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.     

Legionella pneumophila suppresses interleukin-12 production by macrophages

In vitro infection of macrophages with Legionella pneumophila induced interleukin-1alpha (IL-1alpha), IL-10, monocyte chemotactic protein 1 (MCP-1), and MCP-3 but not IL-12. The lipopolysaccharide (LPS)-induced production of IL-12 was down-regulated by infection with virulent L. pneumophila, but other cytokines were not affected. In contrast, avirulent L. pneumophila or UV-killed, virulent L. pneumophila did not induce any suppression of IL-12. The IL-12 suppression occurred at the level of mRNA accumulation for IL-12 genes in response to LPS stimulation, but the infection induced a marked accumulation of mRNA for both MCP-1 and MCP-3, which are known to suppress IL-12 production in LPS-stimulated macrophages. However, pretreatment of macrophages with MCP-1 did not suppress LPS-induced IL-12 production at the concentrations induced by L. pneumophila infection. These results suggest that L. pneumophila selectively suppresses IL-12 production induced by LPS from macrophages in vitro by an MCP-independent mechanism.     

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.     

Phenotypic modulation by Legionella pneumophila upon infection of macrophages.

Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.     

Salmonella typhimurium-induced cytokine production and surface molecule expression by murine macrophages.

The influence of Salmonella enterica serovar Typhimurium (S. typhimurium) on co-stimulatory molecule expression, cytokine production and induction of nitric oxide synthase (iNOS) by murine macrophages (Mphi), as well as the influence of the phoP locus on these aspects of S. typhimurium-Mphi interaction, was characterized. Pulsing Mphi with S. typhimurium resulted in increased surface expression of MHC-I, MHC-II, CD86 and CD54. Furthermore, co-incubating S. typhimurium with Mphi resulted in interleukin (IL)-12p40, IL-6 and tumor necrosis factor-alpha production as well as iNOS induction while IL-12p70 was not detectable. Finally, although phoP did not influence the level of surface molecule expression or cytokine production by S. typhimurium-pulsed Mphi phoP did influence the level of iNOS induction. Together these data show that S. typhimurium interaction with Mphi activates these cells in ways that may enhance their ability to productively stimulate Salmonella-specific T cells following phagocytic processing and presentation of Salmonella antigens.     

Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens.

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.     

Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.     

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.     

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.     

Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.     

A potassium ion channel is involved in cytokine production by activated human macrophages

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.     

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.     

Legionella pneumophila catalase-peroxidases are required for proper trafficking and growth in primary macrophages

Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H(2)O(2) compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.     

Protein profiles of Legionella pneumophila Philadelphia-1 grown in macrophages and characterization of a gene encoding a novel 24 kDa Legionella protein

Legionella pneumophila Philadelphia-1 strain was grown in cultured macrophages of guinea pigs, hamsters, and A/J mice and the bacteria were purified by Percoll density gradient centrifugation without any detergent. Patterns of the bacterial proteins were compared by SDS-PAGE with those of organisms cultured in vitro. A 24 kDa protein was a major protein of intracellularly grown bacteria: its expression was about four times as much as a 24 kDa protein of agar-grown bacteria. At least three novel proteins (100, 65, and 16 kDa) that were not found on agar-grown bacteria were also observed. In this paper, we focused on the 24 kDa major Legionella protein expressed within macrophages. Western blot and N-terminal amino acid analysis revealed that this protein is a novel protein different from Legionella proteins previously reported, including a 24 kDa macrophage infectivity potentiator protein (Mip). On the basis of amino acid sequence (MQRIKKI and IANAQGK), two kinds of oligonucleotides were synthesized and radiolabeled. Using these oligonucleotides as DNA probes, a 7.2 kb EcoRI-digested DNA fragment encoding the 24 kDa protein was cloned into λZAP II phage vector in Escherichia coli XL1-Blue. A 0.9 kb DNA fragment from the 7.2 kb fragment was further subcloned into pUC118 in E. coli CSR603 for maxicell analysis or XL1-Blue for DNA sequencing. Maxicells which carry recombinant plasmids consisting of the 0.9 kb DNA fragment and vector plasmid pUC118 expressed the 24 kDa protein. When the DNA fragment encoding the protein was sequenced, an open reading frame of 555 base pairs was identified. The inferred polypeptide had a molecular weight of 20 kDa and an estimated isoelectric point of 8.14. Both the nucleotide sequence and the deduced amino acid sequence were distinct from those of bacterial proteins reported previously, suggesting that the protein is a novel Legionella protein.     

Identification of a cytotoxin produced by Legionella pneumophila.

Culture filtrates of Legionella pneumophila were cytotoxic for Chinese hamster ovary cells. The cytotoxin was found to be methanol soluble, heat stable, and stable from pH 5 through 8.     

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.     

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.