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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2010 November 3.
Published in final edited form as:
PMCID: PMC2972186
NIHMSID: NIHMS59675

Localized Reactive Oxygen and Nitrogen Intermediates Inhibit Escape of Listeria monocytogenes from Vacuoles in Activated Macrophages1

Abstract

Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of non-activated macrophages within 30 min of infection. Macrophages activated with IFN-γ, LPS, IL-6, and a neutralizing antibody against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91phox−/− and NOS2−/− mice showed that vacuolar retention required ROI and was augmented by RNI. Live-cell imaging with the fluorogenic probe dihydro-2′,4,5,6,7,7′-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the combined actions of ROI and RNI in a small space.

Keywords: macrophage, nitric oxide, lipopolysaccharide, bacterial

Introduction

A critical stage in the life history and pathogenesis of Listeria monocytogenes (Lm)3 occurs just after it enters a macrophage by phagocytosis. During a successful infection, Lm perforates the membranous vacuole that contains it, and escapes into the macrophage cytoplasm. There it can grow, divide, and eventually nucleate host cell actin in a process that facilitates transfer to neighboring cells (1). However, if the macrophage is activated, by IFN-γ, bacterial products, and other cytokines produced during the immune response to infection, then escape from vacuoles is inhibited and bacteria are killed (24).

Various molecules have been implicated in the listericidal activities of activated macrophages, but their relative effects on escape and killing have not been defined. Chief among these are reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which figure in defense against numerous pathogens (59). Activated macrophages produce nitric oxide by inducible nitric oxide synthase encoded by the NOS2 gene, and ROI by the NADPH oxidase complex. The GTPase Rab5a has been implicated in listericidal activity, possibly via regulation of Rac2 and assembly of the oxidase complex (10, 11). Both ROI and RNI contribute to murine resistance to Lm infection, and to the listericidal activities of activated macrophages (2, 1216). However, it is not known if ROI and RNI affect escape from the vacuole or subsequent microbicidal functions.

The present studies examined the contribution of ROI and RNI to Lm retention in vacuoles. The timing of Lm escape from vacuoles was measured, then gp91phox−/− and NOS2−/− knockout mice, which are unable to generate superoxide and nitric oxide, respectively (17, 18), were used to define the relative contributions of ROI and RNI to inhibition of escape in activated macrophages. Finally, fluorescent methods were used to measure the timing of ROI and RNI generation in vacuoles. These studies demonstrate that escape occurs within the first 30 min after entry, and that the combined actions of ROI and RNI inhibit Lm escape from vacuoles in activated macrophages.

Materials and Methods

Bacterial preparation

Listeria monocytogenes 10403S (gift of D. Portnoy) was maintained on brain-heart infusion agar plates. For experiments, one or two bacterial colonies were added to 5 ml of brain-heart infusion broth and shaken overnight at room temperature, diluted 1:6 the following morning, and shaken at 37° for 1.5 hours to obtain an O.D.600 of 0.500. Bacteria were washed by pelleting and resuspending in Ringer’s buffer (155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 2 mM NaH2PO4, 10 mM HEPES, 10 mM glucose, pH 7.2) 3X prior to addition to macrophages.

Macrophages

Female NOS2−/−, gp91phox−/−, and homozygous wild type (C57BL/6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). NOS2−/− and gp91phox−/− had been backcrossed for at least 10 generations onto a C57BL/6 background. Bone marrow-derived macrophages were cultured as previously described (19). After 5–9 days of growth, cells were replated into 6-, 24-, or 96-well tissue culture dishes overnight in DMEM, plus 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Gibco-BRL, Gaithersburg, MD). Macrophages were activated as previously described (20). Briefly, IFN-γ (100 U/ml; R & D Systems, Minneapolis, MN), LPS (100 ng/ml; List Biological, Campbell, CA), and a neutralizing antibody against IL-10 (α-IL-10, 5 μg/ml; R & D Systems) were included in the overnight incubation medium, followed by the presence of α-IL-10 and 5 ng/ml IL-6 (Calbiochem, San Diego, CA) during the course of the experiment.

Vacuole Escape Assay

Macrophages were plated onto 13-mm circular coverslips (7.5 × 104/coverslip) in a 24-well plate and activated overnight. Macrophages were washed twice with Ringer’s buffer, then incubated 15 min at 37° with Lm in DMEM +10% FBS without antibiotics (MOI ~ 0.1). For experiments determining timing of vacuole escape, bafilomycin A1 (BFA1; Sigma) was added to cells at a concentration of 500 nM, at various times after infection. Superoxide dismutase (SOD; 150 U/ml; Sigma), catalase (1500 U/ml; Sigma), and 5,10,15, 20-tetrakis(4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS; 100 μM; Calbiochem) were included during the infection as noted. For experiments involving the use of NG-monomethyl-L-arginine (1 mM; L-NMMA; Calbiochem), or diphenyleneiodonium (10 μM; DPI; Molecular Probes), cells were pretreated with the inhibitor for 15 min. L-NMMA or DPI were then included in the media for the duration of the experiment. Following infection, cells were washed 4X with Ringer’s buffer and incubated in DMEM + 10% FBS + 25 μg/ml gentamicin for 3.5 h. Cells were then fixed for 15 min at room temperature in cystoskeletal fix (30 mM HEPES, 10 mM EGTA, 0.5 mM EDTA, 5 mM MgSO4, 33 mM potassium acetate, 5% polyethylene glycol 400, 4% paraformaldehyde) followed by washing 3X with PBS + 2% goat serum, and permeabilization with 0.3% Triton X-100 in PBS for 5 min. Permeabilized cells were then washed 3X in PBS + goat serum for 5 min each and incubated for 15 min in PBS + goat serum with Texas Red-phalloidin (TR-phalloidin; 2 U/ml from 200 U/ml stock in methanol; Molecular Probes, Eugene, OR) and TR, 4′,6-diamidino-2-phenylindole (DAPI; 2 μg/ml, Molecular Probes). Cells were washed 3X for 5 min with PBS + goat serum and mounted on glass slides with Prolong Antifade (Molecular Probes). For each coverslip, 50 macrophages with DAPI-labeled bacteria were scored for colocalization of bacteria with filamentous actin.

Imaging with DHFF-BSA

Macrophages were plated onto 25-mm circular coverslips (2.5 × 105/coverslip) overnight and then mounted in a temperature-controlled stage at 37°, mounted on an inverted microscope (TE-300; Nikon, Tokyo, Japan) equipped with a cooled CCD camera (Quantix; Photometrics, Tuscon, AZ), filter wheel (Lambda 10-2; Sutter Instruments, Novato, CA), and a phase-contrast 100× oil objective (N.A.1.4). Cells were pulsed 5 min with Lm (MOI ~1) along with dihydro-2′,4,5,6,7,7′-hexafluorofluorescein covalently linked to bovine serum albumin (1 mg/ml; DHFF-BSA; Molecular Probes). Texas Red-Dextran (TR-Dextran, MW 10,000, 0.1 mg/ml) was included in the pulse to verify uptake of the non-fluorescent DHFF-BSA into phagosomes. Coverslips were then washed 3X with 5 ml Ringer’s buffer. MetaMorphtm software (Universal Imaging, Downington, PA) was used to create macros that sequentially acquired phase-contrast and fluorescence images exciting with 485 nm (F485) and 580 nm light (F580) using a multichroic beam-splitter (Omega Optical, Brattleboro, VT). Once an Lm-containing vacuole was located, images were acquired every 30s for about 10 min to assemble time-lapse sequences. Otherwise, coverslips were scanned over a period of 25 min, using phase-contrast microscopy to locate vacuoles containing Lm. Phase-contrast and fluorescence images were then acquired of each vacuole.

MetaMorphtm was used to quantitate fluorescent conversion of DHFF-BSA. Regions were traced around phase-contrast images of Lm-containing phagosomes. The corresponding region was copied to the F485 and F580 images and the average F485 and F580 pixel-intensities of the region were logged into a Microsoft Excel spreadsheet along with the time the image was acquired. To calculate the average amount of conversion of DHFF-BSA (Fig 6C), the individual phagosomal fluorescence intensities were pooled and averaged for each condition and a background value of 127 (average F485 in a phagosome without probe; calculated in a separate experiment) was subtracted. Values were then displayed as a percentage of the response measured in wild type, activated macrophages.

Figure 6
Rapid conversion of DHFF-BSA in Lm phagosomes. Images of phagosomes containing Lm, DHFF-BSA, and TR-Dextran were taken over a period of 25 min following a 5 min infection. The average fluorescence intensity (ex. 485 nm) was recorded in activated macrophages ...

For experiments comparing oxidation of DHFF-BSA with phagosomal area (Fig 7), F485, F580, time, and pixel area were recorded for Lm phagosomes of activated macrophages. Phagosomes were grouped into three arbitrary size groupings representing small, medium and large phagosomes. F485 intensity was divided by F580 intensity to normalize for the amount of probe within the phagosome. Student’s T test (2-tailed distribution) was used to calculate statistical significance of the differences between samples.

Figure 7
DHFF-BSA converted more efficiently in smaller phagosomes. Representative images of phagosomes, with areas of 132 (A), 264 (B), and 531 (C) pixels, respectively (bar = 2 μm). Fluorescence was normalized for the amount of ingested dye by dividing ...

Listericidal Assay

Macrophages were plated onto 13 mm coverslips and incubated overnight in medium alone (Ctrl) or medium plus one of the following combinations of ingredients: IFN-γ and LPS, IFN-γ, LPS, plus IL-6 added during the experiment, or IFN-γ, LPS, and α-IL-10, plus IL-6 added during the experiment. Macrophages were then incubated with Lm for 30 min, washed, and incubated for the indicated times in media with gentamicin (50 μg/ml) for 0.5–7.5 h, after which macrophages were fixed and stained with DAPI to determine the number of bacteria per infected macrophage.

Results

L. monocytogenes escapes from vacuoles within 30 min

To visualize the chemistries involved in the listericidal response of activated macrophages, it was important to know the time frame in which those chemistries would be active. A drop in pH is required for Lm to escape from the vacuole into the cytosol (21). The proton ATPase inhibitor BFA1 prevents endocytic vacuole acidification and can be used to prevent phagosomal escape of Lm (22). Non-activated macrophages were treated with BFA1 at various times after infection with Lm. Bacteria that had escaped into the cytosol were identified by their association with filamentous actin, as indicated by staining with TR-phalloidin. When cells were treated with BFA1 immediately following infection, only about 20% of bacteria engulfed by macrophages managed to escape into the cytoplasm (Fig 1). Later additions of BFA1 showed an increased amount of escape that leveled off after 30 min, at which point addition of BFA1 had no measurable effect on the escape of Lm. Therefore, nearly all vacuolar escape occurred within 30 min of infection. The mechanisms used by activated macrophages to retain Lm within the vacuole must be active during this brief period following entry.

Figure 1
Lm escape from phagosomes within 30 min. Macrophages were infected with Lm for 15 min followed by incubation in media + gentamicin (25 μg/ml) for 2 h. BFA1 (500 nM) was added the indicated times after the infection. Cells were fixed and stained ...

Activation of bone marrow-derived macrophages

Overnight treatment of peritoneal macrophages with IFN-γ and LPS is sufficient to control the growth of Lm (4). The same treatment of bone marrow-derived macrophages, however, does not lead to as high a degree of listericidal activity (23). Therefore, we sought to enhance activation of bone marrow-derived macrophages with additional factors to augment the standard IFN-γ and LPS method of activation. A neutralizing antibody against IL-10, a cytokine secreted by macrophages that downregulates activation (24), was included in an overnight activation medium along with IFN-γ and LPS. IL-6 was also included during the infection because it has been shown to increase the listericidal activity of macrophages when added at the start of infection (25). Activation of macrophages with IFN-γ and LPS alone initially reduced the number of bacteria per macrophage, as measured by staining infected cells with DAPI and counting the number of bacteria per cell, but bacterial numbers increased over 8 h (Fig 2). In contrast, macrophages activated with c controlled the growth and replication of bacteria for the duration of the experiment. Because activation with this cocktail of ingredients resulted in more efficient containment of Lm, this activation protocol was used for all further experiments.

Figure 2
Effects of macrophage activation on growth of Lm. Bone marrow-derived macrophages activated with IFN-γ, LPS, IL-6, and α-IL-10 were infected for 15 min with Lm, then washed and chased for the indicated times in gentamicin (25 μg/ml). ...

ROI and RNI both inhibit vacuolar escape by L. monocytogenes

The ability of activated macrophages to inhibit Lm escape was used to develop assays for the microbicidal activities of ROI and RNI. Macrophages were infected at a low MOI (~0.1) such that each infected macrophage initially contained only one bacterium. 2 h after infection, cells were fixed and stained with TR-phalloidin, and the percentage of infected macrophages with actin-positive bacteria was determined. The effect of various RNI and ROI inhibitors on the ability of Lm to escape from the vacuole into the cytoplasm was tested (Fig 3). Activation of macrophages with IFN-γ, LPS, IL-6, and α-IL-10 reduced Lm access to cytoplasm. Pre-treatment of activated macrophages with the nitric oxide synthase inhibitor L-NMMA led to a dramatic increase in the escape of Lm from vacuoles, indicating a role for nitric oxide in the prevention of Lm escape. Treatment of activated macrophages with the superoxide scavenger SOD produced a small but significant (p > 0.05) increase in Lm escape. The hydrogen peroxide scavenger catalase, alone or in combination with SOD, did not significantly increase vacuolar escape, suggesting that hydrogen peroxide did not contribute to the retention of Lm in vacuoles of activated macrophages. Treatment of activated macrophages with L-NMMA, SOD, and catalase together resulted in the greatest amount of escape from the vacuole, similar to the amount of escape observed in non-activated macrophages.

Figure 3
The effect of ROI and RNI inhibitors on escape of Lm from phagosomes. Cytoplasmic localization of Lm was determined by colocalization with TR-phalloidin. The percent of infected macrophages with TR-positive bacteria was recorded in non-activated and macrophages ...

As a separate approach for analyzing contributions of ROI and RNI to inhibition of Lm escape, vacuolar escape was measured in macrophages from NOS2−/− and gp91phox−/− mice where elimination of RNI, or ROI, respectively, was specific and complete (Fig 4A). Lm escape in non-activated macrophages from NOS2−/− and gp91phox−/− mice was not significantly different than escape in wild type macrophages. However, although activation of wild type macrophages greatly reduced vacuolar escape of Lm, escape from activated gp91phox−/− macrophages was only slightly less than non-activated gp91phox−/− macrophages. These results differed significantly from data obtained with ROI scavengers SOD and catalase (Fig 3), indicating that they may not have inhibited ROI completely. Activation of NOS2−/− macrophages led to a partial reduction in Lm escape. In all instances, data obtained with L-NMMA-treated macrophages was similar to the data obtained from NOS2−/− mice, indicating that it was an effective RNI-inhibitor. To examine the role of peroxynitrite, a microbicidal molecule formed by the reaction of superoxide and nitric oxide, on Lm escape, macrophages were treated with the peroxynitrite-specific scavenger FeTPPS (26) (Fig 4B). FeTPPS had no effect on phagosomal escape in non-activated macrophages, but escape in activated macrophages treated with the scavenger was approximately double that observed in control cells (p< 0.01).

Figure 4
Enhanced phagosomal escape of Lm in gp91phox−/− and NOS2−/− macrophages. The percent of infected macrophages with TR-positive bacteria was determined in activated and non-activated macrophages from wild type, gp91phox−/−, ...

In summary, the Lm escape studies indicated significant contribution of both ROI and RNI to the inhibition of escape from activated macrophages. The inability of macrophages from gp91phox−/− mice to inhibit escape suggests that ROI are essential. The substantial, albeit incomplete, inhibition in NOS2−/− mice indicated its secondary importance. And the partial inhibition by FeTPPS indicated a role for peroxynitrite.

Localization of oxidative activity using DHFF-BSA

To visualize the timing and localization of the oxidative chemistries generated within the Lm vacuole, live-cell imaging was performed with the probe DHFF-BSA. This is a relatively non-fluorescent molecule conjugated to BSA that, when oxidized, becomes highly fluorescent (27). Macrophages were incubated for 5 min with Lm, DHFF-BSA, and TR-Dextran, a fluorescent indicator of the volume of fluid taken into vacuoles and phagosomes. Time-lapse sequences of Lm vacuoles containing DHFF-BSA showed a rapid conversion of the relatively non-fluorescent probe to a fluorescein-like molecule (Fig 5A,B). Dye conversion was restricted to vacuoles containing bacteria. Probe-loaded macropinosomes, identifiable by their labeling with TR-Dextran, generally exhibited little DHFF-BSA fluorescence, even in cells containing vacuoles with very bright DHFF-BSA signals (Fig 5C–E). On rare occasions, fluorescent vacuoles with no apparent bacteria were observed; the fluorescence in these vacuoles was less than those that contained bacteria. It was unclear if these vacuoles contained unseen factors such as bacterial proteins, which generated a response by the macrophage.

Figure 5
Localization of phagosomal oxidative activity with DHFF-BSA. Macrophages were infected for 5 min in the presence of DHFF-BSA and TR-Dextran. A time-lapse series showing, at 30 s intervals, phase-contrast (A) and fluorescence images (ex. 485 nm) (B) of ...

One concern was that repeated exposure to 485 nm light in time-lapse sequences contributed to the oxidation of DHFF-BSA. To circumvent this problem, data were collected by scanning the coverslip using phase-contrast optics to identify Lm vacuoles, then taking only one set of images for each vacuole (phase-contrast, F485, F580). Quantitative data were plotted as a function of time after infection, then analyzed as a population of vesicles.

Fluorescent conversion of the probe was detectable almost as soon as images could be acquired (Fig 6A). DPI, an inhibitor of flavoproteins that prevents generation of both ROI and RNI (28, 29), completely abrogated fluorescent conversion of DHFF-BSA in activated macrophages (Fig 6B). Thus, chemistries restricted to the phagosomes were converting DHFF-BSA very soon after vacuole formation.

These chemistries were quantified by measuring the fluorescence in digital images. The degree of DHFF-BSA conversion was determined by calculating the average fluorescence intensity under various conditions (Fig 6C). Non-activated macrophages were capable of generating fluorescent vacuoles, although their average fluorescence-intensity was less than that of Lm vacuoles in activated macrophages.

Specificity of DHFF-BSA oxidation was examined using gp91phox−/− and NOS2−/− macrophages. Phagosomes from gp91phox−/− macrophages exhibited minimal dye conversion; their average fluorescence was similar to that observed in macropinosomes and in Lm vacuoles of DPI-treated macrophages. Thus, ROI were required for the oxidation of DHFF-BSA, and RNI alone were not sufficient to oxidize DHFF-BSA. Phagosomes from NOS2−/− macrophages, as well as macrophages treated with L-NMMA, demonstrated a reduced average phagosomal fluorescence compared to wild type activated macrophages. Hence, RNI, were not essential, but rather enhanced fluorescent conversion of the probe, indicating either that some dye conversion resulted from oxidation by peroxynitrite, or that nitric oxide somehow enhanced ROI generation.

Although most Lm vacuoles of activated macrophages inhibited escape, a smaller percentage showed dye conversion. We hypothesized that although the oxidative chemistries affecting Lm escape and dye conversion were qualitatively similar, their effects on Lm escape were more efficient than their ability to convert the dye. Moreover, we speculated that smaller, more tightly fitting phagosomes would oxidize DHFF-BSA more efficiently since, presumably, oxidative chemistries could be more concentrated in a smaller volume. To determine if fluorescent conversion of DHFF-BSA was affected by vacuolar size, the data of Fig. 6A were reexamined, measuring the area of Lm vacuoles, along with the intensity of DHFF-BSA and TR-Dextran fluorescence. Phagosomes were divided into three arbitrary size categories that were typical of tight-fitting, medium, and spacious phagosomes (Fig 7A–C). DHFF-BSA fluorescence was normalized for the amount of probe in the phagosome by dividing the DHFF-BSA fluorescence (F485, indicating conversion) by the TR-fluorescence (F580, indicating the volume internalized). Conversion of DHFF-BSA was significantly higher in small, tight-fitting phagosomes (0–200 pixels) than in large, spacious phagosomes (> 400 pixels; p < 0.02). Medium-sized phagosomes (200–400 pixels) also oxidized DHFF-BSA more efficiently than large phagosomes, but this difference was less significant (p < 0.07).

Discussion

Activated macrophages, which are critical components of the cell-mediated immune response against Lm (3033), control the growth and replication of Lm by retaining the bacteria within the phagocytic vacuole (4). ROI and RNI are important microbicidal mediators and contribute to the listericidal activity of macrophages (4). We show here that ROI and RNI directly affect retention of Lm in vacuoles of activated macrophages.

The role of ROI in the inhibition of escape was characterized using gp91phox−/− macrophages as well as the ROI-scavenging enzymes SOD and catalase. Activation did not improve the ability of gp91phox−/− macrophages to retain Lm within vacuoles (Fig 4A), thereby indicating a requirement for ROI in enhanced vacuolar retention by activated macrophages. ROI alone (L-NMMA-treated or NOS2−/− macrophages) could also reduce Lm escape, although this reduction was not as great as when RNI were also present. Results obtained with SOD and catalase did not produce a phenotype as pronounced as when gp91phox−/− macrophages were used. Acidification of the vacuolar compartment may have decreased the activity of SOD and catalase. Alternately, the rapid, nitric oxide-dependent conversion of superoxide to peroxynitrite, which should occur more rapidly than the scavenging of superoxide by SOD (34), may have consumed superoxide before it could be scavenged by SOD.

RNI also aided in the retention of Lm in vacuoles of activated macrophages. Activated NOS2−/− or L-NMMA-treated macrophages showed decreased Lm phagosomal escape, but these effects were not as complete as in wild type macrophages (Fig. 4A). RNI alone were not sufficient to retain Lm within vacuoles, because gp91phox−/− macrophages, producing RNI but not ROI, could not inhibit escape after activation. In all instances, the nitric oxide inhibitor L-NMMA produced results comparable to those obtained with NOS2−/− macrophages. However, preincubation of cells with L-NMMA for 15 min was necessary for complete suppression of nitric oxide generation (unpublished data).

Our data are consistent with a model in which two types of chemistries contribute to retention of Lm in vacuoles; one mediated by ROI alone, and another ROI-mediated chemistry that is also dependent on the presence of RNI. This is similar to the RNI-dependent and RNI-independent listericidal activities observed by Muller et al (12). One mechanism by which RNI could have enhanced the activity of ROI was via the generation of peroxynitrite, which is formed by the reaction of superoxide and nitric oxide. Peroxynitrite is highly reactive and microbicidal (3437). Evidence for peroxynitrite-mediated vacuolar retention was demonstrated by the increased percentage of cytoplasmic Lm in activated macrophages treated with FeTPPS, a peroxynitrite scavenger with little SOD-mimetic activity (26). Attempts to localize nitrotyrosine, a reaction product of peroxynitrite chemistry, have not succeeded. It is also possible, however, that RNI enhance ROI-mediated phagosomal retention by peroxynitrite-independent means. Along with being a microbicidal molecule, nitric oxide is also commonly employed as a signaling molecule. It is possible that nitric oxide-mediated chemistries primed the macrophage to deliver a more potent oxidative burst.

If peroxynitrite does have a role in the vacuolar retention of Lm, then superoxide and nitric oxide must be concomitantly present in the vacuole within 30 min of infection, before the bacteria had escape from the vacuole. Therefore, it was necessary to determine if the localization and timing of the generation of oxidative chemistries within the Lm vacuole were consistent with the generation of peroxynitrite. An imaging method was developed, using the fluorogenic probe DHFF-BSA, to study the timing and localization of the oxidative chemistries generated by macrophages into the Lm vacuole. While it is relatively common to measure the oxidative burst with fluorogenic probes either extracellularly or throughout the cytoplasm, to our knowledge, ROS/RNS have not previously been measured within a bacterial phagosome, the location where oxidative chemistries are most likely to have an effect. Fluorescent conversion of DHFF-BSA is reported to be a result of oxidation by hydrogen peroxide (27). DHFF-BSA oxidation was ROI-dependent, as judged by the lack of fluorescence generated by gp91phox−/− macrophages (Fig. 6C). RNI alone were insufficient for probe oxidation, but fluorescent conversion of DHFF-BSA was reduced in both NOS2−/− and L-NMMA-treated macrophages, indicating RNI contribute to the ROI-mediated oxidation of DHFF-BSA. Although the reactivity of DHFF-BSA with peroxynitrite has not been previously examined, 2′,7′-dichlorofluorescein, a molecule similar to DHFF, is readily oxidized by peroxynitrite (38, 39) suggesting the RNI-mediated enhancement of probe oxidation could be due to reaction with peroxynitrite. Conditions that led to fluorescent conversion of DHFF-BSA were similar to those that retained Lm within the vacuole, with the exception of NOS2−/− and L-NMMA-treated macrophages. In that case, escape was at an intermediate level between activated and non-activated control macrophages (Fig. 4A) whereas fluorescent conversion of DHFF-BSA in those cells was at the same level as in non-activated macrophages (Fig 6C), raising the possibility that there might be a small ROI/RNI independent contribution to vacuolar retention of Lm that does not lead to fluorescent conversion of DHFF-BSA. Overall, however, DHFF-BSA oxidation was a good indicator of the chemistries that led to vacuolar retention of Lm and demonstrated a rapid generation of oxidative chemistries localized to Lm phagosomes.

Many Lm vacuoles were not fluorescent, or were only slightly more fluorescent than background. This may have been a result of loss of probe, via fusion with lysosomes, or leakage through pores formed by the pore-forming toxin LLO. Also, vacuoles that were only partially formed after the 5-min pulse would have lost probe when the non-internalized bacteria were washed off. Still, many non-converting vacuoles must have contained probe, since they were TR-Dextran positive. These vacuoles may have been imaged before ROI/RNI were generated within the vacuole. Another possibility is that the concentrations of the oxidative chemistries required to retain Lm in the vacuole are less than those concentrations required to convert DHFF-BSA. Accordingly, some Lm vacuoles will have had ROI/RNI levels sufficient to block Lm escape, but insufficient to convert DHFF-BSA

The rapid and localized response of the oxidative burst within Lm vacuoles suggests recognition of the bacteria by the macrophage, and invites speculation as to how the signals are generated that result in this response. In the absence of opsonization by antibodies or complement, pattern recognition receptors are likely to contribute to the generation of an immune response. The most likely candidates for the start of the signaling cascade are Toll-like receptors (40, 41). It remains to be seen if oxidation of DHFF-BSA is a result of Toll-like receptor signaling.

Interestingly, DHFF-BSA was oxidized more efficiently in small, tight phagosomes than in large spacious ones. Anti-microbial chemistries generated into a tight-fitting phagosome would presumably be more concentrated than if they were generated within a larger, spacious phagosome. The formation of spacious phagosomes by Lm may be a defense mechanism to reduce the effectiveness of oxidative chemistries within the phagosome. Another possibility that remains to be tested is whether activated macrophages are better able to restrict phagosomal size and therefore enhance the effectiveness of ROI and RNI.

Although it is clear now that the combined actions of ROI and RNI are necessary for retention of Lm in vacuoles, their mechanism of action remains unknown. They could act cooperatively (e.g. generating peroxynitrite) or independently. For example, superoxide could inactivate LLO to slow escape while peroxynitrite kills the bacteria. It is also possible that nitric oxide enhances the function of the NADPH oxidase complex, either by direct chemical modification of component proteins, or by helping to create tight-fitting vacuoles that increase the efficacy of ROI delivered into that space.

Acknowledgments

The authors would like to thank Brenda Byrne and Lynne Shetron-Rama for technical assistance in the culture of macrophages.

Footnotes

1This work was supported by grants from the National Institutes of Health

3Abbreviations used in this paper: Lm, Listeria monocytogenes; ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates; α-IL-10, IL-10 neutralizing antibody; BFA1, bafilomycin A1; SOD, superoxide dismutase; FeTPPS, 5,10,15, 20-tetrakis(4-sulfonatophenyl) porphyrinato iron (III); L-NMMA, NG-monomethyl-L-arginine; DPI, diphenyleneiodonium; TR, texas red; DAPI, 4′,6-diamidino-2-phenylindole; DHFF-BSA, dihydro-2′,4,5,6,7,7′-hexafluorofluorescein coupled to BSA.

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