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Exacerbates Primary and Secondary Pulmonary Histoplasmosis by Differential Mechanisms1
Division of Infectious Diseases, University of Cincinnati College of Medicine, Cincinnati, OH 45267, and Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45220
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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modulates host defenses during experimental primary and secondary
pulmonary infection with Histoplasma capsulatum (Hc).
Neutralization of TNF- in vivo resulted in increased CFU and 100%
mortality in naive and immune mice challenged with Hc intranasally.
Levels of IFN-
and granulocyte macrophage-CSF were elevated in
TNF--neutralized naive mice, whereas IL-4, -6, -10 and TGF-ß did
not differ from controls. In contrast, in secondary histoplasmosis,
significant elevations of IL-4 and -10 were observed in
TNF--depleted mice. Alveolar macrophages (M
) did not exert
fungistatic activity against Hc after exposure to recombinant murine
TNF-, recombinant murine IFN-, or both. The increase in
susceptibility to primary Hc infection was associated with diminished
production of reactive nitrogen intermediates by alveolar M from
TNF--depleted mice, whereas production of nitric oxide during
secondary histoplasmosis was similar in both groups. Upon secondary
challenge, TNF--depleted mice were rescued by concomitant
neutralization of IL-4 and IL-10, but not either cytokine alone. Thus,
TNF- is critical for controlling primary and secondary infection
with Hc, and the mechanisms that lead mice to succumb to primary or
secondary infection when endogenous TNF- is blocked are different. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), within which
they multiply. Resolution of infection depends primarily on
cell-mediated immunity and requires the coordinated cooperation between
M and T cells (4).
Activation of M, the principal effector cells, are central to the
immune response against Hc (4). One mechanism to achieve this state is
by release of cytokines from immunocompetent cells. M produce
monokines that play an important part in the generation and
amplification of the host response to this fungus. One monokine that
affects protective immunity to Hc is IL-12, and its effects are
mediated by induction of IFN- production (5). Although IL-12 is an
absolute requisite for survival in primary infection with Hc, it does
not alter mortality in secondary histoplasmosis (6).
TNF- is involved in the immune response to Hc. This contention is
supported by observations that murine M infected with Hc produce
abundant TNF-, and by reports demonstrating that depletion of
TNF- in vivo increased mortality of mice infected i.v. with Hc (5, 7, 8). Although neutralization of TNF- renders mice highly
susceptible to primary infection with Hc, in vitro treatment of murine
M with recombinant murine (rm)TNF- does not induce these cells to
exert antihistoplasmal activity (9). Thus, the mechanism by which
TNF- modulates host defenses in histoplasmosis remains a matter of
speculation.
The objective of the present report was to examine the mechanisms by
which endogenous TNF- affects host susceptibility and influences the
production of cytokines, including those associated with a Th1 and Th2
type. The studies were conducted in an established murine pulmonary
model, which best mimics the human condition, since the lung is the
primary site of infection (1). We sought to elucidate the effects of
TNF- on both primary and secondary infection. The latter studies are
particularly relevant to the human condition, because individuals
residing in endemic areas are frequently reexposed to Hc (1).
The experiments revealed that TNF- is required for host resistance
to Hc in both primary and secondary pulmonary infection.
TNF--deficient naive mice were not impaired in their ability to
exhibit a Th1-dominated immune response. However, generation of
reactive nitrogen intermediates (RNI) was impaired in alveolar M in
naive TNF--neutralized mice although inducible nitric oxide synthase
(iNOS) expression in lung tissue was not altered. In contrast, in
secondary histoplasmosis, the exaggerated susceptibility was associated
with a predominant Th2-type response, and host resistance was restored
by neutralization of both IL-4 and IL-10.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 mice, 6 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Athymic nude mice were purchased from the 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 (6). 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 (6). The fungus burden was expressed as mean CFU per whole organ ± SD.
Antibodies
Ascites-derived rat anti-mouse TNF- mAb and rat
anti-mouse IL-10 mAb were produced from the hybridomas XT 22-11
(rat IgG1) and JES 2A5 (rat IgG2b), respectively. These cell lines were
obtained from Dr. J. Abrams (DNAX Research Institute, Palo Alto, CA).
The concentration of rat IgG in ascites was assessed by ELISA and
calculated by linear regression from a rat IgG (Organon Teknika,
Durham, NC) standard curve. All Abs contained <5 ng/ml of endotoxin as
determined by Limulus amebocyte lysate test (BioWhittaker,
Walkersville, MD). Rat anti-mouse IL-4 (11B11, rat IgG1) mAb was
provided by the Biological Response Modifiers Program (National Cancer
Institute).
Treatment of mice with cytokine neutralizing mAb
Mice were injected i.p. with 2 mg of anti-TNF- mAb 2
h before challenge with Hc and were given 1 mg of mAb each week
thereafter. In studies with anti-IL-4 and anti-IL-10 mAb, mice
received 2 and 1 mg, respectively, on day 0 of infection and were
boosted with 1 mg each wk. Control animals received an equal amount of
rat IgG concomitantly.
Cytokine measurement
Lungs from mice (n = 5–6) were removed
on days 3, 5, and 7 postinfection, and the tissue was homogenized in 2
ml of RPMI 1640, 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 granulocyte macrophage (GM)-CSF (Endogen, Cambridge, MA). IL-12 was
assayed by sandwich ELISA (PharMingen, San Diego, CA), specific for
mouse IL-12 p70 protein. The sensitivity was >100 pg/ml. 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
was >90%.
Histology
Lungs were removed and tissues were fixed in 10% formalin and embedded in paraffin blocks. Sections (5 µm) were stained with hematoxylin and eosin or with silver for fungal elements. Inflammatory cells within lung sections were quantified in a blinded fashion by examining 3 hpf per lung (n = 3 mice). Data are expressed as mean percent ± SD.
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 was performed by instilling PBS in 1-ml aliquots. Approximately 5 ml of lavage fluid were retrieved 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 was 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 mAbs (PharMingen): anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-Ly-6G (GR-1), (clone RB6-8C5, which recognizes polymorphonuclear cells (PMN)), or isotype-matched rat IgG mAb. The samples were washed and fixed in 2% paraformaldehyde until analyzed on a flow cytometer.
Assay of murine alveolar and peritoneal M fungistatic activity
against Hc
Intracellular growth of Hc in M was quantified by the
incorporation of [3H]leucine into viable yeast cells
(10). M monolayers, cultured in 96-well plates at 37°C, were
exposed to rmTNF- (R&D Systems) or rmIFN- (Genzyme, Cambridge,
MA) for 18 h. The supernatant was removed and 5 x
103 viable yeasts in 0.1 ml of RPMI 1640 containing 10%
heat-inactivated FCS and 10 µg of gentamicin per ml were added to
each well. After incubation for 24 h at 37°C, the plates were
centrifuged at 1000 x g, and the supernatants aspirated
through a 27-gauge needle. Fifty microliters of
[3H]leucine in sterile water (1 µCi) and 5 µl of a
10 x yeast nitrogen broth (Difco, Detroit, MI) were added to each
well and plates were incubated for an additional 24 h at 37°C.
To each well were then added 50 µl of L-leucine (10
mg/ml) and 50 µl of sodium hypochlorite. Cell contents were harvested
onto glass fiber filters using an automated harvester (Skatron,
Sterling, VA). The data are presented as percent inhibition, which is
defined as 1 - (cpm in activated M/cpm in control M) x
100. All experimental procedures were performed in triplicate.
Western blot analysis of iNOS
Lungs from infected TNF--depleted and rat IgG-treated mice
were perfused with PBS, removed, and homogenized in saline. The
material was centrifuged at 3,000 x g to remove
insoluble material. Protein concentration of the samples was determined
by the Bradford method (11). In all, 20 µg of homogenates or 5 µg
of mouse M lysate, prepared from the RAW 264.7 cell line, as a
positive control was analyzed on 5% SDS-PAGE gels. The resolved
proteins were electroblotted overnight to nitrocellulose membranes and
incubated with a 1:10,000 dilution of murine anti-iNOS mAb
(Transduction Laboratories, Lexington, KY). Anti-iNOS was detected
using a 1:1,000 dilution of alkaline-phosphatase-labeled goat
anti-mouse heavy and light chain Ab (Kirkegaard & Perry
Laboratories, Gaithersburg, MD). Blots were developed with BCIP and NBT
as chromogenic substrates (Life Technologies). The correspondent iNOS
proteins were quantified using the National Institutes of Health image
program in conjunction with a Fotodyne image analysis (Eagle I;
Fotodyne, Hartland, WI). Data are presented as mean pixel density
± SD (n = 6).
Macrophage RNI assay
BAL cells from rat-IgG-treated and TNF--depleted mice
(n = 6/group) were obtained on days 3, 5, and 7
of primary and secondary infection. After washing twice, cells were
plated at 105 cells per well in 96-well plates in DMEM
supplemented with 10% FBS. Nonadherent cells were removed after 2
h and monolayers were stimulated with either LPS (Sigma, St. Louis, MO)
and rmIFN- (1 µg and 100 ng/ml, respectively), or rmIFN- and
rmTNF- (100 ng/ml each). Supernatants were collected 48 h after
seeding, filtered, and nitrite was measured by Griess reaction using
Cayman’s nitrate/nitrite assay kit (Alexis, San Diego, CA). Data are
presented as 
NO2 (NO2 from stimulated
cells - NO2 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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Naive mice were injected i.p. with 2 mg of anti-TNF- mAb or
an equivalent amount of rat IgG 2 h before a sublethal i.n.
challenge with Hc. On day 7 postinfection, mice were sacrificed, and
their lungs, livers, and spleens were cultured for Hc. Lungs of
TNF--neutralized mice demonstrated a dramatic increase
(p < 0.01) in CFU (514 ± 370 x
105) when compared with controls (23 ± 9 x
105). Interestingly, CFU in either livers (12.2 ±
0.8 x 105) or spleens (2.4 ± 0.9 x
105) of TNF--depleted mice did not significantly exceed
(p > 0.05) the number of CFU recovered from
controls in livers (11 ± 0.9 x 105) or spleens
(2.1 ± 1.1 x 105).
Intranasal instillation of 104 Hc yeasts produces a host
response that manifests in accelerated clearance of the organisms upon
rechallenge (data not shown). Although TNF- is known to play an
important role in primary infection with a variety of pathogens
including Hc, its role in secondary immune response to Hc remains
unclear (8, 12, 13, 14). Therefore, we sought to determine the effect of
endogenous TNF- in secondary histoplasmosis. Six weeks after i.n.
inoculation with 104 Hc, mice were depleted with 2 mg of
anti-TNF- mAb on the day of rechallenge and burden determined on
day 7. CFU in lungs from TNF--neutralized mice were more than
200-fold increased (32.0 ± 11.1 x 106) when
compared with that in lungs from infected controls (0.1 ±
0.2 x 106) (p < 0.001).
Fungal burden in livers (0.9 ± 0.2 x 105) and
spleens (0.3 ± 0.1 x 105) from TNF--depleted
mice were significantly increased (p < 0.01)
when compared with livers (0.1 ± 0.0 x 105) and
spleens (0.01 ± 0.0 x 105) from infected
control mice (p < 0.01). The data demonstrate
that TNF- is required for clearance of both primary and secondary
pulmonary infection with Hc. Moreover, the findings suggest that during
primary infection with Hc, the absence of TNF- results in an
unopposed fungal growth in lungs, whereas spleens and livers appear to
be less affected in their ability to restrict growth. On the other
hand, during secondary infection, neutralization of TNF-
consistently impaired clearance of Hc in each examined organ.
Neutralization of TNF- accelerates mortality in naive and immune
C57BL/6 mice following i.n. Hc infection
Next, we determined whether neutralization of TNF- would alter
mortality during pulmonary histoplasmosis. Naive mice were treated with
2 mg of anti-TNF- or rat IgG and infected i.n. with 2.5 x
106 Hc. All animals depleted of TNF- died by day 12
of infection, whereas all control mice survived up to 45 days
(Fig. 1
A).
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on survival
during secondary histoplasmosis. Neutralization of endogenous TNF-
resulted in an increased mortality; all anti-TNF--treated mice
succumbed by day 10 whereas control animals survived throughout the
observation period of 45 days (Fig. 1
B). Thus,
TNF- is required for resolution of primary and secondary pulmonary
histoplasmosis, and death is associated with elevated CFU in lungs in
naive and lungs, livers, and spleens in immune mice.
Lung cytokine production during primary and secondary infection
with Hc in TNF--depleted compared with control mice
We determined whether neutralization of TNF- altered protein
levels of cytokines known to be involved in the immune response to Hc
(5, 6, 8, 10). Previously unexposed or pre-exposed mice were treated
with either 2 mg of anti-TNF- mAb or rat IgG 2 h before
i.n. infection with 2.5 x 106 Hc yeasts. On days
3, 5, and 7 postinfection, lungs were removed and cytokine production
assayed in total lung homogenates. We chose to measure the cytokines
during the acute infection and before the onset of the moribund state
in mice given anti-TNF- mAb.
The production of IFN- in lungs of naive and immune control mice
demonstrated a sharp increase by day 5 of infection followed by a
decline at day 7. Naive mice depleted of TNF- exhibited a continuous
rise of IFN- levels reaching a sixfold difference compared with
controls by day 7 of infection (p < 0.001)
(Fig. 2A), whereas
IFN- in immune mice depleted of TNF- declined steadily (Fig. 2B). While lungs of immune control mice displayed
higher IFN- levels on day 5 (p < 0.05),
anti-TNF--treated animals exhibited statistically significant
higher levels on day 7 of infection (p <
0.001).
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C, IL-12 levels in naive infected
controls reached a maximum on day 3 and declined thereafter. In
contrast, levels in TNF--depleted mice rose between days 3 and 5 and
diminished on day 7. The levels of IL-12 in infected control mice were
three- to fourfold higher compared with anti-TNF--treated mice
on day 3 of infection (p < 0.005); this time
point was the only one in which there were significant differences
(Fig. 2C). During secondary infection, measurement of
IL-12 (Fig. 2D) revealed increased levels in lungs of
control mice at day 3 compared with TNF--depleted animals
(p < 0.01). On day 5, amounts of IL-12 were
elevated in TNF--neutralized animals compared with control mice
(p < 0.02). No differences were present at day
7 of secondary infection.
During primary infection, IL-4 and IL-10 levels (Fig. 2, E
and G) did not differ between controls and
TNF--deficient mice at each time point. Conversely, in secondary
histoplasmosis, amounts of IL-4 in TNF--depleted mice were
considerably increased (p < 0.001) on days 5
and 7 of infection when compared with controls (Fig. 2F). Likewise, the concentrations of IL-10 in lungs
of TNF--neutralized mice increased steadily and exceeded those of
controls (p < 0.01) on days 5 and 7
postinfection (Fig. 2H).
TNF- induces the production of GM-CSF, which in turn has been
implicated in the protective immune response against Hc by enhancing
the fungistatic activity of M (10, 15). We reasoned that depletion
of TNF- may alter GM-CSF levels and thus diminish the ability of
M to exert antifungal activity. As demonstrated in Figure 3A, protein levels of GM-CSF
in naive control mice were highest on day 3 of infection, and declined
modestly on days 5 and 7. In TNF--deficient mice, GM-CSF levels were
elevated on day 3; they decreased by day 5 and rose on day 7
postinfection. At this time point, the amount of GM-CSF was
significantly elevated when compared with infected controls
(p < 0.05). In secondary infection, assessment
of GM-CSF in lungs of infected controls revealed a modest decline over
7 days, whereas levels in TNF--depleted mice rose by day 5 of
reinfection and were significantly higher than those of control mice
(p < 0.001, Fig. 3B).
Although levels in anti-TNF- mAb-treated mice declined by day 7,
the amounts remained significantly elevated compared with control mice
(p < 0.001).
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exert opposing effects on M functions: the
former inhibits M activation, and the latter increases their state
of activation (16). We hypothesized that upon depletion of TNF-,
TGF-ß protein levels might be elevated in lungs of mice, and
therefore contribute to the loss of host resistance apparent in the
lungs. A significant difference between TNF--depleted and infected
control mice during primary infection was not detected at any time
point (Fig. 3C). In contrast, by day 5 postinfection
of secondary histoplasmosis, TGF-ß levels had increased sharply and
were approximately fivefold higher in TNF--depleted animals compared
with controls (p < 0.001), but levels had
declined in these mice by day 7 and were similar to those observed in
control mice (Fig. 3D).
In primary infection, IL-6 levels in lungs of controls and
TNF--depleted animals increased from days 3 to 5 and declined by day
7. No significant differences between the controls and TNF--depleted
mice were detected (Fig. 3E). In secondary infection,
protein concentrations of IL-6 in control lungs did not differ from
those measured in TNF--neutralized mice on day 3. Although amounts
of IL-6 did not change dramatically in control animals on days 5 and 7
postinfection, levels in mice depleted of TNF- had risen by day 5
and were significantly different when compared with controls on days 5
and 7 (p < 0.01, Fig. 3F).
Thus, primary Hc infection in TNF--neutralized mice is associated
with increased IFN- and GM-CSF levels, but not IL-4, IL-6, IL-10, or
TGF-ß. In contrast, administration of anti-TNF- altered levels
of several cytokines in lungs of mice with secondary Hc infection,
including those that are associated with a Th2-type response (IL-4 and
IL-10).
Histopathologic changes in lungs of TNF--neutralized naive and
immune mice
Histopathologic changes in lungs following treatment with
anti-TNF- mAb were examined on day 7 of infection.
TNF--neutralized and control mice exhibited a diffuse granulomatous
pneumonia with destruction of the lung parenchyma (Fig. 4, A and B,
respectively). There was moderate to severe peribronchiolar and
perivascular lymphoid cuffing with moderate debris in the bronchiolar
lumen. In TNF--neutralized mice, inflammatory regions were composed
of 48 ± 3% PMN, 21 ± 3% lymphocytes, and 32 ± 3%
M, whereas in infected controls 52 ± 1% PMN, 18 ± 2%
lymphocytes, and 32 ± 3% M were detected.
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-deficient
mice revealed a severe, diffuse granulomatous pneumonia with severe
perivascular lymphoid cuffing and marked destruction of alveoli (Fig. 4C). In contrast, lungs of control mice exhibited a
mild focal granulomatous infiltration of the alveoli with mild to
moderate perivascular lymphoid cuffing (Fig. 4D). In
areas of inflammation, the percentage of lymphocytes in infected
controls was 79 ± 9%; M, 14 ± 3%; and PMN, 7 ±
4%. In the tissue of TNF--depleted mice, lymphocytes composed
81 ± 11%, M-12 ± 5, and PMN-8 ± 3%.
Depletion of endogenous TNF- does not dramatically change the
inflammatory character in infected lungs in primary and secondary
histoplasmosis. However, the extent of the inflammatory response in
secondary histoplasmosis was substantially greater in TNF--depleted
mice, whereas it did not differ from controls during primary infection
with Hc, despite the fact that there was increased fungal burden.
FACS analysis of BAL cells from TNF--deficient mice vs controls
following primary or secondary infection with Hc
CD4+ cells are pivotal in controlling infection with
Hc, and lymphocytes constitute a major immune effector cell found in
lung alveoli (1, 4). To determine the effect of neutralization of
TNF- on inflammatory cells, lungs of infected, naive mice were
lavaged on day 7 of infection and cells analyzed by flow cytometry. PMN
(GR-1+) constituted the majority of cells in BAL in
both groups, composing up to 67 ± 11% (mean ± SD) of the
total cell population in TNF--depleted mice compared with 60 ±
7% of infected controls. The absolute numbers of PMN in BAL fluid from
TNF--depleted mice (3.4 ± 2.3 x 106)
differed (p = 0.02) from that of infected
controls (0.5 ± 0.2 x 106). The percentage of
both CD4+ (2 ± 1%) and CD8+ (1 ±
0.5%) cells in BAL fluid from TNF--depleted mice did not differ
from that of infected controls (CD4+, 2 ± 0.5% and
CD8+, 2 ± 0.5%). However, the absolute number of
CD4+ and CD8+ cells was increased in
anti-TNF--treated mice, since there was an increased number of
inflammatory cells present in BAL. In TNF--depleted mice, 4.8
± 4 x 103 BAL cells were CD4+ and
3.8 ± 3 x 103 were CD8+ compared
with 2.3 ± 2 x 103, CD4+ and
2.1 ± 2 x 103 CD8+, respectively,
in infected controls (p > 0.05). These data
demonstrate that administration of anti-TNF- mAb does not
significantly perturb the numbers of CD4+ or
CD8+ cells in BAL fluid of mice during primary infection
with Hc.
We next analyzed the composition of BAL cells in immune mice to
determine whether blockade of TNF- altered the numbers of
inflammatory cells during secondary histoplasmosis. On day 7
postinfection, lungs were lavaged and cells stained for GR1, CD4, and
CD8 and analyzed by FACS. The percentage of GR-1+ cells in
TNF--depleted mice was 17 ± 5% compared with 18 ± 5%
in infected controls. The absolute numbers in mice given
anti-TNF- (2.7 ± 1.6 x 105) was
significantly greater (p = 0.03) than that of
controls (0.9 ± 0.5 x 105). The percentage of
CD4+ cells was higher (60 ± 12%) in TNF--depleted
mice compared with infected controls (42 ± 17%), whereas the
percentage of CD8+ cells did not differ between the groups
(8 ± 4% in TNF--neutralized mice vs 10 ± 4% in
infected controls). The absolute number of both T cell subsets was
increased approximately two- to threefold (p <
0.05) between anti-TNF--treated mice (6.2 ± 3.0 x
105 for CD4+, and 0.8 ± 0.5 x
105 for CD8+) and rat IgG-treated mice
(2.5 ± 2.0 x 105 for CD4+ and
0.45 ± 0.1 x 105 for
CD8+).
In vitro growth inhibition of Hc following activation of alveolar
or peritoneal M with rmTNF- and rmIFN-
Neutralization of TNF- inhibits the microbicidal activity of
M against Listeria monocytogenes (17). In some models,
cooperation between IFN- and TNF- is essential to enhance M
function in a synergistic fashion (18, 19).
In vitro experiments were conducted to determine whether TNF- and
IFN- would act synergistically to augment the anti-Hc activity
of alveolar M. Cells from normal mice were cultured in the presence
of either LPS, TNF-, IFN- alone, or combinations thereof and
assayed for fungistatic activity against Hc yeasts. None of the factors
activated alveolar M to inhibit the intracellular growth of the
yeasts. As an example, in one of four experiments, exposure to IFN-,
LPS, or TNF- at 10 ng/ml increased [3H]leucine
incorporation of Hc by 23, 14, and 38%, respectively, rather than
diminish intracellular growth. Moreover, the admixture of IFN- plus
TNF- at 10 ng/ml each did not stimulate inhibition of Hc growth
within alveolar M and enhanced [3H]leucine
incorporation of Hc by 7%. IFN- (10 ng/ml) plus LPS (10 ng/ml)
caused a 15% reduction in Hc growth.
In contrast, resident peritoneal M from normal mice cocultured with
IFN- at a concentration of 10 ng/ml exhibited 78% inhibition of Hc
growth. Addition of TNF- or LPS did not increase the inhibitory
activity of peritoneal M. Fungistatic activity remained unchanged
with 68% inhibition for IFN- and LPS at 10 ng/ml and 51% for
IFN- and TNF- at 10 ng/ml. In contrast, culture of peritoneal
M with either LPS or TNF- (10 ng/ml) alone promoted growth of Hc
by 63 and 14%, respectively.
Effect of TNF- neutralization on iNOS expression in lungs and
RNI production by alveolar M during primary and secondary infection
with Hc
A major mechanism for the killing of intracellular pathogens is
the formation of nitric oxide (NO) (20). TNF- synergizes with
IFN- to stimulate M and enhance nitrogen intermediates that are
essential in the destruction of Leishmania major
(18, 21). Expression of iNOS protein can be correlated with generation
of NO (22). We sought to determine whether neutralization of TNF-
altered expression of iNOS in lungs of naive and immune mice infected
with Hc. The amount of iNOS protein in lungs was determined on day 7
postinfection. iNOS expression in lungs of rat IgG-treated mice
(174 ± 7.3 pixel density) did not differ
(p > 0.05) from that found in TNF--depleted
(165.7 ± 7.3) animals in primary histoplasmosis. Moreover, the
amount of iNOS was similar in TNF--neutralized (164.3 ± 8.6;
pixel density ± SD) and rat IgG-treated mice (164.5 ± 11.4)
in secondary infection with Hc.
Although Western blot analysis indicated that lungs of
TNF--deficient mice produced comparable quantities of iNOS, we
sought to measure RNI by alveolar M, since a recent report suggested
that expression of iNOS in lungs does not necessarily correlate with
production of NO (23). Alveolar M were obtained on days 3, 5, and 7
of primary and secondary Hc infection and cultured in endotoxin-free
media alone or stimulated in vitro with IFN- plus LPS or IFN-
plus TNF-. After 48 h, the supernatants were tested for the
presence of nitrite (Fig. 5,
A–D). Unstimulated M from rat IgG-treated and
TNF--depleted mice during primary infection demonstrated the release
of RNI, which ranged from 24 to 42 and 23 to 36 µM, respectively, in
one experiment (Fig. 5A), and 42 to 85 (controls) and
41 to 90 µM (TNF--depleted) in the second (Fig. 5B). In two experiments, RNI production by in
vitro-stimulated alveolar M from TNF--depleted mice was
strikingly less than that of control cells on days 5 and 7
postinfection. In fact, generation of RNI by these M declined over
time. In contrast, M from controls exhibited either a progressive
rise in RNI production with maximum levels at day 7 postinfection (Fig. 5A) or plateaued at days 5 and 7 of infection (Fig. 5B). To determine whether the weak generation of RNI
by M from TNF--depleted mice was caused by a lower number of
cells, we enumerated the percentage of M in BAL. The percentage of
M recovered from control and anti-TNF--treated mice ranged
from 23 to 38% and 23 to 32%, respectively, and thus cannot account
for the difference measured in RNI production.
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did not differ between
TNF--neutralized and control mice upon in vitro stimulation with LPS
and IFN- or IFN- and TNF- in two experiments (Fig. 5,
C and D). Interestingly, both groups
demonstrated a decline in RNI release at day 5 followed by an increase
at day 7 postinfection. The release of RNI in unstimulated alveolar
M from control and TNF--depleted mice ranged from 75 to 138 µM
and 79 to 140 µM, respectively. M constituted 21 to 34% in
control and 16 to 27% in anti-TNF--treated mice.
Concomitant neutralization of IL-4 and IL-10 abrogates mortality in
secondary histoplasmosis in mice lacking endogenous TNF-
The elevated protein concentrations of IL-4 and IL-10 during
secondary histoplasmosis prompted us to examine whether these cytokines
contributed in part to the increase in susceptibility of
TNF--depleted mice. Six weeks after i.n. infection with
104 yeast cells, groups of mice were depleted on the
day of secondary challenge with either 2 mg of anti-TNF-, or
treated with the same amount of anti-TNF- plus 2 mg of
anti-IL-4 and 1 mg anti-IL-10 mAb. Additional groups received
either TNF- mAb plus anti-IL-4 or anti-IL-10 mAb. As
demonstrated in Figure 6, all control
mice and mice depleted of IL-4 and IL-10 alone survived secondary
challenge over an observation period of 45 days. In contrast, all mice
depleted of TNF- alone or combined with either anti-IL-4 or
anti-IL-10 mAb succumbed to infection by day 11 postchallenge.
Concomitant neutralization of IL-4 and IL-10 rescued 75% of mice from
the inimical effects of anti-TNF- mAb treatment. Thus, depletion
of IL-4 and IL-10 reverses the deleterious effects of treatment with
anti-TNF- mAb and restores host resistance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during
primary and secondary pulmonary histoplasmosis. Depletion of TNF-
resulted in a high mortality rate of naive mice infected with Hc and
was associated with increased CFU in lungs at 1 wk postinfection.
Interestingly, fungus burden in livers and spleens of TNF--depleted
mice was not elevated when compared with infected controls at day 7.
These results contrast with the findings in mice administered
anti-IL-12 mAb or in IFN- knockout mice in which CFU in lungs,
livers, and spleens were considerably elevated at wk 1 of Hc infection.
These mice were challenged with the same inoculum size and the same
strain of Hc (6, 24). Thus, the influence of endogenous TNF- in
experimental Hc infection appears to be compartmentalized. Disparate
effects of endogenous TNF- in individual organs also have been
observed in experimental tuberculosis. In TNF--depleted mice
infected i.v. with Mycobacterium tuberculosis, pronounced
tissue necrosis was detected only in lungs and not in liver or spleen
(12). Taken together, these findings suggest the existence of a
differential requirement among visceral organs for endogenous TNF-
in inflammation and host resistance mechanisms.
Histopathologically, the components of the inflammatory response in
lung parenchyma did not differ between TNF--deficient and control
mice. Both groups exhibited granulomatous infiltration composed of
lymphocytes, M, and PMN. However, mice depleted of TNF-
manifested more extensive inflammation, most likely a consequence of
increased fungal burden. Prior work has suggested the necessity of
endogenous TNF- for the generation of granulomatous inflammation in
response to bacillus Calmette-Guérin (25) and to
Schistosoma mansoni eggs (26). Other reports, however, have
provided evidence that this cytokine is not universally required for
this type of inflammatory response. Mice infected with Brucella
abortus and depleted of TNF- form granulomas (27). Likewise,
mice lacking the TNF receptor p55 develop granulomas in response to
M. tuberculosis (12). Therefore, it is reasonable to
conclude that the necessity of this cytokine for the development of
granulomatous inflammation varies with the pathogen.
In experimental cryptococcosis, administration of
anti-TNF- mAb results in a marked perturbation in the
inflammatory response (14). CD4+ cells, PMN, and M were
sharply decreased in lungs. Our findings contrast with those of
cryptococcosis. In experimental Hc, the percentage of CD4+
and CD8+ cells, PMN, and M in BAL fluid did not differ
significantly between TNF--neutralized and infected control mice.
However, the absolute numbers of CD4+, CD8+,
and PMN were elevated over controls. This finding is surprising, since
TNF- is a prominent proinflammatory cytokine (28). In immune mice,
the elevated levels of the proinflammatory cytokine IL-6 may have
contributed to the increased recruitment of cells to the lungs.
However, this observation does not explain the findings in naive mice.
It is possible, therefore, that other cytokines stimulated egress of
inflammatory cells into the lungs of TNF--depleted mice. Thus, in
experimental cryptococcosis, TNF- regulates recruitment of
inflammatory cells, whereas in Hc infection, the contribution of this
cytokine as a stimulus for cellular infiltration appears to be less
prominent.
Depletion of TNF- was not associated with an impairment of IFN-
generation. In fact, lung levels of IFN- were markedly increased in
TNF--deficient mice. Hence, the presence of TNF- during pulmonary
histoplasmosis was not obligatory for the in vivo production of
IFN-. Although this cytokine is important for clearance of Hc, its
presence, even in elevated levels compared with controls, was not
sufficient for resolution of infection. We have demonstrated previously
that IFN- knockout mice succumb to pulmonary Hc infection although
high levels of TNF- are present in their lungs (24). Thus, it
appears that production IFN- and TNF- are independently regulated
in Hc infection and that both must be active in vivo for optimal
clearance.
Based on the in vivo observations, we endeavored to demonstrate that
exposure of alveolar M to TNF- and/or IFN- would activate
these cells to express anti-Hc activity. M incubated with either
cytokine alone or an admixture failed to manifest anti-Hc activity
although peritoneal M exhibited pronounced anti-Hc activity when
exposed to IFN- alone. The inability to demonstrate convincingly
that alveolar M exert anti-Hc activity in vitro when exposed to
both IFN- and TNF- suggests that yet another cytokine or
cytokines must be synthesized in vivo that lead to their activation. It
is unlikely to be GM-CSF, IL-6, TGF-ß, IL-4, or IL-10, since they
were produced in amounts equal to or greater than those found in
controls while mice succumbed to overwhelming histoplasmosis. Thus,
these cytokines failed to compensate for inadequate host resistance
mechanisms.
Another salient point is that much of the prior data has indicated that
cytokine activation of human or murine M results in fungistatic, not
fungicidal, activity (10, 29, 30). However, since mice with sufficient
amounts of both TNF- and IFN- sterilize tissues, actual killing
must be mediated by a cytokine whose production requires engagement of
both TNF- and IFN-.
Accumulated data indicate that NO is a potent killing mechanism for
several intracellular pathogens (20). Moreover, this nitrogen
intermediate has been implicated in the elimination of Hc (9). Since
TNF- contributes to the production of NO (31), we compared iNOS
expression in lungs of TNF--depleted mice to that of infected
controls. We hypothesized that the progression of infection in mice
receiving anti-cytokine mAb could be explained by alterations in
iNOS expression and, thus, NO production. However, we did not detect
any differences in iNOS expression in TNF--depleted and control
mice, during both primary and secondary histoplasmosis.
A recent report has suggested that iNOS expression does not necessarily
correlate with NO production (23). Therefore, we measured RNI
production by alveolar M in response to LPS plus IFN- or to
IFN- plus TNF-, both potent stimuli. The inability to control
infection was associated with a marked decrement in the production of
RNI by stimulated alveolar M from TNF--neutralized naive mice. In
addition, the failure of alveolar M from TNF--depleted mice to
produce RNI upon stimulation with IFN- plus TNF- ex vivo
indicates that the cells manifest a persistent defect in cytokine
responsiveness. These results suggest that the receptors for these
cytokines are dysfunctional or that signal transduction has been
perturbed. In contrast, RNI production was not altered during secondary
histoplasmosis, and these findings indicate that accelerated mortality
is not accompanied by decreased NO production.
Analysis of the cytokines detected in lungs of naive and immune
mice exposed to Hc revealed pronounced differences, particularly in the
production of IL-4 and IL-10. Whereas levels of these Th2-associated
cytokines in TNF--depleted mice were no different from controls
during primary infection, significant differences between immune mice
treated with anti-TNF- mAb and controls were noted.
Up-regulation in IL-4 and IL-10 production was not associated with
decrements in IFN- by immune mice depleted of endogenous TNF-. In
fact, IFN- levels in lungs of TNF--depleted mice re-infected with
Hc were higher than controls.
In some models of infectious diseases, high levels of IL-4 and/or IL-10
have been associated with progressive disease (32, 33). As an example,
unrestricted growth of L. major in susceptible mice is
accompanied by high production of IL-4 and low to nonexistent levels of
IFN- (34). As a corollary, neutralization of IL-4 cures mice
infected with L. major or ameliorates listeriosis in mice
lacking the IFN- receptor gene (35, 36). Furthermore, neutralization
of IL-4 during primary pulmonary infection with Hc abrogates mortality
in mice depleted of IL-12 (6).
Since the levels of IL-4 and IL-10 were elevated in immune
animals given anti-TNF- mAb, we sought to determine the in vivo
influence of these two cytokines on host resistance mechanisms in these
mice. Administration of either anti-IL-4 or anti-IL-10 mAb in
TNF--depleted mice did not restore protective immunity. However,
75% of TNF--neutralized mice given both mAb survived for 45 days.
Thus, blockade of both Th2-ssociated cytokines was necessary to
restore host resistance in mice treated with anti-TNF- mAb.
These results indicate that both of these cytokines contribute to the
down-regulation of protective immunity in immune mice deficient in
endogenous TNF-.
In secondary histoplasmosis, restoration of protective immunity
by treatment with anti-IL-4 and anti-IL-10 mAb in
TNF--depleted mice may seem surprising since the IFN- levels were
high. However, it can be more informative to examine the balance
between Th1 and Th2 cytokines than absolute values. In this regard, the
ratio of IFN- to IL-4 or to IL-10 was 1000 and 350, respectively, in
TNF--neutralized mice on day 7 of secondary infection. These values
were greater in TNF--depleted naive animals (IFN- to IL-4 =
1600 and IFN- to IL-10 = 1000). This finding also was true for
day 5 of infection. Although the relative increase in Th2 cytokines
between primary and secondary histoplasmosis may not appear markedly
dissimilar, it is possible that even slight differences in the ratio of
Th2 to Th1 cytokines impair protective immunity to Hc. Nevertheless,
IL-4 and IL-10 contribute to disease exacerbation in secondary
infection if TNF- is deficient. The importance of the concerted
action of IL-4 and IL-10 in disease exacerbation also has been reported
in an experimental model of L. donovani (37), although the
precise mechanisms by which these cytokines act to impair host defenses
are unknown.
Recently, it has been shown that administration of anti-TNF-
does not alter the course of a secondary systemic infection with Hc in
immunocompetent C57BL/6 mice (38). These results differ considerably
from the findings herein. Although the precise reasons for the
differences have not been determined, one of the most likely reasons is
that in our study Hc was delivered i.n. and in the other study it was
inoculated i.v. Thus, the route of exposure may have a significant
impact on the constituents that are required for a protective immune
response. It is also possible that Hc strain differences may be
involved, since two different isolates were employed in these
studies.
In summary, TNF- is pivotal in controlling primary and secondary
pulmonary histoplasmosis. Based on accumulated data, it is reasonable
to conclude that host resistance to Hc during primary infection
requires the presence of both IFN- and TNF-. Furthermore, in
primary infection, the mounting evidence is that at least one other
soluble mediator, if not more, must be involved in the activation of
M to express fungicidal activity. Alternatively, the lack of TNF-
during secondary infection with Hc biases the host to a Th2-type
response. The elevated levels of both IL-4 and IL-10 contribute to the
increased susceptibility of animals depleted of endogenous TNF-.
Thus, the mechanisms that underlie host susceptibility to Hc in the
absence of TNF- appear disparate and remain to be completely
elucidated.
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Acknowledgments |
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studies. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. George S. Deepe Jr., Department of Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0560. E-mail address:
3 Abbreviations used in this paper: Hc, Histoplasma capsulatum; M, macrophage; BAL, bronchoalveolar lavage; NO, nitric oxide; iNOS, inducible nitric oxide synthase; RNI, reactive nitrogen intermediate; PMN, neutrophils; i.n., intranasal; GM, granulocyte macrophage; rm, recombinant murine.
Received for publication September 24, 1997. Accepted for publication February 17, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Finkel-Jimenez, M. Wuthrich, T. Brandhorst, and B. S. Klein The WI-1 Adhesin Blocks Phagocyte TNF-{{alpha}} Production, Imparting Pathogenicity on Blastomyces dermatitidis J. Immunol., February 15, 2001; 166(4): 2665 - 2673. [Abstract] [Full Text] [PDF]
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F. J. Gomez, E. O. Woodward, R. Pilcher-Roberts, R. S. Gibbons, and G. S. Deepe Jr. V{{beta}}6+ and V{{beta}}4+ T Cells Exert Cooperative Activity in Clearance of Secondary Infection with Histoplasma capsulatum J. Immunol., February 15, 2001; 166(4): 2855 - 2862. [Abstract] [Full Text] [PDF]
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L. A. Gildea, R. E. Morris, and S. L. Newman Histoplasma capsulatum Yeasts Are Phagocytosed Via Very Late Antigen-5, Killed, and Processed for Antigen Presentation by Human Dendritic Cells J. Immunol., January 15, 2001; 166(2): 1049 - 1056. [Abstract] [Full Text] [PDF]
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R. Allendoerfer and G. S. Deepe Jr. Regulation of Infection with Histoplasma capsulatum by TNFR1 and -2 J. Immunol., September 1, 2000; 165(5): 2657 - 2664. [Abstract] [Full Text] [PDF]
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G. S. Deepe Jr., R. Gibbons, and E. Woodward Neutralization of Endogenous Granulocyte-Macrophage Colony-Stimulating Factor Subverts the Protective Immune Response to Histoplasma capsulatum J. Immunol., November 1, 1999; 163(9): 4985 - 4993. [Abstract] [Full Text] [PDF]
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Y. Rongrungruang and S. M. Levitz Interactions of Penicillium marneffei with Human Leukocytes In Vitro Infect. Immun., September 1, 1999; 67(9): 4732 - 4736. [Abstract] [Full Text] [PDF]
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R. Allendorfer, G. D. Brunner, and G. S. Deepe Jr. Complex Requirements for Nascent and Memory Immunity in Pulmonary Histoplasmosis J. Immunol., June 15, 1999; 162(12): 7389 - 7396. [Abstract] [Full Text] [PDF]
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