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Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo
Cell Biology International 27 (2003) 853–861
Cell Biology International www.elsevier.com/locate/cellbi
Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo ´ vila 1, Henrique C. Teixeira 2, Rossana C.N. Melo 1*, Daniela L. Fabrino 1, Heloisa D’A 2 Ana Paula Ferreira 1
Laboratory of Cellular Biology, Institute of Biological Sciences, Federal University of Juiz de Fora (UFJF), 36036-330, Juiz de Fora-MG, Brazil 2 Laboratory of Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora (UFJF), 36036-330, Juiz de Fora-MG, Brazil Received 3 February 2003; revised 27 May 2003; accepted 14 July 2003
Abstract Acute Chagas’ disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2 production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2 production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load. 2003 Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Chagas’ disease; Hydrogen peroxide; Myocarditis; Monocytes/macrophages; Electron microscopy
1. Introduction Chagas’ disease is caused by intracellular protozoan Trypanosoma cruzi, parasite of many mammalian species, and is characterized by an acute phase in which the parasite circulates in the boodstream as trypomastigotes and proliferates within the cytoplasm of a variety of cells as amastigotes. The rupture of amastigote nests provokes cell destruction and a potent inflammatory process, especially in the heart, a target organ of the disease. Approximately 18–20 million people in the Latin America are chronically infected with T. cruzi (reviewed in Ropert et al., 2002). * Corresponding author. Present address: Harvard Medical School/Beth Israel Deaconess Medical Center, DA-617, 330 Brookline Avenue, Boston, MA 002215, USA. Tel.: +1-617-667-3307; fax: +1-617-667-5541 E-mail address: [email protected] (R.C.N. Melo). 1065-6995/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00173-2
The host resistance to acute infection is expressed mainly by cell-mediated reactions involving sensitized T cells and macrophages (reviewed in Brener and Gazzinelli, 1997). However, there is no general agreement on the immunoregulatory mechanisms involved and the precise role of these immune effector cells in the control of parasite (Abrahamsohn and Coffman, 1996; Sun and Tarleton, 1993). During the acute phase of Chagas’ disease, we have observed, in vivo, a dramatic increase in the number of both monocytes in the peripheral blood and macrophages in the heart of infected rats. These cells exhibited clear morphological signs of activation and were directly involved in inhibiting parasite multiplication in the myocardium (Melo and Machado, 2001; Melo et al., 2003). The ultrastructural features of the macrophage activation such as increase in size, surface rufflings and amount of cytoplasmic organelles are recognized as an
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accurate indication of high phagocytic and microbicidal activities of these cells both in humans and experimental models (Anosa et al., 1997; El Shewemi et al., 1996; Takemura et al., 1989), including Chagas’ disease (Melo and Machado, 2001; Melo et al., 2003). On the other hand, biochemical changes, as the ability of secreting hydrogen peroxide (Nathan, 1982; Nogueira and Cohn, 1978; Reed et al., 1987), tumor necrosis factor-alpha (Silva et al., 1995), nitric oxide (Vespa et al., 1994; Petray et al., 1995; Rodrigues et al., 2000) and interleukin-12 (Antunez and Cardoni, 2000) have been considered important markers of macrophage activity. In spite of several authors had evaluated the hydrogen peroxide-releasing capacity of macrophages upon T. cruzi challenge, the results remain contrasting (Borges et al., 1995; Mccabe and Mullins, 1990; Nathan et al., 1979; Reed et al., 1987; Russo et al., 1989). The majority of these studies were carried out in vitro using murine peritoneal macrophages. When mouse macrophages were cultured with the parasite, the hydrogen peroxide production was significant (Nathan et al., 1979; Reed et al., 1987) or very low (Mccabe and Mullins, 1990). On the other hand, the in vivo release of H2O2 by activated macrophages has not been well established during the T. cruzi infection. The amount of H2O2 secreted by peritoneal macrophages during the chagasic infection of susceptible mice was higher than that observed in the resistant mice (Russo et al., 1989). In addition, Borges et al. (1995) have shown that there is not a correlation between the in vivo levels of gamma interferon, a lymphocyte cytokine considered as the major stimulus for the activation of macrophages, and the H2O2 production by murine peritoneal macrophages, during the acute infection. Regarding monocytes, their ability to produce in vivo H2O2 has not been studied in the course of acute infection with T. cruzi. In vitro, peripheral blood monocytes are able to destroy the parasite after ingestion (Villalta and Kierszenbaum, 1984) and exhibit activated morphology when cultured with T. cruzi (Van Voorhis, 1992). We have observed, in vivo, a great increase in the number of both monocytes in the peripheral blood and macrophages in the heart, emphasizing the importance of the newly formed monocyte-derived macrophages in resistance to acute Chagas’ disease (Melo, 1999; Melo and Machado, 2001). These findings and the conflicting views about the H2O2 production by macrophages during chagasic infection in mice, render the whole subject to be worthy of re-investigation. In addition, studies regarding H2O2 production during infectious diseases may be focus of great interest since H2O2 can be responsible for chemokine expression and the development of inflammation (Jaramillo and Olivier, 2002). The present work describes, for the first time, the respiratory burst responses of rat peripheral blood monocytes to in vivo acute T. cruzi infection. The
responses of different types of macrophages (from peritoneum and spleen) were also evaluated in an attempt to clarify the previous contradictory reports. In parallel, we have examined the ultrastructure of heart inflammatory macrophages and the cardiomyocyte parasitism. The Holtzman rat has been used in our lab as an excellent experimental model since the infection in these animals faithfully mimics the human acute phase, triggering a prominent parasitemia, heart parasitism, and myocarditis (Melo, 1999; Melo and Machado, 1998, 2001). 2. Materials and methods 2.1. Animals Female Holtzman rats aged 27–30 days comprised noninfected and infected groups. The animals were sacrificed under exsanguination after ether anesthesia at day 12 of infection. Each group contained four to seven animals. 2.2. T. cruzi infection The rats were inoculated intraperitoneally with a single inoculum of 150 µl of mouse blood containing 3105 trypomastigotes of T. cruzi, Y strain (Silva and Nussenszweig, 1953). Fresh blood sampled from the tail showed living trypomastigotes in all animals 6 or 12 days after inoculation. 2.3. Peripheral blood leukocyte quantification Samples of blood were taken from the tail of the controls and infected animals at 6 and 12 days postinfection. The total number of leukocytes was estimated by hemocytometer counts (20 µl of blood diluted in 2% acetic acid and 0.01% methylene blue). The percentage of each white blood cell in air-dried smears stained with May-Gru¨nwald/Giemsa solution allowed an estimate of the total number of each leukocyte. The leukocyte percentage was determined after counting 200 cells per rat. 2.4. Cell isolation and H2O2 production by peripheral blood mononuclear cells (PBMC), splenic and peritoneal cells The H2O2 production by PBMC, splenic and peritoneal cells was evaluated in controls and infected animals at 12 days of infection by horseradish peroxidedependent phenol red oxidation method (Pick and Mizel, 1981) slightly modified by us as follows. The peripheral blood was drawn from left axillary artery. PBMC were isolated from heparinized pool (5–6 ml/ group of 7 animals) of peripheral blood (20 µl heparin/ml blood) with Histopaque-1077 (Sigma, St Louis, MO, USA) by density gradient centrifugation at
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2000 rpm for 30 min, at room temperature. Spleens were homogenized individually in 5 ml RPMI using a teflon coated tissue homogenizer (Gas-Col Apparatus Co. Terre Haute, IN, USA). PBMC and splenic cells were washed twice in PBS and ressuspended in RPMI 1640 medium containing 5.5 mM dextrose, 0.56 mM phenol red and 8.5U ml1 horseradish peroxide type II (1500 rpm for 10 min at 4 (C). Peritoneal cells were obtained from peritoneal washings with phosphate-buffered saline (PBS) (5 ml/animal) and washed with PBS at 1000 rpm for 10 min at 4 (C. Viability of the cells was assessed by trypan blue test and the total number was estimated by hemocytometer counts. The concentration of all collected cells was adjusted to 2106 cells per ml with fresh medium RPMI 1640. Volumes (100 µl) of the cell suspensions were plated on to each well of 96-well flat-bottom tissue culture plates (Costar, Cambridge, MS, USA). The plates were incubated in humidified atmosphere at 37 (C for 1 h. Subsequently, 10 µl of 1 N NaOH was added to stop the reaction and the absorbance was determined at 620 nm using a Spectra Max 190 (Molecular Devices, USA). Conversion of absorbance to micromoles of H2O2 was deduced from a standard curve. All determinations were performed in quadruplicate and expressed as micromoles of H2O2/2106 cells.
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Fig. 1. Number of monocytes in the peripheral blood of rats at day 6 and 12 of infection with T. cruzi and in uninfected controls. Animals were infected i.p. with 300,000 trypomastigotes of Y strain. On both days, the monocyte numbers (P<0.05) increased in the blood compared to control values. The monocyte numbers found at day 12 were increased (P<0.05) in comparison to numbers found at day 6. The data are expressed as meanSD and are representative of three independent experiments, with four to seven rats per group.
2.5. Light microscopy Fragments of atria and ventricles from controls and infected animals (at day 12 of infection) were fixed in 4% paraformaldehyde in buffered phosphate, pH 7.3, 0.1 M, for 24 h, processed routinely and embedded in glycol methacrilate (Leica). Semi-serial 5-µm-thick sections stained by haematoxylin and eosin or toluidine bluebasic fuchsin (Abreu et al., 1993) were examined for qualitative evaluation of the inflammatory and degenerative processes and quantification of parasitism. The number of amastigote nests in cardiomyocytes was evaluated at 70 µm interval for avoiding parasite recounting (Hanson and Roberson, 1974). For each group of animals were analyzed 1600 fields (800 from atria and 800 from ventricles) at 400. 2.6. Electron microscopy Fragments of the atria of controls and infected rats (12 days) were fixed in the phosphate-buffered 1% paraformaldehyde and 1% glutaraldehyde, pH 7.3, overnight at 4 (C (Karnovsky, 1965). The tissues were postfixed in a mixture of 1% phosphate-buffered osmium tetroxide and 1.5% potassium ferrocyanide (final concentration), for 1 h and processed for resin embedding (PolyBed 812, Polysciences). The ultrathin sections were stained with uranyl acetate and lead citrate before examination in a Zeiss EM 10 at 60 kV. In addition to qualitative observations, quantitative study was made in
electron micrographs of the heart macrophages. The size variation of these cells was obtained by diameter measurements of the macrophages in which appeared the nucleus. Since most macrophages have an irregular shape, 10 measurements of the diameter were made for each macrophage, being analyzed a total of 15 cells from samples of the normal and infected groups. From the mean diameter obtained for each cell, the data were calculated and expressed as mean diameterstandard deviation for each group. 2.7. Statistical analysis Data from different groups were compared by the Mann–Whitney “U” test (P<0.05).
3. Results 3.1. Peripheral blood leucocyte numbers The number of monocytes in the peripheral blood (Fig. 1) showed significant increase (98%) at day 6 of T. cruzi infection in comparison to values from uninfected controls. At day 12, this number (Fig. 1) was dramatically increasead in comparison to control values (1180%) and to data found at day 6 of infection (478%). Analyzes of the blood smears of the infected animals revealed 1–3 monocytes per each field observed. The lymphocyte, neutrophil and eosinophil numbers and the
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Fig. 2. Hydrogen peroxide (H2O2) production by peripheral blood monocytes in rats at day 12 of infection with T. cruzi and in uninfected controls. The H2O2 levels increased at day 12 (85%) in comparison to control values. The data are expressed as meanSD and are representative of two independent experiments, with four to seven rats per group.
total number of peripheral blood leukocytes were not significantly different from the control values on both days analyzed. 3.2. Evaluation of H2O2 release by peripheral blood monocytes and spleen and peritoneal macrophages The acute T. cruzi infection induced H2O2 release by peripheral blood monocytes. The H2O2 levels of these cells increased 85% at day 12 in comparison to control values (Fig. 2). Spleen macrophages of the infected animals also released H2O2 at this time (76% of increase compared to controls) (Fig. 3). This increase was accompanied by 4.5 fold enhancement of the total number of spleen cells (Fig. 4). In contrast, the infection did not promote the release of H2O2 by peritoneal macrophages (Fig. 3). At day 12, this production was similar to that of the controls. In accordance, the total number of peritoneal cells of the infected group was also similar to the controls (Fig. 4). 3.3. Light microscopic findings At day 12 of infection, multiplying forms of T. cruzi (Fig. 5B) were observed within cardiomyocytes in all infected rats. In the atria, the meanstandard deviation of the amastigote nests/group of four animals was 539.2391.2 and in ventricles was 356.7300.2. Para-
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0DFURSKDJHV Fig. 3. Hydrogen peroxide (H2O2) production by splenic and peritoneal macrophages in rats at day 12 of infection with T. cruzi and in uninfected controls. The H2O2 levels increased in splenic macrophages (76%) in comparison to controls, but the infection did not promote the release of H2O2 by peritoneal cells. The data are expressed as meanSD and are representative of two independent experiments, with four to seven rats per group.
sitized cardiomyocytes, mainly in the atria, were frequently dissociated and exhibited degenerating signs (Fig. 5C). In addition, the intersticial tissue was edematous. Usually, diffuse myocarditis was present in the atria (moderate to intense grade) and ventricles (moderate grade) of the infected group, 12 days postinfection. Inflammatory cells were predominantly mononuclear (Fig. 5B and C). Scattered macrophage-like cells (Fig. 5C) were always present in the myocardium of the infected animals, sometimes invading amastigote nests. 3.4. Ultrastructural findings A few macrophage-like cells occurred in the heart of the control animals. These resident macrophages were difficult to be found out and showed size significantly smaller than macrophages seen in infected animals, 4.91.1 (meanstandard deviation) (range 4.0–6.6) compared with 9.43.2 (meanstandard deviation) (range 5.3–16.6). Furthermore, the control macrophages exhibited few surface extensions, lesser amount of
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Fig. 4. Total number of the cells isolated from spleen and peritoneum in rats at day 12 of infection with T. cruzi and in uninfected controls. The total number of cells increased (P<0.05) in the spleen in comparison to controls, but the infection did not promote the increase of total number of peritoneal cells. The data are expressed as meanSD and are representative of two independent experiments, with four to seven rats per group.
cytoplasmic organelles and a more heterochromatic nucleus (Fig. 6A). A high macrophage influx was present in the heart of all infected animals. These cells had different morphological phenotypes and showed ultrastructural signs of activation being more voluminous with a striking increase in cytoplasmic organelles compared to controls. Many surface rufflings, pseudopodia, arrays of rough endoplasmic reticulum, polysomes, lysosomes, mitochondrion profiles and vesicles were present (Fig. 6B). In contrast to normal macrophages, the nucleus occupied a small part of the cell and was irregular in outline. (Fig. 6B). Numerous macrophages were often seen ingesting T. cruzi forms and showed many phagolysosomes with varying sizes and electrondensities containing amorphous or granular materials, cell debris and amastigotes (Fig. 6B). Cell-to-cell interactions mainly between macrophages or macrophages and lymphocytes were frequently observed as previously reported (Melo and Machado, 2001). Occasionally, multinucleated macrophages were detected.
4. Discussion Macrophages subjected to appropriate stimuli undergo morphological and biochemical changes consistent with enhanced activity. Activated macrophages produce and release numerous compounds that regulate the immune response and participate in the destruction of invading pathogens (Turpin and Lopez-Berestein, 1993). The present findings show that clear monocyte/ macrophage mobilization and activation occurred
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earlier in the acute infection in parallel to a great increase in the number of these cells respectively in the blood and heart. According to previous studies in rats (Melo and Machado, 2001; Melo et al., 2003), heart inflammatory macrophages depicted a wide range of ultrastructural variations in cytoplasmic organelles and cellular membrane when challenged with the parasite in vivo. Interestingly, these macrophages showed prominent increase in the number and size of lipid bodies, specialized intracellular domains involved in generating inflammatory mediators (Melo et al., 2003). In the present work, we extended these morphological observations demonstrating, for the first time, that the size of heart inflammatory macrophages increases significantly during the acute phase of Chagas’ disease, other evidence of powerful activation of these cells (Anosa et al., 1997; El Shewemi et al., 1996; Takemura et al., 1989). Our data support previous studies, both in humans and in the murine model emphasizing the importance of the innate immunity, precisely cells from the macrophage lineage to host resistance to acute T. cruzi infection (reviewed in Ropert et al., 2002). In response to parasite, peripheral blood monocytes and spleen macrophages of the infected animals showed increase in the H2O2 production. On the other hand, the infection did not induce in vivo release of H2O2 by peritoneal macrophages. These data are consistent with in vivo results obtained in mice showing a very low release of H2O2 by peritoneal macrophages in the initial phase of the acute T. cruzi infection, in spite of the authors have found a high gamma interferon level in the serum (Borges et al., 1995). We speculate that the observed low macrophage activation may be associated with a diminished number of parasites remaining in the peritoneum on day analyzed. Therefore, peritoneal macrophages may not have been sufficiently activated to produce appreciable amounts of H2O2. In addition, the total number of peritoneal cells in infected animals was similar to controls, indicating a reduced influx of inflammatory cells at least at day 12. Since inflammatory but not resident macrophages release high levels of antimicrobial products (Nathan et al., 1979), the low production of H2O2 may also be related to the few number of these cells in the peritoneum. On the other hand, the high level of trypomastigotes in blood and amastigotes in tissues, a known characteristic of the acute T. cruzi infection (Andrade, 1991; Tanowitz et al., 1992), may have induced the releasing of H2O2 by, respectively, peripheral blood monocytes and spleen macrophages as showed in the present work. In accordance, the number of peripheral blood monocytes and inflammatory spleen cells was significantly increased. The importance of parasite load for macrophage activation in the acute infection was demonstrated in lymphocyte-depleted rats by gamma irradiation (Melo and Machado, 1998).
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Fig. 5. Light micrographs of atrial tissue in normal (A) and infected (B and C) rats, at day 12 of infection with T. cruzi. Acute myocarditis characterized by a diffuse mononuclear infiltrate, macrophage-like cells (arrows) and parasite nests (arrowheads) are clearly seen in B and C compared to A. In B, a degenerating cardiomyocyte (*). Scale bar, 10 µm. The data are representative of two independent experiments. Four to six rats per group.
These animals showed the heart with a high number of macrophages exhibiting typical morphology of activated cells in parallel to an increased parasitism. Accordingly,
recent report has highlighted the importance of parasitederived products to activation of host innate immunity (Ropert et al., 2002).
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Fig. 6. Electron micrographs of atrial tissue in normal (A) and in infected rat (B) at day 12 of infection with T. cruzi. In A, a resident macrophage is seen close to a cardiomyocyte (c). In B, activated macrophage close to cardiomyocyte (c) shows nucleus (n) irregular in outline, voluminous cytoplasm rich in organelles, parasite amastigote forms (*) and many surface rufflings (arrowheads). Scale bar, 1 µm. The data are representative of two independent experiments. Four to six rats per group.
Whereas there are very few studies focused on H2O2 production by macrophages during the in vivo T. cruzi infection, the in vitro reports are contrasting. Most authors have demonstrated that T. cruzi triggers a respiratory burst by activated peritoneal macrophages
(Celentano and Gonza´lez-Cappa, 1992; Nathan et al., 1979; Nathan, 1982; Nogueira and Cohn, 1978; Reed et al., 1987; Villalta and Kierszenbaum, 1984) and have pointed out H2O2 as being relevant to the cytotoxic mechanism (Nathan, 1982; Tanaka et al., 1983; Villalta
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and Kierszenbaum, 1984). Mccabe and Mullins (1990), however, reported that these cells, when cultured with the parasite, released less than 10% of H2O2 indicating the importance of oxygen-independent antimicrobial mechanisms. Results obtained in vitro are difficult to be interpreted since the assay conditions are very variable and only permit the interaction of parasite with macrophages which does not take place in studies operating in vivo. In the present work, the enhanced H2O2 respiratory burst in monocytes at day 12 of infection coexisted with great number of these cells in the blood and parasites in the heart. To our knowledge this is the first study showing activation of peripheral blood monocytes (as measured by H2O2 production) during the in vivo acute chagasic infection. As previously pointed out by us, at day 12, the acute disease triggers a plentiful production of monocytes from bone marrow precursor cells so as to control T. cruzi replication in the target tissues such as the myocardium (Melo and Machado, 2001). The present data showing a high monocytosis since day 6 of infection reinforces the occurrence of a rapid and powerful mobilization of the monocyte/macrophage system by Chagas’ disease. That activated macrophages are effective in controlling parasitism is supported by experiments showing that the myocardial parasite load was increased in macrophage-depleted rats (Melo and Machado, 2001) and was reduced in recombinant IFNgamma treated rats (Revelli et al., 1998). In cultures, the intracellular replication of T. cruzi was inhibited in human peripheral blood monocytes stimulated by granulocyte/macrophage colonystimulating factor (GM-CSF) or gamma-interferon (Reed et al., 1987), indicating that activated monocytes are able to kill the parasite. In fact, even without a stimulus by cytokine or an apparent lymphocyte contribution, the human peripheral blood monocytes cultured with the parasite assume activated morphology, produce interleukin–1 beta, tumor necrosis factor-alpha and interleukin-6 (Van Voorhis, 1992) and may uptake and destroy parasites (Villalta and Kierszenbaum, 1984) which demonstrate the T. cruzi ability to activate these cells. Similarly, human blood monocytes may be directly stimulated for H2O2 generation by tumour cells in vitro, but not by untransformed cells, denoting the monocyte citotoxicity after activation (Mytar et al., 1999). Despite extensive experimental investigations, the mechanisms used by activated macrophages to kill T. cruzi are not fully understood. The nitric oxide synthesis, for instance, has been claimed as a relevant molecule related to host defense against the parasite (Petray et al., 1995; Rodrigues et al., 2000; Vespa et al., 1994). However, recent data showed that nitric oxide production is not essential to control the whole period of acute phase of T. cruzi infection in mice (Saeftel et al., 2002) and rats (Fabrino, personal communication). Our work showing
an important in vivo increase in H2O2 production by infiltrating activated monocytes/macrophages suggests that this product may have a major role in the T. cruzi clearance process and/or in the development of inflammation. In fact, a novel role for hydrogen peroxide has recently been discussed (reviewed in Reth, 2002). Upon stimulation, cells produce hydrogen peroxide and seem use it as an intercellular messenger, involved especially in lymphocyte activation. Cells from monocytic lineage are known to form the first line of defense against intruding pathogens and are dramatically increased in number during acute Chagas’ disease (Melo et al., 2003). In response to T. cruzi invasion, both blood monocytes and specific macrophages produce high amounts of H2O2 during the oxidative burst reaction, as demonstrated in the present work. So far, it is thought that the only role for the oxidative burst is to kill pathogens, but it is possible that the oxidative burst is not primarily a killing device but rather a mechanism for activating the macrophage and neighboring lymphocytes (Reth, 2002). The present data stress the importance of innate immunity to host resistance during the acute phase of T. cruzi infection. A novel finding of our study is that H2O2 production seems related to specific types of monocytes/ macrophages that are able to release this agent when in the presence of high parasite load.
Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-Proc. 521209/97-8) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG—Proc. CBS 2238/96). The authors are grateful to Dr Fa´bio Roland for his helpful discussions, Dr Jane Azevedo da Silva for statistical assistance, Dr C. R. S. Machado for supplying the infected animals and to the Centro de Microscopia Eletroˆnica (CEMEL, ICB/UFMG) for the use of its facilities.
References Abrahamsohn IA, Coffman RL. Trypanosoma cruzi: IL-10, TNF, IFN-gamma, and IL-12 regulate innate and acquired immunity to infection. Exp Parasitol 1996;84(2):231–44. Abreu MA, Baroza LGV, Rossi MA. Toluidine Blue-Basic Fuchisin stain for glycolmetacrylate embedded tissue. J Histotechnol 1993;16(2):139–40. Andrade ZA. Pathogenesis of Chagas’ disease. Res Immunol 1991; 142(2):126–51. Anosa VO, Loganhenfrey LL, Wells CW. The haematology of Trypanosoma cruzi congolense infection in cattle. 2. Macrophage structure and function in the bone marrow of Boran cattle. Comp Haemat Int 1997;7(1):23–9.
R.C.N. Melo et al. / Cell Biology International 27 (2003) 853–861 Antunez MI, Cardoni RL. IL-12 and IFN-gamma production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections. Immunol Lett 2000;71(2):103–9. Brener Z, Gazzinelli RT. Immunological control of Trypanosoma cruzi infection and pathogenesis of Chagas’ disease. Int Arch Allergy Immunol 1997;114:103–10. Borges MM, Vassao R, Andrade SG, Pereira CA, Kloetzel JK. Interferon-Gama levels during the course of Trypanosoma cruzi infection of Calomys callosus (Rodentia-Cricetidae) and Swiss mice. Parasitol Res 1995;81:498–504. Celentano AM, Gonza´lez-Cappa SM. Induction of macrophage activation and opsoning antibodies by Trypanosoma cruzi subpopulations. Parasite Immunol 1992;14:155–67. El Shewemi S, Alshammary F, Alzamel F, Soliman R. Ultrastructure of resting and rh-GMCSF-treated human macrophages derived from blood monocytes. J Electron Microsc 1996;45(5):388–94. Hanson WL, Roberson EL. Density of parasites in various organs and the relation to numbers of trypomastigotes in blood during acute infections of Trypanosoma cruzi in mice. J Protozol 1974; 21(4):512–7. Jaramillo M, Olivier M. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signalregulated kinase- and cyclic adenosine 5#- Monophosphate (cAMP)-dependent pathways: involvement of NF-B, activator protein 1, and cAMP response element binding protein. J Immunol 2002;169:7026–38. Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 1965;27:137A–8A. Mccabe RE, Mullins BT. Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. J Immunol 1990; 144:2384–8. Melo RCN. Depletion of immune effector cells induces myocardial damage in the acute experimental Trypanosoma cruzi infection: ultrastructural study in rats. Tissue Cell 1999;31(3):281–90. Melo RCN, Machado CRS. Depletion of radiosensitive leukocytes exacerbates the heart sympathetic denervation and parasitism in experimental Chagas’ disease in rats. J Neuroimmunol 1998; 84:151–7. Melo RCN, Machado CRS. Trypanosoma cruzi: peripheral blood monocytes and heart macrophages in the resistance to acute experimental infection in rats. Exp Parasitol 2001;97:15–23. Melo RCN, D’Avila H, Fabrino DL, Almeida PE, Bozza PT. Macrophage lipid body induction by Chagas’ disease in vivo: putative intracellular domains for eicosanoid formation during infection. Tissue Cell 2003;35(1):59–67. Mytar B, Siedlar M, Woloszyn M, Ruggiero I, Pryjma J, Zembala M. Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity. Br J Cancer 1999;79(5–6):737–43. Nathan CF. Secretion of oxygen intermediates: role in effector functions of activated macrophages. Fed Proc 1982;41:2206–11. Nathan C, Nogueira N, Juanghanich C, Ellis J, Cohn Z. Activation of macrophages in vivo and in vitro. J Exp Med 1979;149:1056–68. Nogueira N, Cohn ZA. Trypanosoma cruzi: in vitro induction of macrophage microbicidal activity. J Exp Med 1978;148:288–300. Petray P, Castanosvelez E, Grinstein S, Orn A, Rottenberg ME. Role of nitric-oxide in resistance and histopathology during experimental infection with Trypanosoma cruzi. Immunol Lett 1995; 47(1–2):121–6. Pick E, Mizel D. Rapid microssays for a the measurement of superoxide and hydrogen peroxide production by macrophages in culture using na automatic enzime immunoassay reader. J Immunol Methods 1981;46:211–26.
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Reed SG, Nathan CF, Pihl DL, Rodricks P, Shanebeck K, Conlon PJ et al. Recombinant granulocyt/macrophage colony-stimulating factor activates macrophages to inhibit Trypanosoma cruzi and release hydrogen peroxide. Comparison with interferon . J Exp Med 1987;166:1734–46. Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. 2002. Nat Immunol 2002;3(12):1129–34. Revelli S, Didoli G, Roggero E, Moreno H, Bernabo J, Wietzerbin J et al. Macrophage activity, IL-6 levels, antibody response and heart histology in rats undergoing an attenuated Trypanosoma cruzi acute infection upon treatment with recombinant interferon gamma. Cyt Cell Mol Ther 1998;4(3):153–9. Rodrigues MM, Ribeira˜o M, Boscardin SB. CD4 Th1 but not Th2 clones efficiently activate macrophages to eliminate Trypanosoma cruzi through a nitric oxide dependent mechanism. Immunol Lett 2000;73(1):43–50. Ropert C, Ferreira LRP, Campos MAS, Procopio DO, Travassos LR, Ferguson MAJ et al. Macrophage signaling by glycosylphatylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes. Microbes Infect 2002; 4:1015–25. Russo M, Starobinas N, Ribeiro dos Santos R, Minoprio PO, Hontebeyre-Joskowicz M. Susceptible mice present higher macrophage activation than resistant mice during infection with myotropic strains of Trypanosoma cruzi. Parasite Immunol 1989; 11:385–95. Saeftel M, Fleischer B, Hoerauf A. Stage-dependent role of nitric oxide in control of Trypanosoma cruzi infection. Infect Immun 2002;69(4):2252–9. Silva LHP, Nussenszweig V. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Fol Clin Biol 1953;20:191–208. Silva JS, Vespa GNR, Cardoso MAG, Aliberti JCS, Cunha FQ. Tumor-Necrosis-Factor-Alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric-oxide production in infected gamma-interferon-activated macrophages. Infect Immun 1995;63(12):4862–7. Sun J, Tarleton RL. Predominance of CD8+ T lymphocytes in the inflammatory lesions of mice with acute Trypanosoma cruzi infection. Am J Trop Med Hyg 1993;48:161–9. Takemura T, Rom WN, Ferrans VJ, Crystal RG. Morphologic characterization of alveolar macrophages from subjects with occupational exposure to inorganic particles. Amn Rev Resp Dis 1989; 140:1674–85. Tanaka Y, Tanowitz H, Bloom BR. Growth of Trypanosoma cruzi in a cloned macrophage cell line and in a variant defective in oxygen metabolism. Infect Immun 1983;41(3):1322–31. Tanowitz HB, Kirchhoff LV, Simon D, Morris SA, Weiss LM, Wittner M. Chagas’ disease. Clin Microbiol Rev 1992;5(4):400–19. Turpin JA, Lopez-Berestein G. Differentiation, maturation, and activation of monocytes and macrophages: functional activity is controlled by a continuum of maturation. In: Lopez-Berestein G, Klostergaard J, editors. Mononuclear phagocytes in cell biology. Boca Raton (FL): CRC Press, 1993;71–99. Van Voorhis W. Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production. J Immunol 1992;148(1):239–48. Vespa GNR, Cunha FQ, Silva JS. Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. Infect Immun 1994;62(11):5177–82. Villalta F, Kierszenbaum F. Role of inflammatory cells in Chagas’ disease. II. Interactions of mouse macrophages and human monocytes with intracellular forms of Trypanosoma cruzi: uptake and mechanism of destruction. J Immunol 1984;133(6):3338–43.
Cell Biology International www.elsevier.com/locate/cellbi
Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo ´ vila 1, Henrique C. Teixeira 2, Rossana C.N. Melo 1*, Daniela L. Fabrino 1, Heloisa D’A 2 Ana Paula Ferreira 1
Laboratory of Cellular Biology, Institute of Biological Sciences, Federal University of Juiz de Fora (UFJF), 36036-330, Juiz de Fora-MG, Brazil 2 Laboratory of Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora (UFJF), 36036-330, Juiz de Fora-MG, Brazil Received 3 February 2003; revised 27 May 2003; accepted 14 July 2003
Abstract Acute Chagas’ disease triggers potent inflammatory reaction characterized by great increase of peripheral blood monocyte (PBM) and macrophage numbers. We studied the respiratory burst responses of PBM and peritoneal and splenic macrophages to in vivo infection (rats). The ultrastructure of heart inflammatory macrophages was also investigated. The infection increased the hydrogen peroxide (H2O2) production by PBM and splenic macrophages but not by peritoneal macrophages. Accordingly, the PBM and spleen cell numbers increased but the total number of peritoneal cells was similar to controls. Heart macrophages of infected rats exhibited increase (number and size) and activated morphology in parallel to high cardiomyocyte parasitism. Our data highlight the importance of innate immunity and H2O2 production to host resistance during acute phase of T. cruzi infection. A novel finding is that H2O2 production seems related to specific types of monocytes/macrophages that are able to release this agent when in presence of high parasite load. 2003 Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Chagas’ disease; Hydrogen peroxide; Myocarditis; Monocytes/macrophages; Electron microscopy
1. Introduction Chagas’ disease is caused by intracellular protozoan Trypanosoma cruzi, parasite of many mammalian species, and is characterized by an acute phase in which the parasite circulates in the boodstream as trypomastigotes and proliferates within the cytoplasm of a variety of cells as amastigotes. The rupture of amastigote nests provokes cell destruction and a potent inflammatory process, especially in the heart, a target organ of the disease. Approximately 18–20 million people in the Latin America are chronically infected with T. cruzi (reviewed in Ropert et al., 2002). * Corresponding author. Present address: Harvard Medical School/Beth Israel Deaconess Medical Center, DA-617, 330 Brookline Avenue, Boston, MA 002215, USA. Tel.: +1-617-667-3307; fax: +1-617-667-5541 E-mail address: [email protected] (R.C.N. Melo). 1065-6995/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00173-2
The host resistance to acute infection is expressed mainly by cell-mediated reactions involving sensitized T cells and macrophages (reviewed in Brener and Gazzinelli, 1997). However, there is no general agreement on the immunoregulatory mechanisms involved and the precise role of these immune effector cells in the control of parasite (Abrahamsohn and Coffman, 1996; Sun and Tarleton, 1993). During the acute phase of Chagas’ disease, we have observed, in vivo, a dramatic increase in the number of both monocytes in the peripheral blood and macrophages in the heart of infected rats. These cells exhibited clear morphological signs of activation and were directly involved in inhibiting parasite multiplication in the myocardium (Melo and Machado, 2001; Melo et al., 2003). The ultrastructural features of the macrophage activation such as increase in size, surface rufflings and amount of cytoplasmic organelles are recognized as an
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accurate indication of high phagocytic and microbicidal activities of these cells both in humans and experimental models (Anosa et al., 1997; El Shewemi et al., 1996; Takemura et al., 1989), including Chagas’ disease (Melo and Machado, 2001; Melo et al., 2003). On the other hand, biochemical changes, as the ability of secreting hydrogen peroxide (Nathan, 1982; Nogueira and Cohn, 1978; Reed et al., 1987), tumor necrosis factor-alpha (Silva et al., 1995), nitric oxide (Vespa et al., 1994; Petray et al., 1995; Rodrigues et al., 2000) and interleukin-12 (Antunez and Cardoni, 2000) have been considered important markers of macrophage activity. In spite of several authors had evaluated the hydrogen peroxide-releasing capacity of macrophages upon T. cruzi challenge, the results remain contrasting (Borges et al., 1995; Mccabe and Mullins, 1990; Nathan et al., 1979; Reed et al., 1987; Russo et al., 1989). The majority of these studies were carried out in vitro using murine peritoneal macrophages. When mouse macrophages were cultured with the parasite, the hydrogen peroxide production was significant (Nathan et al., 1979; Reed et al., 1987) or very low (Mccabe and Mullins, 1990). On the other hand, the in vivo release of H2O2 by activated macrophages has not been well established during the T. cruzi infection. The amount of H2O2 secreted by peritoneal macrophages during the chagasic infection of susceptible mice was higher than that observed in the resistant mice (Russo et al., 1989). In addition, Borges et al. (1995) have shown that there is not a correlation between the in vivo levels of gamma interferon, a lymphocyte cytokine considered as the major stimulus for the activation of macrophages, and the H2O2 production by murine peritoneal macrophages, during the acute infection. Regarding monocytes, their ability to produce in vivo H2O2 has not been studied in the course of acute infection with T. cruzi. In vitro, peripheral blood monocytes are able to destroy the parasite after ingestion (Villalta and Kierszenbaum, 1984) and exhibit activated morphology when cultured with T. cruzi (Van Voorhis, 1992). We have observed, in vivo, a great increase in the number of both monocytes in the peripheral blood and macrophages in the heart, emphasizing the importance of the newly formed monocyte-derived macrophages in resistance to acute Chagas’ disease (Melo, 1999; Melo and Machado, 2001). These findings and the conflicting views about the H2O2 production by macrophages during chagasic infection in mice, render the whole subject to be worthy of re-investigation. In addition, studies regarding H2O2 production during infectious diseases may be focus of great interest since H2O2 can be responsible for chemokine expression and the development of inflammation (Jaramillo and Olivier, 2002). The present work describes, for the first time, the respiratory burst responses of rat peripheral blood monocytes to in vivo acute T. cruzi infection. The
responses of different types of macrophages (from peritoneum and spleen) were also evaluated in an attempt to clarify the previous contradictory reports. In parallel, we have examined the ultrastructure of heart inflammatory macrophages and the cardiomyocyte parasitism. The Holtzman rat has been used in our lab as an excellent experimental model since the infection in these animals faithfully mimics the human acute phase, triggering a prominent parasitemia, heart parasitism, and myocarditis (Melo, 1999; Melo and Machado, 1998, 2001). 2. Materials and methods 2.1. Animals Female Holtzman rats aged 27–30 days comprised noninfected and infected groups. The animals were sacrificed under exsanguination after ether anesthesia at day 12 of infection. Each group contained four to seven animals. 2.2. T. cruzi infection The rats were inoculated intraperitoneally with a single inoculum of 150 µl of mouse blood containing 3105 trypomastigotes of T. cruzi, Y strain (Silva and Nussenszweig, 1953). Fresh blood sampled from the tail showed living trypomastigotes in all animals 6 or 12 days after inoculation. 2.3. Peripheral blood leukocyte quantification Samples of blood were taken from the tail of the controls and infected animals at 6 and 12 days postinfection. The total number of leukocytes was estimated by hemocytometer counts (20 µl of blood diluted in 2% acetic acid and 0.01% methylene blue). The percentage of each white blood cell in air-dried smears stained with May-Gru¨nwald/Giemsa solution allowed an estimate of the total number of each leukocyte. The leukocyte percentage was determined after counting 200 cells per rat. 2.4. Cell isolation and H2O2 production by peripheral blood mononuclear cells (PBMC), splenic and peritoneal cells The H2O2 production by PBMC, splenic and peritoneal cells was evaluated in controls and infected animals at 12 days of infection by horseradish peroxidedependent phenol red oxidation method (Pick and Mizel, 1981) slightly modified by us as follows. The peripheral blood was drawn from left axillary artery. PBMC were isolated from heparinized pool (5–6 ml/ group of 7 animals) of peripheral blood (20 µl heparin/ml blood) with Histopaque-1077 (Sigma, St Louis, MO, USA) by density gradient centrifugation at
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2000 rpm for 30 min, at room temperature. Spleens were homogenized individually in 5 ml RPMI using a teflon coated tissue homogenizer (Gas-Col Apparatus Co. Terre Haute, IN, USA). PBMC and splenic cells were washed twice in PBS and ressuspended in RPMI 1640 medium containing 5.5 mM dextrose, 0.56 mM phenol red and 8.5U ml1 horseradish peroxide type II (1500 rpm for 10 min at 4 (C). Peritoneal cells were obtained from peritoneal washings with phosphate-buffered saline (PBS) (5 ml/animal) and washed with PBS at 1000 rpm for 10 min at 4 (C. Viability of the cells was assessed by trypan blue test and the total number was estimated by hemocytometer counts. The concentration of all collected cells was adjusted to 2106 cells per ml with fresh medium RPMI 1640. Volumes (100 µl) of the cell suspensions were plated on to each well of 96-well flat-bottom tissue culture plates (Costar, Cambridge, MS, USA). The plates were incubated in humidified atmosphere at 37 (C for 1 h. Subsequently, 10 µl of 1 N NaOH was added to stop the reaction and the absorbance was determined at 620 nm using a Spectra Max 190 (Molecular Devices, USA). Conversion of absorbance to micromoles of H2O2 was deduced from a standard curve. All determinations were performed in quadruplicate and expressed as micromoles of H2O2/2106 cells.
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Fig. 1. Number of monocytes in the peripheral blood of rats at day 6 and 12 of infection with T. cruzi and in uninfected controls. Animals were infected i.p. with 300,000 trypomastigotes of Y strain. On both days, the monocyte numbers (P<0.05) increased in the blood compared to control values. The monocyte numbers found at day 12 were increased (P<0.05) in comparison to numbers found at day 6. The data are expressed as meanSD and are representative of three independent experiments, with four to seven rats per group.
2.5. Light microscopy Fragments of atria and ventricles from controls and infected animals (at day 12 of infection) were fixed in 4% paraformaldehyde in buffered phosphate, pH 7.3, 0.1 M, for 24 h, processed routinely and embedded in glycol methacrilate (Leica). Semi-serial 5-µm-thick sections stained by haematoxylin and eosin or toluidine bluebasic fuchsin (Abreu et al., 1993) were examined for qualitative evaluation of the inflammatory and degenerative processes and quantification of parasitism. The number of amastigote nests in cardiomyocytes was evaluated at 70 µm interval for avoiding parasite recounting (Hanson and Roberson, 1974). For each group of animals were analyzed 1600 fields (800 from atria and 800 from ventricles) at 400. 2.6. Electron microscopy Fragments of the atria of controls and infected rats (12 days) were fixed in the phosphate-buffered 1% paraformaldehyde and 1% glutaraldehyde, pH 7.3, overnight at 4 (C (Karnovsky, 1965). The tissues were postfixed in a mixture of 1% phosphate-buffered osmium tetroxide and 1.5% potassium ferrocyanide (final concentration), for 1 h and processed for resin embedding (PolyBed 812, Polysciences). The ultrathin sections were stained with uranyl acetate and lead citrate before examination in a Zeiss EM 10 at 60 kV. In addition to qualitative observations, quantitative study was made in
electron micrographs of the heart macrophages. The size variation of these cells was obtained by diameter measurements of the macrophages in which appeared the nucleus. Since most macrophages have an irregular shape, 10 measurements of the diameter were made for each macrophage, being analyzed a total of 15 cells from samples of the normal and infected groups. From the mean diameter obtained for each cell, the data were calculated and expressed as mean diameterstandard deviation for each group. 2.7. Statistical analysis Data from different groups were compared by the Mann–Whitney “U” test (P<0.05).
3. Results 3.1. Peripheral blood leucocyte numbers The number of monocytes in the peripheral blood (Fig. 1) showed significant increase (98%) at day 6 of T. cruzi infection in comparison to values from uninfected controls. At day 12, this number (Fig. 1) was dramatically increasead in comparison to control values (1180%) and to data found at day 6 of infection (478%). Analyzes of the blood smears of the infected animals revealed 1–3 monocytes per each field observed. The lymphocyte, neutrophil and eosinophil numbers and the
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Fig. 2. Hydrogen peroxide (H2O2) production by peripheral blood monocytes in rats at day 12 of infection with T. cruzi and in uninfected controls. The H2O2 levels increased at day 12 (85%) in comparison to control values. The data are expressed as meanSD and are representative of two independent experiments, with four to seven rats per group.
total number of peripheral blood leukocytes were not significantly different from the control values on both days analyzed. 3.2. Evaluation of H2O2 release by peripheral blood monocytes and spleen and peritoneal macrophages The acute T. cruzi infection induced H2O2 release by peripheral blood monocytes. The H2O2 levels of these cells increased 85% at day 12 in comparison to control values (Fig. 2). Spleen macrophages of the infected animals also released H2O2 at this time (76% of increase compared to controls) (Fig. 3). This increase was accompanied by 4.5 fold enhancement of the total number of spleen cells (Fig. 4). In contrast, the infection did not promote the release of H2O2 by peritoneal macrophages (Fig. 3). At day 12, this production was similar to that of the controls. In accordance, the total number of peritoneal cells of the infected group was also similar to the controls (Fig. 4). 3.3. Light microscopic findings At day 12 of infection, multiplying forms of T. cruzi (Fig. 5B) were observed within cardiomyocytes in all infected rats. In the atria, the meanstandard deviation of the amastigote nests/group of four animals was 539.2391.2 and in ventricles was 356.7300.2. Para-
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sitized cardiomyocytes, mainly in the atria, were frequently dissociated and exhibited degenerating signs (Fig. 5C). In addition, the intersticial tissue was edematous. Usually, diffuse myocarditis was present in the atria (moderate to intense grade) and ventricles (moderate grade) of the infected group, 12 days postinfection. Inflammatory cells were predominantly mononuclear (Fig. 5B and C). Scattered macrophage-like cells (Fig. 5C) were always present in the myocardium of the infected animals, sometimes invading amastigote nests. 3.4. Ultrastructural findings A few macrophage-like cells occurred in the heart of the control animals. These resident macrophages were difficult to be found out and showed size significantly smaller than macrophages seen in infected animals, 4.91.1 (meanstandard deviation) (range 4.0–6.6) compared with 9.43.2 (meanstandard deviation) (range 5.3–16.6). Furthermore, the control macrophages exhibited few surface extensions, lesser amount of
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Fig. 4. Total number of the cells isolated from spleen and peritoneum in rats at day 12 of infection with T. cruzi and in uninfected controls. The total number of cells increased (P<0.05) in the spleen in comparison to controls, but the infection did not promote the increase of total number of peritoneal cells. The data are expressed as meanSD and are representative of two independent experiments, with four to seven rats per group.
cytoplasmic organelles and a more heterochromatic nucleus (Fig. 6A). A high macrophage influx was present in the heart of all infected animals. These cells had different morphological phenotypes and showed ultrastructural signs of activation being more voluminous with a striking increase in cytoplasmic organelles compared to controls. Many surface rufflings, pseudopodia, arrays of rough endoplasmic reticulum, polysomes, lysosomes, mitochondrion profiles and vesicles were present (Fig. 6B). In contrast to normal macrophages, the nucleus occupied a small part of the cell and was irregular in outline. (Fig. 6B). Numerous macrophages were often seen ingesting T. cruzi forms and showed many phagolysosomes with varying sizes and electrondensities containing amorphous or granular materials, cell debris and amastigotes (Fig. 6B). Cell-to-cell interactions mainly between macrophages or macrophages and lymphocytes were frequently observed as previously reported (Melo and Machado, 2001). Occasionally, multinucleated macrophages were detected.
4. Discussion Macrophages subjected to appropriate stimuli undergo morphological and biochemical changes consistent with enhanced activity. Activated macrophages produce and release numerous compounds that regulate the immune response and participate in the destruction of invading pathogens (Turpin and Lopez-Berestein, 1993). The present findings show that clear monocyte/ macrophage mobilization and activation occurred
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earlier in the acute infection in parallel to a great increase in the number of these cells respectively in the blood and heart. According to previous studies in rats (Melo and Machado, 2001; Melo et al., 2003), heart inflammatory macrophages depicted a wide range of ultrastructural variations in cytoplasmic organelles and cellular membrane when challenged with the parasite in vivo. Interestingly, these macrophages showed prominent increase in the number and size of lipid bodies, specialized intracellular domains involved in generating inflammatory mediators (Melo et al., 2003). In the present work, we extended these morphological observations demonstrating, for the first time, that the size of heart inflammatory macrophages increases significantly during the acute phase of Chagas’ disease, other evidence of powerful activation of these cells (Anosa et al., 1997; El Shewemi et al., 1996; Takemura et al., 1989). Our data support previous studies, both in humans and in the murine model emphasizing the importance of the innate immunity, precisely cells from the macrophage lineage to host resistance to acute T. cruzi infection (reviewed in Ropert et al., 2002). In response to parasite, peripheral blood monocytes and spleen macrophages of the infected animals showed increase in the H2O2 production. On the other hand, the infection did not induce in vivo release of H2O2 by peritoneal macrophages. These data are consistent with in vivo results obtained in mice showing a very low release of H2O2 by peritoneal macrophages in the initial phase of the acute T. cruzi infection, in spite of the authors have found a high gamma interferon level in the serum (Borges et al., 1995). We speculate that the observed low macrophage activation may be associated with a diminished number of parasites remaining in the peritoneum on day analyzed. Therefore, peritoneal macrophages may not have been sufficiently activated to produce appreciable amounts of H2O2. In addition, the total number of peritoneal cells in infected animals was similar to controls, indicating a reduced influx of inflammatory cells at least at day 12. Since inflammatory but not resident macrophages release high levels of antimicrobial products (Nathan et al., 1979), the low production of H2O2 may also be related to the few number of these cells in the peritoneum. On the other hand, the high level of trypomastigotes in blood and amastigotes in tissues, a known characteristic of the acute T. cruzi infection (Andrade, 1991; Tanowitz et al., 1992), may have induced the releasing of H2O2 by, respectively, peripheral blood monocytes and spleen macrophages as showed in the present work. In accordance, the number of peripheral blood monocytes and inflammatory spleen cells was significantly increased. The importance of parasite load for macrophage activation in the acute infection was demonstrated in lymphocyte-depleted rats by gamma irradiation (Melo and Machado, 1998).
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Fig. 5. Light micrographs of atrial tissue in normal (A) and infected (B and C) rats, at day 12 of infection with T. cruzi. Acute myocarditis characterized by a diffuse mononuclear infiltrate, macrophage-like cells (arrows) and parasite nests (arrowheads) are clearly seen in B and C compared to A. In B, a degenerating cardiomyocyte (*). Scale bar, 10 µm. The data are representative of two independent experiments. Four to six rats per group.
These animals showed the heart with a high number of macrophages exhibiting typical morphology of activated cells in parallel to an increased parasitism. Accordingly,
recent report has highlighted the importance of parasitederived products to activation of host innate immunity (Ropert et al., 2002).
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Fig. 6. Electron micrographs of atrial tissue in normal (A) and in infected rat (B) at day 12 of infection with T. cruzi. In A, a resident macrophage is seen close to a cardiomyocyte (c). In B, activated macrophage close to cardiomyocyte (c) shows nucleus (n) irregular in outline, voluminous cytoplasm rich in organelles, parasite amastigote forms (*) and many surface rufflings (arrowheads). Scale bar, 1 µm. The data are representative of two independent experiments. Four to six rats per group.
Whereas there are very few studies focused on H2O2 production by macrophages during the in vivo T. cruzi infection, the in vitro reports are contrasting. Most authors have demonstrated that T. cruzi triggers a respiratory burst by activated peritoneal macrophages
(Celentano and Gonza´lez-Cappa, 1992; Nathan et al., 1979; Nathan, 1982; Nogueira and Cohn, 1978; Reed et al., 1987; Villalta and Kierszenbaum, 1984) and have pointed out H2O2 as being relevant to the cytotoxic mechanism (Nathan, 1982; Tanaka et al., 1983; Villalta
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and Kierszenbaum, 1984). Mccabe and Mullins (1990), however, reported that these cells, when cultured with the parasite, released less than 10% of H2O2 indicating the importance of oxygen-independent antimicrobial mechanisms. Results obtained in vitro are difficult to be interpreted since the assay conditions are very variable and only permit the interaction of parasite with macrophages which does not take place in studies operating in vivo. In the present work, the enhanced H2O2 respiratory burst in monocytes at day 12 of infection coexisted with great number of these cells in the blood and parasites in the heart. To our knowledge this is the first study showing activation of peripheral blood monocytes (as measured by H2O2 production) during the in vivo acute chagasic infection. As previously pointed out by us, at day 12, the acute disease triggers a plentiful production of monocytes from bone marrow precursor cells so as to control T. cruzi replication in the target tissues such as the myocardium (Melo and Machado, 2001). The present data showing a high monocytosis since day 6 of infection reinforces the occurrence of a rapid and powerful mobilization of the monocyte/macrophage system by Chagas’ disease. That activated macrophages are effective in controlling parasitism is supported by experiments showing that the myocardial parasite load was increased in macrophage-depleted rats (Melo and Machado, 2001) and was reduced in recombinant IFNgamma treated rats (Revelli et al., 1998). In cultures, the intracellular replication of T. cruzi was inhibited in human peripheral blood monocytes stimulated by granulocyte/macrophage colonystimulating factor (GM-CSF) or gamma-interferon (Reed et al., 1987), indicating that activated monocytes are able to kill the parasite. In fact, even without a stimulus by cytokine or an apparent lymphocyte contribution, the human peripheral blood monocytes cultured with the parasite assume activated morphology, produce interleukin–1 beta, tumor necrosis factor-alpha and interleukin-6 (Van Voorhis, 1992) and may uptake and destroy parasites (Villalta and Kierszenbaum, 1984) which demonstrate the T. cruzi ability to activate these cells. Similarly, human blood monocytes may be directly stimulated for H2O2 generation by tumour cells in vitro, but not by untransformed cells, denoting the monocyte citotoxicity after activation (Mytar et al., 1999). Despite extensive experimental investigations, the mechanisms used by activated macrophages to kill T. cruzi are not fully understood. The nitric oxide synthesis, for instance, has been claimed as a relevant molecule related to host defense against the parasite (Petray et al., 1995; Rodrigues et al., 2000; Vespa et al., 1994). However, recent data showed that nitric oxide production is not essential to control the whole period of acute phase of T. cruzi infection in mice (Saeftel et al., 2002) and rats (Fabrino, personal communication). Our work showing
an important in vivo increase in H2O2 production by infiltrating activated monocytes/macrophages suggests that this product may have a major role in the T. cruzi clearance process and/or in the development of inflammation. In fact, a novel role for hydrogen peroxide has recently been discussed (reviewed in Reth, 2002). Upon stimulation, cells produce hydrogen peroxide and seem use it as an intercellular messenger, involved especially in lymphocyte activation. Cells from monocytic lineage are known to form the first line of defense against intruding pathogens and are dramatically increased in number during acute Chagas’ disease (Melo et al., 2003). In response to T. cruzi invasion, both blood monocytes and specific macrophages produce high amounts of H2O2 during the oxidative burst reaction, as demonstrated in the present work. So far, it is thought that the only role for the oxidative burst is to kill pathogens, but it is possible that the oxidative burst is not primarily a killing device but rather a mechanism for activating the macrophage and neighboring lymphocytes (Reth, 2002). The present data stress the importance of innate immunity to host resistance during the acute phase of T. cruzi infection. A novel finding of our study is that H2O2 production seems related to specific types of monocytes/ macrophages that are able to release this agent when in the presence of high parasite load.
Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-Proc. 521209/97-8) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG—Proc. CBS 2238/96). The authors are grateful to Dr Fa´bio Roland for his helpful discussions, Dr Jane Azevedo da Silva for statistical assistance, Dr C. R. S. Machado for supplying the infected animals and to the Centro de Microscopia Eletroˆnica (CEMEL, ICB/UFMG) for the use of its facilities.
References Abrahamsohn IA, Coffman RL. Trypanosoma cruzi: IL-10, TNF, IFN-gamma, and IL-12 regulate innate and acquired immunity to infection. Exp Parasitol 1996;84(2):231–44. Abreu MA, Baroza LGV, Rossi MA. Toluidine Blue-Basic Fuchisin stain for glycolmetacrylate embedded tissue. J Histotechnol 1993;16(2):139–40. Andrade ZA. Pathogenesis of Chagas’ disease. Res Immunol 1991; 142(2):126–51. Anosa VO, Loganhenfrey LL, Wells CW. The haematology of Trypanosoma cruzi congolense infection in cattle. 2. Macrophage structure and function in the bone marrow of Boran cattle. Comp Haemat Int 1997;7(1):23–9.
R.C.N. Melo et al. / Cell Biology International 27 (2003) 853–861 Antunez MI, Cardoni RL. IL-12 and IFN-gamma production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections. Immunol Lett 2000;71(2):103–9. Brener Z, Gazzinelli RT. Immunological control of Trypanosoma cruzi infection and pathogenesis of Chagas’ disease. Int Arch Allergy Immunol 1997;114:103–10. Borges MM, Vassao R, Andrade SG, Pereira CA, Kloetzel JK. Interferon-Gama levels during the course of Trypanosoma cruzi infection of Calomys callosus (Rodentia-Cricetidae) and Swiss mice. Parasitol Res 1995;81:498–504. Celentano AM, Gonza´lez-Cappa SM. Induction of macrophage activation and opsoning antibodies by Trypanosoma cruzi subpopulations. Parasite Immunol 1992;14:155–67. El Shewemi S, Alshammary F, Alzamel F, Soliman R. Ultrastructure of resting and rh-GMCSF-treated human macrophages derived from blood monocytes. J Electron Microsc 1996;45(5):388–94. Hanson WL, Roberson EL. Density of parasites in various organs and the relation to numbers of trypomastigotes in blood during acute infections of Trypanosoma cruzi in mice. J Protozol 1974; 21(4):512–7. Jaramillo M, Olivier M. Hydrogen peroxide induces murine macrophage chemokine gene transcription via extracellular signalregulated kinase- and cyclic adenosine 5#- Monophosphate (cAMP)-dependent pathways: involvement of NF-B, activator protein 1, and cAMP response element binding protein. J Immunol 2002;169:7026–38. Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 1965;27:137A–8A. Mccabe RE, Mullins BT. Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. J Immunol 1990; 144:2384–8. Melo RCN. Depletion of immune effector cells induces myocardial damage in the acute experimental Trypanosoma cruzi infection: ultrastructural study in rats. Tissue Cell 1999;31(3):281–90. Melo RCN, Machado CRS. Depletion of radiosensitive leukocytes exacerbates the heart sympathetic denervation and parasitism in experimental Chagas’ disease in rats. J Neuroimmunol 1998; 84:151–7. Melo RCN, Machado CRS. Trypanosoma cruzi: peripheral blood monocytes and heart macrophages in the resistance to acute experimental infection in rats. Exp Parasitol 2001;97:15–23. Melo RCN, D’Avila H, Fabrino DL, Almeida PE, Bozza PT. Macrophage lipid body induction by Chagas’ disease in vivo: putative intracellular domains for eicosanoid formation during infection. Tissue Cell 2003;35(1):59–67. Mytar B, Siedlar M, Woloszyn M, Ruggiero I, Pryjma J, Zembala M. Induction of reactive oxygen intermediates in human monocytes by tumour cells and their role in spontaneous monocyte cytotoxicity. Br J Cancer 1999;79(5–6):737–43. Nathan CF. Secretion of oxygen intermediates: role in effector functions of activated macrophages. Fed Proc 1982;41:2206–11. Nathan C, Nogueira N, Juanghanich C, Ellis J, Cohn Z. Activation of macrophages in vivo and in vitro. J Exp Med 1979;149:1056–68. Nogueira N, Cohn ZA. Trypanosoma cruzi: in vitro induction of macrophage microbicidal activity. J Exp Med 1978;148:288–300. Petray P, Castanosvelez E, Grinstein S, Orn A, Rottenberg ME. Role of nitric-oxide in resistance and histopathology during experimental infection with Trypanosoma cruzi. Immunol Lett 1995; 47(1–2):121–6. Pick E, Mizel D. Rapid microssays for a the measurement of superoxide and hydrogen peroxide production by macrophages in culture using na automatic enzime immunoassay reader. J Immunol Methods 1981;46:211–26.
861
Reed SG, Nathan CF, Pihl DL, Rodricks P, Shanebeck K, Conlon PJ et al. Recombinant granulocyt/macrophage colony-stimulating factor activates macrophages to inhibit Trypanosoma cruzi and release hydrogen peroxide. Comparison with interferon . J Exp Med 1987;166:1734–46. Reth M. Hydrogen peroxide as second messenger in lymphocyte activation. 2002. Nat Immunol 2002;3(12):1129–34. Revelli S, Didoli G, Roggero E, Moreno H, Bernabo J, Wietzerbin J et al. Macrophage activity, IL-6 levels, antibody response and heart histology in rats undergoing an attenuated Trypanosoma cruzi acute infection upon treatment with recombinant interferon gamma. Cyt Cell Mol Ther 1998;4(3):153–9. Rodrigues MM, Ribeira˜o M, Boscardin SB. CD4 Th1 but not Th2 clones efficiently activate macrophages to eliminate Trypanosoma cruzi through a nitric oxide dependent mechanism. Immunol Lett 2000;73(1):43–50. Ropert C, Ferreira LRP, Campos MAS, Procopio DO, Travassos LR, Ferguson MAJ et al. Macrophage signaling by glycosylphatylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes. Microbes Infect 2002; 4:1015–25. Russo M, Starobinas N, Ribeiro dos Santos R, Minoprio PO, Hontebeyre-Joskowicz M. Susceptible mice present higher macrophage activation than resistant mice during infection with myotropic strains of Trypanosoma cruzi. Parasite Immunol 1989; 11:385–95. Saeftel M, Fleischer B, Hoerauf A. Stage-dependent role of nitric oxide in control of Trypanosoma cruzi infection. Infect Immun 2002;69(4):2252–9. Silva LHP, Nussenszweig V. Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Fol Clin Biol 1953;20:191–208. Silva JS, Vespa GNR, Cardoso MAG, Aliberti JCS, Cunha FQ. Tumor-Necrosis-Factor-Alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric-oxide production in infected gamma-interferon-activated macrophages. Infect Immun 1995;63(12):4862–7. Sun J, Tarleton RL. Predominance of CD8+ T lymphocytes in the inflammatory lesions of mice with acute Trypanosoma cruzi infection. Am J Trop Med Hyg 1993;48:161–9. Takemura T, Rom WN, Ferrans VJ, Crystal RG. Morphologic characterization of alveolar macrophages from subjects with occupational exposure to inorganic particles. Amn Rev Resp Dis 1989; 140:1674–85. Tanaka Y, Tanowitz H, Bloom BR. Growth of Trypanosoma cruzi in a cloned macrophage cell line and in a variant defective in oxygen metabolism. Infect Immun 1983;41(3):1322–31. Tanowitz HB, Kirchhoff LV, Simon D, Morris SA, Weiss LM, Wittner M. Chagas’ disease. Clin Microbiol Rev 1992;5(4):400–19. Turpin JA, Lopez-Berestein G. Differentiation, maturation, and activation of monocytes and macrophages: functional activity is controlled by a continuum of maturation. In: Lopez-Berestein G, Klostergaard J, editors. Mononuclear phagocytes in cell biology. Boca Raton (FL): CRC Press, 1993;71–99. Van Voorhis W. Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production. J Immunol 1992;148(1):239–48. Vespa GNR, Cunha FQ, Silva JS. Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. Infect Immun 1994;62(11):5177–82. Villalta F, Kierszenbaum F. Role of inflammatory cells in Chagas’ disease. II. Interactions of mouse macrophages and human monocytes with intracellular forms of Trypanosoma cruzi: uptake and mechanism of destruction. J Immunol 1984;133(6):3338–43.