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Departments of
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Molecular Genetics, Biochemistry, and Microbiology;
Medicine, Division of Infectious Diseases; and
Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages (Mø) play a dual role in host defense against Hc. Initially, Mø provide the fungus with an intracellular environment in which they can replicate (9, 10). Hc yeasts reside within nonactivated Mø phagosomes in which they multiply until the Mø are destroyed. Upon induction of cell-mediated immunity, activated Mø act as the final effector cells and destroy the yeasts (11). During their residence in Mø, Hc yeast does not appear to secrete any factors that globally affect Mø function, but rather control their intraphagosomal environment (12, 13).
In the P388D1 mouse Mø cell line, phagolysosomal (PL) fusion occurs normally (14), but the pH of Hc-containing phagosomes is 6.5 (13). In contrast, phagosomes containing zymosan or methanol-killed Hc acidify normally, maintaining a pH of 5.0 (13). Results of studies in mouse peritoneal Mø, as well as in the Mø cell line J774.2, support this conclusion (15). In contrast, in human Mø that have ingested Hc yeasts, PL fusion is fivefold less than that which occurs upon ingestion of Saccharomyces cerevisiae (Sc) (16), but the pH in the phagosome still is 6.5 (S. L. N. unpublished observations).
In human Mø, raising the pH of Hc-containing phagosomes with the weak base chloroquine results in killing and digestion of the intracellular Hc (17). The effect of chloroquine is reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin that releases iron only in an acidic environment (17). That these in vitro findings are applicable in vivo is demonstrated by the fact that chloroquine reduces the number of organisms in the spleens and livers of Hc-infected mice in a dose-dependent manner, and that chloroquine can protect mice from a lethal inoculum of Hc yeasts (17). Taken together, these data suggest that, to survive and multiply within Mø phagosomes, Hc requires phagosomal acidification low enough to acquire iron from transferrin, but also high enough to minimize the activity of lysosomal hydrolases.
The enzyme responsible for generating and maintaining the acidic pH in phagosomal and endosomal pathways is vacuolar ATPase (V-ATPase) (18, 19, 20). Current data suggest that there are two possible mechanisms that Hc might utilize to control phagosomal pH. First, Hc might alkalinize the phagosome to counteract the effects of the V-ATPase. This mechanism might operate in murine peritoneal Mø or P388D1 Mø in which PL fusion occurs normally. Second, Hc might inhibit the accumulation of functional VATPase in the phagosomal membrane either by inactivating the V-ATPase present in the phagosomal membrane (by direct interactions or by enhancing degradation of the enzyme) or by inhibiting fusion with vesicles containing the V-ATPase. In this case, the yeasts would be required to slightly acidify their environment to obtain a pH of 6.5. This mechanism might operate in human Mø in which PL fusion is limited (16).
To distinguish between these two mechanisms, bafilomycin, a specific inhibitor of the V-ATPase, was used to block V-ATPase function in RAW Mø. The results of these experiments demonstrate that Hc yeasts do not require the phagosomal V-ATPase to acidify the phagosome, and that they inhibit accumulation of the V-ATPase in the phagosomal membrane. Furthermore, we find that RAW Mø are similar in function to human Mø in that PL fusion does not occur normally after ingestion of Hc yeasts. Finally, we demonstrate that Hc-containing phagosomes have diminished access to the late endocytic pathway, but full access to the fluid phase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bafilomycin was the generous gift of Prof. K. Altendorf (Universitat Osnabruck, Osnabruck, Germany). Fluorescein-conjugated and biotinylated secondary Abs were from Vector Laboratories (Burlingame, CA). HRP-coupled secondary Abs were from Amersham (Arlington Heights, IL). Paraformaldehyde and glutaraldehyde were from Electron Microscopy Sciences (Washington, PA). Eponate 12 was from Ted Pella (Redding, CA). All sera were from Hyclone (Logan, UT). All other reagents were reagent grade or better and were from Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO).
Mø cell culture
RAW 264.7 cells (BALB/c mouse macrophage transformed with Abelson leukemia virus) from the American Type Culture Collection (Manasses, VA) were cultured in DMEM containing 4.5 g/L glucose supplemented with 10% heat-inactivated FBS (complete medium) at 37°C, 5% CO2.
Hc and Sc
Hc strain G217B was maintained on brain heart infusion agar and cultured in quantity at 37°C in Hams F12 supplemented with cystine (8.4 µg/L), HEPES (6 g/L), glutamic acid (1 g/L), and glucose (18.2 g/L) (21). The yeast phase was maintained at 37°C. Sc was maintained on YEPD (1% yeast extract, 2% peptone, 2% dextrose) plates and cultured in quantity in the identical medium used to culture Hc.
FITC-labeled Sc and Hc
Yeasts were cultured to midlog phase and then collected by centrifugation. The yeast pellet was resuspended in 0.5 M carbonate/bicarbonate buffer, pH 9.4, containing 10 µg/ml FITC, and incubated in the dark for 15 min at room temperature. For certain experiments, the yeasts then were washed twice and resuspended in Hypo-K buffer (hyposmotic—150 mOsm): 71 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, 0.17 mM K2HPO4, 0.175 mM KH2PO4, 0.405 mM MgSO4, and 2 mg/ml BSA (pH 7.2)). In other experiments, the yeasts were washed and suspended in HBSS containing 0.2% BSA (HBSA). FITC-labeled yeast and unlabeled yeast were always greater than 98% viable as determined by trypan blue dye exclusion.
Binding and ingestion of FITC-labeled yeasts
FITC-labeled yeasts were incubated with monolayers of Mø for 1 h in HBSA in the presence or absence of 25 nM bafilomycin. Unbound yeast was then removed by washing, and the monolayers were incubated for 10 min at room temperature in 0.1% trypan blue in HBSA. The cells then were washed with HBSA and fixed overnight at 4°C in HBSA containing 1% paraformaldehyde. Ingested vs bound yeasts were quantified as described previously (22).
Intracellular replication of Hc
The intracellular growth of Hc was quantified by RIA as
described previously (17). Mø were cultured in 96-well plates and used
when approximately 50% confluent. Prior to infection, aggregated Hc
yeasts were pelleted (500 x g for 5 min) and
discarded. A total of 5 x 103 yeasts in complete
medium were incubated with Mø for 1 h to allow for phagocytosis,
and then postendocytic inhibitors were added (10 µM chloroquine or 25
nM bafilomycin). Monolayers were cultured for 24 h, after which
the medium was aspirated and 5 µl 10x yeast nitrogen broth and 50
µl of water containing 1.0 µCi of [3H]leucine were
added to each well. The cultures then were incubated at 37°C for an
additional 24 h, after which 50 µl of unlabeled leucine (10
mg/ml) and 50 µl of bleach were added to each well. The contents of
the wells then were harvested onto glass fiber filters, unincorporated
tritium was removed by extensive washing, and the
incorporated tritium was quantified via scintillation
counting. The data are presented as the percentage of inhibition of
growth which is defined as:
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"Synchronized" infection of Mø with yeasts
In certain experiments the ingestion of yeast was synchronized by permitting binding but blocking internalization with hypo-K buffer (23). Monolayers were washed with hypo-K buffer and then were incubated with washed yeast in hypo-K buffer for 1 h at 37°C. Unless stated otherwise the ratio of Hc to Mø was 10:1. The monolayers then were washed a minimum of five times with hypo-K buffer to remove unbound yeast. The absence of unbound yeast was confirmed by microscopy. The infected monolayers then were cultured in complete medium under normal growth conditions for varying periods of time.
Quantitation of pH in yeast-containing phagosomes
Mø cultured on coverslips were incubated with FITC-labeled yeasts in hypo-K buffer for 1 h at 37°C. Unbound yeasts were washed away and the Mø then were cultured in complete medium for 2 h to allow ingestion and phagosomal maturation. After the 2-h maturation period, bafilomycin was added to one-half of the cells at a final concentration of 25 nM. Mø were incubated in complete medium for 2 h longer, after which the cells were washed with HBSS lacking phenol red and placed on ice. Prior to fluorometry the monolayers were rinsed with ice-cold 10 mM phosphate buffer at pHs ranging from 4.0 to 7.0. The fluorescence was determined on a Perkin-Elmer (Norwalk, CT) spectrofluorometer at both 450-nm and 490-nm excitation wavelengths and an emission wavelength of 515 nm. After the fluorescence of the intact phagosomes had been determined, the phagosomes were permeabilized with 0.1% Triton X-100. The fluorescence of phagosomes permeabilized in different pH buffers was used to generate a pH curve from which the pH of intact phagosomes was determined.
Acid phosphatase
Mø were infected with yeasts for 1 h at 37°C in hypo-K buffer, unbound yeast was washed away, and the phagosomes were allowed to mature for 4.5 h or 24 h in complete medium. Cells were washed with HBSS, fixed for 30 min at 4°C with 2% glutaraldehyde in cold sodium cacodylate buffer (SCB) (0.1 M sodium cacodylate, 0.25 M sucrose, pH 7.4), and then washed again with SCB. Washing was followed by two 30-min incubations in acid phosphatase reaction buffer (0.1 M sodium acetate, 1 mM glycerophosphate, and 2 mM CeCl3), pH 5.2, at 37°C with gentle shaking. Thereafter the cells were rinsed three times with the acid phosphatase reaction buffer, and refixed in 3% glutaraldehyde in SCB for 1 h at 4°C. After three more washes in SCB, monolayers were postfixed with 1% osmium tetroxide in cold SCB. The cells then were washed, dehydrated, and embedded in Epon. Ultrathin sections were cut and subsequently viewed on a Joel 100CX electron microscope.
Postembedding immunoelectron microscopy
Mø were infected with yeasts for 1 h in hypo-K buffer. At each time point, yeast-containing Mø were rinsed twice with hypo K buffer and then fixed in PLP (2% paraformaldehyde, 0.01 M periodate, 0.075 M lysine, and 0.075 M phosphate buffer, made fresh) for 2 h at room temperature after which the cells were washed with SCB, postfixed with freshly reduced 1% osmium tetroxide, and then dehydrated and embedded in Epon. Ultrathin sections were cut and the plastic etched by exposure to 5% NaIO4 (twice, 30 min each). The samples were blocked in PBS containing 10% normal goat serum and 1% fish gelatin followed by an overnight incubation with the indicated Ab (rabbit anti-mouse LAMP2, rabbit anti-V-ATPase subunit E, rabbit anti-V-ATPase subunit Ac115, or rabbit anti-horse spleen ferritin as a negative control) at room temperature in a humidified chamber. The sections then were washed extensively and incubated with biotinylated goat anti-rabbit IgG (in block at 50 µg/ml) for 1 h at room temperature. The sections were extensively washed and incubated with 10 nM gold coupled to Streptavidin (24) for 1 h at room temperature. The sections again were washed vigorously, stained with 2% uranyl acetate for 30 min, and then rinsed with water. The sections then were viewed on a Joel 100CX electron microscope. Gold particles were counted in the proximity of the yeast cell wall only if the yeast appeared to be in intact cells.
V-ATPase abs
For the V-ATPase subunit E, rabbit polyclonal antiserum was raised to a fusion of the T7 gene 9 protein with the entire coding region of the mouse subunit E (25). The serum was immunoadsorbed with immobilized T7 gene 9 protein and then affinity purified with immobilized subunit E fusion protein prior to use (26). For Ac115, a fusion protein consisting of a conserved region corresponding to amino acids 661–717 of the rat protein fused to T7 gene 9 was used to immunize rabbits. Serum was collected and immunoadsorbed with immobilized T7 gene 9 protein and then affinity purified with immobilized Ac115 fusion protein prior to use (26).
Cationized ferritin
Mø monolayers were infected with yeasts for 1 h in hypo-K buffer and the phagosomes allowed to mature for 4.5 h. Cationized ferritin was added to the monolayers at a concentration of 0.5 mg/ml in complete medium for 30 min. The monolayers then were washed twice with SCB, fixed for 2 h in PLP at 25°C, and then processed for electron microscopy. Phagosomes in intact cells were scored for the presence or absence of ferritin.
PL fusion
Mø monolayers growing on coverslips were cultured overnight in the presence of 50 µg/ml of rhodamine-coupled dextran (70,000 m.w.; Sigma). Excess dextran then was removed by three washes with HBSS, and the cells were cultured in complete medium for 5 h. The Mø then were infected with FITC-labeled yeasts in hypo-K buffer for 1 h after which unbound yeasts were removed, and the infected monolayer was incubated in complete medium. At 5 h postinfection the Mø were washed twice in HBSS and then fixed overnight at 4°C with 1% paraformaldehyde in hypo-K buffer. Mø then were rinsed, mounted, and viewed on a Zeiss fluorescent microscope. Phagosomes were scored positive for lysosomal fusion when a red rim surrounded green yeast.
Statistical analysis
Statistical significance was determined using Student’s t test or a Kruskal-Wallis nonparametric ANOVA test, depending on the data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first sought to determine if disruption of V-ATPase activity
affected the capacity of Hc yeasts to replicate within RAW Mø. Mø
were infected with Hc, bafilomycin was added at 1 h postinfection,
and the cells were cultured for an additional 24 h. After the 24-h
incubation, the number of viable yeasts was quantified by the
incorporation of [3H]leucine as described in
Materials and Methods. Remarkably, bafilomycin had no effect
on the intracellular growth of Hc yeasts (Fig. 1
). In contrast, coculture with the weak
base chloroquine inhibited intracellular replication by 70%. The
effect of chloroquine was identical to that observed in human Mø
(17).
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To determine the effect of bafilomycin on phagosomal pH, Hc was labeled
with the pH-sensitive dye FITC. FITC-labeled methanol-killed Hc and
live Sc served as controls. The labeled yeasts were incubated with Mø
for 1 h, after which unbound yeasts were removed by washing.
Because acidification is required for phagosomal maturation (27, 28, 29),
the yeast-containing phagosomes were allowed to mature for 2 h
prior to the addition of bafilomycin followed by a 2-h incubation in
the presence of bafilomycin. In agreement with previous studies (13),
in the absence of bafilomycin, phagosomes containing either dead Hc or
live Sc acidified normally to a pH of approximately 4.8, whereas
phagosomes containing live Hc were at a pH of about 6.3 (Fig. 2). In the presence of bafilomycin the pH
of phagosomes containing dead Hc or viable Sc increased to neutral,
indicating that bafilomycin had disrupted phagosomal V-ATPase activity
(Fig. 2). In contrast, the pH of phagosomes containing live Hc was
unaffected by bafilomycin. These data suggest that Hc does not require
the V-ATPase to acidify the phagosome.
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As phagosomes mature, they accrue V-ATPase and become more acidic
(18, 19, 20). As the Hc-containing phagosome is held at pH 6.3, but does
not require the phagosomal V-ATPase to maintain this pH, we next sought
to quantify the relative levels of V-ATPase present in the phagosomal
membranes of RAW Mø by immunoelectron microscopy using Abs specific
for the V-ATPase subunits Ac115 and E. Mø were infected with Hc for
1 h in hypo-K buffer, which permitted the yeasts to bind to Mø
but prevented internalization (23). After 1 h, the unbound yeasts
were removed, and ingestion and maturation allowed to proceed for
4.5 h whereupon monolayers were fixed and processed using
affinity-purified V-ATPase antisera as described in Materials and
Methods. The results obtained with the two Abs were identical and
demonstrated that the phagosomal membranes surrounding live Hc
contained V-ATPase at 3- to 4-fold lower levels than phagosomes
containing Sc (Fig. 3). Phagosomes
containing methanol-killed Hc contained an intermediate level of
V-ATPase. The level of V-ATPase in phagosomes containing live Hc
remained unchanged up to 24 h postinfection (data not shown).
There were no detectable killed Hc- or Sc-containing phagosomes at time
points later than 8 h, as all of the yeasts were digested.
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As the V-ATPase was inhibited from accumulating in Hc-containing
phagosomes, we next reexamined the question of PL fusion in RAW Mø.
Previous studies with the P388D1 mouse Mø cell line (14) and murine
resident peritoneal Mø (15) reported normal PL fusion upon
phagocytosis of Hc yeasts. In contrast, human Mø demonstrated minimal
PL fusion after phagocytosis of Hc (16). To quantify PL fusion,
FITC-labeled yeasts were incubated in hypo-K buffer with Mø whose
lysosomes were pre loaded with rhodamine B-isothiocyanate-labeled
dextran (RITC-dextran). At 5 h postinfection, 20% of
Hc-containing phagosomes had a red rim, indicating fusion with an
RITC-dextran containing lysosome (Fig. 4). No increase in PL fusion was observed
up to 24 h postinfection (data not shown). In contrast, greater
than 90% of Sc-containing phagosomes fused with lysosomes at 5 h
postinfection, and phagosomes containing dead Hc showed an intermediate
phenotype (Fig. 4).
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The percentage of Hc-containing phagosomes that were acid phosphatase
positive was unchanged at 24 h postinfection in Mø containing
viable Hc, indicating that Hc remained viable after PL fusion. Thus,
RAW Mø appear to behave like human Mø (16) in that PL fusion is
minimal compared with the PL fusion that occurs upon ingestion of Sc
yeasts.
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All of the viable Hc-containing phagosomes contained equivalent levels
of LAMP2, indicating that the decreased levels of LAMP in the
phagosomal membrane is not the result of a subset of phagosomes that do
not mature.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surprisingly, the V-ATPase specific inhibitor, bafilomycin, did not affect the ability of intracellular Hc to survive, nor did it cause a change in intraphagosomal pH. In contrast, methanol-fixed Hc and viable Sc were found in acidic phagosomes (approximately pH 4.8) and the pH increased to neutral in the presence of bafilomycin. Similar results were seen using antisense oligonucleotides directed to various subunits of the V-ATPase (data not shown). These data suggest that Hc actively senses pH and maintains it at 6.3. Interestingly, while externally replicating Hc alkalinizes the medium (data not shown), it does not buffer it to pH 6.3, suggesting that unique mechanisms are involved when the fungus is inside of the Mø phagosome. An understanding of the sensing mechanisms involved as well as the mechanisms used to maintain phagosomal pH should shed significant insight into the survival strategy of the fungus as well as providing possible drug targets.
The fact that Hc-containing phagosomes take up cationized ferritin indicates that they have full access to the early endocytic pathway. In contrast, the low levels of LAMP2, V-ATPase, lysosomal fusion, and acid phosphatase in Hc-containing phagosomes demonstrate decreased maturation compared with Sc-containing phagosomes. The differences in levels of expression of these markers are not the result of some macrophages killing Hc while in other macrophages the phagosomes do not mature at all. In each case, the variability in expression was the same when examining multiple phagosomes within a macrophage or comparing phagosomes from different macrophages. It is unclear why all of the phagosomes examined expressed low levels of LAMP2, but only one-fourth fused with lysosomes or contained lysosomal enzymes. These observations suggest that Hc may differentially modulate fusion with distinct vesicle populations.
There is contradictory data regarding the extent to which Hc-containing phagosomes fuse with lysosomes. In mouse peritoneal Mø, J774.2 Mø, and P388D1 Mø, PL fusion occurs normally (14, 15) compared with Sc, whereas in human Mø, PL fusion is inhibited compared with Sc (16). In the present experiments with RAW Mø, we found that Hc-containing phagosomes fuse with lysosomes at a low level compared with Sc and that fusion had no apparent effect on the viability of the Hc. Furthermore, approximately the same percentage of phagosomes contained acid phosphatase as fused with RITC-dextran-loaded lysosomes. Thus, RAW Mø function similarly to human Mø with respect to inhibition of PL fusion (16).
Although these disparate results might be caused by different methods
used to quantify lysosomal fusion or by Hc strain variations, the most
likely explanation is that the different Mø populations simply
function differently with respect to their interaction with Hc yeasts.
Indeed, this is a common theme in the Hc literature, and the literature
of other pathogenic microorganisms. Thus, RAW (30, 31) and mouse
peritoneal Mø (32, 33) are activated by IFN-
to inhibit the
intracellular growth of Hc yeasts through the production of nitric
oxide, whereas human Mø (34, 35) and the murine Mø cell lines P388D1
and IC-21 (36) are not activated to an antihistoplasma state by
IFN-, presumably because of their inability to produce nitric oxide
under the experimental conditions studied. Furthermore, phagocytosis of
unopsonized Hc yeasts stimulates a vigorous respiratory burst in human
Mø (37), but not in murine Mø (38, 39). However, phagocytosis of
IgG-opsonized Hc yeasts by murine Mø does stimulate the respiratory
burst (40). It is important to note that the differences in the
interactions between Mø and Hc yeasts do not sort out according to
species, or whether the Mø is "normal" or a tumor cell line. These
observations, along with others, reinforce the fact that data generated
from individual Mø pathogen studies cannot necessarily be interpreted
globally.
The intermediate phenotype exhibited by Mø phagosomes containing methanol-fixed Hc yeasts is intriguing but its cause is unknown. RAW Mø phagosomes containing methanol-killed Hc acidify normally at 4.5 h postinfection, but have intermediate levels of V-ATPase, lysosomal fusion, and acid phosphatase, compared with viable Hc- and Sc-containing phagosomes. One possible explanation relates to the signal transduction pathway that is activated during phagocytosis of Hc yeasts. Although uncharacterized, ligands on Hc that bind to Mø CD18 (22, 37) presumably survive methanol fixation as fixed yeasts are ingested as rapidly as viable yeasts. Thus, observed levels of V-ATPase, acid phosphatase, and PL fusion may be the result of intracellular signaling induced via interaction with Mø CD18. However, whatever causes the initial inhibition of phagosomal maturation seen with methanol-fixed Hc, the inhibition is short lived, as the yeasts are fully digested by 8 h.
Although intracellular pathogens of Mø have evolved unique survival mechanisms, the strategies utilized by Hc and Mycobacterium spp. (Mb) are remarkably similar. Both Hc and Mb exist in a phagosome with a pH of approximately 6.3 (13, 41, 42), retain the ability to fuse with early endosomes (Refs. 41 and 43–45; this paper), and fuse with lysosomes at decreased levels (Refs. 16, 42, 43, and 45–47; this paper). The major difference between Mb- and Hc-containing phagosomes is that Mb-containing phagosomes have no detectable V-ATPase (41, 45, 47, 48), whereas phagosomes containing Hc contain V-ATPase albeit at diminished levels relative to Sc. However, the V-ATPase is not necessary to maintain the pH of phagosomes containing Hc as treatment with bafilomycin does not alter phagosomal pH. Intriguingly, while there is no detectable V-ATPase in Mb-containing phagosomes, exposure to bafilomycin does raise the phagosomal pH to neutral (49). While it is unclear why bafilomycin treatment raises the pH of Mb-containing phagosomes, it is clear that the phagosomal V-ATPase is not required for the acidification of phagosomes containing Hc, indicating that the pathogens use different mechanisms to modify phagosomal pH.
While the V-ATPase is the enzyme that generates the acidic pH of most phagosomes (18, 19, 20), proton pumping by the V-ATPase is not the only mechanism by which phagosomes could acidify. Other proteins that are present in the plasma membrane may be present and active in the phagosome and could generate an acidic pH in the phagosome. Hc could use the Na+/H+ exchanger (NHE) to acidify phagosomes. Interestingly, NHE is present in Mb-containing phagosomes, but is not necessary for generating or maintaining the phagosomal pH (49). For NHE to acidify the phagosome, it would likely require the presence of the Na+/K+ ATPase, but this enzyme is not detectable in Mb-containing phagosomes (49). We speculate that Hc may somehow recruit the Na+/K+ ATPase to the phagosomal membrane or otherwise activate NHE (or another protein) to acidify the phagosome. Alternatively, Hc may directly acidify the phagosome itself. Given the delicate balance required for Hc to access iron while minimizing the activity of acid hydrolases, Hc likely has a pH sensor with which it monitors the pH of its environment, responding as necessary to changes in pH.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jane Strasser, Division of Infectious Diseases, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address:
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; Mø, macrophage; Sc, Saccharomyces cerevisiae; Mb, Mycobacterium spp.; V-ATPase, vacuolar ATPase; NHE, Na+H+ exchanger; PL fusion, phagolysosomal fusion; HBSA, HBSS containing 0.2% BSA; RITC-dextran, rhodamine B-isothiocyanate-labeled dextran; SCB, sodium cacodylate buffer.
Received for publication November 5, 1998. Accepted for publication March 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon stimulates macrophages of the RAW cell line to inhibit intracellular growth of Histoplasma capsulatum. Infect. Immun. 62:680.. Infect. Immun. 55:1014.. J. Infect. Dis. 161:143.[Medline]
interferon fail to inhibit the intracellular growth of Histoplasma capsulatum. Infect. Immun. 57:2903.This article has been cited by other articles:
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  L. Shi, P. C. Albuquerque, E. Lazar-Molnar, X. Wang, L. Santambrogio, A. Gacser, and J. D. Nosanchuk A Monoclonal Antibody to Histoplasma capsulatum Alters the Intracellular Fate of the Fungus in Murine Macrophages Eukaryot. Cell, July 1, 2008; 7(7): 1109 - 1117. [Abstract] [Full Text] [PDF]
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 S. L. Newman, L. Gootee, J. Hilty, and R. E. Morris Human Macrophages Do Not Require Phagosome Acidification to Mediate Fungistatic/Fungicidal Activity against Histoplasma capsulatum J. Immunol., February 1, 2006; 176(3): 1806 - 1813. [Abstract] [Full Text] [PDF]
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 L. A. Gildea, G. M. Ciraolo, R. E. Morris, and S. L. Newman Human Dendritic Cell Activity against Histoplasma capsulatum Is Mediated via Phagolysosomal Fusion Infect. Immun., October 1, 2005; 73(10): 6803 - 6811. [Abstract] [Full Text] [PDF]
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 R. A. Cox and D. M. Magee Coccidioidomycosis: Host Response and Vaccine Development Clin. Microbiol. Rev., October 1, 2004; 17(4): 804 - 839. [Abstract] [Full Text] [PDF]
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M. M. Timmerman and J. P. Woods Potential Role for Extracellular Glutathione-Dependent Ferric Reductase in Utilization of Environmental and Host Ferric Compounds by Histoplasma capsulatum Infect. Immun., December 1, 2001; 69(12): 7671 - 7678. [Abstract] [Full Text] [PDF]
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M. G. Rittig, M.-T. Alvarez-Martinez, F. Porte, J.-P. Liautard, and B. Rouot Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes Infect. Immun., June 1, 2001; 69(6): 3995 - 4006. [Abstract] [Full Text] [PDF]
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50%
confluent. At 1 h postinfection, chloroquine (10 µM) or
bafilomycin (25 nM) was added to the cultures. After a further 24
h of culture, intracellular replication was quantified as described in
Materials and Methods. Results are the mean ± SEM of
three separate experiments.
