Trypanosoma cruzi: Phagolysosomal Fusion after Invasion into Non Professional Phagocytic Cells

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1 CELL STRUCTURE AND FUNCTION 12, (1987) c by Japan Society for Cell Biology Trypanosoma cruzi: Phagolysosomal Fusion after Invasion into Non Professional Phagocytic Cells Maria de Nazareth Leal de Meirelles, Tania C. de Araujo Jorge, Wanderley de Souza*, Andre Luiz Moreira and Helene Santos Barbosa Departmento de Ultraestrutura e Biologia Celular, Institute Oswaldo Cruz, Fundacao Oswaldo Cruz, Av. Brasil 4365-Manguinhos, Rio de Janeiro-R.J , Brasil and *Laboratorio de Ultraestrutura Celular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, R.J.-Brasil ABSTRACT. Parasite-containing endocytic vacuoles are formed during the process of in vitro interiorization of the trypomastigote forms of Trypanosoma cruzi by primary culture of mouse fibroblasts, heart and skeletal muscle cells. Fusion of these vacuoles with host cell lysosomes takes place. The process of T. cruzi-muscle cell interaction was analysed by ultrastructural cytochemistry. Two lysosomal enzymes, acid phosphatase and aryl sulphatase and the fusion of peroxidase-labeled secondary lysosomes with the parasitophorus vacuoles were studied. These finding indicate that the basic mechanism of interaction of T. cruzi with the so called non phagocytic cells is similar to that which occurs with phagocytic cells. Chagas' disease affects about 12 million people in Latin America, and its causative agent, Trypanosoma cruzi, infects preferentially the heart muscle tissue (19). The mechanism by which T. cruzi infects non professional phagocytic cells** is unknown. Previous studies in vitro with macrophages have shown that endocytosis is the main process of interiorization of T. cruzi and that secondary lysosomes fuse with parasitecontaining endocytic vacuoles after T. cruzi interiorization (3, 8, 11, 15, 18). Our recent observation in vitro on the interaction of the trypomastigote form of T. cruzi with heart mouse muscle cells show that after penetration the parasite is found within a membrane bounded vacuole (14). In this paper we will present some cytochemical evidence showing that the lysosomes of the muscle cells and fibroblasts fuse with parasite-containing endocytic vacuoles, a fact that supports the idea of endocytosis as a mechanism of T. cruzi invasion also in non professional phagocytic cells. MATERIALS AND METHODS Primary heart and skeletal muscle cell cultures. Hearts from eighteen days old mouse embryos were dissected, minced and incubated for 10 min at 37 Ž in 0.05 % Trypsin (DIFCO) and 0.01 % Collagenase (Worthington) following the experimental protocol for heart muscle cell (HMC) cultures already described (14). Skeletal muscle cell (SMC) cultures ** This term is used in view of the recent observations showing that many cells can ingest large particles if appropriately stimulated (6). 387

2 388 M. de N.L. de Meirelles et al. were prepared using embryo thigh muscle as described elsewhere (2). The fibroblasts used in the experimental assay are obtained from the first plating of our primary heart or skeletal muscle cell cultures. Parasites. Bloodstream trypomastigotes of the Y and Colombiana strains were obtained at the peak of parasitemia (7 and 20 days, respectively). Metacyclic trypomastigotes of the Dm 28c clone was used in some experiments (4). Host cell-t. cruzi interaction. Parasites were suspended in Dulbecco's Modified Eagle Medium in order to achieve a parasite concentration such that a ratio of 10 parasite/cell was obtained when 0.25 ml of the suspension was added to the host cell culture. The parasites were maintained in contact with the cells for periods varying from 2 to 18 h. After interaction the cultures were rinsed with Ringer's solution to remove extracellular parasites and processed for electron microscopy. Transmission electron microscopy. The infected cultures were fixed either in 2.5 % glutaraldehyde (GA) or 1 % paraformaldehyde (PFA)+1 % GA in 0.1 M Na cacodylate buffer, ph 7.2, for 1 h or 15 min respectively, rinsed in the same buffer, and left overnight at 4 Ž. The cells were then gently scraped off with a "rubber policeman", collected by centrifugation, post-fixed with 1 % 0s04, dehydrated in acetone and embedded in Epon. Sections were observed either unstained or stained with uranyl acetate and lead citrate, in a EM 10B Zeiss Microscope. Ultrastructural cytochemistry: Horseradish peroxidase. (E.C )-(HRP)-For ultrastructural studies HMC, SMC or fibroblasts were incubated in the presence of 2 mg/ml HRP (Sigma, Type II) for 2 h at 37 Ž after which the cells were rinsed with PBS and then incubated with parasites. After parasite-host cell interaction, they were fixed in 2.5 % GA on 0.1 M Na cacodylate buffer ph 7.2, rinsed and left in this buffer for 24 h at 4 Ž. The cells were then incubated for 30 min at room temperature in a medium containing 3,3'- diaminobenzidine (0.5 mg/ml) in 0.05 M Tris (hydroxymethyl) aminomethane-hcl buffer, ph 7.6 and 0.01 % H2O2 for detection of peroxidase activity (5), following routine procedure for transmission electron microscopy. Lysosomal enzymes: Acid phosphatase. (E.C )-After parasite-host cell incubation the cells were fixed in 1 % PFA +1 % GA for 20 min at 4 Ž, washed with buffer and incubated in the medium described by Ryters & Bowers (17) with the following composition : 0.05 M acetate buffer, ph 5, 0.2 mm CaCl2, 11.5 mm Na-ƒÀ-glycerophosphate, 1 mm cerium hloride. In controls, cells were incubated in a medium (a) without the substrate cor (b) containing 10 mm NaF. Aryl Sulphatase. (E.C )-The same process for fixation was used and after that the cells were washed and incubated in a modified medium described by Hopsu-Havu (7) containing : 0.1 M Acetate buffer, ph 6.0, 5 mm p-nitrocathecolsulphate and 1 mm cerium chloride. The control was as in (a) above. Fig. 1. Adhesion of a metacyclic trypomastigote form of Dm 28c clone of T. cruzi to skeletal muscle cell before cellular invasion (arrow). Bar-0.2 ƒêm. In all the figures the value of the Bar is 0.2 ƒêm. Fig. 2. A typical trypomastigote form of T. cruzi is seen within an endocytic vacuole of a muscle cell after 18 h of infection. Note the membrane of the endocytic vacuole (thick arrow) and the membrane of the parasite (thin arrow). Fig. 3. Lysosomes of an skeletal muscle cell containing an electron dense deposit indicative of aryl sulphatase activity are seen in the cell's cytoplasm (arrows). Fig. 4. A typical heart muscle fiber exhibiting sarcomere with Z line (Z), figures of sarcoplasmatic reticulum (SR), T Tubule (t). Small vesicles near the plasma membrane present a positive aryl sulphatase reaction (arrow). Fig. 5. Parasitized skeletal muscle cell with the Y strain of T. cruzi show vesicles containing acid phosphatase. A small vesicle is seen in close proximity of the parasite (arrow); parasite (p).

3 T. cruzi: Phagolysosomal Fusion in Muscle 389

4 390 M. de N.L. de Meirelles et al. Fig. 6. Cytochemical localization of acid phosphatase within a vacuole of a heart muscle cell containing interiorized trypomastigotes forms of T. cruzi. (P) strain Colombiana (arrows). Fig. 7. A phagosome containing a trypomastigote form of Dm 28c clone of T. cruzi in a fibroblast show a strong electron dense reaction positive to aryl sulphatase activity (arrow). P-parasite Fig. 8. Fusion of peroxidase-labeled vacuoles with T. cruzi-(strain Colombiana) endocytic vacuoles in the heart muscle cell. An electron dense reaction is seen inside the phagosome (asterisks) and in vesicles located in cell's cytoplasm (asterisks). P-parasite, thick arrow-membrane of the endocytic vacuole, thin arrow-membrane of the parasite.

5 T. cruzi: Phagolysosomal Fusion in Muscle 391 RESULTS During the process of T. cruzi-host cell interaction the parasite is initially attached to the cell surface (Fig. 1) and is then interiorized, remaining for some time within a membrane-bounded cytoplasmic vacuole (Fig. 2). The morphological observations made using parasites from three different strains and three different host cells (skeletal and heart muscle cells and fibroblasts) were basically the same. As described previously however, the kinetics of parasite-host cell interaction varied according to the parasite strain and the host cells (2, 14). Electron dense reaction product indicative of the presence of aryl sulphatase was found in between the myofibers in skeletal muscle cells (Fig. 3), and in cytoplasmic vesicles located at the periphery of heart muscle cell (Fig. 4). Infected skeletal muscle cells showed the presence of a positive vacuole of acid phosphatase activity located near the endocytic vacuole which contained interiorized parasites (Y strain of T. cruzi) (Fig. 5). In many cases a strong reaction product indicative of acid phosphatase activity was seen within the endocytic vacuoles surrounding the parasites (Colombiana strain of T. cruzi) in heart muscle cells (Fig. 6). Aryl sulphatase activity was also found within endocytic vacuoles of a fibroblast which contained the trypomastigote form of T. cruzi (Dm 28c clone) (Fig. 7). Incubation of the host cell in the presence of horseradish peroxidase led to the ingestion of this macromolecule by the cells where it was concentrated in cytoplasmic vesicles and vacuoles similar to those which showed acid phosphatase or aryl sulphatase activity. Reaction product, indicative of peroxidase activity, was seen within parasite-containing endocytic vacuoles of cells labeled with peroxidase before incubation in the presence of parasites. (Fig. 8). All the controls showed no positive reaction. DISCUSSION At present there is much evidence showing that endocytosis is the mechanism by which the trypomastigote forms of T. cruzi infects macrophages (9, 10, 16). In the case of other cells, however, there is controversy. Our previous observations on the process of interaction of T. cruzi with heart muscle cell in vitro, which are the cells most frequently infected by T. cruzi in the vertebrate host (1) showed clearly that immediately after interiorization, the trypomastigote forms of T. cruzi were found within a membrane-bounded cytoplasmic vacuole (3). This vacuole resembles that found in macrophages. As already shown, the cytochemical characterization of plasma membrane enzymatic markers such as (a) adenylate cyclase, (b) Ca++Mg++ ATPase and (c) anionic sites labelled with cationized ferritin gave similar results. These components were neither found in the membrane which lines the endocytic vacuole in macrophages nor in muscle cells (12, 13, 14). In our opinion these observations indicate that T. cruzi is interiorized by muscle cells and fibroblasts through a process of endocytosis. Results obtained in recent years show that even non professional phagocytic cells can ingest large particles provided that they stimulate a receptor-mediated endocytic process (6). Our present observations show, for the first time in non phagocytic cells, that reaction product indicative of enzymatic activity of aryl sulphatase and acid phosphatase, two classical lysosomal markers, can be seen within the vacuole which

6 392 M. de N.L. de Meirelles et al. contain the interiorized parasites. This observation strongly suggests that, as previously described with phagocytic cells, the lysosomes found in muscle cells also fuse with endocytic vacuoles containing T. cruzi. Recent studies (18) have shown that after an 18 h uptake period, horseradish peroxidase was found in lysosomes of chinese hamster ovary cells (CHO) by cell fractionation in Percoll gradients and by cytochemistry. Labelling lysosomes of muscle cells and fibroblasts with horseradish peroxidase and following the infection of these cells with T. cruzi for periods up to 18 h we could confirm that muscle cell lysosomes discharge their content within the endocytic vacuoles. The mechanisms by which T. cruzi resists the lysosomal enzyme attack, as well as how the parasites are released from the vacuole to the cytoplasm, remain to be elucidated both for macrophage and for non professional phagocytic cells. In conclusion, the cytochemical observation reported in this paper in association with those reported previously strongly suggests that T. cruzi is interiorized into non phagocytic cells by a process of endocytosis and that lysosomes found in these cells fuse with the parasite-containing vacuoles. Acknowledgments. The authors thank Dr. Samuel Goldenberg for providing the Dm 28c of T. cruzi, Drs. H. Meyer and H. Momem for reading the manuscript, and Mrs. Maria Jose Couto for secretarial assistance. This work was supported by Grants from UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and FINEP. REFERENCE 1. ANDRADE, S.G. Caracterizagdo de cepas de Trypanosoma cruzi isoladas do Reconcavo Baiano. Tese 129 p. Universidade Federal da Bahia, ARAUJO JORGE, T.C., H.S. BARBOSA, A.L. MOREIRA, W. DE SOUZA and M.N.L. MEIRELLES. On the interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle. Z. Parasitenkd 72, , CARVALHO, R.G.M., M.N.L. MEIRELLES, W. DE SOUZA and W. LEON. Isolation of the intracellular stage of Trypanosoma cruzi and its interaction with mouse macrophages in vitro. Infect. Immun. 33, , CONTRERAS, V.T., J.M. SALLES, N. THOMAS, C.M. MOREL and S. GOLDENBERG. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol. Biochem. Parasitol. 16, , GRAHAM, R.C. and M.J. KARNOVSKY. The early stages of absortion of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new techniques. J. Histochem. Cytochem. 14, , GRINNEL, F. Fibroblast spreading and phagocytosis : similar cell responses to different-sized substrates. J. Cell Physiol. 119, 58-64, HOPSU-HAVU, V.K., A.U. ARSTILA, H.J. HELMINER, 0. KALIMO HANNU and G.G. GLENNER. Improvements in the method for the electron microscopic localization aryl sulphatase activity. Histochemie 8, 54-64, KRESS, Y., B. BLOOM, M. WITTNER, A. ROWIN and H. TANOWITZ. Resistence of Trypanosoma cruzi to killing by macrophages. Nature 357, , MARIA, T.A., A. ALCANTARA and Z. BRENER. Ultrastructural studies on the in vitro interaction of Trypanosoma cruzi bloodstream forms and mouse peritoneal macrophages. Acta Trop. 39, , MEIRELLES, M.N.L., T.C. ARAUJO JORGE and W. DE SOUZA. Interaction of Trypanosoma cruzi with macrophages in vitro: dissociation of the attachment and internalization phases by

7 T. cruzi: Phagolysosomal Fusion in Muscle 393 low temperature and Cytochalasin B. Z. Parasitenkd 68, 7-14, MEIRELLES, M.N.S.L. and W. DE SOUZA. Interaction of lysosomes with endocytic vacuoles in macrophages simultaneously infected with Trypanosoma cruzi and Toxoplasma gondii. J. Submicrosc. Cytol. 15, , MEIRELLES, M.N.L., T. SOUTO-PADRON and W. DE SOUZA. Participation of cell surface anionic sites in the interaction between Trypanosoma cruzi and macrophages. J. Submicrosc. Cytol. 16, , MEIRELLES, M.N.L. and W. DE SOUZA. The fate of plasma membrane macrophage enzyme markers during endocytosis of Trypanosoma cruzi, J. Submicrosc. Cytol. 18, , MEIRELLES, M.N.L., T.C. ARAUJO JORGE, C.F. MIRANDA, W. DE SOUZA and H.S. BARBOSA. Interaction of Trypanosoma cruzi with heart muscle cells: Ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro. Eur. J. Cell Biol. 41, , MILDER, R.T. and J. KLOETZEL. The development of Trypanosoma cruzi in macrophages in vitro. Interaction with lysosomes and host cell fate. Parasitol. 80, , NOGUEIRA, N. and Z. COHN. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cell. J. Exp. Med. 143, , RYTERS, A. and B. BOWERS. Localization of acid phosphatase in Acanthamoeba castellanii with light and electron microscopy during growth and after phagocytosis. J. Ultrastruct. Res. 57, , STORRIE, B., M. SACKDEVA and V.S. VIERS. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins. Mol. Cell Biol. 4, , ZINGALES, B. and W. COLLI. Trypanosoma cruzi: Interaction with host cells. Curr. Top. Microbiol. Immunol. 171, , 1985 (Received for publication, May 13, 1987)