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Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de Sao Paulo, Sao Paulo, Brazil
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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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We have previously characterized an isogenic murine model in which polar forms of the disease were reproduced. The B10.A mouse strain was found to be susceptible while the A/Sn strain was extremely resistant to Pb infection. Resistant mice are able to control the infection, while susceptible animals develop a progressive disseminating disease with increased recovery of fungal cells from several organs (4). Although the mechanism(s) involved in resistance to Pb infection is poorly understood, it appears that macrophages are fundamental for protection, as mice with blockade of macrophage functions by colloidal carbon are extremely susceptible to infection (5).
A key antimicrobial molecule involved in the killing of intracellular
pathogens is NO, which is generated from the amino acid
L-arginine by the inducible isoform of NO synthase (iNOS or
NOS2). Although NO production is involved in Pb killing
(6), it can also be involved in immunosuppression, because
NO inhibits lymphoproliferation, TNF-
production, and MHC class II
molecule expression (7, 8).
We have previously shown that in susceptible (B10.A), but not in
resistant (A/Sn), mice, Pb infection is accompanied by suppression of
the Ab response to unrelated Ags and an impaired delayed-type
hypersensitivity reaction (9, 10). In the present work, we
selected these two polar mouse strains to ascertain by comparison which
of the macrophage-derived products could be associated with resistance
or susceptibility to Pb infection. We quantified the production of NO
and other macrophage-derived metabolites, such as hydrogen peroxide and
TNF-, in infected B10.A and A/Sn mice. We also determined the
macrophage expression of MHC class II molecule
(IAk), as these two strains are identical in the
IA region. In this study, we show that the major difference in
macrophage responses observed in these strains was related to NO and
TNF- production. Macrophages from susceptible mice released high and
persistent levels of NO throughout the infection, while macrophages
from resistant mice presented persistent TNF- production. Incubation
of resident macrophages from susceptible or resistant mice with Pb
resulted in NO or TNF production, respectively. TNF- production by
B10.A macrophages appeared to be suppressed by NO, because the addition
of an NOS2 inhibitor, aminoguanidine hemisulfate (AG), restored TNF-
production. These results indicate that NO might play a negative role
in Pb infection. However, B6 mice with NOS2 deficiency were more
susceptible to infection than NOS2-sufficient mice. Moreover, treatment
with AG during the first 10 days of infection resulted in increased
fungal load and more severe infection in both susceptible and resistant
animals.
Therefore, we conclude that NOS2-derived NO is essential to confer resistance to paracoccidioidomycosis regardless of the animal genetic background. However, high levels and persistent NO production are associated with susceptibility, indicating that in this situation NO plays a negative role in paracoccidioidomycosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse strains susceptible (B10.A) and resistant (A/Sn) to paracoccidioidomycosis were obtained from our Isogenic Breeding Unit (Department of Immunology, Instituto de Ciências Biomédicas, Universidade de Sao Paulo, Sao Paulo, Brazil). C57BL/6 NOS2-deficient and NOS2-sufficient mice were used in one set of experiments. Animals were age and sex matched, fed sterilized food and acidified water, and treated according to Instituto de Ciências Biomédicas, Universidade de Sao Paulo Animal Welfare guidelines.
Fungus
A highly virulent isolate of P. brasiliensis, designated Pb18, was used throughout this study. To ensure the maintenance of its virulence, the isolate was used after three serial animal passages. Pb18 yeast cells were then maintained by weekly subcultivation in semisolid Fava Netto’s culture medium at 35°C and used on the seventh day in culture (11). The yeasts were washed in PBS (pH 7.2) and counted in a hemocytometer. The viability of fungal suspensions, determined by Janus Green B vital dye (Merck, Darmstadt, Germany) (12), was always >80%.
Macrophages
Peritoneal exudate cells were obtained from control or infected mice by washing their peritoneal cavities with 5-ml ice-cold PBS. The cells from individual mice were centrifuged (160 x g, 10 min, 4°C), resuspended in complete RPMI 1640 medium, and adjusted to 2 x 106 cells/ml. Total and differential cell counts were performed on fixed and stained cell suspensions with 0.05% crystal violet dissolved in 3% acetic acid (final solution). Cell suspensions used in all experiments consisted of at least 85% macrophages. For culturing, 100-µl cell suspension was plated onto each well of 96-well flat-bottom tissue culture plates (Corning, Corning, NY), and cultures were incubated at 37°C in 5% CO2/95% air.
Experimental protocol
Mice were infected i.p. with 5 x 106 yeast cells of P. brasiliensis. Control animals received sterile PBS. The animals were sacrificed 1, 2, 3, or 4 wk postinfection (p.i.). For in vitro infection, yeast cells were adjusted to 2 x 106 cells/ml and opsonized with complement by incubation for 10 min at 37°C with 10% fresh mouse serum in RPMI medium (13). Thereafter, 10 µl of this suspension was added to 96-well macrophage cultures at a 1:10 ratio (Pb-macrophages). In preliminary experiments, we found that one yeast cell per macrophage induced cell death, while a 1:100 ratio was marginally effective in stimulating macrophages. Plates were incubated for 24, 48, and 72 h for determinations of NO and TNF levels.
AG hemisulfate treatment
To inhibit NO production, mice were given a 2.5% solution of AG (Sigma-Aldrich, St. Louis, MO) in sterilized drinking water for 10 consecutive days beginning 1 day before P. brasiliensis infection. In macrophage cultures, 1 mM AG was used (14).
Macrophage hydrogen peroxide (H2O2) release
H2O2 production was measured by the modified HRP-dependent phenol red oxidation microassay (15, 16). After peritoneal cells were harvested, as described earlier, the cell suspensions were centrifuged for 10 min at 150 x g and adjusted to 2 x 106 cells/ml in freshly prepared, ice-cold Dulbecco’s PBS containing 5.5 mM dextrose, 0.56 mM phenol red (Sigma-Aldrich), and 8.5 U/ml HRP type II (Sigma-Aldrich). One hundred microliters of the cell suspension was plated onto each well of a 96-well flat-bottom tissue culture plate (Corning), and 10 µl PMA (1 µg/ml; Sigma-Aldrich) was added to each well. The plates were incubated in humidified atmosphere at 37°C for 1 h. Thereafter, 10 µl 1 N NaOH was added to stop the reaction. The absorbance at 620 nm was determined with a microplate reader. The conversion of absorbance to micromolar concentrations of H2O2 was deduced from a standard curve. All determinations were performed in quadruplicate and expressed as micromolar concentrations of H2O2.
Macrophage MHC class II molecule (IAk) expression
IAk expression by the peritoneal macrophages was determined by cell ELISA using a mouse H-39.487.7 (IgG2a) anti-IAk (17) mAb immediately after cell harvesting. Briefly, 2 x 105 cells (100 µl/well) were incubated in PBS/10% FCS for 1 h; thereafter, when nonadherent cells, mainly lymphocytes, were washed off by three washes with PBS/5% FCS. The remaining adherent cells were fixed with 1% paraformaldehyde in PBS for 15 min, washed twice with PBS/0.05% Tween 20, and incubated for 30 min with 1 µg/ml biotinylated mAb anti-IAk. The bound Abs were revealed by ExtrAvidin-peroxidase (Sigma-Aldrich), followed by o-ortophenyldiamine (Sigma-Aldrich) in sodium citrate buffer (pH 5.0) and H2O2. The enzymatic reaction was stopped by the addition of 50 µl 4 N sulfuric acid. The absorbance at 492 nm was determined with a microplate reader. All determinations were performed in quadruplicate. The biotinylated anti-IAk was prepared by reacting 1 ml Ab in PBS (1 mg/ml) that was dialyzed at 4°C overnight against 0.2 M borate buffer (pH 8.5) with 100 ml N-hydroxysuccinimidobiotin in DMSO (4 mg/ml) for 4 h at room temperature, followed by overnight dialysis against PBS at 4°C (18).
Macrophage NO production
NO production was quantified by the accumulation of nitrite (as a stable end product) in the supernatants by the standard Griess reaction. Briefly, 50 µl of the supernatants were removed from 96-well plates and incubated with an equal volume of Griess reagent (1% sulfanilamide/0.1% naphthylene diamine dihydrocloride/2.5% H3PO4) at room temperature for 10 min. The absorbance at 550 nm was determined with a microplate reader. Conversion of absorbance to micromolar concentrations of NO was deduced from a standard curve using a known concentration of NaNO2 diluted in RPMI medium. All determinations were performed in quadruplicate and expressed as micromolar concentrations of NO.
TNF activity
TNF activity was evaluated by measuring TNF levels in supernatants from cultured macrophages or by direct macrophage cytotoxicity against L929 tumor cells as follows. TNF levels were determined by the standard L929 cytotoxic assay (19). Briefly, 100 µl of the diluted samples were pipetted into 96-well microtiter plates containing target cells (3.5 x 104/100 µl) in the presence of actinomycin D (2 µg/ml). The samples were incubated with the cells for 20 h at 37°C in 5% CO2. Thereafter, the remaining viable adherent cells were fixed and stained with 0.05% crystal violet and 3% acetic acid (final dilution) for 10 min. Excess stain was removed by rinsing with tap water, and the plate was air-dried. A volume of 100 µl absolute methanol was added to each well to dissolve the stain. The absorbance was read at 620 nm (Titertek Multiskan; Helsinki, Finland). Macrophage cytotoxicity was determined by coculturing L929 cells with different concentrations (10–105) of peritoneal macrophages for 4 h. Then the cocultures were treated with actinomycin D (2 µg/ml), and after 20 h of coculture the viable cells were fixed, stained, and processed as described earlier.
The TNF titer (units per milliliter) was defined as the reciprocal of the dilution that induced 50% L929 cell lysis. The macrophage cytolytic activity was determined as the percentage of tumor cytotoxicity, where the percent cytotoxicity = 1 - (OD of L929 cells cocultured with macrophages/OD of control L929 cells) x 100. One unit of TNF-dependent cell cytotoxicity was defined as the number of macrophages that induced 50% lysis of L929 cells. Cytotoxic activity was expressed as TNF units per 103 cells. The specificity of the reaction was evaluated in both assays by the addition of 20 µg/ml polyclonal anti-TNF Abs (Endogen, Cambridge, MA).
CFU assay
The number of viable micro-organisms in the lungs, liver, and spleen was determined by counting the number of CFU. Six to eight animals from each group were sacrificed at 4 wk p.i., and the enumeration of viable organisms in the three organs was performed as previously described (20). Briefly, aliquots (100 µl) of the cellular suspension of each organ were plated on brain-heart infusion agar (Difco, Detroit, MI) supplemented with 4% (v/v) horse serum (Instituto Butantan, Sao Paulo, Brazil) and 5% Pb192 culture filtrate, the latter constituting a source of growth-promoting factor. Plates were incubated at 35°C, and colonies were counted daily until no increase in counts was observed. The number of viable P. brasiliensis colonies per gram of tissue was expressed as the mean ± SEM.
Histopathologic analysis
Livers were removed from different groups of mice and fixed in 10% phosphate-buffered formalin for 24 h and then in 70% ethanol until embedding in paraffin. Tissues were sliced, and 5-µm sections were stained by H&E for light microscopic examination. The size (area) and number of granulomatous lesions in the liver were quantified by histocytometry using an image analyzer (BioScan/OPTIMAS; Media Cybernetics, Silver Spring, MD). Values were expressed as the mean ± SEM of triplicate sections.
Statistical analysis
The data showing normal distribution were submitted to Student’s t test when comparing two groups or ANOVA for more than two groups. The fungal load data were analyzed using the Mann-Whitney U test. All analyses were made using the Instat Program (GraphPad software, San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As TNF is considered a key molecule in granuloma formation
(21), we evaluated the capacity of macrophages from
infected mice to release this cytokine. Fig. 1
A shows
that TNF activity was not detected in supernatants obtained from
macrophage cultures of susceptible mice. Conversely, macrophages
obtained from resistant mice released significant levels of TNF at all
time periods examined (Fig. 1A). To further explore the
TNF activity, we cocultured macrophages from infected mice with L929
tumor cells and determined the tumor cell cytotoxicity. As shown in
Fig. 1B, macrophages from susceptible mice did not exhibit
TNF-dependent cell cytotoxicity, while macrophages from resistant mice
were able to kill L929 tumor cells at 1, 3, and 4 wk p.i. These results
document that TNF production is impaired in susceptible mice.
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It is known that TNF is involved in the generation of NO, whereas
NO may suppress TNF production (7). Thus, we next
determined NO production by macrophages from susceptible and resistant
mice. Strikingly, susceptible (B10.A) macrophages presented persistent
NO production, while NO production by resistant (A/Sn) macrophages was
only detected at 1 wk p.i. (Fig. 2).
However, at this time point, the NO levels of susceptible animals were
significantly higher (
2-fold) than those of resistant mice.
Moreover, except for wk 2 p.i., NO release by susceptible
macrophages was prominent (>35 µM), while NO release by resistant
mice was <20 µM (Fig. 2). Thus, it is apparent that persistent and
exacerbated NO production is associated with susceptibility.
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A previous study indicated that Pb infection inhibited MHC class
II Ag expression (8). As susceptible and resistant animals
in our experimental model share the same IAk
molecule in MHC class II complex, we could compare macrophage
IAk expression during Pb infection in these mouse
strains. Fig. 3A shows that
during Pb infection IAk expression increased in
both mouse strains except at 4 wk p.i., when IAk
expression of A/Sn mice returned to control levels. Surprisingly, the
expression of IAk molecule by macrophages from
B10.A mice was higher than that presented by A/Sn animals, except at 2
wk p.i. (Fig. 3A). These results indicate that macrophage
IAk expression is not impaired in susceptible
animals.
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B shows that macrophage
H2O2 production reached
maximum levels at 1 wk p.i., decreased at 2 wk, and reached control
values at 3 wk p.i. Although
H2O2 production was
significantly higher in resistant mice at 1 wk p.i., macrophages from
susceptible mice produced significant amounts of
H2O2, indicating that the
difference in H2O2
production by these strains might not influence the outcome of
infection. Differences in NO and TNF production by susceptible and resistant macrophages after infection with P. brasiliensis in vitro
Data obtained in vivo indicated that NO and TNF
production is polarized when comparing susceptible and resistant mice.
To further investigate whether this polarization could also be
reproduced in macrophage cultures, we established an in vitro infection
model. Resident macrophages were infected with yeast cells, and TNF and
NO levels in supernatants were quantified at 24, 48, and 72 h of
coculture. As shown in Fig. 4A, significant NO production
by susceptible (B10.A) macrophages was apparent after 24 h and
persisted thereafter, whereas NO production by resistant (A/Sn)
macrophages was <1 µM at all time points examined. Regarding TNF
production, Pb-infected macrophages from both mouse strains released
detectable levels of TNF at 48 and 72 h, but not at 24 h, of
culture (Fig. 4B). However, the production of TNF by
resistant macrophages was significantly higher (5- and 3-fold) than
that of susceptible animals at 48 and 72 h, respectively (Fig. 4B). Thus, the macrophage-polarized responses were also
reproduced in vitro.
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To identify the isoenzyme responsible for NO production in
susceptible macrophages and to ascertain whether NO might have a
suppressive effect on TNF production (7), we added AG, a
more selective NOS2 substrate inhibitor (22), to
Pb-infected macrophage cultures. As shown in Fig. 5, the addition of AG blocked NO
production (A) and concomitantly increased TNF release
substantially (B). These results indicate that NOS2-derived
NO inhibits TNF production and suggest that NO might have a deleterious
effect on Pb infection.
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To evaluate the role played by NOS2 in vivo, we compared the
outcome of infection of B6 NOS2-deficient with B6 control mice.
Infected animals were analyzed at 4 wk p.i. In line with the results
obtained with B10.A mice, macrophages from Pb-infected B6 mice released
significant levels of NO, while, as expected, NO production was absent
in NOS2-deficient mice (Fig. 6A). We next determined the
fungal load (monitored by CFU counts) in lungs, liver, and spleen. Fig. 6B shows that NOS2-deficient mice are more susceptible to Pb
infection, as CFU counts of lungs, spleen, and liver were,
respectively, 2-, 3-, and 4-fold higher than those in B6 mice. The
increased susceptibility of NOS2-deficient mice was also apparent when
the number and size of granulomatous lesions were determined. As shown
in Fig. 6, C and D, the number and size of
granulomas in liver were roughly 2- and 4-fold higher in NOS2-deficient
mice than in NOS2-sufficient mice. Thus, NOS2-derived NO appears to be
essential for the control of fungal growth.
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Our results indicated that either persistent NOS2 activity seen in
B10.A mice or persistent NOS2 deficiency appears to be associated with
increased susceptibility to Pb infection. To elucidate this apparent
paradox, we asked whether transient inhibition of NOS2-derived NO would
have a beneficial effect on Pb infection. To test this hypothesis,
susceptible mice were treated with 2.5% AG solution in drinking water
for 10 consecutive days beginning 1 day before Pb infection. In
parallel experiments, resistant mice received a similar treatment. At 4
wk p.i., the fungal loads of lungs and liver were evaluated. As shown
in Fig. 7A, transient
inhibition of NO production in B10.A mice resulted in a greater fungal
load in these organs compared with nontreated mice. In resistant mice,
AG treatment also increased the fungal load compared with nontreated
animals (Fig. 7B). However, the fungal load of B10.A mice
was significantly higher than that of A/Sn mice when comparing
nontreated or AG-treated animals (Fig. 7, A and
B). Transient inhibition of NOS2 in Pb-infected B10.A
animals resulted in higher NO production at 4 wk p.i. than that in
nontreated animals (Fig. 7C). Interestingly, although AG
treatment increased the fungal load of resistant mice, the macrophage
NO production was still very low and did not differ significantly from
that of nontreated animals. Finally, the effect of AG treatment on Pb
infection in both mouse strains was evaluated by histology (Fig. 8, A–C) and histocytometry
(Fig. 8, E–F) of liver sections. Liver sections from
nontreated susceptible mice presented small granulomatous lesions
(roughly two lesions per 10 mm2), while in
resistant mice no tissue lesions were observed (Fig. 8, A
and B, and E and F). Conversely, after
AG treatment, the number and size of tissue lesions of susceptible mice
increased drastically (25 granulomas/10 mm2 with
a mean area of 0.8 mm2) as shown in Fig. 8, C, E, and F. Although in resistant
mice AG treatment resulted in fungal dissemination to the liver, both
the number and the size of granulomas were significantly lower (three
granulomas per 10 mm2) than in susceptible mice
(Fig. 8, B, E, and F). The above
results indicate that transient suppression of NOS2-derived NO
production during early infection increases the severity of the
disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regarding APC function it will be of interest to ascertain whether the
Ag presentation of freshly explanted macrophages from susceptible mice
is different from that of resistant mice.
We have previously shown that heat-killed yeast cells were as
potent as PMA in triggering macrophage
H2O2 release
(26). This fact suggests that fungal cells might actively
induce the release of reactive oxygen intermediates at inflammatory
sites. Association between reactive oxygen intermediates release and
resistance to infection has been observed with other parasites. For
instance, H2O2 release by
macrophages, following in vivo challenge with BCG, was higher in
resistant than in susceptible animals (27). Furthermore,
macrophages from resistant mice infected with Leishmania
donovani were shown to be superior
H2O2 producers, following
IFN- treatment, than susceptible macrophages (28).
However, in Pb infection, fungal killing seems to be independent of
H2O2 production
(29). In the present work, we demonstrated that the
resistance to Pb infection is not directly correlated with
H2O2 levels, because its
production was almost similar in both mouse strains. Moreover, we could
not detect any significant killing when yeast cells were incubated with
different concentrations of
H2O2 and HRP (data not
shown).
Accumulating evidence points to TNF and/or NO as major molecules involved in resistance to infections by bacteria (Mycobacterium tuberculosis and Salmonella spp.), protozoa (Leishmania major, Plasmodium chabaudi, Toxoplasma gondii, and Trypanosoma cruzi) and helminthes (Schistosoma mansoni) (24, 30 ; reviewed in Refs. 31 and 32). Regarding Pb mycosis, TNF could participate in granuloma formation that may restrict fungal dissemination (23), while NO might participate in the killing process (6) or in down-regulation of immune responses (7). The fact that we used mouse strains with polarized phenotypes to Pb infection allowed us to test these possibilities. We found that the most striking difference between susceptible and resistant mice was related to NO and TNF production. Macrophages from susceptible mice produced prominent and persistent NO levels throughout the infection, while in resistant mice, Pb infection elicited a low and transient NO production at 1 wk p.i. that was terminated at 2 wk p.i. Conversely, TNF production predominated in resistant animals, but was almost absent in susceptible animals. Strikingly, similar results were obtained when peritoneal cells from resistant or susceptible mice were infected in vitro with Pb. Thus, the differential phenotypic expression of susceptible and resistant macrophages is established during the initial contact of Pb with the host cells. We found that the addition of AG, an NOS2 inhibitor, to macrophage cultures of susceptible mice resulted in significant TNF production, indicating that NO was down-regulating this cytokine. Our results are in consonance with a previous report showing that during Pb infection, inhibition of NO production increased TNF production (7). Similarly, AG treatment increased TNF release of peripheral blood monocytes obtained from premenopausal women (33). We suggest that high and persistent NO production might be detrimental to the host, probably due to inhibition of TNF production. Our results clearly indicate that macrophage TNF production is associated with resistance to Pb infection. Indeed, TNF receptor-deficient mice are extremely susceptible to Pb infection (23).
To further define the role of the NOS2 enzyme in Pb infection, we examined the outcome of infection in B6 mice that are genetically NOS2 deficient. It should be noted that B6 mice are relatively more susceptible than resistant to Pb infection (4). Our results showed that NOS2-deficient mice were more susceptible to Pb infection, as revealed by extensive granulomatous lesions in lungs and in the parenchyma of liver in these animals. Thus, it is clear that NO plays a fundamental role in the effector mechanism underlying resistance.
As NOS2 deficiency permanently impairs NO production, we next examined
the effect of transient inhibition of NOS2 activity in susceptible or
resistant mice. For this, we treated the animals with AG starting 1 day
before infection and continued the treatment for 9 consecutive
days. At 4 wk p.i. it was found that in both mouse strains, the
transient NOS2 inhibition exacerbated the infection, i.e., the
infection disseminated extensively to lungs and liver. These data
suggest that regardless of the mouse strain, NO plays an essential role
in the control of fungal dissemination. Our results confirm previous
reports showing that NO is able to kill fungus and hence control
parasite multiplication (6, 7). In addition, we show that
paradoxically, macrophages from susceptible mice produce very high
levels of NO. It is likely that this high and sustained NO production
may be involved in immunosuppression. Indeed, this "two-edged
sword" nature of NO (protection vs immunosuppression) has been
described in Salmonella, Toxoplasma, and
Trypanosoma infections as well as in tumors (32, 34, 35). We have previously shown that Pb infection in susceptible
mice leads to immunosuppression of humoral and cellular responses
(9). More recently, it was found that the low
lymphoproliferative response of B6 mice infected with Pb was prevented
by treatment with
N
-nitro-L-arginine,
an inhibitor of NOS1 and NOS2, during infection (7). It is
noteworthy that even after AG treatment, the differences between
susceptible and resistant mice were still apparent. It will be of
interest to determine whether the relative difference between
susceptible and resistant mice will decrease after prolonged treatment
with AG.
NO production by B10.A macrophages was higher in AG-treated animals than in nontreated animals, while TNF production was not detected. As transient AG treatment increased fungal multiplication at 4 wk of infection, it is possible that the increased fungal load stimulated macrophages to produce more NO. These results indicate that transient inhibition of NOS2 activity quantitatively altered NO production but did not qualitatively change the macrophage responses of susceptible mice. It is noteworthy that the augmented NO levels observed in AG-treated mice did not result in enhanced resistance to Pb infection. We have recently shown that NO is necessary, but not sufficient, to induce macrophage leishmanicidal activity, because the addition of PGE2 to macrophages infected with Leishmania amazonensis, increased NO production, but abolished macrophage leishmanicidal activity (36).
Collectively, our results indicate that macrophage responses are genetically determined and that distinct phenotypic patterns of responses, mainly related to NO and TNF production, are established during the infection. Moreover, although NO derived from NOS2 activity is critical for the control of fungal multiplication in susceptible and resistant mice, high and persistent NO production is the hallmark of susceptibility, while persistent TNF production is associated with resistance to Pb infection. It seems that in susceptible animals, NO production can be viewed in two ways; it controls fungal multiplication, but at the same time inhibits TNF production, which is also essential for the control of fungal dissemination (23). This interpretation is in line with the idea that NO may exert positive and negative effects on the regulation of host defense (32, 37).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Momtchilo Russo, Departamento de Imunologia, Instituto de Ciências Biomédicas IV, Universidade de Sao Paulo, Avenida Prof. Lineu Prestes 1730, CEP 05508-900, Sao Paulo SP, Brazil. E-mail address: momrusso{at}icb.usp.br
3 Abbreviations used in this paper: Pb, Paracoccidioides brasiliensis; AG, aminoguanidine; KO, knockout; NOS, NO synthase; p.i., postinfection.
Received for publication November 5, 2001. Accepted for publication February 20, 2002.
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 C. Arruda, R. C. Valente-Ferreira, A. Pina, S. S. Kashino, R. A. Fazioli, C. A. C. Vaz, M. F. Franco, A. C. Keller, and V. L. G. Calich Dual Role of Interleukin-4 (IL-4) in Pulmonary Paracoccidioidomycosis: Endogenous IL-4 Can Induce Protection or Exacerbation of Disease Depending on the Host Genetic Pattern Infect. Immun., July 1, 2004; 72(7): 3932 - 3940. [Abstract] [Full Text] [PDF]
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A. Pina, R. C. Valente-Ferreira, E. E. W. Molinari-Madlum, C. A. C. Vaz, A. C. Keller, and V. L. G. Calich Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with Impaired Th2 Response Infect. Immun., April 1, 2004; 72(4): 2369 - 2378. [Abstract] [Full Text] [PDF]
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 M.C. Fornazim, A. Balthazar, R. Quagliato Jr, R.L. Mamoni, C. Garcia, and M.H.S.L. Blotta Evaluation of bronchoalveolar cells in pulmonary paracoccidioidomycosis Eur. Respir. J., December 1, 2003; 22(6): 895 - 899. [Abstract] [Full Text] [PDF]
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D. Rodriguez, A. C. Keller, E. L. Faquim-Mauro, M. S. de Macedo, F. Q. Cunha, J. Lefort, B. B. Vargaftig, and M. Russo Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity J. Immunol., July 15, 2003; 171(2): 1001 - 1008. [Abstract] [Full Text] [PDF]
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