Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Legionella pneumophila growth in macrophages from susceptible mice is genetically controlled

Growth of the intracellular opportunistic bacterium Legionella pneumophila in macrophages from A/J mice is a vigorous as growth in macrophages from susceptible guinea pigs and human monocytes, whereas growth is inhibited in macrophages from other mouse strains, such as nonpermissive BALB/c mice. Permissiveness versus nonpermissiveness of macrophages from A/J versus BALB/c mice appeared to be controlled by a genetic mechanism dependent upon a single gene or a closely clustered family of genes. Susceptibility versus resistance of macrophages from F1 offspring of these two strains of mice and macrophages from backcrossed mice prepared from F1 hybrids and the original parental strain showed a segregation of permissiveness for growth of Legionella in vitro, consistent with genetic control.     

Fate of Legionella pneumophila in macrophages of C57BL/6 chronic granulomatous disease mice

We compared the intracellular survival and growth of Legionella pneumophila Philadelphia-1 in peritoneal macrophages obtained from A/J, C57BL/6, and X-linked chronic granulomatous disease (CGD) mice produced from C57BL/6 strain. The initial killing was observed in A/J and C57BL/6 macrophages at 2, 4 and 6 hr after in vitro phagocytosis, but not in the CGD macrophages. Thereafter, there was a 10-fold increase of CFU in A/J macrophages. The bacteria, however, did not proliferate in C57BL/6 and CGD macrophages at 24 or 48 hr after in vitro phagocytosis. These results suggest that effector molecules for the initial killing are a superoxide anion and its metabolites, and Lgn1 gene product inhibits the intracellular growth of L. pneumophila independently of NADPH oxidase.     

Enhanced growth of Legionella pneumophila in tetrahydrocannabinol-treated macrophages.

Legionella pneumophila is an opportunistic intracellular pathogen that infects macrophages, both in vivo and in vitro. Tetrahydrocannabinol is a major psychoactive component of marijuana and can affect the functional activity of macrophages. In the present study, it was found that the treatment of macrophage cultures from permissive A/J mice with THC enhanced the growth of Legionella in these cells. Legionella grew much better in macrophages treated with low doses of THC, which caused no alteration in the number or viability of macrophages, as compared with growth in untreated cells. Furthermore, lipopolysaccharide-treated A/J mouse macrophages restricted the growth of Legionella, but this growth restriction was overcome by the addition of THC to LPS-treated macrophage cultures after infection. Thus, it is apparent that THC has the ability to enhance the growth of the intracellular opportunistic pathogen Legionella that grows in A/J mouse macrophages.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila.

Peritoneal macrophages obtained from lipopolysaccharide (LPS)-low responder C3H/HeJ mice (J) permitted the intracellular growth of the bacterium in macrophages of (J x N) F1 progeny was between the parent strains, showing that the traits were co-dominantly expressed. Correlation between intracellular bacterial growth in macrophages and LPS response of spleen cells was examined. Negative correlation was found between the two factors in F2, (J x F1) backcross and (N x F1) backcross progeny. This result implies that Lps gene controls the innate resistance of murine macrophages against the bacteria. Although macrophages of A/J strain also permit intracellular growth of L. pneumophila, gene complementation analysis of A/J and C3H/HeJ mice made clear that the gene control in C3H/HeJ differs from that of A/J strain. Macrophages of C57BL/10ScN, which is LPS-low responder line obtained from C57BL/10, were also defective in controlling the bacterial growth when compared to C57BL/10 mice. We suggest that the Lps gene also controls the natural resistance of murine macrophages against L. pneumophila.     

Tetrahydrocannabinol treatment suppresses growth restriction of Legionella pneumophila in murine macrophage cultures.

Legionella pneumophila is a facultative intracellular pathogen which readily grows in human and guinea pig macrophages and in peritoneal exudate macrophages from A/J mice. Macrophage cultures capable of supporting the growth of Legionella can be used to test the potency of biologically active substances suspected of modulating host mechanisms of resistance to infection. Accordingly, this model was used to evaluate the influence of delta-9-tetrahydro-cannabinol (THC) on macrophage resistance to infection with an intracellular pathogen. Pretreatment of the macrophages with THC in the concentration range of 2.5 micrograms/ml (8 microM) to 5.0 micrograms/ml (16 microM) had little if any effect on the ability of the macrophages to either ingest or support the replication of Legionella. However, THC treatment of cells following Legionella infection resulted in increased numbers of bacteria recoverable from the macrophage cultures. Stimulation of the macrophage cultures with the activating agent lipopolysaccharide (LPS) was effective in reducing the ability of Legionella to grow in the cells. However, treatment of the LPS activated macrophages with THC resulted in greater growth of the Legionella in the cultures, indicating that the drug abolished the LPS induced enhanced resistance. These results demonstrate that THC treatment of macrophages following infection rather than before infection with Legionella promotes the replication of the bacteria within the macrophages. In addition, drug treatment suppresses the growth restricting potential of macrophages activated by LPS.     

Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway

Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-gamma. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-alphabeta) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-alphabeta protective effect on permissive macrophages was comparable to that induced by IFN-gamma. Even low doses of either IFN-alpha or IFN-beta alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-alphabeta significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-alphabeta in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-gamma-neutralizing Ab did not restore Legionella growth in IFN-alpha- or IFN-beta-treated A/J or CD1 permissive macrophages, indicating that IFN-alphabeta effect was not mediated by IFN-gamma. This observation was further confirmed by the finding that IFN-alphabeta was effective in inhibiting L. pneumophila replication in macrophages from IFN-gamma receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-alphabeta in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.     

Immunomodulation of macrophages by radio-detoxified lipopolysaccharide of Salmonella typhimurium

Lipopolysaccharide (LPS) and ratio-detoxified LPS (Rd-LPS) from Salmonella typhimurium were analysed for their ability to stimulate murine peritoneal exudate cells (PEC) and macrophages. Rd-LPS induced much more inflammatory response as compared to LPS. PEC numbers/mouse obtained were significantly higher (3-fold) in response to Rd-LPS than LPS. The haemorrhage was induced in mice by LPS but not by Rd-LPS. Activation of macrophages in vivo by Rd-LPS was significantly higher as compared to LPS. This was evident from the increase levels of their lysosomal enzymes and cytokines. Rd-LPS induced 10-fold increase in acid phosphatase contents of macrophages as compared to controls while only 7-fold increase was obtained with LPS. Arylsulfatase and beta-glucuronidase increased by about 2-fold by Rd-LPS and LPS. Macrophages incubated with Rd-LPS in vitro showed 16-fold and 20-fold increase in the cell associated levels of arylsulfatase and beta-glucuronidase respectively as compared to unstimulated cells. On the other hand, only 6-fold increase was observed in response to LPS in the levels of both the enzymes. TNF-[symbol: see text] and IL-1 secreted by macrophages increased considerably in response to Rd-LPS as compared to those released by LPS. Rd-LPS, thus seems to be a better immunomodulator than untreated LPS.     

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.     

Macrophage activation to kill Leishmania tropica: characterization of P/J mouse macrophage defects for lymphokine-induced antimicrobial activities against Leishmania tropica amastigotes

Macrophages from P/J mice demonstrated both quantitative and qualitative defects in lymphokine (LK)-induced activated macrophage antileishmanial effector reactions: a) these cells recognized the same LK signals that generated resistance to infection in responsive C3H/HeN macrophages, but more signal was required to observe maximal activity; b) LK-induced intracellular destruction of Leishmania tropica by P/J macrophages was minimal (less than 20%), and was induced by only one of three LK signals that regulate antimicrobial activities in C3H/HeN macrophages. The defective microbicidal activity of P/J macrophages observed with LK activation in vitro could also be demonstrated in vivo. Macrophages from P/J mice exposed to the macrophage-activating agent Mycobacterium bovis strain BCG in vivo were capable of restricting the intracellular replication of L. tropica but could not eliminate intracellular parasites, even with further incubation with LK during the 72-hr culture period. The defect of P/J macrophages for intracellular destruction of L. tropica, then, occurred in the activation sequence before the triggering stage that characterizes the macrophage defect of C3H/HeJ mice. Genetic regulation of the P/J macrophage defect appears to be by a single autosomal gene, with defective microbicidal activity as a recessive trait in these animals.     

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.     

Stimulation of antimycobacterial activity in mouse peritoneal macrophages by priming with trehalose dimycolate (TDM).

We examined the potential of two bacterial immunomodulators, trehalose dimycolate (TDM) and lipopolysaccharide (LPS), to stimulate the capacity of mouse peritoneal macrophages to control the growth of the intracellular bacterium, Mycobacterium tuberculosis BCG. Macrophages were obtained from mice innately susceptible (Bcgs) or resistant (Bcgr) to BCG infection. In all mouse strains tested (Bcgr and Bcgs), with the exception of BALB/c (Bcgs), TDM was sufficient to elicit macrophages with strong antimycobacterial activity in vitro. In BALB/c mice, the induction of anti-BCG activity required two signals, TDM and LPS, given in sequence. Our data suggest that additional gene(s), besides the Bcg locus, control macrophage resistance to BCG.     

Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin

Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway.     

Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.     

Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila

BACKGROUND: Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease. Legionella produces disease because it can replicate inside a specialized compartment of host macrophages. Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella. A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified. RESULTS: Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella. Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation. Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains. A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness. In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages. CONCLUSIONS: We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication. We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins.     

Defective tumoricidal capacity of macrophages from A/J mice. I. Characterization of the macrophage cytotoxic defect after in vivo and in vitro activation stimuli.

Macrophages from A/J mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that activate cells from C3H/HeN mice. Peritoneal macrophages from A/J mice treated i.p. with viable Mycobacterium bovis, strain BCG, killed Corynebacterium parvum, or pyran copolymer fail to develop in vitro tumoricidal activity; varying the numbers of macrophages from treated mice added to target cells, or the dose and time of treatment, or the treatment schedule of these in vivo activation stimuli did not evoke cytotoxic activity. Moreover, cytotoxic activity by macrophages from A/J mice was not observed with any of four target cell lines derived from three different mouse strains. In vitro treatment of peritoneal exudate macrophages from A/J mice with lymphokine-rich supernatants, bacterial endotoxins, or T cell mitogens was also ineffective; varying the numbers of treated macrophages added to target cells, the dose of in vitro activation stimuli, or the time of treatment did not evoke cytotoxic activity. Thus, A/J mice exhibit a profound defect in macrophage tumoricidal capacity to both in vivo and in vitro activation stimuli over a wide range of experimental conditions.     

Ultrastructural, immunocytochemical and flow cytometry study of mouse peritoneal cells stimulated with carrageenan

In the present paper we performed a morphological characterization of mouse peritoneal cells stimulated in vivo for 24 h with carrageenan (CAR) and lipopolysaccharide (LPS) by ultrastructural and flow cytometry analysis. In all samples, the flow cytometry studies showed the presence of three major populations consisting of monocytes, macrophages and lymphocytes. A special recruitment of monocytes was detected in CAR-injected mice. Macrophages and monocytes from CAR-treated mice displayed a characteristic phenotype, with a larger number of cytoplasmic vacuoles and numerous membrane projections, as compared to the cells collected from LPS- and PBS-injected mice. The induction of vacuolization was also confirmed upon in vitro treatment with CAR for 15 min to 24 h. The in vivo CAR-induced vacuoles were not related to lipid storage as judged by the lack of lipidic labeling after imidazole treatment at the ultrastructural level. In order to investigate the acidic nature of the vacuoles we used acidothropic probes, Lysotracker Yellow (LY) and Acridine Orange (AO). CAR injection activated the ability of peritoneal cells to incorporate LY around 2-5 times higher than control cells. However, the AO incorporation was 10-fold lower in CAR-stimulated cells than in LPS-stimulated ones. It is possible that the increase in intracellular vacuolization observed in CAR-stimulated cells could be related to exocytosis, since in most vacuoles the inflammatory protein MRP-14 was immunolocalized. The presence of MRP-14 in the culture supernatant of adherent peritoneal cells from CAR-injected mice was further comfirmed by ELISA, suggesting the discharge of MRP-14 enriched vacuole contents in the extracellular medium. We concluded that the morphological characteristics of activated monocytes and macrophages may depend on the nature of the triggering stimuli. Our observations reflect different functional phenotypes of monocytes/macrophages after in vivo stimulation with inflammatory agents such as CAR and LPS.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.