Comparison between the collagen intensity and mast cell density in the lingual muscles and myocardium of autopsied

chronic chagasic and nonchagasic patients Jos A.Roldo, Marcela Beghini, Luciana S.Ramalho, Carla Souza Porto, Denise B.R.Rodrigues, Vicente P.A.Teixeira, et al.
Parasitology Research Founded as Zeitschrift fr Parasitenkunde ISSN 0932-0113 Volume 111 Number 2 Parasitol Res (2012) 111:647-654 DOI 10.1007/s00436-012-2882-1

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Parasitol Res (2012) 111:647654 DOI 10.1007/s00436-012-2882-1

ORIGINAL PAPER

Comparison between the collagen intensity and mast cell density in the lingual muscles and myocardium of autopsied chronic chagasic and nonchagasic patients
Jos A. Roldo & Marcela Beghini & Luciana S. Ramalho & Carla Souza Porto & Denise B. R. Rodrigues & Vicente P. A. Teixeira & Sanvia A. de Lima Pereira

Received: 5 May 2011 / Accepted: 27 February 2012 / Published online: 13 March 2012 # Springer-Verlag 2012

Abstract In chronic Chagas' disease (CD), an increase in collagen intensity and mast cell density has been described individually in the myocardium and tongue muscles. The aim of this study was to compare the percentage of collagen, mast cell tryptase (MCT) density, and mast cell chymase (MCH) density in the lingual muscles and myocardium from autopsied chagasic (CP) and nonchagasic patients (NCP). The selected cases were divided into two groups: (1) CP (n 0 10) and (2) NCP (n 0 10). Fragments were removed from the tongue and heart. After histological processing, the slices were stained with picrosirius, and immunohistochemistry was performed for MCH and MCT. The CP group showed the highest MCH and MCT densities and the highest percentage of collagen in the lingual muscles and myocardium when compared with the NCP group (p <0.05). A significant positive correlation was observed between the collagen intensity and MCH density in the myocardium of the CP group. Although there are no reports in the literature of MCT and MCH in CD, its higher densities as well as higher percentage of
J. A. Roldo : M. Beghini : C. S. Porto : D. B. R. Rodrigues : S. A. de Lima Pereira (*) Laboratory of Biopathology and Molecular Biology, University of Uberaba (UNIUBE), Av. Nen Sabino, 1801, Bairro Universitrio, Cep 38.055-500 Uberaba, MG, Brazil e-mail: sanivia.pereira@uniube.br L. S. Ramalho : V. P. A. Teixeira General Pathology Division, Tringulo Mineiro Federal University (UFTM), Uberaba, MG, Brazil S. A. de Lima Pereira Cefores, Tringulo Mineiro Federal University (UFTM), Uberaba, MG, Brazil

collagen were found in the lingual muscles and myocardium in the CP group, suggesting that tryptase and chymase are associated with the pathogenesis of CD in these organs. Furthermore, the positive and significant correlation between the percentage of collagen and MCH density in the myocardium of the CP group suggests that the chymase is associated with fibrosis in CD, as demonstrated in other diseases.

Introduction Chagas' disease (CD) is an infectious disease caused by the protozoan parasite, Trypanosoma cruzi (Tanowitz et al. 1992). T. cruzi is often transmitted to humans by the bite of triatomines (Tartarotti et al. 2004) and is capable of invading and replicating in different cell types (Tan and Andrews 2002). CD is a serious public health problem (Carod-Artal et al. 2007) and the leading cause of heart failure in Latin America (Petersen and Burleigh 2003). Despite the existence of programs for vector control in order to restrict the disease, the World Health Organization estimates that about 15 million people are infected in the Americas (Coura and Dias 2009), and of those, 10 million are Latin Americans (Schofield et al. 2006). In Brazil, the number of T. cruzi-infected persons is estimated at around 3.5 million, with approximately 600,000 being residents in the state of Minas Gerais (Oliveira et al. 2006). In Brazil, from 10,000 to 20,000 new cases of Chagas' disease appear annually, accounting for 20,000 deaths per year in the last two decades (Tartarotti et al. 2004). It is known that cardiac lesions in the chronic phase of CD usually affect the left ventricle (Milei et al. 1991; Caliari et al. 2002), being characterized by the destruction of heart muscle

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648 Parasitol Res (2012) 111:647654

cells with inflammatory infiltrate, collagen neoformation (Elizari 1999; Lpez et al. 2006) being associated with an increase in the number of mast cells (Meuser-Batista et al. 2008). Mast cells have intracytoplasmic granules containing proteins, with 25 % of these proteins being neutral proteases known as tryptase and chymase (Horny et al. 2003). Thus, human mast cells are classified into two types, depending on the content of these two proteases: mast cell chymase (MCH) and mast cell tryptase (MCT) (Lindstedt et al. 2007). In addition to the heart muscles, other muscles can be infected in human CD, such as the gastrocnemius (Lugo de Yarbuh et al. 2006) and lingual muscles (Barbosa and Andrade 1984). Recent studies by our group have shown that the tongues of individuals with CD exhibit more significant morphological changes when compared with patients without CD (de Lima Pereira et al. 2006, 2007, 2009). Therefore, there are studies that have described changes restricted to the myocardium and tongue in chronic CD. However, no studies were found in the literature that compared the morphological changes of the tongue and heart of patients with CD. Although there are studies comparing the collagen density between chagasic and nonchagasic patients both in the myocardial (Barretto et al. 1989) and lingual muscles (de Lima Pereira et al. 2007), there are no reports on MCH and MCT in any organ in patients with CD. As there are reports on other diseases demonstrating that the MCH and the MCT are associated with the increased density of collagen (Jones et al. 2003), it was hypothesized that in patients with CD, there would be higher densities of MCH, MCT, and collagen in both the lingual muscles and heart muscles. Therefore, the aim of this study was to compare the percentage of collagen, MCT density, and MCH density in the lingual muscles and myocardium from autopsied chronic chagasic and nonchagasic patients.

non-Caucasian) were collected. They were divided in two groups: (1) chagasic patients (n 0 10) and (2) nonchagasic patients (n 0 10). This classification was based on autopsy reports and the subsequent reactions of indirect immunofluorescence, hemagglutination, and enzyme-linked immunosorbent assay for CD, conducted in the pericardium liquid collected during autopsies. Subjects were considered chagasic in the chronic phase when they showed positive reactions and had morphological findings suggestive of CD such as cardiomegaly, left ventricular lesion, and visceromegalies (megas). Histopathological analysis Samples of the tongue were collected by a longitudinal section in the median lingual sulcus from the base to the apex of the tongue. This fragment was subsequently sectioned into five parts. The central fragment that corresponded to the third fragment was used to perform the analysis. Fragments of the heart were removed in the region of the myocardium, the middle portion of the left ventricle. The 2.01.00.5-cm-thick samples obtained were paraffin embedded and processed for histopathological analysis. Sixmicrometer sagittal sections were mounted on glass slides and stained with picrosirius for quantification of collagen. For morphometric assessment of collagen in the lingual muscle and myocardium, slides stained with picrosirius were used, in addition to a light microscope, camera to capture the image, computer, and KS300 software (Zeiss, Germany). A 5 objective and a polarizing filter were used. In the polarized image, the collagen birefringence was shown in reddish yellow, facilitating quantification. In each case, 40 fields were evaluated, 20 being fields of lingual and 20 fields of myocardium muscles. For this quantification, each slide was divided into four quadrants. The four fields from the edges of each quadrant and the central field were evaluated, totaling 20 fields per case. The results were expressed as a percentage. This analysis was performed blind by the same examiner (J.A. R) who was previously trained and calibrated. Immunohistochemical analysis Additional sections of myocardium and tongue were mounted on glass slides pre-treated with 3-aminopropyltriethoxy-silane (Sigma Chemical, St. Louis, MO, USA) and used for immunohistochemical analysis. Citrate buffer antigen retrieval, pH 6.0, was performed for 20 min. Sections were treated with 3 % hydrogen peroxide (H2O2) for 15 min to block endogenous peroxidase. After this, incubation with the primary antibodies against tryptase (1:200) and chymase (1:2,000) was done overnight. Biotinylated link antibody and streptavidinperoxidase antibody (DAKO, LSAB kit, Glostrup, Denmark) were incubated for 40 min at 22C. The specific reaction to

Materials and methods Patient selection This descriptive, retrospective analysis of archival tissues, designed to evaluate morphological changes developed in vivo (during the life of autopsied individuals), was deemed to be ethical according to the Brazilian guidelines (resolution 196 of the National Health Council, 1996), and the protocol was approved by the Human Research Ethics Committee of the University of Uberaba (UNIUBE; process 004/03), Uberaba, Minas Gerais (MG), Brazil. A study was conducted analyzing reports from complete autopsies performed at the General Pathology Division, Tringulo Mineiro Federal University, Uberaba, MG, Brazil, in the period of January 2008 to December 2010. A total of 20 patients older than 24 years were selected, and data related to age, gender, and ethnicity (Caucasian and

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each antibody was visualized using 3,3 diaminobenzidine, which was applied and incubated in the dark for 7 min. Then, the slides were counterstained with Mayer's hematoxylin, dehydrated through a series of graded ethanol, cleared in xylene, and mounted with Enthelan (Merck, Darmstadt, Germany). Negative controls were obtained by omission of the primary antibodies. The immunolabelled cells were evaluated in all fields of the slides. The area of each field (in square micrometers) was calculated with an ocular micrometer, and the immunolabelled density was expressed as cells per square millimeter. The analyses were performed by the same blinded examiner (M.B.) who was previously trained and calibrated. Statistical analysis Statistical analysis data were analyzed using statistical software GraphPad Prism 4 (San Diego, CA, USA), and the KolmogorovSmirnov test was used for the normality evaluation. Student's t test was used to compare two groups using quantitative variables with normal distribution. The results were expressed as mean standard deviation. For the proportions female/male and Caucasian/non-Caucasian, the exact Fisher test was used. For the correlations, BioEstat 5.0 software was used (Sociedade Civil Mamirau, CNPq, Brazil), to perform Spearman's correlation test. The level of significance assumed was 5 % (p <0.05).

When the MCH density in the myocardium and lingual muscles and the MCT density in the myocardium and lingual muscles were analyzed together, the chagasic patient group showed the highest density both for MCH and MCT, in comparison with the nonchagasic patient group, with significant difference, the p values being 0.020 and 0.041, respectively (Table 2; Figs. 1a, b, c, d and 2a, b, c, d). In the chagasic patient group, the MCT density was significantly higher in the lingual muscles when compared with the myocardium (p 0 0.035). The MCT density in the lingual muscles was significantly higher in the chagasic patient group when compared with the nonchagasic patient group (p 0 0.041) (Table 2). When the collagen densities in the myocardium and lingual muscles were analyzed together, the chagasic patient group showed the highest collagen density when compared with the nonchagasic patient group, with significant difference (p 0 0.034). In the chagasic patient group, the percentage of collagen was significantly higher in the lingual muscles when compared with the percentage in the myocardium (p 0 0.004). In the lingual muscle, the chagasic patient group had a higher percentage of collagen when compared with the nonchagasic patient group, with significant difference (p 0 0.034) (Table 2; Figs. 3a, b, c, d and 4a, b, c, d). A significant positive correlation was observed between the collagen intensity and MCH density in the myocardium of the chagasic patient group (p 0 0.002) (Fig. 5). The other correlations were not significant.

Results Discussion The demographic data of the groups of chagasic and nonchagasic patients, respectively, were: Caucasian (C) (10 versus 9) and non-Caucasian (NC) (0 versus 1), males (7 versus 8) and females (3 versus 2). The mean age standard deviation was 59.5113.33 in the chagasic patient group and 58.8118.78 in the nonchagasic patient group. No statistically significant differences in ethnicity, gender, and age were observed between CP and NCP, showing homogeneous distribution between the two groups (Table 1). The physiological role of chymase and tryptase is still uncertain since the activity of these enzymes is observed only in damaged tissues (Takai et al. 2004). Chymase is a serine protease originating from a family of proteins that plays an essential role in apoptosis and inflammation (Heutinck et al. 2010). The apoptosis is partially inhibited by chymase and TNF-alpha inhibitors. In addition, chymase inhibited the activation of NF-kappaB (p65) and activated caspase-8 and caspase-9. Therefore, activated mast cells induce apoptosis by a combined action of chymase and tumor necrosis factoralpha (Heikkil et al. 2008) and by a mechanism involving fibronectin degradation (Ebihara et al. 2005). In CD, studies have shown an increase in TGF-beta and the incidence of apoptosis (Zhao et al. 2008). In this disease, apoptosis seems to be an important mechanism to contain the infection by T. cruzi (Leskinen et al. 2006). In the present study, when the MCH densities in the myocardium and tongue muscles were analyzed together, the chagasic patient group showed the highest MCH density when compared with the nonchagasic patient group, with significant difference. Several studies have shown an increase in mast cells in tissues from patients with

Table 1 Demographic characteristics of the chagasic and nonchagasic patient groups CP (n 0 10) Ethnicity (C/NC)* Gender (M/F) Age (years; meanSD)***
**

NCP (n 0 10) 9:1 8:2 58.8118.78

10:0 7:3 59.5113.33

SD Standard deviation, M male, F female, C Caucasian, NC non-Caucasian

*

p 0 1, Fisher exact test; ** p 0 1, Fisher exact test; *** p 0 0.850, Student's t test

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650 Parasitol Res (2012) 111:647654 Table 2 Density of mast cells immunostained with anti-chymase (MCH), density of mast cells immunostained with anti-tryptase (MCT), and percentage of collagen in chagasic patients and nonchagasic patients

Student's t test : a, p 0 0.035; b, p 0 0.041; c, p 0 0.004; d, p 0 0.034. Same letters and asterisks indicate statistically significant differences *p 0 0.020; **p 0 0.041; ***p 0 0.034

CD (Almeida et al. 1975). However, there are no reports in the literature showing an increase of MCH in CD, but it is believed that the increase of these cells in the chagasic patient group would play an important role in containing the infection by inducing apoptosis of the infected cells. Mast cells containing tryptase represent mucosal mast cells and are usually found in the lungs and intestinal mucosa (Lindstedt et al. 2007). However, in this study, MCT was found in both myocardium and lingual muscles, demonstrating that

these cells can also be found in these regions of muscles. Although no published reports describing MCT in the lingual muscles and myocardium of patients with CD were found, in the chagasic patient group, the MCT density was significantly higher in lingual muscle in comparison with the myocardium, perhaps because of the presence of lingual mucosa, where MCT is found more frequently (Kleinschmidt et al. 2010). Because these cells are important for the recruitment of leukocytes by selectin release, adhesion molecules, and chemotactic

Fig. 1 Immunohistochemical staining for MCH and MCT in the myocardium. Positive staining for MCH was observed in the myocardium from the chagasic patient group (a) and nonchagasic patient group (b). Positive staining for MCT was observed in the myocardium from the chagasic patient group (c) and nonchagasic patient group (d) (1,600)

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Parasitol Res (2012) 111:647654 Fig. 2 Immunohistochemical staining for MCH and MCT in the lingual musculature. Positive staining for MCH was observed in the tongue from the chagasic patient group (a) and nonchagasic patient group (b). Positive staining for MCT was observed in the lingual musculature from the chagasic patient group (c) and nonchagasic patient group (d) (1,600) 651

factors (Rech and Graa 2006), in CD, these cells might have migrated from the submucosal region to muscle in response to T. cruzi antigens. The human cutaneous scar tissue is populated by a mast cell subpopulation that is chymase, avidin, and tryptase+, most probably reflecting an increased immigration and/or
Fig. 3 Intensity of collagen in the myocardium. a Chagasic patient group (picrosirius image not polarized), b CP group (picrosirius-polarized image), c nonchagasic patient group (picrosirius image not polarized), d nonchagasic patient group (picrosiriuspolarized image (800)

proliferation of immature mast cells and their precursors (Hermes et al. 2000). Furthermore, it has been demonstrated that human mast cell tryptase may be a potent stimulus of microvascular leakage. The activation of mast cells by this proteinase may represent an amplification process in inflammation (He and Walls 1997). Because the disease is an

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652 Fig. 4 Intensity of collagen in the lingual muscle. a Chagasic patient group (picrosirius image not polarized), b chagasic patient group (picrosiriuspolarized image), c nonchagasic patient group (picrosirius image not polarized), d nonchagasic patient group (picrosiriuspolarized image, 800) Parasitol Res (2012) 111:647654

inflammatory disease, the highest MCT density in the chagasic patient group found in this study could have occurred as a response to T. cruzi antigens and collaborated with changes in the microcirculation of chronic inflammation responsible for the persistent response to the parasite. In a previous study, greater intensity of inflammation in the muscles of the tongue of patients with CD was shown, when compared with patients without CD (de Lima Pereira et al. 2006). In the present study, the chagasic patient group had significantly higher MCT

Fig. 5 Correlation between collagen intensity and MCH density in the myocardium of the chagasic patient group

density in the lingual muscles when compared with nonchagasic patients, which suggests that in CD, MCT also plays a role in the inflammation of the lingual muscles. In CD, human collagen deposition has been demonstrated in various organs, such as the heart (Palomino et al. 2000; Rossi 2001; Lpez et al. 2006), pancreas (Saldanha et al. 2001), spleen (Pereira et al. 2002), esophagus (Cabral 2000), colon (Pinheiro et al. 2003), and tongue (de Lima Pereira et al. 2006). When the collagen densities in the myocardium and tongue were analyzed together, the chagasic patient group showed a significant increase in collagen density when compared with the nonchagasic patient group, which is consistent with other studies in patients with chronic CD who were shown to have more collagen in the organs infected with T. cruzi (Coura and Borges-Pereira 2010). In the lingual muscle of the chagasic patient group, there was a significantly higher percentage of collagen when compared with the nonchagasic patient group, which is consistent with a previous study by our group, in which more fibrosis was shown in the tongue muscles of CD patients than nonchagasic patients (de Lima Pereira et al. 2006). In the present study, the chagasic patient group showed a higher collagen density in the lingual muscles when compared with the myocardium. Thus, although no published studies that compared changes in the tongue with changes in the heart of CD were found, we believed that in the cases analyzed, the tongue lesions caused by T. cruzi were older than the cardiac lesions and were at a more advanced stage of the tissue repair process, justifying the greater collagen density in the lingual muscles of

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Parasitol Res (2012) 111:647654 653 Caliari MV, do Pilar MR, de Lana M, Caja RA, Carneiro CM, Bahia MT, dos Santos CA, Magalhaes GA, Sampaio IB, Tafuri WL (2002) Quantitative analysis of cardiac lesions in chronic canine chagasic cardiomyopathy. Rev Inst Med Trop So Paulo 44:273 278 Carod-Artal FJ, Horan TA, Vargas AP, Ribeiro LS, Mamare EM (2007) Cardioembolic stroke and ischemic small bowel infarction in a Chagas' disease patient. Eur J Neurol 14:e8 Coura JR, Borges-Pereira J (2010) Chagas disease: 100 years after its discovery. A systemic review. Acta Trop 115:513 Coura JR, Dias JCP (2009) Epidemiology, control and surveillance of Chagas disease100 years after its discovery. Mem Ins Oswaldo Cruz 104:3140 de Lima Pereira SA, Rodrigues DBR, Ferraz LMF, Castro ECC, Reis MA, Teixeira VPA (2006) Inflammation and glandular duct dilatation of the tongue from patients with chronic Chagas disease. Parasitol Res 98:153156 de Lima Pereira SA, Rodrigues DBR, Castro ECC, Reis MA, Teixeira VPA (2007) Quantitative analysis of fibrosis and mast cells in the tongue of chronic chagasic patients: autopsy study. Md Mal Inf 37:229233 de Lima Pereira SA, Severino VO, Kohl NLM, Rodrigues DBR, Alves PM, Clemente-Napimoga JT, Reis MA, Teixeira VPA, Napimoga MH (2009) Expression of cytokines and chemokines and microvasculature alterations of the tongue from patients with chronic Chagas' disease. Parasitol Res 105:10311039 Doggrell SA, Wanstall JC (2004) Vascular chymase: pathophysiological role and therapeutic potential of inhibition. Cardiovasc Res 61:653 662 Ebihara N, Funaki T, Murakami A, Takai S, Miyazaki M (2005) Mast cell chymase decreases the barrier function and inhibits the migration of corneal epithelial cells. Curr Eye Res 30:10611069 Elizari MV (1999) Chagasic myocardiopathy: historical perspective. Med (B Aires) 59:2540 Fukushima H, Ohsawa M, Ikura Y, Naruko T, Sugama Y, Suekane T, Kitabayashi C, Inoue T, Hino M, Ueda M (2006) Mast cells in diffuse large B-cell lymphoma; their role in fibrosis. Histopathology 49:498505 Garbuzenko E, Nagler A, Pickholtz D, Gillery P, Reich R, Maquart FX, Levi-Schaffer F (2002) Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. Clin Exp Allergy 32:237246 He S, Walls AF (1997) Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation. Eur J Pharmacol 328:8997 Heikkila HM, Latti S, Leskinen MJ, Hakala JK, Kovanen PT, Lindstedt KA (2008) Activated mast cells induce endothelial cell apoptosis by a combined action of chymase and tumor necrosis factor. Arterioscler Thromb Vasc Biol 28:309314 Hermes B, Feldmann-Bddeker I, Welker P, Algermissen B, Steckelings MU, Grabbe J, Henz BM (2000) Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue. J Invest Dermatol 114:5155 Heutinck KM, Ineke JM, Bergeb T, Hackc CE, Hamanna J, Rowshanib AT (2010) Serine proteases of the human immune system in health and disease. Mol Immunol 47:19431955 Horny HP, Greschniok A, Jordan JH, Menke DM, Valent P (2003) Chymase expressing bone marrow mast cells in mastocytosis and myelodysplastic syndromes: an immunohistochemical and morphometric study. J Clin Pathol 56:103106 Jones SE, Kelly DJ, Cox AJ, Zhang Y, Gow RM, Gilbert RE (2003) Mast cell infiltration and chemokine expression in progressive renal disease. Kidney Int 64:906913 Kleinschmidt S, Hardera J, Nolteb I, Marsilioa S, Hewicker-Trautweina M (2010) Phenotypical characterization, distribution and quantification of different mast cell subtypes in transmural biopsies from the

the chagasic patient group. Moreover, as a higher MCT density was found in lingual muscles than in the myocardium, we believed that these cells could have contributed to the fibrogenesis in the inflammatory process, since there is a study showing that tryptase-positive mast cells and fibroblasts are closely associated (Riekki et al. 2004). Mast cells release mediators that stimulate fibroblast proliferation and collagen synthesis (Garbuzenko et al. 2002), being increased in numbers in fibrotic areas (Fukushima et al. 2006). In CD, the mast cells secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as T. cruzi -induced myocardiopathy (Meuser-Batista et al. 2008). Studies in other diseases have shown that chymase may play a role in the release and activation of latent transforming growth factor (TGF)-1, and TGF-beta1 has been identified as the most important profibrotic cytokine (Doggrell and Wanstall 2004; Zhao et al. 2008). In this study, a significant positive correlation was observed between the percentage of collagen and MCH density in the myocardium of the chagasic patient group. Although the underlying mechanism of intracellular signaling remains unclear, one study demonstrates that mast cell-derived chymase is implicated in myocardial fibrosis in CD (Zhao et al. 2008), corroborating the findings of the present study. Although there are no reports in the literature of MCT and MCH in CD, its higher densities as well as higher percentage of collagen were found in the lingual muscles and myocardium in the chagasic patients, suggesting that tryptase and chymase are associated with the pathogenesis of CD in these organs. Furthermore, the positive and significant correlation between the percentage of collagen and MCH density demonstrated in the myocardium of the chagasic patient group suggests that chymase is associated with cardiac fibrosis in patients with chronic CD, as has been demonstrated in other diseases.
Acknowledgments This work was supported by the Programa de Apoio Pesquisa da Universidade de Uberaba (PAPE-UNIUBE) and Programa de Ps-Graduao em Odontologia da UNIUBE, Uberaba, MG, Brazil.

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