Produção Nacional
Revisado por pares
Interferon-γ and Tumor Necrosis Factor-α Determine Resistance to Paracoccidioides brasiliensis Infection in Mice
2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65053-5
ISSN1525-2191
AutoresJaneusa Trindade de Souto, Florêncio Figueiredo, Alessandra Furlanetto, Klaus Pfeffer, Marcos A. Rossi, João S. Silva,
Tópico(s)Toxoplasma gondii Research Studies
ResumoTo investigate the role of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-γ (GKO) or TNF-α receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-α levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-α but higher levels of IFN-γ. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO. resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-γ to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-α, acting through the p55 receptor, and IFN-γ mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness. To investigate the role of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-γ (GKO) or TNF-α receptor p55 (p55KO) were infected with the parasite. GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively. Typical inflammatory granulomas were found only in WT mice. In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice. Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs. Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-α levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-α but higher levels of IFN-γ. Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO. resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice. On the contrary, the addition of IFN-γ to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation. Taken together, these findings suggests that endogenous TNF-α, acting through the p55 receptor, and IFN-γ mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness. Paracoccidioidomycosis (PCM), the major systemic mycosis in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (Pb). The infection is acquired by inhalation of airborne propagules produced by the fungal mycelium form, which then change into the pathogenic yeast form when at core body temperature.1McEwen J Bedoya V Patiño MM Salazar ME Restrepo AM Experimental murine paracoccidioidomycosis induced by inhalation of conidia.J Med Vet Mycol. 1987; 25: 165-167Crossref PubMed Scopus (180) Google Scholar The yeast forms in the mammalian host can be eliminated by immune competent cells or disseminate to tissues by the lymphatic and hematogenous route. After infection there are no immediate signs or symptoms of the disease, characterizing silent PCM but the host develops a specific immune response against fungal antigens which can be detected by a skin test. In endemic areas, up to 60% of asymptomatic individuals may be skin test positive.2Restrepo A The ecology of Paracoccidioides brasiliensis, a puzzle still unsolved.Sabouraudia. 1985; 23: 323-334Crossref PubMed Scopus (219) Google Scholar In the acute form of clinically manifest infection, there is an involvement of the reticuloendothelial system, a nonspecific hypergammaglobulinemia, a depressed cellular immune response, a diffuse inflammatory granulomatous response, and concomitant fungus dissemination. The chronic form presents a broad spectrum of clinical manifestations with frequent damage of the lung and oropharyngeal mucosa and a profound impairment of the immune response.3Franco M Montenegro MR Mendes RP Marques AS Dillon NL Mota NS Paracoccidioidomycosis: a recently proposed classification of its clinical forms.Rev Soc Bras Med Trop. 1987; 20: 129-132Crossref PubMed Scopus (238) Google Scholar In this phase of infection, an epithelioid inflammatory granulomatous reaction is known to prevent the dissemination of the fungus.4Franco M Host-parasite relationships in paracoccidioidomycosis.J Med Vet Mycol. 1986; 25: 5-18Crossref Scopus (216) Google Scholar Athymic mice do not control dissemination of the fungus, suggesting that cell-mediated immunity is an important host defense mechanism against Pb infection.5Miyaji M Nishimura K Granuloma formation and killing functions of granuloma in congenitally athymic nude mice infected with Blastomyces dermatitides and Paracoccidioides brasiliensis.Mycopathologia. 1983; 82: 129-141Crossref PubMed Scopus (43) Google Scholar, 6Burger E Vaz CCA Sano A Calich VLG Singer-Vermes LM Xidieh CF Kashino SS Nishimura K Miyaji M Paracoccidioides brasiliensis infection in nude mice: studies with isolates differing in virulence and definition of their T cell-dependent and T cell-independent components.Am J Trop Med Hyg. 1996; 55: 391-398PubMed Google Scholar However, there is a T cell dysfunction in patients and infected mice that may be related to alterations in the ratio of T cell subpopulations, suppressive effects of fungal components, and an imbalance in the levels of cytokine production.7Restrepo A Restrepo M Restrepo F Aristizabal LH Moncada LH Velez H Immune response in paracoccidioidomycosis. A controlled study of 16 patients before and after treatment.Sabouraudia. 1978; 16: 151-163Crossref PubMed Scopus (54) Google Scholar, 8Benard G Hong MA Del-Negro GMB Batista L Shikanai-Yasuda MA Duarte AJS Antigen-specific immunosuppression in paracoccidioidomycosis.Am J Trop Med Hyg. 1996; 54: 7-12PubMed Google Scholar, 9Benard G Mendes-Giannini MJS Juvenale M Miranda ET Duarte AJ Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response.J Infect Dis. 1997; 175: 1263-1267Crossref PubMed Scopus (71) Google Scholar, 10Silva M Bocca AL Ferracini Jr, R Figueiredo F Silva CL Cellular requirements for immunomodulatory effects caused by cell wall components of Paracoccidioides brasiliensis on antibody production.Clin Exp Immunol. 1997; 109: 261-271Crossref PubMed Scopus (7) Google Scholar The cellular immune response against fungal antigens that occurs in resistant mice results in macrophage activation and granuloma formation.11Figueiredo F Alves LMC Silva CL Tumor necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.Clin Exp Immunol. 1993; 93: 189-194Crossref PubMed Scopus (45) Google Scholar Tumor necrosis factor-α (TNF-α) produced by macrophages in response to the Pb cell wall component11Figueiredo F Alves LMC Silva CL Tumor necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.Clin Exp Immunol. 1993; 93: 189-194Crossref PubMed Scopus (45) Google Scholar is required for macrophage accumulation and differentiation into epithelioid cells, and for persistence of well-formed granulomas.12Kindler V Sappino AP Grau GE Piguet PF Vassali P The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection.Cell. 1989; 56: 731-740Abstract Full Text PDF PubMed Scopus (1082) Google Scholar Interferon-γ (IFN-γ) can activate infected macrophages to secret TNF-α and to inhibit the replication of Pb.13Brummer E Hanson LH Restrepo A Stevens DA In vivo and in vitro activation of pulmonary macrophage by IFN-γ for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatidis.J Immunol. 1988; 140: 2786-2789PubMed Google Scholar Recently, experiments performed on Pb-infected mice treated with anti-IFN-γ revealed an exacerbation of pulmonary infection and earlier fungal dissemination.14Cano LE Kashino SS Arruda C André D Xidieh CF Singer-Vermes L Vaz CAC Burger E Calich VLG Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis.Infect Immun. 1998; 66: 800-806Crossref PubMed Google Scholar In the present study we evaluated the role of IFN-γ and TNF-α in the resistance to Pb infection in mice. Using mice genetically deficient in IFN-γ (GKO) or TNF-α receptor p55 (p55KO), we could demonstrate that both cytokines are involved in the resistance to Pb infection, granuloma formation, and control of fungus dissemination. In addition, our data suggest that IFN-γ and TNF-α modulate the production of cytokines and nitric oxide (NO) and the T-cell proliferative response in Pb-infected mice. Breeding pairs of mice with targeted disruption of the IFN-γ (GKO mice)15Dalton DK Pitts-Meek S Keshav S Figari IS Bradley A Stewart TA Multiple defects of immune cell function in mice with disrupted interferon-gamma genes.Science. 1993; 259: 1739-1742Crossref PubMed Scopus (1514) Google Scholar and TNF-α receptor p55 (p55KO. genes16Pfeffer K Matsuyama T Kündig TM Wakeham A Kishihara K Shahinian A Wiegmann K Ohashi PS Krönke M Mak TW Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1564) Google Scholar were obtained from Jackson Laboratories (Bar Harbor, ME) and Amgen Institute (Toronto, Canada), respectively. Breeding stock backcrossed on C57Bl/6 were obtained and the genotype of p55KO and GKO mice determined by polymerase chain reaction of DNA as previously described.16Pfeffer K Matsuyama T Kündig TM Wakeham A Kishihara K Shahinian A Wiegmann K Ohashi PS Krönke M Mak TW Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1564) Google Scholar, 17Qian Q Cutler JE Gamma interferon is not essential in host defense against disseminated candidiasis in mice.Infect Immun. 1997; 65: 1748-1753PubMed Google Scholar Male C57Bl/6 wild-type (WT), GKO, and p55KO mice, 6 to 8 weeks old, were bred and maintained in microisolator cages in the animal housing facility of the Department of Immunology, University of São Paulo, Ribeirão Preto, Brazil. Yeast cells of virulent Pb 18 strains were cultured at 37°C in Fava-Netto's medium18Fava-Neto C Vegas VS Sciannamea IM Guarnieri DB Antígeno polissacarídico do Paracoccidioies brasiliensis. Estudo do tempo de cultivo do P. brasiliensis necessário ao preparo do antigeno.Rev Inst Med Top São Paulo. 1969; 11: 177-181PubMed Google Scholar for 7 to 14 days. The yeast cells were harvested and washed three times in phosphate-buffered saline (PBS), pH 7.2. Viability of yeast cells was determined as previously described.19Calich VLG Purchio A Paula CRA A new fluorescent viability test for fungi cell.Mycophatologia. 1979; 66: 175-177Crossref PubMed Scopus (142) Google Scholar The animals were infected intravenously with 1 × 106 viable yeast cells in 100 μl of PBS. The dissemination of fungus in lungs, livers, and spleens was assayed as previously described.20Cano LE Singer-Vermes LM Vaz CAC Russo M Calich VLG Pulmonary paracoccidioidomycosis in resistant and susceptible mice: relationship among progression of infection, bronchoalveolar cell activation, cellular immune response, and specific isotype patterns.Infect Immun. 1995; 63: 1777-1783PubMed Google Scholar The organs were removed, weighed, homogenized in sterile PBS, pH 7.2, and serially diluted. Aliquots of 100 μl were dispensed into Petri dishes, in duplicates, containing brain-heart infusion agar (Difco Laboratories, Detroit, MI) supplemented with 4% (v/v) of normal horse serum and 5% (v/v) of Pb 192 broth yeast culture filtrate from 2-week-old cultures as source of growth-promoting factor.21Singer-Vermes LM Ciavaglia MC Kashino SS Burger E Calich VLG The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis.J Med Mycol. 1992; 30: 261-264Crossref Scopus (90) Google Scholar Plates were incubated at 37°C, and colonies were counted 7 to 14 days later. Results were expressed as numbers of colony-forming units (CFU) ± SE per gram of tissue. Fungus cell walls were obtained as previously described.11Figueiredo F Alves LMC Silva CL Tumor necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.Clin Exp Immunol. 1993; 93: 189-194Crossref PubMed Scopus (45) Google Scholar Briefly, yeast forms were sonicated and lipids from the cell walls were removed by treatment with chloroform/methanol (2:1, v/v) with stirring at room temperature for 2 hours. Extracts were separated by centrifugation at 500 × g for 5 minutes. The resulting insoluble cell residue was named the cell wall fraction. The cell wall was further treated with 1 N NaOH and gently stirred at room temperature for 1 hour. The alkali-insoluble sediment was washed with water until it reached pH 7.0 and then washed with ethanol, followed by acetone and diethyl ether. The resulting white powder was named F1 fraction and contained <0.05 ng/ml of bacterial endotoxin, as determined by the Limulus amebocyte assay (Sigma Chemical Co., St. Louis, MO). Spleen cells from uninfected (day zero) or Pb-infected mice were washed in Hanks' medium and incubated for 4 minutes with lysis buffer (one part of 0.17 mol/L Tris and nine parts of 0.16 mol/L ammonium chloride). The cells were washed and suspended to a concentration of 5 × 106 cells per ml in RPMI 1640 (Flow Laboratories, McLean, VA) supplemented with 5% fetal calf serum (Life Technologies Inc., Bethesda, MD), 5 × 10−2 mol/L 2-β-mercaptoethanol, 2 mmol/L l-glutamine, and antibiotics (all from Sigma). The cells were cultured in flat-bottom 96-well plates (Corning Glass Works, Corning, NY) at 1 × 106/well with or without Con A (2 μg/ml) for 72 hours at 37°C in a humidified 5% CO2 incubator. To assess proliferation, 0.5 μCi/well of [3H]TdR (Amersham Corp., Arlington Heights, IL. was added during the final 18 hours of culture, the cells were harvested and radioactivity was measured in a scintillation counter. In some experiments, to inhibit the inducible nitric oxide synthase (iNOS), we added 200 μmol/L of NG-methyl-l-arginine (LNMMA, Sigma). Data were expressed as means (±SE) of counts per minute of triplicate cultures. For nitrite quantification, splenocytes (2 × 106 cells/ml) from WT, GKO, and p55KO animals were cultured in triplicate for 48 hours in medium alone or medium containing Con A (2 μg/ml) in the presence or absence of 100 U/ml of IFN-γ. Duplicates of nitrite concentrations in each culture supernatant were assayed in a microplate by mixing 0.1 ml of culture supernatant with 0.1 ml of Griess reagent.22Green LC Tannenbaum SR Goldman P Nitrate biosynthesis in germfree and conventional rat.Science. 1981; 212: 56-68Crossref PubMed Scopus (458) Google Scholar The A540 was read 10 minutes later, and the nitrite concentration was determined by reference to a standard curve of 1 to 100 μmol/L NaNO2. Five to seven animals selected at random from each group were sacrificed at 7 and 15 days (GKO) and at 15, 30, and 60 days (p55KO. after infection. The lungs obtained were fixed in 10% formalin for 24 hours and embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) or impregnated with silver for demonstration of reticulum fibers using standard protocols. WT mice were used as a control. Spleen cells (2 × 106 cells/ml) from normal or infected mice were cultured in 24-well tissue culture plates (Corning) with 2 μg/ml of Con A, 40 μg/ml of Pb18 F1 fraction, or medium alone for 48 hours at 37°C in a humidified 5. CO2 incubator. The supernatants were harvested and stored at −20°C until assayed for IFN-γ, TNF-α, interleukin (IL)-10, and IL-12, using a two-sandwich enzyme-linked immunosorbent assay. XMG 1.2 (anti-IFN-γ), XT22.11 (anti-TNF-α), JES5–2A5 (anti-IL-10), and C17.15.10 (anti-IL-12) were used as capture monoclonal antibody (mAb). IFN-γ and TNF-α bound to the mAb were visualized with polyclonal rabbit anti-IFN-γ or anti-TNF-α (both from Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit IgG conjugated with peroxidase (Life Technologies Inc., Gaithersburg, MD), whereas IL-10 and IL-12 were detected using appropriated biotinylated mAbs SXC1 (anti-IL-10) and C15.6 (anti-IL-12), respectively. A standard curve was prepared with specific cytokines (all from Sigma) and enzyme-linked immunosorbent assay sensitivities were 0.625 U/ml for IFN-γ, 312 pg/ml for TNF-α, and 390 pg/ml for IL-10 and IL-12. The results are expressed as the mean ± SE of the indicated number of animals or experiments. Statistical analysis was performed using analysis of variance followed by the parametric Tukey-Kramer test (INSTAT software, GraphPad, San Diego, CA). A P value < 0.05 was considered to indicate statistical significance. To determine the role of IFN-γ and TNF-α in the resistance to Pb infection, IFN-γ and TNF-α receptor p55 knockout mice were infected with yeast cells and their survival was compared with that of littermate controls. We found that whereas 100% of the control littermate mice survived during the 90-day period of the experiment, the infected GKO and p55KO mice succumbed by days 16 and 90 after parasite inoculation, respectively (Figure 1). The infected p55KO mice started to die after day 60 of infection and the mortality rate at day 90 of infection was 100% when compared with 100% of survival in infected WT animals. Because infected GKO mice did not survive beyond 16 days, the fungal loads in these animals were determined on days 4, 7, and 15 postinfection. The results showed a significantly higher amount of fungus in GKO mice than that verified in infected WT mice (P < 0.001; Figure 2, A–C). It is possible that the CFU numbers at days 7 and 15 are underestimated, because the data represent the CFU load of mice that have survived. The fungal loads obtained in the organs from infected p55KO mice (Figure 2, D–F) also were higher than those in control-infected WT mice (P < 0.001), except for the spleen at day 15 after infection, for which a statistically significant difference could not be detected (Figure 2F). Similar fungal loads were also obtained at days 4 and 7 postinfection in p55KO and WT mice (data not shown).Figure 2Lack of resistance of infected GKO and p55KO mice to control P. brasiliensis yeast cell proliferation and dissemination. Colony-forming units (CFU) of lungs (A and D), liver (B and E), and spleen (C and F) of WT (open bars), GKO (black bars), and p55KO mice (hatched bars. were determined on different days, as described in Material and Methods. The scale bars represent the mean ± SE of CFU obtained of duplicates cultures performed in groups of three animals and are representative of two independent experiments. *, P < 0.001 compared with infected WT mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Histopathological examination of lung tissue sections revealed marked differences between granulomatous lesions developed in WT mice compared with the lesions observed in both GKO and p55KO after infection with Pb18. At day 7 postinfection, the WT mice lungs showed a diffuse focal infiltrate of polymorphs and mononuclear cells in association with few yeast forms of the fungus. At day 15, diffuse foci of histiocytes, epithelioid cells, lymphocytes, and a few polymorphs aggregated around yeast cells, identifiable as epithelioid granulomas, could be seen (Figure 3A). The reticulum fiber staining clearly demonstrated the early structures of granuloma (Figure 3B). At day 30, well-organized granulomas composed of histiocytes, epithelioid cells, and multinuclear giant cells aggregated around yeast forms and surrounded by a rim of lymphocytes and fibroblasts, were formed (Figure 3C). The normal pattern of reticulum fibers present in granulomas could be clearly demonstrated (Figure 3D). At day 60 after parasite inoculation, multiple and confluent typical epithelioid granulomas were observed (data not shown). The lungs of GKO mice at day 7 after infection showed pulmonary damage characterized by diffuse microfoci of lung parenchyma consolidation depicted as intra-alveolar edema and hemorrhage and exudation of polymorphs and lymphomononuclear cells. At day 15, in agreement with CFU counts, the number of yeast forms of Pb was remarkably increased in association with an inflammatory infiltrate composed of polymorph, lymphomononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas (Figure 4A). The reticulum fiber pattern clearly showed the structure of these incipient granulomas and loss of pulmonary parenchyma architecture (Figure 4B). At day 15 after infection, the p55KO mice showed diffuse foci of lung tissue consolidation characterized by intra-alveolar exudation of lymphomononuclear cells and a few polymorphs correlated with a great number of yeast forms of the fungus (Figure 5A). The reticulum fiber pattern of the lung parenchyma was preserved (Figure 5B). At day 30, the pulmonary lesions were focal and diffuse, characterized by lymphomononuclear, a few epithelioid, and multinuclear giant cells in association with abundant yeast forms, without granuloma formation (Figure 5C). The reticulum fiber pattern of the lung was fairly well preserved (Figure 5D). In agreement with CFU counts, the number of yeast forms of the fungus was markedly increased at both intervals after infection. Progressively (60 days), the lung parenchyma was severely damaged by a diffuse mononuclear cell infiltration associated with an increased number of yeast forms (data not shown). Splenocytes from infected WT mice cultured with Con A produced less IFN-γ when compared with noninfected mice (day zero corresponds to baseline, before inoculation; Figure 6A) and with infected p55KO mice (Figure 6C). The TNF-α levels in supernatants of cells cultured with Pb F1 fraction increased progressively in infected WT mice on days 7 and 15, whereas infected GKO mice produced less TNF-α during the same period (Figure 6B). On the other hand, splenocytes from infected p55KO and WT mice cultured with Pb F1 fraction produced similar levels of TNF-α during the course of infection (Figure 6D). Cells from infected or uninfected mice cultured with medium only did not produce detectable levels of TNF-α or IFN-γ. Although the level of lipopolysaccharide in the F1 fraction was <0.05 ng/ml, we assayed for TNF-α levels in splenocytes from C3H/HeJ mice that are resistant to lipopolysaccharide. We found that the levels of TNF-α produced by the cells from C3H/HeJ in the presence of F1 fraction was similar to that found in C57Bl/6 mice (data not shown). Because IL-12 potentiates and IL-10 inhibits the IFN-γ production, we assayed for IL-12 and IL-10 in supernatants of splenocytes cultured with or without F1 fraction and Con A, respectively. We found that IL-12 levels in the supernatants of leukocytes from infected WT or knockout mice were similar. For example, on day 30 after infection, while the IFN-γ levels were significantly higher in p55KO than in WT mice (Figure 6C), the IL-12 levels were 923 ± 106 and 724 ± 101 pg/ml in infected WT and p55KO mice, respectively. The levels of IL-10 produced on day 30 after infection were higher in infected WT mice (913 ± 75 pg/ml) than in infected p55KO mice (215 ± 30 pg/ml). Increased levels of IL-10 were also found on day 7 after infection in supernatants of splenocytes from infected WT mice (5080 ± 540 pg/ml) compared with GKO mice (1666 ± 28 pg/ml). As previously described,23Bocca AL Hayashi EE Pinheiro AG Furlanetto AB Campanelli AP Cunha FQ Figueiredo F Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response.J Immunol. 1998; 161: 3056-3063PubMed Google Scholar splenocytes from WT mice infected with Pb18 yeast cells proliferated less than cells from normal mice. Conversely, splenocytes from infected GKO and p55KO mice exhibited a proliferative response similar to that of noninfected mice (day zero corresponds to baseline, before inoculation; Figure 7, A and C). When we made the observation that NO could be involved in the immunosuppression observed during Pb infection,23Bocca AL Hayashi EE Pinheiro AG Furlanetto AB Campanelli AP Cunha FQ Figueiredo F Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response.J Immunol. 1998; 161: 3056-3063PubMed Google Scholar we compared the NO production by spleen cells from infected knockout and WT mice. Interestingly, splenocytes from infected GKO and p55KO mice cultured with Con A produced less NO when compared with their infected littermates (Figure 7, B and D). Considering that abrogation of suppression of Con A-induced proliferate responses are concomitant to absence of IFN-γ and decrease in NO production (Figure 7), we evaluated the role of this cytokine in the control of T cell response and NO production by splenocytes of infected mice. We thus added IFN-γ to splenocytes from normal or infected WT and GKO mice and evaluated the subsequent T-cell proliferative response and NO production. We found that the addition of IFN-γ to splenocytes from infected GKO mice cultured with Con A restored NO production (Figure 8B) to the levels detected in infected WT mice (P < 0.001), and led to a significant inhibition of Con A-induced T cell proliferation (Figure 8A). The addition of IFN-γ to cell cultures from noninfected GKO mice led only to a slight increase in the nitrite synthesis (from 0.2 ± 0.05 to 1.67 ± 0.49 μmol/L), that was not enough to modify the cell proliferation. To verify if NO was involved in the mechanism that mediates inhibition of T cell proliferative responses, we added the iNOS inhibitor LNNMA to cell cultures. In the absence of Con A, the addition of LNMMA did not modify the cell proliferation or nitrite production (data not shown). However, addition of LNMMA to spleen cells from infected WT mice cultured with Con A abrogated the NO production and, therefore, reversed the level of T cell proliferation to that found in cells from infected GKO, as well as uninfected WT and GKO mice. On the contrary, the addition of LNMMA to cells from normal or infected GKO mice did not result in alteration in cell proliferation, thus confirming the role of NO in suppression (Figure 8). Therefore, decreased NO production leads to increased T-cell proliferative response.Figure 8Treatments that restore or abrogate NO production by splenocytes from Pb-infected mice inhibit and reverse T cell proliferation, respectively. Spleen cells from noninfected (NI) WT (striped bars. and GKO (hatched bars) mice or WT (open bars. and GKO (black bars) mice at 15 days after infection (I) were cultured with Con A (2 μg/ml) with or without IFN-γ (100 U/ml) or LNMMA (200 μmol/L) and the T-cell proliferative response (A) and NO production (B. were evaluated (for details see Materials and Methods). Splenocytes cultured with medium only were used as a control. Each scale bar represents the mean ± SE of three mice per group in an experiment representative of two separate experiments. *, P < 0.001 compared with infected WT mice. **, P < 0.01 compared with GKO infected mice in absence of IFN-γ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Disseminated PCM in GKO and p55KO mice as well as the kinetics of lesion development in lungs after Pb infection were described. The pattern of cytokine response in the absence of IFN-γ and TNF-α receptor p55 in infected mice was also evaluated. Our results suggest that IFN-γ and TNF-α are essential for the resistance and survival of Pb-infected mice. Although the role of IFN-γ has been extensively studied in fungal infections such as Candida albicans18Fava-Neto C Vegas VS Sciannamea IM Guarnieri DB Antígeno polissacarídico do Paracoccidioies brasiliensis. Estudo do tempo de cultivo do P. brasiliensis necessário ao preparo do antigeno.Rev Inst Med Top São Paulo. 1969; 11: 177-181PubMed Google Scholar, 24Kaposzta R Tree P Marodi L Gordon S Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans.Infect Immun. 1998; 66: 1708-1717PubMed Google Scholar, 25Balish E Wagner RD Vazque
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