Produção Nacional
Revisado por pares
Mast Cell Function and Death in Trypanosoma cruzi Infection
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.014
ISSN1525-2191
AutoresMarcelo Meuser-Batista, José R. Corrêa, Vinícius Frias Carvalho, Constança Britto, Otacílio C. Moreira, Marcos Meuser Batista, Maurílio José Soares, Francisco Alves Farias Filho, Patrı́cia Silva, Joseli Lannes-Vieira, Robson Coutinho‐Silva, Andrea Henriques‐Pons,
Tópico(s)Trypanosoma species research and implications
ResumoAlthough the roles of mast cells (MCs) are essential in many inflammatory and fibrotic diseases, their role in Trypanosoma cruzi–induced cardiomyopathy is unexplored. In this study, we treated infected CBA mice with cromolyn, an MC stabilizer, and observed much greater parasitemia and interferon-γ levels, higher mortality, myocarditis, and cardiac damage. Although these data show that MCs are important in controlling acute infection, we observed MC apoptosis in the cardiac tissue and peritoneal cavity of untreated mice. In the heart, pericardial mucosal MC die, perhaps because of reduced amounts of local stem cell factor. Using RT-PCR in purified cardiac MCs, we observed that infection induced transcription of P2X7 receptor and Fas, two molecules reportedly involved in cell death and inflammatory regulation. In gld/gld mice (FasL−/−), apoptosis of cardiac, but not peritoneal, MCs was decreased. Conversely, infection of P2X7−/− mice led to reduced peritoneal, but not cardiac, MC death. These data illustrate the immunomodulatory role played by MCs in T. cruzi infection and the complexity of molecular interactions that control inflammatory pathways in different tissues and compartments. Although the roles of mast cells (MCs) are essential in many inflammatory and fibrotic diseases, their role in Trypanosoma cruzi–induced cardiomyopathy is unexplored. In this study, we treated infected CBA mice with cromolyn, an MC stabilizer, and observed much greater parasitemia and interferon-γ levels, higher mortality, myocarditis, and cardiac damage. Although these data show that MCs are important in controlling acute infection, we observed MC apoptosis in the cardiac tissue and peritoneal cavity of untreated mice. In the heart, pericardial mucosal MC die, perhaps because of reduced amounts of local stem cell factor. Using RT-PCR in purified cardiac MCs, we observed that infection induced transcription of P2X7 receptor and Fas, two molecules reportedly involved in cell death and inflammatory regulation. In gld/gld mice (FasL−/−), apoptosis of cardiac, but not peritoneal, MCs was decreased. Conversely, infection of P2X7−/− mice led to reduced peritoneal, but not cardiac, MC death. These data illustrate the immunomodulatory role played by MCs in T. cruzi infection and the complexity of molecular interactions that control inflammatory pathways in different tissues and compartments. Bone marrow–derived mast cell (MC) precursors circulate in the blood and lymphatic vessels and migrate to tissues such as muscle and mucosa and celomatic cavities, where they assume mature morphologic and functional characteristics under the influence of local microenvironmental factors.1Metcalfe D.D. Baram D. Mekori Y.A. 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Fas-beyond death: a regenerative role for Fas in the nervous system.Apoptosis. 2003; 8: 551-562Crossref PubMed Scopus (74) Google Scholar and inflammatory regulation.12Melo de Oliveira G. Lopes Diniz R. Batista W. Meuser Batista M. Bani Correa C. Cremonini de Araujo-Jorge T. Henriques-Pons A. Fas ligand-dependent Inflammatory regulation in acute myocarditis induced by Trypanosoma cruzi infection.Am J Pathol. 2007; 171: 79-86Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Another membrane receptor not only involved in apoptosis but also with a wider range of proinflammatory functions is the ATP-sensitive purinergic P2X7 receptor.13da Cruz C.M. Ventura A.L. Schachter J. Costa-Junior H.M. da Silva Souza H.A. Gomes F.R. Coutinho-Silva R. Ojcius D.M. Persechini P.M. 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The P2X7 receptor: a key player in IL-1 processing and release.J Immunol. 2006; 176: 3877-3883PubMed Google Scholar CD62-L shedding from lymphocytes,16Gu B. Bendall L.J. Wiley J.S. Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases.Blood. 1998; 92: 946-951Crossref PubMed Google Scholar and maturation of T cells.17Tsukimoto M. Maehata M. Harada H. Ikari A. Takagi K. Degawa M. P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways.J Immunol. 2006; 177: 2842-2850Crossref PubMed Scopus (66) Google Scholar Bone marrow–derived MCs and MC lines8Bulanova E. Budagian V. Orinska Z. Hein M. Petersen F. Thon L. Adam D. Bulfone-Paus S. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.J Immunol. 2005; 174: 3880-3890PubMed Google Scholar undergo apoptosis and Ca++ influx through P2X7 activation, recruiting caspases 8 and 3, with phosphorylation of ERK, JAK2, and STAT6 and enhanced secretion of IL-4, IL-13, IL-6, and TNF-α.8Bulanova E. Budagian V. Orinska Z. Hein M. Petersen F. Thon L. Adam D. Bulfone-Paus S. Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells.J Immunol. 2005; 174: 3880-3890PubMed Google Scholar Regarding the heart, MCs have been implicated in cardiovascular dysfunctions, such as ischemic heart disease, experimental myocardial infarction, myocarditis, heart failure, transplant-related fibrosis, and hypertensive heart disease.18Kalesnikoff J. Galli S.J. New developments in mast cell biology.Nat Immunol. 2008; 9: 1215-1223Crossref PubMed Scopus (587) Google Scholar, 19Palladini G. Tozzi R. Perlini S. 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Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.J Card Fail. 2005; 11: 548-556Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar In the particular case of myocarditis induced by T. cruzi infection, inflammatory foci in humans and experimental models are mostly composed of CD8+ T cells, with cardiac dysfunction normally associated with fibrosis as the main cause of death.23Andrade Z.A. Andrade S.G. Sadigursky M. Wenthold Jr., R.J. Hilbert S.L. Ferrans V.J. The indeterminate phase of Chagas' disease: ultrastructural characterization of cardiac changes in the canine model.Am J Trop Med Hyg. 1997; 57: 328-336PubMed Google Scholar, 24Silva J.S. Machado F.S. Martins G.A. The role of nitric oxide in the pathogenesis of Chagas disease.Front Biosci. 2003; 8: s314-s325Crossref PubMed Scopus (79) Google Scholar, 25Henriques-Pons A. Oliveira G.M. Paiva M.M. Correa A.F. Batista M.M. Bisaggio R.C. 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Chemokines, inflammation and Trypanosoma cruzi infection.Trends Parasitol. 2002; 18: 262-265Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar For example, macrophages are important in the control of parasite replication, and, apparently, blood eosinophils are an important source of cytokines in patients with cardiodigestive forms of the disease.29Cardoso G.M. Morato M.J. Gomes J.A. Rocha M.O. Bonfim I.P. Williams-Blangero S. VandeBerg J.L. Reis M.R. Magalhães E.F. Correa-Oliveira R. Comparative analysis of cell phenotypes in different severe clinical forms of Chagas' disease.Front Biosci. 2006; 11: 1158-1163Crossref PubMed Scopus (10) Google Scholar Regarding MCs, due to technical limitations, such as a reduced number of cardiac MCs, in situ identification, isolation from the tissue, and purification, most authors use histopathologic analysis and peritoneal MCs to study their relevance in T. cruzi infection. To date, acutely T. cruzi–infected rats present a decrease in cardiac MC numbers,30Chapadeiro E. Beraldo P.S. Jesus P.C. Oliveira Junior W.P. Junqueira Junior L.F. [Cardiac lesions in Wistar rats inoculated with various strains of Trypanosoma cruzi].Rev Soc Bras Med Trop. 1988; 21: 95-103Crossref PubMed Google Scholar whereas analysis of chronic human patients showed an increase in these cell numbers.31Almeida H.O. Pereira F.E. Tafuri W.L. [Mast cells in Chagas' chronic cardiopathy].Rev Inst Med Trop Sao Paulo. 1975; 17: 5-9PubMed Google Scholar, 32Cabral H.R. Novak I.T. Glocker T.M. Castro Viera G.A. Chagas cardiopathy: identification and quantification of infiltrating cells in the hearts of cardiac death patients of different ages [in Spanish].Rev Fac Cien Med Univ Nac Cordoba. 2002; 59: 83-89PubMed Google Scholar The relevance of MCs to heart fibrosis is also a matter of debate31Almeida H.O. Pereira F.E. Tafuri W.L. [Mast cells in Chagas' chronic cardiopathy].Rev Inst Med Trop Sao Paulo. 1975; 17: 5-9PubMed Google Scholar, 33Pinheiro M.C. Beraldo P.S. Junqueira Junior L.F. Lopes E.R. Chapadeiro E. A quantitative analysis of the mastocytes and eosinophilic granulocytes in the myocardium of Wistar rats chronically infected by Trypanosoma cruzi: a contribution to the knowledge of myocardial fibrosis [in Portuguese].Rev Soc Bras Med Trop. 1992; 25: 45-50Crossref PubMed Google Scholar because chronically infected rats and human patients show increased MC counts but not necessarily increased fibrosis. Herein, we aimed to evaluate the functional role of MCs in acute T. cruzi infection by using cromolyn, an MC stabilizer that blocks histamine release, and to observe the aggravation of the disease. Because we observed MC apoptosis in untreated mice, we studied the mechanistic relevance of some membrane death receptors and soluble factors in the control of MC subpopulations after the infection. We observed that MCs control blood and tissue parasitemia, production of interferon-γ (IFN-γ), cardiac inflammation, and susceptibility to infection. Cardiac MCs trigger the transcription of Fas after infection and apparently die after Fas-L engagement. Although P2X7 receptor is expressed in cardiac MCs after infection, it seems to be involved in the death of only peritoneal MCs. Ten- to 12-week-old specific pathogen–free male mice CBA/J, BALB/c, gld/gld (Fas-L−/−) (BALB/c background-susceptible mouse strain), and C57BL/6 (resistant) were obtained from the FIOCRUZ animal facility (CECAL, Rio de Janeiro, Brazil). P2X7−/− mice (C57BL/6 background), derived from Pfizer (Groton, CT) and generated by Solle et al,34Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. Altered cytokine production in mice lacking P2X(7) receptors.J Biol Chem. 2001; 276: 125-132Crossref PubMed Scopus (778) Google Scholar were a gift from Dr. Christopher A. Gabel and were bred at the Transgenic Mice Laboratory, Instituto de Biofísica Carlos Chagas Filho at the Universidade Federal do Rio de Janeiro.34Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. Altered cytokine production in mice lacking P2X(7) receptors.J Biol Chem. 2001; 276: 125-132Crossref PubMed Scopus (778) Google Scholar Mice were housed for at least 1 week before infection at the Animal Experimentation Division in conditions complying with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1985). This project has protocol number 0308/06 at the FIOCRUZ Committee of Ethics in Research, according to resolution 196/96 of the National Health Council of the Brazilian Ministry of Health. For experimental infection, bloodstream trypomastigote forms of Trypanosoma cruzi CL strain were obtained from infected Swiss-Webster mice and were isolated as previously described.35de Araujo-Jorge T.C. The biology of Trypanosoma cruzi-macrophage interaction.Mem Inst Oswaldo Cruz. 1989; 84: 441-462Crossref PubMed Google Scholar Mice were i.p. infected with 1 × 104 parasites in 200 μL of PBS (pH 7.2) (Sigma-Aldrich, St Louis, MO), and age-matched control mice received 200 μL of PBS. Individual parasitemia was counted in 5 μL of blood collected from tail snips on day postinfection (dpi) 17, and mortality was determined daily. For creatine kinase–MB activity (cardiac isoform), plasma was collected on dpi 15 and was analyzed using a commercially available kit (Merck KGaA, Darmstadt, Germany) as described elsewhere.36de Souza A.P. Olivieri B.P. de Castro S.L. Araujo-Jorge T.C. Enzymatic markers of heart lesion in mice infected with Trypanosoma cruzi and submitted to benznidazole chemotherapy.Parasitol Res. 2000; 86: 800-808Crossref PubMed Scopus (39) Google Scholar We used this marker of cardiomyocyte damage owing to its direct correlation with myocarditis.36de Souza A.P. Olivieri B.P. de Castro S.L. Araujo-Jorge T.C. Enzymatic markers of heart lesion in mice infected with Trypanosoma cruzi and submitted to benznidazole chemotherapy.Parasitol Res. 2000; 86: 800-808Crossref PubMed Scopus (39) Google Scholar Data are expressed as a rate of NADPH increase (delta E/min) in five sequential readings using a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 340 nm. To measure serum cytokines by flow cytometry, we used the Cytometric Bead Array kit (flex-inflammation) (BD Biosciences, San Jose, CA) for IL-6, IL-10, monocyte chemotactic protein-1, IFN-γ, TNF, and IL-12 p70, according to the manufacturer's instructions. For glucocorticoid quantification, serum levels of corticosterone were evaluated by means of radioimmunoassay (ICN Pharmaceuticals, Costa Mesa, CA) following the manufacturer's guidelines. Peritoneal cells were collected by washing the cavity of 10 control (noninfected) or 10 infected animals with 5 mL of ice-cold PBS/100 mmol/L cromolyn (MC stabilizing: cromolyn sodium ophthalmic solution; Alcon Laboratories, Fort Worth, TX). The cells were centrifuged at 50 × g for 10 minutes, resuspended in 10% of the individual collected volume, and counted in a Neubauer chamber. For cardiac MC analysis, 20 control or infected mice were euthanized; the hearts were collected, rinsed with ice-cold PBS/cromolyn, and dissected in a Petri dish into 1- to 2-mm fragments. The samples were extensively washed again in cold PBS/cromolyn to remove contaminant blood cells and then were incubated in 6 to 7 mL of PBS/100 mmol/L cromolyn containing 0.2% type IV collagenase (2.6 U/mg; lot 51K8610, Sigma-Aldrich). Tissue dissociation occurred in 5 to 7 cycles of 20-minute incubations under very gentle circular agitation in a water bath at 37°C. After each cycle, isolated cells in the supernatant were centrifuged at 80 × g for 8 minutes at 4°C, washed in cold PBS/cromolyn, pooled for the last centrifugation, resuspended in complete medium [RPMI 1640 (Sigma-Aldrich)/100 mmol/L cromolyn, pH 7.4], and stored on ice. To control the possible contamination of blood cells in tissue samples during enzymatic dissociation, especially for lymphocyte analysis, we labeled all the samples with anti-CD62-L (SouthernBiotech, Birmingham, AL) and proceeded to the purification of MCs (see later herein) only when CD62-L+ cells were <5% of events in flow cytometry (CyAn; DakoCytomation, Fort Collins, CO). Data were analyzed using Summit 4.3 software (DakoCytomation). CBA/J mice were daily treated with cromolyn diluted in PBS from dpi −3 until dpi 20, receiving 100 mg/kg in 200 μL by i.p. injection. Eight mice per group were divided as follows: infected and cromolyn-treated mice (G1), infected and PBS-treated mice (G2), infected mice with no treatment (G3), PBS injection and cromolyn-treated mice (G4), PBS injection and PBS-treated mice (G5), and PBS-injected mice with no treatment (G6). As a control for treatment efficacy, tracheal tissue was collected for histamine quantification as previously described.37Barreto E.O. Carvalho V.F. Lagente V. Lugnier C. Cordeiro R.S. Martins M.A. E Silva P.M. Increased levels of cyclic adenosine monophosphate contribute to the hyporesponsiveness of mast cells in alloxan diabetes.Int Immunopharmacol. 2004; 4: 755-762Crossref PubMed Scopus (11) Google Scholar Briefly, this method consists of sample dilution with 0.1 N of HCl followed by 0.8 N of NaOH and further addition of the substrate o-phthaldialdehyde. After 4 minutes of incubation, the reaction was stopped with 3 N of HCl, and fluorescence was measured in a Shimadzu RF1501 spectrofluorophotometer (Shimadzu Corp., Kyoto, Japan) (excitation at 360 nm; emission at 450 nm). Control and infected mice were euthanized at the time points indicated in the figure legends, and the hearts were removed, sagittally divided, embedded in Tissue-Tek (OCT, Milles-Inc., Zoeterwoude, The Netherlands), and frozen at −70°C. The hearts were then cut into 16- to 18-μm-thick slices (unfixed samples) for MC identification as described later herein or into 5-μm-thick slices for H&E staining. MCs (isolated or in tissue slices) were stained with either 1% toluidine blue (TB) or a mixed solution of 0.36% Alcian Blue (AB)/0.02% safranin (S)/0.01% TB (AB/S/TB) in acetate buffer (pH 1.42).38Meuser-Batista M. Correa J.R. Soares M.J. Henriques-Pons A. Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection.Tissue Cell. 2008; 40: 309-316Crossref PubMed Scopus (12) Google Scholar AB was previously cleared39McAuliffe W.G. A note on the purification of Alcian blue.Stain Technol. 1983; 58: 374-376PubMed Google Scholar by stirring for 1 hour in 90% acetone and then filtering through number 2 filter paper. The residue was recovered from the filter, air-dried, and immediately added to the mixed solution. This protocol identifies mucosal MCs (MMCs) as AB-positive cells, connective tissue MCs (CTMCs) as S- and/or TB-positive cells, and hybrid MCs.38Meuser-Batista M. Correa J.R. Soares M.J. Henriques-Pons A. Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection.Tissue Cell. 2008; 40: 309-316Crossref PubMed Scopus (12) Google Scholar Note that quantification of MC numbers (average) was performed by scanning whole cardiac tissue sections, thus reflecting variations in MC numbers in different areas of the organ. These cells are mostly found in pericardium and perivascular areas, but very few MCs are observed in endocardium, reducing the final value. For qualitative results we show pericardial areas. For peritoneal MCs, 5 mL of total peritoneal cells was overlaid on a 20-mL isotonic cromolyn-supplemented Percoll column (Sigma-Aldrich) at 72% and was centrifuged at 340 × g for 25 minutes. Pelleted cells were collected, washed twice with PBS/100 mmol/L cromolyn, and then resuspended in ice-cold complete medium until use. For cardiac MCs, 5 mL of enzymatically dissociated total cells was overlaid on a 20-mL column of Percoll/cromolyn divided into two parts of 70% and 60% and centrifuged at 700 × g for 1 hour. Pelleted cells were also resuspended in ice-cold complete medium, and all the samples were used with at least 98% purity, as previously described.38Meuser-Batista M. Correa J.R. Soares M.J. Henriques-Pons A. Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection.Tissue Cell. 2008; 40: 309-316Crossref PubMed Scopus (12) Google Scholar Total mRNA was extracted from purified MCs using the RNeasy kit (Qiagen, Germantown, MD) and was converted to cDNA by reverse transcription using the iScript kit (Bio-Rad, Hercules, CA) as recommended by the manufacturer. Specific primers were used to amplify cDNA fragments by PCR as follows: 5′-CCAGGTTGTCTCCTGCGACT-3′ forward and 5′-ATACCAGGAAATGAGCTTGACAAAGT-3′ reverse to GAPDH; 5′-GTCCTGCCTCTGGTGCTTGCT-3′ forward and 5′-AGTGTCTGGGGTTGATTTTCC-3′ reverse to Fas, and 5′-CGAGTTGGTGCCAGTGTGGA-3′ forward and 5′-CCTGCTGTTGGTGGCCTCTT-3′ reverse to P2X7 receptor. The primers flanked conserved regions of genes and were used to amplify cDNA fragments by PCR under high stringency conditions. PCR amplification was performed in a final volume of 10 μL, with 1 ng of target cDNA, 5 pmol of each primer, 200 μmol/L of each deoxyribonucleotide triphosphate (Promega, Madison, WI), and 0.8 U of taqDNA polymerase (Cembiot, RS, Brazil) in a saline buffer (10 mmol/L Tris-HCl, 50 mmol/L KCl, and 1.5 mmol/L MgCl2; pH 8.5). All the samples were amplified in a Mastercycler thermocycler (Eppendorf AG, Hamburg, Germany) using 26 cycles as follows: denaturation step at 95°C for 3 minutes, annealing at 56°C for 1 minute, and extension at 72°C for 30 seconds, then 25 cycles of denaturation at 95°C for 45 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, and the last extension cycle was changed to 5 minutes. A negative control (no cDNA) was included in all the experiments. PCR products were visualized in 6% silver–stained polyacrylamide gels (Sigma-Aldrich), and digital gel images were obtained using ImageScanner III (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Hearts from control (G6) and infected (G3) mice on dpi 7 and 17 were extensively reperfused through the aorta with PBS. Total mRNA was extracted from fragments of left ventriculum using the RNeasy kit (Qiagen) and treated with DNase to exclude DNA contamination. RNA was converted to cDNA by reverse transcription using the high-capacity cDNA kit, as recommended by the manufacturer (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed on an ABI Prism 7500 fast sequence detection system using SYBR Green PCR fast master mix (Applied Biosystems) following the manufacturer's protocols. The following primers and concentrations were used: chymase sense (200 nmol/L), 5′-TTGCCAGCCTGTGAGGAAA-3′; chymase antisense (200 nmol/L), 5′TACAGACAGGCCAGATCGCAT-3′; tryptase sense (200 nmol/L), 5′-CGACATTGATAATGACGAGCCTC-3′; tryptase antisense (200 nmol/L), 5′-ACAGGCTGTTTTCCACAATGG-3′; GAPDH sense (200 nmol/L), 5′-CCAGGTTGTCTCCTGCGACT-3′; and GAPDH antisense (200 nmol/L), 5′-ATACCAGGAAATGAGCTTGACAAAGT-3′. The conditions for the PCR were as follows: 95°C for 20 seconds, followed by 40 cycles at 95°C for 3 seconds and 60°C for 30 seconds. Each of these primer sets gave a unique product. PCR assays were tripled, and the data were pooled. Sample quantification was obtained by relative standard curve normalized by GAPDH. Hearts were collected and frozen as previously described,37Barreto E.O. Carvalho V.F. Lagente V. Lugnier C. Cordeiro R.S. Martins M.A. E Silva P.M. Increased levels of cyclic adenosine monophosphate contribute to the hyporesponsiveness of mast cells in alloxan diabetes.Int Immunopharmacol. 2004; 4: 755-762Crossref PubMed Scopus (11) Google Scholar and 16-μm-thick sections were fixed in acetone at 4°C. Specimens were incubated with 3% H2O2 for 15 minutes to inactivate endogenous peroxidase activity, washed in PBS, and incubated with FcγR blocking solution (inactivated sheep serum 1:10 in PBS supplemented with 4% albumin) (Sigma-Aldrich) for 30 minutes. Heart tissue sections were then incubated for 12 hours at 4°C with antibodies against SCF or IL-3 (Santa Cruz Biotechnology, Santa Cruz, CA), washed with PBS/albumin, incubated with horseradish peroxidase–conjugated anti-goat IgG (R&D Systems, Minneapolis, MN) for 1 hour at room temperature, and revealed with 3-amino-9-ethyl-carbazol (Sigma-Aldrich) for 10 minutes protected from light. The slices were washed in PBS, stained with hematoxylin for 1 minute, and then analyzed by light microscopy using a BX50 Olympus microscope (Olympus, Center Valley, PA). In control experiments, no immunostaining was observed when primary antibody was omitted. For stromal labeling quantification, the microscope was coupled to a video camera (DEI-750; Optronics Engineering, Goleta, CA) with output processed and analyzed using Image-Pro Plus 4 image analyzer software (Media Cybernetics Inc., Bethesda, MD). Hearts were collected and frozen as previously described,37Barreto E.O. Carvalho V.F. Lagente V. Lugnier C. Cordeiro R.S. Martins M.A. E Silva P.M. Increased levels of cyclic adenosine monophosphate contribute to the hyporesponsiveness of mast cells in alloxan diabetes.Int Immunopharmacol. 2004; 4: 755-762Crossref PubMed Scopus (11) Google Scholar and 16-μm-thick sections were fixed in acetone at 4°C for the detection of apoptosis using the TUNEL-POD kit (Roche, Mannheim, Germany) according to the manufacturer's recommendations. After the reaction, the slices were stained with AB/S/TB solution for concomitant identification of MCs. Statistical analysis was performed using one-way analysis of variance followed by the Tukey posttest for nonparametric data, and results were considered significant at P < 0.05. Parasitemia was detectable on dpi 13, peaking on dpi 17 and between dpi 26 and 29 (data not shown). This led us
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