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*
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
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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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), 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, 7, 8, 9, 10, 11, 12, 13). In 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, 20, 21, 22, 23, 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, 26, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 105/ml in RPMI 1640 containing 200 mM glutamine, 50 µM 2-ME (Sigma, St. Louis, MO), 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 50 ng/ml kanamycin (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 x
106/ml in Teflon beakers with RPMI 1640
containing 15% human serum, 10 µg/ml gentamicin (Sigma), 100 U/ml
penicillin, and 100 µg/ml streptomycin (Sigma). M were studied
after 5–7 days in culture.
FACS analysis
DC (5 x 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 fluorochrome-labeled 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 µg/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 anti-mouse 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).
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 x
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
carbonate-bicarbonate 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 x 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 x
106/ml. DC (1 x 106)
were incubated with FITC-labeled HK Hc yeasts (5 x
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 x 106/ml in HBSA.
Fifty microliters of cells were incubated in 12- x 75-mm polypropylene
tubes for 30 min at 4°C with 5 µg of purified mouse anti-human
CD18, CD11a, CD11b, or CD11c mAb (Ancell), either alone or in
combination (15 µg of total mAb), 3 µg of purified mouse
anti-human very late Ag-5 (VLA-5) mAb (Caltag), or HBSA only. After
preincubation with mAbs, 1 x 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;
64), the -chain of
another RGD-independent fibronectin receptor (CD49d;
41), the
Mg2+-dependent collagen receptor (CD49b;
21), and CD29
(1).
Alternatively, for adherent binding assays, M (2.5 x
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 µl of mAb (CD18, CD11a, CD11b, or CD11c at
50 µg/ml; VLA-5 at 17 or 24 µg/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 x
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 x 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 x 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
x 103 viable yeasts. All plates then were
centrifuged, and supernatants were carefully aspirated through a
27-gauge needle. Fifty microliters (1.0 µCi) of
[3H]leucine (sp. act., 153 Ci/nmol; DuPont/New
England Nuclear, Boston, MA) in sterile water and 5 µl of a 10x
yeast nitrogen broth (Difco, Detroit, MI) 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 (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 x 106 each)
were incubated separately in polypropylene tubes for 2 or 24 h at
37°C with 5 x 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 JEOL-100CX 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 x 107/ml in RPMI 1640 containing 5% FCS and 10 µg/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 x 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 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 µCi 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
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 x
106/ml in RPMI 1640 supplemented with 10%
autologous serum and 10 µg/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 
3.
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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Previous studies have shown that immature human DC can phagocytose
some bacteria and protozoans (36, 37, 38, 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.
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, 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).
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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).
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Our original rationale for these experiments was based on the fact
that DC contain high levels of CD18 on their surface. As M use
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 FITC-labeled 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 µg/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).
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, 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;
v3), the
-chain of the laminin receptor (CD104;
64), the
4-chain of the RGD-independent fibronectin
receptor 41
(CD49d), and the 2-chain of the
Mg2+-dependent collagen receptor
21 (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
51, 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).
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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 x 103) than HK yeasts (1 x
106) were required to stimulate optimum
lymphocyte proliferation.
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, 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.
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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 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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, 7, 8, 9, 10, 11, 12, 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 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, 45, 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 Calmette-Guérin bacillus (BCG) mycobacterium (52), in addition to the protozoan Leishmania major (53, 54, 55, 56, 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 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 were with L. major (54, 55) and B. burgdorferi (36).
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 assay. DC from these three individuals that were infected with viable or HK Hc yeasts still were capable of stimulating significant T cell proliferation. Our cautious interpretation of these data is that in the case of these three individuals, T cell proliferation represents a primary immune stimulation, whereas with our Hc-immune blood donors DC stimulation of T cells represents a secondary or memory response.
The ability of DC to phagocytose and kill Hc yeasts and to present Hc Ags to T cells suggests that DC may play an important 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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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.
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.
Received for publication August 8, 2000. Accepted for publication October 11, 2000.
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J.-S. Lin, C.-W. Yang, D.-W. Wang, and B. A. Wu-Hsieh Dendritic Cells Cross-Present Exogenous Fungal Antigens to Stimulate a Protective CD8 T Cell Response in Infection by Histoplasma capsulatum J. Immunol., May 15, 2005; 174(10): 6282 - 6291. [Abstract] [Full Text] [PDF]
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M. Scheckelhoff and G. S. Deepe Jr. A Deficiency in Gamma Interferon or Interleukin-10 Modulates T-Cell-Dependent Responses to Heat Shock Protein 60 from Histoplasma capsulatum Infect. Immun., April 1, 2005; 73(4): 2129 - 2134. [Abstract] [Full Text] [PDF]
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) 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).