveness and also did not have the ability to convert normal spleen cells to nonpermissiveness. Polymyxin B, a known inactivator of LPS activity, did not inhibit the macrophages from the LPS-treated mice from inducing this resistance. It seemed unlikely that free LPS released from the macrophages mediated this effect. The results of this study thus showed that macrophages activated by LPS in vivo can evince nonpermissiveness for Legionella growth in vitro and also can induce macrophages from normal, permissive mice to become nonpermissive for Legionella growth in vitro.     

Differential induction of gamma interferon in Legionella pneumophila-infected macrophages from BALB/c and A/J mice

Gamma interferon (IFN-gamma), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, gammadelta cells, and activated T cells. The autocrine biological functions of IFN-gamma on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-gamma by macrophages was demonstrated to correspond to antibacterial activity. Legionella pneumophila replicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-gamma mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophila activity. Moreover, IFN-gammaneutralization by antibody treatment of the cultures resulted in increased L. pneumophila growth in the macrophages. In contrast, no IFN-gamma mRNA was expressed in TG-macrophages from A/J mice, where L. pneumophila grew unrestricted. As would be expected, IFN-gamma treatment decreased bacterial growth. An IFN-gamma-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 following L. pneumophila infection. Thus, autocrine IFN-gamma is involved in anti-L. pneumophila activity associated with different growth patterns and appears to be important during intracellular infection.     

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.     

Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis.

The potential role of inhaled protozoa as a niche for intrapulmonary replication of Legionella pneumophila was investigated in vivo with mutant strains of L. pneumophila which have reduced virulence for the amoeba Hartmannella vermiformis. L. pneumophila AA488 and AA502 were derived from wild-type strain AA100 after transposon mutagenesis. These mutants have reduced virulence for H. vermiformis but are fully virulent for mononuclear phagocytic cells. A/J mice, which are susceptible to replicative L. pneumophila lung infections, were inoculated intratracheally with L. pneumophila AA100, AA488, or AA502 (10[6] bacteria per mouse) or were coinoculated with one of the L. pneumophila strains (10[6] bacteria per mouse) and uninfected H. vermiformis (10[6] amoebae per mouse). The effect of coinoculation with H. vermiformis on intrapulmonary growth of each L. pneumophila strain was subsequently assessed. In agreement with our previous studies, coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of the parent L. pneumophila strain (AA100). In contrast, intrapulmonary growth of L. pneumophila AA488 or AA502 was not significantly enhanced by coinoculation of mice with H. vermiformis. These studies demonstrate that L. pneumophila virulence for amoebae is required for maximal intrapulmonary growth of the bacteria in mice coinoculated with H. vermiformis and support the hypothesis that inhaled amoebae may potentiate intrapulmonary growth of L. pneumophila by providing a niche for bacterial replication.     

Interferon-gamma induced resistance to Legionella pneumophila in susceptible A/J mouse macrophages

Legionella pneumophila (Lp) grow in cultures in human, guinea pig, and mouse macrophages from A/J strain mice. Because exudate macrophages from this strain of mice have been reported deficient in responsiveness to lymphokines, we thought it of interest to document the extent of responsiveness to interferon-gamma in the context of growth restriction of Lp. Peritoneal exudate macrophages were obtained from A/J mice and cultured in either the presence or absence of recombinant interferon-gamma. These cultures were then infected with Lp and the extent of bacterial growth estimated 48 hr later by means of a colony-forming unit (CFU) assay and electron microscopy. Interferon-gamma treatment significantly restricted the number of CFUs in the culture at concentrations as low as 20 U/ml, but did not affect the uptake of bacteria by macrophages. Furthermore, treatment with interferon induced morphological changes consistent with activated macrophages. The involvement of oxygen-dependent mechanisms in phagocyte killing and growth restriction was examined by the use of inhibitors such as superoxide dismutase (SOD) and catalase. Neither one of these inhibitors of toxic oxygen metabolites affected the interferon-gamma-induced suppression of Lp growth. These results suggest that although thioglycolate-induced exudate macrophages from A/J mice support the growth of Lp, these cells readily respond to the activating influence of interferon-gamma. Furthermore, lymphokine treatment does not inhibit Lp uptake by macrophages and apparently restricts the growth of bacteria by mechanisms independent of the activity of toxic oxygen metabolites.     

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.     

Infection of macrophages with Legionella pneumophila induces phosphorylation of a 76-kilodalton protein.

Infection of peritoneal macrophages from susceptible A/J mice with Legionella pneumophila induced phosphorylation of a 76-kDa protein. The phosphorylation occurred when macrophages were infected with a virulent strain of L. pneumophila but did not occur when they were infected with an avirulent strain or with other bacteria such as either Pseudomonas aeruginosa or Salmonella typhimurium. Also, no phosphorylation of this protein was observed when macrophages were stimulated with either lipopolysaccharide or phorbol myristate acetate. However, phosphorylation did occur in macrophages infected with a virulent strain of L. pneumophila and treated with either erythromycin to inhibit growth or with cytochalasin D to inhibit uptake of L. pneumophila by macrophages. These results support the view that phosphorylation of this protein occurs during the early phases of interaction between L. pneumophila and macrophages. The role of this specific protein in the recognition, intracellular uptake, and growth of L. pneumophila in permissive macrophages remains to be clarified.     

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.     

Genetic control of natural resistance in mouse macrophages regulating intracellular Legionella pneumophila multiplication in vitro.

It is known that Legionella pneumophila proliferates in peritoneal macrophage cultures derived from A/J mice but not in macrophage cultures derived from many other strains, including C57BL/6 mice. To analyze the genetic control of this trait and the location of the Legionella resistance-susceptibility gene, we prepared segregating progeny of A/J and C57BL/6 mice and determined the levels of susceptibility of individual mice. Peritoneal macrophages were collected by injecting thioglycolate medium, and macrophage monolayers were infected in vitro with L. pneumophila Philadelphia-1. Counting of colonies on buffered charcoal yeast extract agar plates and Gimenez staining of macrophage monolayers were carried out daily. There was a 10-fold increase in bacterial burden 1 day after infection and a 100-fold increase after 2 days in A/J (susceptible) macrophages. The increase in bacterial burden was always less than 10-fold in macrophages from C57BL/6 (resistant) progenitors, A/J x C57BL/6 F1 hybrids, and C57BL/6 x F1 backcross progeny. The ratios of resistant individuals to susceptible individuals were 22:6 for F2 progeny and 20:22 for A/J x F1 backcross progeny. The fact that the organism did not proliferate in macrophages from B10.A mice demonstrated that major histocompatibility antigens did not regulate the macrophage resistance of C57BL/6-derived mice. The sex and coat color genes of mice were not linked to the resistance-susceptibility gene. We suggest that resistance and susceptibility are controlled by a single gene or closely linked genes which are autosomal and that the resistance allele is dominant. The results of a comparison of the strain distribution pattern of this trait with the distribution pattern of 185 allelic markers in A/J x C57BL/6 and C57BL/6 x A/J recombinant inbred strains suggest that this susceptibility-resistance gene is located in the proximal part of chromosome 15.     

Specificity of Legionella pneumophila and Coxiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection

Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.     

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.     

Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila

The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.