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Histoplasma capsulatum Yeasts Are
Phagocytosed Via Very Late Antigen-5,
Killed, and Processed for Antigen
Presentation by Human Dendritic Cells
Lucy A. Gildea, Randal E. Morris and Simon L. Newman
J Immunol 2001; 166:1049-1056; ;
doi: 10.4049/jimmunol.166.2.1049
http://www.jimmunol.org/content/166/2/1049
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Copyright © 2001 by The American Association of
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References
Histoplasma capsulatum Yeasts Are Phagocytosed Via Very
Late Antigen-5, Killed, and Processed for Antigen Presentation
by Human Dendritic Cells1
Lucy A. Gildea,*† Randal E. Morris,† and Simon L. Newman2*†
istoplasma capsulatum (Hc)3 is a dimorphic intracellular
fungal pathogen of worldwide importance that is endemic
to the Ohio and Mississippi River Valleys (1). Infection
of the host with Hc occurs by inhalation of microconidia or small
mycelial fragments that accumulate in the terminal bronchioles
and alveoli of the lung. Upon inhalation, the conidia convert to the
pathogenic yeast form that causes the clinical manifestations of
histoplasmosis. The yeast cells are engulfed by resident alveolar
macrophages (M␾), within which they subvert the normal hostile
environment and multiply. The alveolar M␾ are destroyed by the
dividing yeasts and then are phagocytosed by M␾ recruited to the
site of infection. Repetition of this cycle leads to the spread of
infection to lymph organs and other tissues of the body, which is
eventually resolved in the immunocompetent host (1). Although
most infections involving Hc are unapparent, this organism causes
a broad range of disease activity clinically, including progressive
disseminated infections and even death in immunocompromised
patients (2, 3).
Maturation of cell-mediated immunity (CMI) leads to the production of cytokines that either directly or indirectly activate M␾
to inhibit yeast cell proliferation (4, 5). In vivo, in a murine model
of histoplasmosis, IFN-␥, IL-12, TNF-␣, and GM-CSF are critical
cytokines involved in the immune response to Hc yeasts (6 –13). In
H
*Department of Internal Medicine, Division of Infectious Diseases, and †Department
of Anatomy, Cell Biology, and Neurobiology, University of Cincinnati College of
Medicine, Cincinnati, OH 45267
vitro, IFN-␥ activates murine peritoneal M␾ to inhibit the intracellular growth of Hc, although no killing is observed (14). Although IFN-␥ does not activate human M␾ anti-histoplasma activity (15, 16), GM-CSF, IL-3, or M-CSF, when present during the
in vitro maturation of monocytes into M␾, activate human M␾ to
inhibit the growth of Hc. However, the maximum inhibition of
intracellular growth is only 60% (16).
Although M␾ can serve as APCs, dendritic cells (DC) are more
potent Ag presenters than M␾ (17). DC precursors originate in the
bone marrow, enter the blood, and seed nonlymphoid tissues.
These DC are classified as immature and specialize in Ag uptake
and processing (18). The immature DC differentiate into mature
DC as they migrate to tissue-draining secondary lymphoid organs
where they efficiently present Ag to T cells (19 –24). The strategic
location of DC in tissues, the lung in particular, suggests that these
cells can link the innate and adaptive immune responses to foreign
Ags. In the lung, DC are located within the airway epithelium, lung
parenchyma, and submucosa below the airway epithelium; within
alveolar septal walls; and on the alveolar surface (25–27). Because
Hc infects humans via the respiratory route, DC in the lung may
play a key role in host defense against this fungus.
Human M␾ bind and internalize Hc yeasts and conidia via the
CD18 family of integrin receptors (28, 29). As human DC also
express high levels of CD18 receptors on their surface, we hypothesized that immature DC might phagocytose Hc yeasts
through CD18 and subsequently kill and degrade the organism,
process Hc-specific Ags, and stimulate lymphocyte proliferation.
Received for publication August 8, 2000. Accepted for publication October 11, 2000.
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.
Materials and Methods
1
Monocytes were isolated by sequential centrifugation on Ficoll-Hypaque
and Percoll gradients (Amersham Pharmacia LKB, Piscataway, NJ) from
buffy coats obtained from the Hoxworth Blood Center (Cincinnati, OH) or
from blood drawn from normal adult donors in our laboratory (29). To
obtain DC, monocytes were cultured in six-well tissue culture plates (Corning-Costar, Cambridge, MA) at 6.5 ⫻ 105/ml in RPMI 1640 containing 200
mM glutamine, 50 ␮M 2-ME (Sigma, St. Louis, MO), 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 50 ng/ml kanamycin
This work was supported by National Institutes of Health Grants AI37639 and
HL55948.
2
Address correspondence and reprint requests to Dr. Simon L. Newman, Division of
Infectious Diseases, University of Cincinnati College of Medicine, P.O. Box 670560,
Cincinnati, OH 45267-0560. E-mail address: [email protected]
3
Abbreviations used in this paper: Hc, Histoplasma capsulatum; M␾, macrophage(s);
CMI, cell-mediated immunity; DC, dendritic cells; HK, heat-killed; HBSA, HBSS
containing 0.25% BSA; VLA-5, very late Ag-5.
Copyright © 2001 by The American Association of Immunologists
Preparation of human DC and M␾
0022-1767/01/$02.00
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Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As the induction of cell-mediated
immunity to Hc is of critical importance in host defense, we sought to determine whether dendritic cells (DC) could function as
a primary APC for this pathogenic fungus. DC obtained by culture of human monocytes in the presence of GM-CSF and IL-4
phagocytosed Hc yeasts in a time-dependent manner. Upon ingestion, the intracellular growth of yeasts within DC was completely
inhibited compared with rapid growth within human macrophages. Electron microscopy of DC with ingested Hc revealed that
many of the yeasts were degraded as early as 2 h postingestion. In contrast to macrophages, human DC recognized Hc yeasts via
the fibronectin receptor, very late Ag-5, and not via CD18 receptors. DC stimulated Hc-specific lymphocyte proliferation in a
concentration-dependent manner after phagocytosis of viable and heat-killed Hc yeasts, but greater proliferation was achieved
after ingestion of viable yeasts. These data demonstrate that human DC can phagocytose and degrade a fungal pathogen and
subsequently process the appropriate Ags for stimulation of lymphocyte proliferation. In vivo, such interactions between DC and
Hc may facilitate the induction of cell-mediated immunity. The Journal of Immunology, 2001, 166: 1049 –1056.
1050
(Sigma), 1% nonessential amino acids (BioWhittaker, Walkersville, MD),
and 1% pyruvate (BioWhittaker). Human rGM-CSF (115 ng/ml; PeproTech, Rocky Hill, NJ) and human rIL-4 (50 ng/ml; PeproTech) also were
added to each well, and DC were studied after 6 – 8 days of culture.
M␾ were obtained by culture of monocytes at 1 ⫻ 106/ml in Teflon
beakers with RPMI 1640 containing 15% human serum, 10 ␮g/ml gentamicin (Sigma), 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Sigma).
M␾ were studied after 5–7 days in culture.
FACS analysis
Hc yeasts
Hc strain G217B was maintained as previously described (29). Yeasts were
grown in histoplasma macrophage medium (30) at 37°C with orbital shaking at 150 rpm. For binding and phagocytosis assays, 2- to 3-day-old yeasts
were harvested by centrifugation, washed three times in 0.01 M phosphate
buffer, pH 7.2, containing 0.15 M NaCl (PBS), and then heat-killed (HK)
at 65°C for 1 h. Yeasts were sonicated to prepare a single-cell suspension,
and then were stored at 4° in PBS containing 0.05% sodium azide as
described previously (29). To label with fluorescein, HK yeasts were resuspended to 2 ⫻ 108/ml in either 0.01 mg/ml FITC (suspension assays) or
0.1 mg/ml FITC (M␾ Terasaki plate binding assay) in 0.05 M carbonatebicarbonate buffer, pH 9.5. After incubation for 15 min at room temperature in the dark, FITC-labeled Hc yeasts were washed twice with HBSS
containing 0.25% BSA (HBSA) and resuspended to the appropriate concentration in HBSA.
For studies with viable yeasts, 48-h log phase yeasts were harvested by
centrifugation, washed three times in HBSA, and resuspended to 50 ml in
HBSA. Large aggregates were removed by centrifugation at 200 ⫻ g for 5
min at 4°C. The top 10 ml was removed, and the single-cell suspension
obtained was standardized to the appropriate concentration according to the
assay protocol.
Phagocytosis assay
Phagocytosis of Hc yeasts by human DC was quantified by a modification
of our previously published assay for adherent M␾ (29). DC were harvested from six-well plates after 6 – 8 days of culture, washed in HBSA,
and standardized to 2 ⫻ 106/ml. DC (1 ⫻ 106) were incubated with FITClabeled HK Hc yeasts (5 ⫻ 106) in a total volume of 1 ml at 37°C in a water
bath with orbital shaking at 150 rpm for varying periods of time. At the end
of the incubation period, trypan blue (1 mg/ml in PBS) was added for 15
min at 25°C to quench the fluorescence of bound, but uningested, organisms (29). The cells then were washed with HBSA, cytocentrifuged onto
glass slides, and fixed in 1% paraformaldehyde at 4°C. Coverslips were
mounted in 90% glycerol in PBS, and phagocytosis was quantified by
phase contrast and fluorescent microscopy. One hundred DC were counted
per slide, and the number of ingested or bound, but uningested, yeasts was
quantified. Results are expressed as the mean ⫾ SEM of the phagocytic
index (the total number of yeasts ingested per 100 DC) and the percent
ingesting (the percentage of DC containing one or more yeasts).
Binding assays
DC and M␾ were harvested after 6 – 8 days of culture, washed, and resuspended to 4 ⫻ 106/ml in HBSA. Fifty microliters of cells were incubated
in 12- ⫻ 75-mm polypropylene tubes for 30 min at 4°C with 5 ␮g of
purified mouse anti-human CD18, CD11a, CD11b, or CD11c mAb (Ancell), either alone or in combination (15 ␮g of total mAb), 3 ␮g of purified
mouse anti-human very late Ag-5 (VLA-5) mAb (Caltag), or HBSA only.
After preincubation with mAbs, 1 ⫻ 106 FITC-labeled HK yeasts were
added to each tube and incubated for 30 min at 37°C in a water bath with
orbital shaking at 150 rpm. Ten microliters of sample was mounted on a
clean glass slide and coverslipped for immediate quantitation via fluorescent microscopy (Axioscope; Zeiss, Oberkochen, Germany). One hundred
DC or M␾ were counted per slide, and the results are expressed as the
mean ⫾ SEM of the attachment index, the total number of organisms
bound per 100 cells. DC also were tested with mAbs to the vitronectin
receptor (CD51; ␣v␤3), the ␤-chain of the laminin receptor (CD104; ␣6␤4),
the ␣-chain of another RGD-independent fibronectin receptor (CD49d;
␣4␤1), the Mg2⫹-dependent collagen receptor (CD49b; ␣2␤1), and CD29 (␤1).
Alternatively, for adherent binding assays, M␾ (2.5 ⫻ 103) were adhered for 1 h at 37°C in 5% CO2-95% air in the wells of a Terasaki tissue
culture plate (Miles Scientific Division, Naperville, IL) that previously had
been coated with 1% human serum albumin. The cells were washed twice
with HBSA, 5 ␮l of mAb (CD18, CD11a, CD11b, or CD11c at 50 ␮g/ml;
VLA-5 at 17 or 24 ␮g/ml) or HBSA was added to the monolayers, and the
mixture was incubated for 30 min at 4°C. Five microliters of FITC-labeled
HK Hc yeasts (2 ⫻ 107/ml) were added to each well, and the mixture was
incubated for 30 min at 37°C. Unattached organisms were removed by
washing with HBSA, and the monolayers were fixed with 1% paraformaldehyde. Attachment of the yeasts was quantified via fluorescence microscopy on an inverted microscope (Diaphot, Nikon, Melville, NY) by counting 100 M␾ well. Results are expressed as the mean ⫾ SEM of the
attachment index. mAb 3G8 was included in both suspension and adherent
binding assays as an isotype control for nonspecific inhibition.
Quantitation of intracellular growth of Hc yeasts in DC and M␾
Intracellular growth of Hc yeasts in DC and M␾ was quantified by the
incorporation of [3H]leucine as described previously (16). DC were incubated at varying ratios of cells to yeasts (50/1, 10/1, and 5/1) in polypropylene tubes with 5 ⫻ 103 viable Hc yeasts for 48 h at 37°C in a water bath
with orbital shaking at 150 rpm. After 48 h of incubation, the contents of
the tubes were transferred to a 96-well plate (Corning-Costar, Cambridge,
MA). Simultaneously with DC, M␾ cultured in Teflon beakers were harvested, washed, and adhered (6 ⫻ 104) in a 96-well plate for 1 h at 37°C
in HBSA containing 2% aprotinin (Sigma). After adherence, M␾ were
washed twice in RPMI and incubated for 48 h with 5 ⫻ 103 viable yeasts.
All plates then were centrifuged, and supernatants were carefully aspirated
through a 27-gauge needle. Fifty microliters (1.0 ␮Ci) of [3H]leucine (sp.
act., 153 Ci/nmol; DuPont/New England Nuclear, Boston, MA) in sterile
water and 5 ␮l of a 10⫻ yeast nitrogen broth (Difco, Detroit, MI) were
added to each well. After further incubation for 24 h at 37°C, 50 ␮l of
L-leucine (10 mg/ml) and 50 ␮l of sodium hypochlorite were added to each
well. The contents of the wells were harvested onto glass-fiber filters using
an automated harvester (Skatron, Sterling, VA). The filters were placed
into scintillation vials, scintillation cocktail was added, and the vials were
counted in a Beckman LS 6500 liquid scintillation spectrometer (Beckman
Instruments, Fullerton, CA). The results are expressed as the mean ⫾ SEM
of the counts per minute incorporated by remaining viable Hc yeasts in DC
and M␾. Experiments were performed in triplicate, and five to seven experiments were performed with cells from different donors.
Electron microscopy
Cultured DC and M␾ (1 ⫻ 106 each) were incubated separately in polypropylene tubes for 2 or 24 h at 37°C with 5 ⫻ 106 viable Hc yeasts in a water
bath with orbital shaking at 150 rpm. After phagocytosis, the tubes were
washed once in HBSA, fixed immediately, and then processed for electron
microscopy (31, 32). After polymerization of the samples, ultrathin sections were cut with a diamond knife (Diatome U.S., Ft. Washington, PA)
on a Reichert-Jung Ultracut E ultramicrotome (Cambridge Instruments,
Buffalo, NY). Samples were picked up on 300-mesh copper grids, stained
with uranyl acetate and lead citrate for contrast, and viewed on a JEOL100CX electron microscope (Peabody, MA) operating at 80 kV.
T cell isolation
On day 6 of the DC culture, blood was obtained from the same donor, and
mixed mononuclear cells were isolated on Ficoll-Hypaque gradients (29).
The mononuclear cells were standardized to 7.5 ⫻ 107/ml in RPMI 1640
containing 5% FCS and 10 ␮g/ml gentamicin, warmed to 37°C, and passed
over a nylon wool column at 37°C. After 1 h of incubation on the column,
the first 15 ml of column flow-through was collected and cultured at 2 ⫻
106/ml overnight in a T-flask (Corning-Costar). After 24 h of culture, the
nonadherent cells were collected, washed in Dulbecco’s PBS containing
2% FCS, and incubated for 1 h on ice with mouse mAbs to human CD56
(Becton Dickinson) and human CD16 (Medarex, Annandale, CA) to eliminate any remaining NK cells. The cells then were washed in Dulbecco’s
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DC (5 ⫻ 105) were incubated with primary mAbs at 4°C for 45 min. After
two washes in PBS containing 1% BSA (PBS-BSA), the cells either were
fixed in 1% paraformaldehyde or were incubated with a fluorochromelabeled secondary Ab for an additional 45 min at 4°C. The cells then were
washed twice with PBS-BSA and fixed overnight with 1% paraformaldehyde before analysis by flow cytometry on a FACSCalibur flow cytometer
(Becton Dickinson, San Jose, CA) with standard optics and filter. The
acquired data were analyzed with CellQuest software (Becton Dickinson).
Nonspecific Ab binding was blocked by preincubation of DC with 250
␮g/ml human IgG (Sigma) for 30 min at 4°C before the addition of primary
mAb. The following mAbs were used: FITC-labeled CD11a, CD11b,
CD11c, CD80, CD18, CD14, CD40, and mouse IgG1, IgG2a, and IgM
(Ancell, Bayport, MN); unconjugated CD19, CD3, CD16, and goat antimouse IgG/IgM (Ancell); CD83 (Serotec, Raleigh, NC); CD86 (PharMingen, San Diego, CA); HLA-DR (Caltag, South San Francisco, CA); CD1a
(BioSource International, Menlo Park, CA); and CD56 (Tcell Diagnostics,
Woburn, MA).
DENDRITIC CELL INTERACTION WITH Histoplasma capsulatum
The Journal of Immunology
PBS containing 2% FCS and incubated for 1 h at 4°C on a rocking shaker
(Thermolyne, Dubuque, IA) with goat anti-mouse IgG magnetic beads
(Perseptive Biosystems, Framingham, MA) at 10 beads/cell. Purified T
cells were obtained after incubation of the cell/bead suspension on a
MPC-1 magnet (Dynal, Oslo, Norway) for 10 min. The T cells remaining
in suspension were collected and used in the Ag presentation assays as
described below. T cells were 98.5% CD3⫹ by FACS analysis.
Ag presentation assays
Ag presentation by DC to T cells was quantified by the incorporation of
[3H]thymidine. DC were cultured with either HK or viable Hc yeasts for
1 h at 37°C in a 96-well plate to allow for phagocytosis of the yeasts.
Autologous T cells then were added to each well, and the plate was cultured at 37°C for 7 days. On day 7 of culture, 1.0 ␮Ci of [3H]thymidine (sp.
act., 6.7 Ci/mmol; DuPont/New England Nuclear) in RPMI 1640 was
added to each well. After further incubation for 24 h at 37°C, the contents
of the wells were harvested onto glass-fiber filters and counted in a liquid
scintillation counter as described above. The results are expressed as the
mean ⫾ SEM of the log counts per minute incorporated by T cells in the
presence of varying amounts of DC and Hc yeasts. All donors were tested
for Hc responsiveness as described below.
Donor screens for CMI to Hc
Statistics
Statistical analysis of the data was performed using SigmaStat (Jandel Scientific, San Rafael, CA). Student’s t test was used in all experiments, and
the results were considered significant at p ⬍ 0.05.
Results
Phenotypic analysis of monocyte-derived DC
Human DC were derived from the differentiation of peripheral
blood monocytes in the presence of GM-CSF and IL-4. After several days of culture, nonadherent clusters of cells with typical processes and veils were observed by phase-contrast microscopy. By
days 6 – 8 of culture, many of these clustered veiled cells were free
in the medium and comprised the majority of the cell population.
Analysis of the nonadherent cells for DC surface markers revealed
that the cells displayed staining patterns similar to those described
by Romani et al. (34) and Sallusto et al. (35). The DC expressed
high levels of CD18, CD11b, CD11c, HLA-DR, and VLA-5, and
moderate levels of CD11a, CD1a, and CD86. DC were negative
for CD14, CD3, CD16, CD19, CD80, and CD83 (Table I).
Table I. Phenotypic analysis of human DC
Surface Ag
CD14
CD18
CD11a
CD11b
CD11c
CD1a
CD3
CD83
CD86
CD80
HLA-DR
VLA-5
CD16
CD19
Mean % Positive (Range)a
7 (0–34)
99 (98–100)
39 (3–75)
80 (11–99)
93 (59–99)
46 (4–92)
11 (4–19)
4 (2–6)
43 (11–89)
14 (6–36)
99 (96–100)
99 (98–99)
9 (3–23)
3 (0–12)
a
DC (5 ⫻ 105) were incubated at 4°C for 45 min with each mAb. If the primary
mAb was not labeled with FITC, a second incubation was performed with FITClabeled goat anti-mouse IgG/IgM for an additional 30 min at 4°C. FACS analysis was
performed as described in Materials and Methods. Data are representative of 5–35
individual donors.
observed within DC as early as 2 h, as indicated by the separation
of the yeast cell wall from the phagosome membrane (Fig. 2, A and
B). By 24 h there were numerous empty vacuoles that previously
had contained yeasts (Fig. 2C). Partially digested yeasts were
never observed at any time point in human M␾ (data not shown);
this is consistent with previous reports that viable Hc yeasts multiply rapidly within human M␾ (15, 16, 40).
To confirm that Hc yeasts did not multiply within DC, the intracellular growth of Hc was quantified by the incorporation of
[3H]leucine into remaining viable yeasts. DC and M␾ prepared
from the same donors were infected with Hc yeasts and cultured
for 48 h. Compared with their rapid intracellular growth within
M␾, Hc yeast growth within DC was inhibited by ⬎90% at all
DC/Hc ratios tested (Fig. 3). GM-CSF and IL-4 were not present
during the 48 h of culture of DC with Hc, and the addition of these
cytokines had no effect on the results obtained (data not shown).
Binding of Hc yeasts to human DC is mediated by VLA-5
Our original rationale for these experiments was based on the fact
that DC contain high levels of CD18 on their surface. As M␾ use
Phagocytosis and killing of Hc yeasts by human DC
Previous studies have shown that immature human DC can phagocytose some bacteria and protozoans (36 –39). Therefore, we next
examined the ability of human DC to phagocytose the pathogenic
fungus Hc. DC were incubated in suspension with unopsonized
FITC-labeled HK Hc yeasts for varying periods of time. Fig. 1
shows that DC ingested Hc yeasts in a time-dependent manner.
After 1 h 49% of DC had ingested one or more yeasts, with an
average of 3.5 yeasts/DC. By 6 h 75% of DC had ingested an
average of 4.7 yeasts/DC.
Phagocytosis of viable Hc yeasts was confirmed by electron
microscopy. Following phagocytosis the yeasts were found in
membrane-bound phagosomes. Remarkably, degraded yeasts were
FIGURE 1. DC phagocytose Hc yeasts in a time-dependent manner.
Day 7 DC were incubated with FITC-labeled HK Hc yeasts (5/1 Hc/DC
ratio) for varying periods of time. At the end of each time period 1 mg/ml
trypan blue was added to each sample for 15 min to quench the fluorescence of uningested organisms. An aliquot of cells was cytocentrifuged
onto glass slides, and phagocytosis was quantified by phase and fluorescent
microscopy. The data are presented as the percent ingesting (percentage of
DC containing at least one ingested organism) and the phagocytic index
(the total number of organisms ingested/100 DC). The data are the mean ⫾
SEM of eight experiments performed in duplicate with different donors.
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The responsiveness of donor lymphocytes to HK Hc yeasts was determined
for donors used in the Ag presentation assays as described previously (33).
HK yeasts are used in this screen because viable Hc yeasts overgrow the
culture and destroy the monocytes. Mixed mononuclear cells were isolated
from peripheral blood by Ficoll-Hypaque centrifugation and standardized
to 1 ⫻ 106/ml in RPMI 1640 supplemented with 10% autologous serum
and 10 ␮g/ml gentamicin. One hundred microliters of cells were incubated
for 7 days at 37°C in a 96-well plate either alone or with HK Hc yeasts
(103–106). On day 7 the cells were pulsed for 24 h with [3H]thymidine and
then were harvested onto glass-fiber filters before counting in a liquid scintillation counter. The results are expressed as the mean counts per
minute ⫾ SD. A donor is considered nonresponsive with counts per minute
of 5000 and below. This usually corresponds to a stimulation index of ⱕ3.
1051
1052
DENDRITIC CELL INTERACTION WITH Histoplasma capsulatum
binding of Hc to DC. As shown in Fig. 5, binding of Hc to DC was
Ca2⫹ dependent, but not Mg2⫹ dependent. DC express high levels
of the fibronectin receptor VLA-5 (41) (Table I), and binding to
VLA-5 is known to be divalent cation dependent (42). Therefore,
we next considered the possibility that DC VLA-5 mediated recognition of Hc yeasts. Preliminary studies revealed that binding of
Hc yeasts to DC was inhibited by soluble fibronectin in a concentration-dependent manner (data not shown). Fig. 6 shows that mAb
to VLA-5 (␣5 chain) inhibited Hc yeast binding to DC by 90%,
whereas mAbs to the ␣-chain of the vitronectin receptor (CD51;
␣v␤3), the ␤-chain of the laminin receptor (CD104; ␣6␤4), the
FIGURE 2. DC phagocytose and degrade viable Hc yeasts. DC were
incubated with viable Hc yeasts for either 2 h (A and B) or 24 h (C), and
the cell pellets were processed for electron microscopy as described in
Materials and Methods. Note that the yeasts are contained in a phagosome
and are partially degraded by 2 h. By 24 h many internalized yeasts had
been completely digested, leaving only empty phagocytic vacuoles. Data
are representative of three experiments.
CD11/CD18 to recognize and phagocytose unopsonized Hc yeasts
(28, 29), we hypothesized that human DC CD18 might perform the
same function. To test this hypothesis, DC were preincubated with
mAbs to CD11/CD18, and their subsequent capacity to bind FITClabeled HK Hc yeasts was quantified. mAb to CD18 inhibited the
attachment of yeasts to M␾, but not to DC (Fig. 4). Further, mAb
to CD18 failed to inhibit binding of Hc to DC even when the
amount of mAb was tripled to 75 ␮g/ml (data not shown). Because
M␾ in suspension bind fewer yeasts than adherent M␾, we performed the experiment with adherent M␾ as an additional control.
mAbs to the ␣-chains of the CD18 family (CD11a, CD11b, and
CD11c), either alone or all together, also failed to inhibit the binding of yeasts to DC (data not shown). Consistent with previous
data (28), a cocktail of all three ␣-chain mAbs was required to
inhibit binding of Hc yeasts to M␾ to the same degree as the CD18
mAb (data not shown).
As DC CD18 did not appear to mediate recognition of Hc
yeasts, we next determined the requirement for divalent cations for
FIGURE 4. DC do not bind Hc yeasts via CD18 receptors. DC or M␾
were preincubated with HBSA or anti-CD18 mAb for 30 min at 4°C and
then incubated with FITC-labeled HK Hc yeasts for 30 min at 37°C in a
shaking water bath. The adherent assay was performed similarly, except
that M␾ were adhered in a Terasaki plate before the addition of mAb and
Hc yeasts. Bound yeasts were quantified by fluorescent microscopy, and
the data are presented as the attachment index (the total number of organisms bound per 100 cells). The data are the mean ⫾ SEM of four experiments with M␾ and seven experiments with DC performed in duplicate.
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FIGURE 3. DC inhibit the intracellular growth of Hc yeasts. Viable Hc
yeasts (5 ⫻ 103) were incubated with varying ratios of DC in suspension
or 6 ⫻ 104 adherent M␾ for 48 h at 37°C. After the incubation period the
DC and Hc were transferred to the 96-well plate that contained the M␾, and
the cells were pulsed for an additional 24 h with [3H]leucine to quantify
remaining viable yeasts as described in Materials and Methods. The data
are the mean ⫾ SEM of five to seven experiments performed in triplicate.
The Journal of Immunology
␣4-chain of the RGD-independent fibronectin receptor ␣4␤1
(CD49d), and the ␣2-chain of the Mg2⫹-dependent collagen receptor ␣2␤1 (CD49b) all failed to inhibit binding of Hc yeasts to
DC. mAb to CD29, the ␤-chain that complexes with ␣5 to form the
fibronectin receptor heterodimer ␣5␤1, also failed to inhibit binding of Hc yeast to DC, indicating that Hc binds specifically to the
␣-chain (Fig. 6). Finally, anti-VLA-5 did not inhibit binding of
yeasts to M␾ in either adherent or suspension assays (data not
shown).
Ag presentation by DC
As DC phagocytosed and killed Hc yeasts, we next tested the
ability of DC to process and present Hc Ag to T cells. DC (104)
were incubated for 1 h with varying concentrations of HK or viable
Hc yeasts and then were cultured for 1 wk with autologous CD3⫹
T cells. Lymphocyte proliferation over the last 24 h was quantified
by the incorporation of [3H]thymidine. As shown in Fig. 7, T cell
proliferation was stimulated by both viable and HK yeasts in a
concentration-dependent manner. Most interesting was the fact
that 200-fold fewer viable yeasts (5 ⫻ 103) than HK yeasts (1 ⫻
106) were required to stimulate optimum lymphocyte proliferation.
In a second series of experiments varying concentrations of DC
were infected for 1 h with viable (5 ⫻ 103) or HK (1 ⫻ 106) Hc
yeasts, and then further cultured with either 104 or 105 T cells for
7 days. As shown in Fig. 8, all concentrations of DC tested stimulated lymphocyte proliferation, with optimal proliferation
achieved at 104 DC. Further, concentrations of DC ⬎104 actually
resulted in a decrease in T cell proliferation.
The lymphocyte proliferation data presented in Figs. 7 and 8
were performed using cells obtained from Hc-responsive donors as
determined by our screening method that used HK yeasts and
mixed mononuclear cells (33). Thus, the T cell proliferation data
presumably represent a secondary immune response to Hc Ags. To
determine whether DC that had phagocytosed Hc yeasts could
present Hc Ags to T cells in a putative primary response, we
sought normal donors that were Hc Ag unresponsive when their
mononuclear cells were cultured with HK yeasts. As shown in
FIGURE 6. DC recognize Hc yeasts via the fibronectin receptor,
VLA-5. DC were preincubated with mAbs for 30 min at 4°C, and then
incubated with FITC-labeled HK Hc yeasts for 30 min at 37°C in a shaking
water bath. The mAbs were used at 25 ␮g/ml, except for anti-VLA-5,
which was used at 15 ␮g/ml. The data are presented as the mean ⫾ SEM
of the attachment index of three to six experiments performed in duplicate.
Table II, we identified three individuals who incorporated ⬍3,000
cpm of [3H]thymidine upon culture of 105 mixed mononuclear
cells with 106 HK yeasts for 7 days. In contrast, normal Hc-immune donors had an average counts per minute of ⬃28,000. However, when DC from these putative naive donors were incubated
with either viable or HK Hc yeasts, the counts per minute obtained
were 5- to 50-fold greater than that obtained with mixed mononuclear cells (Table II).
Discussion
In murine models of histoplasmosis IL-12, IFN-␥, TNF-␣, and
GM-CSF are critical cytokines required for survival and resolution
of the disease, demonstrating that protective immunity to Hc requires a Th1-type response (6 –13). In vitro experiments demonstrate that IFN-␥ activates mouse peritoneal M␾ to inhibit the intracellular growth of Hc yeasts (14), whereas IFN-␥ and LPS are
required to stimulate splenic M␾ anti-histoplasma activity (43).
Thus, other as yet unidentified cytokines may be required for M␾
activation in vivo.
In contrast to the murine system, IFN-␥ does not activate human
M␾ anti-histoplasma activity (15, 16), and the only cytokines that
activate human M␾ to inhibit the intracellular growth of Hc are the
colony-stimulating factors GM-CSF, IL-3, and M-CSF (16). Furthermore, M␾ infected with HK Hc yeasts stimulate lymphocyte
proliferation, but the supernatants generated do not contain factors
that activate human M␾ to inhibit the intracellular growth of Hc
(S. L. Newman, unpublished observations). Consequently, the cytokines required to activate human M␾ anti-histoplasma activity
remain obscure.
DC are the most potent APC of the immune system and are vital
for the initiation of primary T cell-mediated immune responses
that are the hallmark of CMI (17). As host defense against Hc
requires the induction of CMI, we sought to determine a role for
DC in this process. The data presented herein demonstrate that
human DC avidly ingest the pathogenic fungus Hc and that serum
is not required for recognition and phagocytosis. After 6 h of incubation 75% of DC had ingested at least one unopsonized yeast,
indicating that the majority of DC, and not a subpopulation, are
phagocytic. These experiments confirm and extend a previous
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FIGURE 5. Hc yeast binding to DC is Ca2⫹ dependent, but not Mg2⫹
dependent. DC were incubated with FITC-labeled HK Hc yeasts for 30 min
at 37°C in HBSA devoid of divalent cations or in HBSA containing Ca2⫹,
Mg2⫹, or both. The data are presented as the mean ⫾ SEM of the attachment index of four experiments performed in duplicate.
1053
1054
DENDRITIC CELL INTERACTION WITH Histoplasma capsulatum
study (38) that reported that human DC phagocytosed serum opsonized HK yeasts. However, in the latter study ingested vs bound
organisms were not distinguished, quantitative data were not presented, and viable yeasts were not studied.
Although early reports suggested that DC had weak or no endocytic activity (44 – 46), recent data indicate that DC can ingest a
number of microbial pathogens. Thus, in vitro studies demonstrated that mouse DC and murine-derived DC cell lines ingested
bacteria such as Bordetella bronchiseptica (47, 48), Listeria monocytogenes (49), Chlamydia trachomatis (50, 51), and CalmetteGuérin bacillus (BCG) mycobacterium (52), in addition to the protozoan Leishmania major (53–57). In vivo, murine DC containing
internal Leishmania donovani (58) and L. major (59) also have
been observed. Human DC have been reported to phagocytose
Borrelia burgdorferi (36) and Mycobacterium tuberculosis (37),
influenza (60), and measles (61) viruses and the protozoans
Trypanosoma cruzi (39) and L. donovani (38).
A surprising finding was that binding of Hc yeasts to DC was
mediated by the fibronectin receptor, VLA-5, rather than the CD18
receptors as was found for M␾ (28, 29). This difference in receptor
usage between these cells may account for the ability of DC to
inhibit the intracellular growth of Hc, whereas M␾ are permissive
for intracellular growth. Thus, phagocytosis via VLA-5 may trigger an intracellular signaling cascade that allows DC to counter the
ability of Hc yeasts to inhibit or reduce phagolysosomal fusion
(62). Indeed, we have found that there is considerable phagolysosomal fusion in DC that have phagocytosed viable Hc yeasts (L.
Gildea, manuscript in preparation), in contrast to the minimal
amount of phagolysosomal fusion that occurs in human M␾ (62).
The reason for the preferential binding of Hc to VLA-5 on DC
and to CD18 on M␾ is unknown. Indeed, it is peculiar, particularly
because the numbers of CD18 and VLA-5 expressed on the surface
of DC and M␾ are roughly equivalent (L. Gildea, unpublished
observations). As mobility of CD18 within the M␾ membrane is a
FIGURE 8. DC stimulate T cell proliferation in a concentration-dependent manner. Varying concentrations of DC were incubated with either 5 ⫻ 103
viable or 1 ⫻ 106 HK Hc yeasts for 1 h 37°C to allow for phagocytosis and then were cultured for 7 days with 104 (F) or 105 (E) T cells. The wells were
pulsed with [3H]thymidine to quantify T cell proliferation. The data are presented as the mean ⫾ SEM of the log counts per minute. Each data point was
derived from 4 –11 donors. The counts per minute in control wells containing 104 DC and 105 T cells was 1781 ⫾ 511 (mean ⫾ SEM).
Downloaded from http://www.jimmunol.org/ by guest on August 2, 2017
FIGURE 7. DC infected with viable and HK Hc yeasts stimulate T cell proliferation. DC (104) were incubated with varying concentrations of either
viable or HK Hc yeasts for 1 h at 37°C to allow for phagocytosis. Purified CD3⫹ T cells then were added to the wells, and the mixtures were cultured for
an additional 7 days. On day 7 the wells were pulsed with [3H]thymidine to quantify T cell proliferation. The data are presented as the mean ⫾ SEM of
log counts per minute. Each data point was derived from 4 –11 donors. The counts per minute in control wells containing DC and T cells only was 1975 ⫾
1046 (mean ⫾ SEM).
The Journal of Immunology
1055
Table II. DC from naive blood donors stimulate T cell proliferation
upon incubation with viable or HK Hc yeasts
CPM (mean ⫾ SD)
DC (104)b
Donor
1
2
3
MM Screena
Hc (V)
Hc (HK)
2,330 ⫾ 1,793
1,774 ⫾ 1,412
2,048 ⫾ 595
20,602 ⫾ 10,689
10,386 ⫾ 4,575
52,245 ⫾ 36,593
66,704 ⫾ 9,096
10,079 ⫾ 5,978
103,873 ⫾ 12,528
a
Mixed mononuclear (MM) cells 105 were incubated for 7 days with 106 HK Hc
yeasts and then pulsed with [3H]thymidine for an additional 24 h. The mean cpm ⫾
SD for eight Hc-responsive donors tested in the MM screen was 28,653 ⫾ 7,784 for
an average stimulation index of 40.
b
DC 104 were incubated for 1 h with either 5 ⫻ 103 viable (V) or 106 HK Hc
yeasts, and then incubated for another 7 days with CD3⫹ T cells. The cells then were
pulsed with [3H]thymidine as described in Materials and Methods.
Acknowledgments
We thank Georgianne Ciraolo for technical assistance with electron microscopy, Dan Marmer for technical assistance with flow cytometry, and
Carlos Subauste for informative discussion.
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requirement for binding and subsequent phagocytosis of Hc yeasts
by M␾ (29), it is possible that the CD18 of DC is immobile, thus
driving Hc yeasts to bind DC via VLA-5. Another possibility for
the different receptor usage is that the particular topology of CD18
on DC or VLA-5 on M␾ may not be optimal to promote binding
of Hc yeasts.
Another striking aspect of this study is that unlike human M␾
that permit rapid intracellular growth of Hc yeasts, human DC
inhibited the intracellular growth of Hc and even killed and degraded many of the organisms. In contrast, T. cruzi survive and
multiply intracellularly within human DC (39), and L. monocytogenes (49) and B. bronchiseptica (47) apparently survive within
the murine DC cell line CB1. Electron microscopy revealed both
viable and degraded Chlamydia in murine DC (50, 51) and both
viable and degraded B. burgdhorferi in human DC (36). Further,
murine DC, collected after in vivo infection with L. major, contained viable parasites that caused the development of lesions upon
reinjection into BALB/c mice (54).
Although killing of a micro-organism would seem to be a necessary prerequisite to obtain efficient presentation of Ags, Moll and
colleagues (54) found that although murine DC contained viable,
virulent L. major parasites, infected DC still were capable of stimulating lymphocyte proliferation. Because the DC that stimulated
proliferation contained viable organisms, possibly a small number
of intracellular parasites were actually degraded, and Ag processed
and deposited on the surface of the infected DC before presentation to lymphocytes. Alternatively, Ags could be processed and
regurgitated by other infected phagocytes and then transferred to
DC for presentation. In fact, the propinquity between M␾ and DC
in the lung might suggest this route of Ag transfer for inhaled
micro-organisms (26). However, attempts to demonstrate that mycobacterial Ags could be transferred from infected M␾ to DC were
unsuccessful (63). Whether DC might acquire Ags from other microbial pathogens via this route remains to be determined.
An interesting observation in our studies was that DC that had
phagocytosed viable Hc yeasts actually were more efficient at stimulating T cell proliferation than DC that had ingested HK yeasts.
Thus, ingestion of 200-fold fewer viable yeasts than HK yeasts led
to equivalent stimulation of lymphocyte proliferation. These results are analogous to original experiments that demonstrated that
inoculation of mice with Hc yeasts engenders protection against a
subsequent exposure to Hc, and that viable yeasts conferred better
protection than nonviable yeasts (64). Thus, these data suggest that
the heat inactivation process may destroy important immunogenic
Ags and has implications for the design and use of DC in vaccine
strategies. The only other reports that DC stimulated lymphocyte
proliferation after phagocytosis of a viable microbial pathogen
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Our laboratory is in an area that is indigenous for histoplasmosis, and, therefore, one would expect that ⬎90% of our normal
blood donors have been exposed to Hc and have developed specific CMI. We confirmed this expectation by a standard in vitro
lymphocyte proliferation screening assay (33). However, we also
identified three individuals who recently came to Cincinnati from
overseas who did not have CMI to Hc as defined in the screening
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T cell proliferation. Our cautious interpretation of these data is that
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a primary immune stimulation, whereas with our Hc-immune
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role in the host response to Hc infection by coordinating the development of CMI. Current efforts are directed toward delineating
the mechanism by which human DC inhibit the intracellular
growth of Hc yeasts and to identify the cytokines induced upon DC
phagocytosis of Hc yeasts.
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