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*
Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and
Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45267
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß TCR+ or
CD4+ T cell-depleted mice infected with the fungus
Histoplasma capsulatum is associated with impairment of
IFN-
production. Upon secondary infection, mice concomitantly
depleted of CD4+ and CD8+ cells exhibit
decreased survival beyond day 25 of rechallenge, whereas elimination of
either T cell subset or B cell deficiency does not result in
accelerated mortality compared with controls. Remarkably, despite a
decrease of H. capsulatum CFU in lungs of
CD4+ plus CD8+-deficient mice, a progressive
increase in spleen CFU is observed. The ability to control fungus
growth in lungs is associated with vigorous TNF-, but not IFN-,
production by bronchoalveolar lavage cells. In contrast, spleen cells
from CD4+ plus CD8+-deficient mice are unable
to produce TNF-. Thus, the cellular and molecular requirements for
protective immunity vary between primary and secondary infection.
Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn
between lungs and spleens in their reliance upon T cells to control
fungal replication. The opposing activities of these organs can be
ascribed in part to differential production of
TNF-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell mediated immunity is a major host factor promoting resistance to initial infection and reinfection with intracellular pathogens (4, 5). Numerous reports indicate that CD4+ and CD8+ cells contribute to immunity against Listeria monocytogenes and Toxoplasma gondii during primary and secondary infection (6, 7). Optimum protection in these models is dependent upon the coordinated interplay between the different T cell subsets.
There is ample experimental evidence that CD4+ and CD8+ T cells exert important functions in host resistance to systemic infection with Hc. Mice congenitally deficient in T cells are more susceptible to histoplasmosis (8). Depletion of CD4+ cells followed by i.v. infection with Hc leads to accelerated mortality in naive mice. ß2-microglobulin knockout (ko) mice, or mice depleted of CD8+ T cells, exhibit impaired clearance of the fungus after systemic challenge, but do not succumb to primary or secondary infection (9, 10). However, there is limited evidence about the role of T cells during primary and secondary pulmonary histoplasmosis.
Resolution of Hc requires cytokines that activate macrophages, the
principal effector cells in host resistance (11). One critical cytokine
involved in the immune response to primary infection with Hc is IL-12
through its induction of IFN- (12, 13). Depletion of IL-12 in naive
mice infected with Hc is associated with accelerated mortality. In
contrast, during secondary infection, an effective immune response
can be maintained in the absence of IL-12, whereas reinfection in
IFN- -/- mice is associated with decreased survival (14).
TNF- production is pivotal in primary and secondary immunity to
pulmonary infection with Hc (15). The mechanisms by which TNF-
exerts its effects during secondary histoplasmosis are distinct from
that of primary infection. Macrophages of naive mice depleted of
TNF- exhibit impaired release of reactive nitrogen intermediates;
during secondary infection, up-regulation of Th2-type cytokines ensues
with deleterious effects that are abrogated by neutralization of these
cytokines.
Because cytokines and T cells are key to immunity to Hc, we explored
the role of distinct T cell subsets in regulating cytokine responses
during primary and secondary pulmonary histoplasmosis. We demonstrate
that /ß TCR+ and, in particular, CD4+
cells are crucial in sustaining survival during primary infection,
while elimination of CD8+ cells subvert the efficiency of
clearance of Hc. The defective host responses in naive mice depleted of
/ß TCR+ and CD4+ can be attributed in part
to deficient IFN- production. The modest impairment detected in mice
devoid of CD8+ cells is IFN--independent. In secondary
infection, all mice in whom CD4+ or CD8+ cells
were abolished survive rechallenge. Mice depleted of both subsets
succumb to Hc. Surprisingly, in the absence of CD4+ and
CD8+, the lung restricts Hc growth, whereas progressive
growth in lymphoid tissue transpires. The organ-specific regulation of
host resistance is correlated with differential production of TNF-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-week-old male C57BL/6, µMT (with a disruption of the IgM gene) ko mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). Six-week-old athymic nude mice were purchased from National Cancer Institute (Frederick, MD) and used to produce ascites. All animal experiments were done in accordance with the Animal Welfare Act guidelines of the National Institutes of Health.
Preparation of Hc and infection of mice
Hc yeasts were prepared as described (12). To produce a sublethal infection in naive mice, animals were infected intranasally (i.n.) with 2.5 x 106 Hc yeasts in a 50-µl volume. For secondary histoplasmosis, mice were initially inoculated with 104 yeasts i.n. Six weeks later, previously exposed animals were rechallenged i.n. with 2.5 x 106 yeasts.
Organ culture for Hc
Recovery of Hc was performed as described previously (12). The fungus burden was expressed as mean CFU per whole organ ± SE.
Antibodies
mAb were ascites-derived or generated via tissue culture. Rat
anti-mouse CD8+ (clone 2.43, rat IgG2b) and
anti-CD4+ (GK 1.5, rat IgG2b) mAb were used to deplete
CD8+ and CD4+ T cells, respectively. Hybridoma
H57 (hamster IgG) was used to eliminate /ß TCR+ T
cells. The concentration of mAb was assessed by ELISA and calculated by
linear regression from an IgG (Organon Teknika, Durham, NC) standard
curve. All mAb contained <5 ng/ml of endotoxin, as determined by
Limulus amebocyte lysate test (BioWhittaker, Walkersville,
MD).
Treatment of mice with mAb
To deplete T cell subsets, mice were injected i.p. with 100 µg
of mAb to CD8+ cells or 300 µg of mAb to CD4+
cells. To eliminate /ß TCR+ cells, 300 µg was
administered i.p. (9, 10). To abolish both CD4+ and
CD8+ T cells, 300 µg and 100 µg were given i.p.
concomitantly. The schedule for injection of each dose of mAb was as
follows: days -7, -3, and at the time of i.n. challenge. mAb were
given each week thereafter. Control animals received an equal amount of
rat or hamster IgG (a control for mAb to /ß TCR+
cells) concomitantly.
Cytokine measurement
Lungs and spleens from mice (n = 5–6) were
removed on days 3, 5, and 7 postinfection, and the tissue was
homogenized in 5 ml of HBSS, centrifuged at 1500 x g,
filter sterilized, and stored at -70°C until assayed. Commercially
available ELISA kits were used to measure IFN-, IL-4, IL-10, IL-6,
and GM-CSF (Endogen, Cambridge, MA). IL-12, specific for mouse IL-12
p70 protein was assayed by sandwich ELISA (PharMingen, San Diego, CA).
The sensitivities were >100 and 45 pg/ml, respectively. TGF-ß was
measured by two-site ELISA (R&D Systems, Minneapolis, MN). The
sensitivity was >10 pg/ml, and the cross-reactivity with murine
TGF-ß is >90%.
Bronchoalveolar lavage (BAL) and FACS analysis
BAL was performed on day 7 of primary and secondary infection to obtain inflammatory cells. The trachea was exposed and intubated using a 1.7-mm OD polyethylene catheter. BAL fluid was collected by instilling PBS in 1 ml aliquots and retrieving it. Approximately 5 ml of lavage fluid was obtained per mouse. Differential counts were performed after cytocentrifugation of 5 x 104 BAL cells/slide and staining with Leuko Stat (Fisher Scientific, Pittsburgh, PA). The remainder of BAL cells were adjusted to 5 x 105/200 µl in HBSS containing 10% FBS and 0.02% sodium azide and stained with 0.5 µg of one of the following FITC-labeled mAb (PharMingen): anti-CD4+ (clone RM4-5), anti-CD8+ (clone 53-6.7). The samples were washed and fixed in 2% paraformaldehyde until analyzed on a flow cytometer. Depletion of either alveolar or splenic T cells caused a 95–98% reduction of the respective T cell subset.
In vitro activation of alveolar macrophages and spleen cells
BAL or spleen cells from CD4+ plus
CD8+-depleted and rat IgG-treated mice (n =
5–6/group) were obtained on days 7, 14, and 25 of secondary infection.
Cells were washed twice and adjusted in DMEM supplemented with 10% FBS
to 105 or 106 cells per well in 96- and 24-well
plates, respectively. Nonadherent and adherent cells were stimulated
with either Con A (0.2, 1, and 5 µg/ml) or LPS (at 1 µg/ml) (Sigma,
St. Louis, MO). The supernatants were harvested 24 and 48 h later,
and IFN- and TNF- levels were determined by ELISA. Data are
presented as 
IFN- or TNF- (IFN- or TNF- from stimulated
cells - IFN- or TNF- from unstimulated cells).
Statistical analyses
The log rank test was used to analyze differences in survival; the Wilcoxon rank sum test was employed to analyze differences in cytokine production and fungal burden of organs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß
TCR+, CD4+, or CD8+ T cells
Naive mice were infected i.n. with a sublethal dose of Hc, and
survival was monitored. All mice depleted of /ß TCR+ T
cells succumbed to infection by day 12 with a mean survival time of
9 ± 3 days (Fig. 1
A). Animals lacking
CD4+ T cells also exhibited accelerated mortality and died
by day 20 (mean survival time 17 ± 3), whereas
CD8+-depleted mice and controls survived up to 40 days.
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/ß TCR+ cells before rechallenge with Hc was
associated with the death of >80% of mice. This high mortality was
unexpected and certainly not observed in naive animals. The mAb
contained <5 pg/ml of LPS and was sterile. Mice became noticeably ill
2–3 days after injection of mAb and died 1–2 days later. Although the
cause is obscure, the clinical course suggests either immune complex
disease or massive T cell activation by the mAb.
To overcome that difficulty, we injected mAb to CD4+ and
CD8+ cells concomitantly to eliminate T cells in
reinfection histoplasmosis. Interestingly, mice depleted of both
CD4+ and CD8+ T cells appeared healthy until
day 25 postinfection. Subsequently, they became immobile, their fur
ruffled, and they lost weight. They began to succumb to histoplasmosis
after day 25 with a mean survival time of 29 ± 2 days (Fig. 1
B). Elimination of either CD4+ or
CD8+ T cells alone did not alter survival upon rechallenge
with Hc compared with controls; all mice lived beyond 35 days.
Growth of Hc in infected naive and rechallenged mice deficient in
/ß TCR+, CD4+, or CD8+ T cells
To determine whether accelerated mortality in naive mice was
accompanied by an inability to control secondary Hc, animals were
sacrificed and their lungs and spleens removed on days 7, 14, and 21 of
infection. As shown in Fig. 2A, lungs of mice lacking
CD4+, CD8+, and /ß TCR+ cells
contained significantly higher CFU (p < 0.01)
than lungs of controls on day 7 postinfection. On day 14, CFU in lungs
of CD4+ or CD8+-depleted mice exceeded
(p < 0.01) those of controls. Anti-/ß
TCR+ mAb-treated animals had all died by this time. By day
21, the fungal load in lungs of both CD8+-depleted animals
and controls had decreased 
1000-fold.
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/ß TCR+ mAb-treated mice
were increased compared with all other experimental groups
(p < 0.01) on day 7 of infection (Fig. 2B). On day 14, CFU in spleens of CD4+-depleted
mice were elevated (p < 0.01) compared with
those given mAb to CD8+ cells and controls. The splenic
burden decreased in mice lacking CD8+ cells and controls by
day 21 postinfection; however, clearance was not as efficient in mice
lacking CD8+ cells as compared with infected controls
(p < 0.02).
On day 7 of secondary infection, CFU in lungs (Fig. 2C) of
control, CD4+, CD8+, and CD4+ plus
CD8+ T cell-depleted mice were comparable to each other
(p > 0.05). On day 14, all groups exhibited a
significant decrease in lung burden when compared with their respective
group of day 7 (p < 0.01), but the decline was
more prominent (>2 logs) in infected controls and in those given mAb
to CD8+ cells. CFU in lungs of mice lacking both
CD4+ and CD8+ cells exceeded
(p < 0.01) that of three other groups at this
time. By day 25, there was a continued decrease in CFU in lungs in all
groups, although much less marked in mice whose CD4+ and
CD8+ cells had been eliminated. In infected controls, CFU
were below the limit of detection (
100). The lungs of mice that were
devoid of both subsets harbored far more CFU (p
< 0.001) than the other groups. In CD4+-depleted animals,
the lungs contained more CFU (p < 0.01) than
controls and mice deficient in CD8+ cells.
Recovery of Hc from spleens of mice rechallenged with yeasts (Fig. 2D) was similar between controls, anti-CD4+,
or anti-CD8+ mAb-treated mice on day 7 postinfection
(p > 0.05). On day 14 of reinfection, spleens
of control mice exhibited a dramatic decrease (2 logs) in CFU;
clearance of Hc in spleens of CD4+ or
CD8+-depleted mice was less striking. By contrast, in
CD4+ plus CD8+-depleted mice, CFU were not
different from day 7; in fact, they were static. By day 25, no CFU were
detectable in spleens of controls and CD8+-depleted mice.
Conversely, CFU from spleens of anti-CD4+ plus
anti-CD8+ mAb-treated mice had risen 1000-fold from day
14, and there existed a 5 log-fold difference between this group and
controls. Thus, an effective host defense is preserved in mice devoid
of either CD4+ or CD8+ cells. On the other
hand, if both T cell subsets are depleted, mice succumb to systemic
infection with Hc, although fungal growth is restrained within lungs.
Survival and fungal growth of Hc infected rechallenged B cell-deficient mice depleted of CD4+ and CD8+ T cells
Since lungs of mice controlled infection in the absence of both T cell subsets, we assessed whether B cells contributed to this protective effect. Exploratory experiments revealed that B cell-deficient and control mice infected with 2.5 x 106 yeasts survived primary infection for 45 days, at which time, the experiments were terminated. In addition, B cell ko mice did not exhibit altered clearance of Hc in lungs (log10 CFU: 6.7 ± 0.1) or spleens (5.4 ± 0.2) when compared with control lungs (6.6 ± 0.2) or spleens (5.6 ± 0.2) at day 7 after primary infection.
In secondary histoplasmosis, B cell-deficient mice and controls were
immunized with 104 Hc yeasts, and, 6 wk later, treated with
mAb to CD4+ and CD8+ cells or rat IgG and then
rechallenged with 2.5 x 106 yeasts. B cell ko mice
depleted of CD4+ and CD8+ T cells began to by
die by day 30, whereas B cell ko and control mice survived at least 45
days. As demonstrated in Fig. 3, culture
studies performed at days 7, 14, and 25 postinfection revealed
increased CFU in lungs (p < 0.02 on day 7;
p < 0.001 on day 30) and spleens
(p < 0.01 on day 7; p <
0.0001 on day 30) of anti-CD4+ plus
anti-CD8+-treated B cell ko mice, compared with the
fungal load in lungs and spleens of B cell ko and control mice, which
were similar. Thus, the capacity of lungs from T cell-deficient mice to
control infection was independent of B cells.
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We hypothesized that individual T cell subsets would alter the cytokine profile in lungs of naive and rechallenged mice infected with Hc and, therefore, assayed cytokine levels at selected time points during primary and secondary histoplasmosis. Mice were depleted of T cells, and, on days 3, 5, and 7 postinfection, cytokines were assayed in lung homogenates.
During primary infection, IFN- production dramatically increased in
control and CD8+-depleted mice by day 5 of infection (Fig. 4). Although the amount of IFN- in
control mice declined by day 7, levels of IFN- in
CD8+-depleted mice remained high and reached an 2-fold
difference compared with controls (p < 0.02).
In contrast, IFN- production in CD4+-depleted mice was
blunted. It was significantly lower than that of controls or
CD8+-depleted mice at day 5 (p <
0.01). By day 7, levels in lungs of these mice were similar to those
found in controls, but significantly lower than IFN- levels of
CD8+-depleted mice (p < 0.02).
Depletion of ß-TCR+ cells was associated with
depressed amounts of IFN- on days 5 and 7 of infection, which were
significantly lower (p < 0.001) than controls
and mice lacking either CD4+ or CD8+ cells.
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levels in lungs were elevated by
day 3 of infection in control and CD8+-deficient animals
compared with CD4+- and CD4+ plus
CD8+-depleted mice (p < 0.0001).
The levels in lungs of controls were unchanged on day 5 postinfection
and declined by day 7. In contrast, there was a steady decrease of
IFN- in lungs of CD8+-depleted mice. Lungs of mice
administered either mAb to CD4+ cells or to
CD4+ plus CD8+ cells exhibited significantly
depressed levels (p < 0.01) of IFN-
compared with controls and CD8+-deficient animals on days 3
and 5 of secondary infection.
There are several critical regulatory cytokines in histoplasmosis,
among those IL-12, which is the key modulator in controlling primary
infection with Hc. We ascertained whether alterations in IL-12
generation were apparent upon elimination of distinct T cell subsets.
As shown in Fig. 4, IL-12 levels did not differ among all groups during
either primary or secondary histoplasmosis. A similar pattern was seen
in the production of TNF-, which is another pivotal cytokine in
resolution of primary and secondary pulmonary histoplasmosis, except
for a 2- to 3-fold decrease in CD4+-depleted naive mice
compared with other experimental groups on day 3 postinfection
(p < 0.02). Neither IL-4 nor IL-10
demonstrated any differences (p > 0.05) among
the groups of mice in primary or secondary infection. We quantified
additional cytokines associated with alterations in host defenses to
infection with pathogenic organisms including Hc, such as GM-CSF,
TGF-ß, and IL-6 (16, 17). None of those cytokines exhibited dramatic
differences between depleted and control mice in either primary or
secondary histoplasmosis (data not shown).
Elevated and prolonged IFN- production in
CD8+-depleted mice compared with CD4+-depleted
and control mice during primary infection
Surprisingly, IFN- levels in CD8+-depleted naive
animals were higher than controls, although they manifested impaired
clearance of Hc. To determine whether the elevation persisted beyond
day 7, we measured IFN- at additional time points. As shown in Fig. 5, IFN- levels in
anti-CD8+ mAb-treated mice remained elevated beyond day
3 (p < 0.001), compared with
CD4+-depleted and control mice. In contrast, IFN- was
diminished in anti-CD4+ mAb-treated mice compared with
controls, but at only one time point, day 5 (p
< 0.01).
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in BAL vs mixed spleen cells of
CD4+ plus CD8+-depleted mice during secondary
infection
The finding that lungs of mice devoid of T cells constrained
fungal growth, while progressive infection transpired in spleens,
prompted the hypothesis that the dissimilar growth of Hc was a result
of organ-specific differences in generation of a particular
cytokine(s). Since we had previously shown that TNF- is instrumental
in controlling secondary pulmonary histoplasmosis (15), we stimulated
either BAL or spleens cells obtained from depleted and control mice at
days 7, 14, and 25 post secondary infection with Con A or LPS and
measured IFN- and TNF- content in supernatants. The levels of IFN
from in vitro-stimulated BAL cells of T cell-deficient mice were
decreased compared with controls at all time points measured. Amounts
of IFN- from spleen cells were diminished at days 7 and 14 of
infection, but were comparable to controls on day 25 (Fig. 6, A and B). In
contrast, TNF- levels from LPS-stimulated BAL cells (Fig. 6C) were similar between controls and depleted mice on days
7 and 14; indeed, amounts were increased in T cell-deficient mice on
day 25 of infection compared with controls (p
< 0.01). Conversely, spleen cells from CD4+ plus
CD8+-depleted mice did not generate any TNF- beyond
spleen cells stimulated with media alone, whereas cells from control
mice mounted a vigorous TNF- response on days 7 and 14, which
decreased by day 25 of infection (Fig. 6D). Thus, the
discordant generation of TNF- between lungs and spleens correlates
with control of fungal growth in the corresponding organs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß TCR+ cells,
yet, in secondary infection, only a lack of both T cell subsets
abrogated the capacity to survive. Depletion of CD8+ cells
impaired clearance in primary, but not secondary, infection. Although
elimination of both T cell subsets in naive and rechallenged mice
caused death, there was a pronounced difference in length of survival.
Naive animals succumbed by day 12, whereas previously exposed animals
did not die before day 25 postinfection. Thus, in secondary
histoplasmosis, the disease is less aggressive in mice deficient in
CD4+ or both T cell subsets. The findings indicate that
host resistance in secondary infection is not entirely dysfunctional
after elimination of T cells.
The protective immune response mediated by T cells is mainly via
release of cytokines, most notably IFN-, which is an important
regulator of killing in models of intracellular infections, such as
listeriosis and leishmaniasis (18, 19, 20). As a corollary, in vivo
treatment with mAb to IFN- increases susceptibility to a variety of
intracellular pathogens, including Hc (13, 21, 22, 23, 24). In the present
studies, depletion of /ß TCR+ or CD4+
cells in primary pulmonary infection caused a significant decrease only
in IFN- levels in lungs. The elimination of these cells and the
subsequent decrease in IFN- was accompanied by increased CFU and
100% mortality. Thus, the cause of death in animals that are deficient
in either CD4+ or /ß TCR+ can be
attributed to a failure to generate IFN-. Because IFN- production
was nearly abrogated by elimination of T cells, the results strongly
suggest that NK cells do not play a prominent role in IFN-
production in Hc infection. This finding is divergent from models of
listeriosis in which NK cells are the major source of IFN-
production in innate immune response (19).
The most marked difference in IFN- levels among controls and animals
lacking /ß TCR+ or CD4+ cells was noted on
day 5. Although it would appear that such a temporal decrease may not
be meaningful to survival, previous data suggest otherwise. Mice unable
to generate IFN- within the first 5 days of pulmonary Hc infection
manifest a high mortality (12). Thus, the ability to generate IFN-
within 5 days of the initiation of infection is key to the development
of protective immunity in primary infection.
In contrast to the elimination of CD4+ cells, depletion of
CD8+ cells was associated with a rise in IFN- levels
that exceeded those of infected controls beyond the acute phase of
infection. The modest elevation of IFN- observed in
CD8+-deficient mice implies that these cells regulate
production of IFN-. Since elevated levels of IFN- have been
associated with massive inflammation, CD8+ cells may
prevent overproduction of this cytokine and the inimical consequences
associated with this event (25, 26). The data also demonstrate that, in
the absence of CD8+ cells, an effective, but not optimal,
protective immune response can be maintained. The fact that impaired
clearance in CD8+-deficient mice is associated with high
levels of IFN- indicates that another cytokine or functional
activity of CD8+ cells must contribute to the development
of a protective immune response to Hc.
Additional cytokines, especially IL-12 and TNF-, are necessary for
host resistance in both the systemic and pulmonary model of primary
infection with Hc. Neutralization of either cytokine will result in
diminished survival of infected animals in primary histoplasmosis (12, 13, 15, 27). Protein measurements of either cytokine in lungs of T
cell-depleted mice did not exhibit any differences when compared with
controls. The selective decrease of IFN- in lungs of
TCR-ß+ or CD4+-deficient mice was not
caused by a reduction in levels of IL-12, which is important in IFN-
generation in Hc infection (12, 13). Furthermore, the decrement in
IFN- did not impact levels of IL-12 or TNF- as has been described
in infection with Bacille Calmette-Guérin or Leishmania
sp. (28, 29). The findings strengthen our previous contention that
two independent pathways exist, one IFN--, the other
TNF--dependent, and blockade of either one does not alter production
of the other.
During secondary histoplasmosis, depletion of either CD4+ or CD8+ cells was not associated with increased mortality. Mice deficient in both T cell subsets exhibited a decrease in survival after an interval in which these animals controlled infection. In the lungs of these mice there was a progressive decline in CFU, although not as brisk as infected controls. In spleens, however, the course of infection was strikingly different. There was at least a 3-log increase in CFU over the 25 days of observation followed shortly by death of animals. Hence, in reinfection histoplasmosis, T cells are dispensable in lungs, but the presence of both subsets is requisite for clearance in spleens. These results share similarities and discrepancies with a report in which T cell-deficient mice are capable of controlling secondary infection with Listeria monocytogenes in spleens. In that model, both T cell subsets are required to achieve optimum protection against listeriosis (6).
One of the mechanisms that could explain prolonged survival in mice lacking both T cell subsets is the production of Abs. A recent report reveals that B cells play an important role during primary and secondary chlamydial infection in lungs. Accordingly, µMt mice that are devoid of B cells are more susceptible to challenge with mouse pneumonitis agent (30). In addition, B cells are required for in vivo priming of T cells. In the present study, B cell ko mice did not exhibit any differences in survival or infectious burden compared with controls in either primary or secondary histoplasmosis. Thus, neither the production of Abs nor the Ag-presenting capacity of B cells contributed to the protective immune response. It should be emphasized that the effect of B cells on protective immunity may be dependent on the size of the inoculum of Hc or the route of infection.
IFN- levels in lungs of rechallenged mice depleted of
CD4+ and CD4+ plus CD8+ cells were
diminished as was observed in primary infection. Amounts of other
cytokines, in particular TNF-, were comparable among groups.
Although a decrease of IFN- occurred in both groups of animals,
there was a marked difference in outcome. In CD4+-deficient
animals, survival was not altered in secondary histoplasmosis. On the
other hand, CD4+ plus CD8+-deficient mice
succumbed to infection with Hc. The results indicated that the blunted
IFN- response could not simply account for the demise of mice
lacking both T cell subsets. The findings raised the possibility that
the spleens failed to release a mediator involved in the protective
immune response or that another cytokine was exacerbating disease.
Endogenous TNF- is a key regulator of host resistance to secondary
histoplasmosis; neutralization of it is associated with disease
exacerbation and decreased survival (15). Since concomitant depletion
of CD4+ and CD8+ cells in secondary
histoplasmosis caused a dramatic increase in Hc CFU in spleens but not
lungs, we determined whether production of TNF- differed between
these two organs. Generation of TNF- from BAL and spleen cells was
strikingly different between the cell types. Whereas TNF- levels of
T cell-depleted BAL cells were comparable to levels from control BAL
cells, spleen cells of CD4+ plus CD8+ -depleted
mice did not produce any TNF- upon stimulation in comparison to
controls. As anticipated, production of IFN- by BAL and spleen cells
was diminished in T cell-deficient mice as compared with controls.
Thus, the capacity to control secondary infection with Hc is directly
correlated with generation of TNF-. Furthermore, the findings also
indicate that cellular requirements for production of TNF- differ
between organs. In spleens, the presence of T cells is pivotal, which
is not the case in lungs. The necessity of T cells for TNF- release
by spleen cells is highlighted by the fact that adherent splenocytes
that were devoid of T cells from infected controls did not produce this
cytokine (data not shown).
The present studies substantiate the role of T cells in host resistance
against Hc infection and highlight the complexities of innate and
acquired immune response to intracellular pathogens. Our data
demonstrate that there are differential T cell requirements for
clearance of infection with Hc in primary and secondary histoplasmosis.
In addition, the results employing the pulmonary model provide strong
evidence that resolution of reinfection with Hc in immune-deficient
mice is compartmentalized and dependent upon the ability of the
specific organ to generate TNF-. Studies are underway to completely
elucidate cellular and molecular requirements for clearance of Hc
within the organ-specific sites.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. George S. Deepe, Jr., Division of Infectious Diseases, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0560. E-mail address:
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; ko, knockout; BAL, bronchoalveolar lavage; i.n., intranasal.
Received for publication January 12, 1999. Accepted for publication April 2, 1999.
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  G. S. Deepe Jr, R. S. Gibbons, and A. G. Smulian Histoplasma capsulatum manifests preferential invasion of phagocytic subpopulations in murine lungs J. Leukoc. Biol., September 1, 2008; 84(3): 669 - 678. [Abstract] [Full Text] [PDF]
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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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 E. Lazar-Molnar, A. Gacser, G. J. Freeman, S. C. Almo, S. G. Nathenson, and J. D. Nosanchuk The PD-1/PD-L costimulatory pathway critically affects host resistance to the pathogenic fungus Histoplasma capsulatum PNAS, February 19, 2008; 105(7): 2658 - 2663. [Abstract] [Full Text] [PDF]
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 G. S. Deepe Jr. and R. S. Gibbons TNF-{alpha} Antagonism Generates a Population of Antigen-Specific CD4+CD25+ T Cells That Inhibit Protective Immunity in Murine Histoplasmosis J. Immunol., January 15, 2008; 180(2): 1088 - 1097. [Abstract] [Full Text] [PDF]
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D. M. Lindell, M. N. Ballinger, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Immunologic Homeostasis during Infection: Coexistence of Strong Pulmonary Cell-Mediated Immunity to Secondary Cryptococcus neoformans Infection While the Primary Infection Still Persists at Low Levels in the Lungs J. Immunol., October 1, 2006; 177(7): 4652 - 4661. [Abstract] [Full Text] [PDF]
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H. L. Allen and G. S. Deepe Jr. B Cells and CD4-CD8- T Cells Are Key Regulators of the Severity of Reactivation Histoplasmosis J. Immunol., August 1, 2006; 177(3): 1763 - 1771. [Abstract] [Full Text] [PDF]
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D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Generation of Antifungal Effector CD8+ T Cells in the Absence of CD4+ T Cells during Cryptococcus neoformans Infection J. Immunol., June 15, 2005; 174(12): 7920 - 7928. [Abstract] [Full Text] [PDF]
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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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J. Rivera, O. Zaragoza, and A. Casadevall Antibody-Mediated Protection against Cryptococcus neoformans Pulmonary Infection Is Dependent on B Cells Infect. Immun., February 1, 2005; 73(2): 1141 - 1150. [Abstract] [Full Text] [PDF]
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L. F. Barton, H. A. Runnels, T. D. Schell, Y. Cho, R. Gibbons, S. S. Tevethia, G. S. Deepe Jr., and J. J. Monaco Immune Defects in 28-kDa Proteasome Activator {gamma}-Deficient Mice J. Immunol., March 15, 2004; 172(6): 3948 - 3954. [Abstract] [Full Text] [PDF]
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G. S. Deepe Jr. and R. S. Gibbons Protective and Memory Immunity to Histoplasma capsulatum in the Absence of IL-10 J. Immunol., November 15, 2003; 171(10): 5353 - 5362. [Abstract] [Full Text] [PDF]
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 F. X. McCormack, R. Gibbons, S. R. Ward, A. Kuzmenko, H. Wu, and G. S. Deepe Jr. Macrophage-independent Fungicidal Action of the Pulmonary Collectins J. Biol. Chem., September 19, 2003; 278(38): 36250 - 36256. [Abstract] [Full Text] [PDF]
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 M. Wuthrich, H. I. Filutowicz, T. Warner, G. S. Deepe Jr., and B. S. Klein Vaccine Immunity to Pathogenic Fungi Overcomes the Requirement for CD4 Help in Exogenous Antigen Presentation to CD8+ T Cells: Implications for Vaccine Development in Immune-deficient Hosts J. Exp. Med., June 2, 2003; 197(11): 1405 - 1416. [Abstract] [Full Text] [PDF]
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 K. L. Wood, C. A. Hage, K. S. Knox, M. B. Kleiman, A. Sannuti, R. B. Day, L. J. Wheat, and H. L. Twigg III Histoplasmosis after Treatment with Anti-Tumor Necrosis Factor-{alpha} Therapy Am. J. Respir. Crit. Care Med., May 1, 2003; 167(9): 1279 - 1282. [Abstract] [Full Text] [PDF]
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M. Wuthrich, H. I. Filutowicz, T. Warner, and B. S. Klein Requisite Elements in Vaccine Immunity to Blastomyces dermatitidis: Plasticity Uncovers Vaccine Potential in Immune-Deficient Hosts J. Immunol., December 15, 2002; 169(12): 6969 - 6976. [Abstract] [Full Text] [PDF]
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M. Scheckelhoff and G. S. Deepe Jr. The Protective Immune Response to Heat Shock Protein 60 of Histoplasma capsulatum Is Mediated by a Subset of V{beta}8.1/8.2+ T Cells J. Immunol., November 15, 2002; 169(10): 5818 - 5826. [Abstract] [Full Text] [PDF]
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