participation of concanavalin a binding sites in the interaction

J. Cell Sri. 62, 287-299 (1983) 287Printed in Great Britain © The Company of Biologists Limited 1983

PARTICIPATION OF CONCANAVALIN A BINDINGSITES IN THE INTERACTION BETWEENTRYPANOSOMA CRUZI AND MACROPHAGES

M. N. L. MEIRELLESFundaqao Oswaldo Cruz, Avenida Brasil 4365, Rio de Janeiro, Brasil

A. MARTINEZ-PALOMOCentra 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 Universitaria,21910, Rio de Janeiro, Brasil

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 trypo-mastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA in-creased or decreased their uptake by macrophages, respectively. Treatment of the macrophages withConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypo-mastigotes, respectively. These results are discussed in relation to previous studies on the mobilityof ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA weobserved that ConA binding sites located on the plasma membrane of macrophages are internalizedduring endocytosis of T. cruzi, and observed in association with the membrane of the endocyticvacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, whilethese sites were distributed in patches in vacuoles containing parasites. These results, in associationwith others previously reported, suggest the involvement of glycoproteins and/or glycolipidslocalized on the cell surface of T. cruzi and macrophages during the T. em27-macrophage inter-action.

INTRODUCTION

Trypanosoma cruzi is a pathogenic protozoon that is able to infect different celltypes, including macrophages. On the basis of experiments in which the process ofendocytosis in the macrophages was blocked by treatment of the cells withcytochalasin B or by incubation at 4°C, it has been suggested that endocytosis is themain mechanism by which T. cruzi infects macrophages (Alexander, 1975; Nogueira& Cohn, 1976; Meirelles, Araiijo-Jorge & De Souza, 1982). Previous studies haveshown that the fate of the parasite within the macrophage depends on the develop-mental stage of the parasite that was endocytosed. While epimastigotes are destroyedinside the endocytic vacuole, trypomastigotes survive and, by an as yet unclarifiedmechanism, disrupt the membrane of the endocytic vacuole and enter in contact withthe cytoplasm of the macrophage (Nogueira & Cohn, 1976).

•Author for correspondence.

288 M. N. L. Meirelles and others

In the process of endocytosis the parasite first interacts with the surface of the hostcell. As in other cell-to-cell interaction processes, the surface properties of both cellsmay play a fundamental role in the interaction. Previous studies have shown thatepimastigote and trypomastigote forms of T. cruzi have different surface properties(De Souza et al. 1977; De Souza, Martinez-Palomo & Gonzales-Robles, 1978; Szarf-man, Queiroz & De Souza, 1980), and different kinetics of interaction withmacrophages (Meirelles, Araujo-Jorge & De Souza, 1980).

During endocytosis part of the plasma membrane of the macrophage is internalizedto form the membrane of the endocytic vacuole. Since T. cruzi, as well as otherintracellular parasites, interacts with the membrane of the endocytic vacuole it is ofinterest to determine which components of the plasma membrane of the macrophageare internalized and will be part of the membrane of the vacuole. In this paper wereport results that show the distribution of concanavalin A (ConA) binding sites of theplasma membrane of macrophages during endocytosis of T. cruzi, as well as the effectof ConA on the T. craai'-macrophage interaction.

MATERIALS AND METHODS

ParasitesThe 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 withthe Y strain. The blood was collected with 3-8 % sodium citrate as an anticoagulant and centrifugedat 150 g for lOmin, after which the tube was incubated at 37 °C for 30min so that some trypo-mastigotes could move from the pellet to the supernatant. The pellet was discarded and the super-natant fluid, which contained the parasites, was collected and centrifuged at 900^for lOmin. Thepellet was washed in Tyrode's solution (composition in g/1: NaCl, 8-0; KC1, 0'2; MgCb, 0*1;NaHCO3) 1-0; glucose, 1-0; NaH2PO4.H20, 005 ; CaCl2, 0-2).

MacrophagesPeritoneal 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 calfserum, and the macrophages removed from there were plated on 300 mm2 glass coverslips, whichhad been introduced into Leighton tubes. The cells were allowed to adhere to the glass surface for1 h at 37 °C, after which time the saline solution and the non-adherent cells were removed and culturemedium (199 medium plus 10% foetal calf serum) was added. After incubation at 37 °C for 24 hmacrophage cultures were rinsed with Ringer's solution (0-015 M-NaCl, 0 - 0 7 M - K C 1 , 0-05 M-CaCl2) and used for the experiments.

Macrophage-parasite interactionBloodstream 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 solutionin order to achieve a parasite concentration such that a ratio of 10 parasites/macrophage wasobtained when 025 ml of the suspension was added to the macrophage cultures. The number ofmacrophages in the preparations was estimated by counting 20 microscopic fields. This numbervaried from 500-1000 cells/mm2. The parasites were maintained in contact with the cells for periodsranging from 5 min to 2h, according to the experimental procedure, after which time the cultureswere rinsed with Ringer's solution to remove extracellular parasites and processed for light,fluorescence or electron microscopy.

Concanavalin AThe macrophage monolayer was rinsed twice with Ringer's solution at 4°C and incubated for

Interaction between Trypanosoma and macrophages 289

IS min at 4°C in a solution containing 100/ig/ml ConA (dissolved in Ringer's solution). Afterincubation the cultures were rinsed three times with Ringer's solution and incubated for IS min at4°C in a solution containing 50/<g/ml horseradish peroxidase, after which time the cultures wererinsed, incubated in the presence of parasites and maintained for 2 h at 37°C. After macrophage-T.cruzi interaction the cultures were washed twice and fixed for 30 min in 2-5 % glutaraldehyde in 0-1M-cacodylate buffer (pH 7-2). After fixation, the cultures were washed and incubated in a mediumcontaining 5 mg/ml diaminobenzidine, 001 % H2O2 in 0'05 M-Tris-HCl buffer (pH7 6) for 30 minat room temperature for detection of the peroxidase activity (Graham & Karnovsky, 1966). Afterincubation the cultures were rinsed and mounted in Permount to be observed by light microscopy.

Fluorescein—concanavalin ACoverslips bearing the macrophages were rinsed and incubated for 15 min at 4°C in the presence

of 17^g/ml of fluorescein-labelled ConA (Sigma Chemical Co.). After incubation the cultures wererinsed twice and incubated at 37 CC for periods varying from 5-120 min. The incubation wasinterrupted by fixation for 10 min in 10% formaldehyde (freshly prepared from paraformaldehyde)in PBS (pH 7-2), after which the cultures were rinsed and mounted over a drop of 10% glycerol inPBS and observed with a Zeiss photomicroscope. In some experiments the macrophages, previouslylabelled with ConA at 4°C, were incubated in the presence of T. cruzi.

Electron microscopyFor ultrastructural studies macrophages were plated in glass flasks, cultivated as described above,

and incubated with parasites. After interaction the cultures were fixed in 2-5 % glutaraldehyde in0-1 M-cacodylate buffer (pH7-2) with 3-5% sucrose, rinsed in cacodylate buffer with 3-5%sucrose, and left in this buffer for 24 h at 4°C. The cells were then gently scraped off with a rubberpoliceman, post-fixed with 1 % OsCs , dehydrated in acetone, and embedded in Epon. Thin sectionswere stained with uranyl acetate and lead citrate, and observed with a Jeol 100C-X electronmicroscope.

Concanavalin A-ferritinThe macrophage monolayer was rinsed at 4°C with Ringer's solution and incubated with

0-5 mg/ml ConA-ferritin (Sigma Chemical Co.) for IS min at 4 °C, after which the cells were rinsedagain with Ringer's solution. The ConA was dissolved in Ringer's solution. The interaction ofmacrophages with epimastigote and trypomastigote forms of T. cruzi was allowed to take place for2h at 37 °C. Following incubation of the macrophages with the parasites the cultures were rinsedtwice with Ringer's solution, fixed in 2-5 % glutaraldehyde in 0-1 M-cacodylate buffer (pH 7-2) for1 h and processed for electron microscopy as described previously. Thin sections were observedeither unstained or after staining with uranyl acetate and lead citrate.

Effect of concanavalin A on the T. cruzi-macrophage interactionMacrophages were cultivated on glass coverslips as described previously. Before interaction either

the macrophages or the parasites or both were washed and then incubated at 4°C for 20 min in thepresence of ConA (50^g/ml of Ringer's solution for the macrophages and 10^g/ml for theparasites), after which they were rinsed and left to interact for 2 h at 37 °C. After the interaction thecells were washed in PBS, fixed with Bouin's fixative and stained with May-Grunwald Giemsa. Thepercentage of infected macrophages was determined by examining at least 200 cells. Controlsconsisted of systems in which the cells were incubated in the absence of ConA or in the presence ofConA + lOmM-a-methyl-D-mannoside.

RESULTS

Macrophages previously fixed in glutaraldehyde and then incubated in the presenceof labelled ConA (with peroxidase, fluorescein or ferritin) showed a uniform


distribution of ConA receptors over the whole cell surface (Figs 1, 7). WhenConA-ferritin was used a uniform layer of ferritin particles was seen on the plasmamembrane (Fig. 7). The same distribution of ConA—ferritin particles was seen inliving cells incubated in the presence of the labelled lectin at 4°C before glutaral-dehyde fixation (not shown).

We did not observe the process of capping of the ConA receptors when livingmacrophages, labelled with ConA at 4°C, were incubated at 37°C. Most, if not all,of the lectin binding sites were internalized, appearing associated with the membraneof the endocytic vacuoles (Fig. 2). Observation of the vacuoles by electron microscopyshowed that the ConA binding sites were uniformly distributed on the membrane ofthe endocytic vacuoles (Fig. 8).

Macrophages incubated at 4°C in the presence of ConA, washed and then in-cubated at 37°C in the presence of T. cntzi, were able to ingest the parasites (Figs 3,5). We observed that the ConA binding sites were internalized during the process ofendocytosis of epimastigote and trypomastigote forms of T. cruzi (Figs 4, 6). How-ever, the pattern of distribution of the ConA binding sites in the membrane of endo-cytic vacuoles containing parasites differed from those vacuoles that did not containparasites. As shown in Figs 9-11, ConA binding sites appeared uniformly distributedin the membrane of vacuoles without parasites, while in those containing parasites theConA binding sites appeared in clusters.

Concanavalin A interfered with the kinetics of ingestion of T. cruzi by macrophages(Fig. 12). However, its interference varied according to the developmental stage ofthe parasite and according to whether parasite or host cell was incubated in thepresence of the lectin before the parasite-macrophage interaction. Usually the per-centage of macrophages that ingested epimastigotes was higher than that ofmacrophages that ingested trypomastigotes of T. cruzi. Incubation of epimastigotesin the presence of unlabelled ConA (at a concentration that did not induce the forma-tion of clumps of parasites) before the interaction with macrophages, doubled thepercentage of macrophages that ingested the parasites. Incubation of bloodstreamtrypomastigotes under the same conditions did not change the percentage ofmacrophages that ingested them. Incubation of the macrophages in the presence ofunlabelled ConA before interaction with T. cruzi reduced by about 70%, and in-creased by about five times, the percentage of macrophages that ingested epimastigote

Fig. 1. Macrophages fixed in glutaraldehyde and then incubated in the presence offluorescein-labelled ConA. ConA binding sites are distributed evenly over the cell surface.X1600.

Fig. 2. Macrophages labelled with fluorescein-ConA at 4°C and then incubated for10 min at 37 °C. Most of the lectin binding sites were internalized, appearing in associationwith cytoplasmic vesicles. X1600.Figs 3-6. Macrophages first labelled with fluorescein-ConA and then incubated in thepresence of T. cruzi at 4°C, after which they were incubated for 15 min at 37 °C and fixed.The same cells are observed by phase contrast (Figs 3, 5) and immunofluorescence (Figs4, 6). The arrows indicate vacuoles containing parasites. Fluorescence, indicative of thepresence of ConA binding sites, is seen at the periphery of the vacuoles. X 1600.


Figs 1-6


ev

Fig. 7. Macrophage fixed in glutaraldehyde and then incubated in the presence of ferritin-labelled ConA. Ferritin particles are seen homogeneously distributed over the cell surface(arrow), m, macrophage. X33 200.

Fig. 8. Macrophage labelled with ferritin-ConA at 4°C and then incubated at 37 °C for30min. All particles were internalized, appearing homogeneously distributed in associa-tion with the membrane of endocytic vacuoles (see small arrow) inside the vesicle. Noferritin particles are seen on the macrophage surface (large arrow), ev, endocytic vacuole.X 20 000.


and trypomastigote forms, respectively. When both the parasites and themacrophages were incubated in the presence of the lectin before interaction, adecrease in the percentage of macrophages that ingested epimastigotes and an increasein the percentage of macrophages that ingested trypomastigotes were observed.

In all conditions in which either the macrophages or the parasites were incubatedin the presence of ConA, binding of the lectin to the cell surface occurred, as couldbe visualized by light and electron microscopy. The effect of ConA on the kinetics ofinteraction between T. cruzi and macrophages was considered to be specific, since itwas completely abolished if a-methyl-D-mannoside was added to the incubationmedium.

DISCUSSION

Previous experiments have shown that treatment of macrophages with cytochalasinB or their incubation at 4°C almost completely blocks the ingestion of epimastigoteand trypomastigote forms of T. cruzi by the macrophage (Alexander, 1975; Nogueira& Cohn, 1976; Meirelles et al. 1982). As in other processes of endocytosis, it was alsopossible to distinguish two steps in the process of incorporation of T. cruzi bymacrophages: attachment and ingestion (Meirelles et al. 1982).

In all processes of endocytosis the particle to be ingested interacts first with thesurface of the macrophage. As a consequence of this interaction, complex changes inthe organization of the cytoskeleton and the plasma membrane components occur,leading to the internalization of the particle, which then appears surrounded by amembrane that originated from the plasma membrane of the macrophage. One impor-tant point to be clarified concerns the similarities or differences that exist between theplasma membrane of the macrophage and the membrane of the endocytic vacuole.Since the plasma membrane is fluid, it is possible that only some of its componentsare internalized while others remain in the plasma membrane. We cannot exclude thepossibility that the nature of the components that are internalized during endocytosisis determined by the surface properties of the particle to be ingested. In view of thefact that many intracellular parasites interact with the membrane of endocyticvacuoles, at least during part of their life-cycle, it is important to know more aboutthe structure and composition of this membrane. In the case of T. cruzi this isespecially interesting, since the trypomastigote form of this parasite has developed thecapacity to penetrate the membrane of the endocytic vacuole, allowing the parasite tomultiply in direct contact with the cytoplasm of the host cell (Nogueira & Cohn,1976).

Our results indicate that ConA binding sites located on the plasma membrane ofmacrophages are internalized during the process of endocytosis of epimastigotes andtrypomastigotes of T. cruzi. Our observations on cells incubated in the presence of thelectin at 4°C or after glutaraldehyde fixation indicated, as already shown by others(Bar-Shavit & Goldman, 1976; Walter, Berlin, Pfeiffer & Oliver, 1980), thatmacrophages have ConA binding sites uniformly distributed throughout the cellsurface. When macrophages previously labelled with ConA were incubated at 37 °C


Figs 9-11

Interaction between Trypanosoma and macrophages100

90

(0

I 80si

295

a.2

70

'c"oa>a>2auID

Q . .

60

5 0

40

30

20

10Epimastigote Trypomastigote

Fig. 12. Effect of ConA on the endocytosis of epimastigote and trypomastigote forms ofT. cntzi by macrophages. Either the parasites or the macrophages or both were incubatedin the presence of ConA before interaction. After 2 h of interaction the cultures were fixed,stained with Giemsa and observed in the light microscope. ( • ) control; (S) macrophageincubated in the presence of 50^g/ml ConA before interaction; (BE) parasites incubatedin the presence of ConA before interaction; (W) both macrophages and parasites incubatedin the presence of ConA before interaction.

an intense process of endocytosis took place and all the ConA could be visualized inthe cytoplasmic vacuoles. These results are in agreement with those of other authors(Walter el al. 1980), who did not find capping of ConA receptors on the surface ofmacrophages, as usually occurs with other cells.

Fluorescence, which is indicative of the presence of ConA binding sites interiorizedduring endocytosis, was also observed in those endocytic vacuoles that containedparasites. However, when ferritin-labelled ConA was used, we could see that vacuoles

Figs 9-11. Macrophages first labelled with ferritin-ConA at 4 °C, incubated with T. cruziat 4°C and then incubated for 30min at 37°C, after which the cultures were fixed. Theferritin particles (arrows in Figs 10, 11) associated with the membrane of vacuoles withoutparasites are uniformly distributed, showing a patchy distribution in those vacuoles thatcontain parasites. Fig. 10 is a high magnification of the area marked with an asterisk in Fig.9, showing in detail the membranes of two different vacuoles. hen, host cell nucleus; p,parasite; pvc, parasite-containing vacuole. Arrow in Fig. 9 indicates absence of ferritinparticles on the macrophage surface. Fig. 9, X13200; Figs 10, 11, X60000.


without parasites showed a uniform distribution of ConA binding sites, while in thosevacuoles containing parasites the ConA binding sites were distributed in clusters.This result indicates that the distribution of the ConA receptors was changed eitherduring or after the process of endocytosis of T. cruzi. It is possible that the clustersare formed as a consequence of the interaction of surface components of the parasitewith the surface of the macrophage. Membrane-associated components of the parasitewould act as a second ligand when interacting with the ConA bound to its receptorson the plasma membrane of the macrophage. Although a large number of sectionswere examined we were not able to detect the exact moment at which the clusters ofConA receptors appear in the membrane of the endocytic vacuole.

Previous studies have shown that during the process of endocytosis plasma-membrane transport carriers are selectively retained in the plasma membrane and arenot observed in the membrane of the endocytic vacuole (Tsan & Berlin, 1971). It wasreported that the binding of [125I]ConA to the surface of macrophages was reducedby 25-30% immediately after endocytosis of latex beads (Lulton, 1973). These dataare in accordance with our cytochemical observations, which show that ConA recep-tors are internalized, thus explaining the decrease in the number of these sites on thecell surface. Freeze-fracture studies have also shown the existence of differencesbetween the plasma membrane of Dictyostelium discoideum and the membrane of itsendocytic vacuoles formed after ingestion of yeast or latex beads (Favard-Sereno,Ludosiky & Ryter, 1981).

Our results show that the incorporation of epimastigote and trypomastigote formsof T. cruzi by macrophages is affected by previous incubation of either the protozoonor the macrophage with ConA. It is well known that ConA interacts with receptor sitescontaining a-mannopyranose-like residues (Goldstein, Hollerman & Smith, 1965;Lis & Sharon, 1973), which exist in both T. cruzi (Alves & Colli, 1974; Chiari et al.1978; Pereira, Loures, Villalta & Andrade, 1980; Araiijo, Handman & Remington,1980) and macrophages (Bar-shavit & Goldman, 1976; Walter et al. 1980). It hasbeen shown that incubation of yeast cells (Bar-shavit & Goldman, 1976) or thetrypanosomatidHerpetomonas samuelpessoai (Oda, Alviano, Angluster & De Souza,1982) with ConA increases their uptake by macrophages. Similar results are obtainedif the macrophages are incubated in the presence of the lectin before interaction withthese two cell types. In the case of T. cruzi, however, the influence of ConA on theinteraction between the parasite and the macrophages was more complex than thatwhich occurs with yeast cells or with H. samuelpessoai. While incubation of epimas-tigotes with ConA increased their incorporation by the macrophages, ConA did notincrease the ingestion of trypomastigotes. Previous studies have shown that bothepimastigote and trypomastigote forms of T. cruzi have ConA binding sites on theircell surface as could be observed by ConA-induced agglutination and localization ofConA binding sites by light and electron microscopy (AJves & Colli, 1974; Chiari etal. 1978; Pereira et al. 1980; Araiijoe^a/. 1980). These studies indicated that T. cruzihas ConA binding sites homogeneously distributed throughout the cell surface. Whenliving trypomastigotes are labelled with ConA at 4°C and then incubated at 37 °Cthere is aggregation of the ConA binding sites, which form a cap, thus indicating that


the ConA receptors are mobile in the plasma membrane. In contrast, capping was notobserved when similar experiments were carried out with epimastigotes (Szarfmanefal. 1980). Similar results were observed when antibodies against T. cruzi surfaceantigens were used (Schmunis, Szarfman, De Souza & Langenbach, 1980). It ispossible that the increase in the ingestion of epimastigotes of T. cruzi covered byConA is the result of an opsonizing effect of the lectin. According to the zipperhypothesis for receptor-mediated endocytosis, the whole particle should be coveredwith the opsonin, which then interacts with the receptors localized on the surface ofthe macrophage (Griffin, Griffin, Lieber & Silverstein, 1975). Since the ConA bind-ing sites on the surface of trypomastigotes are mobile, a process of endocytosismediated by ConA receptors would not take place, thus explaining our results, whichdid not show any effect of ConA on the uptake of trypomastigotes. On the other hand,incubation of macrophages only in the presence of ConA before interaction with theparasites increased considerably the endocytosis of epimastigotes. These results can-not be explained, as has been pointed out for other systems (Bar-shavit & Goldman,1976; Oda et al. 1982), in terms of a stimulation in the process of macrophageendocytosis by ConA. Indeed, ConA induces vacuolization of the macrophages(Goldman, 1976). However, if this is the case we would expect an increase in theuptake of both epimastigote and trypomastigote forms. We cannot exclude thepossibility that the endocytosis induced by ConA leads to a decrease in the numberof some, as yet undetermined, component of the macrophage's plasma membraneinvolved in the ingestion of epimastigotes. In the case of trypomastigotes it is interest-ing to note that the increase observed by us in their uptake by ConA-treatedmacrophages is similar to that observed when the parasites are incubated in thepresence of trypsin (Kipnis et al. 1981; Nogueira, Chaplan & Cohn, 1980) orneuraminidase (Araiijo Jorge & De Souza, 1981) before interaction. It is possible thatConA blocks some component localized on the surface of the macrophage that recog-nizes the anti-phagocytic component localized on the surface of trypomastigotes of T.cruzi, which is removed by mild enzymic treatment. Further studies are necessary totest this possibility.

In conclusion, our results suggest that glycoproteins and/or glycolipids localizedon the cell surfaces of T. cruzi and macrophages may play some role in their inter-action. They also indicate that differences between epimastigote and trypomastigoteforms in the mobility of plasma membrane components may affect the process ofendocytosis. These data, which indicate participation of glycoproteins and glycolipidsin the T. cruzi— host cell interaction, are in accordance with those that show thatinfection of non-professional phagocytic cells by T. cruzi trypomastigotes can beinhibited by some lectins (Henriquez, Piras & Piras, 1981) and some monosac-charides, mainly Af-acetyl-glucosamine (Colli, Andrews & Zingales, 1981; Crane &Dvorak, 1982). We also show that during the process of endocytosis of T. cruzi ConAreceptors localized in the plasma membrane of the macrophage are internalized toform part of the membrane of the endocytic vacuole.

This work has been supported by UNDP/World Bank/WHO Special Programm for Researchand Training in Tropical Diseases, the Rockefeller Foundation, CNPq, FINEP and CEPG-UFRJ.


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{Received 11 August 1982-Accepted 31 January 1983)

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participation of concanavalin a binding sites in the interaction