199 (M. A. Bioproducts, Walkersville, Md.). placed on 22-mm2 cover slips and incubated at 37 C in

Transcription

1 INFECTION AND IMMUNITY, June 1981, p /81/ $02.00/0 Vol. 32, No. 3 Interaction of Leishmania donovani Promastigotes with Human Monocyte-Derived Macrophages: Parasite Entry, Intracellular Survival, and Multiplication RICHARD D. PEARSON,* ROSEANN ROMITO, PHILIP H. SYMES, AND JANE L. HARCUS Department of Medicine, Division of Geographic Medicine, University of Virginia School ofmedicine, Charlottesville, Virginia Leishmania donovani promastigotes were incubated with human monocytederived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, % of the macrophages were infected with a mean of parasites per infected cell. After 6 days of incubation, % of the macrophages were infected with 15.9 ± 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 ± 0.8 x 105 to 1.8 ± 0 x 106 (P < 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication. Leishmania donovani, a dimorphic protozoan, is an important cause of human morbidity and mortality in numerous areas in the developing world. In humans, it resides within mononuclear phagocytes as a round or oval, 2- to 4-,um amastigote without a flagellum visible by light microscopy. It exists as an extracellular, 3- to 4-tLm by 15- to 20-nm, spindle-shaped, flagellated promastigote in the gut of the Phlebotomus sandfly, its insect vector. Little is known about the sequence of events that follows inoculation of promastigotes into a susceptible host. The finding of amastigotes within mononuclear phagocytes in an established infection suggests that macrophages play an important role in the early stage of infection by phagocytizing promastigotes and protecting them from other potentially effective humoral (11) and phagocytic host defense mechanisms (R. D. Pearson and R. T. Steigbigel, Clin. Res. 28: 376, 1980). To assess the role of macrophages in the early stage of visceral leishmaniasis, L. donovani promastigotes were incubated with human monocyte-derived macrophages. Promastigotes attached to macrophages with either their flagellar or aflagellar ends. Macrophages subsequently phagocytized promastigotes, permitted the conversion of promastigotes to an amastigote-like state, and supported intracellular multiplication. MATERIALS AND METHODS Mononuclear ceus were obtained by Ficoll-sodium diatrizoate (Histopaque; Sigma Chemical Co., St. Louis, Mo.) sedimentation of peripheral blood from five healthy adult donors and were suspended in medium 199 (M. A. Bioproducts, Walkersville, Md.). Mononuclear cells (5 x 106) in 0.5 ml of medium were placed on 22-mm2 cover slips and incubated at 37 C in 5% C02-95% room air. After 2 h, 1.5 ml of medium 199 supplemented with penicillin (100 U/ml), gentamicin (50 [Lg/ml), and 20% autologous human serum was added. Nonadherent cells were removed after 24 h. The monolayers were washed with warm (37 C) medium 199 and then flooded with fresh medium, which contained 13% autologous serum and antibiotics. Cell monolayers were used for study on day 5 after being washed with medium 199. The number of macrophages on each cover slip was determined by visual enumeration. Each cover slip contained between 1.5 x 105 and 5.0 x 105 adherent cells. More than 98% of the adherent cells were found to be esterase positive, which indicated that they were macrophages. Promastigotes were derived from amastigotes by methods previously described (11). In short, a Sudanese strain of L. donovani was maintained by serial intracardiac inoculation of amastigotes into Syrian hamsters. Amastigotes were obtained from the spleens of hamsters infected 4 to 6 weeks previously and were suspended in a modification of minimal essential medium described by Berens et al. (4), to which 10% heatinactivated fetal calf serum, penicillin, and gentamicin 1249

2 1250 PEARSON ET AL. had been added. Incubation was carried out at 26 C in 5% C02-95% room air. Promastigotes were harvested on days 3 through 8 and were used for study after two washes in phosphate-buffered saline, or they were passaged into fresh medium and used for study before day 14. At the time of study, monocyte-derived macrophages were incubated with promastigotes at a parasite-to-phagocyte ratio of 20:1 in 1 ml of modified minimal essential medium containing 10% heat-inactivated (56 C, 30 min) autologous serum at 37 C in 5% C02-95% room air. Early promastigote-phagocyte interactions were characterized by scanning electron and light microscopy at 30 and 60 min. After 2 h of incubation, residual extracellular promastigotes were removed, and the monolayers were flooded with medium 199 which contained 13% fresh autologous serum. Fresh serum has been shown to lyse cell-free promastigotes (11). The monolayers were cultivated at 37 C and the medium was changed at 72 h. At days 1, 2, 3, 5 and 7, duplicate monolayers were fixed and stained with Giemsa. The percentage of infected macrophages, the number of parasites per infected macrophage, the INFECT. IMMUN. number of parasites per 100 total macrophages, and the number of parasites per monolayer were determined by blind examination of -200 macrophages on each cover slip. RESULTS Promastigotes attached to monocyte-derived macrophages with either their flagellar or aflagellar end. Long, thin pseudopodia formed around promastigotes (Fig. 1). At 2 h, cell-associated parasites looked like promastigotes under the light microscope. After 24 h, the parasites appeared morphologically similar to the small, ovoid amastigotes seen in infected hamster spleens. Transmission electron micrographs demonstrated that the parasites were within vacuoles (Fig. 2). Dividing forms were observed in some vacuoles. Intracellular parasites were morphologically indistinguishable by light and electron microscopy from amastigotes obtained from infected hamster spleens. Downloaded from on July 11, 2018 by guest FIG. 1. Scanning electron micrograph demonstrating the formation ofa macrophage (m)pseudopod (arrow) around the flagellum of an L. donovani promastigote (Ld). Human monocyte-derived macrophages were incubated with 10%o heat-inactivated (56 C, 30 min) serum and promastigotes at a parasite-to-macrophage ratio of 20:1 at 37 C in 5% C02-95% room air for 30 min.

3 A4 r.1 1 '".- pp. -' p.., 'I 9 0r `7',. I'flSf' p AMt pit' / 4,s. 'A * I, A Uo $4 1 o_jb *,, - jt,,-4 A. F-l 5p FPir> Downloaded from f _.e,.-a ~ ~ F-'': u*.:. p on July 11, 2018 by guest VE z f. _!,' 4LL,* ; 41sU ti. II I.- s i' ~va W';- FIG. 2. Transmission electron micrographs demonstrating L. donovani within monocyte-derived macrophages. (A) Multiple parasites (arrows) were observed within single macrophages. (B) Parasites (Ld) were located within vacuoles in macrophages (m). Macrophages were incubated with promastigotes at a parasiteto-macrophage ratio of 20:1 in the presence of 10%o heat-inactivated (560C, 30 min) serum and antibiotics at 37 C in 5% C02-95% room air. Extracellular parasites were removed after 2 h, and medium 199 with 13%7o fresh autologous serum was added. Five days after infection, monolayers were fixed in 2%o glutaraldehyde and prepared for transmission electron microscopy.

4 1252 PEARSON ET AL. The monocyte-derived macrophages from five donors phagocytized promastigotes and supported intracellular multiplication (Table 1). There was a significant increase in the number of parasites per macrophage (P < 0.05, paired t test) and per monolayer (P < 0.05) from day 1 to day 7. Histograms of the data from individual experiments demonstrated a shift toward greater numbers of parasites per infected cell (Fig. 3). On days 5 and 7, some macrophages had -30 parasites per cell. Such heavily parasitized cells were not observed on day 1. The percentage of infected macrophages also increased, but the difference was not significant. Parasites free from phagocytes were not observed in any sample. There was a gradual loss of macrophages from monolayers after day 3. On day 5, the mean number of macrophages per cover slip had decreased by % and on day 7, by %. Cells of one donor were studied on two occasions; there was multiplication of intracellular parasites on one occasion, but on the other occasion, there was a progressive decline in the percentage of infected macrophages and the number of parasites per macrophage. Only data from the first experiment were included in Table 1. DISCUSSION The results indicate that human monocytederived macrophages have the capacity to engulf L. donovani promastigotes, permit the intracellular conversion of the promastigotes to an amastigote-like state, and support intracellular multiplication. Macrophage pseudopodia formed around promastigotes after attachment and enclosed them in vacuoles. Fusion of the phagosomes with lysosomes was not studied, but phagosome-lysosome fusion after phagocytosis of amastigotes by hamster peritoneal (8) and human monocyte-derived macrophages (5) has been demonstrated. Intracellular parasites assumed the morphological characteristics of amastigotes. Additional study will be necessary to determine whether the parasites have the TABLE 1. Association of L. donovani with human monocyte-derived macrophagesa Macro- L. dono- L. donovani phages infected per 100 vani total per No. of L. donovani Day with L. macro- macro- per monodonovani (') phage phages layer (105) (no.) (no.) ± ± ± ± ±72 5.0± ± ± ± afor details, see text. Values represent the mean ± standard error of the mean with macrophages from five donors. I- z w U- a: w H 50 H H 20 H I 0 INFECT. IMMUN. [1 isuijieeei I 0 1-D E W2j >30 PARASITES /MACROPHAGE FIG. 3. Histogram of data from a representative experiment illustrating frequency distribution of L. donovani within macrophages on day 1 (open bars) and day 7 (hatched bars) of infection. The percentage of infected macrophages is given as a function of the number ofparasites per macrophage. antigenic and biochemical characteristics of amastigotes obtained from in vivo preparations. Parasites multiplied over the period of study. There was a significant increase in the number of parasites per infected macrophage and in the total number of parasites per monolayer. The actual increase in cell-associated parasites may have been greater than was observed. Some parasites may have been missed in the heavily parasitized cells found late in the experiment. In addition, macrophage attrition occurred over the course of the study, and parasites associated with macrophages which had released from the monolayers were not counted. The rate of intracellular parasite multiplication in our study was similar to that reported by Berman et al. (5) for human monocyte-derived macrophages infected with L. donovani amastigotes obtained from hamster spleens. We also observed dividing parasite forms within phagocytic vacuoles. This suggests, but does not prove, intracellular multiplication. Dividing forms are found among axenically grown promastigotes. Phagocytosis of a dividing promastigote followed by intracellular conversion to an amastigote-like state without completion of cell division could simulate intracellular amastigote division. Failure of intracellular multiplication occurred in one experiment with cells from a single donor. In a second experiment, macrophages from that donor supported multiplication. It is possible that the promastigotes used in the first experiment were damaged during preparation or that the macrophages were activated by an unidentified stimulus and killed the parasites. Chang (6) observed that hamster peritoneal

5 VOL. 32, 1981 macrophages can engulf L. donovani promastigotes, but he found that intracellular parasites degenerated in phagosomes. Poor survival of Leishmania promastigotes in animal macrophages has been reported by others (2, 9, 12) as well. Chang (7) recently described a murine macrophage tumor cell line in which promastigotes of Leishmania mexicana convert to an amastigote-like state and multiply. It is difficult to generalize from the preceding studies to the in vivo situation in humans because intracellular survival of Leishmania promastigotes may have species-specific characteristics, as Mauel et al. (10) demonstrated with Leishmania enriettii, and because tumor cell lines may differ from normal macrophages in microbicidal activity. In vitro infection of human monocyte-derived macrophages avoids these variables and serves as an excellent model for study of the initial sequence of events in visceral leishmaniasis. In humans, macrophages may provide a sanctuary in which promastigotes escape destruction by serum factors and polymorphonuclear leukocytes. Promastigotes are subject to destruction by fresh nonimmune serum (1, 3, 13) via classical pathway activation of the complement membrane attack complex (C5b-C9) (11). Promastigotes are also rapidly phagocytized and killed by human polymorphonuclear leukocytes in the presence of fresh, but not heat-inactivated (560C, 30 min), nonlimmune serum which lacks complement activity (Pearson and Steigbigel, Clin. Res. 28:376, 1980). Phagocytosis of promastigotes by human monocyte-derived macrophages occurred in the absence of complement in our study. In nature, promastigotes may be inoculated at a cutaneous site where complement is not active or is locally inactivated by sandfly factors, such as saliva. Thus, the parasite would be protected from destruction by complement and polymorphonuclear leukocytes, and the likelihood would increase that it would safely reach the confines of the macrophage where it has been shown to prosper. Further study is needed to determine the sequence of events in vivo. L. DONOVANI: MACROPHAGE INTERACTION 1253 ACKNOWLEDGMENTS The project was supported in part by a grant from The Rockefeller Foundation and by a Biomedical Research Support Grant to the University of Virginia School of Medicine from the National Institutes of Health. We are indebted to Richard L. Guerrant for his helpful suggestions; to Sydney S. Breese, Associate Director of the Central Electron Microscope Facility at the University of Virginia School of Medicine, for assistance in obtaining electron micrographs; and to Margaret A. Busch for help in preparing this manuscript. LITERATURE CITED 1. Adler, S Attempts to transmit visceral leishmaniasis to man. Trans. R. Soc. Trop. Med. Hyg. 33: Akiyama, H. J., and N. K. McQuillen Interaction and transformation of Leishmania donovani within in vitro cultured cells. An electron microscopical study. Am. J. Trop. Med. Hyg. 21: Ben Rachid, M. S Action lytique du serum humain normal vis-a-vis de Leishmania infantum. Arch. Inst. Pasteur Tunis 44: Berens, R. L., R. Brun, and S. M. Krassner A simple monophasic medium for axenic culture of hemoflagellates. J. Parasitol. 62: Berman, J. D., D. M. Dwyer, and D. J. Wyler Multiplication of Leishmania in human macrophages in vitro. Infect. Immun. 26: Chang, K.-P Leishmania donovani: promastigotemacrophage surface interactions in vitro. Exp. Parasitol. 48: Chang, K.-P Human cutaneous leishmania in a mouse macrophage line: propagation and isolation of intracellular parasites. Science 209: Chang, K.-P., and D. M. Dwyer Leishmania donovani-hamster macrophage interactions in vitro: cell entry, intracellular survival, and multiplication of amastigotes. J. Exp. Med. 147: Lewis, D. H., and W. Peters The resistance of intracellular Leishmania parasites to digestion by lysosomal enzymes. Ann. Trop. Med. Parasitol. 71: Mauel, J., R. Behin, Biroum-Noerjasin, and B. Holle Studies on protective cell-mediated mechanisms in experimental Leishmania infections, p In R. Van Furth (ed.), Mononuclear phagocytes. Blackwell Scientific Publications, Ltd., Oxford. 11. Pearson, R. D., and R. T. Steigbigel Mechanism of serum lytic activity against Leishmania donovani. J. Immunol. 125: Rahman, A. A., and K. K. Sethi Intracellular behavior of Leishmania enriettii within murine macrophages. Experientia 34: Taub, J The effect of normal serum of Leishmania. Bull. Res. Counc. Isr. 6E:55-57.