Paraaitology (1979), 78, 89-98 With 1 plate and 1figurein the text
89
Active entry of bloodstream forms of Trypanosoma cruzi into macrophages T. L. KIPNIS, V. L. G. CALICH and W. DIAS DA SILVA Departamento de Microbiologia e Imunologia, Institute de Ciencias BiomMicas, Universidade de Sao Paulo, Sao Paulo, Brasil (Received 1 March 1978) SUMMARY
The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi were not able to differentiate into amastigotes. Tneir uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.
INTBODTTCTION
There are two observations with respect to the entry of Trypanosoma cruzi into macrophages. Studies with non-purified culture forms of T. cruzi demonstrated that pre-treatment of macrophages with cytochalasin B (Cyt B) partially inhibits the uptake of the organisms (Alexander, 1975); when however, purified culture forms of T. cruzi were used, the uptake by mammalian cells of both epimastigote and trypomastigote forms was inhibited by Cyt B (Noqueira & Cohn, 1976). There is no information concerning the interactions between trypomastigote bloodstream forms and macrophages. The experiments reported here were undertaken (a) to compare the interaction of trypomastigote bloodstream forms of two different stocks of T. cruzi with mouse peritoneal macrophages; (6) to verify the effect of pre-treatment of these trypanosomes with specific antibodies and/or complement (C) on these interactions; (c) to explore the effect of substances which interfere with microfilaments 0031-1820/79/0078-0408 $01.00 © 1979 Cambridge University Presa
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T. L. KIPNIS AND OTHERS
and with the membrane receptors of the macrophages; and finally, (d) to study the influence of the functional state of these phagocytes on the uptake and fate of the parasites. MATERIALS AND METHODS
Mice Swiss outbred, male mice were housed in plastic cages and allowed free access to both food and water. Inbred DBA/2J mice housed in the same way were used to supply serum deficient in C5. Parasites
Y and CL stocks of T. cruzi were maintained in mice, the Y stock by weekly intraperitoneal blood passages and the CL stock by passages done at intervals of 16 days. Blood was collected without anticoagulant and was defibrinated with glass beads. An equal volume of Hanks' balanced salt solution (BSS) containing 1 % bovine serum albumin (BSA-fraction V, Nutritional Biochemical Corporation, Cleveland, Ohio), 5 units heparin (Prod. Roche Quim. e Farm. R. J. Brasil), 100 units penicillin and 100 fig streptomycin/ml were added. The blood was centrifuged at 1500 x g for 1 min and allowed to stand at 37 °C for 60 min. The supernatant serum containing most of the T. cruzi was centrifuged at 1200 x g for 15 min. The sediment was resuspended in 3 ml of medium 199 (Flow Laboratories, Rockville, Md, USA) containing 10% inactivated foetal calf serum (Flow Laboratories, Rockville, Md, USA), 100 units penicillin, 100 fig streptomycin and buffered with 1 x 10~3 M iVr-2-hydroxyethylpiperazine-i^'-2-ethansulfonic acid (SIGMA Chemical Company, St Louis, Mo, USA). This will be designated supplemented medium 199. The number offlagellateswas determined in a Neubauer chamber and the parasite concentration adjusted to 1 x 106 organisms/ml. Culture forms
The parasites were grown in liver infusion-tryptose-serum medium (LIT), prepared as previously described (Camargo, 1964) and subcultured weekly. Parasites were harvested from 5 to 7-day-old cultures, washed twice in phosphate buffered saline (PBS), pH 7-4, /i = 0-15 at 750 xg for 10 min, resuspended in supplemented medium 199, counted and adjusted to 1 x 106 organisms/ml. Macrophage culture
Cells were collected from the peritoneal cavities of outbred, uninfected mice upon injection of 5 ml of supplemented medium 199. A sample of 0-5 ml of a suspension containing about 2 x 106/ml mononuclear cells was placed into Leighton tubes containing flying cover-slips. After incubating for 1 h at 35 °C, under 5 % of CO2, the non-adherent cells were removed, the macrophage monolayers washed 3 times in BSS and finally overlaid with supplemented medium 199. All cultures were maintained in a 5 % CO2 atmosphere at 35 °C. Cell viability, as determined by Trypan blue exclusion, was greater than 95 %.
Macrophage interaction with T. cruzi
91
Macrophage stimulating agents Lyophilized lipopolysaccharide B (LPS) (Escherichia coli 055: B5 lipopolysaccharide B ; Difco Laboratories, Mich, USA) was suspended in a sterile 5 % glucose solution and stored frozen at a concentration of 100/ig/ml. Mice were injected intravenously with 50 fig of LPS, 24 h before collecting the peritoneal exudate. Brewer's thioglycollate medium and proteose-peptone (Difco La/boratories, Detroit, USA) were reconstituted as recommended by the vendor. Mice were injected intraperitoneally with 0-6 ml of either of the reconstituted reagents and cells were harvested 4 days later. Sera and antisera C5-deficient mouse serum was obtained from DBA/2J mice by axillary bleeding, just before use. The blood was kept at 37 °C for 60 min, centrifuged at 4 °C at 1200x g for 15 min and the serum collected. Immune serum was obtained from mice chronically infected with T. cruzi stock Y, after 4 weekly intraperifconeal injections of 1 x 103 blood forms of trypanosomes. The blood and serum were obtained as described above. The titre of these antisera was 1:20 as determined by passive haemagglutination assay and by indirect immunofluorescence test. Rabbit anti-mouse macrophage serum with a titre of 1:320 as tested by immunofluorescence, was kindly donated by Dr R. R. dos Santos (Departamento de Microbiologia, Imunologia e Parasitologia da Faculdade de Medicina de Ribeirao Preto, SP). This antiserum was inactivated at 56 °C for 30 min before use. A rabbit anti-sheep red blood cell serum rich in IgM (IgM-SRBC) was prepared according to the method previously described (Mayer, 1961) whereas IgG-SRBC was prepared in mice (Lay & Nussenzweig, 1968). E{IgM) and E (IgG) A 5 % SRBC (E) suspension was incubated with sub-agglutinating dilutions of rabbit anti-SRBC IgM-rich serum or mouse anti-SRBC IgG-rich serum for 30 min at 37 °C. The cells were washed twice with BSS, resuspended in supplemented medium 199 and the suspension adjusted to a concentration of 5 % . E(IgMC) A 5 % suspension of E(IgM) was incubated with C5-deficient mouse serum at 1:10 dilution for 10 min at 37 °C. The cells were washed and the suspension adjusted as described above. Trypsin treatment of macrophages Macrophage monolayers were incubated for 30 min at 37 °C with 500 jug/ml trypsin in supplemented medium 199, and washed 3 times with 500 fig/ml soybean trypsin inhibitor (SBTI) (Sigma Chemical Company, St Louis, Mo, USA) in supplemented medium 199 to stop trypsin digestion.
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Treatment of macrophages vrith cytochalasin B Macrophage cultures (24 h) were pre-fcreated with 10 jig Cyt B (Calbiochem, San Diego, Ca, USA, Lot 501132) for 1 h at 35 °C before the addition of the T. cruzi. A stock solution at 1 mg/ml in supplemented medium 199 was prepared by diluting the Cyt B in a small amount of dimethylsulfoxide (Merck Laboratories, Germany). The Cyt B was maintained in. the culture medium during the entire period of incubation with T. cruzi. It was seen in preliminary experiments that the Cyt B (10 /tg/ml) did not affect the trypomastigote bloodstream forms for at least 24 h of contact. Treatment of macrophages with rabbit anti-mouse macrophage serum Macrophage monolayers were incubated for 60 min at 37 °C with 1:5 dilution of anti-mouse macrophage serum which h.ad been previously heated at 56 °C for 30 min. After this treatment the cells were washed twice in BSS. In the experiments in which the macrophages were treated with trypsin, Cyt B or anti-macrophage serum, the trypomastigote bloodstream forms were added immediately after washing whereas the E(IgG), E(IgMC) or culture forms were added 23 h later. Antibody treatment of T. cruzi Trypomastigote bloodstream forms or culture forms of T. cruzi were treated with 1:40 dilution of immune serum for 60 min at 37 °C. The parasites were washed twice in BSS, resuspended in supplemented medium 199 and the suspension adjusted to 1 x 106 organisms/ml. Complement treatment of T. cruzi Trypomastigote bloodstream forms or culture forms of T. cruzi, unsensitized or coated with specific antibodies, were incubated for 10 min at 37 °C with C5deficient mouse serum diluted 1:10. After incubation the parasites were washed twice in BSS, resuspended in supplemented medium 199 and the suspension adjusted to 1 x 106 organisms/ml. Experimental design Except when otherwise indicated, macrophages cultured for 24 h were infected with 0-5 to 1-0 x 106 parasites. The culture forms were allowed to remain in contact with the macrophages at 35 °C for 1 h and. the trypomastigote bloodstream forms for a period of 24 h. After these periods of contact the macrophages were washed 3 times with BSS to remove the extracellular parasites. The macrophages cultivated with culture forms werefixedin Bouin solution and stained with haematoxylin and eosin. The macrophages incubated -with trypomastigote bloodstream forms were cultivated for an additional period of 24-48 h,fixedand stained as described above. All experiments were run in triplicate and repeated 3 times. The percentage of macrophages showing culture forms inside vacuoles or amastigote forms was determined by counting 1 x 103 cells/slide. The results are presented as the means + standard error.
0 29-2 ±1-6
Y
CL Y
Trypomastigotes (bloodstream) 0 100
0 97-8+1-4
0
C
0 100
0
l-4±0-3 0
9-4 ±0-7
Immune serum + C Untreated
4-0 ±0-5 0
50-4 ±1-1
Immune serum
1-3 + 0-3 0
31-3 ±4-8
C
4-2 ±1-0 0
51-2±2-6
Immune serum +C
Percentage of macrophages showing amastigote format
* The figures are the means + standard error. t The macrophages were fixed and stained after 1 h contact with T. cruzi. | The macrophages were in contact with T. cruzi for 24 h. The cultures were washed and re-incubated for a further 24 h period, when the cells were fixed and stained.
Epimastigotes (culture forms)
0
Stock
Immune Untreated serum
Percentage of macrophages containing ingested parasitest
Forms
Trypanosoma cruzi
Table 1. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of previous treatment of the trypanosomes with specific antiserum, complement (C) or both*
CO
re
t
II
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T. L. KIPNIS AND OTHEES
Table 2. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of different agents that 'stimulate' macrophages* Percentage of macrophages containing intracellular trypanosomesf Peritoneal exudate cells f obtained after stimulation -with 0-15MNEC1
Brewer thioglycollate Peptone LPS
Stock Y Stock CL trypomaatigotes % trypomastigotest (bloodstream forms) (bloodstream forms) 9-4 + 0-7 4 0 ±0-2 27-4 + 4-4 5-8 + 0-2 25-0 + 0-7 11-8 ±0-6 28-0+1-7 2-5 ±0-3
Stock Y epimastigotes§ (culture forms) 36-8+1-0 29-2 ±1-6 66-5 ± 3 0 64-8 ±2-9
* The figures are the means ± standard error. t Brewer thioglycollate and peptone were injected intraperitoneally; LPS was injected intravenously. t The macrophages were in contact with T. cruzi for 24 h. The cultures were washed and re-incubated for a further 24 h period, when the cells were fixed and. stained. § Macrophages infected with epimastigote forms were counted after 1 h of contact. KESTTLTS
Entry and fate of the parasites in the macrophages
In preliminary experiments not represented in this paper, we verified that the number of macrophages containing amastigote forms of T. cruzi increases with the time of contact between trypomastigotes and the cells. Maximal values were found when the contact period was prolonged to 24 h (9-4 ± 0-7 for the Y stock and 1-4 + 0-3 for the CL stock) (Table 1 and PL 1A). In some experiments, in which the incubation of the infected macrophages was extended to 72 h instead of 48 h, about 26 x lC^/ml trypomastigote forms appeared free in the fluid phase. With respect to the culture forms, the parasites were found inside vacuoles after 1 h of contact with the macrophages (Table 1, PI. IB) but they had disappeared from the cell cytoplasm by 24 h of incubation. Pre-treatment of trypomastigotes with immune serum, C or both did not potentiate the uptake of the parasites by macrophages during 1 h incubation. Nevertheless, the number of macrophages containing amastigote forms was higher with the Y stock after 24 h of contact. With respect to trypomastigote bloodstream forms of the CL stock, although immune serum produced a potentiation, treatment with C alone was without effect. On the other hand, those treatments potentiated the uptake of culture forms. These later results are presented in Table 1. In order to determine the effect of treatments which non-specifically stimulate macrophages, on the entry and fate of T. cruzi, mice were pre-treated with Brewer thioglycollate, peptone or LPS. As is shown in Table 2, these treatments produced the following effects: (a) all treatments potentiated the entry of Y stock; (6) peptone increased the entry of CL stock, and (c) peptone and LPS but not thioglycollate potentiated the uptake of culture forms. In order to determine the participation of the known plasma membrane receptors on the uptake of T. cruzi, 24 h cultured macrophages were treated with trypsin which
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95
Table 3. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of svbstances which destroy or block the macrophage receptors involved in phagocytosis* Percentage of macrophages showing intracellular trypanosomest Phagocytosis or adherence E(IgG) Treatment^ Control 100 Trypsin 100 Babbit anti0 macrophage serum
E(IgM)C 100 0 0
Stock Y trypomastigotes (bloodstream forms) 9-4 ±0-7 15-5±0-7 260 + 2-1
Stock CL Stock Y trypomastigotes epimastigotes (bloodstream (bloodstream forms) forms) l-4±0-0 7-7±M 5-6 ±0-5
36-8 ±1-0 0 0
* The figures are the means + standard error. f Macrophages infected with trypomastigotes were counted after 24 h contact and 48 h incubation, whereas those infected with epimastigotes were counted after 1 h contact. X Macrophages obtained from mice previously treated with Brewer thioglycollate 4 days before, were treated with trypsin or rabbit anti-macrophage serum and washed before addition of T. cruzi. 100
E(lgG)
Culture forms
TBF Y stock
TBF CL stock
Fig. 1. Effect of Cyt B on the uptake by macrophages of culture forms and trypomastigote bloodstream (TBP) forms of Trypanosoma cruzi. Comparison with E(IgG). Peritoneal macrophages were obtained from mice stimulated 4 days previously with Brewer thioglycollate. Trypanosomes or E(IgG) were incubated with the macrophages in the absence (open columns) or in the presence of Cyt B at a final concentration of 10 fig/ml (solid columns). Bars indicate standard error. 7-2
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T. L. KIPNIS AND OTHERS
destroys the C3b receptors (Lay & Nussenzweig, 1968), or with antiserum to mouse macrophages which blocks the Pc receptors (Holland, Holland & Conn, 1972). Table 3 shows that previous trypsinization of the macrophages inhibited the attachment of the E(IgMC) and the uptake of culture forms of T. cruzi, but did not interfere with the entry of the trypomastigote bloodstream forms of both Y and CL stocks. Macrophages pre-treated with anti-macrophage serum, although unable to ingest either E(IgG), E(IgMC) or culture forms of T. cruzi, still became infected with the trypomastigote bloodstream forms of both Y and CL stocks of T. cruzi. Since phagocytosis is inhibited by drugs which affect the microfilaments, macrophages were incubated with trypomastigote bloodstream forms of both Y and CL stocks of T. cruzi in the presence of Cyt B during the entire 24 h period of contact. E(IgG) and culture forms were added after 23 h of contact between the macrophages and Cyt B. It was found that Cyt B did not significantly affect the uptake of both Y and CL stocks of trypomastigote bloodstream forms untreated or pre-treated with specific antiserum. Under those conditions, the uptake of E(IgG) and culture forms was completely inhibited (Fig. 1). DISCUSSION
The present work was designed in order to provide a precise experimental assessment of the mode of entry of T. cruzi into macrophages. The two trypomastigotes bloodstream forms used, the Y and CL stocks, are morphologically and biologically different (Brenner, 1975; Takehara & Dias da Silva, 1975). With both stocks, the number of macrophages containing amastigote forms increases with the time of contact between the trypomastigote bloodstream forms and the cells. The situation with the culture forms was, however, distinct since a number of these organisms was found inside phagocytic vacuoles even after short periods of contact with the macrophages. This latter observation is consistent with previous reports (Noqueira & Cohn, 1976). The observations reported in this paper indicate that the trypomastigote bloodstream forms of T. cruzi enter macrophages actively. Previous opsonization of the trypomastigote bloodstream forms with specific antiserum, C or both produced an increase in the number of macrophages containing amastigote forms in 48 h cultures. Our experimental conditions did not permit the determination of the precise rate of penetration of trypomastigote bloodstream forms as we could not enumerate, at 1 h of contact, the number of parasitized macrophages. Thus, the question remains as to whether these treatments potentiate the entry or the intracellular survival of the trypanosomes. The specific antiserum and C should increase the attachment of the parasites through the macrophage receptors for the Fc or the C3b fragment respectively. The complement could be activated by the host antibodies already adhered to the parasites (Kloetzel & Deane, 1977) or through the alternative pathway (Kierszenbaum, Ivany & Budzko, 1976; Krettly & Nussenzweig, 1977). The role of these receptors in mediating the attachment and subsequent entry of trypo-
Macrophage interaction with T. cruzi
97
mastigote bloodstream forms appears not to be essential. For example, previous treatment of the macrophages with trypsin, which destroys the C3b receptors (Lay & Nussenzweig, 1969) or with rabbit anti-mouse macrophage serum which blocks the Fc receptors did not interfere with the entry of the trypomastigote bloodstream forms. Since treatment of the macrophages with trypsin inhibits attachment and ingestion of the trypomastigote culture forms (Noqueira & Cohn, 1976), a marked difference in the cell surface components of these two trypomastigote forms can be suggested, or, alternatively the mode of entry of the parasite into the cells. Phagocytosis and the other membrane activities can be disturbed by agents that interfere with the function of the microfilaments, (Allison, Davies & de Petris, 1971). With respect to the effect of Cyt B, our results, obtained from several experiments, indicated that the penetration of trypomastigote bloodstream forms into the macrophages was not blocked. These results were the same regardless of whether the parasites were pre-treated with specific antibodies. The functional state of peritoneal macrophages can be enhanced by local or systemic administration of LPS (Rabinovitch & DeStefano, 1973), or intraperitoneal inoculation of thioglycollate broth (Werb & Cohn, 1971). In macrophages thus stimulated, lysosomal constituents (Werb & Cohn, 1971), spreading activity (Rabinovitch, Manejias, Russo & Abbey, 1976) and the capacity to ingest and kill micro-organisms (Nelson, 1976) are greatly enhanced. Besides, the C3b receptors of macrophages stimulated with thioglycollate acquire the capacity to mediate the ingestion of E(IgMC) (Bianco, Griffin & Silverstein, 1975). The experiments described here clearly show that previously stimulated macrophages, although more susceptible to invasion by the trypomastigote bloodstream forms, are unable to interfere with the further differentiation of the parasite. An indirect argument that trypomastigote bloodstream forms can actively penetrate macrophages comes from the observations that the Y stock are capped when incubated at 37 °C with specific antibodies (Kloetzel & Deane, 1977; Cappa, Kloetzel & Ribeiro dos Santos, 1977; Schmunis, Szarfman & Souza, 1977). As we have shown in the present paper, trypanosomes submitted to this treatment are able to penetrate the macrophages to an even greater amount as compared to non-opsonized parasites. According to the 'Zipper Hypothesis' for phagocytosis (Griffin, Griffin & Silverstein, 1976) if the opsonized trypanosomes are in fact capped, they are in an unfavourable condition to be ingested by macrophages. We wish to thank Dr M. Mariano for many helpful discussions of this work and Dr A. M. Saliba for the opportunities offered in his laboratory. We are also indebted to Miss Kazuko Okabe for her technical assistance. This work was supported by Grant Nos SIP/08-080 and SIP/08-074 from the FINEP-CNPq and from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brasil. KEFERENCES ALEXANDER, J. (1975). Effect of the antiphagocytic agent cytochalasin B on macrophage invasion by Leishmania mexicana promastigotes and Trypanosoma cruzi epimastigotes. Journal of Protozoology 22, 237-40.
A. C, DAVIES, P. & DE PETRIS, S. (1971). Role of contractile microfilaments in macrophage movement and endocytosis. Nature, New Biology 232, 153-5.
ALLISON,
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C , GRIFFIN, F. M. & SHVERSTEIN, S. C. (1975). Studies of macrophage complement receptors: alteration of receptor function upon macrophage activation. Journal of Experimental Medicine 141, 1278-90. BRENNER, Z. (1975). Significance of morphologic variation of bloodstream forms. !New approaches in American trypanosomiasis research. Pan American Health Organization. Scientific Publication No. 318, 127-31. CAMARGO, E. P. (1964). Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Revista do Instituto de Medicina Tropical, Sdo Paulo 93-100. CAPPA, S. M. G., KLOETZEL, J. & RIBEIRO DOS SANTOS, R. (1977). Antibody-induced modulation of Trypanosoma cruzi surface membrane antigens. Pesquisa bdsica em Doenpa de Chagaa. IV. Reuniao Anual, Caxambu, Brasil. GRIFFIN, F. M. J R . , GRIFFIN, J. A. & SHATEBSTEIN, S. C. (1976). Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes. Journal of Experimental Medicine 144, 788-809. HOT.TIAKD, P., HOLLAND, N. H. & COHN, Z. A. (1972). The selective inhibition of macrophage phagocytic receptors by antimembrane antibodies. Journal of Experimental Medicine 135, 458-75. KIBKSZENBATJM, F., IVANY, J. & BTJDZKO, D. G. (1976). Mechanisms of natural resistance to trypanosomal infection. Role of complement in avian resistance to Trypanosoma cruzi infection. Immunology 30, 1-6. BIANCO,
KLOETZEI/, J. & DEANE, M. P. (1977). Presence of immunoglobulins on the surface of blood-
stream T. cruzi. Capping during differentiation in culture. Revistado Instituto de Medicina Tropical, Sao Paulo 19, 397-402. KRETTLY, A. U. & NUSSENZWEIG, R. (1977). A surface coat of immunoglobuuns on the bloodstream forms of Trypanosoma cruzi. Pesquisa Bdsica em Doenca de Ohagas. IV. Reuniao Anual Caxambu, Brasil. LAY, W. H. & NTJSSENZWEIG, V. (1968). Receptors for complement on leukocytes. Journal of Experimental Medicine 128, 991-1009. LAY, W. H. & NTTSSENZWEIG, V. (1969). Ca++-dependent binding of antigen-19S antibody complexes to macrophages. Journal of Immunology 102, 1172-8. MAYER, M. M. (1961). Complement and complement fixation. In Experimental Imrnunochemistry (ed. E. A. Kabat) Chapter 4, Springfield, 111.: Charles C. Thomas. NELSON, D. S. (1976). Immunobiology of the Macrophage. New York: Academic Press. NOQTJEIRA, N. & COHN, Z. (1976). Trypanosoma cruzi: mechanism of entry and intracellular fate in mammals cells. Journal of Experimental Medicine 143, 1402—20. RABINOVICH, M. & DESTEFANO, M. J. (1973). Macrophage spreading in vitro. I. Inducers of spreading. Experimental Cell Research 77, 323-34. RABINOVICH, M., MANEJIAS, R. E., RTTSSO, M. & ABBEY, E. E. (1976). Increased spreading of macrophages from mice treated with interferon inducers. Cellular Immunology 29, 86-95. SCHMTJNIS, G. A., SZARFMAN, H. & SOUZA, W. (1977). Antibody -induced modulation of Trypanosoma cruzi surface membrane antigens. Pesquisa Bdsica em Doenca de Ghagas. TV. Reuniao Anual, Caxambu, Brasil. TAKEHARA, H. & DIAS DA SILVA, W. (1975). Mechanism of resistance against T. cruzi in animals vaccinated with L. pessoai. New Approaches in American Trypanosomiasis. Pan American Health Organization. Scientific Publication No. 318, 174-8. WEBB, Z. & COHN, Z. A. (1971). Cholesterol metabolism in macrophages. 1. The regulation
of cholesterol compartments and exchange in other cell types. Journal of Experimental Medicine 134, 1570-90. EXPLANATION OF PLATE 1 A. Peritoneal macrophages showing amastigote forms of Trypanosoma cruzi after 48 h incubation. B. Peritoneal macrophages containing epimastigote culture forms within vacuoles after 1 h incubation. Sections stained with haematoxylin and eosin. ( x 500.) Printed in Cheat Britain
Parasitology, Vol. 78, Part 1
T. L. KIPX1S AND OTHERS
Plate 1
(Facing p. 98)
89
Active entry of bloodstream forms of Trypanosoma cruzi into macrophages T. L. KIPNIS, V. L. G. CALICH and W. DIAS DA SILVA Departamento de Microbiologia e Imunologia, Institute de Ciencias BiomMicas, Universidade de Sao Paulo, Sao Paulo, Brasil (Received 1 March 1978) SUMMARY
The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi were not able to differentiate into amastigotes. Tneir uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.
INTBODTTCTION
There are two observations with respect to the entry of Trypanosoma cruzi into macrophages. Studies with non-purified culture forms of T. cruzi demonstrated that pre-treatment of macrophages with cytochalasin B (Cyt B) partially inhibits the uptake of the organisms (Alexander, 1975); when however, purified culture forms of T. cruzi were used, the uptake by mammalian cells of both epimastigote and trypomastigote forms was inhibited by Cyt B (Noqueira & Cohn, 1976). There is no information concerning the interactions between trypomastigote bloodstream forms and macrophages. The experiments reported here were undertaken (a) to compare the interaction of trypomastigote bloodstream forms of two different stocks of T. cruzi with mouse peritoneal macrophages; (6) to verify the effect of pre-treatment of these trypanosomes with specific antibodies and/or complement (C) on these interactions; (c) to explore the effect of substances which interfere with microfilaments 0031-1820/79/0078-0408 $01.00 © 1979 Cambridge University Presa
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T. L. KIPNIS AND OTHERS
and with the membrane receptors of the macrophages; and finally, (d) to study the influence of the functional state of these phagocytes on the uptake and fate of the parasites. MATERIALS AND METHODS
Mice Swiss outbred, male mice were housed in plastic cages and allowed free access to both food and water. Inbred DBA/2J mice housed in the same way were used to supply serum deficient in C5. Parasites
Y and CL stocks of T. cruzi were maintained in mice, the Y stock by weekly intraperitoneal blood passages and the CL stock by passages done at intervals of 16 days. Blood was collected without anticoagulant and was defibrinated with glass beads. An equal volume of Hanks' balanced salt solution (BSS) containing 1 % bovine serum albumin (BSA-fraction V, Nutritional Biochemical Corporation, Cleveland, Ohio), 5 units heparin (Prod. Roche Quim. e Farm. R. J. Brasil), 100 units penicillin and 100 fig streptomycin/ml were added. The blood was centrifuged at 1500 x g for 1 min and allowed to stand at 37 °C for 60 min. The supernatant serum containing most of the T. cruzi was centrifuged at 1200 x g for 15 min. The sediment was resuspended in 3 ml of medium 199 (Flow Laboratories, Rockville, Md, USA) containing 10% inactivated foetal calf serum (Flow Laboratories, Rockville, Md, USA), 100 units penicillin, 100 fig streptomycin and buffered with 1 x 10~3 M iVr-2-hydroxyethylpiperazine-i^'-2-ethansulfonic acid (SIGMA Chemical Company, St Louis, Mo, USA). This will be designated supplemented medium 199. The number offlagellateswas determined in a Neubauer chamber and the parasite concentration adjusted to 1 x 106 organisms/ml. Culture forms
The parasites were grown in liver infusion-tryptose-serum medium (LIT), prepared as previously described (Camargo, 1964) and subcultured weekly. Parasites were harvested from 5 to 7-day-old cultures, washed twice in phosphate buffered saline (PBS), pH 7-4, /i = 0-15 at 750 xg for 10 min, resuspended in supplemented medium 199, counted and adjusted to 1 x 106 organisms/ml. Macrophage culture
Cells were collected from the peritoneal cavities of outbred, uninfected mice upon injection of 5 ml of supplemented medium 199. A sample of 0-5 ml of a suspension containing about 2 x 106/ml mononuclear cells was placed into Leighton tubes containing flying cover-slips. After incubating for 1 h at 35 °C, under 5 % of CO2, the non-adherent cells were removed, the macrophage monolayers washed 3 times in BSS and finally overlaid with supplemented medium 199. All cultures were maintained in a 5 % CO2 atmosphere at 35 °C. Cell viability, as determined by Trypan blue exclusion, was greater than 95 %.
Macrophage interaction with T. cruzi
91
Macrophage stimulating agents Lyophilized lipopolysaccharide B (LPS) (Escherichia coli 055: B5 lipopolysaccharide B ; Difco Laboratories, Mich, USA) was suspended in a sterile 5 % glucose solution and stored frozen at a concentration of 100/ig/ml. Mice were injected intravenously with 50 fig of LPS, 24 h before collecting the peritoneal exudate. Brewer's thioglycollate medium and proteose-peptone (Difco La/boratories, Detroit, USA) were reconstituted as recommended by the vendor. Mice were injected intraperitoneally with 0-6 ml of either of the reconstituted reagents and cells were harvested 4 days later. Sera and antisera C5-deficient mouse serum was obtained from DBA/2J mice by axillary bleeding, just before use. The blood was kept at 37 °C for 60 min, centrifuged at 4 °C at 1200x g for 15 min and the serum collected. Immune serum was obtained from mice chronically infected with T. cruzi stock Y, after 4 weekly intraperifconeal injections of 1 x 103 blood forms of trypanosomes. The blood and serum were obtained as described above. The titre of these antisera was 1:20 as determined by passive haemagglutination assay and by indirect immunofluorescence test. Rabbit anti-mouse macrophage serum with a titre of 1:320 as tested by immunofluorescence, was kindly donated by Dr R. R. dos Santos (Departamento de Microbiologia, Imunologia e Parasitologia da Faculdade de Medicina de Ribeirao Preto, SP). This antiserum was inactivated at 56 °C for 30 min before use. A rabbit anti-sheep red blood cell serum rich in IgM (IgM-SRBC) was prepared according to the method previously described (Mayer, 1961) whereas IgG-SRBC was prepared in mice (Lay & Nussenzweig, 1968). E{IgM) and E (IgG) A 5 % SRBC (E) suspension was incubated with sub-agglutinating dilutions of rabbit anti-SRBC IgM-rich serum or mouse anti-SRBC IgG-rich serum for 30 min at 37 °C. The cells were washed twice with BSS, resuspended in supplemented medium 199 and the suspension adjusted to a concentration of 5 % . E(IgMC) A 5 % suspension of E(IgM) was incubated with C5-deficient mouse serum at 1:10 dilution for 10 min at 37 °C. The cells were washed and the suspension adjusted as described above. Trypsin treatment of macrophages Macrophage monolayers were incubated for 30 min at 37 °C with 500 jug/ml trypsin in supplemented medium 199, and washed 3 times with 500 fig/ml soybean trypsin inhibitor (SBTI) (Sigma Chemical Company, St Louis, Mo, USA) in supplemented medium 199 to stop trypsin digestion.
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Treatment of macrophages vrith cytochalasin B Macrophage cultures (24 h) were pre-fcreated with 10 jig Cyt B (Calbiochem, San Diego, Ca, USA, Lot 501132) for 1 h at 35 °C before the addition of the T. cruzi. A stock solution at 1 mg/ml in supplemented medium 199 was prepared by diluting the Cyt B in a small amount of dimethylsulfoxide (Merck Laboratories, Germany). The Cyt B was maintained in. the culture medium during the entire period of incubation with T. cruzi. It was seen in preliminary experiments that the Cyt B (10 /tg/ml) did not affect the trypomastigote bloodstream forms for at least 24 h of contact. Treatment of macrophages with rabbit anti-mouse macrophage serum Macrophage monolayers were incubated for 60 min at 37 °C with 1:5 dilution of anti-mouse macrophage serum which h.ad been previously heated at 56 °C for 30 min. After this treatment the cells were washed twice in BSS. In the experiments in which the macrophages were treated with trypsin, Cyt B or anti-macrophage serum, the trypomastigote bloodstream forms were added immediately after washing whereas the E(IgG), E(IgMC) or culture forms were added 23 h later. Antibody treatment of T. cruzi Trypomastigote bloodstream forms or culture forms of T. cruzi were treated with 1:40 dilution of immune serum for 60 min at 37 °C. The parasites were washed twice in BSS, resuspended in supplemented medium 199 and the suspension adjusted to 1 x 106 organisms/ml. Complement treatment of T. cruzi Trypomastigote bloodstream forms or culture forms of T. cruzi, unsensitized or coated with specific antibodies, were incubated for 10 min at 37 °C with C5deficient mouse serum diluted 1:10. After incubation the parasites were washed twice in BSS, resuspended in supplemented medium 199 and the suspension adjusted to 1 x 106 organisms/ml. Experimental design Except when otherwise indicated, macrophages cultured for 24 h were infected with 0-5 to 1-0 x 106 parasites. The culture forms were allowed to remain in contact with the macrophages at 35 °C for 1 h and. the trypomastigote bloodstream forms for a period of 24 h. After these periods of contact the macrophages were washed 3 times with BSS to remove the extracellular parasites. The macrophages cultivated with culture forms werefixedin Bouin solution and stained with haematoxylin and eosin. The macrophages incubated -with trypomastigote bloodstream forms were cultivated for an additional period of 24-48 h,fixedand stained as described above. All experiments were run in triplicate and repeated 3 times. The percentage of macrophages showing culture forms inside vacuoles or amastigote forms was determined by counting 1 x 103 cells/slide. The results are presented as the means + standard error.
0 29-2 ±1-6
Y
CL Y
Trypomastigotes (bloodstream) 0 100
0 97-8+1-4
0
C
0 100
0
l-4±0-3 0
9-4 ±0-7
Immune serum + C Untreated
4-0 ±0-5 0
50-4 ±1-1
Immune serum
1-3 + 0-3 0
31-3 ±4-8
C
4-2 ±1-0 0
51-2±2-6
Immune serum +C
Percentage of macrophages showing amastigote format
* The figures are the means + standard error. t The macrophages were fixed and stained after 1 h contact with T. cruzi. | The macrophages were in contact with T. cruzi for 24 h. The cultures were washed and re-incubated for a further 24 h period, when the cells were fixed and stained.
Epimastigotes (culture forms)
0
Stock
Immune Untreated serum
Percentage of macrophages containing ingested parasitest
Forms
Trypanosoma cruzi
Table 1. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of previous treatment of the trypanosomes with specific antiserum, complement (C) or both*
CO
re
t
II
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T. L. KIPNIS AND OTHEES
Table 2. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of different agents that 'stimulate' macrophages* Percentage of macrophages containing intracellular trypanosomesf Peritoneal exudate cells f obtained after stimulation -with 0-15MNEC1
Brewer thioglycollate Peptone LPS
Stock Y Stock CL trypomaatigotes % trypomastigotest (bloodstream forms) (bloodstream forms) 9-4 + 0-7 4 0 ±0-2 27-4 + 4-4 5-8 + 0-2 25-0 + 0-7 11-8 ±0-6 28-0+1-7 2-5 ±0-3
Stock Y epimastigotes§ (culture forms) 36-8+1-0 29-2 ±1-6 66-5 ± 3 0 64-8 ±2-9
* The figures are the means ± standard error. t Brewer thioglycollate and peptone were injected intraperitoneally; LPS was injected intravenously. t The macrophages were in contact with T. cruzi for 24 h. The cultures were washed and re-incubated for a further 24 h period, when the cells were fixed and. stained. § Macrophages infected with epimastigote forms were counted after 1 h of contact. KESTTLTS
Entry and fate of the parasites in the macrophages
In preliminary experiments not represented in this paper, we verified that the number of macrophages containing amastigote forms of T. cruzi increases with the time of contact between trypomastigotes and the cells. Maximal values were found when the contact period was prolonged to 24 h (9-4 ± 0-7 for the Y stock and 1-4 + 0-3 for the CL stock) (Table 1 and PL 1A). In some experiments, in which the incubation of the infected macrophages was extended to 72 h instead of 48 h, about 26 x lC^/ml trypomastigote forms appeared free in the fluid phase. With respect to the culture forms, the parasites were found inside vacuoles after 1 h of contact with the macrophages (Table 1, PI. IB) but they had disappeared from the cell cytoplasm by 24 h of incubation. Pre-treatment of trypomastigotes with immune serum, C or both did not potentiate the uptake of the parasites by macrophages during 1 h incubation. Nevertheless, the number of macrophages containing amastigote forms was higher with the Y stock after 24 h of contact. With respect to trypomastigote bloodstream forms of the CL stock, although immune serum produced a potentiation, treatment with C alone was without effect. On the other hand, those treatments potentiated the uptake of culture forms. These later results are presented in Table 1. In order to determine the effect of treatments which non-specifically stimulate macrophages, on the entry and fate of T. cruzi, mice were pre-treated with Brewer thioglycollate, peptone or LPS. As is shown in Table 2, these treatments produced the following effects: (a) all treatments potentiated the entry of Y stock; (6) peptone increased the entry of CL stock, and (c) peptone and LPS but not thioglycollate potentiated the uptake of culture forms. In order to determine the participation of the known plasma membrane receptors on the uptake of T. cruzi, 24 h cultured macrophages were treated with trypsin which
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95
Table 3. Percentage of macrophages showing intracellular forms of Trypanosoma cruzi. Effect of svbstances which destroy or block the macrophage receptors involved in phagocytosis* Percentage of macrophages showing intracellular trypanosomest Phagocytosis or adherence E(IgG) Treatment^ Control 100 Trypsin 100 Babbit anti0 macrophage serum
E(IgM)C 100 0 0
Stock Y trypomastigotes (bloodstream forms) 9-4 ±0-7 15-5±0-7 260 + 2-1
Stock CL Stock Y trypomastigotes epimastigotes (bloodstream (bloodstream forms) forms) l-4±0-0 7-7±M 5-6 ±0-5
36-8 ±1-0 0 0
* The figures are the means + standard error. f Macrophages infected with trypomastigotes were counted after 24 h contact and 48 h incubation, whereas those infected with epimastigotes were counted after 1 h contact. X Macrophages obtained from mice previously treated with Brewer thioglycollate 4 days before, were treated with trypsin or rabbit anti-macrophage serum and washed before addition of T. cruzi. 100
E(lgG)
Culture forms
TBF Y stock
TBF CL stock
Fig. 1. Effect of Cyt B on the uptake by macrophages of culture forms and trypomastigote bloodstream (TBP) forms of Trypanosoma cruzi. Comparison with E(IgG). Peritoneal macrophages were obtained from mice stimulated 4 days previously with Brewer thioglycollate. Trypanosomes or E(IgG) were incubated with the macrophages in the absence (open columns) or in the presence of Cyt B at a final concentration of 10 fig/ml (solid columns). Bars indicate standard error. 7-2
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T. L. KIPNIS AND OTHERS
destroys the C3b receptors (Lay & Nussenzweig, 1968), or with antiserum to mouse macrophages which blocks the Pc receptors (Holland, Holland & Conn, 1972). Table 3 shows that previous trypsinization of the macrophages inhibited the attachment of the E(IgMC) and the uptake of culture forms of T. cruzi, but did not interfere with the entry of the trypomastigote bloodstream forms of both Y and CL stocks. Macrophages pre-treated with anti-macrophage serum, although unable to ingest either E(IgG), E(IgMC) or culture forms of T. cruzi, still became infected with the trypomastigote bloodstream forms of both Y and CL stocks of T. cruzi. Since phagocytosis is inhibited by drugs which affect the microfilaments, macrophages were incubated with trypomastigote bloodstream forms of both Y and CL stocks of T. cruzi in the presence of Cyt B during the entire 24 h period of contact. E(IgG) and culture forms were added after 23 h of contact between the macrophages and Cyt B. It was found that Cyt B did not significantly affect the uptake of both Y and CL stocks of trypomastigote bloodstream forms untreated or pre-treated with specific antiserum. Under those conditions, the uptake of E(IgG) and culture forms was completely inhibited (Fig. 1). DISCUSSION
The present work was designed in order to provide a precise experimental assessment of the mode of entry of T. cruzi into macrophages. The two trypomastigotes bloodstream forms used, the Y and CL stocks, are morphologically and biologically different (Brenner, 1975; Takehara & Dias da Silva, 1975). With both stocks, the number of macrophages containing amastigote forms increases with the time of contact between the trypomastigote bloodstream forms and the cells. The situation with the culture forms was, however, distinct since a number of these organisms was found inside phagocytic vacuoles even after short periods of contact with the macrophages. This latter observation is consistent with previous reports (Noqueira & Cohn, 1976). The observations reported in this paper indicate that the trypomastigote bloodstream forms of T. cruzi enter macrophages actively. Previous opsonization of the trypomastigote bloodstream forms with specific antiserum, C or both produced an increase in the number of macrophages containing amastigote forms in 48 h cultures. Our experimental conditions did not permit the determination of the precise rate of penetration of trypomastigote bloodstream forms as we could not enumerate, at 1 h of contact, the number of parasitized macrophages. Thus, the question remains as to whether these treatments potentiate the entry or the intracellular survival of the trypanosomes. The specific antiserum and C should increase the attachment of the parasites through the macrophage receptors for the Fc or the C3b fragment respectively. The complement could be activated by the host antibodies already adhered to the parasites (Kloetzel & Deane, 1977) or through the alternative pathway (Kierszenbaum, Ivany & Budzko, 1976; Krettly & Nussenzweig, 1977). The role of these receptors in mediating the attachment and subsequent entry of trypo-
Macrophage interaction with T. cruzi
97
mastigote bloodstream forms appears not to be essential. For example, previous treatment of the macrophages with trypsin, which destroys the C3b receptors (Lay & Nussenzweig, 1969) or with rabbit anti-mouse macrophage serum which blocks the Fc receptors did not interfere with the entry of the trypomastigote bloodstream forms. Since treatment of the macrophages with trypsin inhibits attachment and ingestion of the trypomastigote culture forms (Noqueira & Cohn, 1976), a marked difference in the cell surface components of these two trypomastigote forms can be suggested, or, alternatively the mode of entry of the parasite into the cells. Phagocytosis and the other membrane activities can be disturbed by agents that interfere with the function of the microfilaments, (Allison, Davies & de Petris, 1971). With respect to the effect of Cyt B, our results, obtained from several experiments, indicated that the penetration of trypomastigote bloodstream forms into the macrophages was not blocked. These results were the same regardless of whether the parasites were pre-treated with specific antibodies. The functional state of peritoneal macrophages can be enhanced by local or systemic administration of LPS (Rabinovitch & DeStefano, 1973), or intraperitoneal inoculation of thioglycollate broth (Werb & Cohn, 1971). In macrophages thus stimulated, lysosomal constituents (Werb & Cohn, 1971), spreading activity (Rabinovitch, Manejias, Russo & Abbey, 1976) and the capacity to ingest and kill micro-organisms (Nelson, 1976) are greatly enhanced. Besides, the C3b receptors of macrophages stimulated with thioglycollate acquire the capacity to mediate the ingestion of E(IgMC) (Bianco, Griffin & Silverstein, 1975). The experiments described here clearly show that previously stimulated macrophages, although more susceptible to invasion by the trypomastigote bloodstream forms, are unable to interfere with the further differentiation of the parasite. An indirect argument that trypomastigote bloodstream forms can actively penetrate macrophages comes from the observations that the Y stock are capped when incubated at 37 °C with specific antibodies (Kloetzel & Deane, 1977; Cappa, Kloetzel & Ribeiro dos Santos, 1977; Schmunis, Szarfman & Souza, 1977). As we have shown in the present paper, trypanosomes submitted to this treatment are able to penetrate the macrophages to an even greater amount as compared to non-opsonized parasites. According to the 'Zipper Hypothesis' for phagocytosis (Griffin, Griffin & Silverstein, 1976) if the opsonized trypanosomes are in fact capped, they are in an unfavourable condition to be ingested by macrophages. We wish to thank Dr M. Mariano for many helpful discussions of this work and Dr A. M. Saliba for the opportunities offered in his laboratory. We are also indebted to Miss Kazuko Okabe for her technical assistance. This work was supported by Grant Nos SIP/08-080 and SIP/08-074 from the FINEP-CNPq and from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brasil. KEFERENCES ALEXANDER, J. (1975). Effect of the antiphagocytic agent cytochalasin B on macrophage invasion by Leishmania mexicana promastigotes and Trypanosoma cruzi epimastigotes. Journal of Protozoology 22, 237-40.
A. C, DAVIES, P. & DE PETRIS, S. (1971). Role of contractile microfilaments in macrophage movement and endocytosis. Nature, New Biology 232, 153-5.
ALLISON,
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C , GRIFFIN, F. M. & SHVERSTEIN, S. C. (1975). Studies of macrophage complement receptors: alteration of receptor function upon macrophage activation. Journal of Experimental Medicine 141, 1278-90. BRENNER, Z. (1975). Significance of morphologic variation of bloodstream forms. !New approaches in American trypanosomiasis research. Pan American Health Organization. Scientific Publication No. 318, 127-31. CAMARGO, E. P. (1964). Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Revista do Instituto de Medicina Tropical, Sdo Paulo 93-100. CAPPA, S. M. G., KLOETZEL, J. & RIBEIRO DOS SANTOS, R. (1977). Antibody-induced modulation of Trypanosoma cruzi surface membrane antigens. Pesquisa bdsica em Doenpa de Chagaa. IV. Reuniao Anual, Caxambu, Brasil. GRIFFIN, F. M. J R . , GRIFFIN, J. A. & SHATEBSTEIN, S. C. (1976). Studies on the mechanism of phagocytosis. II. The interaction of macrophages with anti-immunoglobulin IgG-coated bone marrow-derived lymphocytes. Journal of Experimental Medicine 144, 788-809. HOT.TIAKD, P., HOLLAND, N. H. & COHN, Z. A. (1972). The selective inhibition of macrophage phagocytic receptors by antimembrane antibodies. Journal of Experimental Medicine 135, 458-75. KIBKSZENBATJM, F., IVANY, J. & BTJDZKO, D. G. (1976). Mechanisms of natural resistance to trypanosomal infection. Role of complement in avian resistance to Trypanosoma cruzi infection. Immunology 30, 1-6. BIANCO,
KLOETZEI/, J. & DEANE, M. P. (1977). Presence of immunoglobulins on the surface of blood-
stream T. cruzi. Capping during differentiation in culture. Revistado Instituto de Medicina Tropical, Sao Paulo 19, 397-402. KRETTLY, A. U. & NUSSENZWEIG, R. (1977). A surface coat of immunoglobuuns on the bloodstream forms of Trypanosoma cruzi. Pesquisa Bdsica em Doenca de Ohagas. IV. Reuniao Anual Caxambu, Brasil. LAY, W. H. & NTJSSENZWEIG, V. (1968). Receptors for complement on leukocytes. Journal of Experimental Medicine 128, 991-1009. LAY, W. H. & NTTSSENZWEIG, V. (1969). Ca++-dependent binding of antigen-19S antibody complexes to macrophages. Journal of Immunology 102, 1172-8. MAYER, M. M. (1961). Complement and complement fixation. In Experimental Imrnunochemistry (ed. E. A. Kabat) Chapter 4, Springfield, 111.: Charles C. Thomas. NELSON, D. S. (1976). Immunobiology of the Macrophage. New York: Academic Press. NOQTJEIRA, N. & COHN, Z. (1976). Trypanosoma cruzi: mechanism of entry and intracellular fate in mammals cells. Journal of Experimental Medicine 143, 1402—20. RABINOVICH, M. & DESTEFANO, M. J. (1973). Macrophage spreading in vitro. I. Inducers of spreading. Experimental Cell Research 77, 323-34. RABINOVICH, M., MANEJIAS, R. E., RTTSSO, M. & ABBEY, E. E. (1976). Increased spreading of macrophages from mice treated with interferon inducers. Cellular Immunology 29, 86-95. SCHMTJNIS, G. A., SZARFMAN, H. & SOUZA, W. (1977). Antibody -induced modulation of Trypanosoma cruzi surface membrane antigens. Pesquisa Bdsica em Doenca de Ghagas. TV. Reuniao Anual, Caxambu, Brasil. TAKEHARA, H. & DIAS DA SILVA, W. (1975). Mechanism of resistance against T. cruzi in animals vaccinated with L. pessoai. New Approaches in American Trypanosomiasis. Pan American Health Organization. Scientific Publication No. 318, 174-8. WEBB, Z. & COHN, Z. A. (1971). Cholesterol metabolism in macrophages. 1. The regulation
of cholesterol compartments and exchange in other cell types. Journal of Experimental Medicine 134, 1570-90. EXPLANATION OF PLATE 1 A. Peritoneal macrophages showing amastigote forms of Trypanosoma cruzi after 48 h incubation. B. Peritoneal macrophages containing epimastigote culture forms within vacuoles after 1 h incubation. Sections stained with haematoxylin and eosin. ( x 500.) Printed in Cheat Britain
Parasitology, Vol. 78, Part 1
T. L. KIPX1S AND OTHERS
Plate 1
(Facing p. 98)
