ABSTRACT
Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellular immunity and gamma interferon (IFN-γ) production. To assess the role of interleukin-4 (IL-4), a Th2-inducing cytokine, pulmonary paracoccidioidomycosis was studied in IL-4-deficient (IL-4−/−) and wild-type (WT) C57BL/6 mice at the innate and acquired phases of immune response. Forty-eight hours after infection, equivalent numbers of viable Paracoccidioides brasiliensis yeast cells were recovered from the lungs of IL-4−/− and WT mice intratracheally infected with one million fungal cells. Alveolar macrophages from infected IL-4−/− mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secreted higher levels of nitric oxide. Compared with WT mice, IL-4−/− animals presented increased levels of pulmonary IFN-γ and augmented polymorphonuclear leukocyte influx to the lungs. Decreased pulmonary fungal loads were characterized in deficient mice at week 2 postinfection, concomitant with diminished presence of IL-10. At week 8, lower numbers of yeasts were recovered from lungs and liver of IL-4−/− mice associated with increased production of IFN-γ but impaired synthesis of IL-5 and IL-10. However, a clear shift to a Th1 pattern was not characterized, since IL-4−/− mice did not alter delayed-type hypersensitivity anergy or IL-2 levels. In addition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophage colony-stimulating factor, IL-3, monocyte chemotactic protein 1, and specific antibody isotypes. In IL-4−/− mice, well-organized granulomas restraining fungal cells replaced the more extensive lesions containing high numbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice. These results clearly showed that genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.
Paracoccidioides brasiliensis is a pathogenic fungus restricted to Latin America. The natural route of infection is the inhalation of fungal particles, which usually leads to an asymptomatic infection. The disease presents a wide range of clinical and immunological manifestations, varying from benign and localized to severe and disseminated forms (9). Classical studies on the immune responses developed by patients with polar forms of paracoccidioidomycosis (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 (20). More recent works have demonstrated a Th1-biased immune response in the asymptomatic and mild forms of PCM, whereas a Th2 pattern has been associated with the severe disease. When compared with patients with the mild form of the disease, patients presenting the more disseminated infection produce higher levels of type 2 cytokines (interleukin-4 [IL-4], IL-5, IL-10, and transforming growth factor β) and antibodies (immunoglobulin E [IgE], IgG4, and IgA) in addition to eosinophilia and impaired secretion of gamma interferon (IFN-γ) (3, 5, 28, 36, 41).
A greater insight into the immune response elicited by P. brasiliensis has come from the use of murine models. Our laboratory developed an isogenic murine model of PCM where B10.A mice were susceptible to and A/Sn mice were resistant to mouse strains of P. brasiliensis. Although resistance of A/Sn mice was linked with the secretion of T helper 1 cytokines (IL-2 and IFN-γ), susceptibility was not clearly associated with a Th2 pattern, since IL-4 was not found in the supernatants of antigen-stimulated lymph node cells from infected B10.A mice (11, 29). When infected by the intratracheal (i.t.) route, B10.A and A/Sn mice maintained the same resistant and susceptible pattern observed after the intraperitoneal infection (15). Both strains of mice secrete type 1 and type 2 cytokines in the lungs, but the progressive disease developed by susceptible B10.A mice appears to be governed by type 2 cytokines (11, 14-16). Furthermore, previous results with the murine model of infection showed that depletion of IFN-γ or genetic deficiency of this cytokine led to exacerbated disease (14, 50). It was also verified that administration of recombinant IL-12 is protective to susceptible mice which presented with a less-disseminated disease but a high inflammatory reaction in the lungs (2).
Since IL-4 directs Th2 development during the innate phase of immune response (23, 46) and has been shown to inhibit Th1-dominated immunity, we assumed that IL-4-deficient (IL-4−/−) mice would develop a more-efficient cellular response and protective immunity to P. brasiliensis infection than their IL-4-competent counterparts. Thus, the effect of IL-4 was investigated in the innate (48 h after infection) and acquired (weeks 2 and 8 of infection) phases of immune response to P. brasiliensis infection. After intratracheal infection with one million P. brasiliensis yeast cells, the severity of infection was monitored by several immunological parameters. PCM in IL-4−/− mice was less severe than in IL-4-normal mice and was associated with an impaired Th2 immune response, leading to enhanced fungicidal activity of alveolar phagocytes.
MATERIALS AND METHODS
Animals.Breeding pairs of homozygous IL-4-deficient (IL-4−/−) and wild-type (WT) control C57BL/6 mice (intermediate susceptibility to P. brasiliensis) were bred at the University of São Paulo animal facilities under specific-pathogen-free conditions in enclosed-top cages. Clean food and water were given ad libitum. Mice were 8 to 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.The P. brasiliensis 18 isolate, which is highly virulent, was used throughout this study. To ensure the maintenance of its virulence, the isolate was used after three serial animal passages (30). P. brasiliensis 18 yeast cells were then maintained by weekly subcultivation in semisolid Fava Netto culture medium (18) at 35°C and used on the seventh day of culture. The fungal cells were washed in phosphate-buffered saline (PBS; pH 7.2) and counted in a hemocytometer, and the concentration was adjusted to 20 × 106 fungal cells ml−1. The viability of fungal suspensions, determined by Janus Green B vital dye (Merck, Darmstadt, Germany), was always higher than 80%.
P. brasiliensis infection.Mice were anesthetized and submitted to i.t. P. brasiliensis infection as previously described (15). Briefly, after intraperitoneal anesthesia, the animals were infected with 106P. brasiliensis 18 yeast cells, contained in 50 μl of PBS, by surgical i.t. inoculation, which allowed dispensing of the fungal cells directly into the lungs. The skins of the animals were then sutured, and the mice were allowed to recover under a heat lamp. Mice were studied during an early period (48 h after infection) and a late period (at week 8).
Bronchoalveolar lavage fluid (BALF).Forty-eight hours after i.t. infection, mice were lavaged after cannulation of the 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 of PBS) and normal mice of both mouse strains. The lungs were lavaged by repeated injections of 0.5 ml of sterile PBS (final volume, 2.0 ml). The recovered fluid was spun at 1,200 rpm, the supernatant was removed, and cells were analyzed for fungicidal activity and leukocyte subsets.
Assessment of leukocyte population.For differential counts, samples of lung cell suspensions were cytospun (Shandon Cytospin, Pittsburgh, Pa.) onto glass slides and stained by the Diff-Quik blood stain (Baxter Scientific, Miami, Fla.). A total of 200 to 400 cells were counted from each sample. The absolute number of a leukocyte subset was calculated by multiplying the percentage of each subset in an individual sample by the total number of lung leukocytes in that mouse.
Fungicidal activity of alveolar macrophages.The BALFs obtained from individual mice were centrifuged, and pellets resuspended in RPMI containing 10% fetal calf serum, 2 mM l-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Cell suspensions were adjusted at 4 × 105 cells/ml of culture medium, and 0.5 ml was dispensed in 24-well tissue culture plates for a 2-h adhesion step. Nonadherent cells were transferred to another plate. Plates containing adherent and nonadherent cells were centrifuged, supernatants were discarded, and cells were incubated with 0.5 ml of culture medium supplemented or not with 100 U of IFN-γ (Pharmingen, San Diego, Calif.)/ml. 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. Wells were then washed five times with 0.5 ml of distilled water, and suspensions were collected in individual tubes. Cells were centrifuged and resuspended in culture medium, and aliquots (100 μl) and serial dilutions were assayed for the presence of viable yeasts.
Assay for CFU.The numbers of viable microorganisms in BALF, cell cultures, and infected organs (lungs, liver, and spleen) from experimental and control mice were determined by counting the number of CFU. Animals from each group were sacrificed, and the enumeration of viable organisms was done as previously described (47). Briefly, aliquots (100 μl) of the cellular suspensions and serial dilutions were plated on brain heart infusion agar (Difco, Detroit, Mich.) supplemented with 4% (vol/vol) horse serum (Instituto Butantan, São Paulo, Brazil) and 5% P. brasiliensis 192 culture filtrate, the latter constituting a source of growth-promoting factor. 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 colonies are expressed as means ± standard errors.
Histopathologic analysis.The left lung of each mouse was removed, fixed in 10% formalin, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin for an analysis of the lesions and silver stained for fungal evaluation. Pathological changes were analyzed based on the size, morphology, and cell composition of granulomatous lesions, presence of fungi, and intensity of the inflammatory infiltrates.
Measurement of cytokines.Mice were infected i.t. with P. brasiliensis, and their right lungs were removed aseptically and individually disrupted in 4.0 ml of RPMI 1640 medium (Gibco BRL). 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, Mass.), and stored at −70°C. The levels of IL-2, IL-4, IL-5, IL-10, IFN-γ, IL-12, tumor necrosis factor alpha (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from Pharmingen. Reagents for monocyte chemotactic protein 1 (MCP-1) were obtained from R&D Systems (Minneapolis, Minn.). The ELISA procedure was performed according to the manufacturer's protocol. The concentrations of cytokines were determined with reference to a standard curve for serial twofold dilutions of murine recombinant cytokines. The lower limits of detection standard curves were 32.4, 7.8, 7.0, 7.8, 20.0, 15.0, 31.2, 15.3, 7.8, and 31.2 pg/ml for IL-2, IL-4, IL-5, IL-10, IFN-γ, IL-12, TNF-α, GM-CSF, IL-3, and MCP-1, respectively. As an additional control, lung homogenates were added to recombinant cytokines used to obtain standard curves; no interference was detected, indicating the absence of inhibitory substances (e.g., soluble cytokine receptors).
NO production.NO production was quantified by the accumulation of nitrite (as a stable end product) in the supernatants by a standard Griess reaction. Briefly, 50 μl of supernatants was removed from 24-well plates and incubated with an equal volume of Griess reagent (1% sulfanilamide-0.1% naphthylene diamine dihydrochloride-2.5% H3PO4) at room temperature for 10 min. The absorbance at 550 nm was determined with a microplate reader. The conversion of absorbance to micromolar NO was deduced from a standard curve by using a known concentration of NaNO2 diluted in RPMI medium. All determinations were performed in duplicate and expressed as micromolar NO.
DTH assay.The DTH reactions were always evaluated just before sacrifice of the same animals used in the CFU assays by the footpad test according to previously determined conditions (19). Briefly, mice were inoculated with 25 μl of Fava Netto antigen (18), and the footpad thickness was measured with a caliper (Mitutoyo Corporation, 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.
Measurement of serum P. brasiliensis-specific isotypes.Specific isotypes levels (total IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3) were measured by a previously described ELISA (15) employing a cell-free antigen (12) prepared by using a pool of different P. brasiliensis isolates (339, 265, and 18). The average of the optical densities obtained with sera from control mice (PBS inoculated), diluted 1:20, was considered the cutoff for each respective isotype. Optical densities for each dilution of experimental sera were compared to the control values. The titer for each sample was expressed as the reciprocal of the highest dilution that presented an absorbance higher than the cutoff.
Statistical analysis.Data were analyzed by Student's t test or two-way analysis of variance depending on the number of experimental groups (54). P values under 0.05 were considered significant.
RESULTS
Early in infection, lung tissue and BALF from IL-4−/− and control mice present equivalent fungal loads and nitrite concentrations.Groups (n = 6 to 8) of IL-4−/− C57BL/6 mice and their WT controls (IL-4+/+) were infected i.t. with one million P. brasiliensis yeast cells, and equivalent fungal counts were detected in lung tissue and BALF 48 h after infection. In addition, both groups of mice presented similar levels of nitrite in lung homogenates (Fig. 1).
(A) Number of viable yeasts (CFU counts) recovered from lung tissue and BALF of IL-4−/− and WT C57BL/6 mice 48 h after P. brasiliensis infection with one million yeast cells; (B) nitrite concentrations in lung homogenates. No nitric oxide was detected in BALF supernatants. Data are expressed as means ± standard errors. Similar results were obtained from two separate experiments.
IL-4 deficiency determines early increased leukocyte recruitment into lungs.To better characterize the early phase of pulmonary infection, leukocyte recruitment to the alveolar spaces of P. brasiliensis-infected IL-4−/− and IL-4+/+ mice was compared. Groups of sham-infected and normal mice were included as additional controls. As can be seen in Fig. 2A, no differences in the relative number of mononuclear and polymorphonuclear (PMN) cells were found between the WT and IL-4−/− groups. The same result was obtained with untreated groups of normal WT and IL-4-deficient mice (data not shown). Compared with WT mice, a significantly augmented number of total cells, mainly due to the influx of PMN leukocytes, was observed in the BALF of IL-4-deficient mice (Fig. 2B).
Relative (A) and absolute (B) numbers of leukocytes in BALF of mice inoculated i.t. with one million P. brasiliensis yeast cells. Lungs of IL-4−/− and WT mice (n = 6 to 8) were lavaged with PBS 48 h after infection, and cell suspensions were cytospun onto glass slides and stained with the Diff-Quik blood stain. The absolute number of a leukocyte subset (PMN neutrophils or mononuclear cells [MNC]) was calculated as described in Materials and Methods. Data are expressed as means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with WT controls).
Adherent cells from BALF of IL-4−/− mice present increased fungicidal activity and nitric oxide production.To determine the role of IL-4 in the fungicidal activity of inflammatory cells recruited early into the lungs, adherent and nonadherent BALF cells were cultivated for 72 h in the presence or absence of exogenously added IFN-γ and recovered viable P. brasiliensis yeasts were enumerated. Figure 3A shows the increased fungicidal ability of adherent cells (macrophages) of IL-4-deficient mice when compared with their normal counterparts. On the contrary, the killing ability of nonadherent cells (mostly PMN leukocytes) was similar for both mouse strains (Fig. 3B). In agreement, adherent cells of IL-4-deficient mice secreted higher levels of NO than those produced by IL-4+/+ animals. The addition of IFN-γ to cell cultures increased fungicidal activity and NO production of adherent cells but did not alter fungal growth and NO secretion by nonadherent cells (Fig. 3C and D).
Fungicidal activity (A and B) and nitric oxide production (C and D) by macrophages (A and C) and PMN leukocytes (B and D) from BALF stimulated or not by IFN-γ (100 U/ml) and cultivated in vitro for 72 h. Supernatants and cell suspensions were collected and analyzed for nitrite content and the presence of viable yeast cells, respectively. Data are expressed as means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared with WT controls). MNC, mononuclear cells.
Early in infection, IL-4-deficient mice present increased amounts of pulmonary IFN-γ.To determine whether the increased fungicidal ability of inflammatory cells was associated with a different milieu of pulmonary cytokines, we characterized the secretion of pro- and anti-inflammatory cytokines in the lungs of mice at the early phase of infection (Fig. 4). IFN-γ production was significantly increased in IL-4−/− mice compared to than in IL-4+/+ mice after 48 h of infection. The former strain also presented a trend to diminished levels of Th2 cytokines (IL-5 and IL-10), but these differences did not reach significant levels.
IL-4 deficiency is associated with early increased levels of pulmonary IFN-γ. Forty-eight hours after P. brasiliensis infection, lungs from IL-4−/− and WT control C57BL/6 mice were collected and disrupted in 4.0 ml of RPMI 1640 medium, and supernatants were analyzed for cytokine content by capture ELISA. The bars depict means ± standard errors of the means of cytokine levels (n = 6 to 8). *, P < 0.05 (compared with WT controls).
IL-4−/− mice develop less-severe PCM.After analyzing the early phase of infection, we compared the severity of disease developed by IL-4-deficient and control mice at the acquired phase of immunity. Diminished fungal growth was detected in the lungs of IL-4−/− mice at week 2 postinfection. At week 8, the lungs and livers of these mice presented diminished fungal loads compared to those of IL-4-normal mice (Fig. 5).
Recovery of viable yeasts (CFU) from lungs, livers, and spleens of IL-4-deficient (IL-4−/−) and IL-4-sufficient (WT) mice at weeks 2 (A) and 8 (B) after i.t. infection with 106 yeast cells. The bars depict means ± standard errors of the means of log10 CFU obtained from groups of 6 to 8 mice. *, P < 0.05 (compared with WT controls).
Levels of pulmonary cytokines in the acquired phase of immunity.As at the innate phase of immunity to pulmonary PCM increased amounts of IFN-γ were found in the lungs of IL-4-deficient mice, we sought to determine whether this fact would have altered the secretion of pulmonary cytokines at later phases of infection. At week 2, decreased levels of IL-10 and MCP-1 were found in lung homogenates of IL-4-deficient mice (Fig. 6A). By week 8, augmented levels of IFN-γ but not IL-2 were concomitant with decreased amounts of IL-5, IL-10, MCP-1, IL-12, IL-3, and GM-CSF (Fig. 6B) in the lungs of IL-4−/− mice.
Levels of cytokines in lung homogenates. At 2 (A) and 8 (B) weeks after i.t. infection with 106 yeast cells of P. brasiliensis, lungs from IL-4-deficient and WT control mice were collected and disrupted in 4.0 ml of RPMI 1640 medium, and supernatants were analyzed for cytokine content by capture ELISA. The bars depict means ± standard errors of the means of cytokine levels (6 to 8 animals per group). *, P < 0.05; **, P < 0.01 (compared with WT controls).
Absence of IL-4 does not revert DTH anergy of C57BL/6 mice.As CFU counts demonstrated the protective role of genetic deficiency of IL-4 in murine PCM, we asked whether absence of this cytokine would also have the ability to modulate DTH reactions. At both periods of acquired immunity studied, WT C57BL/6 mice infected i.t. with P. brasiliensis did not present positive DTH reactions as well as their IL-4-deficient counterparts (data not shown). Thus, IL-4 deficiency did induce a less-severe PCM but was not able to eliminate the DTH anergy of C57BL/6 mice.
IL-4 deficiency results in mild lung pathology.Forty-eight hours after P. brasiliensis infection, an acute and diffuse inflammatory infiltrate mainly composed of PMN cells and recently migrated monocytes was detected in the lungs of infected mice (n = 7). The whole inflammatory picture was similar in both experimental groups, although in some IL-4-deficient mice (3 of 7) a more peribronchial and perivascular focal pattern was observed and appears to predict the more-organized lesions developed at week 8 after infection. At this time point, when compared with their normal counterparts, IL-4−/− mice presented small, more-compact granulomas, which were infiltrated with a diminished number of inflammatory cells causing a mild disruption of the pulmonary parenchyma (Fig. 7C). Grocott staining allows the demonstration of budding yeast cells at the center of granulomas of IL-4−/− mice (Fig. 7D), suggesting that this morphology is sufficient to avoid fungal dissemination. In contrast, the more-severe pulmonary lesions developed by IL-4-normal mice were randomly distributed and composed of isolated or confluent granulomas of various sizes containing many fungal cells surrounded by macrophages, lymphocytes, and plasma cells (Fig. 7A and B). An equivalent cellular composition was observed in the IL-4−/− strain. However, granulomas of IL-4−/− mice showed a more prominent presence of PMN leukocytes, usually at the center of lesions in close proximity to fungal cells.
Photomicrographs of granulomatous lesions from IL-4−/− and WT control C57BL/6 mice i.t. infected with 106P. brasiliensis cells. Pulmonary lesions of WT (A and B) and IL-4−/− (C and D) mice at week 8 postinfection are shown. At this period of infection, the granulomatous inflammation is more extensive and confluent in WT mice than in IL-4−/− animals. (A and C) hematoxylin and eosin stain (magnification, ×100); (B and D) Grocott stain (magnification, ×100).
Absence of IL-4 diminishes production of P. brasiliensis-specific isotypes.We also studied the influence of IL-4 on the production of P. brasiliensis-specific antibodies by IL-4-deficient and control mice (Fig. 8). As expected, due to the IL-4 influence on B-cell growth and antibody class switch (48, 49), IgG, IgA, and IgM antibodies were synthesized at lower levels by IL-4-deficient mice than by WT mice. Among the IgG subclasses, IgG1, IgG2b, and IgG3 were produced at decreased levels by IL-4-deficient mice. IgG2a, however, was detected at similar levels in the serum of both mouse strains.
Levels of P. brasiliensis-specific antibodies in sera of IL-4−/− and WT control mice at week 8 after i.t. infection with 106 yeast cells. Sera were assayed for total Ig, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 by using an isotype-specific ELISA as detailed in Materials and Methods. The bars depict means (log2) ± standard errors of serum titers (6 to 8 mice per group). *, significant difference (P < 0.05) from WT group.
DISCUSSION
This work showed that IL-4 can exert a deleterious role in the pulmonary infection caused by P. brasiliensis. Using IL-4- deficient mice and their IL-4-sufficient counterparts, both the onset and later periods of infection were explored, with an emphasis on trying to understand the influence of IL-4 on the innate and acquired immunity developed by C57BL/6 mice infected with yeast cells. At 48 h after infection, equivalent number of viable P. brasiliensis cells and NO production were detected in the lungs and bronchoalveolar spaces of IL-4-deficient and -sufficient strains. IL-4 deficiency, however, resulted in increased inflammatory reactions in the lungs, with elevated numbers of PMN cells recovered from alveolar spaces at this early phase of infection. When cultivated in vitro, in the presence or not of exogenous IFN-γ, nonadherent cells (mostly PMN leukocytes) of WT mice showed fungicidal ability equivalent to that presented by those of IL-4−/− mice. As deficient mice presented a twofold increase of inflammatory cells in the lungs mainly due to the PMN cell influx, we can suppose that, at the onset of infection, PMN leukocytes from IL-4−/− mice could exert a more-efficient clearance of yeast cells. Yet, compared with those of IL-4-deficient mice, adherent cells of WT mice demonstrated a lower ability to secrete NO, and this diminished production correlated with impaired fungicidal activity. The addition of exogenous IFN-γ to cell cultures increased the NO production and killing ability of adherent cells of both mouse strains but did not revert the macrophage-inhibiting activity of IL-4 in WT cells. As the main P. brasiliensis killing mechanism of PMN cells is oxygen dependent (8) and that of macrophages is nitric oxide mediated (8, 21), we can suppose that both cell types can exert a synergistic effect on the control of fungal growth. However, other effector mechanisms could play a role at this period of innate immunity. The inflammatory milieu of the lungs seems to activate phagocytic cells, but at this time point, the enhanced killing ability of these cells was not sufficient to significantly decrease fungal loads recovered from the lung parenchyma. When cultivated in vitro for a further 72 h, macrophages from IL-4 knockout (KO) mice could show their enhanced ability to kill yeast cells and secrete NO. The difference between cells from WT and IL-4 KO mice appears to be due to the increased levels of IFN-γ present in lung homogenates of the latter strain. As at this early period of infection the adaptive immune response is not well established, we can speculate that the IFN-γ production was mainly due to the cytokine secretion by other cells of innate immunity such as NK cells (53).
As in murine PCM, La Flamme et al. (34) detected an enhanced ability of IL-4−/− macrophages to secrete NO and observed a higher number of PMN leukocytes in the lesions of IL-4-deficient mice infected with Schistosoma mansoni than in the WT control. It was also verified that macrophages, lymphocytes, and PMN cells were able to secrete NO in culture supernatants, although the former cells were the main producers of this reactive species. The low levels of NO secreted by PMN leukocytes from P. brasiliensis-infected mice could also play a role in fungal killing, although this mechanism was not yet described for murine PCM. However, whatever the mechanisms PMN cells use to kill P. brasiliensis, its abundance and constant presence near yeast cells at the center of lesions lead us to suppose they have a very important role in the in vivo control of fungal growth.
The fact that IL-4-deficient mice infected with several pathogens present impaired Th2 responses but enhanced or not Th1 responses (33, 38, 42) led us to characterize the pattern of cytokines in the lungs at weeks 2 and 8 of P. brasiliensis infection. The impaired fungal growth in the lungs of IL-4-deficient mice at the former period was associated with decreased production of IL-10, a well-known macrophage-deactivating cytokine. The diminished number of viable yeast cells recovered from the lungs and liver of IL-4−/− mice at week 8 was concomitant with increased production of pulmonary IFN-γ and significantly impaired secretion of IL-5 and IL-10. As equivalent levels of IL-2 and anergy in DTH reactions were found in both mouse strains, a shift to a Th1 pattern of immune response could not be characterized. However, the high amounts of IFN-γ associated with diminished or absent levels of Th2 cytokines (IL-4, IL-5, and IL-10) appear to have determined a cytokine milieu deviated to a more proinflammatory pattern. This new cytokine balance would favor phagocyte activation and better control of fungal growth. Although a positive correlation between the presence of DTH reactions and protective immunity to murine and human PCM is usually found (10, 20), in some experimental conditions this association is controversial and some researchers have claimed they are not causally related (27). Indeed, in pulmonary PCM of susceptible B10.A mice, depletion of CD8+ T cells led to a more-severe disease concomitant with prominent DTH reactions, indicating a suppressive activity of CD8+ T lymphocytes on the cellular immunity mediated by CD4+ T cells (13). More importantly, the increased production of NO shown by IL-4 KO mice may be responsible for the impaired cellular immunity as previously described for murine PCM of C57BL/6 mice (7, 39). Thus, there are different mechanisms that can lead to DTH anergy, and these manifestations are dependent on the genetic pattern of hosts. Furthermore, IL-4 appears to be important or necessary in the inductive phase of cellular immunity. Most studies on IL-4 neutralization during infections caused by intracellular pathogens revealed the Th2-inducing activity of this cytokine and its suppressive effect on DTH reactions. However, under certain conditions, IL-4 induces the development of Th1-mediated DTH reactions (45, 51).
The lower CFU counts in IL-4−/− mice were associated with decreased secretion of several cytokines. Interestingly, IL-12 was detected at lower levels in lung homogenates at week 8. As previously reported, IL-4 can prime macrophages for synthesis of IL-12 (37, 44) and could be acting as a priming factor for IL-12 secretion after P. brasiliensis infection only in IL-4-sufficient mice. The reduced inflammatory reaction associated with diminished levels of IL-12 in lung homogenates is also consistent with previous results on the proinflammatory effect of IL-12 administration to susceptible mice. Despite the less-disseminated infection, the early treatment with exogenous IL-12 induced a highly increased inflammatory reaction that remained through week 8 after infection (2).
Interestingly, IL-4 deficiency was accompanied by decreased levels of IL-5, IL-3, and GM-CSF, whose genes, together with that coding for IL-4, map on the cytokine gene cluster on mouse chromosome 11 (35). The concomitant rise and fall of these cytokines is not an unusual fact, and coordinate regulation of cytokine genes clustered on the same chromosome was seen in other pathologies, such as the late-phase cutaneous reaction in atopic subjects (31) and in Th2 clones (32) as well as in the more-severe pulmonary PCM induced by in vivo depletion of CD8+ T cells or IL-12 by specific monoclonal antibodies (V. L. G. Calich, T. Alves, and E. E. W. Molinari-Madlum, unpublished data).
Although the protective effect of IL-3 and GM-CSF was not described for murine PCM, Allendoerfer et al. (1) demonstrated the protective function of GM-CSF in murine histoplasmosis. Furthermore, the stimulatory activity of IL-3 and GM-CSF on the fungicidal activity of phagocytes was also described previously (40). So, it is difficult to understand the less-severe PCM associated with low levels of these mediators. However, we can speculate that in IL-4−/− mice these activities were replaced by the increased production of IFN-γ, unchanged secretion of TNF-α, and stimulatory activity on phagocytes for fungal killing (14, 50). On the other hand, the decreased production of several pro- and anti-inflammatory cytokines could be due to the less-severe infection observed in IL-4-deficient mice.
MCP-1 is a C-C chemokine produced by several cell types and is chemotactic for monocytes and lymphocytes (22, 43). MCP-1 is produced in the lungs of mice infected i.t. with Cryptococcus neoformans, and the in vivo neutralization or absence of its receptor (CCR2) results in a dramatic reduction in macrophage and CD8+-T-lymphocyte recruitment and elimination of fungal cell clearance (26, 52). At weeks 2 and 8 of infection, WT C57BL/6 mice infected with P. brasiliensis produced MCP-1 in higher amounts than their IL-4-deficient counterparts. The decreased production of this chemotactic chemokine was associated with decreased recruitment of inflammatory cells, as detected by lung histology, indicating that this mediator could be involved in the organization of P. brasiliensis granulomas. The concomitant diminished production of MCP-1 and Th2 cytokines (IL-5 and IL-10) detected in IL-4−/− mice infected with P. brasiliensis is consistent with the previously reported Th2-inducing activity of this chemokine (24). As a whole, the pattern of cytokines detected in IL-4-deficient mice indicated that IL-4 may play a role in the pathogenesis of P. brasiliensis infection by indirect regulation of fungal growth and control of other cytokines secreted at the site of infection. This new lung environment appears to lead to a more-efficient phagocyte activation and inflammatory reaction that impairs fungal dissemination to the liver and diminishes lung pathology by decreasing the recruitment of inflammatory cells to pulmonary lesions.
Another prominent feature of PCM in IL-4-deficient mice was the less-severe lung pathology, where well-organized granulomas containing yeast cells circumscribed by small numbers of inflammatory leukocytes replace the more exuberant inflammatory reactions developed by WT mice. This inflammatory architecture developed by IL-4−/− mice appears to be highly efficient in avoiding fungal growth outside the lesion and preserving the pulmonary tissue. The increased levels of IFN-γ detected in lung homogenates appear to contribute to the lesion morphology, since in previous studies with IFN-γ-depleted (14) and KO mice (50), the focal granulomatous reaction was replaced by nonorganized inflammatory exudate that destroys the normal lung parenchyma. In murine schistosomiasis, a well-studied Th2 disease, granuloma size was also reduced in IL-4−/− infected mice, which, however, display a more-severe disease that correlates with increased production of NO by lesion cells isolated from liver granulomas (17).
In contrast with these results with C57BL/6 mice, previous studies with the B10.A strain, highly susceptible to P. brasiliensis, showed that IL-4 can exert a protective function in pulmonary PCM. In vivo depletion of IL-4 did not alter the pattern of cytokines in the lungs but led to increased fungal load in the lungs of infected B10.A mice (10, 16). In the C57BL/6 strain, however, equivalent treatment with anti-IL-4 monoclonal antibodies induced a less-severe pathology associated with increased production of pulmonary IFN-γ (C. Arruda, R. C. Valente-Ferreira, A. Pina, and V. L. G. Calich, unpublished data). This result clearly shows that IL-4 is an important mediator of susceptibility in the C57BL/6 strain, but other mechanisms, such as excessive NO production, could account for B10.A susceptibility (39). The deleterious effect of IL-4 in the C57BL/6 strain is similar to that obtained by Hostetler et al. (25) with BALB/c mice depleted with high doses of anti-IL-4 monoclonal antibodies. Thus, in murine PCM, IL-4 can play a protective or exacerbating effect on the infection depending on the genetic pattern of the host. A dual role for IL-4 has already been described for other experimental pathologies. In murine candidiasis, IL-4-deficient mice were more resistant to early Candida albicans infection but failed to mount a protective Th1 immune response in the late phase and succumbed due to the impaired ability to produce efficient amounts of IFN-γ and IL-12 (37). Biedermann et al. (6) clearly showed the opposing effects of IL-4 in murine leishmaniasis. The presence of IL-4 during the initial activation of dendritic cells induces the production of IL-12 by these cells and development of Th1 lymphocytes; when present later, during the period of T-cell priming, IL-4 induced a Th2 immune response. Furthermore, the overlapping functions of several mediators of immune response and its different manifestation in the context of different genetic backgrounds were demonstrated. Indeed, studying resistance to infection with Trichuris muris, it was verified that IL-4−/− mice in a C57BL/6 background are susceptible, whereas IL-4−/− mice in a BALB/c background are resistant (4).
Previous studies from our group have demonstrated the prevalence of Th2-regulated isotypes (IgG2b, IgG1, and IgA) in the most severe forms of the murine disease and a preferential production of the Th1-controlled isotype (IgG2a) in the benign infection (10, 15, 29). In this investigation, we verified that all isotypes of specific antibodies, except IgG2a, were produced in lower amounts by IL-4-deficient mice than by IL-4-sufficient C57BL/6 mice. This lack of effect on the level of IgG2a could be due to the increased secretion of IFN-γ, a well-characterized IgG2a-switching factor (46). Despite the lower levels, IL-4−/− mice secreted all anti-P. brasiliensis isotypes, demonstrating that IL-4 activity could be replaced by other cytokines.
In conclusion, the genetically determined absence of IL-4 is protective to P. brasiliensis infection of C57BL/6 mice, and its effect can be indirectly seen as early as 48 h after fungal inoculation by the increased ability of IL-4−/− lung phagocytes to kill P. brasiliensis yeast cells. The precocious increased production of pulmonary IFN-γ, the enhanced killing ability of alveolar macrophages, and the augmented inflammatory PMN influx into the lungs appear to lead to a more efficient protective immunity which, however, does not switch to the Th1 phenotype, as evidenced by the lack of reversal of DTH anergy and no increase of IL-2 levels.
ACKNOWLEDGMENTS
We are grateful to T. Alves and B. P. Albe for technical assistance.
This work was supported by a grant (98/13766-0) from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).
FOOTNOTES
- Received 14 July 2003.
- Returned for modification 18 September 2003.
- Accepted 15 January 2004.
- Copyright © 2004 American Society for Microbiology
