Dual Role of Interleukin-4 (IL-4) in Pulmonary Paracoccidioidomycosis: Endogenous IL-4 Can Induce Protection or Exacerbation of Disease Depending on the Host Genetic Pattern

Abstract

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10⁶ Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.

An isogenic murine model of paracoccidioidomycosis (PCM), the most important endemic mycosis of Latin America, was developed. In this model, B10.A mice were susceptible and A/Sn mice were resistant to intraperitoneal (i.p.) Paracoccidioides brasiliensis infection (12). Infections in these mouse strains mimicked the polar forms of the disease. Anergy of delayed-type hypersensitivity reactions, elevated production of immunoglobulin G1 (IgG1) and IgG2b antibodies, impaired macrophage activation, and progressive infection were the main features of P. brasiliensis-infected B10.A mice. The immune response of resistant A/Sn mice was characterized by the preferential production of IgG2a antibodies, the presence of delayed-type hypersensitivity reactions, and efficient macrophage activation (10, 11). When infected by the intratracheal (i.t.) route, which better mimics human pulmonary disease, B10.A and A/Sn mice sustained their progressive and regressive patterns of disease, respectively (17).

Cytokine measurements in the i.p. model of infection showed preferential and sustained secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2) by antigen-stimulated lymphocytes from A/Sn mice. Although a typical Th2 pattern was not found, the absence of IFN-γ production characterized the secretion pattern of B10.A lymphocytes (9, 26). In the pulmonary model, both mouse strains secreted IFN-γ, IL-2, IL-4, IL-5, and IL-10 in the lungs, but these mediators appeared in larger amounts in B10.A mice than in A/SN mice (15, 16). Accordingly, in vivo depletion of IFN-γ was shown to exacerbate pulmonary disease in both susceptible and resistant mice (16). Furthermore, IFN-γ gene knockout C57BL/6 mice developed extremely severe and disseminated PCM, with nonorganized granulomas and precocious death of animals (46). It was also verified that the administration of recombinant IL-12 was protective for susceptible mice that showed decreased dissemination of yeasts to the liver and spleen but a marked inflammatory reaction in the lungs (2).

IL-4 is an immunoregulatory cytokine produced by Th2 lymphocytes, mast cells, basophils, and a subpopulation of NK lymphocytes (1). IL-4 induces the differentiation of naive T cells into Th2 lymphocytes, regulates the switch of B cells to IgE-secreting cells, and is involved in immediate-type hypersensitivity inflammatory reactions (28, 42). In contrast, IL-4 inhibits IFN-γ production and suppresses IFN-γ-mediated macrophage activation (22, 49). For several facultative or obligatory intracellular pathogens, the neutralization of endogenous IL-4 leads to the immunoprotection of susceptible hosts and allows the development of Th1 rather Th2 immune responses (23, 29, 34, 39, 40).

The severity of human PCM was found to correlate with the host's propensity to mount a Th2 immune response characterized by the secretion of high levels of IL-4, IL-5, and IL-10, the presence of blood eosinophilia, and the secretion of IgG4- and IgE-specific antibodies (3, 30, 37). In addition, the secretion of IFN-γ was found to be impaired in patients with the active disease (5, 25, 37).

Earlier work showed that IL-4-deficient C57BL/6 mice were more resistant to P. brasiliensis infection than their non-IL-4-deficient counterparts. Compared with wild-type controls, IL-4-deficient mice had lower pulmonary and hepatic fungal counts, decreased production of Th2 cytokines (IL-5 and IL-10), increased secretion of IFN-γ, and smaller and better organized granulomas (38). In the present study, we examined whether IL-4 is an endogenous mediator of susceptibility to P. brasiliensis infection by comparing the severity of pulmonary PCM in IL-4-depleted and untreated susceptible (B10.A) mice. A specific monoclonal antibody (MAb) (11B11) was used in two experimental protocols to deplete endogenous IL-4 from susceptible mice. We also examined whether in vivo depletion of IL-4 from C57BL/6 mice would lead to less severe PCM like that developed by C57BL/6 mice with a homozygous deletion of the IL-4 gene.

After i.t. infection with 10⁶ yeast cells, IL-4-depleted and untreated mice were studied with regard to the severity of infection in the lungs, liver, and spleen, the production of specific isotypes, the levels of pulmonary and hepatic cytokines, and pulmonary histopathologic findings. Surprisingly, an exacerbation of pulmonary infection was observed in IL-4-depleted B10.A mice, although only minor alterations in their patterns of cellular immunity and humoral immunity were detected. In contrast, PCM in IL-4-depleted C57BL/6 mice was less severe than that in untreated mice and was associated with decreased production of Th2 cytokines in association with enhanced levels of proinflammatory cytokines. As a whole, our results demonstrated that IL-4 has a dual role in pulmonary PCM and that its effects depend on the genetic background of the host.

MATERIALS AND METHODS

Animals.

Groups of five to seven male mice (8 to 11 weeks old) from strains susceptible (B10.A) and intermediate (C57BL/6) to P. brasiliensis infection were used for each period of infection. All of the animals were bred at University of São Paulo animal facilities under specific-pathogen-free conditions. Procedures involving animals and their care were conducted in conformity with national and international laws and policies.

Fungus.

P. brasiliensis isolate 18 (Pb18), 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 (27). Pb18 yeast cells then were maintained by weekly subcultivation in a semisolid culture medium (20) at 35°C and were used on day 7 of culturing. 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 with Janus green B vital dye (Merck, Darmstadt, Germany) (6), was always higher than 80%.

P. brasiliensis infection.

Mice were anesthetized and infected i.t. with P. brasiliensis as previously described (17). Briefly, after i.p. anesthesia, the animals were infected with 10⁶ Pb18 yeast cells, contained in 50 μl of PBS, by a surgical i.t. inoculation that allowed dispensing of fungal cells directly into the lungs. The skins of the mice were sutured, and the mice were allowed to recover under a heat lamp.

Treatment of mice with an anti-IL-4 MAb.

The anti-murine IL-4 hybridoma 11B11 (kindly provided by Robert Coffman, DNAX Research Institute, Palo Alto, Calif.) was grown i.p. in Pristane (Sigma Chemical Co., St. Louis, Mo.)-primed BALB/c athymic mice. The MAb was purified from ascites by using the method of McKinney and Parkinson (32) and was assayed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The biological activity of MAb 11B11 was tested by measuring its activity as a capture antibody in a previously standardized enzyme-linked immunosorbent assay (ELISA).

Two protocols were used to deplete endogenous IL-4. Groups of B10.A mice were given 1 mg of MAb 11B11 or rat IgG (control) in 0.5 ml of PBS by the i.p. route 1 day before i.t. inoculation with P. brasiliensis and 1.0 of MAb or rat IgG weekly thereafter. These mice were studied 4 and 8 weeks after P. brasiliensis infection. C57BL/6 mice were also depleted of IL-4 in vivo by weekly injection of 1 mg of MAb 11B11. Additional B10.A mice were injected i.p. with 8 mg of MAb 11B11 or rat IgG 1 day before infection and were studied 8 weeks later.

Assay for organ CFU.

The numbers of viable microorganisms in the lungs, liver, and spleen of experimental and control mice were determined by counting the numbers of CFU. Animals from each group were sacrificed at adequate intervals after infection, and enumeration of viable organisms in the three organs was done as previously described (43). Briefly, aliquots (100 μl) of cellular suspensions and serial dilutions of samples from each organ were plated on brain heart infusion agar (Difco) supplemented with 4% (vol/vol) horse serum (Instituto Butantan, São Paulo, Brazil) and 5% Pb192 culture filtrate, the latter constituting a source of growth-promoting factor. The plates were incubated at 35°C, and the colonies were counted daily until no increase in counts was observed. The numbers (log₁₀) of viable P. brasiliensis colonies per gram of tissue were expressed as means and standard errors (SEs).

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 analysis of lesions and stained with silver for fungal evaluation. Pathologic findings were analyzed based on the number, size, morphology, and cell composition of granulomatous lesions, the number of fungi, and the intensity of 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), and stored at −70°C. The levels of IL-2, IL-4, IL-5, IL-10, IFN-γ, and IL-12 were measured by capture ELISAs with antibody pairs purchased from Pharmingen (San Diego, Calif.). The concentrations of cytokines were determined by reference to a standard curve for serial twofold dilutions of murine recombinant cytokines. The lower limits of detection for the recombinant standard curves were 32.0, 8.0, 7.0, 8.0, 20.0, and 15.0 pg/ml for IL-2, IL-4, IL-5, IL-10, IFN-γ, and IL-12, respectively.

Measurement of serum P. brasiliensis-specific isotypes.

Levels of specific isotypes (total IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3) were measured by a previously described ELISA (17) with a cell-free antigen (13) prepared from a pool of various P. brasiliensis isolates (Pb339, Pb265, and Pb18). The average of the optical densities obtained for sera collected from control mice (PBS inoculated) and diluted 1:20 was considered the cutoff for each respective isotype. Optical densities for each dilution of experimental sera were compared to control values. The titer for each sample was expressed as the reciprocal of the highest dilution that resulted in an optical density higher than the cutoff.

Statistical analysis.

Data from depletion experiments with 1 mg of MAb/week were analyzed by parametric two-way analysis of variance followed by Tukey comparisons (52). All other data were analyzed by unpaired Student's t test. P values of <0.05 were considered significant.

RESULTS

IL-4-depleted mice have increased pulmonary fungal loads.

With a view to determining whether the production of IL-4, a Th2 cytokine, is responsible for progressive disease in susceptible mice, IL-4-depleted B10.A mice were compared with their counterparts in terms of their ability to control a P. brasiliensis infection initiated by the respiratory route. Two protocols were used to neutralize endogenous IL-4 in B10.A mice. In a first experiment, IL-4 was depleted by weekly injections of 1 mg of an anti-IL-4 MAb, and organs were monitored for fungal growth at weeks 4 and 8 after i.t. infection. Infected B10.A mice inoculated with equivalent doses of normal rat IgG were used as controls. Surprisingly, depletion of IL-4 with this protocol increased the pulmonary fungal burden at week 4 after infection, but at week 8, equivalent numbers of viable yeast cells were recovered from the lungs, liver, and spleen (Fig. 1). Since various investigators (23, 38) have demonstrated the disease-promoting effect of IL-4 and since our protocol could have induced only a partial depletion of IL-4, another schedule was used to neutralize endogenous IL-4. Thus, an additional group of B10.A mice was treated with 8 mg of MAb at day 1 before infection and was studied 8 weeks later. Confirming the results of the first experiment, IL-4-depleted mice had larger number of yeast cells in the lungs than did IgG-treated controls (Fig. 2).

FIG. 1.

Effect of in vivo depletion of IL-4 (1 mg of anti-IL-4/week) in B10.A mice infected i.t. with 10⁶ P. brasiliensis yeast cells. The recovery of CFU from the lungs, liver, and spleen of infected mice treated with normal rat IgG (control) (white bars) or rat MAb 11B11 (1 mg/week) (stippled bars) is shown. The bars depict means and SEs of log₁₀ CFU obtained from groups of five to seven mice at weeks 4 and 8 after infection. Similar results were obtained in two separate experiments. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

FIG. 2.

Effect of in vivo depletion of IL-4 (8 mg of anti-IL-4/week) in B10.A mice infected i.t. with 10⁶ P. brasiliensis (Pb) yeast cells. The recovery of CFU from the lungs, liver, and spleen of infected mice treated with normal rat IgG (control) or rat MAb 11B11 (8 mg 1 day before infection) is shown. The bars depict means and SEs of log₁₀ CFU obtained from groups of five to seven mice at week 8 after infection. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

Since IL-4 was previously shown to be deleterious to C57BL/6 mice (38), the 1 mg-per-week protocol was used in this strain, and mice were assessed in terms of CFU at 8 weeks after infection. Diminished numbers of fungal cells were detected in the lungs of MAb-treated mice (Fig. 3). These results showed that IL-4 plays different roles in pulmonary PCM. In B10.A mice, IL-4 exerts a protective effect in the lungs, but its neutralization in C57BL/6 mice leads to less severe disease at the site of infection.

FIG. 3.

Effect of in vivo depletion of IL-4 in C57BL/6 mice infected i.t. with 10⁶ P. brasiliensis (Pb) yeast cells. The recovery of CFU from the lungs, liver, and spleen of infected mice treated with normal rat IgG (control) or rat MAb 11B11 (1 mg/week) is shown. The bars depict means and SEs of log₁₀ CFU obtained from groups of five to seven mice at week 8 after infection. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

Depletion of IL-4 does not modify the morphology of pulmonary lesions in B10.A mice but results in mild pathologic changes in the lungs of C57BL/6 mice.

Because of the earlier observation of less severe lung pathologic changes in C57BL/6 mice with a disruption of the IL-4 gene (38), we compared pulmonary lesions in IL-4-depleted B10.A and C57BL/6 mice with those in their IgG-treated control counterparts. The lesions that developed in IL-4-depleted B10.A mice (Fig. 4B) were equivalent to those in IgG-treated mice (Fig. 4A). The pulmonary lesions in both groups were poorly organized, randomly distributed, and composed of isolated or confluent granulomas of various sizes and containing many fungal cells surrounded by macrophages, lymphocytes, and plasma cells. Confluent or isolated lesions in control C57BL/6 mice (Fig. 4C) were more organized than those in B10.A mice and were circumscribed by an evident mantle of small lymphocytes. Less intense results were seen in IL-4-depleted C57BL/6 mice (Fig. 4D): the lesions were smaller, better organized, and infiltrated with a diminished number of inflammatory cells, causing a milder disruption of the pulmonary parenchyma.

FIG. 4.

Photomicrographs of pulmonary lesions from B10.A (A and B) and C57BL/6 (C and D) mice. Animals were depleted of IL-4 in vivo with an anti-IL-4 MAb (1 mg/week) (B and D) or received control normal rat IgG (A and C). Control and anti-IL-4 treated mice were infected i.t. with 10⁶ P. brasiliensis yeast cells and studied at week 8 of infection. At this time, no differences in the lesions of treated and untreated B10.A mice were noted, but the granulomatous inflammation was more extensive and confluent in control C57BL/6 mice than in IL-4-depleted mice. Hematoxylin-eosin stain was used; the magnifciation was ×100.

IL-4 depletion does not alter pulmonary cytokine production in B10.A mice but induces decreased Th2 responses in C57BL/6 mice.

Since IL-4 depletion led to different results in the pulmonary disease seen in B10.A and C57BL/6 mice, we examined whether these results were associated with different patterns of pro- and anti-inflammatory cytokines in infected organs. First, we characterized the production of IFN-γ, IL-2, and IL-12 and the production of IL-4, IL-5, and IL-10 in homogenates of lung samples from IL-4-depleted B10.A mice. Neutralization of IL-4 with 1 mg of MAb/week did not change the amounts of Th1 and Th2 cytokines present in the lungs of mice at weeks 4 and 8 after infection (Fig. 5). The administration of a high dose (8 mg) of anti-IL-4 MAb 1 day before infection also did not alter the amounts of both types of cytokines (Fig. 6).

FIG. 5.

IL-4 depletion by treatment with 1 mg of anti-IL-4 MAb/week does not alter Th1 and Th2 cytokines of B10.A mice. At 4 and 8 weeks after P. brasiliensis (Pb) infection, lungs were collected from IL-4-depleted and control B10.A mice and disrupted in 4.0 ml of RPMI 1640 medium. Supernatants were analyzed for cytokine contents by a capture ELISA. The bars depict means and SEs of cytokine levels for five or six mice.

FIG. 6.

IL-4 depletion by treatment with 8 mg of anti-IL-4 MAb 1 day before infection does not alter Th1 and Th2 cytokines of B10.A mice. At 8 weeks after P. brasiliensis (Pb) infection, lungs were collected from IL-4-depleted and control B10.A mice and disrupted in 4.0 ml of RPMI 1640 medium. Supernatants were analyzed for cytokine contents by a capture ELISA. The bars depict means and SEs of cytokine levels for five or six mice.

Since IL-4-depleted C57BL/6 mice developed less intense disease and inflammatory reactions in the lungs, we extended our cytokine study to include the quantification of tumor necrosis factor alpha (TNF-α) in the lungs and liver. Figures 7 and 8 demonstrate that, indeed, the neutralization of endogenous IL-4 in C57BL/6 mice caused decreased production of Th2 cytokines (IL-5 in the lungs and IL-4 and IL-5 in the liver). This finding was associated with increased production of proinflammatory cytokines (TNF-α in the lungs and IL-12 in the liver). However, an evident shift to a Th1 immune response was not detected, since the production of IFN-γ and IL-2, typical Th1 cytokines, did not change in anti-IL-4 MAb-treated mice.

FIG. 7.

IL-4 depletion induces the production of higher levels of TNF-α and diminished levels of IL-5 in the lungs of C57BL/6 mice. At 8 weeks after i.t. infection with 10⁶ yeast cells of P. brasiliensis (Pb), lungs were collected from IL-4-depleted and control mice and disrupted in 4.0 ml of RPMI 1640 medium. Supernatants were analyzed for cytokine contents by a capture ELISA. The bars depict means and SEs of cytokine levels for four or five animals per group. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

FIG. 8.

IL-4 depletion induces the production of higher levels of IL-12 and diminished levels of IL-5 and IL-4 in the livers of C57BL/6 mice. At 8 weeks after i.t. infection with 10⁶ yeast cells of P. brasiliensis (Pb), livers from IL-4-depleted and control mice were collected and disrupted in 4.0 ml of RPMI 1640 medium. Supernatants were analyzed for cytokine contents by a capture ELISA. The bars depict means and SEs of cytokine levels for four or five animals per group. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

Depletion of IL-4 does not modify the humoral immune response of B10.A mice but alters the levels of IgG1 and IgG2a isotypes in C57BL/6 mice.

Since IL-4 is the principal cytokine that stimulates B-cell heavy-chain class switching to the IgE and IgG1 isotypes and inhibits IFN-γ-stimulated switching to the IgG2a isotype in mice (44, 45), we examined whether IL-4 depletion would influence isotype production in B10.A and C57BL/6 mice differently. Confirming the CFU and cytokine data, both schedules of IL-4 depletion did not change the levels of any studied isotypes in B10.A mice (Fig. 9). However, IgG1 antibodies were synthesized at lower levels by IL-4-depleted C57BL/6 mice than by untreated control mice. Conversely, IgG2a was detected at higher levels in the serum of IL-4-depleted C57BL/6 mice.

FIG. 9.

Levels of P. brasiliensis (Pb)-specific antibodies in sera of IL-4-depleted and control B10.A (A) and C57BL/6 (B) mice at week 8 after i.t. infection with 10⁶ yeast cells. Sera were assayed for total immunoglobulin, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 by using an isotype-specific ELISA as detailed in Materials and Methods. The bars depict means (log₂) and SEs of serum titers for five to eight mice per group. Data marked by an asterisk were significantly different (P < 0.05) from those for the control group.

DISCUSSION

This work showed that IL-4 could exert different roles in pulmonary PCM depending on the genetic pattern of the host. Depletion of IL-4 in genetically susceptible B10.A mice led to increased pulmonary fungal burdens, whereas PCM in C57BL/6 mice (intermediate susceptibility to P. brasiliensis) depleted in vivo of endogenous IL-4 was less severe and was associated with the increased production of TNF-α and IL-12 and with the diminished secretion of IL-4 and IL-5. The experiments with IL-4 depletion showed that the susceptibility of B10.A mice to P. brasiliensis is not governed mainly by IL-4. This result is different from those obtained with other systemic mycoses, such as candidiasis, histoplasmosis, and coccidioidomycosis, where in vivo depletion of IL-4 induced immunoprotection and allowed the development of Th1 rather than Th2 immune responses (29, 39, 53).

The increased pulmonary CFU numbers detected in IL-4-depleted B10.A mice were not accompanied by marked differences in lung histopathologic findings, the presence of various cytokines in lung homogenates, and humoral immune responses. It can be argued that IL-4 depletion was not efficient, since no differences in immune responses were noted. However, neutralization of IL-4 increased pulmonary fungal burdens, demonstrating that the MAb in some way reached or influenced the behavior of the lungs. One possible explanation for this result is that IL-4 is not the main mediator that governs susceptibility to pulmonary PCM in B10.A mice. Other cytokines, such as IL-10 and transforming growth factor β, are under investigation in our laboratories. Indeed, PCM in C57BL/6 IL-10 knockout mice is less severe than that in control mice with normal IL-10 and, at week 8 after infection, the lungs of IL-10-deficient mice show almost no pathologic changes compared to those of control mice (T. Alves and V. L. G. Calich, unpublished results). Other immunological mechanisms could be ascribed to the susceptibility of B10.A mice. It was recently demonstrated that nitric oxide is an important molecule associated with both immunoprotection, by enhancing the clearance of fungal cells, and immunosuppression developed in B10.A mice. When challenged by P. brasiliensis yeast cells, macrophages from susceptible B10.A mice secreted large amounts of nitric oxide, whereas those from resistant A/Sn mice secreted TNF-α. The impaired production of TNF-α by macrophages from B10.A mice was reversed by drugs that inhibit nitric oxide production (36). Additional findings indicate that B10.A susceptibility is not governed by CD4⁺ Th2 responses; in vivo depletion of CD4⁺ T cells does not abolish the progressive nature of the disease, and the relative immunoprotection developed by this strain of mice is mediated by IFN-γ-secreting CD8⁺ T cells (8).

The increased fungal burdens detected in IL-4-depleted mice are not totally unexpected. Indeed, Flesh and Kaufmann (21) described increased bacteriostactic activity of IL-4-treated macrophages. Furthermore, IL-4 has been reported to enhance murine macrophage mannose receptor activity (47) and to stimulate phagocytosis and microbial killing by neutrophils (7) and macrophages (51). In other experimental models of immunoprotection, IL-4 was also found to be important in controlling pathology. Thus, IL-4 is a protective cytokine against tumor cells (41, 50), adenoviruses (18), mycobacteria (48), and Trypanosoma brucei (24), among others. In an elegant study, Mencacci et al. (33) also demonstrated a dual role for IL-4 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 an impaired ability to produce sufficient amounts of IFN-γ and IL-12.

In contrast to the findings obtained with B10.A mice, the results obtained with IL-4-depleted C57BL/6 mice demonstrated the therapeutic effect of IL-4 neutralization and are consistent with the results obtained by Hostetler et al. (23) showing the deleterious role of IL-4 in murine PCM of BALB/c mice. Altogether, these data clearly show the influence of the genetic background on the effect of endogenous IL-4 and demonstrate that different mechanisms can account for host susceptibility to PCM. The diminished numbers of viable yeast cells recovered from the lungs of IL-4-depleted C57BL/6 mice were concomitant with a diminished Th2 immune response, as revealed by decreased levels of IL-5 in the lungs and IL-5 and IL-4 in the liver. The less severe disease was associated with the increased production of TNF-α in the lungs and IL-12 in the liver of IL-4 depleted mice. The decreased fungal loads observed could have been ascribed, at least partially, to cytokines that were previously shown to be immunoprotective against murine PCM (19, 36, 46). Another prominent feature of PCM in IL-4-depleted mice was the less severe lung pathologic changes, where well-organized granulomas containing yeast cells circumscribed by small numbers of inflammatory leukocytes replaced the more exuberant inflammatory reactions developed by control mice. This inflammatory architecture appears to be highly efficient in avoiding fungal growth outside the center of the lesion. The increased production of TNF-α detected locally in the lungs appeared to contribute to this type of lesion, since in previous studies with TNF-α receptor knockout mice, the focal granulomatous reaction developed by control wild-type C57BL/6 mice was substituted by a nonorganized inflammatory exudate that destroyed the normal lung parenchyma. We can speculate that in IL-4-depleted mice, the increased production of TNF-α and its stimulatory activity on phagocytes for fungal killing contributed to the immunological control of P. brasiliensis growth.

The humoral immune response to P. brasiliensis did not change after depletion of IL-4 in B10.A mice with 1 mg of MAb/week. It can be argued that the amount of MAb used was not sufficient to inhibit the B-cell-activating function of IL-4. However, even with 8 mg of MAb at day 1 before infection, no differences were noted in antibody production. Thus, for PCM in B10.A mice, the residual activity of IL-4 could be sufficient to regulate antibody production or this cytokine could be replaced by another mediator, such as IL-13, whose activity is similar to that of IL-4. This situation is not unusual, and in a murine model of granulomas induced by Schistosoma mansoni eggs, the use of IL-4 and IL-13 double knockout mice clarified the cooperative and overlapping actions of IL-4 and IL-13 in initiating Th2 cell-driven responses (31). In parallel with decreased levels of Th2 cytokines (IL-4 and IL-5), smaller amounts of IgG1 antibodies were secreted by IL-4-depleted C57BL/6 mice than by untreated control mice. The increased titers of IgG2a could have been due to the maintained secretion of IFN-γ, a well-characterized IgG2a switching factor (44, 45) associated with decreased levels of Th2 cytokines. Similar alterations of IgG1 and IgG2a antibodies were obtained in C57BL/6 IL-4 knockout mice compared with their wild-type control counterparts (38).

Overlapping functions of several mediators of immune responses and their different manifestations in the context of different genetic backgrounds have been described. Indeed, in a study of resistance to infection with Trichuris muris, it was demonstrated that IL-4 knockout mice in a C57BL/6 background were susceptible, whereas IL-4 knockout mice in a BALB/c background were resistant (4). Similar findings have been described for the development of hapten-induced contact hypersensitivity. IL-4 played an important role in the onset of hapten-induced reactions in BALB/c mice but not in C57BL/6 mice (35).

As a whole, our results demonstrated that IL-4 is an important cytokine that can modulate positively or negatively the patterns of susceptibility of hosts to P. brasiliensis infection. The antagonistic effects of IL-4 in hosts of different genetic backgrounds demonstrate that several immunological mechanisms can lead to susceptibility to pulmonary infection by P. brasiliensis and, more than that, the Th1-Th2 paradigm does not explain all of the immunological mechanisms that determine disease outcome. Our findings also open new perspectives for analyzing and trying to understand the behavior of the severe form of the disease in many patients who do not show an immune response polarized to the Th2 pattern. Consistent with our findings, a recent study showed that Fas-Fas ligand (CD95-CD95L) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) engagement is an important mechanism associated with T-cell unresponsiveness in patients with severe PCM (14).