Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with...

INFECTION AND IMMUNITY, Apr. 2004, p. 2369�2378 Vol. 72, No. 40019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.4.2369–2378.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Absence of Interleukin-4 Determines Less Severe PulmonaryParacoccidioidomycosis Associated with Impaired Th2 Response

Adriana Pina, Rita C. Valente-Ferreira, Eugenia E. W. Molinari-Madlum,Celideia A. C. Vaz, Alexandre C. Keller, and Vera L. G. Calich*Departamento de Imunologia, Instituto de Ciencias Biomedicas da Universidade

de Sao Paulo, Sao Paulo, Brazil

Received 14 July 2003/Returned for modification 18 September 2003/Accepted 15 January 2004

Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellularimmunity and gamma interferon (IFN-�) production. To assess the role of interleukin-4 (IL-4), a Th2-inducingcytokine, 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 infectedIL-4�/� mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secretedhigher levels of nitric oxide. Compared with WT mice, IL-4�/� animals presented increased levels of pulmonaryIFN-� and augmented polymorphonuclear leukocyte influx to the lungs. Decreased pulmonary fungal loadswere 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 withincreased production of IFN-� but impaired synthesis of IL-5 and IL-10. However, a clear shift to a Th1 patternwas not characterized, since IL-4�/� mice did not alter delayed-type hypersensitivity anergy or IL-2 levels. Inaddition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophagecolony-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 highnumbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice. These results clearly showedthat genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.

Paracoccidioides brasiliensis is a pathogenic fungus restrictedto Latin America. The natural route of infection is the inha-lation of fungal particles, which usually leads to an asymptom-atic infection. The disease presents a wide range of clinical andimmunological manifestations, varying from benign and local-ized to severe and disseminated forms (9). Classical studies onthe immune responses developed by patients with polar formsof paracoccidioidomycosis (PCM) demonstrated that the be-nign forms of the disease were associated with production oflow levels of antibodies and positive delayed-type hypersensi-tivity (DTH) reactions, whereas the severe disseminated formswere associated with high levels of antibodies and anergy inDTH reactions (20). More recent works have demonstrated aTh1-biased immune response in the asymptomatic and mildforms of PCM, whereas a Th2 pattern has been associated withthe severe disease. When compared with patients with the mildform of the disease, patients presenting the more disseminatedinfection produce higher levels of type 2 cytokines (interleu-kin-4 [IL-4], IL-5, IL-10, and transforming growth factor �)and antibodies (immunoglobulin E [IgE], IgG4, and IgA) inaddition to eosinophilia and impaired secretion of gamma in-terferon (IFN-�) (3, 5, 28, 36, 41).

A greater insight into the immune response elicited by

P. brasiliensis has come from the use of murine models. Ourlaboratory developed an isogenic murine model of PCM whereB10.A mice were susceptible to and A/Sn mice were resistantto mouse strains of P. brasiliensis. Although resistance of A/Snmice was linked with the secretion of T helper 1 cytokines(IL-2 and IFN-�), susceptibility was not clearly associated witha Th2 pattern, since IL-4 was not found in the supernatants ofantigen-stimulated lymph node cells from infected B10.A mice(11, 29). When infected by the intratracheal (i.t.) route, B10.Aand A/Sn mice maintained the same resistant and susceptiblepattern observed after the intraperitoneal infection (15). Bothstrains of mice secrete type 1 and type 2 cytokines in the lungs,but the progressive disease developed by susceptible B10.Amice appears to be governed by type 2 cytokines (11, 14–16).Furthermore, previous results with the murine model of infec-tion showed that depletion of IFN-� or genetic deficiency ofthis cytokine led to exacerbated disease (14, 50). It was alsoverified that administration of recombinant IL-12 is protectiveto susceptible mice which presented with a less-disseminateddisease but a high inflammatory reaction in the lungs (2).

Since IL-4 directs Th2 development during the innate phaseof immune response (23, 46) and has been shown to inhibitTh1-dominated immunity, we assumed that IL-4-deficient(IL-4�/�) mice would develop a more-efficient cellular re-sponse and protective immunity to P. brasiliensis infection thantheir IL-4-competent counterparts. Thus, the effect of IL-4 wasinvestigated in the innate (48 h after infection) and acquired(weeks 2 and 8 of infection) phases of immune response toP. brasiliensis infection. After intratracheal infection with one

* Corresponding author. Mailing address: Departamento de Imuno-logia, Instituto de Ciencias Biomedicas da Universidade de Sao Paulo,Av. Prof. Lineu Prestes 1730, CEP 05508-900, Sao Paulo, SP, Brazil.Phone: 55-11-30917397. Fax: 55-11-30917224. E-mail: [email protected].

2369

on Novem

ber 14, 2015 by guesthttp://iai.asm

.org/D

ownloaded from

http://iai.asm.org/

million P. brasiliensis yeast cells, the severity of infection wasmonitored by several immunological parameters. PCM in IL-4�/� mice was less severe than in IL-4-normal mice and wasassociated with an impaired Th2 immune response, leading toenhanced 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) werebred at the University of Sao Paulo animal facilities under specific-pathogen-freeconditions 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 involvinganimals and their care were conducted in conformity with national and interna-tional policies.

Fungus. The P. brasiliensis 18 isolate, which is highly virulent, was usedthroughout this study. To ensure the maintenance of its virulence, the isolate wasused after three serial animal passages (30). P. brasiliensis 18 yeast cells were thenmaintained 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 washedin 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 viabilityof fungal suspensions, determined by Janus Green B vital dye (Merck, Darm-stadt, Germany), was always higher than 80%.

P. brasiliensis infection. Mice were anesthetized and submitted to i.t. P. bra-siliensis infection as previously described (15). Briefly, after intraperitoneal an-esthesia, the animals were infected with 106 P. brasiliensis 18 yeast cells, con-tained in 50 �l of PBS, by surgical i.t. inoculation, which allowed dispensing ofthe fungal cells directly into the lungs. The skins of the animals were thensutured, and the mice were allowed to recover under a heat lamp. Mice werestudied during an early period (48 h after infection) and a late period (at week8).

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 tosham-infected (submitted to surgical stress and injected with 50 �l of PBS) andnormal mice of both mouse strains. The lungs were lavaged by repeated injec-tions of 0.5 ml of sterile PBS (final volume, 2.0 ml). The recovered fluid was spunat 1,200 rpm, the supernatant was removed, and cells were analyzed for fungi-cidal activity and leukocyte subsets.

Assessment of leukocyte population. For differential counts, samples of lungcell suspensions were cytospun (Shandon Cytospin, Pittsburgh, Pa.) onto glassslides and stained by the Diff-Quik blood stain (Baxter Scientific, Miami, Fla.). Atotal of 200 to 400 cells were counted from each sample. The absolute numberof a leukocyte subset was calculated by multiplying the percentage of each subsetin an individual sample by the total number of lung leukocytes in that mouse.

Fungicidal activity of alveolar macrophages. The BALFs obtained from indi-vidual mice were centrifuged, and pellets resuspended in RPMI containing 10%fetal calf serum, 2 mM L-glutamine, 100 U of penicillin/ml, and 100 �g ofstreptomycin/ml. Cell suspensions were adjusted at 4 � 105 cells/ml of culturemedium, and 0.5 ml was dispensed in 24-well tissue culture plates for a 2-hadhesion step. Nonadherent cells were transferred to another plate. Plates con-taining adherent and nonadherent cells were centrifuged, supernatants werediscarded, and cells were incubated with 0.5 ml of culture medium supplementedor not with 100 U of IFN-� (Pharmingen, San Diego, Calif.)/ml. After 72 h ofculture 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 pres-ence 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 andresuspended in culture medium, and aliquots (100 �l) and serial dilutions wereassayed 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 controlmice were determined by counting the number of CFU. Animals from eachgroup were sacrificed, and the enumeration of viable organisms was done aspreviously described (47). Briefly, aliquots (100 �l) of the cellular suspensionsand serial dilutions were plated on brain heart infusion agar (Difco, Detroit,Mich.) supplemented with 4% (vol/vol) horse serum (Instituto Butantan, SaoPaulo, Brazil) and 5% P. brasiliensis 192 culture filtrate, the latter constituting asource of growth-promoting factor. The plates were incubated at 35°C, andcolonies 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 in10% formalin, and embedded in paraffin. Five-micrometer sections were stainedwith hematoxylin and eosin for an analysis of the lesions and silver stained forfungal evaluation. Pathological changes were analyzed based on the size, mor-phology, and cell composition of granulomatous lesions, presence of fungi, andintensity of the inflammatory infiltrates.

Measurement of cytokines. Mice were infected i.t. with P. brasiliensis, and theirright lungs were removed aseptically and individually disrupted in 4.0 ml ofRPMI 1640 medium (Gibco BRL). Supernatants were separated from cell debrisby centrifugation at 2,000 � g for 15 min, passed through 0.22-�m-pore-sizefilters (Millipore, Bedford, Mass.), and stored at �70°C. The levels of IL-2, IL-4,IL-5, IL-10, IFN-�, IL-12, tumor necrosis factor alpha (TNF-�), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 were measured bycapture enzyme-linked immunosorbent assay (ELISA) with antibody pairs pur-chased from Pharmingen. Reagents for monocyte chemotactic protein 1(MCP-1) were obtained from R&D Systems (Minneapolis, Minn.). The ELISAprocedure was performed according to the manufacturer’s protocol. The con-centrations of cytokines were determined with reference to a standard curve forserial twofold dilutions of murine recombinant cytokines. The lower limits ofdetection standard curves were 32.4, 7.8, 7.0, 7.8, 20.0, 15.0, 31.2, 15.3, 7.8, and31.2 pg/ml for IL-2, IL-4, IL-5, IL-10, IFN-�, IL-12, TNF-�, GM-CSF, IL-3, andMCP-1, respectively. As an additional control, lung homogenates were added torecombinant cytokines used to obtain standard curves; no interference was de-tected, indicating the absence of inhibitory substances (e.g., soluble cytokinereceptors).

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 �l of supernatants was removed from 24-well plates and incubatedwith an equal volume of Griess reagent (1% sulfanilamide–0.1% naphthylenediamine dihydrochloride–2.5% H3PO4) at room temperature for 10 min. Theabsorbance at 550 nm was determined with a microplate reader. The conversionof absorbance to micromolar NO was deduced from a standard curve by using aknown concentration of NaNO2 diluted in RPMI medium. All determinationswere performed in duplicate and expressed as micromolar NO.

DTH assay. The DTH reactions were always evaluated just before sacrifice ofthe same animals used in the CFU assays by the footpad test according topreviously determined conditions (19). Briefly, mice were inoculated with 25 �lof Fava Netto antigen (18), and the footpad thickness was measured with acaliper (Mitutoyo Corporation, Tokyo, Japan) immediately before and 24 h afterantigen inoculation. The increase in thickness was calculated and expressed inmillimeters. Noninfected mice submitted to the footpad test were used as con-trols.

Measurement of serum P. brasiliensis-specific isotypes. Specific isotypes levels(total IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3) were measured by apreviously described ELISA (15) employing a cell-free antigen (12) prepared byusing a pool of different P. brasiliensis isolates (339, 265, and 18). The average ofthe optical densities obtained with sera from control mice (PBS inoculated),diluted 1:20, was considered the cutoff for each respective isotype. Optical den-sities 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 dilutionthat presented an absorbance higher than the cutoff.

Statistical analysis. Data were analyzed by Student’s t test or two-way analysisof variance depending on the number of experimental groups (54). P valuesunder 0.05 were considered significant.

RESULTS

Early in infection, lung tissue and BALF from IL-4�/� andcontrol mice present equivalent fungal loads and nitrite con-centrations. Groups (n 6 to 8) of IL-4�/� C57BL/6 mice andtheir WT controls (IL-4�/�) were infected i.t. with one millionP. brasiliensis yeast cells, and equivalent fungal counts weredetected in lung tissue and BALF 48 h after infection. Inaddition, both groups of mice presented similar levels of nitritein lung homogenates (Fig. 1).

IL-4 deficiency determines early increased leukocyte re-cruitment into lungs. To better characterize the early phase ofpulmonary infection, leukocyte recruitment to the alveolar

2370 PINA ET AL. INFECT. IMMUN.

on Novem


.org/D

ownloaded from

http://iai.asm.org/

spaces of P. brasiliensis-infected IL-4�/� and IL-4�/� mice wascompared. Groups of sham-infected and normal mice wereincluded as additional controls. As can be seen in Fig. 2A, nodifferences in the relative number of mononuclear and poly-morphonuclear (PMN) cells were found between the WT andIL-4�/� groups. The same result was obtained with untreatedgroups of normal WT and IL-4-deficient mice (data not shown).Compared with WT mice, a significantly augmented number oftotal cells, mainly due to the influx of PMN leukocytes, wasobserved in the BALF of IL-4-deficient mice (Fig. 2B).

Adherent cells from BALF of IL-4�/� mice present in-creased fungicidal activity and nitric oxide production. Todetermine the role of IL-4 in the fungicidal activity of inflam-matory cells recruited early into the lungs, adherent and non-adherent BALF cells were cultivated for 72 h in the presenceor absence of exogenously added IFN-� and recovered viableP. brasiliensis yeasts were enumerated. Figure 3A shows theincreased fungicidal ability of adherent cells (macrophages) ofIL-4-deficient mice when compared with their normal coun-terparts. On the contrary, the killing ability of nonadherentcells (mostly PMN leukocytes) was similar for both mousestrains (Fig. 3B). In agreement, adherent cells of IL-4-deficientmice secreted higher levels of NO than those produced byIL-4�/� animals. The addition of IFN-� to cell cultures in-creased fungicidal activity and NO production of adherent cellsbut did not alter fungal growth and NO secretion by nonad-herent cells (Fig. 3C and D).

Early in infection, IL-4-deficient mice present increasedamounts of pulmonary IFN-�. To determine whether the in-creased fungicidal ability of inflammatory cells was associatedwith a different milieu of pulmonary cytokines, we character-ized the secretion of pro- and anti-inflammatory cytokines inthe lungs of mice at the early phase of infection (Fig. 4). IFN-�production was significantly increased in IL-4�/� mice com-pared to than in IL-4�/� mice after 48 h of infection. Theformer strain also presented a trend to diminished levels ofTh2 cytokines (IL-5 and IL-10), but these differences did notreach significant levels.

IL-4�/� mice develop less-severe PCM. After analyzing theearly phase of infection, we compared the severity of diseasedeveloped by IL-4-deficient and control mice at the acquiredphase of immunity. Diminished fungal growth was detected inthe lungs of IL-4�/� mice at week 2 postinfection. At week 8,the lungs and livers of these mice presented diminished fungalloads compared to those of IL-4-normal mice (Fig. 5).

Levels of pulmonary cytokines in the acquired phase ofimmunity. As at the innate phase of immunity to pulmonaryPCM increased amounts of IFN-� were found in the lungs ofIL-4-deficient mice, we sought to determine whether this factwould have altered the secretion of pulmonary cytokines atlater phases of infection. At week 2, decreased levels of IL-10and MCP-1 were found in lung homogenates of IL-4-deficientmice (Fig. 6A). By week 8, augmented levels of IFN-� but notIL-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 ofIL-4�/� mice.

Absence of IL-4 does not revert DTH anergy of C57BL/6mice. As CFU counts demonstrated the protective role ofgenetic deficiency of IL-4 in murine PCM, we asked whetherabsence of this cytokine would also have the ability to modu-late DTH reactions. At both periods of acquired immunitystudied, WT C57BL/6 mice infected i.t. with P. brasiliensis didnot present positive DTH reactions as well as their IL-4-defi-cient counterparts (data not shown). Thus, IL-4 deficiency didinduce a less-severe PCM but was not able to eliminate theDTH anergy of C57BL/6 mice.

IL-4 deficiency results in mild lung pathology. Forty-eighthours after P. brasiliensis infection, an acute and diffuse inflam-matory infiltrate mainly composed of PMN cells and recentlymigrated monocytes was detected in the lungs of infected mice(n 7). The whole inflammatory picture was similar in bothexperimental groups, although in some IL-4-deficient mice (3of 7) a more peribronchial and perivascular focal pattern wasobserved and appears to predict the more-organized lesionsdeveloped at week 8 after infection. At this time point, whencompared with their normal counterparts, IL-4�/� mice pre-

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 afterP. brasiliensis infection with one million yeast cells; (B) nitrite concentrations in lung homogenates. No nitric oxide was detected in BALFsupernatants. Data are expressed as means � standard errors. Similar results were obtained from two separate experiments.

VOL. 72, 2004 PULMONARY PCM IN IL-4-DEFICIENT MICE 2371

on Novem


.org/D

ownloaded from

http://iai.asm.org/

sented small, more-compact granulomas, which were infiltrat-ed with a diminished number of inflammatory cells causing amild disruption of the pulmonary parenchyma (Fig. 7C). Gro-cott staining allows the demonstration of budding yeast cells atthe center of granulomas of IL-4�/� mice (Fig. 7D), suggestingthat this morphology is sufficient to avoid fungal dissemination.In contrast, the more-severe pulmonary lesions developed byIL-4-normal mice were randomly distributed and composed ofisolated or confluent granulomas of various sizes containingmany fungal cells surrounded by macrophages, lymphocytes,and plasma cells (Fig. 7A and B). An equivalent cellular com-position was observed in the IL-4�/� strain. However, granu-lomas of IL-4�/� mice showed a more prominent presence ofPMN leukocytes, usually at the center of lesions in close prox-imity to fungal cells.

Absence of IL-4 diminishes production of P. brasiliensis-specific isotypes. We also studied the influence of IL-4 on theproduction of P. brasiliensis-specific antibodies by IL-4-defi-cient and control mice (Fig. 8). As expected, due to the IL-4influence on B-cell growth and antibody class switch (48, 49),IgG, IgA, and IgM antibodies were synthesized at lower levelsby IL-4-deficient mice than by WT mice. Among the IgG sub-classes, IgG1, IgG2b, and IgG3 were produced at decreasedlevels by IL-4-deficient mice. IgG2a, however, was detected atsimilar levels in the serum of both mouse strains.

DISCUSSION

This work showed that IL-4 can exert a deleterious role inthe pulmonary infection caused by P. brasiliensis. Using IL-4-deficient mice and their IL-4-sufficient counterparts, both theonset and later periods of infection were explored, with anemphasis on trying to understand the influence of IL-4 on theinnate and acquired immunity developed by C57BL/6 miceinfected with yeast cells. At 48 h after infection, equivalentnumber of viable P. brasiliensis cells and NO production weredetected in the lungs and bronchoalveolar spaces of IL-4-deficient and -sufficient strains. IL-4 deficiency, however, re-sulted in increased inflammatory reactions in the lungs, withelevated numbers of PMN cells recovered from alveolar spacesat this early phase of infection. When cultivated in vitro, in thepresence or not of exogenous IFN-�, nonadherent cells (mostlyPMN leukocytes) of WT mice showed fungicidal ability equiv-alent to that presented by those of IL-4�/� mice. As deficientmice presented a twofold increase of inflammatory cells in thelungs mainly due to the PMN cell influx, we can suppose that,at the onset of infection, PMN leukocytes from IL-4�/� micecould exert a more-efficient clearance of yeast cells. Yet, com-pared with those of IL-4-deficient mice, adherent cells of WTmice demonstrated a lower ability to secrete NO, and thisdiminished production correlated with impaired fungicidal ac-tivity. The addition of exogenous IFN-� to cell cultures in-creased the NO production and killing ability of adherent cellsof both mouse strains but did not revert the macrophage-inhibiting activity of IL-4 in WT cells. As the main P. brasil-iensis killing mechanism of PMN cells is oxygen dependent (8)and that of macrophages is nitric oxide mediated (8, 21), wecan suppose that both cell types can exert a synergistic effect onthe control of fungal growth. However, other effector mecha-nisms could play a role at this period of innate immunity. Theinflammatory milieu of the lungs seems to activate phagocyticcells, but at this time point, the enhanced killing ability of thesecells was not sufficient to significantly decrease fungal loadsrecovered from the lung parenchyma. When cultivated in vitrofor a further 72 h, macrophages from IL-4 knockout (KO) micecould show their enhanced ability to kill yeast cells and secreteNO. The difference between cells from WT and IL-4 KO miceappears to be due to the increased levels of IFN-� present inlung homogenates of the latter strain. As at this early period ofinfection the adaptive immune response is not well established,we can speculate that the IFN-� production was mainly due tothe cytokine secretion by other cells of innate immunity such asNK cells (53).

As in murine PCM, La Flamme et al. (34) detected anenhanced ability of IL-4�/� macrophages to secrete NO and

FIG. 2. Relative (A) and absolute (B) numbers of leukocytes inBALF 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 PBS48 h after infection, and cell suspensions were cytospun onto glassslides and stained with the Diff-Quik blood stain. The absolute numberof a leukocyte subset (PMN neutrophils or mononuclear cells [MNC])was calculated as described in Materials and Methods. Data are ex-pressed as means � standard errors. *, P 0.05; **, P 0.01; ***, P 0.001 (compared with WT controls).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

FIG. 3. Fungicidal activity (A and B) and nitric oxide production (C and D) by macrophages (A and C) and PMN leukocytes (B and D) fromBALF stimulated or not by IFN-� (100 U/ml) and cultivated in vitro for 72 h. Supernatants and cell suspensions were collected and analyzedfor 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.

FIG. 4. IL-4 deficiency is associated with early increased levels of pulmonary IFN-�. Forty-eight hours after P. brasiliensis infection, lungs fromIL-4�/� and WT control C57BL/6 mice were collected and disrupted in 4.0 ml of RPMI 1640 medium, and supernatants were analyzed for cytokinecontent by capture ELISA. The bars depict means � standard errors of the means of cytokine levels (n 6 to 8). *, P 0.05 (compared with WTcontrols).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

observed a higher number of PMN leukocytes in the lesions ofIL-4-deficient mice infected with Schistosoma mansoni than inthe WT control. It was also verified that macrophages, lym-phocytes, and PMN cells were able to secrete NO in culturesupernatants, although the former cells were the main produc-ers of this reactive species. The low levels of NO secreted byPMN leukocytes from P. brasiliensis-infected mice could alsoplay a role in fungal killing, although this mechanism was notyet described for murine PCM. However, whatever the mech-anisms PMN cells use to kill P. brasiliensis, its abundance andconstant presence near yeast cells at the center of lesions leadus to suppose they have a very important role in the in vivocontrol of fungal growth.

The fact that IL-4-deficient mice infected with several patho-gens present impaired Th2 responses but enhanced or not Th1responses (33, 38, 42) led us to characterize the pattern ofcytokines in the lungs at weeks 2 and 8 of P. brasiliensis infec-tion. The impaired fungal growth in the lungs of IL-4-deficientmice at the former period was associated with decreased pro-duction of IL-10, a well-known macrophage-deactivating cyto-kine. The diminished number of viable yeast cells recoveredfrom the lungs and liver of IL-4�/� mice at week 8 was con-

comitant with increased production of pulmonary IFN-� andsignificantly impaired secretion of IL-5 and IL-10. As equiva-lent levels of IL-2 and anergy in DTH reactions were found inboth mouse strains, a shift to a Th1 pattern of immune re-sponse could not be characterized. However, the high amountsof IFN-� associated with diminished or absent levels of Th2cytokines (IL-4, IL-5, and IL-10) appear to have determined acytokine milieu deviated to a more proinflammatory pattern.This new cytokine balance would favor phagocyte activationand better control of fungal growth. Although a positive cor-relation between the presence of DTH reactions and protec-tive immunity to murine and human PCM is usually found (10,20), in some experimental conditions this association is con-troversial and some researchers have claimed they are notcausally related (27). Indeed, in pulmonary PCM of susceptibleB10.A mice, depletion of CD8� T cells led to a more-severedisease concomitant with prominent DTH reactions, indicatinga suppressive activity of CD8� T lymphocytes on the cellularimmunity mediated by CD4� T cells (13). More importantly,the increased production of NO shown by IL-4 KO mice maybe responsible for the impaired cellular immunity as previouslydescribed 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 patternof hosts. Furthermore, IL-4 appears to be important or neces-sary in the inductive phase of cellular immunity. Most studieson IL-4 neutralization during infections caused by intracellularpathogens revealed the Th2-inducing activity of this cytokineand its suppressive effect on DTH reactions. However, undercertain conditions, IL-4 induces the development of Th1-me-diated DTH reactions (45, 51).

The lower CFU counts in IL-4�/� mice were associated withdecreased secretion of several cytokines. Interestingly, IL-12was detected at lower levels in lung homogenates at week 8. Aspreviously reported, IL-4 can prime macrophages for synthesisof IL-12 (37, 44) and could be acting as a priming factor forIL-12 secretion after P. brasiliensis infection only in IL-4-suf-ficient mice. The reduced inflammatory reaction associatedwith diminished levels of IL-12 in lung homogenates is alsoconsistent with previous results on the proinflammatory effectof IL-12 administration to susceptible mice. Despite the less-disseminated infection, the early treatment with exogenousIL-12 induced a highly increased inflammatory reaction thatremained through week 8 after infection (2).

Interestingly, IL-4 deficiency was accompanied by decreasedlevels of IL-5, IL-3, and GM-CSF, whose genes, together withthat coding for IL-4, map on the cytokine gene cluster onmouse chromosome 11 (35). The concomitant rise and fall ofthese cytokines is not an unusual fact, and coordinate regula-tion of cytokine genes clustered on the same chromosome wasseen in other pathologies, such as the late-phase cutaneousreaction in atopic subjects (31) and in Th2 clones (32) as wellas in the more-severe pulmonary PCM induced by in vivodepletion of CD8� T cells or IL-12 by specific monoclonalantibodies (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 notdescribed for murine PCM, Allendoerfer et al. (1) demon-strated the protective function of GM-CSF in murine his-toplasmosis. Furthermore, the stimulatory activity of IL-3 and

FIG. 5. Recovery of viable yeasts (CFU) from lungs, livers, andspleens of IL-4-deficient (IL-4�/�) and IL-4-sufficient (WT) mice atweeks 2 (A) and 8 (B) after i.t. infection with 106 yeast cells. The barsdepict means � standard errors of the means of log10 CFU obtainedfrom groups of 6 to 8 mice. *, P 0.05 (compared with WT controls).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

GM-CSF on the fungicidal activity of phagocytes was alsodescribed previously (40). So, it is difficult to understand theless-severe PCM associated with low levels of these mediators.However, we can speculate that in IL-4�/� mice these activitieswere replaced by the increased production of IFN-�, un-changed secretion of TNF-�, and stimulatory activity onphagocytes for fungal killing (14, 50). On the other hand, thedecreased production of several pro- and anti-inflammatorycytokines could be due to the less-severe infection observed inIL-4-deficient mice.

MCP-1 is a C-C chemokine produced by several cell typesand is chemotactic for monocytes and lymphocytes (22, 43).MCP-1 is produced in the lungs of mice infected i.t. withCryptococcus neoformans, and the in vivo neutralization orabsence of its receptor (CCR2) results in a dramatic reductionin macrophage and CD8�-T-lymphocyte recruitment and elim-ination of fungal cell clearance (26, 52). At weeks 2 and 8 of

infection, WT C57BL/6 mice infected with P. brasiliensis pro-duced MCP-1 in higher amounts than their IL-4-deficientcounterparts. The decreased production of this chemotacticchemokine was associated with decreased recruitment of in-flammatory cells, as detected by lung histology, indicating thatthis mediator could be involved in the organization of P. bra-siliensis granulomas. The concomitant diminished productionof MCP-1 and Th2 cytokines (IL-5 and IL-10) detected inIL-4�/� mice infected with P. brasiliensis is consistent with thepreviously 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 patho-genesis of P. brasiliensis infection by indirect regulation offungal growth and control of other cytokines secreted at thesite of infection. This new lung environment appears to lead toa more-efficient phagocyte activation and inflammatory reac-tion that impairs fungal dissemination to the liver and dimin-

FIG. 6. 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 fromIL-4-deficient and WT control mice were collected and disrupted in 4.0 ml of RPMI 1640 medium, and supernatants were analyzed for cytokinecontent 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).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

ishes lung pathology by decreasing the recruitment of inflam-matory cells to pulmonary lesions.

Another prominent feature of PCM in IL-4-deficient micewas the less-severe lung pathology, where well-organized gran-ulomas containing yeast cells circumscribed by small numbersof inflammatory leukocytes replace the more exuberant inflam-matory reactions developed by WT mice. This inflammatoryarchitecture developed by IL-4�/� mice appears to be highlyefficient in avoiding fungal growth outside the lesion and pre-serving the pulmonary tissue. The increased levels of IFN-�detected in lung homogenates appear to contribute to thelesion morphology, since in previous studies with IFN-�-de-pleted (14) and KO mice (50), the focal granulomatous reac-tion was replaced by nonorganized inflammatory exudate thatdestroys the normal lung parenchyma. In murine schistosomi-asis, a well-studied Th2 disease, granuloma size was also re-duced in IL-4�/� infected mice, which, however, display amore-severe disease that correlates with increased productionof NO by lesion cells isolated from liver granulomas (17).

In contrast with these results with C57BL/6 mice, previousstudies with the B10.A strain, highly susceptible to P. brasil-iensis, showed that IL-4 can exert a protective function inpulmonary PCM. In vivo depletion of IL-4 did not alter thepattern of cytokines in the lungs but led to increased fungalload in the lungs of infected B10.A mice (10, 16). In theC57BL/6 strain, however, equivalent treatment with anti-IL-4monoclonal antibodies induced a less-severe pathology associ-ated with increased production of pulmonary IFN-� (C. Ar-ruda, 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, butother mechanisms, such as excessive NO production, couldaccount for B10.A susceptibility (39). The deleterious effect ofIL-4 in the C57BL/6 strain is similar to that obtained byHostetler et al. (25) with BALB/c mice depleted with highdoses of anti-IL-4 monoclonal antibodies. Thus, in murinePCM, IL-4 can play a protective or exacerbating effect on theinfection depending on the genetic pattern of the host. A dualrole for IL-4 has already been described for other experimentalpathologies. In murine candidiasis, IL-4-deficient mice weremore resistant to early Candida albicans infection but failed tomount a protective Th1 immune response in the late phase andsuccumbed due to the impaired ability to produce efficientamounts of IFN-� and IL-12 (37). Biedermann et al. (6) clearlyshowed the opposing effects of IL-4 in murine leishmaniasis.The presence of IL-4 during the initial activation of dendriticcells induces the production of IL-12 by these cells and devel-opment of Th1 lymphocytes; when present later, during theperiod of T-cell priming, IL-4 induced a Th2 immune response.Furthermore, the overlapping functions of several mediators ofimmune response and its different manifestation in the contextof different genetic backgrounds were demonstrated. Indeed,studying resistance to infection with Trichuris muris, it wasverified that IL-4�/� mice in a C57BL/6 background are sus-ceptible, whereas IL-4�/� mice in a BALB/c background areresistant (4).

Previous studies from our group have demonstrated theprevalence of Th2-regulated isotypes (IgG2b, IgG1, and IgA)in the most severe forms of the murine disease and a prefer-ential production of the Th1-controlled isotype (IgG2a) in the

FIG. 7. Photomicrographs of granulomatous lesions from IL-4�/� and WT control C57BL/6 mice i.t. infected with 106 P. 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 granulomatousinflammation 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).


on Novem


.org/D

ownloaded from

http://iai.asm.org/

benign infection (10, 15, 29). In this investigation, we verifiedthat all isotypes of specific antibodies, except IgG2a, wereproduced in lower amounts by IL-4-deficient mice than byIL-4-sufficient C57BL/6 mice. This lack of effect on the level ofIgG2a could be due to the increased secretion of IFN-�, awell-characterized IgG2a-switching factor (46). Despite thelower levels, IL-4�/� mice secreted all anti-P. brasiliensis iso-types, demonstrating that IL-4 activity could be replaced byother cytokines.

In conclusion, the genetically determined absence of IL-4 isprotective to P. brasiliensis infection of C57BL/6 mice, and itseffect can be indirectly seen as early as 48 h after fungalinoculation by the increased ability of IL-4�/� lung phagocytesto kill P. brasiliensis yeast cells. The precocious increased pro-duction of pulmonary IFN-�, the enhanced killing ability ofalveolar macrophages, and the augmented inflammatory PMNinflux into the lungs appear to lead to a more efficient protec-tive immunity which, however, does not switch to the Th1phenotype, as evidenced by the lack of reversal of DTH anergyand 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 Fundacao

de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP).

REFERENCES

1. Allendoerfer, R., G. P. Boivin, and G. S. Deepe, Jr. 1997. Modulation ofimmune responses in murine pulmonary histoplasmosis. J. Infect. Dis. 175:905–914.

2. Arruda, C., M. F. Franco, S. S. Kashino, F. R. F. Nascimento, R. A. Fazioli,C. A. C. Vaz, M. Russo, and V. L. G. Calich. 2002. IL-12 protects mice againstdisseminated infection caused by Paracoccidioides brasiliensis but enhancespulmonary inflammation. Clin. Immunol. 103:185–195.

3. Baida, H. B., P. J. C. Biselli, M. Juvenale, G. M. B. Del Negro, M. J. S.Mendes-Giannini, A. J. S. Duarte, and G. Benard. 1999. Differential anti-body isotype expression to the major Paracoccidioides brasiliensis antigen injuvenile and adult form paracoccidioidomycosis. Microbes Infect. 1:273–278.

4. Bancroft, A. J., D. Artis, D. D. Donaldson, J. P. Sypek, and R. K. Grencis.2000. Gastrointestinal nematode expulsion in IL-4 knockout mice is IL-13dependent. Eur. J. Immunol. 30:2083–2091.

5. Benard, G., C. C. Romano, C. R. Cacere, M. Juvenale, M. J. Mendes-Giannini, and A. J. Duarte. 2001. Imbalance of IL-2, IFN-gamma and IL-10secretion in the immunosuppression associated with human paracoccidioid-omycosis. Cytokine 13:248–252.

6. Biedermann, T., S. Zimmermann, H. Himmelrich, A. Gumy, O. Egeter, A. K.Sakrauski, I. Seegmuller, H. Voigt, P. Launois, A. D. Levine, H. Wagner, K.Heeg, J. A. Louis, and M. Rocken. 2001. IL-4 instructs Th1 responses andresistance to Leishmania major in susceptible BALB/c mice. Nat. Immunol.2:1054–1060.

7. Bocca, A. L., E. E. Hayashi, A. G. Pinheiro, A. B. Furianetto, A. P. Cam-panelli, F. Q. Cunha, and F. Figueiredo. 1988. Treatment of Paracoccidioidesbrasiliensis infected mice with a nitric oxide inhibitor prevents the failure ofcell-mediated immune responses. J. Immunol. 162:3056–3063.

8. Brummer, E. 1994. Interaction of Paracoccidioides brasiliensis with host de-fense cells, p. 213–223. In M. Franco, C. S. Lacaz, A. Restrepo, and G. DelNegro (ed.), Paracoccidioidomycosis. CRC Press, Boca Raton, Fla.

9. Brummer, E., E. Castaneda, and A. Restrepo. 1993. Paracoccidioidomycosis:an update. Clin. Microbiol. Rev. 6:89–117.

10. Calich, V. L. G., C. A. C. Vaz, and E. Burger. 1998. Immunity to Paracoc-cidioides brasiliensis infection. Res. Immunol. 149:407–416.

11. Calich, V. L. G., and S. S. Kashino. 1998. Cytokines produced by susceptibleand resistant mice in the course of Paracoccidioides brasiliensis infection.Braz. J. Med. Biol. Res. 31:615–623.

12. Camargo, Z. P., C. P. Taborda, E. G. Rodrigues, and L. R. Travassos. 1991.The use of cell-free antigens of Paracoccidioides brasiliensis in serologicaltests. J. Med. Vet. Mycol. 29:31–38.

13. Cano, L. E., L. M. Singer-Vermes, J. A. Mengel, C. F. Xidieh, C. Arruda,D. C. Andre, C. A. C. Vaz, E. Burger, and V. L. G. Calich. 2000. Depletionof CD8 T cells in vivo impairs host defense of resistant and susceptible miceto pulmonary paracoccidioidomycosis. Infect. Immun. 68:352–359.

14. Cano, L. E., S. S. Kashino, C. Arruda, D. Andre, C. F. Xidieh, L. M.Singer-Vermes, C. A. C. Vaz, E. Burger, and V. L. G. Calich. 1998. Protectiverole of gamma interferon in experimental pulmonary paracoccidioidomyco-sis. Infect. Immun. 66:800–806.

15. Cano, L. E., L. M. Singer-Vermes, C. A. C. Vaz, M. Russo, and V. L. G.Calich. 1995. Pulmonary paracoccidioidomycosis in resistant and susceptiblemice: relationship among progression of infection, bronchoalveolar cell ac-tivation, cellular immune response, and specific isotype patterns. Infect.Immun. 63:1777–1783.

16. Deepe, G. S., L. Romani, V. L. G. Calich, G. Huffnagle, C. Arruda, E. E. I. W.Molinari-Madlum, and J. R. Perfect. 2000. Knockout mice as experimentalmodels of virulence. Med. Mycol. 38:87–98.

17. Fallon, P. G., E. J. Richardson, G. J. MacKenzie, and A. N. McKenzie. 2000.Schistosome infection of transgenic mice defines distinct and contrastingpathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J. Immunol.164:2585–2591.

18. Fava Netto, C., V. S. Vegas, I. M. Sciannamea, and E. D. B. Guarnieri. 1969.Antígeno polissacarídico do Paracoccidioides brasiliensis. Estudo do tempode cultivo do P. brasiliensis necessario ao preparo do antígeno. Rev. Inst.Med. Trop. Sao Paulo 11:177–181.

19. Fazioli, R. A., L. M. Singer-Vermes, S. S. Kashino, E. Burger, M. Franco, M.Moschardi-Bacchi, and V. L. G. Calich. 1994. Delayed-type hypersensitivityresponse in an isogenic murine model of paracoccidioidomycosis. Myco-pathologia 126:137–146.

20. Franco, M. F., R. P. Mendes, M. Moscardi-Bachi, M. T. Reskallah-Iwasso,and M. R. Montenegro. 1989. Paracoccidioidomycosis. Bailliere’s Clin. Trop.Med. Commun. 4:185–220.

21. Gonzalez, A., W. de Gregori, D. Velez, A. Restrepo, and L. E. Cano. 2000.Nitric oxide participation in the fungicidal mechanism of gamma interferon-activated murine macrophages against Paracoccidioides brasiliensis. Infect.Immun. 68:2546–2552.

22. Gu, L., B. Rutledge, J. Fiorillo, C. Ernst, I. Grewal, R. Flavell, R. Gladue,and B. Rollins. 1997. In vivo properties of monocyte chemoattractant pro-tein-1. J. Leukoc. Biol. 62:577–580.

23. Hickey, M. J., D. N. Granger, and P. Kubes. 1999. Molecular mechanisms

FIG. 8. Levels of P. brasiliensis-specific antibodies in sera of IL-4�/� and WT control mice at week 8 after i.t. infection with 106 yeastcells. Sera were assayed for total Ig, IgM, IgA, IgG1, IgG2a, IgG2b,and IgG3 by using an isotype-specific ELISA as detailed in Materialsand Methods. The bars depict means (log2) � standard errors of serumtiters (6 to 8 mice per group). *, significant difference (P 0.05) fromWT group.


on Novem


.org/D

ownloaded from

http://iai.asm.org/

underlying IL-4-induced leukocyte recruitment in vivo: a critical role for thealpha-4 integrin. J. Immunol. 163:3441–3448.

24. Hogaboam, C. M., N. W. Lukacs, S. W. Chensue, R. M. Strieter, and S. L.Kunkel. 1998. Monocyte chemoattractant protein-1 synthesis by murine lungfibroblasts modulates CD4� T cell activation. J. Immunol. 160:4606–4614.

25. Hostetler, J. S., E. Brummer, R. I. Coffman, and D. Stevens. 1993. Effect ofanti-IL-4, interferon-gamma and antifungal triazole (SCH42427) in paracoc-cidioidomycosis: correlation of IgE levels with outcome. Clin. Exp. Immunol.94:11–16.

26. Huffnagle, G. B., R. M. Strieter, T. J. Standiford, R. A. McDonald, M. D.Burdick, S. L. Kunkel, and G. B. Toews. 1995. The role of monocyte che-motactic protein-1 (MCP-1) in the recruitment of monocytes and CD4� Tcells during a pulmonary Cryptococcus neoformans infection. J. Immunol.155:4790–4797.

27. Johnson, C. M., A. M. Cooper, A. A. Frank, and I. M. Orme. 1998. Adequateexpression of protective immunity in the absence of granuloma formation inMycobacterium tuberculosis-infected mice with a disruption in the intracel-lular adhesion molecule 1 gene. Infect. Immun. 66:1666–1670.

28. Karhawi, A. S. K., A. L. Colombo, and R. Salomao. 2000. Production ofIFN-� is impaired in patients with paracoccidioidomycosis during activedisease and is restored after clinical remission. Med. Mycol. 38:225–229.

29. Kashino, S. S., R. A. Fazioli, C. Cafalli-Favati, L. H. Meloni-Bruneri,C. A. C. Vaz, E. Burger, and V. L. G. Calich. 2000. Resistance to Paracoc-cidiodes brasiliensis infection is linked to a preferential Th1 immune responsewhereas susceptibility is associated with absence of IFN-� production. J. In-terferon Cytokine Res. 20:89–97.

30. Kashino, S. S., L. M. Singer-Vermes, V. L. G. Calich, and E. Burger. 1990.Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate donot correlate with its in vitro growth. Mycopathologia 111:173–180.

31. Kay, A. B., S. Ying, V. Varney, M. Gaga, S. R. Durham, R. Moqbel, A. J.Wardlaw, and Q. Hamid. 1991. Messenger RNA expression of the cytokinegene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophagecolony-stimulating factor, in allergen induced late-phase cutaneous reactionsin atopic subjects. J. Exp. Med. 173:775–778.

32. Kelly, B. L., and R. M. Locksley. 2000. Coordinate regulation of the IL-4,IL-13 and IL-5 cytokine cluster in the TH2 clones revealed by allelic expres-sion patterns. J. Immunol. 165:2982–2986.

33. Kopf, M., G. Le Gros, A. J. Coyle, M. Kosko-Vilbois, and F. Brombacher.1995. Immune responses of IL-4, IL-5 and IL-6 deficient mice. Immunol.Rev. 148:45–69.

34. La Flamme, A. C., E. A. Patton, B. Bauman, and E. J. Pearce. 2001. IL-4plays a crucial role in regulating oxidative damage in the liver during schis-tosomiasis. J. Immunol. 166:1903–1911.

35. Lee, J. S., H. D Campbell, C. A. Kozak, and I. G. Young. 1989. The IL-4 andIL-5 genes are closely linked and are part of a cytokine gene cluster onmouse chromosome 11. Somat. Cell Mol. Genet. 15:143–152.

36. Mamoni, R. L. R., A. M. S. Nouer, S. A. Oliveira, C. C. Musatti, C. L. Rossi,Z. P. Camargo, and M. H. S. L. Blotta. 2002. Enhanced production ofspecific IgG4, IgE, IgA and TGF-� in sera from patients with the juvenileform of paracoccidioidomycosis. Med. Mycol. 40:1–7.

37. Mencacci, A., G. Del Sero, E. Cenci, C. F. d’Ostiani, A. Bacci, C. Montagnoli,M. Kopf, and L. Romani. 1998. Endogenous interleukin-4 is required for

development of protective CD4� T helper type 1 cell responses to Candidaalbicans. J. Exp. Med. 187:307–317.

38. Metwali, A., D. Elliot, A. M. Blum, J. Li, M. Sandor, R. R. Lynch, N.Noben-Trauth, and J. V. Weinstock. 1996. The granulomatous response inmurine Shistosomiasis mansoni does not switch to Th1 in IL-4-deficientC57BL/6 mice. J. Immunol. 157:4546–4553.

39. Nascimento, F. R. F., V. L. G. Calich, D. Rodrigues, and M. Russo. 2002.Dual role of nitric oxide in paracoccidioidomycosis: essential for resistancebut overproduction associated with susceptibility. J. Immunol. 168:4593–4600.

40. Newman, S. L., and L. Gootee. 1992. Colony-stimulating factors activatehuman macrophages to inhibit intracellular growth of Histoplasma capsula-tum yeasts. Infect. Immun. 60:4593–4597.

41. Oliveira, S. J., R. L. Mamoni, C. C. Musatti, P. M. Papaiodarnau, andM. H. S. L. Blotta. 2002. Cytokines and lymphocyte proliferation in juvenileand adult forms of paracoccidioidomycosis: comparison with infected andnon-infected controls. Microbes Infect. 4:139–144.

42. Pearce, E. J., A. Cheever, S. Leonard, M. Covalesky, R. Fernandez-Botran,G. Kohler, and M. Kopf. 1996. Schistosoma mansoni in IL-4-deficient mice.Int. Immunol. 8:435–444.

43. Rollins, B. J. 1996. Monocyte chemoattractant protein 1: a potential regu-lator of monocyte recruitment in inflammatory disease. Mol. Med. Today2:198–204.

44. Roy, S., R. Charboneau, D. Melnyk, and R. A. Barke. 2000. Interleukin-4regulates macrophage interleukin-12 protein synthesis through a c-fos me-diated mechanism. Surgery 128:219–224.

45. Salerno, A., F. Dieli, G. Sireci, A. Bellavia, and G. L. Asherson. 1995.Interleukin-4 is a critical cytokine in contact sensitivity. Immunology 84:404–409.

46. Seder, R. A., and W. E. Paul. 1994. Acquisition of lymphokine producingphenotype by CD4� T cells. Annu. Rev. Immunol. 22:1179–1184.

47. Singer-Vermes, L. M., M. C. Ciavaglia, S. S. Kashino, E. Burger, andV. L. G. Calich. 1992. The source of the growth-promoting factor(s) affectsthe plating efficiency of Paracoccidioides brasiliensis. J. Med. Vet. Mycol.30:261–264.

48. Snapper, C. M., and W. E. Paul. 1987. Interferon-� and B stimulatoryfactor-I reciprocally regulate Ig isotype production. Science 236:944–947.

49. Snapper, C. M., K. B. Marcu, and P. Zelazowsky. 1997. Immunoglobulinclass switch: beyond accessibility. Immunity 6:217–223.

50. Souto, J. F., F. Figueiredo, A. Furlanetto, K. Pfeffer, M. A. Rossi, and J. S.Silva. 2000. Interferon-� and tumor necrosis factor–� determines resistanceto Paracoccidioides brasiliensis infection. Am. J. Pathol. 156:1811–1820.

51. Traidl, C., F. Jugert, T. Krieg, H. Merk, and N. Hulzelmann. 1999. Inhibitionof allergic contact dermatitis to DNCB but not oxazolone in interleukin-4-deficient mice. J. Investig. Dermatol. 112:476–482.

52. Traynor, T. R., W. A. Kuziel, G. B. Toews, and G. B. Huffnagle. 2000. CCR2expression determines T1 versus T2 polarization during pulmonary Crypto-coccus neoformans infection. J. Immunol. 164:2021–2027.

53. Trinchieri, G., and P. Scott. 1995. Interleukin 12: a proinflammatory cyto-kine with immunoregulatory functions. Res. Immunol. 146:423–431.

54. Zar, J. H. 1984. Biostatistical analysis, 2nd ed. Prentice Hall, Inc., Engle-wood Cliffs, N.J.

Editor: S. H. E. Kaufmann


on Novem


.org/D

ownloaded from

http://iai.asm.org/

Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with...

Documents

Transcript of Absence of Interleukin-4 Determines Less Severe Pulmonary Paracoccidioidomycosis Associated with...