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Originally published online as doi:10.1189/jlb.1107738 on February 15, 2008

Published online before print February 15, 2008
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(Journal of Leukocyte Biology. 2008;83:1088-1099.)
© 2008 by Society for Leukocyte Biology

Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection

Adriana Pina*,{dagger}, Simone Bernardino* and Vera L. G. Calich*,1

* Departamento de Imunologia do Instituto de Ciências Biomédicas and
{dagger} Departamento de Análises Clínicas da Faculdade de Ciências Farmacêuticas da Universidade de São Paulo, São Paulo, Brazil

1Correspondence: Departamento de Imunologia, Instituto de Ciências Biomédicas da Universidade de São Paulo, Av. Prof. Lineu Prestes 1730, CEP 05508-900, São Paulo, SP, Brazil. E-mail: vlcalich{at}icb.usp.br

ABSTRACT

Alveolar macrophages (AM) are the first host cells to interact with Paracoccidioides brasiliensis (Pb), a primary human pathogen that causes severe pulmonary infections in Latin America. To better understand innate immunity in pulmonary paracoccidioidomycosis, we decided to study the fungicidal and secretory abilities of AM from resistant (A/J) and susceptible (B10.A) mice to infection. Untreated, IFN-{gamma} and IL-12 primed AM from B10.A and A/J mice were challenged with P. brasiliensis yeasts and cocultured for 72 h. B10.A macrophages presented an efficient fungicidal ability, were easily activated by both cytokines, produced high levels of nitric oxide (NO), IL-12, and MCP-1 associated with low amounts of IL-10 and GM-CSF. In contrast, A/J AM showed impaired cytokine activation and fungal killing, secreted high levels of IL-10 and GM-CSF but low concentrations of NO, IL-12, and MCP-1. The fungicidal ability of B10.A but not of A/J macrophages was diminished by aminoguanidine treatment, although only the neutralization of TGF-β restored the fungicidal activity of A/J cells. This pattern of macrophage activation resulted in high expression of MHC class II antigens by A/J cells, while B10.A macrophages expressed elevated levels of CD40. Unexpectedly, our results demonstrated that susceptibility to a fungal pathogen can be associated with an efficient innate immunity, while a deficient innate response can ultimately favor the development of a resistant pattern to infection. Moreover, our data suggest that different pathogen recognition receptors are used by resistant and susceptible hosts to interact with P. brasiliensis yeasts, resulting in divergent antigen presentation, acquired immunity, and disease outcomes.

Key Words: Paracoccidioidomycosis • fungicidal activity • innate immunity • fungal infection • nitric oxide • cytokines

INTRODUCTION

Paracoccidioides brasiliensis, a primary fungal pathogen from Latin America, infects individuals from endemic areas via respiratory route. Inhaled fungal spores or particles reach alveolar spaces, where they are ingested by resident macrophages [1 , 2 ]. This initial interaction appears to govern the subsequent mechanisms of innate and adaptative immunity, which result in localized infection or overt disease. Paracoccidioidomycosis (PCM) infection has been characterized as the Th1 pole of the immune response since healthy infected individuals present positive delayed-type hypersensitivity reactions (DTH) and their antigen-stimulated lymphocytes secrete IL-2 and IFN-{gamma}. The juvenile or acute form of the disease affects young adults of both sexes, is usually severe, and induces profound anergy of DTH reactions. Their lymphocytes behave as Th2 cells due to the enhanced secretion of IL-4, IL-5, and IL-10 [3 , 4 ]. The most common form of PCM, the adult form, has a chronic evolution and preferentially affects male individuals. The chronic mild cases are more frequently associated with a Th1-skewed pattern of immunity, whereas a more prominent Th2 profile in observed in the most severe cases [3 4 5 ].

We described an experimental model of pulmonary PCM where A/J and B10.A mice behaved as resistant and susceptible hosts to P. brasiliensis infection, respectively [6 ]. The progressive and disseminated disease of B10.A mice was associated with DTH anergy, absence of macrophage activation, and high fungal loads in nonorganized lesions mimicking the severe forms of the human disease. On the contrary, A/J mice presented a regressive disease with well-organized lesions containing a low number of yeasts, positive cellular immunity, and macrophage activation, resembling the benign forms of PCM [6 ]. Studies on pulmonary cytokines demonstrated that both mouse strains are able to secrete proinflammatory and anti-inflammatory cytokines, but higher levels of IFN-{gamma}, IL-5, and IL-10 were present in the lungs of susceptible mice [7 , 8 ]. Using gene knockout or cytokine-depleted mice, protective immunity to Pb infection was shown to be mediated by IFN-{gamma}, IL-12, and TNF-{alpha} [9 10 11 12 ], all macrophage-activating cytokines. Studies on the main T cell subsets involved in protective immunity to P. brasiliensis infection demonstrated that susceptibility of B10.A mice is associated with CD4+ T cell anergy and prevalent CD8+ T cell response. In resistant mice, immunity is mediated by CD4 and CD8 T cells, which secrete a mixed pattern of type 1 and type 2 cytokines [13 ].

Macrophages participate in the innate and acquired phases of immunity, and their activation is fundamental to control pathogen growth. Furthermore, as tissue macrophages exist in higher numbers than dendritic cells, they can exert an important regulatory role of innate immunity. Normal macrophages of mice are permissive to P. brasiliensis growth, while cytokine-activated macrophages are able to restrain P. brasiliensis multiplication (1). The fungicidal activity of macrophages was shown to be mediated by nitric oxide production as well as by the depletion of fundamental metabolites for fungal growth [14 15 16 17 ]. Despite its involvement in fungal killing, NO secretion was also clearly associated with suppression of T cell immunity [12 , 18 ].

In a previous work, we analyzed the behavior of alveolar macrophages at the adaptative phase of immunity of susceptible and resistant mice infected i.t. with 1 million yeasts. No hydrogen peroxide production was detected with cells from susceptible mice, whereas macrophages from resistant mice produced high levels of this metabolite [6 ]. These different activities paralleled the DTH anergy and the evident cutaneous reactivity developed by susceptible and resistant mice, respectively. As alveoloar macrophages (AM) were not previously studied at the innate phase of pulmonary infection, we have undertaken studies to characterize the secretory and fungicidal abilities of these cells upon P. brasiliensis infection. Unexpectedly, AM from susceptible mice presented a better control of P. brasiliensis growth than those of resistant hosts. The enhanced fungicidal ability of B10.A macrophages was associated with elevated secretion of NO and IL-12 and could be abolished by NO inhibition. On the contrary, A/J macrophages secreted high levels of IL-10 and GM-CSF, produced low amounts of NO, and presented a negligible microbicidal activity that was reverted by neutralization of endogenous TGF-β. In addition, P. brasiliensis infection of B10.A macrophages induced enhanced expression of CD40, whereas A/J macrophages expressed high levels of MHC class II molecules. This diverging pattern of macrophage activation appears to exert a profound influence in antigen presentation and in the subsequent adaptative immunity developed by resistant and susceptible mice.

MATERIALS AND METHODS

MICE
Susceptible (B10.A) and resistant (A/J) mouse strains to P. brasiliensis infection were obtained from our Isogenic Breeding Unit (Departmento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil) and used at 8 to 11 weeks of age. SPF mice were fed sterilized laboratory chow and water ad libitum. The experiments were approved by the ethics committee on animal experiments of Institute of Biomedical Sciences of University of São Paulo.

Fungus
P. brasiliensis Pb18, a highly virulent isolate [19 ], was used throughout this investigation. To ensure the maintenance of its virulence, the isolate was used after three animal passages [8 ]. Pb18 yeast cells were then maintained by weekly subcultivation in semisolid Fava Netto’s culture medium [20 ] at 35°C and used on day 7 after culture. The yeast cells were washed in phosphate-buffered saline (PBS) (pH 7.2) and adjusted to 20 x 106 cells/ml based on hemocytometer counts. Viability was determined with Janus Green B vital dye (Merck, Darmstadt, Germany) and was always higher than 80%.

Alveolar macrophages
Normal B10.A and A/J mice were lavaged after the canulation of trachea with polyethylene tubing, which was attached on a tuberculin syringe. The lungs were lavaged by repeated injections of 0.5 ml of sterile PBS (final volume, 2.0 ml). The bronchoalveolar lavage fluids (BALFs) were centrifuged, and pellets were resuspended in RPMI containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ìg/ml streptomycin. Cell count and viability were determined by hemocytometer and trypan blue exclusion (Sigma Chemical Co., St. Louis, MO, USA). AM were isolated by adherence (2 h at 37°C in 5% CO2) to plastic-bottom tissue-culture plates (2x105 cells/well in 96-well plates for fungicidal assays) or plated onto 13-mm-round glass cover slips (4x105 cells/slip in 24-well plates) for phagocytosis. Thereafter, macrophages were washed twice with PBS to remove nonadherent cells and cultivated overnight with fresh complete media RPMI 1640 with 10% fetal bovine serum in the presence or absence of three different concentrations of recombinant IFN-{gamma}, IL-12, or both cytokines (50, 10, or 2 ng/ml in culture medium, PharMingen, San Diego, CA, USA). For TGF-β measurements, AM were cultivated in serum-free medium (Sigma) to avoid the basal levels of this cytokine usually present in calf sera. In selected experiments, cytokine-treated and -untreated-macrophages were cultivated in the presence of 10 or 20 µg of 2A5 anti-IL-10 monoclonal antibodies or 20 µg of polyclonal neutralizing antibodies against TGF-β, (R&D Systems, Minneapolis, MN, USA). Cultures were also treated with 1 mM aminoguanidine (AG; Sigma-Aldrich), an inhibitor of inducible nitric oxide synthase (iNOS). All compounds were also added to the wells following the washing steps

Phagocytosis
Macrophage cultures were infected with P. brasiliensis yeasts at a yeast:macrophage ratio of 1:50. The cells were incubated with yeasts for 2 h at 37°C in 5% CO2 to allow fungi adhesion and ingestion. Media were then removed, cells were washed twice with PBS to remove any noningested or nonadhered yeasts, and samples were processed for microscopy. Cells were fixed with methanol and stained with Giemsa (Sigma). Experimental conditions were performed in triplicate, and the number of phagocytosed or adhered yeasts per 200 AM was counted on at least three separate slides.

Fungicidal activity of alveolar macrophages
Cytokine-primed and unprimed macrophage cultures were infected with P. brasiliensis yeasts as above described. After 2 h of interaction, cells were washed twice with PBS to remove any noningested, nonadhered yeasts and fresh medium with or without cytokines was added. After 72 h of culture at 37°C in a CO2 incubator, plates were centrifuged (400 g, 10 min, 4°C), and supernatants were stored at –70°C and further analyzed for the presence of nitrite and cytokines. The wells were washed 5 times with 0.5 ml of distilled water to lyse macrophages, and suspensions were collected in individual tubes. Cell homogenates were centrifuged and resuspended in culture medium; aliquots (100 µl) were assayed for the presence of viable yeasts. All assays were done with three wells per condition in more than three independent experiments. Fungicidal activity was calculated according the following formula: [100 – (experimental CFUx100/ control CFU), where CFU is colony forming units.

Assay for CFU
Coculture homogenates (100 µl) were plated on brain heart infusion agar (Difco, San Diego, CA, USA), which contained 4% (vol/vol) normal horse serum (Instituto Butantan, São Paulo, Brazil) and 5% P. brasiliensis 192 culture filtrate, the latter constituting the source of growth-promoting factor [21 ]. When necessary, dilutions were made in sterile PBS. The plates were incubated at 35 C, and colonies were counted daily until no increase in counts was observed. The numbers (log10) of viable P. brasiliensis per gram of tissue are expressed as the means ± SE.

Measurement of cytokines
Supernatants were separated from cell debris by centrifugation at 2,000 g for 15 min, passed through 0.22-µm-pore-size filters (Millipore, Bedford, MA, USA), and stored at –70°C. The levels of IL-12, IL-10, GM-CSF, TGF-β, and MCP-1, were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from PharMingen. Latent plus active TGF-β was measured using commercially available kits from R&D Systems. The ELISA procedure was performed according to the manufacturer’s protocol, and absorbances were measured with Versa Max Microplate Reader (Molecular Devices). The concentrations of cytokines were determined with reference to a standard curve for serial twofold dilutions of murine recombinant cytokines.

Flow cytofluorometry
P. brasiliensis yeasts were cocultivated with IFN-{gamma}-primed (10 ng/ml) or unprimed B10.A and A/J macrophages for 72 h in 24-well culture plates. Supernatants were removed, cultures were washed, and macrophages were detached from plastic with fresh cold medium and a rubber cell scraper. Cells were adjusted to 5 x 105 viable cells/ml and stained with phycoerythrin-conjugated anti-CD40 and fluorescein isothiocyanate-labeled anti-IAK monoclonal antibodies (PharMingen). The stained cells were analyzed immediately on a FACScan equipment using the PC-Lysys software (Becton and Dickinson, San Jose, CA, USA), gating on macrophages, as judged from forward and side light scatter. Ten thousand macrophages were counted, and the data were expressed as the percentage of CD40 or IAK-positive cells.

Statistical analysis
Data were expressed as means ± SEM and analyzed by Student’s t test or two-way ANOVA depending on the number of experimental groups using GraphPad Prism ver. 3 for Windows (GraphPad Software). P values under 0.05 were considered significant.

RESULTS

Alveolar macrophages from susceptible mice have a more efficient fungicidal ability than those from resistant mice
Before performing fungicidal studies, we asked whether the initial interaction between P. brasiliensis yeasts and alveolar macrophages from B10.A and A/J mice was equivalent. Macrophages cultures (2x105/well), performed in round glass coverslips, were preactivated or not with IFN-{gamma} (10 ng/ml) and infected with 4 x 103 viable yeasts (1:50 fungus:macrophage ratio). After 2 h of incubation, supernatants were aspirated, the monolayers were gently washed with PBS, and the cells were stained with Giemsa. An average of 200 macrophages was counted, and the number of ingested and adherent yeasts was determined. No significant differences were found between B10.A and A/J macrophages (Fig. 1 ). Additional experiments were performed using another yeast:macrophage ratio (1:12) and, again, no differences between B10.A and A/J AM were found (data not shown).


Figure 1
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  		Figure 1. In vitro infection of B10.A and A/J alveolar macrophages (AM) by P. brasiliensis yeasts. Alveolar macrophages (2x105/well) stimulated or not by 10.0 ng/ml of IFN-{gamma}, were cultivated overnight over round glass coverslips in 24-well flat-bottom plates. Cells were incubated with P. brasiliensis yeasts (1:50, fungus:macrophage ratio) during 2 h at 37°C in 5% CO2. Supernatants were aspirated, monolayers were gently washed, cells were fixed and stained with Giemsa. An average of 200 AM was analyzed, and the number of macrophages with adhered or ingested yeasts was determined. Data are the mean ± SEM of 3 independent experiments performed in triplicate.

AM were primed or not with three different concentrations (50, 10, and 2 ng/ml) of IFN-{gamma}, IL-12, or both cytokines and cultivated with P. brasiliensis yeasts for an additional 72-h period. Supernatants were removed and assayed for the presence of nitric oxide and cytokines, and cell homogenates were plated for CFU determinations. As shown in Fig. 2A and 2B , AM from susceptible mice presented a more prominent fungicidal activity than those from resistant mice. Of note is the smaller CFU counts recovered from B10.A control, nonstimulated, macrophages when compared with control A/J cells (Fig. 2A ). The three doses of IFN-{gamma}, IL-12, or both cytokines employed, led to decreased numbers (51–97%) of viable yeasts recovered from B10.A macrophages when compared with control cells. The same was not observed with A/J AM. Only the higher concentration of IFN-{gamma} induced a significant fungal killing (97%). Compared with A/J cells, B10.A macrophages secreted higher levels of NO (Fig. 2C ). In addition, for both mouse strains, IL-12 was a poor NO inducer, whereas the combination of both cytokines resulted in effector activity similar to those of IFN-{gamma}-primed macrophages.


Figure 2
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  		Figure 2. AM from susceptible mice (B10.A) have a better fungicidal ability than those from resistant mice (A/J). AM (2x105/well) were cultivated overnight in the presence of three different concentrations (50, 10, and 2 ng/ml) of IFN-{gamma}, IL-12, or both cytokines. The cells were challenged with viable P. brasiliensis yeasts (1:50, fungus:macrophage ratio) during 2 h at 37°C in 5% CO2. Cultures were then gently washed, fresh medium, containing or not cytokines, added, and cells cocultivated for 72 h. Supernatants were removed, AM were lysed, and viable fungi in cell homogenates were determined by a CFU assay (A). Fungicidal activity (B) was calculated according the following formula: [100 – (experimental CFUx100/control CFU)]. Nitric oxide (C) was measured in culture supernatants by the Griess reaction. Data are the mean ± SEM of triplicate samples from one experiment representative of 3 independent determinations. *, P < 0.05 vs. control group (C).

AM from resistant and susceptible mice secrete different levels of IL-10 and IL-12, resulting in diverse IL-10/IL-12 ratios
To better characterize the diverse pattern of macrophage activation, cocultivation supernatants were assayed for the presence of a macrophage activating cytokine (IL-12) and a deactivating cytokine (IL-10). As depicted in Fig. 3 , compared with A/J cells, AM from susceptible mice secreted high levels of IL-12 and low levels of IL-10, whereas A/J macrophages presented a preferential production of IL-10 associated with low concentrations of IL-12. As can also be seen in Fig. 3 , in all experimental conditions (untreated and all cytokine-treated preparations), a very higher IL-10/IL-12 ratio was seen with A/J cells. This finding indicated that a prevalent action of proinflammatory cytokines appeared to govern the behavior of B10.A macrophages, whereas a prevalent anti-inflammatory profile was seen with the resistant strain. Thus, the profile of cytokines secreted by B10.A and A/J cells was shown to be different and appeared to reflect the diverse fungicidal ability of these mice.


Figure 3
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  		Figure 3. AM from resistant and susceptible mice to P. brasiliensis infection secrete different levels of IL-10 and IL-12, resulting in diverse IL-10/IL-12 ratios . Adherent AM from B10.A and A/J mice, primed or not with three different concentrations (50, 10, and 2 ng/ml) of IFN-{gamma}, IL-12, or both cytokines, were cocultivated with P. brasiliensis yeasts for 72 h at 37°C in 5% CO2. Supernatants were obtained, and cytokines were measured by ELISA and compared with P. brasiliensis-infected control macrophages (C). Data (pg/ml) were also used to calculate the IL-10/IL-12 ratios. Data are the means ± SEM of triplicate samples from one experiment representative of 3 independent determinations. *, P < 0.05 vs. control group (C).

We have also measured MCP-1 (CCL2), a Th2-inducing chemokine involved in mononuclear cell chemotaxis [22 ] and GM-CSF, a growing factor that promotes the differentiation and maturation of antigen-presenting cells [23 ]. These latter mediators were included because of their possible influence in the impaired cellular immunity displayed by susceptible mice and the balanced Th1/Th2 response mounted by resistant mice [6 , 13 ]. Again, a diverse pattern of cytokine secretion was detected. The susceptible strain produced high levels of MCP-1, while high amounts of GM-CSF were secreted by the resistant mice (Fig. 4 ).


Figure 4
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  		Figure 4. AM from susceptible mice (B10.A) to P. brasiliensis infection secrete high levels of MCP-1, while those from resistant mice (A/J) preferentially secrete GM-CSF. Adherent AM from B10.A and A/J mice, primed or not with three different concentrations (50, 10, and 2 ng/ml) of IFN-{gamma}, IL-12, or both cytokines, were cocultivated with P. brasiliensis yeasts for 72 h at 37°C in 5% CO2. Supernatants were obtained and cytokines were measured by ELISA and compared with P. brasiliensis infected control macrophages (C). Data are the means ± SEM of triplicate samples from 1 experiment representative of 3 independent determinations. *, P < 0.05 vs. control group (C).

Aminoguanidine treatment inhibits the fungicidal ability of B10.A but not of A/J macrophages
We further asked whether the higher fungicidal ability of B10.A macrophages was associated with the high levels of NO produced. Hence, cocultivation experiments were performed in the presence or absence of 1 mM aminoguanidine, an iNOS inhibitor. As can be seen in Fig. 5 , the addition of AG did not affect the behavior of control macrophages. AG treatment, however, abolished the enhanced killing activity of IFN-{gamma}-primed macrophages of B10.A mice, although no effect was detected with A/J cells. As expected, in both mouse strains, IFN-{gamma} treatment caused increased NO production, and AG significantly reduced these levels. The NO concentrations reached by B10.A AM, however, were always higher that that displayed by A/J cells.


Figure 5
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  		Figure 5. Aminoguanidine (AG) treatment decreases the fungicidal activity of B10.A macrophages but does not modify the behavior of A/J AM. Adherent B10.A and A/J AM macrophages, primed or not with 10 ng/ml of IFN-{gamma}, were treated with AG (1.0 mM) and infected with P. brasiliensis yeasts. After 72 h of cocultivation, CFU counts were determined in cell homogenates and NO in cell supernatants. Data are the means ± SEM of triplicate samples from 1 experiment representative of 3 independent determinations. **, P < 0.01, ***, P < 0.001 vs. control group (C); and +++, P < 0.001 vs. IFN-{gamma}-treated AM.

Anti-IL-10 treatment does not modify the microbicidal activity of AM from both mouse strains
As A/J AM were shown to produce higher levels of IL-10 than B10.A cells, and this cytokine is one important macrophage-deactivating cytokine, we further study the effect of IL-10 neutralization in the fungicidal and secretory activity of AM. Thus, AM from both mouse strains were primed or not with IFN-{gamma}, and some cultures were concomitantly treated with 20 ng/ml of anti-IL-10 mAb (2A5). In both mouse strains, the addition of anti-IL-10 antibodies did not change the behavior of AM, i.e., B10.A cells retained their high fungicidal and NO secretion ability, while no killing activity was seen with A/J macrophages, even with the higher NO secretion induced by IL-10 neutralization (Fig. 6 ). Equivalent results were obtained with other doses of anti-IL-10 mAb (10 and 30 ng/ml; data not shown).


Figure 6
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  		Figure 6. Anti-IL-10 treatment does not alter the fungicidal ability of alveolar macrophages of susceptible (B10.A) and resistant (A/J) mice. Adherent AM from B10.A and A/J mouse strains were primed or not with IFN-{gamma} (10 ng/ml), and some cultures were concomitantly treated with 20 µg/ml of anti-IL-10 mAb (2A5). Cells were infected with P. brasiliensis yeasts and 72 h after cocultivation, CFU counts were determined in cell homogenates and NO in supernatants. Equivalent results were obtained with 10 and 30 µg/ml of anti-IL-10 mAb (data not shown). Data are expressed as the means ± SEM of triplicate samples from 1 experiment representative of 3 independent determinations. *, P < 0.05, **, P < 0.01, vs. control group (C); ++, P < 0.01 vs. IFN-{gamma}-treated AM.

Levels of IL-12 and IL-10 were assessed in cocultivation supernatants to further characterize the effect of anti-IL-10 treatment. As shown in Fig. 7 , increased IL-12 was detected in anti IL-10-treated A/J macrophages. In addition, the diminished levels of IL-10 detected in the anti-IL-10-treated supernatants demonstrated the adequate inhibitory activity of our mAb preparation (Fig. 7) .


Figure 7
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  		Figure 7. Effect of anti-IL-10 treatment on the production of cytokines by alveolar macrophages of B10.A and A/J mice. AM were treated and infected as described in Fig. 6 . Supernatants were obtained and cytokines measured by ELISA. Data are the mean ± SEM of triplicate samples *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. control group (C); +, P < 0.05 and ++, P < 0.01 vs. IFN-{gamma}-treated AM.

Endogenous TGF-β inhibits the fungicidal ability of A/J but not B10.A macrophages
As IL-10 neutralization had no effect on macrophages behavior, and TGF-β is another well-known macrophage inhibitory cytokine [24 , 25 ], we performed cocultivation experiments in the presence of anti-TGF-β polyclonal antibodies. Neutralization of TGF-β did not alter the NO secretion and fungicidal activity of normal and IFN-{gamma}-primed B10.A macrophages. This treatment, however, had an important effect on A/J AM. Anti-TGF-β antibodies rescue the killing activity of normal and IFN-{gamma}-primed A/J cells demonstrating its major inhibitory activity in the resistant strain. This treatment increased the NO synthesis of IFN-{gamma}-primed A/J macrophages, and this fact was associated with a further improvement in fungal killing (Fig. 8 ).


Figure 8
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  		Figure 8. Anti-TGF-β treatment rescues the fungicidal ability of alveolar macrophages from resistant mice. Adherent AM of B10.A and A/J mouse strains were primed or not with IFN-{gamma} (10 ng/ml) and some cultures were concomitantly treated with 20 µg/ml of anti-TGF-β polyclonal antibodies. Cells were infected with P. brasiliensis yeasts (50:1) and 72 h after co-cultivation, CFU counts were determined in cell homogenates and NO in supernatants. Data are the mean ± SEM of triplicate samples from one experiment representative of three independent determinations. *, P < 0.05, **, P < 0.01 vs. control group (C); +, P < 0.05 vs. IFN-{gamma}-treated AM.

To obtain further information on the effects of anti-TGF-β treatment, cytokines were dosed in culture supernatants. As shown in Fig. 9 , TGF-β neutralization led to more evident effects in IFN-{gamma}-treated AM resulting in both mouse strains in decreased IL-12 levels, although altered IL-10 synthesis was seen only with A/J cells. The effectiveness of anti-TGF-β treatment was confirmed by the diminished levels of this cytokine detected in the anti-cytokine-treated cultures (Fig. 9) .


Figure 9
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  		Figure 9. Influence of anti-TGF-β treatment on the production of pro-inflammatory and anti-inflammatory cytokines by alveolar macrophages. Adherent AM from B10.A and A/J mice were primed or not with IFN-{gamma} (10 ng/ml), and some cultures were concomitantly treated with 20 µg/ml of anti-TGF-β polyclonal antibodies. Cells were infected with P. brasiliensis yeasts (50:1), and 72 h after cocultivation, cytokines were measured in cell supernatants. Data are expressed as the mean ± SEM of triplicate samples. *, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. control group (C); +, P < 0.01 vs. IFN-{gamma}-treated AM.

P. brasiliensis-infected B10.A macrophages present a prevalent expression of CD40, whereas A/J cells express high levels of MHC class II antigen
As in the course of infection, resistant mice to P. brasiliensis infection developed cell-mediated immunity, whereas susceptible mice presented CD4+ T cell anergy besides elevated antibody production [6 , 13 ], we asked whether the different pattern of AM activation here observed would lead to diverse expression of costimulatory molecules involved in antigen presentation. Thus, normal and IFN-{gamma} primed B10.A and A/J macrophages were infected with P. brasiliensis, and 72 h after cocultivation, the expression of the costimulatory molecule CD40 and MHC class II (IAK) antigen was assayed by flow cytometry. As can be seen in Fig. 10 , CD40 was preferentially expressed by B10.A macrophages, whereas higher MHC class II antigen expression was seen with A/J cells.


Figure 10
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  		Figure 10. Alveolar macrophages from susceptible mice (B10.A) express higher levels of CD40, whereas MHC class II molecules are preferentially expressed by A/J AM. Flow cytometry profiles for CD40 (upper panels) and MHC class II (lower panels) on normal and IFN-{gamma}-primed (10 ng/ml) AM infected with P. brasiliensis yeasts for 72 h. AM were stained with FITC-conjugated anti-IAK or PE-conjugated anti-CD40. Figure panels are representative of 2 independent experiments.

DISCUSSION

AM are the first cells to interact with P. brasiliensis cells, but few studies have been addressed to determine the consequences of such interaction [16 ]. Taking advantage of the experimental model developed in our laboratory, where genetically resistant mice develop the mild, regressive form of the disease, and susceptible mice present a severe, progressive disease, we asked whether studies on the interaction between AM and P. brasiliensis could give some clues on the early events that result in resistance or susceptibility to this pathogen. Although no differences in the ingestion/adherence rates between macrophages from resistant and susceptible mice were detected (Fig. 1) , studies on the fungicidal activity of cytokine-primed and unprimed AM, showed, unexpectedly, that cells from susceptible mice have a better ability to control P. brasiliensis growth than those of resistant mice. These differences were detected with normal, unprimed B10.A macrophages and further confirmed with cytokine-activated cells. Indeed, all IFN-{gamma} and IL-12 doses employed to stimulate B10.A macrophages were able to induce significant fungicidal ability, whereas with A/J AM, only the higher IFN-{gamma} dose employed was able to induce an efficient killing activity (Fig. 2) .

The intrinsic differences herein observed with AM from resistant and susceptible mice probably reflect the use of different pathogen recognition receptors (PRR) in their interaction with P. brasiliensis agonists. Indeed, the activation of different PRR can result in opposite cell signaling, which can activate a preferential proinflammatory response as that mediated by Toll like receptor-4 and lipopolysaccharide or an anti-inflammatory response due to the overproduction of IL-10 as that induced TLR2 or mannose receptor, for instance [26 , 27 ]

For B10.A macrophages, the fungicidal ability paralleled the NO production, and the inhibitory effect of the AG treatment indicated the participation of NO in the process (Fig. 5) . Those results are in accordance with previous reports showing the NO-dependent mechanisms employed by murine macrophages to kill P. brasiliensis yeasts [6 , 14 ]. The high killing ability and NO production of B10.A AM were associated with prevalent synthesis of IL-12 and MCP-1, despite the concomitant production of IL-10 and TGF-β, which, however, were not able to abrogate the priming effect of IFN-{gamma} or IL-12. This was probably because of the low IL-10/IL-12 ratios observed with B10.A cells, in contrast with the very high ratios observed with A/J cells, resulting from the enhanced synthesis of IL-10 and probable synergistic effect of endogenous TGF-β. Although IL-12 is well known to be involved in Th1 induction [29 ], this cytokine does not appear to have a prevalent role in the T cell immunity of B10.A mice since this mouse strain, early in the infection, develop CD4+ T cell anergy [6 , 13 ]. In addition, MCP-1 (CCL2), a chemokine essential for Th2 responses, whose overexpression is associated with defects in cellular immunity [22 ], could contribute to the suppression of T cell immunity of B10.A mice.

In contrast with B10.A cells, A/J macrophages secreted high levels of IL-10 and GM-CSF, presented a poor fungicidal ability, and needed high IFN-{gamma} concentrations to be activated and efficiently kill P. brasiliensis yeasts (Figs. 2 3 4) . The impaired microbicidal ability of A/J AM was reverted by anti-TGF-β antibodies (Fig. 8) but not by NO inhibition (Fig. 5) or IL-10 neutralization (Fig. 6) , demonstrating that endogenous TGF-β has a more important regulatory activity on A/J macrophages than IFN-{gamma} priming.

Interestingly, experiments with both anti-IL-10 and anti TGF-β antibodies indicate that A/J AM have a NO-independent mechanism of P. brasiliensis killing. As can be seen in Fig. 6 , NO concentrations even higher than 30 µM induced by IL-10 neutralization were not sufficient to promote fungal killing; in addition, the rescued fungicidal ability of normal A/J macrophages induced by anti-TGF-β antibodies occurred in the absence of increased NO production (Fig. 7) . Consistent with those observations, studies on the fungicidal ability of TNF-{alpha}-activated murine macrophages suggested the existence of NO-independent mechanisms of P. brasiliensis killing [14 ] The same is true for human macrophages whose fungicidal ability was shown to be dependent on the production of oxygen, but not NO radicals [30 ]. As TGF–β was shown to be the most important inhibitory cytokine for A/J macrophages, we can suppose that the higher NO levels induced by anti-IL-10 treatment was not sufficient to abolish the inhibitory activity of TGF-β. Perhaps the fungicidal ability of A/J cells was totally TNF-{alpha}-dependent and NO-independent, and the anti-IL-10 treatment was not sufficient to abolish the intrinsic inhibitory activity of TGF-β on TNF-{alpha} production.

In human PCM, IL-10 is an important regulatory cytokine, has an inhibitory activity on IFN-{gamma} secretion by antigen-stimulated lymphocytes [31 ], and was associated with the severe forms of the disease [4 ]. In murine models of the disease, IL-10 was consistently observed in the lungs of resistant mice which, in contrast to the T cell anergy of susceptible mice, develop an equilibrated protective Th1/ Th2 immunity [6 , 13 ]. Experiments on IL-10 neutralization here reported appear to indicate that this cytokine has a minor role in the innate immunity of both mouse strains but do not exclude its regulatory activity in the adaptive immunity of infected hosts. Indeed, the early synthesis of IL-10, despite its Th2 enhancing ability, was shown to be required to the development of protective Th1 immunity against Candida albicans infection by IL-12-deficient mice [32 ]. Despite its adverse effect in innate immunity, IL-10 could provide optimal costimulatory activity to A/J macrophages by its inhibitory effect on IL-4 secretion and CTLA4/B7-2 interaction [33 ].

TGF-β is a broadly expressed cytokine that has regulatory effects on several cell types of the immune system. This cytokine inhibits the acquisition of effector functions by naïve CD4+ and CD8+ T cells, but the differentiation of Th2 cells seems to be more sensitive to TGF-β inhibition than Th1 differentiation [34 ]. In addition to the earlier reports showing the inhibitory activity of TGF-β on the activation of macrophages and their production of nitric oxide and proinflammatory cytokines [35 , 36 ], more recent studies have shown its preventive effect on dendritic cell maturation and antigen-presenting cell activity [37 ]. Of particular interest is the regulatory effect of TGF-β on T cell responses to self and pathogen antigens through its ability to control the activity of regulatory T cells [38 ]. Despite those well-described inhibitory activities of immune responses, in murine candidiasis, TGF-β was associated with the development of protective mechanisms. In nonhealer mice, administration of exogenous TGF-β led to less severe disease, while in healing mice TGF-β neutralization increased susceptibility to infection. Administration of TGF-β to healer mice, however, results in diminished ability to develop protective immunity, clearly indicating that a rigorous control of TGF-β levels is necessary to reach protective immunity [39 ]. In this context, early TGF-β secretion by resistant macrophages could be viewed as a protective mechanism. Its inhibitory effect on NO secretion [34 ] would impair the inhibitory activity of this mediator on T cell responses. Indeed, in murine PCM production of high levels of NO was associated with anergy of cellular immunity [12 , 18 ] and TGF-β production would rescue T immunity. Thus, as in murine candidiasis, the early synthesis of TGF-β in PCM could be correlated with the protective T cell immunity developed by resistant mice [6 , 13 ].

Data here reported on the behavior of AM are unexpected for P. brasiliensis infection since this fungus is considered a facultative intracellular pathogen [17 ]. Indeed, immunoprotection to PCM is typically associated with the production of type 1 cytokines (IL-12, IFN-{gamma}) and disease exacerbation with type 2 or anti-inflammatory cytokines [5 , 40 ]. Although unusual, our result explains some previous findings that characterize the genetic model of resistance and susceptibility to P. brasiliensis infection. When described, it was demonstrated that during the first weeks of infection, the lung of susceptible mice presented lower fungal loads than that of resistant animals [6 ]. This pattern was lately reverted when A/J mice develop positive DTH reactions, their lymph node cells secrete IL-2 and IFN-{gamma}, and pulmonary macrophages acquire fungicidal ability [6 , 8 , 9 ]. In susceptible mice, the early control of fungal growth was, however, followed by anergy in DTH reaction, unrestrained increase of yeasts multiplication in the lungs and further dissemination to liver and spleen. The results here presented appear to explain this unusual behavior and demonstrate that susceptibility to P. brasiliensis infection is associated with an efficient innate immune response. Indeed, other results from our laboratory confirmed such behavior: B10.A mice secrete increased levels of leukotrienes, and this lipid mediator is used to increase the rate of P. brasiliensis phagocytosis and increased fungal loads, despite the high macrophage activation [5 , 28 , 41 ]. In the same line, Toll-like receptors 2 and 4 were shown to be used by P. brasiliensis yeasts to gain entry into macrophages and to activate macrophages which, however, do not control fungal multiplication [42 ]. In addition, early production of NO, although leading to diminished fungal loads, results in impaired T cell immunity [12 , 18 ], indicating that the early control of P. brasiliensis growth can result in impaired protective cellular immunity.

A clear difference between B10.A and A/J macrophages can be seen if the IL-10/IL-12 ratio is considered. While low ratios were found for B10.A macrophages, indicating the preferential activation of a proinflammatory pattern, the behavior of A/J cells favor an anti-inflammatory pattern, probably due to the synergistic action of IL-10 and TGF-β. The behavior of B10.A AM (prevalent production of IL-12, NO) is similar to that described for classically activated (Ca), M1, or reductive macrophages [43 44 45 46 ]. This behavior is usually associated with efficient killing of intracellular pathogens via production of oxygen or nitrogen radicals [43 ] and was shown to induce preferential Th1 immunity, a fact that does not occur with susceptible animals [6 , 13 ]. In fact, the adaptative immunity of B10.A mice was shown to be mediated by CD8+ T cells and anergy or deletion of the CD4+ T cell subset [6 , 7 , 13 ].

In contrast with B10.A cells, A/J AM secrete low levels of NO, high levels of IL-10, TGF-β, and GM-CSF, exert a poor fungicidal activity and behave as deactivated macrophages. So, A/J cells present some features of the alternative activation induced by IL-4/IL-13/GM-CSF and could be considered as M2 macrophages [43 44 45 ]. Further characterization of arginase activity will help us to verify whether the low NO production by A/J cells could be attributed to the arginine consumption by the arginase activity induced by GM-CSF [46 ]. Interestingly, this growing factor was shown to be involved in the differentiation, maturation, and increased expression of MHC class II antigens by antigen presenting cells, such as activated macrophages and dendritic cells [23 ] and was consistently secreted by A/J macrophages. As previously reported [12 ], A/J macrophages have also an efficient ability of TNF-{alpha} secretion, and this could further improve their antigen-presenting cell (APC) function. Thus, despite the deactivated behavior in innate immunity, AM of A/J mice could develop into efficient APC and induce T cell immunity. In agreement, resistant mice were shown to mount a balanced Th1/Th2 CD4+ T cell immunity besides a protective, type 1, CD8+ T cell response [5 , 6 , 13 , 47 ]. Consistent with these findings, a recent work showed that dendritic cells from resistant mice induce a better activation of naïve T cells than those from susceptible mice [48 ].

Trying to reconcile results here reported for innate immunity with the major features of the adaptative immunity described previously for our model [5 , 13 , 47 ], we further studied the expression of some costimulatory molecules on P. brasiliensis-infected AM. As can be seen in Fig. 10 , 72 h after cocultivation with yeast cells, B10/A AM expressed increased levels of CD40, whereas A/J cells presented prevalent expression of MHC class II antigens. This is in accordance with the proinflammatory milieu produced by B10.A cells and the class II-inducing activity of GM-CSF [23 ] secreted by A/J AM. Interestingly, the elevated expression of costimulatory molecules (CD40, for example) by macrophages was reported to directly activate CD8+ T cells without the help of CD4+ T lymphocytes [49 , 50 ], and this behavior would explain the prevalent CD8+ T cell response of susceptible mice in the absence of CD4+ T cell immunity [5 , 13 ]. On the other hand, A/Sn mice, despite the inefficient innate microbicidal activity, develop CD4+ and CD8+ T cell responses [13 ], which could be linked to the enhanced APC activity induced by innate immunity. Furthermore, it is tempting to suppose that the high pulmonary fungal loads due to the early failure of A/Sn AM would permit precocious fungal dissemination to distant organs. This fact, indeed, has been detected in some experimental circumstances previously reported [6 , 51 ]. The systemic presence of P. brasiliensis associated with the increased APC activity of A/J AM could result in systemic T cell immunity that could result in mild disease, controlled fungal growth, and organized lesions.

In conclusion, this work revealed the unexpected behavior of AM from resistant and susceptible mice to P. brasiliensis infection, in which efficient innate immunity characterized the susceptible mouse strain, while an unrestrained control of fungal growth was displayed by the resistant mice. Thus, in contrast to the major paradigm of immunoprotection to facultative intracellular pathogens, in pulmonary PCM the proinflammatory milieu induced by innate immunity results in anergy of CD4+ T-cell responses and progressive disease. Furthermore, the early unchecked growth of P. brasiliensis at the site of infection does not appear to be deleterious to the hosts and can result in antigen presentation to T cells, adequate cellular immunity, and regressive disease as demonstrated by the resistant mouse strain.

ACKNOWLEDGEMENTS

We are grateful to Tania A. Costa for technical assistance. This work was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Pesquisas.

Received November 8, 2007; revised December 17, 2007; accepted December 20, 2007.

REFERENCES

    1
  1. Brummer, E. (1994) Interaction of Paracoccidioides brasiliensis with host defense cells Franco, M. Silva Lacaz, C. Restrepo Moreno, A. Del Negro, G. eds. Paracoccidioidomycosis ,213-223 CRC Press Boca Raton, FL.
    2. 2
  2. Franco, M. (1987) Host-parasite relationship in paracoccidioidomycosis J. Med. Vet. Mycol. 25,5-18[Medline]
    3. 3
  3. Benard, G., Mendes-Giannini, M. J. S., Juvenale, M., Miranda, E. T., Duarte, A. J. S. (1997) Immunosuppression in Paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response J. Infect. Dis. 175,1263-1267[Medline]
    4. 4
  4. Mamoni, R. L., Nouer, R. A. M. S., Oliveira, S. A., Musatti, C. C., Rossi, C. L., Camargo, Z. P., Blotta, M. H. S. L. (2002) Enhanced production of specific IgG4, IgE, IgA and TGF-β in sera from patients with the juvenile form of paracoccidioidomycosis Med. Mycol. 40,153-159[Medline]
    5. 5
  5. Calich, V. L. G., Blotta, M. H. S. L. (2005) Pulmonary paracoccidioidomycosis Fidel, P.L. Huffnagle, G.B. eds. Fungal Immunology, From an Organ Perspective ,201-228 Springer New York, NY.
    6. 6
  6. Cano, L. E., Singer-Vermes, L. M., Vaz, C. A., Russo, M., Calich, V. L. G. (1995) Pulmonary paracoccidioidomycosis in resistant and susceptible mice, relationship among progression of infection, bronchoalveolar cell activation, cellular immune response, and specific isotype patterns Infect. Immun. 63,1777-1783[Abstract]
    7. 7
  7. Cano, L. E., Singer-Vermes, L. M., Mengel, J. A., Xidieh, C. F., Arruda, C., André, D. C., Vaz, C. A. C., Burger, E., Calich, V. L. G. (2000) Depletion of CD8+ T cells in vivo impairs host defense of resistant and susceptible mice to pulmonary paracoccidioidomycosis Infect. Immun. 68,352-359[Abstract/Free Full Text]
    8. 8
  8. Kashino, S. S., Fazioli, R. A., Cafalli-Favati, C., Meloni-Bruneri, L. H., Vaz, C. A. C., Burger, E., Singer, L. M., Calich, V. L. G. (2000) Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-{gamma} production J. Interferon Cytokine Res. 20,89-97[CrossRef][Medline]
    9. 9
  9. Cano, L. E., Kashino, S. S., Arruda, C., André, D., Xidieh, C. F., Singer-Vermes, L. M., Vaz, C. A. C., Burger, E., Calich, V. L. G. (1998) Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis Infect. Immun. 66,800-806[Abstract/Free Full Text]
    10. 10
  10. Arruda, C., Franco, M. F., Kashino, S. S., Nascimento, F. R., Fazioli, R. A., Vaz, C. A., Russo, M., Calich, V. L. G. (2002) Interleukin-12 protects mice against disseminated infection caused by Paracoccidioides brasiliensis but enhances pulmonary inflammation Clin. Immunol. 103,185-195[CrossRef][Medline]
    11. 11
  11. Deepe, G. S., Jr, Romani, L., Calich, V. L. G., Huffnagle, G., Arruda, C., Molinari-Madlum, E. E., Perfect, E. E., Jr (2000) Knockout mice as experimental models of virulence Med. Mycol. 38,87-98[Medline]
    12. 12
  12. Nascimento, F. R. F., Calich, V. L. G., Rodríguez, D., Russo, M. (2002) Dual role for nitric oxide in paracoccidioidomycosis, essential for resistant, but overproduction associated with susceptibility J. Immunol. 168,4593-4600[Abstract/Free Full Text]
    13. 13
  13. Chiarella, A. P., Arruda, C., Pina, A., Costa, T. A., Ferreira, R. C., Calich, V. L. G. (2007) The relative importance of CD4+ and CD8+ T cells in immunity to pulmonary paracoccidioidomycosis Microbes Infect. 9,1078-1088[CrossRef][Medline]
    14. 14
  14. Gonzalez, A., Aristizabal, B. H., Gomez, E. C., Restrepo, A., Cano, L. E. (2004) Short report: Inhibition by tumor necrosis factor-{alpha}-activated macrophages of the transition of Paracoccidioides brasiliensis conidia to yeast cells through a mechanism independent of nitric oxide Am. J. Trop. Med. Hyg. 71,828-830[Abstract/Free Full Text]
    15. 15
  15. Cano, L. E., Gómez, B., Brummer, E., Restrepo, A., Stevens, D. A. (1994) Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin Infect. Immun. 62,1494-1496[Abstract/Free Full Text]
    16. 16
  16. Brummer, E., Hanson, L. H., Restrepo, A., Stevens, D. A. (1988) In vivo and in vitro activation of pulmonary macrophages by IFN-{gamma} for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis J. Immunol. 140,2786-2789[Abstract]
    17. 17
  17. Brummer, E., Hanson, L. H., Restrepo, A., Stevens, D. A. (1989) Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages Infect. Immun. 57,2289-2294[Abstract/Free Full Text]
    18. 18
  18. Bocca, A. L., Hayasshi, E. E., Pinheiro, A. G., Furianetto, A. B., Campanelli, A. P., Cunha, F. Q., Figueiredo, F. (1998) Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response J. Immunol. 161,3056-3063[Abstract/Free Full Text]
    19. 19
  19. Kashino, S. S., Calich, V. L., Burger, E., Singer-Vermes, L. M. (1985) In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains Mycopathologia 92,173-178[CrossRef][Medline]
    20. 20
  20. Netto, C. F., Vegas, V. S., Sciannaméa, I. M., Guarnieri, E. D. B. (1969) Antígeno polissacarídico do Paracoccidioides brasiliensis. Estudo do tempo de cultivo do P. brasiliensis necessário ao preparo do antígeno Rev. Inst. Med. Trop. Sao Paulo 11,177-181[Medline]
    21. 21
  21. Singer-Vermes, L.M., Burger, E., Franco, M.F., Moscardi-Bacchi, M., Mendes- Giannini, M.J.S., Calich, V.L.G. (1998) Evaluation of the pathogenicity and immunogenicity of seven Paracoccidioides brasiliensis isolates in susceptible inbred mice J. Med. Vet. Mycol. 27,71-82[CrossRef]
    22. 22
  22. Gu, L., Tseng, S., Horner, R. M., Tam, C., Loda, M., Rollins, B. J. (2000) Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1 Nature 404,407-411[CrossRef][Medline]
    23. 23
  23. Goth, S. R., Chu, R. A., Pessah, I. N. (2006) Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression J. Immunol. Methods 308,179-191[CrossRef][Medline]
    24. 24
  24. Vodovotz, Y., Bogdan, C., Paik, J., Xie, Q. W., Nathan, C. (1993) Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor β J. Exp. Med. 178,605-613[Abstract/Free Full Text]
    25. 25
  25. Tsunawaki, S., Sporn, M., Ding, A., Nathan, C. (1988) Deactivation of macrophages by transforming growth factor β Nature 334,260-262[CrossRef][Medline]
    26. 26
  26. Netea, M. G., Van Der Graaf, C. A. A., Vonk, A. G., Verschueren, I., Van Der Meer, J. W. M., Kullberg, B. J. (2002) The role of toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis J. Infect. Dis. 186,1377-1379[Medline]
    27. 27
  27. Netea, M. G., Van der Meer, J. W., Kullberg, B. J. (2006) Role of dual interaction of fungal pathogens with pattern recognition receptors in the activation and modulation of host defense Clin. Microbiol. Infect. 12,404-409[CrossRef][Medline]
    28. 28
  28. Calich, V. L. G., Costa, T. A., Felonato, M., Arruda, C., Bernardino, S., Loures, F. V., Ribeiro, L. R. R., Valente-Ferreira, R. C., Pina, A. (2008) Innate Immunity to Paracoccidioides brasiliensis infection Mycopathologia In press
    29. 29
  29. Trinchieri, G., Kubin, M., Bellone, G., Cassatella, M. A. (1993) Cytokine cross-talk between phagocytic cells and lymphocytes, relevance for differentiation/activation of phagocytic cells and regulation of adaptive immunity J. Cell Biochem. 53,301-308[CrossRef][Medline]
    30. 30
  30. Carmo, J. P., Dias-Melicio, L. A., Calvi, S. A., Peraçoli, M. T., Soares, A. M. (2006) TNF-{alpha} activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism Med. Mycol. 44,363-368[Medline]
    31. 31
  31. Romano, C. C., Mendes-Giannini, M. J., Duarte, A. J., Benard, G. (2005) The role of interleukin-10 in the differential expression of interleukin-12p70 and its β2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects Clin. Immunol. 114,86-94[CrossRef][Medline]
    32. 32
  32. Mencacci, A., Cenci, E., Del Sero, G., Fé d' Ostiani, C., Mosci, P., Trinchieri, G., Adorini, L., Romani, L. (1998) IL-10 is required for development of protective Th1 responses in IL-12-deficient mice upon Candida albicans infection J. Immunol 161,6228-6237[Abstract/Free Full Text]
    33. 33
  33. Spaccapelo, R., Del Sero, G., Mosci, P., Bistoni, F., Romani, L. (1997) Early T cell unresponsiveness in mice with candidiasis and reversal by IL-2: effect on T helper cell development J. Immunol. 158,2294-2302[Abstract]
    34. 34
  34. Gorelik, L., Flavell, R. A. (2002) Transforming growth factor-β in T cell biology Nat. Rev. Immunol. 2,46-53[CrossRef][Medline]
    35. 35
  35. Bogdan, C., Nathan, C. (1993) Modulation of macrophage function by transforming growth factor β, interleukin-4, and interleukin-10 Ann. N. Y. Acad. Sci. 685,713-739[Medline]
    36. 36
  36. Vodovotz, Y., Bogdan, C. (1994) Control of nitric oxide synthase expression by transforming growth factor-β: implications for homeostasis Prog. Growth Factor Res. 5,341-351[CrossRef][Medline]
    37. 37
  37. Yamaguchi, Y., Tsumura, H., Miwa, M., Inaba, K. (1997) Contrasting effects of TGF-β 1 and TNF-{alpha} on the development of dendritic cells from progenitors in mouse bone marrow Stem Cells 15,144-153[Abstract/Free Full Text]
    38. 38
  38. Nakamura, K., Kitani, A., Strober, W. (2001) Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor β J. Exp. Med. 194,629-644[Abstract/Free Full Text]
    39. 39
  39. Spaccapelo, R., Romani, L., Tonnetti, L., Cenci, E., Mencacci, A., Del Sero, G., Tognellini, R., Reed, S. G., Puccetti, P., Bistoni, F. (1995) TGF-β is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans J. Immunol. 155,1349-1360[Abstract]
    40. 40
  40. Borges-Walmsley, M. I., Chen, D., Shu, X., Walmsley, A. R. (2002) The pathobiology of Paracoccidioides brasiliensis Trends Microbiol. 10,80-87[CrossRef][Medline]
    41. 41
  41. Ribeiro, L. R. R. (2005) Caracterização do papel dos leucotrienos na paracoccidioidomicose (PCM) pulmonar e na atividade fungicida e secretora de macrófagos peritoneais infectados pelo Paracoccidioides brasiliensis. (M. Sc. dissertation) Instituto de Ciências Biomédicas, Universidade de São Paulo São Paulo.
    42. 42
  42. Loures, F. V. (2007) Caracterização da função dos lipídios do Paracoccidioides brasiliensis e do receptor TLR4 na interação fungo-hospedeiro (M. Sc. Dissertation) Instituto de Ciências Biomédicas, Universidade de São Paulo São Paulo.
    43. 43
  43. Nathan, C., Shiloh, M. U. (2000) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens Proc. Natl. Acad. Sci. USA 97,8841-8848[Abstract/Free Full Text]
    44. 44
  44. Mills, C. D., Kinkaid, K., Alt, J. M., Heilman, M. J., Annete, M. H. (2000) M1/M2 macrophages paradigm J. Immunol. 164,6166-6173[Abstract/Free Full Text]
    45. 45
  45. Murata, Y., Shimamura, T., Hamuro, J. (2002) The polarization of T(h)1/T(h)2 balance is dependent on the intracellular thiol redox status of macrophages due to the distinctive cytokine production Int. Immunol. 14,201-212[Abstract/Free Full Text]
    46. 46
  46. Gordon, S. (2003) Alternative activation of macrophages Nat. Rev. Immunol. 3,23-35[CrossRef][Medline]
    47. 47
  47. Arruda, C., Vaz, C. A., Calich, V. L. G. (2007) Aseptic cure of pulmonary paracoccidioidomycosis can be achieved after a previous subcutaneous immunization of susceptible but not resistant mice Microbes Infect. 9,704-713[CrossRef][Medline]
    48. 48
  48. Ferreira, K. S., Bastos, K. R., Russo, M., Almeida, S. R. (2007) Interaction between Paracoccidioides brasiliensis and pulmonary dendritic cells induces interleukin-10 production and toll-like receptor-2 expression: possible mechanisms of susceptibility J. Infect. Dis. 196,1108-1115[CrossRef][Medline]
    49. 49
  49. Albert, M. L., Sauter, B., Bhardwaj, N. (1998) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs Nature 392,86-89[CrossRef][Medline]
    50. 50
  50. Ridge, J. P., Di Rosa, F., Matzinger, P. (1998) A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell Nature 393,474-478[CrossRef][Medline]
    51. 51
  51. Pina, A., Saldiva, P. H., Restrepo, L. E., Calich, V. L. G. (2006) Neutrophil role in pulmonary paracoccidioidomycosis depends on the resistance pattern of hosts J. Leukoc. Biol. 79,1202-1213[Abstract/Free Full Text]




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