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Originally published online as doi:10.1189/jlb.0106052 on April 7, 2006

Published online before print April 7, 2006
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(Journal of Leukocyte Biology. 2006;79:1202-1213.)
© 2006 by Society for Leukocyte Biology

Neutrophil role in pulmonary paracoccidioidomycosis depends on the resistance pattern of hosts

Adriana Pina*,{dagger}, Paulo Hilário Nascimento Saldiva{ddagger}, Luz Elena Cano Restrepo* and Vera L. G. Calich*,1

* Departamentos de Imunologia do Instituto de Ciências Biomédicas,
{dagger} Análises Clínicas da Faculdade de Ciências Farmacêuticas, e
{ddagger} Patologia da Faculdade de Medicina, Universidade de 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

The immunoprotective and immunomodulatory role of neutrophils during pulmonary infection of resistant (A/J) and susceptible (B10.A) mice to Paracoccidioides brasiliensis was investigated. First, comparative studies about early cellular influx to the lungs demonstrated higher numbers of neutrophils in susceptible rather than in resistant mice. Neutrophil depletion resulted in decreased survival times of susceptible but not resistant mice. In both mouse strains, depletion led to increased fungal burdens at Week 1 of infection; however, only susceptible mice remained with increased pulmonary fungal loads and presented a dramatic fungal dissemination to liver and spleen. At Week 1 of infection, treated and untreated B10.A and A/J mice were negative for delayed-type hypersensitivity (DTH) reactions, which remained negative for the susceptible strain. In contrast, from the second week onward, control and neutrophil-depleted, resistant mice became positive for DTH reactions. In B10.A mice, neutrophil depletion resulted in increased levels of interleukin (IL)-12 and IL-4 in the lungs, high levels of hepatic cytokines, and increased synthesis of T helper cell type 1 (Th1)- and Th2-regulated antibodies [immunoglobulin G1 (IgG1), IgA, and IgG3]. In neutrophil-depleted A/J mice, high levels of pulmonary IL-12 and granulocyte macrophage-colony stimulating factor were concomitant to diminished levels of hepatic cytokines and increased amounts of Th1-regulated isotypes (IgG2a, IgG2b, and IgG3). Differently from primary infection, neutrophil depletion did not alter immunoprotection in secondary paracoccidioidomycosis. As a whole, our data showed that the genetic patterns of hosts exert an important influence on the immunoprotective and immunoregulatory functions of neutrophils, which appear to be essential in situations devoid of cell-mediated immunity.

Key Words: Paracoccidioides brasiliensis • polymorphonuclear leukocyte • innate immunity • adaptative immunity • fungal infection • cytokines

INTRODUCTION

Paracoccidioidomycosis (PCM) is an endemic mycosis present in tropical and subtropical areas of Latin America, with the highest incidence in Brazil. The causative agent is the dimorphic fungus Paracoccidioides brasiliensis [1 , 2 ]. The natural route of infection is the inhalation of fungal particles, which usually leads to an asymptomatic infection. PCM presents a wide spectrum of clinical manifestation, and the severity of the disease is related to the host immune response and to the virulence of the infectious agent [3 ]. Classical studies about the immune responses developed by patients with polar forms of PCM demonstrated that the benign forms of the disease were associated with production of low levels of antibodies and positive delayed-type hypersensitivity (DTH) reactions, whereas the severe, disseminated forms were associated with high levels of antibodies and anergy in DTH reactions [3 ].

In the murine model of PCM, resistant mice (A/Sn, A/J) are assumed to direct the immune response to a preferential T helper cell type 1 (Th1) activation with predominant secretion of interferon-{gamma} (IFN-{gamma}) and interleukin (IL)-2, leading to resolution of the disease. Conversely, susceptible mice (B10.A) present early secretion of IL-10 and IL-5 associated with absent or impaired IFN-{gamma} production, resulting in progressive disease [4 , 5 ]. Studies using athymic BALB/c mice (nu/nu) and their heterozygous counterparts (nu/+) revealed that susceptibility to P. brasiliensis infection is exacerbated in athymic animals, demonstrating that cellular immune responses are fundamental to resistance to P. brasiliensis infection. However, nude mice were still able to control the infection at its initial stages, suggesting that mechanisms of innate immunity play an important role in resistance to PCM [6 ].

One cell type that may be involved with early resistance to P. brasiliensis infection is the polymorphonuclear neutrophil (PMN). One of the most remarkable differences between chronic lesions of resistant and susceptible was the presence of abundant neutrophils in the lesion areas of massive fungal destruction and few viable yeast cells in the resistant strain and a less-intense phenomenon in susceptible mice, indicating that PMN could play a role in PCM resistance. This was confirmed further using PMN cells obtained from subcutaneous (s.c.) air-pouches of resistant and susceptible mice infected with yeast cells. By Day 15 of infection, only PMN leukocytes from the resistant mice showed a significantly increased production of H2O2. Furthermore, at this period of infection, PMN cells from susceptible mice have low fungicidal activity, in contrast to those from resistant mice, which are more efficient in P. brasiliensis yeast cell killing [7 ].

In this study, we examined the in vivo role of PMN leukocytes in the host defense of resistant and susceptible mice following intratracheal (i.t.) infection with P. brasiliensis. We have undertaken a series of studies in neutrophil-depleted B10.A and A/J mice using a PMN-specific monoclonal antibody (mAb; RB6-8C5). We characterized the resulting peripheral blood neutropenia after mAb administration and investigated the progression of pulmonary and extrapulmonary primary infections, the specific DTH reactions, and humoral responses. We also established the effect of neutrophil depletion on cytokine production and survival of resistant and susceptible mice after P. brasiliensis infection. The effect of in vivo depletion of PMN leukocytes on secondary PCM of vaccinated B10.A mice was also investigated. Previous studies in our laboratory showed that the s.c. inoculation of P. brasiliensis in susceptible mice stimulates intense cellular and humoral immune responses as well as immunoprotection to a further intraperitoneal (i.p.) or i.t. challenge [8 ]. As a whole, the results herein obtained clearly indicated that PMN cells play an important role on the innate immunity of susceptible and resistant mice. Their effect, in the later phases of the disease, however, was dependent on the resistant/susceptible patterns of hosts. Furthermore, in secondary PCM, PMN cells do not affect the protective immunity induced by a vaccinating protocol.

MATERIALS AND METHODS

Mice
Susceptible (B10.A) and resistant (A/J) mouse strains to PCM were obtained from our Isogenic Breeding Unit (Departmento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil). Mice were 8–11 weeks of age at the time of infection, and procedures involving animals and their care were conducted in conformity with national and international policies.

Fungus
P. brasiliensis Pb18, a highly virulent isolate [9 ], was used throughout this investigation. To ensure the maintenance of its virulence, the isolate was used after three animal passages [10 ]. Pb18 yeast cells were then maintained by weekly subcultivation in semisolid Fava Netto and co-workers’ culture medium [11 ] 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%.

P. brasiliensis infection
Mice were anesthetized and submitted to i.t. P. brasiliensis infection, as described previously [12 ]. Briefly, after i.p. anesthesia, the animals were infected with 106 P. brasiliensis Pb18 yeast cells, contained in 50 µl PBS by surgical i.t. inoculation, which allowed dispensing of the fungal cells directly into the lungs. The skin was then sutured, and the mice were allowed to recover under a heat lamp.

Bronchoalveolar lavage fluid (BALF)
At several time-points after infection, mice were lavaged after the canulation of trachea with polyethylene tubing, which was attached on a tuberculin syringe. The same procedure was applied to sham-infected (submitted to surgical stress and injected with 50 µl PBS) and normal mice of both mouse strains. The lungs were lavaged by repeated injections of 0.5 ml sterile PBS (final volume, 2.0 ml). The recovered fluid was cytospun at 1200 revolutions per minute (rpm; Shandon Cytospin, Pittsburgh, PA) onto glass slides and stained by the Diff-Quik blood stain (Baxter Scientific, Miami, FL); the supernatant was removed, and cells were analyzed for leukocyte subsets.

Depletion of neutrophils
The RB6-8C5 clone of an anti-PMN hybridoma was a generous gift from R. L. Coffman from DNAx Research Institute (Palo Alto, CA). The rat immunoglobulin G2b (IgG2b) mAb produced by Clone RB6-8C5 reacts with the Gr-1 surface antigen expressed by murine granulocytes but not by monocytes or lymphocytes [13 14 15 ]. Ascites were prepared in pristane-primed BALB/c nude mice, and the mAb was purified by the method of McKinney and Parkinson [16 ] and was assayed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Pharmacia, Uppsala, Sweden). The total protein concentration was determined by bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). To determine the effect of anti-Gr-1 treatment on PCM, mice were injected i.p. with 250 µg mAb, 24 h before the i.t. challenge with P. brasiliensis viable yeasts. This treatment was repeated at Days 2 and 5 after infection. Control mice received equivalent amounts of normal rat IgG. The number of blood PMN cells was evaluated before treatment (at Day –1) and 24 h after the mAb inoculation, immediately before yeast cell inoculation (zero time). Mice were bled through a lateral tail vein, and cell suspensions were cytospun (Shandon Cytospin) onto glass slides and stained by the Diff-Quik blood stain (Baxter Scientific). The numbers of leukocytes were calculated on the basis of the total and differential cell counts. The number of leukocytes was also determined on the day before sacrifice of animals to evaluate the degree of PMN depletion (Days 6, 14, 29, and 119 of infection).

DTH assay
The DTH reactions were always evaluated just before sacrifice of the same animals used in the colony-forming unit (CFU) assays by the footpad test, under previously determined conditions [17 ]. Briefly, mice were inoculated with 25 µl Fava Netto and co-workers’ antigen [11 ], and footpad thickness was measured with a caliper (Mitutoyo Corp., Tokyo, Japan) immediately before and 24 h after antigen inoculation. The increase in thickness was calculated and expressed in millimeters. Noninfected mice submitted to the footpad test were used as controls.

Assay for CFU
The number of viable microorganisms in different organs of infected mice was determined by CFU counts. Several periods after infection (1, 2, 4, and 16 weeks), eight to 10 infected B10.A and A/J mice of RB6-treated and untreated groups were killed, and their lungs, livers, and spleens were removed aseptically, weighed, and homogenized in 5 mL sterile PBS by means of a tissue grinder. The cellular suspensions were centrifuged to 2000 rpm for 5 min, and the pellets were resuspended in 1 ml PBS. Aliquots (100 µl) of each homogenate were plated on brain heart infusion agar (Difco, Detroit, MI), 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 a growth-promoting factor [18 ]. When necessary, dilutions were made in sterile PBS. The plates were incubated at 35ºC, 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.

Histological analysis
The left lung, the highest part of the liver, and half spleen of each mouse were removed, fixed in 10% formalin, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin (H&E) for analysis of lesions. Pathologic findings were qualitatively evaluated to characterize the morphological characteristics on granulomas, in terms of the following parameters: necrosis, density of inflammatory cells, and organization of the lesion (well or badly formed granulomas). In addition, the proportion of the organ affected by granulomas was determined using a standard point-count procedure described previously [19 ]. Briefly, a coherent system of 100 points was applied over five randomly selected, noncoincident microscopic fields (100x), and the proportion of the points hitting granulomas was used to estimate the volume fraction of the organ affected by granulomatous inflammation.

Antibody levels
Specific antibody levels (total Ig, IgM, IgA, IgG1, IgG2a, IgG3, and IgG2b) were measured by a previously described enzyme-linked immunosorbent assay (ELISA) using a cell-free antigen [12 ] prepared from a pool of different P. brasiliensis isolates (Pb339, Pb265, and Pb18). The average of the absorbances obtained with sera from control mice (PBS inoculated) was considered the cutoff for each respective isotype. Absorbances for each dilution of experimental serum were compared with control values. The titer for each sample was expressed as the reciprocal of the highest dilution that presented absorbance higher than the cutoff.

Measurement of cytokines
Mice were infected i.t. with P. brasiliensis, and their right lungs were removed aseptically and individually disrupted in 5.0 ml RPMI-1640 medium (Gibco-BRL, Grand Island, NY). Supernatants were separated from cell debris by centrifugation at 2000 g for 15 min, passed through 0.22 µm pore-size filters (Millipore, Bedford, MA), and stored at –70°C. The levels of IL-2, IL-4, IL-5, IL-10, IFN-{gamma}, IL-12, tumor necrosis factor {alpha} (TNF-{alpha}), and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured by capture ELISA, and antibody pairs were purchased from PharMingen (San Diego, CA). The ELISA procedure was performed according to the manufacturer’s protocol, and absorbances were measured with Versa Max microplate reader (Molecular Devices, Sunnyvale, CA). The concentrations of cytokines were determined with reference to a standard curve for serial twofold dilutions of murine recombinant cytokines.

Statistical analysis
Data were expressed as means ± SE and analyzed by Student’s t-test or two-way ANOVA depending on the number of experimental groups [20 ]. P values under 0.05 were considered significant.

RESULTS

Cellular characterization of BALF in the course of pulmonary infection
We first decided to determine the cellular composition of BALF in the course of infection to verify if a different pattern of cell migration to alveolar spaces would occur in susceptible and resistant mice after P. brasiliensis infection. Mice were i.t.-infected and submitted to BAL at the innate (24, 48, and 96 h after infection) and adaptative [1–16 weeks postinfection (p.i.)] phases of immunity. Compared with resistant mice, a higher inflammatory cell influx was detected in susceptible mice, peaking at 48 h after infection (Fig. 1 ). Differential counts revealed that this increase was mainly a result of increased neutrophil and eosinophil migration to the lungs of B10.A mice. No significant differences were detected regarding mononuclear cell migration; however, at Weeks 1 and 16, A/J mice presented an augmented number of multinucleated giant cells in their BALF.


Figure 1
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  		Figure 1. Number of total cells and leukocyte subsets (mononuclear cells, PMN neutrophils, eosinophils, and giant multinucleated cells) in BALF from susceptible (B10.A) and resistant (A/J) mice inoculated i.t. with 1 million P. brasiliensis yeast cells. Lungs of B10.A and A/J mice (n=6–8) were lavaged with PBS 24, 48, and 96 h and 1–16 weeks after infection, and cell suspensions were cytospun onto glass slides and stained by the Diff-Quik bloodstain. The absolute number of a leukocyte subset was calculated as described in Materials and Methods. Data are expressed as means ± SE of the means. *, Significant at P < 0.05 between strains.

 
Effect of in vivo mAb treatment on PMN cell counts in peripheral blood and BALF
Mice were injected i.p. with anti-Gr-1 mAb or control IgG 24 h before the i.t. challenge with P. brasiliensis yeast cells and at Days 2 and 5 after infection. Peripheral blood was collected through a tiny cut into the tail tip of mAb-treated and untreated B10.A and A/J mice on Days –1, 0, 6, 14, 29, and 119 p.i. An efficient PMN depletion (96%) peaking at Day 6 of infection was seen in B10.A and A/J mice (Fig. 2 ). Compared with IgG-treated, infected controls, RB6 mAb-treated A/J mice presented at Day 14 increased numbers of blood PMN neutrophils. It is noteworthy that at this time-point, infected A/J mice presented twice the numbers of blood PMN cells (9063 cells/mm3) compared with B10.A (4687 cells/mm3) mice. From Days 14 to 119 of infection, an equivalent number of PMN neutrophils were seen in treated and untreated groups of the susceptible strain. To verify if PMN cells were similarly depleted at the site of infection, lung cell influxes in control and PMN-depleted mice were analyzed 72 h after P. brasiliensis infection and demonstrated a 97.5% and 96.0% decrease in PMN leukocyte counts in the airways of susceptible and resistant mice, respectively.


Figure 2
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  		Figure 2. In vivo treatment of B10.A and A/J mice (n=8–10) with RB6-8C5 mAb led to decreased numbers of peripheral blood PMN cells. B10.A and A/J mice were treated in vivo with anti-PMN RB6-8C5 mAb (250 µg/mouse at Days –1, 2, and 5 of infection). Their blood was collected, and number of granulocytes was determined in Diff-Quik blood-stained smears. Data are expressed as means ± SE of the means. *, Significant at P < 0.05 between treatments.

 
CFU counts in neutrophil-depleted and control mice
The evolution of the disease in RB6 mAb-treated and untreated B10.A and A/J was monitored by CFU counts in the lung, liver, and spleen at Weeks 1, 2, 4, and 16 after infection (Fig. 3 ). In the first week of infection, both mouse strains presented significantly increased numbers of pulmonary yeasts when compared with control mice. By weeks 2, 4, and 16 of infection, depleted and nondepleted A/J mice showed similar fungal burdens in the lung and a small number of viable yeasts in liver and spleen. In contrast, at the second week p.i., B10.A animals presented increased fungi number in the lung and spleen. This increased number remained in the lungs by Week 4, but at Week 16, fungal burden was equivalent in depleted and control B10.A mice. However, at this time-point, a marked fungal dissemination to liver and spleen was seen in PMN-depleted B10.A mice: RB6-treated B10.A mice presented 5.52 ± 0.14 log10 CFU in the liver and 4.73 ± 0.22 log10 CFU in the spleen compared with 1.99 ± 0.95 log10 CFU in the liver and 1.98 ± 0.51 log10 in the spleen of IgG controls. Thus, a difference of 3.6 log10 fungal cells was detected in the liver of neutrophil-depleted and nondepleted mice.


Figure 3
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  		Figure 3. Effect of in vivo depletion of PMN cells (mAb RB6-8C5, 250 µg/mouse at Days –1, 2, and 5 of infection) on the severity of infection developed by B10.A and A/J mice i.t.-infected with 1 million P. brasiliensis yeast cells. Recovery of CFU from the lung, liver, and spleen of infected mice treated with normal rat IgG (control) or rat mAb (RB6) was shown. The results are representative of two independent experiments. The bars depict means ± SE of log10 CFU obtained from groups of eight to 10 mice at Weeks 1, 2, 4, and 16 after infection. *, Significantly different (P<0.05) from IgG-treated groups.

 
In susceptible mice, PMN cell depletion led to sustained and increased organ pathology
To further understand the effect of PMN depletion in the severity of PCM developed by susceptible and resistant mice, qualitative and quantitative histopathological analyses of organs were performed in depleted and nondepleted mice. It is interesting that the time-course distribution of lesions presented a similar profile to that observed in CFU data (Fig. 4 ). At Week 1 p.i., all groups of mice presented pulmonary, exudative lesions containing numerous fungi cells. Treated and untreated A/J mice developed, at Weeks 2 and 4 p.i., well-organized, granulomatous lesions, mainly around the bronchi. At Week 16, pulmonary lesions showed central necrosis and decreased fungal loads. In contrast, B10.A mice developed more diffuse pulmonary lesions, and central necrosis was never detected. When compared with controls, B10.A-depleted mice developed, at Weeks 2, 4, and 16 p.i., superior number and size of lesions in the lungs. This fact occurred in A/J-depleted mice only at Week 2 p.i. Furthermore, by 16 weeks, PMN-depleted B10.A mice presented an augmented number and size of lesions in the liver and spleen besides intense fungal dissemination to all analyzed organs (e.g., thymus, heart, and thyroid, among others), leading to precocious death of mice. Figure 5 illustrates diverse patterns of pulmonary and hepatic lesions developed by neutrophil-depleted and control B10.A mice. At Week 16 p.i., extensive and confluent lesions almost replace lung parenquima of RB6-treated mice, whereas in control mice, lesions are smaller and better-defined. Furthermore, the normal livers of control mice contrast those of neutrophil-depleted mice, which contain severe dissemination lesions with an elevated number of yeasts surrounded by discrete, inflammatory exudates. An important feature differentiated B10.A from A/J mice. Treated- and untreated-resistant mice developed increased bronchus-associated lymphoid tissue, mainly at the perivascular tissues, but this fact was not prominent in susceptible mice. These analyses confirm the high susceptibility of B10.A mice to PCM and the more efficient, protective role of PMN leukocytes in this mouse strain.


Figure 4
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  		Figure 4. Quantitative, histological analysis of lesions in the lungs, liver, and spleen of anti-PMN-treated and untreated B10.A and A/J mice at Weeks 1, 2, 4, and 16 after infection. H&E-stained, histological sections of lungs, liver, and spleen of PMN-depleted and untreated B10.A and A/J mice (n=6–8 mice) were analyzed regarding type and size of lesions. *, Significantly different (P<0.05) from IgG-treated groups.

 

Figure 5
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  		Figure 5. Photomicrographs of pulmonary (A and B) and hepatic (C and D) lesions of control (A and C) and neutrophil-depleted (B and D) B10.A mice i.t.-infected with 1 million P. brasiliensis yeasts. At Week 16 of infection, PMN-depleted mice presented extensive and confluent lesions occupying almost all lung parenchyma (B), whereas in control mice, isolated and better-defined lesions affected smaller areas of lungs (A). The livers of control mice presented a normal morphology (C) in contrast to those of neutrophil-depleted mice, which contained severe dissemination lesions with an elevated number of yeasts and discrete inflammatory exudates. H&E, 100x original magnification.

 
PMN depletion decreased survival time of B10.A but not of A/J-infected mice
Mortality of PMN-depleted and nondepleted B10.A and A/J mice was registered daily for a 400-day period, and the median survival time was calculated for each strain. The mortality of RB6-treated B10.A mice was significantly higher than that of IgG-treated controls (Fig. 6 ). The mean survival time of B10.A-treated mice was 158.0 ± 36.22 days, and in the control group, it was 239.5 ± 54.61 days. The deaths of RB6-treated mice began at Day 128, and at Day 238 after infection, all the animals had died. In control B10.A mice, mortality started at Day 161 and ended by Day 291. PMN depletion, however, did not modify the survival time of resistant mice. Only 10% of RB6-treated A/J mice died at Day 3 p.i., and no deaths were registered for the control group.


Figure 6
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  		Figure 6. Survival time of mice after in vivo depletion of PMN leukocytes. B10.A and A/J mice (n=6–8 mice) were depleted or not by anti-PMN mAb and infected by i.t. route with 1 x 106 P. brasiliensis yeast cells, and mortality was registered daily. The results are representative of three independent experiments. *, Significantly different (P<0.05) from control (IgG-treated) group.

 
Depletion of PMN cells does not affect specific DTH reactions of B10.A and A/J mice
As shown in Figure 7 , treated and untreated B10.A animals developed low or absent, cutaneous reactivity to P. brasiliensis antigen at all assayed periods. In contrast, at Weeks 2, 4, and 16 after infection, A/J mice presented positive DTH responses. No significant differences were detected between treated and untreated groups of both mouse strains.


Figure 7
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  		Figure 7. Effect of neutrophil depletion on DTH response of susceptible (B10.A) and resistant (A/J) mice infected i.t. with 106 P. brasiliensis yeast cells. Neutrophil-depleted or nondepleted mice were infected, and DTH reaction was evaluated at Weeks 1, 2, 4, and 16 after infection (n=6–8). Mice were injected intra-footpad with a soluble yeast antigen 24 h before measurement of the footpad response. The bars depict means ± SE of footpad swelling. The dotted line denote means ± 2 SD (confidence interval, 95%) of noninfected B10.A and A/J mice submitted to the footpad test (n=5–8 mice).

 
Effect of neutrophil depletion on serum levels of P. brasiliensis-specific antibodies
As PMN depletion increased the severity of disease developed by susceptible mice, and antibody levels are good markers of PCM severity [1 ], we asked whether this treatment would affect the humoral immune response of susceptible and resistant mice. Figures 8 and 9 demonstrate that some differences were detected between treated and untreated groups of both mouse strains. Compared with untreated animals, increased levels of total Ig (second week), IgA (Weeks 4 and 16), and IgG1 and IgG3 (Week 4) were seen in neutrophil-depleted B10.A groups. No differences in IgG2a and IgG2b levels were detected (Fig. 8) . In A/J mice, IgG2b and IgG3 (Week 2), besides IgG2a (Weeks 2 and 4), appeared in increased levels in PMN-depleted animals (Fig. 9) .


Figure 8
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  		Figure 8. Levels of P. brasiliensis-specific antibodies in the sera of neutrophil-depleted (RB6) and control (IgG-treated) B10.A mice at Weeks 1, 2, 4, and 16 after i.t. infection with 106 yeast cells. Sera were assayed for total Ig (IgT), IgG1, IgG2a, IgA, IgG2b, and IgG3, by using an isotype-specific ELISA as detailed in Materials and Methods. The lines depict means (log2) and SE of serum titers for five to seven mice per group. *, Significantly different (P<0.05) from those for the control group.

 

Figure 9
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  		Figure 9. Levels of P. brasiliensis-specific antibodies in the sera of neutrophil-depleted (RB6) and control (IgG-treated) A/J mice at Weeks 1, 2, 4, and 16 after i.t. infection with 106 yeast cells. Sera were assayed for total Ig, IgG1, IgG2a, IgA, IgG2b, and IgG3, by using an isotype-specific ELISA as detailed in Materials and Methods. The lines depict means (log2) and SE of serum titers for five to seven mice per group. *, Significantly different (P<0.05) from those for the control group.

 
Cytokine profile in the lungs of infected B10.A and A/J mice
Levels of pulmonary cytokines were also altered after neutrophil depletion (Fig. 10 ). B10.A-treated mice presented increased levels of IL-12 and IL-4 at the second and 16th weeks p.i., respectively. Individually, no differences in IFN-{gamma} levels were seen at any p.i. periods; however, the whole curve of IFN-{gamma} secretion was higher than that obtained with the PMN-depleted group. In A/J-depleted mice, augmented levels of IL-12 were detected at the first and 16th week after infection, besides increased amounts of GM-CSF at Week 1. In both mouse strains, PMN depletion did not cause differences in IL-2 and IL-10 production (data not shown).


Figure 10
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  		Figure 10. Levels of cytokines in lung homogenates. At several periods after i.t. infection with 106 yeast cells of P. brasiliensis, lungs from PMN-depleted (RB6) and control B10.A and A/J mice were collected and disrupted in 5.0 mL PBS, and supernatants were analyzed for cytokine content by capture ELISA. The bars depict means ± SE of cytokine levels (six to eight animals per group). *, P < 0.05, compared with control mice.

 
Cytokine profile in the liver of infected B10.A and A/J mice
Only in PMN-depleted B10.A mice, an intense dissemination to liver was seen at the 16th week of infection (5.52±0.14 vs. 1.99±0.95 log10 CFU in depleted vs. untreated B10.A mice, respectively). In contrast, low CFU counts were detected in the liver of treated and untreated A/J mice (0.07±0.07 log10x0.17±0.17 log10, respectively). It is interesting that despite the low, hepatic fungal loads, resistant control mice secreted higher levels of Th1, Th2, and proinflammatory hepatic cytokines than susceptible mice (Figs. 11 and 12 ). In addition, neutrophil depletion led to opposite effects in resistant and susceptible mice, resulting in significantly diminished levels of IFN-{gamma}, IL-2, and GM-CSF in resistant but augmented levels of IL-12, TNF-{alpha}, IFN-{gamma}, and GM-CSF in susceptible mice. Anti-Gr-1 treatment did not alter the already high and low levels of Th2 cytokines secreted by resistant and susceptible mice, respectively (Fig. 12) .


Figure 11
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  		Figure 11. Levels of proinflammatory cytokines in the liver of P. brasiliensis-infected mice. Cell-free supernatants of livers from RB6-treated, IgG-treated, and normal, noninfected B10.A and A/J mice at Week 16 after infection were analyzed to measure cytokines by capture ELISA. The bars depict means ± SE of cytokine levels (six to eight animals per group). *, P < 0.05, compared with IgG-treated mice.

 

Figure 12
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  		Figure 12. Levels of type 1 and type 2 cytokines in the liver of P. brasiliensis-infected mice. Cell-free supernatants of livers from RB6-treated, IgG-treated, and normal, noninfected B10.A and A/J mice at Week 16 after infection were analyzed to measure cytokines by capture ELISA. The bars depict means ± SE of cytokine levels (six to eight animals per group). *, P < 0.05, compared with IgG-treated mice.

 
PMN leukocytes do not play a protective role in a secondary P. brasiliensis infection
Previous studies in our laboratory showed that s.c.-infected B10.A mice but not A/J mice develop a protective and sterilizing immunity to a secondary P. brasiliensis challenge by the i.t. route [8 , 21 ]. To investigate the effect of in vivo depletion of PMN leukocytes in secondary PCM, B10.A mice were s.c.-immunized with yeast cells and 15 days later, were depleted or not of PMN cells (250 µg/mouse RB6 mAb at Days –1, 2, and 5 of infection) and i.t.-challenged with 1 million yeast cells. Eight weeks after infection, no differences were detected in the CFU counts in the lungs, liver, and spleen between IgG-treated and PMN-depleted, vaccinated mice. Furthermore, as previously reported [8 ], both vaccinated groups showed significant immunoprotection and intense DTH reactivity (Fig. 13 ). It is unexpected that neutrophil-depleted mice demonstrated a higher DTH response than IgG-treated, vaccinated mice, although no differences were observed in the levels of specific isotypes and pulmonary cytokines (data not shown).


Figure 13
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  		Figure 13. Effect of neutrophil depletion on secondary PCM. Groups of B10.A mice were infected by the s.c. route with 5 x 106 P. brasiliensis yeast cells and challenged i.t. 15 days later with 106 yeast cells. Control mice received normal rat IgG (s.c./i.t.-IgG), whereas PMN-depleted mice (s.c./i.t-RB6) received antineutrophil mAb at Days –1, 2, and 5 of pulmonary challenge. The i.t. group was previously injected s.c. with saline. Severity of PCM was determined by CFU counts in the lungs, liver, and spleen at Week 8 after i.t. challenge (n=5–6). Before sacrifice, all groups were analyzed regarding DTH reactions by the footpad-swelling test with a soluble P. brasiliensis antigen. The results are representative of two independent experiments. The bars represent the means ± SE of the means. *, P < 0.05, compared with i.t. controls.

 
DISCUSSION

Several studies in human and experimental PCM have suggested the importance of PMN leukocytes in the resistance mechanisms to P. brasiliensis infection. However, to our knowledge, there is no investigation evaluating the in vivo function of these cells. Allied to this, it is well known that PMN cells are important mediators of innate immunity and can influence the adaptative immunity to several pathogens [13 , 22 23 24 ]. These facts led us to investigate the effect of PMN leukocyte depletion in the immunity of genetically resistant and susceptible mice to P. brasiliensis infection. This work shows for the first time that in primary, pulmonary PCM, PMN leukocytes drive the innate immunity of susceptible and resistant mice, but only in the susceptible strain, these cells govern disease outcome. Furthermore, the protective effect of neutrophils does not occur in the secondary PCM of previously s.c.-infected, susceptible animals.

Initially, we characterized the inflammatory cell influx to the lungs of susceptible and resistant mice and verified an increased number of neutrophils and eosinophils in lung cell washings of B10.A mice. This cellular influx may reflect the secretion of different kinds or amounts of cytokines and chemokines governing the expression of endothelial and leukocyte cell adhesion molecules and their ligands [25 , 26 ]. In the course of infection of both mouse strains, no differences in the number of mononuclear cells were found; however, resistant mice presented an increased presence of BALF giant cells, and this fact could be ascribed to the balanced secretion of type 1 and type 2 cytokines by this mouse strain [21 ]. Indeed, IFN-{gamma} and IL-4 secreted by CD4+ T cells have been described as multinucleated giant cell inducers [27 28 29 ].

In vivo treatment of B10.A and A/J mice with anti-Gr-1 mAb led to a decreased number of PMN cells in the peripheral blood of both mouse strains, and this depletion remained until Day 6 after infection. It is interesting that only PMN-depleted A/J mice presented a higher number of PMN cells in the blood at Day 14 of infection, and this could be associated with the increased levels of pulmonary GM-CSF observed at Week 1 of infection. There is also a possibility that this delayed PMN rebound could have protected A/J mice from increased CFU counts and mortality rates induced by anti-RB6 treatment.

The severity of PCM developed by PMN-depleted B10.A and A/J mice was characterized by CFU counts, histopathological analysis, and mortality data. Besides increased fungal loads and organ dissemination, PMN depletion enhanced mortality rates and caused more extensive lesions in susceptible hosts. Altogether, our data strongly indicate that PMN cells exert a protective role in pulmonary PCM at the innate phase of immunity, but their influence at later phases of infection depends on the genetic susceptibility of the host. Using another route of infection, we previously observed a different activation pattern and fungicidal ability of neutrophils obtained from air pouches of resistant and susceptible mice infected s.c. with P. brasiliensis [7 ]. Our findings are also consistent with other microbial infections in which neutrophil function was shown to be dependent on the resistance pattern of hosts [24 , 30 , 31 ].

Despite the differential effect of PMN depletion in B10.A and A/Sn mice, this important leukocyte subpopulation does not appear to play an important role in defining the resistance/susceptibility patterns to P. brasiliensis infection. Neutrophil depletion did not alter the positive and anergic patterns of DTH reactions developed by resistant and susceptible mice, respectively. It is important that the deleterious effect of neutrophil depletion in resistant mice disappears concomitantly with cell-mediated immunity development (as measured by DTH reactions) at Week 2 of infection. Such a recovery was not seen in B10.A mice, in which the harmful effect of neutrophil depletion could not be compensated by T cell immunity. Although DTH reactions are good markers of disease severity and cellular immunity in murine PCM [5 , 12 , 17 ], other studies such as antigen-induced lymphoproliferation, phenotypic characterization inflammatory T cell subsets, and others should be conducted to better characterize the regulatory effect of PMN neutrophils on cellular immunity.

Despite the negligible effect in DTH reactions, PMN cells were shown to exert an immunoregulatory role in antibodies and cytokine secretion in the course of P. brasiliensis infection. In fact, neutrophil-depleted A/J mice produced high titers of IgG2a, IgG3, and IgG2b antibodies, which are Th1- or IFN-{gamma}-regulated isotypes [32 , 33 ], although susceptible mice showed increased levels of specific IgG1, IgA, and IgG3 antibodies, controlled, respectively, by IL-4, transforming growth factor-ß (TGF-ß), and IFN-{gamma} [32 ]. Thus, in resistant mice, PMN depletion skewed some aspects of humoral response to a more pronounced Th1 pattern and indicates that these cells play a partial inhibitory function on Th1 responses. In contrast, enhanced Th1 and Th2 isotypes were secreted by PMN-depleted B10.A mice, and this appears to parallel the observed changes in pulmonary cytokines. Indeed, higher levels of IFN-{gamma}, IL-12, and IL-4 were seen in lung homogenates of PMN-depleted B10.A mice, although in A/J animals, increased levels of IL-12 and GM-CSF were detected. Moreover, the sustained increase in IgA production suggests an increased effect of IL-10 and/or TGF-ß on the B cell switch [32 ] of PMN-depleted, susceptible mice.

In another model of infection, we could previously demonstrate the deleterious and protective roles of PMN cells to susceptible (BALB/c) and resistant (C57BL/6) mice to Leishmania sp. infection, respectively [31 ]. In our P. brasiliensis model, an opposite phenomenon appears to happen, although further work should be done to address this hypothesis. The small, hepatic and splenic fungal loads in control-resistant mice evolved to an even lower yeast number in PMN-depleted mice. In contrast, in the susceptible strain, anti-Gr-1 treatment led to a dramatic fungal dissemination to liver and spleen, besides other organs such as heart, kidney, and others. So, in addition to evidences provided by humoral immunity, the fungal burdens detected in the dissemination organs of B10.A and A/J mice appear to indicate an opposite effect of PMN cells in the adaptative immunity of murine PCM, being protective to susceptible but deleterious to resistant mice.

It is of interest that the enhanced, proinflammatory milieu given by pulmonary cytokines in PMN-depleted B10.A mice could not restrain P. brasiliensis growth. Several investigations have previously demonstrated that macrophage and PMN activation by IFN-{gamma} and other cytokines leads to enhanced fungicidal and fungistatic activity [34 35 36 ]. In our work, however, the enhanced, proinflammatory environment of lung, probably inducing more efficient phagocyte activities, was not sufficient to compensate the deficient PMN cell influx, which resulted, early in infection, in a more severe disease. Moreover, the sustained, deleterious effect caused by PMN leukocyte depletion in B10.A mice appeared to be proportional to the defensive effect they play at the innate phase of immunity.

The increased production of proinflammatory cytokines herein observed could be a result of a diminished ingestion of apoptotic granulocytes by lung macrophages, a phenomenon associated with enhanced secretion of anti-inflammatory mediators such as IL-10, TGF-ß, or prostaglandins [37 , 38 ]. In addition, the early, uncontrolled fungal growth could participate in the increased secretion of cytokines, as our previous work describing the pulmonary model of PCM demonstrated that B10.A mice are good cytokine producers, and early in infection, they secrete elevated levels of pro- and anti-inflammatory cytokines [39 ]. In a recent investigation, we could show that differently from expected, pulmonary macrophages from susceptible mice are easily activated by IFN-{gamma} and secrete high levels of nitric oxide (NO) and IL-12, resulting in efficient fungicidal killing. A/Sn macrophages, conversely, secrete low levels of NO associated with elevated amounts of TGF-ß, which impairs fungal killing [21 , 40 ]. Despite the high efficient innate immunity, the excessive NO production by susceptible mice appears to exert a deleterious effect on adaptative immunity, suppressing T cell-mediated immunity [41 , 42 ]. In this context, the diminished, apoptotic PMN ingestion by pulmonary macrophages as a result of the decreased PMN cell influx induced by RB6 mAb treatment resulted in an increased proinflammatory milieu, which in turn, appears to contribute to the T cell anergy developed by susceptible mice. In addition, the cellular influx governed by this proinflammatory milieu would result in more severe lesions. Our quantitative analysis of fungal lesions appears to support such hypothesis, as more extensive lesions were detected in PMN-depleted B10.A mice. Gazzinelli et al. [43 ] described a similar experimental situation in IL-10-deficient mice infected with a low-virulence Toxoplasma gondii strain. In A/Sn mice, the more proinflammatory milieu resulting from neutrophil depletion could compensate the high levels of endogenous TGF-ß secreted by alveolar macrophages [20 ] and drive the immune response to a more defined type 1 pattern.

Comparing cytokine levels present in liver homogenates of infected control mice, we could verify that despite the low number of fungal cells, A/J mice produce high levels of type 1 and type 2 cytokines, and this could be ascribed to the efficient T cell immunity developed by these mice [12 , 21 ]. On the contrary, in the livers of B10.A mice, the high fungal loads were associated with low levels of cytokines. Furthermore, depletion of PMN cells resulted in increased amounts of IL-12, GM-CSF, IFN-{gamma}, and TNF-{alpha} in B10.A mice, and in A/J mice, levels of GM-CSF, IFN-{gamma}, and IL-2 appeared in lower levels than that observed in controls. Thus, it appears that high fungal loads in the livers of T cell-unresponsive B10.A mice led to an uncontrolled, inflammatory response, whereas in A/J mice, which are able to mount a well-balanced Th1/Th2 response [12 , 21 ], fungal clearance results in lowered immunity and inflammatory response.

The observed alterations in cytokines production and antibody isotypes could be a result of the increased fungal loads as well as by the altered pattern of innate immunity caused by PMN depletion. Indeed, the immune system is so rich of interconnected elements that an initial alteration in the inflammatory response can affect the immunological reactivity that further develops. This would affect control of fungal growth and correspondent antigen presentation to T and B cells that occurs at draining lymph nodes. In our work, the increased fungal growth and the altered environment of innate immunity caused by PMN depletion could have changed some characteristics of antigen presentation to lymphocytes resulting in different patterns of adaptative immunity.

Differently from primary infection, neutrophil depletion did not alter the immunoprotective effect conferred by a previous s.c. inoculation of P. brasiliensis yeasts in susceptible mice. As s.c. infection induces high levels of DTH reactivity in susceptible hosts [8 ], in secondary infection, the mAb-induced neutropenia appears to be compensated by T cell-mediated immunity. Our results appear to contrast with those described by McEwen et al. [44 ], showing that only immunologically activated PMN are fungicidal for P. brasiliensis yeasts. However, the different experimental designs (in vivo vs. ex vivo experiments), mouse strains, and inflammatory agents used to elicit PMN migration appear to explain those apparent discrepancies. It is interesting that anti-Gr-1 treatment of s.c.-infected mice led to enhanced DTH reactions. Although the main effect of anti-Gr-1 treatment is neutrophil depletion [13 14 15 ], recent investigations have demonstrated that this antibody also affects other myeloid cell populations expressing Gr-1 antigen, including a suppressive, immature myeloid cell subset, which induces tolerance in CD8 T cells [45 , 46 ]. Further studies are needed to investigate if a suppressive, Gr-1-positive cell plays a regulatory role in pulmonary PCM, as our previous studies on CD8 T cell depletion showed the presence of a CD8 T cell subset with suppressive activity on DTH reactions [39 ].

As a whole, our data about PMN depletion at the innate phase of infection showed that neutrophils are important cells in host defense to P. brasiliensis infection, and their effect depends on the genetic background of the host and is more pronounced in the susceptible strain. Our results also demonstrated that studies using a single isogenic strain of mice or phase of immune response do not reveal the gamut of influences that a particular cell subset can exert on the immunity to a certain microbial pathogen.

ACKNOWLEDGEMENTS

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisas (CNPq). We are grateful to T. Alves and B. P. Albe for technical assistance.

Received January 24, 2006; revised February 23, 2006; accepted February 27, 2006.

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