Fate of Listeria monocytogenes in murine peritoneal macrophage subpopulations.

Peritoneal macrophages derived from CD-1 and C57BL/6 mice were separated into distinct groups based on their buoyant densities on discontinuous gradients of Percoll and assayed for antibacterial activity against Listeria monocytogenes. Subpopulations of peritoneal macrophages derived from Listeria-immune mice present a wide variation in their ability to control intracellular infection. Distinct subsets were found which exhibited bacteriostatic and listericidal activity. The fractionation procedure yielded a population of peroxidase-positive macrophages which were devoid of antilisterial action. Subpopulations of resident and elicited macrophages were also functionally heterogeneous in their ability to restrict intracellular growth of bacterial. In some experiments, subclasses were examined for secretion of plasminogen activator and phagocytosis of latex particles. These activities varied considerably with the status of activation of the macrophages, but failed to correlate with antimicrobial activity within given subpopulations.     

Human phagocytic cell responses to Scedosporium prolificans.

Scedosporium prolificans (SP) is an emerging opportunistic dematiaceous mould that causes serious infections in immunocompromised patients. Antifungal activities of human polymorphonuclear (PMN) and mononuclear (MNC) leukocytes against five SP isolates and Aspergillus fumigatus (AF) were evaluated. While monocyte-derived macrophages (MDM) phagocytosed conidia of both organisms comparably, they inhibited the germination of S. prolificans conidia less efficiently than those of A. fumigatus. Unopsonized hyphae of SP strains decreased the superoxide anion (O2-) produced by both PMN and MNC, whereas opsonized hyphae significantly stimulated it. In comparison to AF, phagocytes generally exhibited equal oxidative burst in response to SP. While PMN- and MNC-induced hyphal damage was similar among SP strains, phagocytes tended to damage SP hyphae to an equal or higher degree than AF hyphae. The susceptibility of SP to phagocytes contrasts with its high resistance to antifungal agents and may be related with the very low pathogenicity of the mould.     

Increased phagocytic activity of splenectomized mice challenged with Listeria monocytogenes.

Adult splenectomized mice exhibit increased resistance to infection with Listeria monocytogenes. Phagocytosis, by reticulo-endothelial cells, of test particles (51Cr-labelled sheep erythrocytes) is the same in splenectomized and control mice. However, 24 h exposure to Listeria, which fails to influence phagocytic activity of normal mice, greatly enhances the blood clearance and liver uptake of the test particles in splenectomized mice. The presence of a cell population and/or product in the spleen which modulates macrophage activation upon the exposure to appropriate stimuli is postulated.     

Activation of mouse peritoneal cells to kill Listeria monocytogenes by T-lymphocyte products.

An in vitro system has been used to demonstrate that glass-adherent mouse peritoneal cells can be activated to kill intracellular Listeria monocytogenes by antigen-stimulated T-lymphocytes derived from immunized mice. The soluble products of such stimulated lymphocyte cultures could only be shown to similarly activate peritoneal cells if the antigen used in both the immunization and lymphocyte stimulation was also present on the target intracellular organism.     

MHC class Jb-restricted cell responses to Listeria monocytogenes infection

Murine infection with Listeria monocytogenes induces CD8+ T cell responses specific for bacterial peptides that are presented on the infected cell surface by MHC class Ia and MHC class Ib molecules. We have used MHC tetramers to demonstrate that CD8+ T cells restricted by the H2-M3 MHC class Ib molecules constitute a substantial portion of the T cell response to L. monocytogenes infection. The in vivo size and kinetics of MHC class Ib-restricted T cell populations suggests that they play a prominent role in bacterial clearance following primary L. monocytogenes infection.     

Relationship between non-specific activity of macrophages and immune responses to Listeria monocytogenes.

Delayed footpad reactions and acquired cellular resistance to Listeria monocytogenes were studied in mice whose mononuclear phagocyte system (MPS) had been blocked or stimulated. Colloidal carbon was used for the blockade of MPS and Corynebacterium parvum used for the stimulation. Strong delayed footpad reactions. On the other hand, the i.v. injection of 3 X 10(1) listeria induced an appreciable level mice, while in MPS-stimulated mice, i.v. injection with even 4 X 10(3) listeria could not induce such strong delayed footpad reaction. On the other hand, the i.v. injection of 3 X 10(1) listeria induced an appreciable level of delayed footpad reaction only in MPS-blocked mice. Acquired cellular resistance was depressed by MPS stimulation, whereas it was augmented by MPS blockade. These results suggested that non-specific activity of MPS modulates subsequent immune responses after inoculation of listeria.     

Pharmacological analysis of guinea-pig macrophage chemiluminescence responses to platelet activating factor and opsonized zymosan

The luminol-dependent chemiluminescence (CL) response in vitro of guinea-pig C. parvum-activated peritoneal macrophages to platelet activating factor (PAF) has been compared with that to opsonized zymosan (OpZ). The response to PAF (5 X 10(-6) mol/l.) reached a peak within 1 min, that to OpZ (0.17 mg/ml) within 10-20 min. Peak responses to both stimuli were dose-dependently inhibited in a similar manner by p-hydroxymercuribenzoate (10(-5) - 10(-3) mol/l), sodium benzoate (10(-5) - 10(-3) mol/l.) and quinacrine (10(-6) - 10(-3) mol/l.). In contrast, the xanthine oxidase inhibitor allopurinol (IC50 vs OpZ, 220 mumol/l.; vs PAF greater than 1000 mumol/l.), the methylation-inhibiting combination homocysteine + 3-deazaadenosine (IC50 vs OpZ, 22 mumol/l.; vs PAF greater than 100 mumol/l.), the phospholipase A2 inhibitor and alkylating agent p-bromophenacylbromide (pBPB; IC50 vs OpZ, 2.6 mumol/l.; vs PAF 15 mumol/l.) and the beta-adrenoceptor agonist isoprenaline (IC50 vs OpZ, 0.1 mumol/l.; PAF greater than 10 mumol/l.) all exerted differential inhibitory effects on the CL responses to the two stimuli, though colour quenching by adrenochrome cannot be ruled out in the differential effect of isoprenaline. In screening studies, carried out with CL responses measured 2 or 5 min after PAF and OpZ, respectively, verapamil (less than or equal to 10(-4) mol/l.), trifluoperazine (less than or equal to 10(5) mol/l.) EDTA (less than or equal to 10(6) mol/l.), mannitol (less than or equal to 10(-2) mol/l.), metyrapone (less than or equal to 10(-5) mol/l.), SQ 22536 (less than or equal to 10 micrograms/ml.), iso-butyl methylxanthine (less than or equal to 10(-5) mol/l.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a chemiluminescent DNA probe assay for the rapid confirmation of Listeria monocytogenes.

A Listeria monocytogenes-specific, acridinium-ester-labelled DNA probe was evaluated in a chemiluminescent homogeneous protection assay (HPA) for the rapid confirmation of suspect L. monocytogenes colonies from blood agar plates. The HPA uses an acridinium-ester-labelled chemiluminescent DNA probe in a free-solution hybridization format. After the DNA probe hybridized with the target ribosomal RNA, the acridinium label on the unhybridized probe was inactivated by a chemical differential hydrolysis step. Formation of a hybrid between probe and target was detected in a luminometer after the addition of a detection reagent. The assay can be completed in 30 to 45 min and allows for simultaneous processing of several (50-100) samples. The probe showed 100% sensitivity and 100% specificity for L. monocytogenes when evaluated in the HPA against L. monocytogenes, other Listeria species and other Gram-positive bacteria. The lower detection limit of the HPA was between 10(4) and 10(5) cells. In an evaluation with 296 bacterial colonies isolated from food, the HPA colony confirmation showed 100% agreement with conventional biochemical characterization. HPA will be useful for the rapid confirmation of L. monocytogenes isolated from food and clinical specimens.     

Antigen-specific T-cell responses during primary and secondary Listeria monocytogenes infection.

Although murine listeriosis is a widely used experimental model for the analysis of cell-mediated immunity, there is little information about individual T-cell antigens of Listeria monocytogenes which are recognized during primary and secondary infection. To study the antigen responses of L. monocytogenes-reactive T cells, somatic and secreted listerial proteins were separated by two-dimensional gel electrophoresis and subsequently divided into 480 liquid fractions. Antigen-specific T cells isolated from mice at different times of primary and secondary listeriosis were tested for their capacity to proliferate with distinct protein fractions. Supernatants of these cultures were screened for the production of gamma interferon, interleukin-4 (IL-4), and IL-10. Proliferation of antigen-specific T cells correlated with the production of high concentrations of gamma interferon, whereas IL-4 and IL-10 production in response to listerial protein fractions could not be detected. During both primary and secondary listeriosis, T cells recognized a multitude of somatic and secreted proteins rather than one or a few dominant antigens. Secreted proteins were recognized before somatic proteins, and T cells recognized different fractions in secreted and somatic proteins.     

Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes.

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon-gamma (IFN-gamma) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone-elicited macrophages from DBA2/J, CD-1, and C3H/HeN mice are listericidal. Activation of these cells in vitro for 20 h by IFN-gamma (20 or 500 U/ml) increased H2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6-h or 7-h culture. Incubation of macrophages with IFN-gamma plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6-h or 7-h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone-elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN-gamma in vitro, but was enhanced by IFN-gamma plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone-elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN-gamma does not improve listericidal activity.     

Role of macrophage scavenger receptors in response to Listeria monocytogenes infection in mice

Type I and type II macrophage scavenger receptors (SR-A I/II) recognize a variety of polyanions including bacterial cell-wall products such as lipopolysaccharide, suggesting a role for SR-A I/II in immunity against bacterial infection. SR-A I/II-deficient (MSR-A-/-) mice were more susceptible to infection with listeriolysin-O (LLO)-producing Listeria monocytogenes. After infection, Kupffer cells in wild-type (MSR-A+/+) mice phagocytized larger numbers of Listeria than those in MSR-A-/- mice. The number and the diameter of hepatic granulomas were larger in MSR-A-/- mice than MSR-A+/+ mice. L. monocytogenes replicated at higher levels in the liver of MSR-A-/- mice compared with MSR-A+/+ mice, and macrophages from MSR-A-/- mice showed impaired ability to kill Listeria in vitro. However, macrophages from MSR-A+/+ and MSR-A-/- mice showed similar levels of listericidal activity against isogenic mutant L. monocytogenes with an inactivated LLO gene. The listerial phagocytic activities of MSR-A+/+ macrophages treated with an anti-SR-A I/II antibody (2F8) and MSR-A-/- macrophages were significantly impaired compared with untreated MSR-A+/+ macrophages, indicating that SR-A I/II function as a receptor for L. monocytogenes. Electron microscopy revealed that most L. monocytogenes had been eliminated from the lysosomes of MSR-A+/+ macrophages in vivo and in vitro. In contrast, L. monocytogenes rapidly lysed the phagosomal membrane and escaped to the cytosol in MSR-A-/- macrophages and in MSR-A+/+ macrophages treated with 2F8 before phagosome-lysosome fusion. These findings imply that SR-A I/II plays a crucial role in host defense against listerial infection not only by functioning as a receptor but also by mediating listericidal mechanisms through the regulation of LLO-dependent listerial escape from the macrophages.     

The chemiluminescent response of human phagocytic cells to mineral dusts.

Luminol-dependent chemiluminescence (CL) was used to assess the in vitro production of reactive oxygen species by human neutrophils and monocytes on exposure to six standard respirable mineral dusts. Every dust caused CL production in both phagocytic cell types, although, for each dust, the two cells showed a different pattern of response. Light output was markedly affected by the presence of serum in the system. While the results illustrated the complexity of the interaction between mineral dusts and monocytes and neutrophils, they did not support the hypothesis that pathogenic dusts would induce the production of more reactive oxygen species than non-pathogenic dusts.     

T-cell-independent macrophage activation in mice induced with rRNA from Listeria monocytogenes and dimethyldioctadecylammonium bromide.

Purified rRNA from Listeria monocytogenes or Pseudomonas aeruginosa injected in combination with dimethyldioctadecylammonium bromide (DDA), protects mice nonspecifically against a lethal challenge of various extra- and intracellular bacteria. In the present study vaccination of BALB/c as well as C57BL/Ka mice with listerial RNA-DDA resulted in activation of fixed-tissue macrophages, as measured by an enhanced in vivo L. monocytogenes killing in spleen and liver. Evidence was found that macrophage activation by vaccination with rRNA-DDA occurred by a T-cell-independent mechanism. Treatment of mice with cyclosporin A had no effect on the enhanced L. monocytogenes killing induced with RNA-DDA; in vitro exposure of RNA-DDA to spleen cell cultures did not give rise to any lymphocyte proliferation. No evidence could be found for a possible adjuvant activity for RNA-DDA in cellular responses; in fact, RNA-DDA had an inhibitory effect on lymphocyte proliferative responses to Listeria antigen and to concanavalin A.     

Phagocytic and chemiluminescent responses of mouse peritoneal macrophages to living and killed Salmonella typhimurium and other bacteria.

In the presence of luminol, resident as well as thioglycolate-induced and immunized macrophages emitted chemiluminescence more efficiently when the cells were exposed to living Salmonella typhimurium than when they were exposed to the same bacterium killed by ultraviolet light or heat. This phenomenon was observed whether or not the bacterium was opsonized. The different response to living and killed bacteria was also found with Escherichia coli, Pseudomonas aeruginosa, Proteus morganii, and Enterobacter aerogenes, but not with Shigella sonnei, Klebsiella pneumoniae, and Propionibacterium acnes. The results suggest that macrophages respond better to living, motile bacteria than to nonmotile or killed bacteria. The experimental results obtained with motility mutants of S. typhimurium, E. coli, and P. aeruginosa confirm that macrophages exposed to the motile bacteria emit chemiluminescence more efficiently and ingest the motile bacteria at a much faster rate than the nonmotile bacteria.     

Human gamma/delta T-cell response to Listeria monocytogenes protein components in vitro.

Listeria monocytogenes is a facultative intracellular pathogen that replicates inside mononuclear phagocytes and induces specific cellular immunity. Listeriosis encompasses many clinical syndromes and meningitis is the most frequent clinical manifestation. Human alpha/beta and gamma/delta T cells have been shown to respond to L. monocytogenes antigens and to play an important role in resistance against listerial infection. We investigated the nature of listerial ligands and the influence of the major virulence factor, listeriolysin (hly), on the stimulation of human gamma/delta T cells from healthy individuals. We found that a listerial somatic protein ligand, which is sensitive to proteinase treatment, stimulated gamma/delta T cells in vitro; the majority of Listeria-responsive gamma/delta T cells expressed V gamma 9V delta 2 T-cell receptor chains and human leucocyte antigen-DR molecules; gamma/delta T-cell responses to hly+ and hly- Listeria strains were comparable; L. monocytogenes strains of different virulence stimulated gamma/delta T cells equally. Thus, protein components of L. monocytogenes unrelated to virulence activate human gamma/delta T cells in vitro.     

Ingestion and killing of Listeria monocytogenes by blood and milk phagocytes from mastitic and normal cattle.

Human listeriosis resulting from consumption of listeria-contaminated dairy products is emerging as a significant public health concern. There is a need to understand better the processes involved in the pathogenesis of Listeria monocytogenes-induced bovine mastitis. In the present report, we describe the results of the in vitro interaction of L. monocytogenes with bovine blood and milk leukocytes. Induction of an experimental L. monocytogenes mastitis resulted in a rapid and dramatic increase in neutrophils in the milk of infected cows. Blood neutrophils and mononuclear cells and milk leukocytes from listeria-infected and uninfected cows readily ingested L. monocytogenes in the presence of serum opsonins. These leukocytes also killed a portion of the ingested listeriae. Ingestion of listeriae evoked a vigorous chemiluminescence response by blood neutrophils and a relatively weak response by blood mononuclear cells. Ingestion, killing, and chemiluminescence by milk leukocytes were directly related to the percentage of neutrophils that were present. Blood neutrophils from healthy donor cattle ingested and killed L. monocytogenes when leukocyte-depleted milk and whey from mastitic cows were the sole sources of opsonins, although fewer listeriae were ingested than when normal bovine serum was present. These results indicate that bovine blood and milk phagocytes, like blood and inflammatory phagocytes from other mammalian species, can ingest and kill L. monocytogenes in vitro.     

Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri.

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.     

Hepatic cells' mitotic and peritoneal macrophage phagocytic activities during Trypanosoma musculi infection in zinc-deficient mice.

The effects of zinc deficiency on hepatic cell mitotic and peritoneal macrophage phagocytic activities were examined in mice infected with Trypanosoma musculi or immunized with parasitic products. On a full-complement or pair-fed diet, infected and homogenate-inoculated mice showed mitotic activity gains of 7.9% to 80.3% and 6.5% to 99.0%, respectively. Infected and homogenate-inoculated mice on a zinc-deficient diet showed 21.8% to 95.7% and 17.2% to 65.2%, respectively, more dividing liver cells compared with controls. In comparison to controls, macrophages isolated from infected and homogenate-immunized mice on full-complement or pair-fed diets had phagocytized 13.4% to 31.4% more latex particles from day 50 to 80. In the zinc-deficient group, macrophages isolated from infected mice had significant numbers of phagocytized latex particles (1.8% to 38.5%) from day 20 to day 80 compared with controls. The homogenate-immunized mice also had increased numbers (18.6 to 30.8%) of phagocytized latex particles.     

Human macrophage function and physical exercise: phagocytic and histochemical studies

Summary Macrophages derived from human connective tissue were assayed for their enzyme content and phagocytic activity after physical exercise. A single exhaustive endurance-running test caused increased phagocytic and enzymatic activities of the macrophages. Thus, an exercise challenge activates the functional status of the cells. This effect of physical exercise on macrophages is inconsistent with the practical experience that high performance athletes suffer more frequently from harmless infectious diseases.     

iactA of Listeria ivanovii, although distantly related to Listeria monocytogenes actA, restores actin tail formation in an L. monocytogenes actA mutant.

A gene homologous to the actA gene of Listeria monocytogenes was cloned from Listeria ivanovii (strain CLIP257) by chromosome walking starting from the ilo gene that encodes the pore-forming toxin ivanolysin. The nucleotide sequence revealed that this gene, named iactA, encodes a protein of 1,044 amino acids (IactA) comprising a central region with seven highly conserved tandem proline-rich repeats of 47 amino acids. Although IactA and ActA share an overall similar structure, these two proteins are only distantly related. Like ActA, IactA migrates aberrantly on sodium dodecyl sulfate gels. When expressed in an L. monocytogenes actA deletion mutant strain, iactA restored actin polymerization.