Parasitol Res (1992) 78:545 552
Parasitnlngy Research
9 Springer-Verlag1992
A cytochemical study of the interaction between Tritrichomonas foetus and mouse macrophages* Neide L. A z e v e d o 1,2 and Wanderley de S o u z a 1
1 Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund/io, 21949, Rio de Janeiro, Brasil 2 Departamento de Histologia, Instituto de Biologia, Universidade to Estado do Rio de Janeiro, Brasil Accepted April 25, 1992
Abstract. Light and electron microscopy were used to
analyse the process of interaction of normal and antibody-coated Tritrichomonasfoetus with resident and activated mouse peritoneal macrophages. Activated macrophages ingest m o r e parasites than do resident macrophages. Previous incubation o f the parasites in the presence of sub-agglutinating concentrations of a polyclonal anti-T, foetus antibody significantly increased their ingestion by the macrophages. Adherence of the parasites to the surface of activated m a c r o p h a g e s triggers the respiratory oxidative burst as revealed by reduction of nitroblue tetrazolium. This process was more evident in antibody-coated parasites. Transmission electron microscopy showed the presence o f reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the portions of the m a c r o p h a g e plasma m e m b r a n e that were in contact with the parasites as well as in the phagocytic vacuoles. Fusion of m a c r o p h a g e lysosomes with parasite-containing phagocytic vacuoles was observed in macrophages labeled with Lucifer yellow and gold-labeled peroxidase as well as by localisation of acid phosphatase.
Tritrichomonas foetus is the causative agent of bovine trichomoniasis. In the female host, it causes a venereal disease characterised by mild endometritis and, occasionally, p y o m e t r a and abortion (Parsonson et al. 1976). In the male host, T.foetus appears to be confined almost exclusively to the prepucial cavity, usually manifesting as an a s y m p t o m a t i c infection ( H a m m o n d and Bartlett 1943). Although a good experimental model for trichomoniasis is not presently available, mice have been used to analyse the pathogenicity of various strains o f Triehomonas tenax and T. vaginalis (Honigberg t961). * This work was supported by Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) Correspondence to .' W. de Souza
In the case of h u m a n trichomoniasis caused by T. circumstantial evidence suggests that the m o n o c y t e - m a c r o p h a g e lineage plays some role in the process of killing o f the parasite (Landolfo et al. 1980). In a previous study we have shown that mouse macrophages are capable of ingesting T.foetus, especially when the parasite surface has been modified by previous treatment with concanavalin A (Azevedo et al. 1991). To analyse further the interaction between T. foetus and macrophages, we investigated (a) whether attachment of the parasites to the m a c r o p h a g e surface would trigger the respiratory burst and (b) whether host-cell lysosomes would fuse with parasite-containing phagocytic vacuoles. The results obtained are described in this paper.
vaginalis,
Materials and methods
Parasites The K strain of Tritrichomonasfoetus was originally isolated by Guida et al. (1982; EMBRAPA, Rio de Janeiro, Brazil) from the urogenital tract of a bull in the state of Rio de Janeiro, Brazil, and was characterised by Silva-Filho et al. (1986). The parasites were placed in TYM medium (Diamond 1957) supplemented with 10% bovine calf serum and then incubated at 37~ C. Parasites obtained from the mid-log phase of growth were centrifuged at 600 g for l0 rain, washed twice in phosphate-buffered saline (PBS, pH 7.2) and counted using a Neubauer chamber, and their viability was evaluated using a trypan blue dye-exclusion test. The parasites were re-suspended in 199 medium (Sigma Chemical Co., USA) in the absence of serum. In other experiments, the living parasites were incubated for 30 min at 4~ C in the presence of heat-inactivated anti-Trichomonasserum at a 1:4000 dilution (sub-agglutinating dilution) prior to their interaction with the macrophages. After being washed with PBS at 4~ C, these parasites were re-suspended in 199 medium.
Resident macrophages Peritoneal macrophages obtained from normal Swiss mice were used. The animals were killed with ether and their peritoneal cavities were washed with Hanks' solution. Cells were plated on glass
546 coverslips in Falcon 24-well tissue-culture plates (Becton & Dickinson Labware, New Jersey) and maintained in a humidified atmosphere containing 5% COz at 37~ C. After 30 min, non-adherent cells were removed and the macrophages were cultivated for 24 h in 199 medium plus 10% foetal calf serum.
Activated macrophages Swiss mice were infected intraperitoneally with 5 x 106 viable noninfective epimastigote forms of Trypanosoma cruzi and were boosted 3 weeks later with an intraperitoneal injection of the homologous antigen. Previous studies have shown that the behaviour of mouse macrophages activated in such a way is similar to that of those activated using Listeria or bacille Calmette-Gu~rin (BCG; Nogueira and Cohn 1978; Carvalho and De Souza 1987). After 3 days, the animals were killed with ether, their peritoneal cavities were washed with 5 ml Hanks' solution in the absence of serum and the collected cells were plated into Falcon 24-well tissue-culture plates for light microscopy and onto the walls of glass flasks for electron microscopy. They were allowed to adhere to the glass surface for 30 min at 37~ and were then washed with Hanks' solution to remove the cells that had not adhered. Culture medium (199 medium plus 10% foetal calf serum) was added and the cells were incubated for 24 h at 37 ~ C, washed with 199 medium in the absence of serum and then used for the experiments.
Parasite-macrophage interaction The parasite-macrophage ratio was adjusted to 5:1 as based on the number of cells adhering to the wells and on the parasite density. In some experiments, the parasites were first incubated for 30 min at 4 ~ C in the presence of a 1:4000 dilution of immune serum (sub-agglutinating concentration) and then rinsed with PBS and allowed to interact with the macrophages. After interaction periods of 15, 30 or 60 min, the parasite-macrophage preparations were rinsed with PBS to remove non-attached parasites and fixed as described below.
Light microscopy The cultures were fixed with Bouin's solution and stained with Giemsa. The percentage of macrophages with associated parasites, the mean number of associated parasites per infected macrophage and the association index were calculated as previously described by Saraiva et al. (1987), whereby all preparations were examined using a Zeiss Universal Photomicroscope equipped with an immersion objective.
Fluorescence assay of phagosome-lysosome fusion Phagosome-lysosome (P-L) fusion in activated mouse macrophages was assayed by monitoring the transfer of a fluorescent dye from lysosomes to phagocytic vacuoles as previously described by Kielian and Cohn (1980). The macrophages were incubated at 37~ C for 2 h in Lucifer yellow (1 mg/ml), washed in 199 medium, and then incubated for 15 min at 37~ with the parasites (parasitemacrophage ratio, 5:1). The cells were examined by fluorescence microscopy. The presence of yellow-stained Trichomonaswas considered to be indicative of P-L fusion.
ed; ratio adjusted to 5:1) in 1 ml 199 medium containing 0.5 mg nitroblue tetrazolium (NBT)/ml (grade 111, Sigma Chemical Co. ; Murray and Cohn 1980). Uningested parasites were removed by washing, and the coverslips were reincubated in medium alone for an additional 30-min period. Intracellular parasites were identified using phase-contrast light microscopy and were then viewed by bright-field microscopy. Macrophages were scored as positive if ingested parasites were stained blue-black by precipitated formazan, the oxygen-dependent reduction product of NBT.
Transmission electron microscopy For transmission electron microscopy the macrophages were plated into 25-cm z glass flasks. After 15 or 30 min interaction between activated or resident macrophages, respectively and the parasites, the cultures were fixed for 60 min at room temperature in a solution containing 4% paraformaldehyde, 1% glutaraldehyde and 5 mM CaClz in 0.1 M cacodylate buffer (pH 7.2). After fixation, the cultures were rinsed in cacodylate buffer and left overnight at 4 ~ C in the same buffer. The cells were gently scraped off with a rubber policeman, post-fixed with 1% OsO, in 0.1 M cacodylate buffer supplemented with 0.8 M potassium ferrocyanide, washed in the same buffer, dehydrated in acetone and embedded in Epon. U1trathin sections were stained with uranyl acetate and lead citrate and observed in a Zeiss EM 900 or EM 902 electron microscope.
Ultrastructural cytochemistry The enzymes reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase and acid phosphatase were used as markers of O f production during the process of destruction of T. foetus by activated macrophages and for labeling of secondary lysosomes, respectively. The labeling of secondary lysosomes was also accomplished using peroxidase-gold. For detection of NAD(P)H-oxidase activity, the medium described by Briggs et al. (1975) was used. After activated macrophage-parasite interaction, the cells were washed at 4 ~ C in 0.1 ~ TRIS-maleate buffer (pH 7.5) containing 7% sucrose and were first incubated for 10 min at 37 ~ C in medium containing 0.1 M TRIS-maleate buffer (pH 7.5) supplemented with 7% sucrose and 1 mM [3-amino-l,2,4]-triazole (AT) and subsequently incubated for 20 rain at 37 ~ C in medium containing 0.1 M TRIS-maleate buffer (pH 7.5) supplemented with 7% sucrose, 0.71 mM NAD(P)H as the substrate, 2 mM CeC13 as the capture agent, and 10 mM AT. The medium used for control preparations lacked the substrate and AT and contained 0.01% catalase.
Acid phosphatase For detection of acid phosphatase activity, the method of Robinson and Karnovsky (1983) was used as follows. After interaction, the cells were fixed for 30 min at 4 ~ C in 1% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH 7.2). They were then rinsed in buffer and pre-incubated at room temperature for 30 rain in medium containing 1 mM CeC13, 5% sucrose and 0.05 N TRIS-maleate buffer (pH 5.0). Thereafter, the cells were incubated at 37~ C for 60 min in medium containing 2 mM CeCla, 7 mM Na-~-glycerophosphate, 5% sucrose and 0.05 M TRIS-maleate buffer (pH 5.0). Control cells were incubated in medium in the absence of the substrate. After incubation, the ceils were rinsed in cacodylate buffer, re-fixed in a solution containing 4% paraformaldehyde and 1% glutaraldehyde in cacodylate buffer (pH 7.2) for 1 h at room temperature and processed for electron microscopy as described above.
Qualitative nitroblue tetrazolium reduction
Gold-labeled peroxidase
Activated macrophages were exposed for 15 min at 37 ~ C (atmosphere, 5% COa/95% air)to T.foetus (control and antibody-coat-
For labeling of secondary lysosomes, the macrophages were incubated at 37~ C for 2 h in medium containing gold-labeled peroxi-
547
dase (Carva[ho et al. 1988) and 199 medium (1:20, v/v). After incubation, the cells were rinsed twicein 199 medium. The interaction between parasites and macrophages (ratio, 5:1) was allowed to proceed at 37~ C for 30 min. Thereafter, the preparations were washed three timeswith PBS and then fixedfor 60 min in a solution containing 1% glutaraldehyde,4% paraformaldehydeand 5 mM CaClz in 0.1 Mcacodylatebuffer (pH 7.2). After fixation, the cultures were processed for electron microscopyas described above. Results
Light microscopy Kinetics of the interaction. Observation of Giemsastained preparations showed that resident and activated macrophages ingested control and antibody-coated parasites. After an interaction period of 30 rain, the ingestion of untreated parasites by activated macrophages was 2-fold that observed for resident macrophages (Fig. 1). For both types of macrophages the ingestion was significantly increased when the parasites had previously been incubated in the presence of a sub-agglutinating concentration of anti-Tritrichomonas foetus polyclonal antibodies. This effect was attributable to an increase in the percentage of macrophages that ingested parasites and in the mean number of ingested parasites per macrophage. Incubation in the presence of the antibody did not induce alterations in the parasite.
Oxidative response. Association of the parasites with macrophages, especially with activated macrophages, led to the induction of respiratory burst as indicated by reduction of nitroblue tetrazolium. A black reaction prod100
R E S I D E NT
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I,.712.,I I,.512.21
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Fig. 1. Effect of an immune serum on the association of Tritricho-
uct was seen in the responsive cells. The percentage of reactive cells was significantly higher ,when antibodycoated parasites were used (Figs. 2, 3).
Lysosome-phagosomefusion. Lucifer yellow-labeled macrophage lysosomes that had been allowed to interact with the parasites were examined by fluorescence microscopy. P-L fusion was clearly indicated by the presence of many labeled vacuoles that contained parasites (Figs. 4, 5). No significant difference in the level of P-L fusion was observed between preparations containing untreated parasites and those containing antibody-treated parasites.
Electron microscopy Ultrastructural observations of the trichomonad-macrophage interaction revealed an intense process of pseudopod formation at sites of parasite-cell contact. After internalisation of the parasites, they were found to be lodged in membrane-bound vacuoles (data not shown). Electron microscopy was also used to analyse two aspects of the process of trichomonad-macrophage interaction: (1) P-L fusion and (2) NAD(P)H-oxidase activation. For further demonstration of fusion of macrophage lysosomes with parasite-containing vacuoles, macrophages were incubated in the presence of gold-labeled peroxidase, which accumulate in the lysosomes. Subsequently, these macrophages were incubated in the presence of the parasites. Figures 6 and 7 clearly show that labeled lysosomes concentrated around the phagocytic vacuole and later discharged their contents into the vacuole (Fig. 7). Acid phosphatase, a classic enzyme marker of lysosomes, was detected within many but not all phagocytic vacuoles. Reaction product, indicative of enzyme activity, was restricted to the peripheral region of the vacuole (Fig. 8). For demonstration of NAD(P)H-oxidase activity, the macrophages were incubated for 15 min in the presence of untreated or antibody-treated parasites and then in a medium designed to locate the enzyme. Reaction product, indicative of enzyme activity, was seen in the portions of the macrophage plasma membrane that were involved in the interaction process, in some cytoplasmic vacuoles and in the membrane lining the phagocytic vacuoles (Figs. 9, 10). In some cases, reaction product was observed on the surface of parasites located within phagocytic vacuoles (Fig. 9) and even in the cytoplasm of digested parasites (Fig. 10). It should be noted that no reaction product was detected in other portions of the macrophage plasma membrane that were not involved in the parasite-macrophage interaction. No reaction product was seen when catalase was added to the incubation medium (data not shown).
monas foetus with resident and activated mouse macrophages as examined using a parasite: macrophage ratio of 5 : 1. The percentage of macrophages associated with parasites, the mean number of associated parasites per macrophage (P/M) and the association index (A/) are shown, n, untreated T. foetus; [] antibody-treated
T. foetus
Discussion
In the present study we analysed the process of interaction of normal and antibody-coated Tritrichomonasfoe-
548
Figs. 2, 3. Light micrographs of the interaction of untreated (Fig. 2) and antibody-treated (Fig. 3) Tritrichomonasfoetus (P) with activated macrophages (M). The black reaction product (arrows) indicates nitroblue tetrazolium reduction, which occurred more frequently when antibody-coated parasites were used. x 65.
Figs. 4, 5. Fluorescence microscopy of the interaction of normal (Fig. 4) and antibody-coated (Fig. 5) T. foetus (P) with activated macrophages (M), showing fusion of Lucifer-yellow-labeled lysosomes with vacuoles containing parasites (arrows). Fig. 4, x 350; Fig. 5, x 230
tus with a mononuclear cell population. These cells were adherent, phagocytic and oxidatively active, characteristics that are typical ofmacrophages. As expected, macrophages ingested many more antibody-coated than untreated parasites. Ingestion of parasites was significantly enhanced by the addition of anti-Trichomonas specific antibody and by the use o f activated macrophages. Several authors (Landolfo et al. 1980; Mantovani etal. 1981; Martinotti et al. 1982, 1983) have suggested that
resident peritoneal cells exhibit very high cell-mediated cytotoxity (CMC) against T. vaginalis in the mouse as well as the human cell system (Mantovani et al. 1981). Some ingested parasites presented significant morphological changes, clearly indicating that they were killed by the macrophages. The oxidative activity of the macrophages was observed by light and transmission electron microscopy. The association of T.foetus with macrophages produced
549
Figs. 6, 7. Electron micrographs of parasites (P) inside macrophages (34). Fig. 6. Parasite ingested by a macrophage. Note the presence of vacuoles (V) containing gold-labeled peroxidase near the parasitophorus vacuole, x 9500. Fig. 7. High magnification
showing the fusion of lysosomes with phagosomes (arrowheads'), x 25000. Fig. 8. Detection of acid phosphatase activity (arrows) in the vacuole containing the parasite (P). M, Macrophage. • 18900
an oxidative burst as revealed by N B T reduction, which was higher in treated than in untreated parasites. Our observations are in agreement with those previously made in m a c r o p h a g e s ingesting Giardia lamblia (Hill and
Pohl 1990) as confirmed by transmission electron microscopy. It is k n o w n that the enzymes N A D H and N A D ( P ) H oxidase, which are localised in the plasma m e m b r a n e
550
Figs. 9, 10. Electron micrographs of antibody-treated parasites (P) allowed to interact with activated macrophages (M), showing the activity of NADH-oxidase localised on part of the macrophage surface (thick arrow, Fig. 10), in some cytoplasmic vacuoles (I0,
on the surface of an apparently intact intracellular parasite (thin arrows, Fig. 9) and in the cytoplasm of a digested parasite (asterisk, Fig. 10). Fig. 9, x45000; Fig. 10, x 27000
o f phagocytic cells, are responsible for the of oxygen intermediate derivatives during (Rossi et al. 1962; Cagan and Karnovsky novsky et al. 1982). Previous studies have
NAD(P)H-oxidase is internalised during endocytosis and retains its hydrogen-peroxide-generating capacity within the phagocytic vacuoles (Carvalho and De Souza 1987). During the process o f interaction between Trypan-
production endocytosis 1964; Karshown that
551 osoma cruzi and activated mouse macrophages, the reaction product indicative of enzyme activity was detected in the portion of the m a c r o p h a g e p l a s m a m e m b r a n e to which the parasites had attached as well as in the parasitophorous vacuole (Carvalho and De Souza 1987). It was suggested that an oxygen-derived product m a y enter into contact with extracellular parasites, which establish physical contact with the m a c r o p h a g e s as well as with intracellular parasites that are located within the parasitophorous vacuole. Reaction product has also been seen in association with m e m b r a n e s that surround vacuoles containing IgGcoated particles (Briggs etal. 1975; K a r n o v s k y etal. 1982; Hirai et al. 1985) and during the ingestion of untreated epimastigotes o f T. cruzi (Hirai et al. 1985) and amastigote and promastigote forms of Leishmania mexicana amazonensis (Pimenta and De Souza 1988). These observations contrast with those previously reported for untreated Toxoplasma gondii, which do not undergo respiratory burst during their interaction with macrophages (Murray and C o h n 1979; Carvalho and De Souza 1989). It is possible that the parasite stimulates enzyme activity during the initial attachment phase and that this activity continues inside the parasitophorous vacuole (Karnovsky et al. 1982; Pimenta and De Souza 1988). Our present observations clearly show that reaction product can be observed in association with parasite-containing vacuoles. In cases in which the parasites were significantly damaged, the reaction product was distributed throughout the parasite cytoplasm. Another potential mechanism of parasite killing by macrophages involves the fusion of phagosomes with lysosomes. We analysed this process using fluorescence and electron microscopy. For fluorescence microscopy, we used Lucifer yellow, a fluorescent dye that concentrates in the lysosomes (Swanson et al. 1983; H a r t et al. 1987). The dye was initially found in vesicles located around the vacuoles containing the parasites. Subsequently, parasite-containing vacuoles were also labeled, indicating that fusion between phagosomes and lysosomes took place. This observation was confirmed by electron microscopy, which revealed that structures that both accumulated gold-labeled peroxidase and corresponded to lysosomes (Carvalho et al. 1988) fused with the trichomonad-containing vacuoles. In addition, we also demonstrated the presence of acid phosphatase, a classic enzyme m a r k e r o f lysosomes (Robinson and Karnovsky 1983), within the parasite-containing phagocytic vacuole. Taken together, these observations represent an unequivocal demonstration that lysosome-phagosome fusion takes place during the T. f o e t u s - m a c r o p h a g e interaction. Our ultrastructural observations indicate that immediately after ingestion of the parasites by macrophages, lysosomes fuse with the phagocytic vacuoles that contain the parasites. At present, the mechanism that initiates killing of the parasites remains unknown. Is this killing triggered by respiratory burst, by fusion of the lysosome and the phagosome, or by the simultaneous action o f both of these mechanisms?
Acknowledgements. The authors thank Dr. F. Costa e Silva Eilho for critically reading the manuscript, Miss N. Rodrigues Gon~alves and Mr. A. de Oliveira for technical assistance, and Miss N. Cardoso de Oliveira for the skillful typing of the manuscript.
References Azevedo NL, Silva Filho FC, De Souza W (1991) The interaction of the Tritrichomonas .foetus and Trichomonas vaginalis with macrophages. J Submicrosc Cytol Pathol 23:319-326 Briggs RT, Darth DB, Karnovsky ML, Karnovsky MJ (1975) Localization of NADH-oxidase on the surface of human polymorphonuclear leucocytes by a new cytochemistry method. J Cell Biol 67 : 566 586 Cagan RH, Karnovsky ML (1964) Enzymatic basis of the respiratory stimulations during phagocytosis. Nature 204:255-256 Carvalho TU, De Souza W (1987) Cytochemical localization of NADH and NAD(P)H-oxidase during interaction of Trypanosoma cruzi with activated macrophages. Parasitol Res 73:213217 Carvalho L, De Souza VV (1989) Cytochemical localization of plasma membrane enzyme markers during interiorization of tachyzoites of Toxoplasma gondii by macrophages. J Protozool 36:164-170 Carvalho TU, Souto-Padr6n T, De Souza W (1988) The use of albumin-gold to follow lysosome-phagosome fusion. J Submicrosc Cytol Pathol 20: 773-776 Diamond LS (1957) The establishment of various trichomonads from animals and man in axenic cultures. J Protozool 43:488490 Guida HG, Ramos AA, Coelho NM, Ramos JA, Mendonza IR (1982) Incidencia de Tritrichomonasfoetus ern reprodutores bovinos da regiao centro-sul do Brasil. Pesq Agrop Bras Ser Vet 7:23-25 Hammond DM, Bartlett DE (1943) The distribution of Trichomonas foetus in the prepucial cavity of infected bulls. Am J Vet Res 4:143-149 Hart PD, Young MR, Gordon AH, Sullivan KH (1987) Inhibition of phagosome-lysosome fusion in macrophages by certain my_ cobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis. J Exp Med 166:933-946 Hill DR, Pohl R (1990) Ingestion of Giardia lamblia trophozoites by murine Peyer's patch macrophages. Infect Immun 58 : 320~ 3207 Hirai K, Ohno Y, Ogawa K (1985) Cytochemistry of hydrogen peroxide production by culture activated macrophages in phagocytosis. Acta Histochem Cytochem 18:69-73 Honigberg BM (1961) Comparative pathogenicity of Trichomonas vaginalis and Trichomonas gallinae to mice: I. Gross pathology, quantitative evaluation of virulence, and some factors affecting pathogenicity. J Parasitol 47:545-571 Karnovsky MJ, Robinson JM, Briggs RT, Karnovsky ML (1982) A cytochemical approach to the function of phagocytic leucocytes. In: Karnovsky ML, Bolls L (eds) Phagocytosis, past and future. Academic Press, New York, pp 215-239 Kielian MC, Cohn ZA (1980) Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages. J Cell Biol 85:754-776 Landolfo S, Martinotti MG, Martinetto P, Forni G (J980) Naturai cell-mediated cytotoxity against Trichomonas vaginalis in the mouse. Tissue, strain age distribution and some characteristics of the effector cells. J Immunol 124: 508-514 Mantovani A, Polentarutti N, Peri G, Martinotti G, Landolfo S (1981) Cytotoxity of human peripheral blood monocytes against Trichomonas vaginalis. Clin Exp Immunol 46:391-396 Martinotti MG, Gallione MA, Martinetto P, Landolfo S (1982) Role of cytoskeleton in natural cell-mediated cytotoxity against Trichomonas vaginalis. Microbiology 5:389-391
552 Martinotti MG, Cofano F, Martinetto P, Landolfo S (1983) Natural macrophage cytotoxity against Trichomonas vaginalis is mediated by soluble lytic factors. Infect Immun 41 : 1144-1149 Murray HW, Cohn ZA (1979) Macrophage oxygen-dependent antimicrobicidal activity : I. Susceptibility of Toxoplasma gondii to oxygen intermediates. J Exp Med 150:938-949 Murray HW, Cohn ZA (1980) Macrophage oxygen-dependent antimicrobial activities: III. Enhanced oxidative metabolism as an expression of macrophage activation. J Exp Med 152:15961609 Nogueira N, Cohn ZA (1978) Trypanosoma cruzi: "in vitro" induction of macrophage microbicidal activity. J Exp Med 148: 288300 Parsonson IM, Clark BL, Duffey JH (1976) Early pathogenesis of pathology of Tritrichomonasfoetus infection in virgin heifers. J Comp Pathol 86 : 59-66 Pimenta PFP, De Souza W (1988) Freeze-fracture and cytochemistry study of the interaction between Leishmania mexicana amazonensis and macrophages. J Submicrosc Cytol 20: 89-99
Robinson IM, Karnovsky MJ (1983) Ultrastructural localization of several phosphatases with cerium. J Histochem Cytochem 31 : 1197-1208 Rossi F, Romero D, Patriarcha P (1962) Mechanism of phagocytosis associated oxidative metabolism in polymorphonuclear leukocytes and macrophages. J Reticuloendothel Soc 12:127-149 Saraiva EMB, Andrade AFB, De Souza W (1987) Involvement of the macrophage mannose-6-phosphate receptor in the recognition of Leishmania mexicana amazonensis. Parasitol Res 73:411416 Silva-Filho FC, Elias CA, De Souza W (1986) Further studies on the surface charge of various strains of Tritrichomonas foetus. Cell Biophys 8:161-176 Swanson JA, Yrinee BD, Silverstein SC (1983) Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol 100:851859
Parasitnlngy Research
9 Springer-Verlag1992
A cytochemical study of the interaction between Tritrichomonas foetus and mouse macrophages* Neide L. A z e v e d o 1,2 and Wanderley de S o u z a 1
1 Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund/io, 21949, Rio de Janeiro, Brasil 2 Departamento de Histologia, Instituto de Biologia, Universidade to Estado do Rio de Janeiro, Brasil Accepted April 25, 1992
Abstract. Light and electron microscopy were used to
analyse the process of interaction of normal and antibody-coated Tritrichomonasfoetus with resident and activated mouse peritoneal macrophages. Activated macrophages ingest m o r e parasites than do resident macrophages. Previous incubation o f the parasites in the presence of sub-agglutinating concentrations of a polyclonal anti-T, foetus antibody significantly increased their ingestion by the macrophages. Adherence of the parasites to the surface of activated m a c r o p h a g e s triggers the respiratory oxidative burst as revealed by reduction of nitroblue tetrazolium. This process was more evident in antibody-coated parasites. Transmission electron microscopy showed the presence o f reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the portions of the m a c r o p h a g e plasma m e m b r a n e that were in contact with the parasites as well as in the phagocytic vacuoles. Fusion of m a c r o p h a g e lysosomes with parasite-containing phagocytic vacuoles was observed in macrophages labeled with Lucifer yellow and gold-labeled peroxidase as well as by localisation of acid phosphatase.
Tritrichomonas foetus is the causative agent of bovine trichomoniasis. In the female host, it causes a venereal disease characterised by mild endometritis and, occasionally, p y o m e t r a and abortion (Parsonson et al. 1976). In the male host, T.foetus appears to be confined almost exclusively to the prepucial cavity, usually manifesting as an a s y m p t o m a t i c infection ( H a m m o n d and Bartlett 1943). Although a good experimental model for trichomoniasis is not presently available, mice have been used to analyse the pathogenicity of various strains o f Triehomonas tenax and T. vaginalis (Honigberg t961). * This work was supported by Financiadora de Estudos e Projetos (FINEP) and Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) Correspondence to .' W. de Souza
In the case of h u m a n trichomoniasis caused by T. circumstantial evidence suggests that the m o n o c y t e - m a c r o p h a g e lineage plays some role in the process of killing o f the parasite (Landolfo et al. 1980). In a previous study we have shown that mouse macrophages are capable of ingesting T.foetus, especially when the parasite surface has been modified by previous treatment with concanavalin A (Azevedo et al. 1991). To analyse further the interaction between T. foetus and macrophages, we investigated (a) whether attachment of the parasites to the m a c r o p h a g e surface would trigger the respiratory burst and (b) whether host-cell lysosomes would fuse with parasite-containing phagocytic vacuoles. The results obtained are described in this paper.
vaginalis,
Materials and methods
Parasites The K strain of Tritrichomonasfoetus was originally isolated by Guida et al. (1982; EMBRAPA, Rio de Janeiro, Brazil) from the urogenital tract of a bull in the state of Rio de Janeiro, Brazil, and was characterised by Silva-Filho et al. (1986). The parasites were placed in TYM medium (Diamond 1957) supplemented with 10% bovine calf serum and then incubated at 37~ C. Parasites obtained from the mid-log phase of growth were centrifuged at 600 g for l0 rain, washed twice in phosphate-buffered saline (PBS, pH 7.2) and counted using a Neubauer chamber, and their viability was evaluated using a trypan blue dye-exclusion test. The parasites were re-suspended in 199 medium (Sigma Chemical Co., USA) in the absence of serum. In other experiments, the living parasites were incubated for 30 min at 4~ C in the presence of heat-inactivated anti-Trichomonasserum at a 1:4000 dilution (sub-agglutinating dilution) prior to their interaction with the macrophages. After being washed with PBS at 4~ C, these parasites were re-suspended in 199 medium.
Resident macrophages Peritoneal macrophages obtained from normal Swiss mice were used. The animals were killed with ether and their peritoneal cavities were washed with Hanks' solution. Cells were plated on glass
546 coverslips in Falcon 24-well tissue-culture plates (Becton & Dickinson Labware, New Jersey) and maintained in a humidified atmosphere containing 5% COz at 37~ C. After 30 min, non-adherent cells were removed and the macrophages were cultivated for 24 h in 199 medium plus 10% foetal calf serum.
Activated macrophages Swiss mice were infected intraperitoneally with 5 x 106 viable noninfective epimastigote forms of Trypanosoma cruzi and were boosted 3 weeks later with an intraperitoneal injection of the homologous antigen. Previous studies have shown that the behaviour of mouse macrophages activated in such a way is similar to that of those activated using Listeria or bacille Calmette-Gu~rin (BCG; Nogueira and Cohn 1978; Carvalho and De Souza 1987). After 3 days, the animals were killed with ether, their peritoneal cavities were washed with 5 ml Hanks' solution in the absence of serum and the collected cells were plated into Falcon 24-well tissue-culture plates for light microscopy and onto the walls of glass flasks for electron microscopy. They were allowed to adhere to the glass surface for 30 min at 37~ and were then washed with Hanks' solution to remove the cells that had not adhered. Culture medium (199 medium plus 10% foetal calf serum) was added and the cells were incubated for 24 h at 37 ~ C, washed with 199 medium in the absence of serum and then used for the experiments.
Parasite-macrophage interaction The parasite-macrophage ratio was adjusted to 5:1 as based on the number of cells adhering to the wells and on the parasite density. In some experiments, the parasites were first incubated for 30 min at 4 ~ C in the presence of a 1:4000 dilution of immune serum (sub-agglutinating concentration) and then rinsed with PBS and allowed to interact with the macrophages. After interaction periods of 15, 30 or 60 min, the parasite-macrophage preparations were rinsed with PBS to remove non-attached parasites and fixed as described below.
Light microscopy The cultures were fixed with Bouin's solution and stained with Giemsa. The percentage of macrophages with associated parasites, the mean number of associated parasites per infected macrophage and the association index were calculated as previously described by Saraiva et al. (1987), whereby all preparations were examined using a Zeiss Universal Photomicroscope equipped with an immersion objective.
Fluorescence assay of phagosome-lysosome fusion Phagosome-lysosome (P-L) fusion in activated mouse macrophages was assayed by monitoring the transfer of a fluorescent dye from lysosomes to phagocytic vacuoles as previously described by Kielian and Cohn (1980). The macrophages were incubated at 37~ C for 2 h in Lucifer yellow (1 mg/ml), washed in 199 medium, and then incubated for 15 min at 37~ with the parasites (parasitemacrophage ratio, 5:1). The cells were examined by fluorescence microscopy. The presence of yellow-stained Trichomonaswas considered to be indicative of P-L fusion.
ed; ratio adjusted to 5:1) in 1 ml 199 medium containing 0.5 mg nitroblue tetrazolium (NBT)/ml (grade 111, Sigma Chemical Co. ; Murray and Cohn 1980). Uningested parasites were removed by washing, and the coverslips were reincubated in medium alone for an additional 30-min period. Intracellular parasites were identified using phase-contrast light microscopy and were then viewed by bright-field microscopy. Macrophages were scored as positive if ingested parasites were stained blue-black by precipitated formazan, the oxygen-dependent reduction product of NBT.
Transmission electron microscopy For transmission electron microscopy the macrophages were plated into 25-cm z glass flasks. After 15 or 30 min interaction between activated or resident macrophages, respectively and the parasites, the cultures were fixed for 60 min at room temperature in a solution containing 4% paraformaldehyde, 1% glutaraldehyde and 5 mM CaClz in 0.1 M cacodylate buffer (pH 7.2). After fixation, the cultures were rinsed in cacodylate buffer and left overnight at 4 ~ C in the same buffer. The cells were gently scraped off with a rubber policeman, post-fixed with 1% OsO, in 0.1 M cacodylate buffer supplemented with 0.8 M potassium ferrocyanide, washed in the same buffer, dehydrated in acetone and embedded in Epon. U1trathin sections were stained with uranyl acetate and lead citrate and observed in a Zeiss EM 900 or EM 902 electron microscope.
Ultrastructural cytochemistry The enzymes reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase and acid phosphatase were used as markers of O f production during the process of destruction of T. foetus by activated macrophages and for labeling of secondary lysosomes, respectively. The labeling of secondary lysosomes was also accomplished using peroxidase-gold. For detection of NAD(P)H-oxidase activity, the medium described by Briggs et al. (1975) was used. After activated macrophage-parasite interaction, the cells were washed at 4 ~ C in 0.1 ~ TRIS-maleate buffer (pH 7.5) containing 7% sucrose and were first incubated for 10 min at 37 ~ C in medium containing 0.1 M TRIS-maleate buffer (pH 7.5) supplemented with 7% sucrose and 1 mM [3-amino-l,2,4]-triazole (AT) and subsequently incubated for 20 rain at 37 ~ C in medium containing 0.1 M TRIS-maleate buffer (pH 7.5) supplemented with 7% sucrose, 0.71 mM NAD(P)H as the substrate, 2 mM CeC13 as the capture agent, and 10 mM AT. The medium used for control preparations lacked the substrate and AT and contained 0.01% catalase.
Acid phosphatase For detection of acid phosphatase activity, the method of Robinson and Karnovsky (1983) was used as follows. After interaction, the cells were fixed for 30 min at 4 ~ C in 1% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH 7.2). They were then rinsed in buffer and pre-incubated at room temperature for 30 rain in medium containing 1 mM CeC13, 5% sucrose and 0.05 N TRIS-maleate buffer (pH 5.0). Thereafter, the cells were incubated at 37~ C for 60 min in medium containing 2 mM CeCla, 7 mM Na-~-glycerophosphate, 5% sucrose and 0.05 M TRIS-maleate buffer (pH 5.0). Control cells were incubated in medium in the absence of the substrate. After incubation, the ceils were rinsed in cacodylate buffer, re-fixed in a solution containing 4% paraformaldehyde and 1% glutaraldehyde in cacodylate buffer (pH 7.2) for 1 h at room temperature and processed for electron microscopy as described above.
Qualitative nitroblue tetrazolium reduction
Gold-labeled peroxidase
Activated macrophages were exposed for 15 min at 37 ~ C (atmosphere, 5% COa/95% air)to T.foetus (control and antibody-coat-
For labeling of secondary lysosomes, the macrophages were incubated at 37~ C for 2 h in medium containing gold-labeled peroxi-
547
dase (Carva[ho et al. 1988) and 199 medium (1:20, v/v). After incubation, the cells were rinsed twicein 199 medium. The interaction between parasites and macrophages (ratio, 5:1) was allowed to proceed at 37~ C for 30 min. Thereafter, the preparations were washed three timeswith PBS and then fixedfor 60 min in a solution containing 1% glutaraldehyde,4% paraformaldehydeand 5 mM CaClz in 0.1 Mcacodylatebuffer (pH 7.2). After fixation, the cultures were processed for electron microscopyas described above. Results
Light microscopy Kinetics of the interaction. Observation of Giemsastained preparations showed that resident and activated macrophages ingested control and antibody-coated parasites. After an interaction period of 30 rain, the ingestion of untreated parasites by activated macrophages was 2-fold that observed for resident macrophages (Fig. 1). For both types of macrophages the ingestion was significantly increased when the parasites had previously been incubated in the presence of a sub-agglutinating concentration of anti-Tritrichomonas foetus polyclonal antibodies. This effect was attributable to an increase in the percentage of macrophages that ingested parasites and in the mean number of ingested parasites per macrophage. Incubation in the presence of the antibody did not induce alterations in the parasite.
Oxidative response. Association of the parasites with macrophages, especially with activated macrophages, led to the induction of respiratory burst as indicated by reduction of nitroblue tetrazolium. A black reaction prod100
R E S I D E NT
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Fig. 1. Effect of an immune serum on the association of Tritricho-
uct was seen in the responsive cells. The percentage of reactive cells was significantly higher ,when antibodycoated parasites were used (Figs. 2, 3).
Lysosome-phagosomefusion. Lucifer yellow-labeled macrophage lysosomes that had been allowed to interact with the parasites were examined by fluorescence microscopy. P-L fusion was clearly indicated by the presence of many labeled vacuoles that contained parasites (Figs. 4, 5). No significant difference in the level of P-L fusion was observed between preparations containing untreated parasites and those containing antibody-treated parasites.
Electron microscopy Ultrastructural observations of the trichomonad-macrophage interaction revealed an intense process of pseudopod formation at sites of parasite-cell contact. After internalisation of the parasites, they were found to be lodged in membrane-bound vacuoles (data not shown). Electron microscopy was also used to analyse two aspects of the process of trichomonad-macrophage interaction: (1) P-L fusion and (2) NAD(P)H-oxidase activation. For further demonstration of fusion of macrophage lysosomes with parasite-containing vacuoles, macrophages were incubated in the presence of gold-labeled peroxidase, which accumulate in the lysosomes. Subsequently, these macrophages were incubated in the presence of the parasites. Figures 6 and 7 clearly show that labeled lysosomes concentrated around the phagocytic vacuole and later discharged their contents into the vacuole (Fig. 7). Acid phosphatase, a classic enzyme marker of lysosomes, was detected within many but not all phagocytic vacuoles. Reaction product, indicative of enzyme activity, was restricted to the peripheral region of the vacuole (Fig. 8). For demonstration of NAD(P)H-oxidase activity, the macrophages were incubated for 15 min in the presence of untreated or antibody-treated parasites and then in a medium designed to locate the enzyme. Reaction product, indicative of enzyme activity, was seen in the portions of the macrophage plasma membrane that were involved in the interaction process, in some cytoplasmic vacuoles and in the membrane lining the phagocytic vacuoles (Figs. 9, 10). In some cases, reaction product was observed on the surface of parasites located within phagocytic vacuoles (Fig. 9) and even in the cytoplasm of digested parasites (Fig. 10). It should be noted that no reaction product was detected in other portions of the macrophage plasma membrane that were not involved in the parasite-macrophage interaction. No reaction product was seen when catalase was added to the incubation medium (data not shown).
monas foetus with resident and activated mouse macrophages as examined using a parasite: macrophage ratio of 5 : 1. The percentage of macrophages associated with parasites, the mean number of associated parasites per macrophage (P/M) and the association index (A/) are shown, n, untreated T. foetus; [] antibody-treated
T. foetus
Discussion
In the present study we analysed the process of interaction of normal and antibody-coated Tritrichomonasfoe-
548
Figs. 2, 3. Light micrographs of the interaction of untreated (Fig. 2) and antibody-treated (Fig. 3) Tritrichomonasfoetus (P) with activated macrophages (M). The black reaction product (arrows) indicates nitroblue tetrazolium reduction, which occurred more frequently when antibody-coated parasites were used. x 65.
Figs. 4, 5. Fluorescence microscopy of the interaction of normal (Fig. 4) and antibody-coated (Fig. 5) T. foetus (P) with activated macrophages (M), showing fusion of Lucifer-yellow-labeled lysosomes with vacuoles containing parasites (arrows). Fig. 4, x 350; Fig. 5, x 230
tus with a mononuclear cell population. These cells were adherent, phagocytic and oxidatively active, characteristics that are typical ofmacrophages. As expected, macrophages ingested many more antibody-coated than untreated parasites. Ingestion of parasites was significantly enhanced by the addition of anti-Trichomonas specific antibody and by the use o f activated macrophages. Several authors (Landolfo et al. 1980; Mantovani etal. 1981; Martinotti et al. 1982, 1983) have suggested that
resident peritoneal cells exhibit very high cell-mediated cytotoxity (CMC) against T. vaginalis in the mouse as well as the human cell system (Mantovani et al. 1981). Some ingested parasites presented significant morphological changes, clearly indicating that they were killed by the macrophages. The oxidative activity of the macrophages was observed by light and transmission electron microscopy. The association of T.foetus with macrophages produced
549
Figs. 6, 7. Electron micrographs of parasites (P) inside macrophages (34). Fig. 6. Parasite ingested by a macrophage. Note the presence of vacuoles (V) containing gold-labeled peroxidase near the parasitophorus vacuole, x 9500. Fig. 7. High magnification
showing the fusion of lysosomes with phagosomes (arrowheads'), x 25000. Fig. 8. Detection of acid phosphatase activity (arrows) in the vacuole containing the parasite (P). M, Macrophage. • 18900
an oxidative burst as revealed by N B T reduction, which was higher in treated than in untreated parasites. Our observations are in agreement with those previously made in m a c r o p h a g e s ingesting Giardia lamblia (Hill and
Pohl 1990) as confirmed by transmission electron microscopy. It is k n o w n that the enzymes N A D H and N A D ( P ) H oxidase, which are localised in the plasma m e m b r a n e
550
Figs. 9, 10. Electron micrographs of antibody-treated parasites (P) allowed to interact with activated macrophages (M), showing the activity of NADH-oxidase localised on part of the macrophage surface (thick arrow, Fig. 10), in some cytoplasmic vacuoles (I0,
on the surface of an apparently intact intracellular parasite (thin arrows, Fig. 9) and in the cytoplasm of a digested parasite (asterisk, Fig. 10). Fig. 9, x45000; Fig. 10, x 27000
o f phagocytic cells, are responsible for the of oxygen intermediate derivatives during (Rossi et al. 1962; Cagan and Karnovsky novsky et al. 1982). Previous studies have
NAD(P)H-oxidase is internalised during endocytosis and retains its hydrogen-peroxide-generating capacity within the phagocytic vacuoles (Carvalho and De Souza 1987). During the process o f interaction between Trypan-
production endocytosis 1964; Karshown that
551 osoma cruzi and activated mouse macrophages, the reaction product indicative of enzyme activity was detected in the portion of the m a c r o p h a g e p l a s m a m e m b r a n e to which the parasites had attached as well as in the parasitophorous vacuole (Carvalho and De Souza 1987). It was suggested that an oxygen-derived product m a y enter into contact with extracellular parasites, which establish physical contact with the m a c r o p h a g e s as well as with intracellular parasites that are located within the parasitophorous vacuole. Reaction product has also been seen in association with m e m b r a n e s that surround vacuoles containing IgGcoated particles (Briggs etal. 1975; K a r n o v s k y etal. 1982; Hirai et al. 1985) and during the ingestion of untreated epimastigotes o f T. cruzi (Hirai et al. 1985) and amastigote and promastigote forms of Leishmania mexicana amazonensis (Pimenta and De Souza 1988). These observations contrast with those previously reported for untreated Toxoplasma gondii, which do not undergo respiratory burst during their interaction with macrophages (Murray and C o h n 1979; Carvalho and De Souza 1989). It is possible that the parasite stimulates enzyme activity during the initial attachment phase and that this activity continues inside the parasitophorous vacuole (Karnovsky et al. 1982; Pimenta and De Souza 1988). Our present observations clearly show that reaction product can be observed in association with parasite-containing vacuoles. In cases in which the parasites were significantly damaged, the reaction product was distributed throughout the parasite cytoplasm. Another potential mechanism of parasite killing by macrophages involves the fusion of phagosomes with lysosomes. We analysed this process using fluorescence and electron microscopy. For fluorescence microscopy, we used Lucifer yellow, a fluorescent dye that concentrates in the lysosomes (Swanson et al. 1983; H a r t et al. 1987). The dye was initially found in vesicles located around the vacuoles containing the parasites. Subsequently, parasite-containing vacuoles were also labeled, indicating that fusion between phagosomes and lysosomes took place. This observation was confirmed by electron microscopy, which revealed that structures that both accumulated gold-labeled peroxidase and corresponded to lysosomes (Carvalho et al. 1988) fused with the trichomonad-containing vacuoles. In addition, we also demonstrated the presence of acid phosphatase, a classic enzyme m a r k e r o f lysosomes (Robinson and Karnovsky 1983), within the parasite-containing phagocytic vacuole. Taken together, these observations represent an unequivocal demonstration that lysosome-phagosome fusion takes place during the T. f o e t u s - m a c r o p h a g e interaction. Our ultrastructural observations indicate that immediately after ingestion of the parasites by macrophages, lysosomes fuse with the phagocytic vacuoles that contain the parasites. At present, the mechanism that initiates killing of the parasites remains unknown. Is this killing triggered by respiratory burst, by fusion of the lysosome and the phagosome, or by the simultaneous action o f both of these mechanisms?
Acknowledgements. The authors thank Dr. F. Costa e Silva Eilho for critically reading the manuscript, Miss N. Rodrigues Gon~alves and Mr. A. de Oliveira for technical assistance, and Miss N. Cardoso de Oliveira for the skillful typing of the manuscript.
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