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The Journal of Immunology, 2006, 176: 1806-1813.
Copyright © 2006 by The American Association of Immunologists

Human Macrophages Do Not Require Phagosome Acidification to Mediate Fungistatic/Fungicidal Activity against Histoplasma capsulatum1

Simon L. Newman2,*, Lisa Gootee*, Jeremy Hilty{dagger} and Randall E. Morris{dagger}

* Department of Medicine, Division of Infectious Diseases, and {dagger} Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (M{phi}). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human M{phi} that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of ~6.4–6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. M{phi} that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by M{phi}, also maintained an intraphagosomal pH of ~6.5 over a period of several hours. Stimulation of human M{phi} fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human M{phi} cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal M{phi} digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse M{phi} fungistatic activity induced by IFN-{gamma}. Thus, human M{phi} do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse M{phi} do require acidification for fungicidal but not fungistatic activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Histoplasma capsulatum (Hc)3 is a dimorphic fungal pathogen that causes a broad spectrum of disease activity. In most cases, immunocompetent individuals develop a mild flulike illness, and only a small number of individuals actually exhibit acute clinical symptoms that require medical attention. However, if the inoculum is large enough, some individuals may develop a chronic cavitary pulmonary infection or a progressive disseminated infection that is life threatening (1). This latter disease progression is commonly observed in individuals whose immune system is compromised either by immunosuppressive agents or infection with HIV (2).

Infection with Hc occurs when conidia living in the soil are aerosolized and inhaled into the terminal bronchioles and alveoli of the lung. Inhaled conidia then convert into the yeast form that is responsible for the pathogenesis of histoplasmosis, and the yeasts are phagocytized by the alveolar macrophages (M{phi}) within which they multiply (1). The replicating yeasts destroy the alveolar M{phi}, and subsequently they are ingested by other resident alveolar M{phi} and by inflammatory phagocytes recruited into the lung. Repetition of this cycle leads to lymphohematogenous spread of infection to lymph nodes and other organs rich in mononuclear phagocytes. The hallmark of host defense against Hc is the development of specific cell-mediated immunity that leads to activation of M{phi} and resolution of the infection (3, 4).

The interaction of Hc with M{phi} is the key event in the pathogenesis of histoplasmosis. M{phi} first provide an environment for fungal replication and dissemination and then subsequently act as the final effector cells to remove the organism from the host. The strategies that Hc yeasts use to survive in M{phi} are only partly known, but it is clear that, in both human and murine M{phi}, the yeasts survive by controlling the intraphagosomal environment (5, 6). Upon ingestion, Hc yeasts activate the respiratory burst of human M{phi} (7) but do not stimulate a respiratory burst when phagocytosed by resident (8, 9) or IFN-{gamma}-activated (10) mouse peritoneal M{phi} (PM). Furthermore, phagocytosis of Hc yeasts by the mouse P388D1 M{phi} cell line (11) and resident PM (12) leads to normal phagolysosomal (PL) fusion. In contrast, there is minimal PL fusion after ingestion of Hc yeasts by human M{phi} (13, 14). As Hc yeasts multiply readily within all of these M{phi} populations (15, 16, 17), the yeasts apparently are able to evade the normally destructive effects of M{phi} lysosomal hydrolases.

The mechanism by which this is accomplished in mouse M{phi} is that Hc yeasts maintain an intraphagosomal pH of ~6.5 (6, 18). In the RAW 264.7 mouse M{phi} cell line, normal acidification of the phagosome is prevented by inhibition of the insertion of the M{phi} vacuolar ATPase (V-ATPase; proton pump) into the phagosomal membrane (18). These data suggest that the yeasts themselves must acidify the phagosome to pH 6.5. This strategy presumably allows the yeasts to avoid killing by lysosomal acid hydrolases and still acquire iron from transferrin that would be half-saturated at pH 6.5 (19), leaving some free iron available for yeast survival and growth.

In the present study, we sought to determine the intraphagosomal pH in human M{phi} under conditions in which Hc yeasts multiply or are killed. To our surprise, we found that, under both conditions, the intraphagosomal pH remains relatively neutral at pH ~6.4–6.5. Thus, human M{phi} lysosomal hydrolases do not require an acid pH to kill and degrade Hc yeasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Bafilomycin was a generous gift from K. Altendorf (Universitat Osnabruck, Osnabruck, Germany). Type I collagen from rat tails (Sigma-Aldrich) was dissolved in 0.1% acetic acid at 1 mg/ml and dialyzed overnight at 4°C in distilled water. Collagen (50 µl) was dispensed into the wells of a 96-well tissue culture plate and exposed to ammonia fumes for 30 min at 25°C. The gels were washed four times with RPMI 1640 before adherence of M{phi}. 3-(2,4-Dinitroanilo)-3'-amino-N-methyldipropylamine (DAMP) was obtained from Oxford Biomedical Research. Zymosan (Sigma-Aldrich) was prepared as described previously (20).

Yeasts

Hc strain G217B was maintained as described previously (21). Yeasts were grown in histoplasma M{phi} medium (22) at 37°C with orbital shaking at 150 rpm. For the digestion assay, log-phase yeasts were heat-killed (HK) at 65°C for 1 h and stored at 4°C in PBS containing 0.05% sodium azide (21). HK yeasts were labeled with FITC (0.1 mg/ml in 0.5 M carbonate-bicarbonate buffer; pH 9.0), washed, and resuspended in HBSS containing 20 mM HEPES and 0.25% BSA (HBSA) as described previously (21). For studies with viable yeasts, 48-h, log-phase yeasts were harvested by centrifugation, washed three times in HBSS, and resuspended to 50 ml in HBSS. Large aggregates were removed by centrifugation at 200 x g for 7 min at 4°C. The top 10 ml was removed, and the single-cell suspension obtained was standardized to the appropriate concentration in RPMI 1640 medium containing 5% FCS and 10 g/ml gentamicin. Saccharomyces cerevisae (Sc) was inoculated into 50 ml of yeast extract-peptone-dextrose broth and cultured for 24 h at 37°C with orbital shaking at 150 rpm. The yeasts were harvested, washed, and standardized following the same procedure as for Hc yeasts.

Preparation of human M{phi}

Human monocytes were purified from buffy coats obtained from the Hoxworth Blood Center (Cincinnati, OH). After partial purification via Ficoll-Hypaque centrifugation, monocytes were separated from lymphocytes by positive selection with anti-CD14 and EasySep magnetic nanoparticles (StemCell Technologies) according to the manufacturer’s instructions. M{phi} were obtained by culture of monocytes at 1 x 106/ml in Teflon beakers in RPMI 1640 medium containing 15% human serum, 10 µg/ml gentamicin (Sigma-Aldrich) and 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich) for 5–7 days (21). The acquisition and use of human blood cells for these studies has been approved by the Internal Review Board of the University of Cincinnati College of Medicine.

Preparation of mouse PM

Mice were sacrificed by CO2 asphyxiation and 10 ml of HBSS injected into the peritoneal cavity. After abdominal massage, PM were harvested by withdrawal of the medium. PM were washed twice and standardized to 1 x 106/ml in RPMI 1640 medium containing 10% FCS and 10 µg/ml gentamicin. Depending on the experiment, either 1 ml of PM was seeded into the wells of a 24-well tissue culture plate containing a 12-mm-diameter round coverglass, or 0.1 ml of PM was seeded into the wells of a 96-well tissue culture plate. PM were cultured for 24 h at 37°C in 5% CO2/95% air before assay. In some experiments, the PM were cultured for 24 h with 5 ng/ml murine IFN-{gamma}. The acquisition and use of mice in these studies has been approved by the Institutional Animal Care and Use Committee of the University of Cincinnati College of Medicine.

Quantitation of intraphagosomal pH by fluorescence ratio

Viable, HK, and paraformaldehyde (1% in PBS)-fixed yeasts (108/ml) were labeled by incubation at 37°C for 1 h in 0.1 mg/ml FITC in 0.5 M carbonate-bicarbonate buffer (pH 9.0). Zymosan (108/ml) was labeled by incubation at 25°C for 30 min in 0.1 mg/ml FITC. All particles were washed twice in HBSA and then resuspended to 108/ml in HBSA. M{phi} were adhered to 10 x 23-mm glass coverslips, and then incubated with 107 FITC-labeled yeasts or zymosan particles at 37°C for 1 h. After removal of nonadherent yeasts, the coverslips were placed into a cuvette containing warm 0.1 M phosphate buffer (pH 7.0), and the cuvette was placed into a thermostatted SPEX spectrofluorometer (Jobin Yvon Horiba) set at 37°C. Intracellular pH then was calculated by linear regression analysis from the fluorescence intensity ratio at Ex495/Ex450 (23) by comparison to a standard curve. Standard curves were generated with yeasts or zymosan alone, and with yeast and zymosan-infected M{phi}. In the former, yeasts or zymosan were suspended at 107/ml in 0.1 M phosphate buffer ranging in pH from 4 to 7.5 in half pH increments. In the latter, adherent M{phi} were infected with yeasts or zymosan as described above. After washing, the monolayers were inserted into the cuvette, and buffer of varying pH was added. The monolayers then were permeabilized by adding 0.1 ml of 3 mM monensin, and the fluorescence intensity ratio at Ex495/Ex450 was determined.

Quantitation of intraphagosomal pH by immunoelectron microscopy

As an alternative method, we quantified M{phi} intraphagosomal pH using DAMP, a weak base that accumulates exclusively in the acidic compartments of cells (24, 25). M{phi} adhered in six-well tissue culture plates were incubated with 30 µM DAMP for 30 min at 37°C. The monolayers were washed with HBSS and then incubated for 1 h with 5 x 106 viable or HK Hc or Sc yeasts to allow for phagocytosis. After removal of unbound yeasts by washing, one set of monolayers was fixed in 4% paraformaldehyde/0.5% glutaraldehyde. The remaining monolayers were incubated in RPMI 1640 medium for an additional 2, 4, 8, and 24 h. At each time point, the monolayers were washed and then fixed in 4% paraformaldehyde/0.5% glutaraldehyde. The monolayers then were washed with 0.1 M sodium cacodylate buffer (pH 7.2) and postfixed in 2% osmium tetroxide. After embedding in Epon, ultrathin sections were cut and put on a nickel grid. The sections then were incubated with a rabbit anti-DNP Ab (Sigma-Aldrich), followed by a biotinylated goat anti-rabbit IgG. After further washing, the sections were developed with streptavidin colloidal gold (5 nm). Pictures were made at a total magnification of x81,000, overlaid with a grid containing 12.5-mm squares, and the number of the gold particles per square micrometer in yeast-containing phagosomes and the cell nucleus (as the neutral control) was quantified. The pH of yeast-containing phagosomes then was calculated by using the formula: pH = 7.0 – log D1/D2, where D1 is the density of DAMP-specific gold particles in yeast-containing phagosomes, and D2 is the density of gold particles in the nucleus (24).

Quantitation of PL fusion

M{phi} were adhered in 24-well tissue culture plates to 12-mm round glass coverslips. Monolayers then were incubated with 200 nM LysoTracker Red (Invitrogen Life Technologies) in RPMI 1640 medium containing 5% FCS for 2 h to label lysosomes. After two washes, the M{phi} were infected with HK and viable Hc yeasts (2 x 106) for 1 h at 37°C. HK Hc was labeled with FITC as described previously (26). After an additional two washes, the M{phi} were cultured in 200 nM LysoTracker for an additional 2 h. After washing, the monolayers were fixed in 3.75% paraformaldehyde for 20 min at 25°C. The M{phi} then were covered in Dulbecco’s PBS containing 5% glucose. Coverslips were mounted cell-side down in 90% glycerol in PBS onto microscope slides, and 100 yeast-containing phagosomes were counted and scored for lysosomal fusion or no fusion. The data are expressed as the mean ± SEM of the percent PL fusion in three or more experiments performed in duplicate.

Quantitation of intracellular growth of Hc yeasts in M{phi}

Intracellular growth of Hc yeasts in human and mouse M{phi} was quantified by the incorporation of [3H]leucine as described previously (27). Human M{phi} cultured in Teflon beakers were harvested, washed, and adhered (1 x 105) in a 96-well plate for 1 h at 37°C in HBSA containing 2% aprotinin (Sigma-Aldrich). After adherence, M{phi} were washed twice in RPMI and incubated for 24 h with 1 x 104 viable yeasts. Mouse PM cultured for 24 h in 96-well plates were infected with Hc yeasts in an identical manner. All plates then were centrifuged and supernatants were carefully aspirated through a 27-gauge needle. Fifty microliters (1.0 µCi) of [3H]leucine (specific activity, 153 Ci/nmol; DuPont/New England Nuclear) in sterile water and 5 µl of a 10x yeast nitrogen broth (Difco Laboratories) were added to each well. After further incubation for 24 h at 37°C, 50 µl of L-leucine (10 mg/ml) and 50 µl of sodium hypochlorite were added to each well. The contents of the wells were harvested onto glass fiber filters using an automated harvester (Molecular Devices). The filters were placed into scintillation vials, scintillation mixture was added, and the vials were counted in a Beckman LS 6500 liquid scintillation spectrometer (Beckman Coulter). The results are expressed as the mean ± SEM of the cpm incorporated by remaining viable Hc yeasts. Experiments were performed in triplicate, and three to five experiments were performed with cells from different donors.

Yeast digestion assay

Human or mouse M{phi} adherent to 12-mm round glass coverslips in a 24-well tissue culture plate were infected with 5 x 106 FITC-labeled HK yeasts for 1 h at 37°C. The monolayers were washed twice to remove noningested yeasts. One pair of coverslips then was fixed in 1% paraformaldehyde, and the remaining monolayers were cultured for 24 h at 37°C in the absence or presence of 25 nM bafilomycin. At the end of the incubation period, the M{phi} were washed and then fixed in 1% paraformaldehyde. Coverslips were mounted cell-side down onto microscope slides, and 100 M{phi} per coverslip were counted via phase and fluorescent microscopy. The data are presented as the association index that is defined as the total number of yeasts bound or ingested per 100 M{phi}. Although we did not specifically discriminate bound from ingested yeasts in these experiments, under the conditions of this assay, >90% of yeasts reside in M{phi} phagosomes after 1 h of phagocytosis, and after additional culture, all yeasts are internalized (21).

Statistics

Statistical analysis of the data was performed using SigmaStat (Jandel). Student’s t test or the Mann-Whitney rank sum test was used to analyze the data, and the results were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intraphagosomal pH of Hc-infected human M{phi}

To quantify intraphagosomal pH, we took advantage of the fact that excitation of FITC is sensitive to pH (23). Viable and HK Hc yeasts were labeled with FITC, and the relationship of fluorescence intensity to pH was used to generate standard curves (Fig. 1). Separate standard curves were generated with M{phi} infected with viable or HK yeasts. In addition, standard curves were generated with viable or HK yeasts alone. In Fig. 1, it is clear that the curves generated by Hc-infected M{phi} and Hc alone were almost identical, confirming that the M{phi} were effectively permeabilized with monensin. FITC labeling of live yeasts did not affect their viability as determined by comparison of their growth to unlabeled yeasts in tissue culture medium (data not shown). A similar standard curve was generated for experiments using paraformaldehyde-fixed Hc yeasts (data not shown).


Figure 1
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  		FIGURE 1. The relationship between fluorescence intensity and pH of FITC-labeled viable and HK Hc yeasts. M{phi} that had ingested FITC-labeled yeasts were placed in buffer of varying pH and permeabilized with monensin so that the pH of the phagosome was the same as the medium. The ratio of fluorescence emissions after excitation at 495 and 450 nm then was determined. The fluorescence intensity ratio of yeasts alone also was determined for comparison. The data are the mean ± SEM of three to four individual experiments.

 
M{phi} were infected with viable or HK Hc yeasts, and then pH was determined at various times postinfection. After 1 h of phagocytosis, the pH in M{phi} phagosomes containing viable Hc was 6.5 (Table I). These data confirm the original observation of Eissenberg et al. (6) with the P388D1 mouse M{phi} cell line, and our own studies with the RAW 264.7 mouse M{phi} cell line (18). Unexpectedly, we found that the pH in phagosomes containing HK Hc was 6.4, and the pH did not decrease over a 4-h period. Likewise, the pH in M{phi} phagosomes containing paraformaldehyde-fixed Hc yeasts was ≥6.5, and again there was no acidification over a 4-h period (Table I). These data contrast with previous studies with P388D1 M{phi} (6) and RAW M{phi} (18) in which the intraphagosomal pH with methanol-fixed yeasts or HK yeasts were reported to be 4.7 and 4.8, respectively.


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  		Table I. Intraphagosomal pH of human M{phi} containing Hc yeasts

 
Intraphagosomal pH of human M{phi} ingesting zymosan

We next sought to determine whether the lack of phagosome acidification was unique to Hc yeasts. M{phi} were allowed to ingest FITC-labeled zymosan particles for 1 h and were pH quantified immediately or after culture for a further 2–4 h. The pH standard curve for monocytes or M{phi} that had ingested FITC-labeled zymosan is shown in Fig. 2. The data in Table II show that M{phi} phagosomes containing zymosan slowly acidified over a 4-h period, dropping from an initial pH of 6.6 to 5.2. By comparison, freshly isolated monocytes acidified phagosomes containing zymosan after 1 h. Thus, the lack of acidification of M{phi} phagosomes containing HK or paraformaldehyde-fixed yeasts was not an artifact of the assay system.


Figure 2
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  		FIGURE 2. The relationship between fluorescence intensity and pH of FITC-labeled zymosan. Freshly isolated monocytes or M{phi} that had ingested FITC-labeled zymosan were placed in buffer of varying pH and permeabilized with monensin so that the pH of the phagosome was the same as the medium. The ratio of fluorescence emissions after excitation at 495 and 450 nm then was determined. The data are the mean ± SEM of five experiments with monocytes and three experiments with M{phi}.

 

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  		Table II. Intraphagosomal pH in human monocytes and M{phi} containing zymosan

 
Intraphagosomal pH in human M{phi} quantified by immunoelectron microscopy

To confirm that human M{phi} do not acidify phagosomes containing HK Hc yeasts, we quantified intraphagosomal pH by immunoelectron microscopy using the DAMP reagent (24, 25). M{phi} were labeled with DAMP and then allowed to phagocytose viable or HK Hc yeasts for 1 h. M{phi} monolayers were fixed and immediately processed for immunoelectron microscopy or were cultured for 2–24 h before fixation. Fig. 3 shows a yeast-containing phagocytic vacuole at 1 h postinfection. The gold particles indicate the localization of DAMP in the phagosome. Fig. 4A shows that from 2 to 8 h postingestion, M{phi} phagosomes containing viable Hc yeasts maintained a pH of ~6.4. Likewise, M{phi} phagosomes containing HK Hc yeasts maintained a similar pH up to 24 h postingestion. These data confirm the results obtained with the fluorescent ratio technique.


Figure 3
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  		FIGURE 3. Electron micrograph of a yeast-containing phagosome labeled with DAMP. The number of gold particles in the phagosome in this micrograph calculates to a pH of 6.4. The total magnification is x27,000, and the bar represents 1 µm. For preparation of this photograph, the gold particles were identified on the negative and enlarged using Adobe PhotoShop software (Adobe Systems). The final image then was optimized for contrast (63 ).

 

Figure 4
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  		FIGURE 4. Quantitation of intraphagosomal pH via immunoelectron microscopy with DAMP. M{phi} were cultured for 30 min with DAMP and then infected with viable or HK Hc yeasts (A) or viable or HK Sc for 2 h (B). Either immediately or after further culture for up to 24 h, M{phi} monolayers were processed as described in Materials and Methods, and the pH was quantified by comparing the number of gold particles in yeast-containing phagosomes with the number of gold particles in the nucleus. The data represent the mean of 15–22 individual phagosomes as shown over each bar.

 
Human M{phi} rapidly kill and degrade the nonpathogenic yeast Sc. Therefore, we sought to determine whether human M{phi} phagosomes would acidify after ingestion of Sc. Fig. 4B shows that, 2 and 4 h after phagocytosis of viable or HK Sc, the pH in yeast-containing phagosomes again was ~6.4.

Bafilomycin does not reverse the fungicidal activity of human M{phi}

Overall, these data suggested that human M{phi} did not require phagosomal acidification to digest HK Hc yeasts or to kill nonpathogenic Sc yeasts. Previously, we identified two conditions under which human M{phi} could be induced to kill viable Hc yeasts. In the first, Hc-infected M{phi} were incubated in the presence of chloroquine (28). As a weak base, chloroquine raises intraphagosomal pH, thereby restricting the availability of iron from transferrin, which is pH dependent (29). Our original interpretation of the data was that after the yeasts became metabolically inactive, at some point thereafter, yeast-containing phagosomes were acidified, and the yeasts then were killed and degraded by lysosomal enzymes that presumably required an acid pH to be active. As the current data suggested otherwise, we tested this hypothesis by culturing Hc-infected M{phi} in the absence and presence of chloroquine and bafilomycin A1, an inhibitor of the V-ATPase (30). As originally reported, culture of Hc-infected M{phi} in the presence of chloroquine caused significant inhibition of the intracellular growth of Hc yeasts (Fig. 5A). The addition of bafilomycin to chloroquine-treated M{phi} did not reverse inhibition of Hc replication, and bafilomycin alone had no effect on the replication of yeasts in control M{phi}.


Figure 5
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  		FIGURE 5. Bafilomycin does not reverse the fungicidal activity of human M{phi} induced by adherence to collagen or by coculture with chloroquine. A, Hc-infected M{phi} were cultured in the absence or presence of chloroquine and with or without bafilomycin for 24 h. B, M{phi} were adhered to collagen matrices, infected with Hc yeasts, and then cultured for 24 h in the absence or presence of bafilomycin. M{phi} adhered to plastic served as the control. The intracellular growth of Hc yeasts then was quantified as described in Materials and Methods. The data are presented as the mean ± SEM of the cpm from four experiments on collagen and five experiments with chloroquine. All experiments were performed in triplicate. A, *, p < 0.001, compared with the control; B, **, p < 0.05, compared with M{phi} on plastic.

 
The second condition known to induce M{phi} fungicidal activity against Hc is by adherence of the M{phi} to type 1 collagen matrices (13). Under these conditions, M{phi} override the yeasts’ ability to inhibit PL fusion. Therefore, M{phi} adhered to collagen gels were infected with Hc yeasts and then cultured for 24 h in the absence and presence of bafilomycin. As shown in Fig. 5B, M{phi} adhered to collagen significantly restricted the replication of Hc yeasts, compared with M{phi} adhered to tissue culture plastic. The addition of bafilomycin to the cultures did not reverse collagen-induced inhibition of intracellular growth.

Bafilomycin inhibits the capacity of mouse PM, but not human M{phi}, to digest HK Hc yeasts

M{phi} easily degrade ingested HK Hc yeasts (5). Therefore, to confirm that phagosomal acidification was not required for this process, M{phi} were allowed to phagocytose FITC-labeled HK Hc yeasts for 1 h. One set of monolayers then was fixed and served as a baseline for the total number of yeasts ingested. The remaining M{phi} were cultured overnight in the absence or presence of bafilomycin. After 1 h of phagocytosis, M{phi} had ingested an average of ~3.5 yeasts/M{phi} (Fig. 6A). After culture for 24 h, virtually all of the yeasts had been digested, and bafilomycin did not inhibit this process. Fig. 6B shows the identical experiment performed with resident mouse PM. Like human M{phi}, PM ingested an average of 3.5 yeasts/M{phi} after 1 h of phagocytosis. PM also completely digested the internalized yeasts after further culture for 24 h. However, the addition of bafilomycin completely inhibited the capacity of mouse PM to digest HK Hc yeasts.


Figure 6
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  		FIGURE 6. Human M{phi}, but not mouse PM, digest HK yeasts in the presence of bafilomycin. Human and mouse M{phi} were incubated with FITC-labeled HK yeasts for 1 h at 37°C. Noningested yeasts were removed by washing, and one set of monolayers was fixed immediately (1 h). The remaining M{phi} were cultured for 24 h in the absence or presence of bafilomycin, and then fixed. The data are presented as the mean ± SEM (n = 4) of the association index, the total number of yeasts bound/ingested per 100 M{phi}. A, Human M{phi}; B, mouse PM. *, p < 0.05, compared with 1-h controls.

 
Bafilomycin does not inhibit the IFN-{gamma}-induced fungistatic activity of mouse PM

IFN-{gamma} activates mouse PM (31), but not human M{phi} (16, 27), to inhibit the replication of Hc yeasts. The mechanism of this inhibition is through the production of NO (32) and the restriction of iron (33). Although Hc yeast replication is completely inhibited in IFN-{gamma}-activated mouse PM, none of the yeasts is killed and degraded (31). This observation is paradoxical, considering that PL fusion occurs even in unactivated mouse PM (12). We have confirmed this previous study using LysoTracker Red to label the lysosomes. After 1 h of phagocytosis, >90% of mouse PM phagosomes containing viable or HK yeasts demonstrated PL fusion (Fig. 7). In contrast, and as reported previously (13, 14), only 8% of human M{phi} phagosomes containing viable Hc yeasts demonstrated PL fusion, whereas >95% of phagosomes containing HK yeasts showed PL fusion (Fig. 7).


Figure 7
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  		FIGURE 7. Inhibition of PL fusion by viable Hc yeasts in human M{phi}. Human and murine M{phi} adhered to 12-mm-diameter coverslips were loaded with LysoTracker Red and then incubated for 1 h with viable (V) or HK Hc yeasts. The percent lysosomal fusion was quantified as described in Materials and Methods. The data are the mean ± SEM of four experiments with mouse PM and six experiments with human M{phi}.

 
Based on the above observations, we hypothesized that the lack of killing of Hc yeasts by IFN-{gamma}-activated mouse PM might be caused by an absence of phagosome acidification. To determine whether this was a viable hypothesis, mouse PM were cultured for 24 h in the absence or presence of IFN-{gamma}. The PM then were infected with Hc yeasts and cultured for an additional 24 h in the absence or presence of bafilomycin. As reported previously (31), PM preincubated with IFN-{gamma} completely inhibited the replication of Hc yeasts (Fig. 8). The addition of bafilomycin did not abrogate the IFN-{gamma}-induced inhibition, and the addition of bafilomycin to control PM was without effect (Fig. 8).


Figure 8
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  		FIGURE 8. Bafilomycin does not inhibit the fungistatic activity of IFN-{gamma}-activated mouse PM. Mouse PM were cultured for 24 h alone (Co) or in the presence of IFN-{gamma} (IFN) and then infected for an additional 24 h with Hc yeasts in the absence or presence of bafilomycin (BAF). Intracellular replication was quantified as described in Materials and Methods. The data are presented as the mean cpm of two experiments performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The interaction between Hc yeasts and human and murine M{phi} differs considerably (reviewed in Ref.34). Phagocytosis of Hc yeasts by murine M{phi} does not stimulate the production of toxic oxygen radicals (8, 9, 10), whereas uptake of yeasts by human M{phi} does stimulate the respiratory burst (7). Regardless, Hc yeasts multiply readily in both human and murine M{phi}. More provocatively, PL fusion occurs normally in some murine M{phi} (11, 12), but not others (18), and PL fusion is inhibited in human M{phi} (Refs.13 and 14 and present paper). The reason(s) for these differences are unclear.

In the P388D1 mouse M{phi} cell line (6) and in the RAW mouse M{phi} cell line (18), phagosomes containing viable Hc yeasts maintain an intraphagosomal pH of ~6.5, whereas phagosomes containing HK or fixed yeasts are acidified. A pH of 6.5 presumably allows Hc yeasts to avoid killing by lysosomal acid hydrolases but still acquire free ferric from transferrin that would be half-saturated at that pH (19). Indeed, culture of Hc-infected human M{phi} with the weak base chloroquine (to raise intraphagosomal pH) induces M{phi} to kill and degrade the yeasts. The effect of chloroquine is reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH (35), but not by holotransferrin, which releases iron only in an acidic environment. Furthermore, chloroquine given i.p. to Hc-infected C57BL/6 mice significantly reduces the growth of Hc in spleens and livers and even protects mice from a lethal inoculum of Hc (28). Thus, iron is of critical importance to the survival of Hc yeasts in vitro in human M{phi} and in vivo in a murine model of histoplasmosis.

In the present study, we sought to determine the intraphagosomal pH in human M{phi} infected with viable or HK Hc yeasts and then to determine the temporal acidification of yeast-containing phagosomes after treatment with chloroquine or upon adherence of M{phi} to type 1 collagen gels, conditions that lead to the killing and digestion of intracellular Hc (13, 28). As was found for P388D1 and RAW M{phi} (6, 18), the intraphagosomal pH in human M{phi} that had phagocytosed viable Hc yeasts was ~6.5. However, to our surprise, we never observed the expected acidification in phagosomes containing HK or fixed Hc yeasts. Rather, we consistently observed an intraphagosomal pH of 6.5, the same as for viable yeasts. This finding was verified using two different techniques to quantify intraphagosomal pH. In addition, human M{phi} failed to acidify phagosomes containing the nonpathogenic yeast Sc, which is easily killed and degraded by M{phi}. These results were not due to a technical complication, because we observed phagosomal acidification in monocytes and M{phi} that had ingested zymosan. Thus, it appears that the acidification of phagosomes in human M{phi} depends either on the target particle or microorganism that is ingested or the specific receptor-initiated signaling pathway that is engaged by the target organism/particle. Additional experiments with other microorganisms and/or inert target particles will be required to sort out this question.

PL fusion with concomitant acidification of the phagosome is considered to be a critical requirement for effective M{phi} antimicrobial activity regardless of whether the encounter is with an extracellular pathogen or an intracellular pathogen. Indeed, a major strategy for survival in M{phi} by intracellular pathogens centers on their ability to inhibit PL fusion and/or phagosome acidification. Thus, Mycobacterium tuberculosis (36, 37, 38, 39, 40), Mycobacterium avium (41, 42, 43), Toxoplasma gondii (44, 45), and Hc (6, 13, 14) all inhibit both PL fusion and acidification. Interestingly, Coxiella burnetii inhibits PL fusion but permits phagosome acidification, because it apparently prefers this low pH (46, 47, 48, 49). Likewise, Brucella suis (50) and Cryptococcus neoformans (51, 52) also prefer an acidic compartment, and raising the intraphagosomal pH causes a marked decrease in their intracellular viability.

Bordetella pertussis is easily killed by M{phi}, whereas Bordetella bronchiseptica remains viable for days. After phagocytosis of both of these microorganisms, M{phi} phagosomes acidify and PL fusion occurs. B. pertussis apparently is sensitive to low pH, because it does not even survive in acidic bacterial growth medium. Interestingly, increasing intraphagosomal pH enhances the survival of B. pertussis but decreases the survival of B. bronchiseptica (53). Overall, these data highlight the fact that each individual microorganism is unique in its nutritional requirements and in its strategy to survive the hostile environment inside M{phi} phagosomes.

Human M{phi} easily digest HK Hc yeasts (5), and based on the present data, the lysosomal hydrolases that degrade the yeasts do so at a relatively neutral pH. Indeed, the addition of bafilomycin to cultures of human M{phi} did not abrogate their ability to digest HK Hc yeasts. Likewise, bafilomycin did not block the ability of chloroquine-treated M{phi} or collagen-adherent M{phi} to inhibit the growth of viable Hc yeasts. In contrast, addition of bafilomycin to cultures of mouse PM completely inhibited their capacity to digest HK Hc yeasts. Thus, it would appear that, unlike the lysosomal enzymes of human M{phi}, the lysosomal hydrolases of murine PM require an acidic pH for their activity.

This information now provides a rationale for why there is normal PL fusion in Hc-infected mouse M{phi}, but PL fusion is inhibited in Hc-infected human M{phi}. As stated earlier, maintenance of a pH of 6.5 in Hc-containing phagosomes in murine M{phi} protects the yeasts from the lethal effect of lysosomal hydrolases that require an acid pH for optimal activity. However, this strategy would not be sufficient in human M{phi}, because the lysosomal enzymes apparently are active at pH 6.5. Thus, Hc yeasts must prevent their exposure to these enzymes by inhibiting PL fusion. In both human and murine M{phi}, the maintenance of a pH of 6.5 in yeast-containing phagosomes permits the acquisition of iron.

As discussed earlier, Hc yeasts must procure intracellular iron to survive in human and murine M{phi} (28, 54). Indeed, part of the mechanism by which IFN-{gamma} activates murine M{phi} anti-Histoplasma activity is via the restriction of iron (33, 55). Therefore, it is curious that IFN-{gamma}-activated murine M{phi} only are fungistatic and not fungicidal, particularly considering the fact that lysosomal hydrolases are present in addition to the production of NO and iron restriction (32, 33, 55, 56). We hypothesized that IFN-{gamma}-activated mouse M{phi} might only be fungistatic because of a lack of acidification of the yeast-containing phagosome, which would prevent lysosomal enzymes from killing and digesting the yeasts. The fact that bafilomycin, an inhibitor of the V-ATPase, did not inhibit the fungistatic activity of IFN-{gamma}-activated mouse PM supports this hypothesis.

In vivo studies in a murine model of histoplasmosis demonstrate that a successful immune response to Hc requires the induction of the cytokines IL-12, IFN-{gamma}, and TNF-{alpha} (57, 58, 59, 60, 61). Furthermore, the secretion of IL-12 is required for the production of IFN-{gamma} (58, 60), which presumably is required for activation of M{phi} to destroy the yeasts. Although TNF-{alpha} production is required for a successful host response in both naive and Hc-immune animals (57, 58, 59, 61, 62), the mechanism of action of TNF-{alpha} is unknown. However, TNF-{alpha} does not appear to directly activate M{phi} (55, 61). Based on the data in the present studies, we hypothesize that there is at least one other as-yet-unidentified cytokine required for M{phi} activation, and this cytokine induces the M{phi} to acidify Hc yeast-containing phagosomes.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by U.S. Public Health Service Grants AI-37639, AI-49358, and AI-061298 from the National Institutes of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Simon L. Newman, Division of Infectious Diseases, University of Cincinnati College of Medicine, P.O. Box 67056, Cincinnati, OH 45267. E-mail address: newmansl{at}email.uc.edu Back

3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; M{phi}, macrophage; PM, peritoneal M{phi}; PL, phagolysosomal; V-ATPase, vacuolar ATPase; DAMP, 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine; HK, heat-killed; Sc, Saccharomyces cerevisae. Back

Received for publication August 1, 2005. Accepted for publication November 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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