Anionic site behavior in and its role in the parasite-macrophage interaction

E. M. B. SARAIVA1'2, M. A. VANNIER-SANTOS1, F. C. SILVA-FILHO1 and W. DE SOUZA1'*

' Laboratdrio de Ullraestnitura Celular, Institute de Biofisica Carlos Chagas Filho and zDepartamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Ilha do Fundao, 21941, Rio de Janeiro, Brasil

* Author for correspondence

Summary

The behavior of cationized ferritin (CF) binding of non-infective promastigotes was decreased by sites on the surface of Leishmania mexicana ama- 26%, while once again the other parasite forms zonensis (amastigotes, infective and non-infective 'were not significantly affected. Transmission elec- promastigotes) and their participation in the inter- tron microscopy of mouse peritoneal macrophage action with macrophages were evaluated. Glutaral- cultures, fixed after interaction with CF-labelled dehyde-fixed parasites treated with CF present a parasites, revealed that both amastigotes and infec- uniform labelling over the whole cell surface. How- tive promastigotes quickly removed bound CF. ever, living parasites displayed CF patches and Therefore CF was seen neither in parasite-macro- caps. Capping was usually seen towards the an- phage attachment areas nor in parasitophorous terior (flagellated) portion of the cells, where shed- vacuoles. On the contrary, non-infective promasti- ding phenomena took place. These processes were gote-macrophage attachment areas were remark- inhibited by sodium azide but not by low tempera- ably large and preferentially comprised CF- ture (4°C). CF treatment of non-infective promasti- labelled membranes. These results strongly suggest gotes led to an increase in their uptake by macro- an important participation of cell surface anionic phages, whereas the uptake of amastigotes or sites in the L. mexicana amazanensis-macrophage infective promastigotes was not significantly interaction. altered. The effect of CF on the parasite surface charge was analyzed by whole-cell microelectro- Key words: Leishmania-macrophage interaction, anionic phoresis. The mean electrophoretic mobility (EPM) sites, cationized ferritin.

Introduction that most cell types have a net negative surface charge, and that cell surface properties can participate in cellular Parasites of the genus Leishmania are of special interest, phenomena such as recognition, adhesion and endo- since they infect cells of the mononuclear-phagocytic cytosis (reviewed by Van Oss, 1978). In the case of system. It is through the contact between the two cell Leishmania, macromolecules such as the lipophosphogly- surfaces that this crucial step in the parasite's life cycle can (LPG) may be relevant because of its highly anionic takes place. Promastigotes inoculated by the invertebrate nature (reviewed by Turco, 1988). The net negative host infect vertebrate phagocytes, in which they are surface charge of macrophages and Leishmania has been transformed and reproduce as amastigotes. The mechan- analyzed by cell electrophoresis (Pimenta & De Souza, isms underlying such phagocyte specificity are still poorly 1983; Silva-Filho et al. 1987; Santos & De Souza, 1983) understood, although different surface receptor-ligand and by labeling with cationic probes (Papadimitriou, systems, involving fibronectin, complement and man- 1982; Pimenta & De Souza, 1983; Santos & De Souza, nose 6-phosphate receptors, have been shown to partici- 1983), but no single work to date has attempted to pate in this recognition process (Wyler et al. 1985; correlate these data with the interaction process. Polyca- Russell &Wilhelm, 1986; Russell & Wright, 1988; Wilson tionic substances such as polylysine (Capo et al. 1981; & Pearson, 1986; Mosser et al. 1987; Saraiva et al. 1987; Matsui et al. 1983), lysozyme (Pruzanski & Saito, 1978) Puentes et al. 1988; Wozencroft et al. 1986). Alterna- and cationized ferritin (Meirelles et al. 1984) enhance tively, parasite-macrophage interaction may be studied endocytosis. Cationized ferritin has the advantage of as a physical surface phenomenon. It is well established being readily identified on electron micrographs, because

Journal of Cell Science 93, 481-489 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 481 of its electron density and particulate appearance. In determination or processed for transmission electron mi- contrast to other cationic substances (Werk et al. 1984) croscopy. For the capping experiments, promastigotes sus- CF can be used at physiological pH (Danon et al. 1972), pended in PBS (lOmM-phosphate buffer; 150mM-NaCl), without interfering with cell viability. pH7-2, were incubated with cationized ferritin (10;/gml~ ) for 30 min at 4°C. The parasites were subsequently washed in PBS Among trypanosomatids, cationized ferritin binding to by centrifugation at 1000g for 10 min at 4°C. After that, one T. cruzi (De Souza et al. 1977; Souto-Padron & De sample was immediately fixed, the others were incubated at Souza, 1986) and to Leishmania (Pimenta & De Souza, 26°C and portions were removed at 10, 20 and 30 min, and fixed 1983; Ayesta et al. 1985) has been shown in aldehyde- as described below. fixed cells, and so the dynamics of this reaction and its possible biological implications remain unknown. The Parasite--macrophage interaction fate of cationized ferritin-binding sites has been studied Interaction experiments were performed as described (Saraiva in T. cruzi and in Leishmama-macrophagz interactions, et al. 1987). Briefly, washed parasites and macrophages were but mostly by focusing the formation of endocytic left in contact for 1 h at 37°C in a parasite: macrophage ratio of vacuoles (Meirelles et al. 1984; Pimenta & De Souza, 50: 1 in Eagle's medium (Sigma Chem. Co., MO, USA). The 1988). cells were then rinsed with Ringer's solution, fixed in Bouin and In this paper we report the behavior of cationized stained with Giemsa. The percentage of infected macrophages ferritin-binding sites on the surface of living Leishmania was determined by randomly counting a minimum of 600 cells in each of the triplicate preparations. Experiments were re- mexicana amazonensis (amastigotes, infective and non- peated at least three times. For transmission electron mi- infective promastigotes) and their participation in the croscopy, the interactions were carried out in Descarplast interaction with macrophages. culture flasks (Rio de Janeiro, Brasil) in a parasite: macrophage ratio of 10:1. After 30 min of interaction the cultures were fixed in 4% paraformaldehyde, 1 % glutaraldehyde and 1 mM-CaCl2 Materials and methods in O'l M-cacodylate buffer, washed overnight in the same buffer and, after removal with a rubber policeman, they were post- Parasites fixed for 1 h in a solution containing 1 % osmium tetroxide, The Josefa strain of Leishmania mexicana amazonensis ob- 0-8% potassium ferricyanide, 1 niM-CaO2 in 0-1 M-cacodylate tained from Dr C. A. Cuba Cuba (Universidade de Brasilia, buffer, washed in the same buffer, dehydrated in acetone and Brasil) in 1981 has been maintained in our laboratory since embedded in Epon. then, in culture and by hamster footpad inoculation. Promasti- gotes cultured for long periods in axenic medium are considered Cell microelectrophoresis non-infective (Saraiva et al. 1986) because they are unable to The electrophoretic mobility (EPM) was determined using a produce visible lesions when inoculated into hamsters or to Zeiss cytopherometer, by timing the passage of cells through a survive within macrophages maintained in vitro. The promasti- calibrated graticule, when a current of 6 mA and a gradient of gotes used for the experiments described here had undergone 5-5 Vcm"1 were applied to the electrophoresis chamber, as more than 450 passages in vitro. Freshly transformed infective described (Silva-Filho et al. 1987). Briefly, fixed parasites (in promastigotes were obtained by culturing amastigotes from 2-5% glutaraldehyde in 0-1 M-phosphate buffer, pH7-2) were hamster lesions for up to five sub-cultures. Both were main- carefully washed and suspended in a NaCl solution (ionic tained at 26°C in a medium consisting of a brain-heart infusion strength 0-145moldm~3, pH7-2) for the EPM determinations. supplemented with 10% fetal calf serum and a 0-2% hemoglo- Measurements of living cells at 4°C were not significantly bin solution, and harvested for the experiments during the different from those obtained at 25 °C with glutaraldehyde-fixed stationary phase of growth. Amastigote purification from ham- cells (Pimenta & De Souza, 1983). The EPM was determined ster footpad lesions has been described in detail (Saraiva et al. using the equation: EPM = d/t X D/V, where d is the distance 1983). (usually 16,um) the cell is conveyed during measurements, / is the time (s) required for the cell to move along d, D is the Macrophages distance (18 cm) between the two electrodes and V is the Peritoneal macrophages from normal 6- to 8-week-old Swiss potential applied to the electrodes. For each experiment the mice were collected in Hank's balanced solution and plated on EPM of at least 50 cells was timed in alternating directions, to glass coverslips in Falcon 24-well tissue-culture plates (Becton avoid electrode polarization. Statistical analysis was done using Dickinson Labware, New Jersey, USA) as described by Saraiva Student's Mest. We considered significant those differences et al. (1987). The cells were allowed to adhere for 30-40 min at above 10% (P^0-05). 37°C in a 5 % CO2 atmosphere, after which time the non- adherent cells were removed and new culture medium (Eagle's plus 10% fetal calf serum) was added. The cells were then Results incubated overnight under the same conditions as above before the interaction experiments. Cationized ferritin binding to the Leishmania surface Glutaraldehyde-fixed parasites treated with cationized Cationized ferritin treatment ferritin (CF) particles present uniform labeling over the Before interaction, either the macrophages or the parasites were whole cell surfaces (Fig. 1). On the contrary, living incubated at 4°C for 30 min in the presence of cationized ferritin parasites preincubated at 4°C with CF, shifted to 25 °C at 100jUgml~ or 10 jig ml" (subagglutinating concentration), respectively. After washing twice with Hank's solution at 4°C and fixed after 10-30 min, showed a non-uniform, patchy cells and parasites were allowed to interact for 1 h at 37°C. The distribution of the particles (Fig. 2). This CF rearrange- parasites were also fixed in 25% glutaraldehyde in 01M- ment was inhibited by azide but not by low temperature phosphate buffer, pH7-2, for the electrophoretic mobility (4°C). Capping of the surface CF particles was seen at

482 E. M. B. Saraiva et al. ^^^*V

4 j Fig. 1. Glutaraldehyde-fixed promastigote of L. mexicana amazonensis treated with lOjUgml"1 cationized ferritin (CF). A uniform CF distribution is observed on both flagellar and cell body membranes./, flagellum; X25 000. Fig. 2. Promastigote preincubated with 10,ugml~ CF and kept at room temperature for lOmin before fixation. Note the uneven, patchy CF distribution at the anterior end of the cell (arrowheads)./, flagellum; X25 000. Fig. 3. CF patching (open arrows) and shedding (filled arrows) from the flagellar basis./, flagellum; X55000. Fig. 4. CF shedding in the presence of 3 mM-azide (see text). Membrane units (arrowheads) can be observed in association with liberated CF particles./, flagellum; X34000.

both poles of the cells, but far more frequently towards Cationized ferritin-labeled free membranes were often the flagellar, anterior end of the parasite. Membrane- found in our preparations, especially with infective bound CF particles were eventually released from flagel- promastigotes, which appeared to remove bound CF lar membranes (Fig. 3) but CF-associated membrane particles much more rapidly than non-infective ones. units were hardly seen (unless the preparation angle was After 30 min at room temperature, most infective cells altered using a goniometer), possibly because particle were almost completely depleted of CF, whereas the non- aggregation caused the twisting of detached membranes. infective cells still presented large amounts of particles. Low azide concentrations (3 mM) also made membrane units more easily observed (Fig. 4), probably because of Effects of cationized ferritin on the the reduced amount of bound CF on shedding mem- Leishmania—macrophage interaction branes. Cationized ferritin (CF) had different effects upon the

interaction 483 Parasite % of infected cells Macrophage Figs 6-12. L. mexicana amazonensis promastigotes were (S.E.M.) 8 treated with 10f«gml~ CF, at 4CC for 30min, before interaction with mouse peritoneal macrophages. 8-96 Fig. 6. Three non-infective promastigotes attached to a (1-24) Hi macrophage, showing CF in the attachment areas. X 12 500. Fig. 7. Parasite bound to two macrophages. CF can be seen 2-51 Control (0-24) only in attachment areas, in, macrophage; p, parasite. X14 000. 1-69 (0-11) Fig. 8. Non-infective promastigote during ingestion by J macrophage. A macrophage pseudopod is observed covering a CF-labelled membrane area. X22500. Inset: macrophage 7-43 pseudopods preferentially bind to CF-labeled areas. X45 500. (1-11) i Fig. 9. CF-labeled flagellum (/) attached to a macrophage. 2-70 d CF-treated x 35 000. (0-46) Leishmania Fig. 10. CF removal (arrow) from bound parasite (see text). 1-65 X 34 000. (0-11) v/m J Fig. 11. Macrophage showing a vacuole (v) with endocytosed CF-labeled membrane. Extracellular CF-labeled membranes are observed attached to the macrophage surface 6-57 (arrowheads). X22000. (0-81) H CF-treated Fig. 12. Larger vacuole (v) showing several CF-labeled 2-51 macrophages vesicles, x22 000. (0-60) 1-64 (0-19) V////AM _ showed different labelling patterns for infective and non- infective cells. Both infective promastigotes and amasti- Fig. 5. Effect of cationized ferritin (CF) on the L. mexicana gotes quickly removed bound CF, whereas non-infective amazonensis (amastigotes, infective and non-infective cells presented CF particles during attachment promastigotes) macrophage interaction. Parasites or (Figs 6-10) and even after endocytosis (Fig. 13). macrophages previously treated with CF (10 or 100 f.ig nil" , Non-infective promastigotes attachment areas were respectively) were allowed to interact for 1 h, fixed and Giemsa stained. Control of parasite-macrophage interaction remarkably large (Figs 6 and 8) and preferentially me- (CF untreated): (D) amastigote; (@) infective promastigote; diated by CF-labelled membranes. The CF layer in the (M) non-infective promastigote. CF-treated parasites- attachment area was usually 4-10 particles thick. Macro- macrophages: (D) amastigotes; (S) infective promastigotes; phage pseudopods were seen in contact particularly with (M) non-infective promastigotes. Parasites-CF-treated CF-labelled parasite regions (Fig. 8). CF removal by macrophages: (•) amastigotes; (H) infective promastigote; macrophages was observed after the attachment phase (M) non-infective promastigote. (Fig. 10). On the contrary, the attachment between macrophages and amastigotes or infective promastigotes interaction of macrophages with amastigotes, and infec- was mediated by unlabelled focal membrane areas tive and non-infective promastigotes of L. mexicana (Figs 14 and 15). Later these parasite forms were seen in amazonensis (Fig. 5). Treatment of the macrophages CF-free endocytic vacuoles. with CF before the interaction significantly increased the After interaction, many macrophages presented uptake of non-infective promastigotes, mainly due to a vacuoles containing CF-labelled vesicles. Small vacuoles higher percentage of infected macrophages. However, (Fig. 11) probably gave rise to larger ones (Fig. 12) by the same treatment did not interfere with the uptake of vacuole fusion. Extracellular CF-labelled membranes amastigotes and infective promastigotes. It is interesting were often seen attached to macrophages (Fig. 11). to note that the mean number of parasites per infected cell, as compared with controls, was not altered for both Electrophoretic mobility of parasites before and after CF promastigotes. In amastigotes the same index decreased treatment by 27 % but the percentage of infected macrophages was All the evolutive forms of L. mexicana amazonensis not affected by the CF treatment of phagocytes. studied exhibit negatively charged surfaces, since the In the same way, only the non-infective promastigote parasites migrate, in random orientation, towards the incubation with CF increased its uptake by macrophages, negative electrode under an applied voltage. again by producing a higher percentage of infected cells. The average mobilities of the parasites are closely At the light microscopy level it is not always possible to similar. Amastigotes have a mean EPM value of distinguish between attachment and actual uptake, but — l-12|iwns~ V~ cm, while infective and non-infective ultrastructural observations of many thin sections suggest promastigotes have mean EPM values of —1-12 and that both processes were remarkably enhanced in CF- — l-14,ums~ V"'cm, respectively. As can be deduced treated non-infective cells. Preincubation with CF of from the populational analysis shown in Fig. 16, the three infective promastigotes and amastigotes did not signifi- parasite forms studied by cell electrophoresis consist of cantly affect their interaction with macrophages. pooled cell suspensions with different EPM values. The Transmission electron microscopy of macrophage cul- modal EPM values fall into the class interval of —1-0 to tures fixed after interaction with CF-labelled parasites — l-2[ims~l V"1 cm for amastigotes, and non-infective

484 E. M. B. Saraiva et al. P

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10 12 and infective promastigotes. Incubation of the parasites in non-infective promastigotes the mean EPM decreased in the presence of cationized ferritin render their surfaces by about 26% after CF treatment. However, as can be less negative. The mean EPM values decreased slightly seen in Fig. 16, this treatment was effective in altering (12%) after treatment of amastigotes and infective pro- the modal EPM values of the parasites. The more mastigotes with the cationic particles. On the other hand, frequent EPM values are now found between — 0"8 and

Leishmania-w«r/o/>/;«£fe interaction 485 1

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Fig. 13. Macrophage showing several endocytosed non-infective promastigotes. Large amounts of bound CF can be seen on interiorized parasites. X 17 000. Fig. 14. Discontinuous attachment area between infective promastigote and macrophage. Parasite cells are completely devoid of CF. X30 000. Fig. 15. Focal attachment between amastigote and macrophage. X52 000.

Amastigotes Non-infective Infective 100, 100 promastigotes JQQ. promastigotes EPM = -l-12/ims"'v"'cm EPM= -l 'v 'cm EPM = — 1-12 funs v cm

50 50 _1—1 50

0/100 0/100 •0/100 CF-treated CF-treated CF-treated EPM = -0-99/«ns~1v-1cm EPM = -0-84 fan s~' v"1 cm EPM= -0-99^ms"'v"1cm

50 50 50 Fig. 16. Populational analysis of the electrophoretic mobilities of different developmental L. m. amazonensis forms before (top) and after (bottom) treatment with 10 fig ml"1 cationized ferritin (CF). 0 0 EPM = mean value of electrophoretic -0-7 -1-4 -0-7 -1-4 -0-7 -1-4 mobility.

486 E. M. B. Saraiva et al. — 1-0/ims V cm. Indeed, new mean class intervals of was energy-dependent and followed by liberation of non-infective and infective promastigotes could be ob- labelled membranes, similar to the antibody-induced tained after measurement of the EPM of parasites from shedding in T. cruzi (Schmunis et al. 1980) and the CF-treated populations. These mean class intervals were ConA-ferritin shedding in L. braziliensis (Ayesta et al. found between —0-6 and —0-8/ims"1 V"1 cm for both 1985). Skutelsky & Danon (1976) suggested that surface non-infective and infective promastigotes. anionic sites were detached from endothelial cells with clustered CF particles, although associated membranes could not be seen. In L. mexicana amazonensis shed Discussion membrane units were hardly seen without altering the preparation angle, except when low azide concentrations Our present observations show CF-induced capping and were used at 4°C to slow down the process, presumably shedding of anionic sites from L. mexicana amazonensis. by diminishing the amount of CF bound to detaching CF rearrangement has been described in several cell membranes, which were then less twisted. types including a free-living protozoan (King & Preston, In contrast to lectin and antibody-induced capping, 1977), mycoplasma (Schiefer et al. 1976) and sporozoan CF-induced capping in L. mexicana amazonensis was parasites (Augustine & Danforth, 1984; Russell & Sin- not inhibited at 4°C. Low temperatures also failed to den, 1981), but to date there are no reports on CF- inhibit antibody-induced capping of host immunoglobu- induced movement of surface ionogenic sites on trypano- lin G (IgG) on T. lewisi (Giannini & D'Alesandro, 1978), somatids. This may be due to the fact that fixed cells were CF-induced reorganization in Naegleria (King & Pres- always used for CF labelling. In previous work no ton, 1977) and rat thyroid cells (Nishiyama et al. 1982). difference in CF-labeling patterns between living and Although CF-binding could be related to infectivity in glutaraldehyde-fixed promastigotes of Leishmania dono- L. braziliensis (Ayesta et al. 1985), we observed no vani, L. tropica and L. braziliensis (Muhlpfordt, 1975) difference between CF-binding to infective and non- and , T. gambiense and T. rho- infective cells. However, it must be noted that infective desiense (Muhlpfordt, 1977) was observed. Such results cells shed CF-binding sites more quickly than non- may be explained by the probable use of agglutinating CF infective ones, being almost completely devoid of CF concentrations, as agglutination was shown to inhibit after 30min. These data suggest that L. mexicana antibody-induced capping in L. donovani (Dwyer, 1976) amazonensis promastigotes may have different surface and T. cruzi (Leon et al. 1979; Schmunis et al. 1980). properties concerning the mobility of anionic sites. The The CF-induced capping phenomenon is similar in role of membrane fluidity and surface component mo- many aspects to other described receptor-ligand systems bility on parasite infectivity remains to be clarified but it in Leishmania, such as specific antibodies (Doyle et al. is interesting to note that opsonized T. cruzi trypomasti- 1974; Dwyer, 1976) and lectins (Doyle et al. 1974). It gotes of the Y strain, which are able to survive in displays remarkable movement polarity of the CF-aggre- macrophages, can readily cap bound antibodies, whereas gated sites towards the flagellated end of the parasites. the CL strain, which takes a longer time to induce cap Antibody- or lectin-induced cap formation in L. ennettii formation, is destroyed inside the phagocytes (Schmunis (Doyle et al. 1974), L. donovani (Dwyer, 1976) and T. et al. 1980; Alcantara & Brener, 1978). cruzi (Szarfman et al. 1980; Schmunis et al. 1980) was CF treatment of non-infective promastigotes produced observed at both poles of the cells, but generally at that a remarkable increase in their uptake by macrophages, pole where the flagellar pocket is found, i.e. the anterior and a reduction in their electrophoretic mobility, prob- in Leishmania, and the posterior in T. cruzi trypomasti- ably because of its slower shedding. Therefore, the gotes (Dwyer, 1976; Szarfman et al. 1980; Schmunis et remaining bound CF could reduce the negative surface al. 1980). In our preparations, CF particles were also charge of these cells, reducing the electrostatic repulsion usually observed aggregated at the anterior (flagellated) between the parasite and the macrophage surfaces, as portion of the cells and CF labelling was rarely seen on attachment was mediated by CF-labelled membranes. posterior ends. Antibody-induced shedding in L. dono- When two macrophages were attached to a parasite, CF vani (Dwyer, 1976) and T. cruzi (Schmunis et al. 1980) could be seen solely in attachment areas (Fig. 7). Several usually takes place in the basal region of the flagellum, Leishmania species (reviewed by Chang, 1983) and L. close to the flagellar pocket. It is noteworthy that the mexicana amazonensis promastigotes, particularly flagellar pocket of trypanosomatids is generally involved (Pimenta & De Souza, 1986), were shown to interact with in several membrane-exchanging phenomena including macrophages predominantly at the flagellar end first. endocytosis and exocytosis (reviewed by De Souza, 1981; Cationized ferritin-treated non-infective promastigotes Coppensefa/. 1987). also presented CF in the flagellar attachment (Fig. 9), Cationized ferritin redistribution in unfixed baby ham- suggesting that anionic sites can also take part in the ster kidney cells (Grinnel et al. 1975) and Ehrlich ascites initial contact between parasite and phagocyte. As amas- tumor cells (Subjeck & Weiss, 1975) did not lead to the tigotes and infective promastigotes quickly removed formation of defined caps, and in the first case it was not bound CF, its effects on EPM and uptake by macro- blocked by metabolic inhibitors, suggesting that the phages were not significant. Meirelles et al. (1984) initial phase of this process is brought about by asymmet- showed that CF treatment of T. cruzi cells reduced the ric lateral diffusion of cross-linked anionic sites. In L. EPM and increased the uptake of trypomastigotes mexicana amazonensis the formation of defined CF caps whereas epimastigotes had neither EPM nor endocytic

heishmama-macmphage interaction 487 indexes altered by CF. Once again, charge reduction is technical assistance and Mrs Alba Valeria Peres for secretarial shown to promote increased endocytosis. assistance. This work has been supported by FINEP, CNPq Pimenta & De Souza (1988) demonstrated that CF and andCEPG-UFRJ. parasites were found in different vacuoles after macro- phage-L. mexicana amazonensis interaction in the pres- ence of CF, suggesting different endocytic pathways. References Our results showing CF removal from prelabeled non- infective promastigotes also indicate that different mech- ALCANTARA, A. & BRENER, Z. (1978). The in vitro interaction of anisms are used for their uptake by the macrophages. Trypaiiosoma cnizi bloodstream forms and mouse peritoneal macrophages. Ada tropica 35, 209-219. Evidence has been accumulated on the role of natural AUGUSTINE, P. C. & DANFORTH, H. D. (1984). Effects of cationized or synthetic polycationic agents during the interaction of ferritin and neuraminidase on invasion of cultured cells by Eimeria pathogens with their hosts, such as in the cases of the meleagrimitis sporozoites. J. Pmtozool. 31, 140-144. penetration of Toxoplasma gondii into host cells (Werk et AYESTA, C, ARGUELLO, C. & HERNANDEZ, A. G. (1985). al. 1984) and the bacterial phagocytosis by polymorpho- Leishmania brazilieiisis: cell surface differences in promastigotes of pathogenic and nonpathogenic strains. Expl Parasitol. 59, 185-191. nuclear leukocytes and macrophages (Pruzanski & Saito, CAPO, C, BONGRAND, P., BENOLIEL, A. M., RYTER, A. &: DEPIEDS, 1978). R. (1981). Particle-macrophage interaction: role of surface Two different mechanisms could explain the increased charges. Amils Immunol. (Inst. Pasteur) 132D, 165-173. phagocytosis of CF-treated cells. First, CF molecules CHANG, K. P. (1983). Cellular and molecular mechanisms of intracellular symbiosis in . Int. Rev. Cvtol. Suppl. 14, may function as cross-linkers between negatively charged 267-305. membranes of the interacting cells. Second, bound CF COPPENS, I., OPPERDOES, F. R., COURTOY, P. J. & BAUDHUIN, P. may reduce the negative surface charge and thus permit (1987). Receptor-mediated endocytosis in the bloodstream form of close apposition of negatively charged cells. Previous Trypanosoma brucei.J. Pmtozool. 34, 465-473. reports (Mutsaers & Papadimitriou, 1988; Denef & DANON, D., GOLDSTEIN, L., MARIKOVSKY, Y. & SKUTELSKY, E. (1972). Use of cationized ferritin as a label of negative charges of Ekholm, 1980) suggest that the second mechanism is cell surfaces. J. Ultrastntct. Res. 38, 500-510. more likely when the CF layer is more than one particle DENEF, J. F. & EKHOLM, R. (1980). Membrane labeling with thick. As demonstrated (Capo et al. 1981), neuramini- cationized ferritin in isolated thyroid follicles. J. Ultrastnict. Res. dase treatment and poly-L-lysine can increase erythrocyte 71, 203-221. DE SOUZA, W. (1981). Cell Biology of Trypanosoma cmzi. Int. Rev. binding to macrophages and produce the formation of Cytol. 86, 197-283. large continuous attachment areas. Therefore, charge DE SOUZA, W., ARGOELLO, C, MARTINEZ-PALOMO, A., TRISSL, D., reduction or neutralization may have similar effects on GONZAL£Z-ROBLES, A. & CHIARI, E. (1977). Surface charge of parasite attachment to macrophages. Larger attachment Tiypanosoma cnizi. Binding of cationized ferritin and areas presenting a layer four to ten particles thick, as well measurement of cellular electrophoretic mobility. J'. Pmtozool. 24, 411-415. as an enhanced endocytic rate, strongly suggest the role of DOYLE, J. J., BEHIN, R., MAUEL, J. & ROWE, D. S. (1974). surface charge reduction in the uptake of CF-treated non- Antibody-induced movement of membrane components of infective promastigotes. Mouse peritoneal macrophages Leishmania enriettii.J. exp. Mecl. 139, 1061-1069. were shown (Skutelsky & Hardy, 1976) to remove bound DWYER, D. M. (1976). Antibody-induced modulation of surface membrane antigens. J. Iminun. 117, 2081-1091. CF by endocytosis and surface detachment and to take a GIANNINI, M. S. & D'ALESANDRO, P. A. (1978). Unusual antibody- few hours to recover the CF binding. Therefore, macro- induced modulation of surface antigens in the cell coat of a phage CF treatment, producing removal of surface an- bloodstream trypanosome. Science 201, 916-918. ionic sites, could increase non-infective promastigote GRINNEL, F., TOBLEMAN, M. Q. & HACKENBROCK, C. R. (1975). uptake by reducing electrostatic repulsion. It remains to The distribution and mobility of anionic sites on the surfaces of baby hamster cells. J. Cell Biol. 66, 470-479. be explained why the other parasite forms were not KING, C. A. & PRESTON, T. M. (1977). Studies of anionic sites on affected by the same procedure. the cell surface of the amoeba Naegleria gmbeii using cationized The electrokinetic behavior of the parasite populations ferritin. J. Cell Sci. 28, 133-149. revealed interesting aspects. Both infective and non- LEON, W., VILLALTA, F., QUF.IROZ, T. & SZARFMAN, A. (1979). Antibody-induced capping of the intracellular stage of infective promastigotes represent highly heterogeneous Tiypanosoma cnizi. Infect. Immun. 26, 1218-1220. populations, since after treatment with cationized ferritin MATSUI, H., ITO, T. & OHNISHI, S. (1983). Phagocytosis by new subpopulations of both promastigotes could be macrophages. III. Effects of heat-labile opsonin and poly-L-lysine. observed. Thus, it is possible that the results of interac- J. Cell Sci. 59, 133-143. MEIRELLES, M. N. L., SOUTO-PADR6N, T. & DE SOUZA, VV. (1984). tion experiments could be due much more to intrinsic Participation of cell surface anionic sites in the interaction between variations in a population than merely to the binding Tiypanosoma cnizi and macrophages. J. sitbmicmsc. Cvtol. 16, efficiency of a non-infective population when compared 533-545. with the infective one. The same reasoning must be MOSSER, D. M., VLASSARA, H., EDELSON, P. J. & CERAMI, A. applied to amastigotes. (1987). Leishmania promastigotes are recognized by the macrophage receptor for advanced glycosylation endproducts. Detailed knowledge about the chemical nature of the J. exp. Mecl. 165, 140-145. surface anionic groups of L. mexiccma amazonensis is MilHLPFORDT, H. (1975). Vergleichende elektronenmikroskopische required for a better understanding of the interaction Untersuchung fiber die Markierung von I^eishmania donovani, L. process. This subject is under investigation. tropica und L. brazilieiisis mit Ferritin. Tropenmed. Parasit. 26, 385-389. MUHLPFORDT, H. (1977). Cell surface labelling of bloodstream The authors thank Dr H. Meyer and Dr Paulo F. P. Pimenta trypanosomes with cationic ferritin. Protozoology 3, 71-74. for reading the manuscript, Mr Antonio de Oliveira for MUTSAERS, S. E. & PAPADIMITRIOU, J. M. (1988). Surface charge of

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The lipophosphoglycan of Leishmania. Parasil. 3(CR3) binds to an Arg-Gly-Asp-containing region of the major Today 4, 255-257. surface glycoprotein, gp63, of Leishmmania promastigotes. J. exp. VAN OSS, C. J. (1978). Phagocytosis as a surface phenomenon. Med. 168," 279-292. A. Rev. Microbiol. 32, 19-39." RUSSELL, D. G. & SINDEN, R. E. (1981). The role of the WERK, R., DUNKER, R. & FISCHER, S. (1984). Polycationic cytoskeleton in the mobility of coccidian sporozoites. J. Cell Sci. polypeptides: a possible model for the penetration-enhancing factor 50, 345-359. in the invasion of host cells by Toxoplasma gondii. J. i>en. RUSSELL, D. G. & WILHELM, I-I. (1986). The involvement of GP63, Microbiol. 130, 927-933. the major surface glycoprotein in the attachment of Leishmania WILSON, M. E. & PEARSON, R. D. (1986). Evidence that leishmania promastigotes to macrophages. J'. Immnn. 136, 2613-2620. donovani utilizes a mannose receptor on human mononuclear SANTOS, A. B. S. & DE SOUZA, W. (1983). Surface charge and phagocytes to establish intracellular parasitism. J'. Immnn. 12, ultrastructure of the cell surface of resident and thioglycolate- 4681-4688. elicited mouse peritoneal macrophages. J. snbmicrosc. Cvtol. 15, WOZENCROFT, A., SAYERS, G. & BLACKWELL, J. M. (1986). 897-911. Macrophage type 3 complement receptors mediate serum- SARAIVA, E. M. B., ANDRADE, A. F. B. & DE SOUZA, W. (1987). independent binding of Leishmania donovani. J. exp. Med. 164, Involvement of the macrophage mannose-6-phosphate receptor in 1332-1340. the recognition of Leishmania mexicana amazonensis. Parasil. WYLER, D. J., SPYEK, J. P. & MCDONALD, J. A. (1985). In vitro Res. 73, 411-416. parasite-monocyte interactions in human leishmaniasis: possible SARAIVA, E. M. B., ANDRADE, A. F. B. & PEREIRA, M. E. A. (1986). role of fibronectin in parasite attachment. Infect. Immnn. 49, A comparative study of infective and non-infective forms of 305-309. leishmania mexicana amazonensis. Eur.J. Cell Biol. 40, 219—225. SARAIVA, E. M. B., PIMENTA, P. F. P., PEREIRA, M. E. A. & DE SOUZA, W. (1983). Isolation and purification of amastigotes of leishmania mexicana amazonensis by a gradient of metrizamide. J. Parasil. 69, 627-629. (Received 26 January I9S9 - Accepted 6 April 1989)

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