main mechanism by which T. cruzi infects macrophages (Alexander, 1975; Nogueira. & Cohn, 1976 ... had been introduced into Leighton tubes. The cells were ...
J. Cell Sri. 62, 287-299 (1983)
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PARTICIPATION OF CONCANAVALIN A BINDING SITES IN THE INTERACTION BETWEEN TRYPANOSOMA CRUZI AND MACROPHAGES M. N. L. MEIRELLES Fundaqao Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, Brasil
A. MARTINEZ-PALOMO Centra de Investigation e de Estudos Avanzados del IPN, Mexico
T. SOUTO-PADRON AND W. De SOUZA* Instituto de Biofisica, Universidade Federal do Rio de Janeiro, ddade 21910, Rio de Janeiro, Brasil
Universitaria,
SUMMARY Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of the macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. em27-macrophage interaction.
INTRODUCTION
Trypanosoma cruzi is a pathogenic protozoon that is able to infect different cell types, including macrophages. On the basis of experiments in which the process of endocytosis in the macrophages was blocked by treatment of the cells with cytochalasin B or by incubation at 4°C, it has been suggested that endocytosis is the main mechanism by which T. cruzi infects macrophages (Alexander, 1975; Nogueira & Cohn, 1976; Meirelles, Araiijo-Jorge & De Souza, 1982). Previous studies have shown that the fate of the parasite within the macrophage depends on the developmental stage of the parasite that was endocytosed. While epimastigotes are destroyed inside the endocytic vacuole, trypomastigotes survive and, by an as yet unclarified mechanism, disrupt the membrane of the endocytic vacuole and enter in contact with the cytoplasm of the macrophage (Nogueira & Cohn, 1976). •Author for correspondence.
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In the process of endocytosis the parasite first interacts with the surface of the host cell. As in other cell-to-cell interaction processes, the surface properties of both cells may play a fundamental role in the interaction. Previous studies have shown that epimastigote and trypomastigote forms of T. cruzi have different surface properties (De Souza et al. 1977; De Souza, Martinez-Palomo & Gonzales-Robles, 1978; Szarfman, Queiroz & De Souza, 1980), and different kinetics of interaction with macrophages (Meirelles, Araujo-Jorge & De Souza, 1980). During endocytosis part of the plasma membrane of the macrophage is internalized to form the membrane of the endocytic vacuole. Since T. cruzi, as well as other intracellular parasites, interacts with the membrane of the endocytic vacuole it is of interest to determine which components of the plasma membrane of the macrophage are internalized and will be part of the membrane of the vacuole. In this paper we report results that show the distribution of concanavalin A (ConA) binding sites of the plasma membrane of macrophages during endocytosis of T. cruzi, as well as the effect of ConA on the T. craai'-macrophage interaction. MATERIALS
AND
METHODS
Parasites The Y strain of T. cruzi was used. Epimastigotes were obtained at the Sth day of cultivation in Warren's medium. Bloodstream trypomastigotes were obtained from mice infected for 7 days with the Y strain. The blood was collected with 3-8 % sodium citrate as an anticoagulant and centrifuged at 150 g for lOmin, after which the tube was incubated at 37 °C for 30min so that some trypomastigotes could move from the pellet to the supernatant. The pellet was discarded and the supernatant fluid, which contained the parasites, was collected and centrifuged at 900^for lOmin. The pellet was washed in Tyrode's solution (composition in g/1: NaCl, 8 - 0; KC1, 0'2; MgCb, 0*1; NaHCO 3 ) 1-0; glucose, 1-0; NaH 2 PO 4 .H 2 0, 0 0 5 ; CaCl2, 0-2).
Macrophages Peritoneal macrophages were collected from Swiss mice weighing 20-25 g. Animals were killed with ether, their peritoneal cavities were washed with 4 ml of Hanks' solution plus 5 % foetal calf serum, and the macrophages removed from there were plated on 300 mm2 glass coverslips, which had been introduced into Leighton tubes. The cells were allowed to adhere to the glass surface for 1 h at 37 °C, after which time the saline solution and the non-adherent cells were removed and culture medium (199 medium plus 10% foetal calf serum) was added. After incubation at 37 °C for 24 h macrophage cultures were rinsed with Ringer's solution (0-015 M-NaCl, 0 - 0 7 M - K C 1 , 0-05 MCaCl2) and used for the experiments.
Macrophage-parasite interaction Bloodstream trypomastigotes as well as epimastigotes of the Y strain were suspended in phosphate-buffered saline (PBS; 0-01 M-phosphate buffer plus 0-15M-NaCl) or Ringer's solution in order to achieve a parasite concentration such that a ratio of 10 parasites/macrophage was obtained when 025 ml of the suspension was added to the macrophage cultures. The number of macrophages in the preparations was estimated by counting 20 microscopic fields. This number varied from 500-1000 cells/mm 2 . The parasites were maintained in contact with the cells for periods ranging from 5 min to 2h, according to the experimental procedure, after which time the cultures were rinsed with Ringer's solution to remove extracellular parasites and processed for light, fluorescence or electron microscopy.
Concanavalin A The macrophage monolayer was rinsed twice with Ringer's solution at 4°C and incubated for
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IS min at 4°C in a solution containing 100/ig/ml ConA (dissolved in Ringer's solution). After incubation the cultures were rinsed three times with Ringer's solution and incubated for IS min at 4°C in a solution containing 50/
