Z eilscbri~[l ~)~t
Z. Parasitenkd. 59, 31-41 (] 979)
Parasitenkunde ParasitDIc~gY Research
9 by Springer-Verlag 1979
In vitro Light and Electron Microscope Studies on Different Virulent Promastigotes of Leishmania donovani in Hamster Peritoneal Macrophages F. Ebert*, E. Buse, and H. Mfihlpfordt Bernhard-Nocht-[ast/tut f~.r Scbiffs- und Tropenkrankheicen, Hamburg, Abteilung ftir ProIozoologie: Bernhard-Nocht-Strage 74, D-2000 Hamburg 4, Federal Republic of Germany
Summary. Hamster peritoneal macrophages were infected with avirulent and virulent promastigotes of a L. donovani strain using various ratios (1:1; I :10) of parasites and peritoneal ceils. Light microscope studies have shown that there was a significant difference in the number of parasites taken up by phagocylic cells between the macrophage cultures infected with avirulent and virulent promastigotes at 4 h as well as during the following 14 days of infection. In both virulent groups the number of anaastigotes were sharply increased. However, the surviving parasites were eliminated continuously when the macrophage cultures were infected with avirulent parasites. Electron microscope examinations of the different infected macrophage cultures did not show any difference in the localization of the surviving parasites. At oae and 24 h post-refection, parasites have beet~ observed in typical parasitophorous vacuoles. However, by day 4, 7, and 14 post-infections, the majority of intact parasites were surrounded by a four-laminar membrane without a space between parasite and vacuole membrane. Besides, some amastigotes were seen in large parasitophorous vacuoles. It seemed as if some of these amastigotes were trying to leave the parasitophorous vacuoles. In all cases acid phosphatase could be demonstrated in the parasitophorous vacuoles and around the parasites indicating that the lysosomes of the host ce~l have been fosed with t'ne parasitophorous vacuole. It is indicated that the virulent L e i s h m a n i a parasites are mare resistant to the digestive system of the macrophages. Introduction The host-parasite relationship of visceral leishmaniasis has been investigated by infecting peritoneal maeraphages in viva and in vitro with L e i s h m a n i a dana*
Present address." Centro de Microscopia Electronica, Funda~gto Oswatdo Cruz, Rio de Janeiro,
Brasil
0044-3255/79/0059/0031/$02.20
32
F. Ebert et al.
vani (Miller and Twonhy, 1967; Akiyama and McQuillen, 1972; Ebert et al.,
1976; Chang and Dwyer, 1978). The results of these investigations are contradictory and the factors explaining the resistance of L. donovani and other species of Leishmania to the digestive system of macrophages remained unclear. Macrophage cultures which have been infected with amastigotes of L. donovani showed that the parasitophorous vacuoles containing Leishmania fuse with secondary lysosomes (Chang and Dwyer, 1978). Similar findings have been observed in vivo with avirulent promastigotes in hamster peritoneal macrophages (Ebert et al., 1976), However, Akiyama and McQuillen (1972) demonstrated that some of the engulfed promastigotes were enclosed within vacuoles of the macrophages before being degraded, whereas other promastigotes escaped vacuolization and were transformed into amastigotes. Moreover, they found that macrophages were not very susceptible host cells in vitro for studying the parasite host interaction of L. donovani, although Leishmania are obligate intracellular parasites of the mononuclear phagocytes. The purpose of the present study was to examine the influence of different virulent Leishmania parasites on their specific host cells and to study the fate of the intracellular parasites at the ultrastructural level.
Materials and Methods Source o f the Parasites P romastigotes of an Indian Le&hmania donovani strain (LRC-L51)I used in the present experiments were collected from two sources. (I) F r o m a modified N a k a m u r a culture continuously maintained in the laboratory since 1973 (Ebert, 1973) and (2) hamster-spleen-derived amastigotes cultured in N N N - m e d i u m for four subcultures at weekly intervals. The cultures were maintained at 26 ~ C. The parasites were harvested during the log-phase, washed in medium 199 containing 20% heat inactivated foetal bovine serum (M199 + 2 0 % F B S ) , and adjusted to the desired concentration. The virulence of promastigotes was examined by intraperitoneal inoculation of 3 x 107 parasites per hamster. Smears of spleen and liver were taken not earlier than four weeks and not later than 10 m o n t h s after infection. Only the hamsters infected by the freshly isolated parasites showed amasfigotes in spleen and liver after 4-6 weeks. Experimental infections with the promastigotes of the N a k a m u r a m e d i u m were without success over a ten-months observation period.
Macrophage Cultures The peritoneal cavity of normal hamsters was washed with 10 ml physiological NaC1 solution. The peritoneal cells were centrifuged and resuspended in M 1 9 9 + 2 0 % FBS and antibiotics (200 gg/ ml Streptomycin, 200 U/ml Penicillin). The ceils were counted in a Neubauer counting chamber and adjusted to 4 • 106 cells/ml. One ml of the cell suspension was plated in a 10 x 35 m m Leighton tube for 4 h at 37 ~ C and then washed with M 1 9 9 + 2 0 % FBS to remove non-adherent cells. The adherent cells were incubated overnight (16-18 h) until used in experiments. 1 The strain was sent by Prof~ A. Zuckermann, Leishmania Reference Centre, Jerusalem, in t971 and was maintained by hamster to hamster passage and in cultures since that time
L. donovani in Macrophages
33
Infection of Macrophages Macrophage monolayers were rinsed with M 199 + 2 0 % FBS before being infected with promastigotes at ratios of 1:1 and 1:10 (peritoneal ceils:parasites). After 30 min the infected macrophage cultures were rinsed again to wash out extracellular parasites.
Preparation oJ"the Cells for Microscopy a) Light Microscopy. The coverslips were harvested at intervals of two days, fixed in 2% OsO, in cacodylate buffer (0.1 M, pH 7.2) for a few seconds, and stained with Giemsa solution. b) Electron Microscopy. Macrophages were cultured in 250 ml flasks. At intervals of one and 24 h and 4, 7, and 14 days after infection, the monolayers were fixed in 2.5% glutaraldehyde in 0.I M cacodyla~e buffer (pH 7.2), washed several times in the same buffer, subjected to the Gomori reaction, and then postfixed in 1% OsO4, T1ae complete process was done in situ. Subseql~ent to postfixation, the cells were gently scraped off the surface of the flasks with a rubber policeman. The procedure of embedding was as described previously (Ebert et al., 1976), The statistical significance of the results was tested by the variance test on homogeneity of the binorninal distribution after Snedecor and Irwin (1933).
Results
Two experiments were performed in which normal hamster peritoneal macrophages were challenged with promastigotes of L. donovani from the two sources using different ratios of parasites and peritoneal cells. There was no prefered position of the first contact of Leishmania to the macrophages. In slides harvested five up to 20 min after inoculation promastigotes were seen to attach themselves to the surface of macr0phages either by means of the flagellum or body of the parasite. The process of engulfment of parasites into macrophages was very rapid. After 5 min of the infection some of the parasites had been phagocytized completely. After 4 h the majority of Leishmania were engulfed by their posterior end and only the flagellum could be seen outside the phagocytic cells (Fig. 1A). At 24 h the surviving parasites had the appearance of amastigotes (Fig. 1 B). The quantitative study is presented in the growth curve of Fig. 2. There was a significant difference in the number of parasites taken up by the phagocytic cells between the macrophage cultures infected with Leishmania of both sources at 4 h as well as during the following 14 days of infection. The figure illustrates that Group C (infected with avirulent parasites, 1:10) had phagocytized a significantly higher percentage of Leishmania parasites (66%) than Group B (46%), infected with virulent parasites, l : l . However, only in Group C was the number of surviving parasites decreasing continuously. Likewise it can be seen in Group D that a strong reduction of the number of amastigotes had taken place during the experiment. In this group parasites were found in less than 1% of macrophages from 7 to 14 days post-infection. It is further seen that in the virulent groups (A and B) the amastigotes were increased as evidenced by the increase of infected macrophages after 14 days of infection, However,
Fig. 1 A Light micrograph of hamster peritoneal macrophages infected with promastigotes of L. donovani (1 : 10) four hours after infection, x 680. B Light micrograph of hamster peritoneal macrophages infected with L. donovani (arrowed) after 7 days of infection, x 710
lO0-
A
8o
o o '~ 60 40 z_
g 20
ll;
~ 4h 1
3
5
7
9
11
13
15
DAYS
Fig. 2. The course of L. donovani infection in hamster peritoneal macrophages with different doses (peritoneal cells:parasites). 9 1 : 10, virulent (A); 9 1 : 1, virulent (B); r 1 : 10, avirulent (C); [] 1:1, avirulent (D)
~60I
VIRULENT ./~
t DAYS
ul uJ
VIRULENT B so
40
I,,2o u.
z
4h 1
3
5
7 DAYS
9
11
13
15
-
-" ~--___,~__.._.~
AVIRULENT C 60,
O 40. uJ cJ M. 20. z
/, h 1
3
5
7 DAYS
g
11
13
15
AVIRULENT I ~ so
120 u. Z
i
.
4h 1
3
-,
5
9
7 DAYS
9
9
11
13
15
Fig. 3 A - D . N u m b e r of L. donovani per hamster peritoneal macrophage after infection with virulent and avirulent promastigotes at rates 1:10 (A and C) and 1:1 (B and D). 9 1 parasite per cell, [] 2 parasites per cell, ~ 3 parasites per cell, [] 5 parasites per cell, 9 more than 5 parasites per cell
36
F. Ebert et al.
Fig. 4. A Leishmania parasites (P) in hamster peritoneal macrophage, 4 h after addition of the parasites. The parasites are located in typical parasitophorous vacuoles (PV). x 20,000. B Histochemical localization of acid phosphatase, 4 h after infection. Black deposits of lead phosphate indicate sites of activity, x22,400. F/, flagellum; Ki, kinetoplast; Nm, nucleus of macrophage; Np, nucleus of parasite; P, parasite; P V, parasitophorous vacuole
L. donovani in Macrophages
37
Fig. 5. A Infected macrophage 7 days after infection. Note the absence of the parasitophorous vacuoles, x 13,200. B Higher magnification showing the typical four-laminar structure of parasitehost-membranes. Note that some areas show only a three-laminar membrane (arrowed). • 103,200. C Intact Leishmania parasite 7 days after infection. A lysosome (L) is located near the parasite. x 28,800. D Phosphatase activity around the intact parasite 7 days after addition of the parasites. x 36,720. Fl, flagellum; Ki, kinetoplast; L, lysosome; Mi, mitochondrium; Nm, nucleus of macrophage; Np, nucleus of parasite; P, parasite
Group A did not show a significant increase in the number of infected macrophages, since its rate of infection was as high as 92% from the beginning. The multiplication of amastigotes found only in the virulent groups can be clearly seen in Fig. 3. This figure represents the number of parasites per macrophage during the experiment. In Group A the numbers of amastigotes representing more than five per host cell were increased from 27 to 64% and in Group B from 2 to 18% during the 14 days of infection. Electron microscopic examinations of macrophage cultures inoculated with virulent and avirulent Leishmania parasites did not show any differences in the localization of survived parasites within their host cell. In specimens prepared
38
F. Ebert et al.
Fig. 6. A Two parasites in a parasitophorous vacuole 7 days after infection. Note the amorphous electron dense material, x 15,750. B Detail of a macrophage infected 7 days showing a parasite changing from the parasitophorous vacuole to the cytoplasm, x 13,500. C Infected macrophage 7 days after infection. One parasite leaving the parasitophorous vacuole. Another parasite is already located in the cytoplasm, x14,250. Inset: high magnification showing the typical four-laminar membrane, x40,000. D Parasitophorous vacuole shows acid phosphatase activity, x 31,500. p, parasite; PV, parasitophorous vacuole one and 24 h post-infection, parasites have been frequently observed in phagosomes which were losely surrounded by host m e m b r a n e s with a clear space between the m e m b r a n e s of the parasite and the p a r a s i t o p h o r o u s vacuole (Fig. 4A). In these p a r a s i t o p h o r o u s vacuoles heavy deposits o f lead p h o s p h a t e were observed indicating that the lysosomes have been fused with the parasitop h o r o u s vacuole (Fig. 4B). By day 4, 7, and 14 post-infection, the majority of intact parasites was surrounded by a four-laminar m e m b r a n e w i t h o u t a
L. donovani in Macrophages
39
space between the membrane of the parasitophorous vacuole and the membrane of the parasite (Fig. 5A and B). Using the Gomori technique, a positive reaction could be shown around the parasites in most cases (Fig. 5 C and D). Moreover, the deposit of lead phosphate was less than that observed one and 24 h postinfection. Besides, some healthy amastigotes in large parasitophorous vacuoles were associated with amorphous electron dense material (Fig. 6A) after 4 and 7 days of infection. It seemed as if some of the amastigotes were trying to leave the parasitophorous vacuole (Fig. 6B, and C). Near the Vacuole, parasites were lying within the cytoplasm of the host cell and were surrounded by a four-laminar membrane as already noticed in the majority of surviving parasites (Fig. 6 C). Acid phosphatase could be demonstrated in the vacuoles which indicate that amastigotes persist within the phagosome-lysosomal complexes of the macrophages (Fig. 6D).
Discussion
The present results show that only virulent Leishmania parasites survived in normal hamster peritoneal macrophages in vitro. The higher attachment of the virulent promastigotes to macrophages in both 1:1 and 1:10 infections at 4 h cannot explain the subsequent higher multiplication of the parasites. Only avirulent parasites were continuously eliminated during the two-weekly infection although in Group C more parasites were engulfed than in Group B at 4 h. Thus the degree of virulence of promastigotes seems to be an important factor in determining the survival of parasites. The differences of attachment between the two Leishmania cultures are not understood. The mobility of the cells was not different. However, the variation of morphology of virulent parasites was much higher than the avirulent ones (unpublished data). Whether or not these morphological different ' stages' of L. donovani promatigotes show different behaviour in macrophages requires further investigations. Our results are not identical with the findings of Akiyama and McQuillen (1972) who reported that only a small percentage of promastigotes of L. donovani survived in hamster peritoneal macrophages although Leishmania with a minimum of in vitro passages was used. The transformation of promastigotes into amastigotes does not necessarily mean that the parasites survived in macrophages because the avirulent promastigotes were transformed after 24 h but their elimination was continued by macrophages. Other investigations of promastigotes of L. donovani and hamster peritoneal macrophages have been carried out (Pulvertaft and Hoyle, 1960; Miller and Twonhy, 1967), however, the observation period was limited. Moreover, all of the strains used were not inoculated in hamsters to examine their virulence. On the other hand, a 7-fold increase of L. donovani in hamster peritoneal macrophages over the period of some days was reported by Chang and Dwyer (1978) when macrophages were infected with amastigotes. Handman and Spira (1977) reported the successful growth of L. tropica promastigotes in mouse macrophages for 26 days.
40
F. Ebert et al.
There is no ultrastructural evidence to explain the significant higher multiplication of virulent Leishmania parasites in macrophages. At 4 and 24 h L. donovani are localized in a typical parasitophorous vacuole of the host cell. Similar findings were reported by Alexander and Vickerman (1975) and Lewis and Peters (1976) using L. mexicana and mouse macrophages. However, at 4, 7, and 14 days post-infection the morphological features of amastigotes have been changed from the vacuole to the cytoplasm, here, surrounded by a four-laminar membrane. There is no doubt that one part of the four-laminar membrane represents the unit membrane of the parasitophorous vacuole and the other one is part of the membrane of the parasite. Moreover, in peritoneal macrophages from highly infected hamsters with L. donovani we found intact amastigotes which were also surrounded by a four-laminar membrane (unpublished data). Rudzinska et al. (1964) described similar observations in their electron microscopic study of amastigotes of L. donovani within spleen ceils. The host cell lysosomes should have been fused with the parasitophorous vacuole as evident by their acid phosphatase. Our results conform with those of other Leishmania spp. (Alexander and Vikerman, 1975; Lewis and Peters, 1976). Recent studies on the intracellular protozoa, Trypanosoma cruzi and Toxoplasma gondii, have shown that other mechanisms are involved. T. cruzi (Nogueira and Cohn, 1976; Kress et al., 1977) has been reported to escape from the parasitophorous vacuole of the macrophage without being surrounded by the host membrane. The growth of T. gondii in mouse macrophages is ascribed to the non-fusion o f lysosomes with the phagocytic vacuole (Jones and Hirsch, 1972). According to our results, Chang and Dwyer (1978) described several types of parasitophorous vacuoles. Parasites in loose vacuoles and amastigotes appear to 'push into' the host cytoplasm as has also been seen in our study (Fig. 6B, and C). Another type was characterized by the close apposition of host-parasite membranes, leaving no space for thorotrast granules. This type could be identical with our one, seen in Fig. 5 A and 6 C. Contrary to Chang and Dwyer we found, however, that in this type acid phosphatase activity could be demonstrated around the amastigotes (Fig. 5 D). The presence of intact Leishmania parasites and the acid phosphatase in all types of parasitophorous vacuoles suggest that amastigotes are resistant to the digestive system of macrophages. The four-laminar membrane surrounding the amastigotes could behave as a functional physical barrier. Acknowledgements. This study was supported by BMZ, Research program-Fiocruz/Rio de Janeiro, BNI/Hamburg. The authors wish to thank Mr. H. Jabionski for his technical assistance and Mr. R. Geister for his help to elaborate statistical analysis. References
Akiyama, H.J., McQuillen, N.K. : Interaction and transformation of Leishmania donovani within in vitro cultured cells. Am. J. Trop. Med. Hyg. 21, 873 879 (1972) Alexander, J., Vickerman, K. : Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexieana infected macrophages. J. Protozool. 22, 502-508 (1975) Chang, K.P., Dwyer, D.M. : Leishmania donovani. Hamster macrophage interactions in vitro. Cell entry, intracellular survival, and multiplicationof amastigotes. J. Exp. Med. 147, 515-529 (1978)
L. donovani in Macrophages
41
Ebert, F.: Charakterisierung von Leishmania donovani-Stfimmen mit der Disk-Elektrophorese. Z. Tropenmed. Parasitol. 24, 517 524 (1973) Ebert, F., Enriquez, G.L., Mfihlpfordt, H. : Electronmicroscopic studies of phagocytosis of Leishmania donovani by hamster peritoneal macrophages and its lysosomal activity in vivo. Behring Inst. Mitt. 60, 65 74 (1976) Handman, E., Spira, D.T.: Growth of Leishmania amastigotes in macrophages from normal and immune mice. Z. Parasitenkd. 53, 78 81 (1977) Jones, T.C., Hirsch, J.G.: The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 136, 1173-1193 (1972) Kress, Y., Tanowitz, H., Bloom, B., Wittner, M.: Trypanosoma cruzi.: Infection of normal and activated mouse macrophages. Exp. Parasitol. 41, 385 396 (1977) Lewis, D.H., Peters, W. : The resistance of intracellular Leishmania parasites to digestion by lysosomal enzymes. Ann. Trop. Med. Parasitol. 71,295-312 (1977) Miller, H.C., Twonhy, D.W. : Infection of macrophages in culture by leptomonads of Leishmania donovani. J. Protozool. 14, 781-789 (1967) Nogueira, N., Cohn, Z. : Trypanosoma cruzi. : Mechanism of entry and intracellular fate in mammalian ceils. J. Exp. Med. 143, 1402 1420 (1976) Pulvertaft, R.J.V., Hoyle, G.F.: Stages in the life cycle of Leishmania donovani. Trans. R. Soc. Trop. Med. Hyg. 54, 191-196 (1960) Rudzinska, N.A., D'Alessandro, P., Trager, W.: The fine structure of Leishmania donovani and the role of kinetoplast in the leishmania-leptomonad transformation. J. Protozool. 11, 166 191 (1964) Snedecor, G.W., Irwin, M.R.: On the Chi-square test for homogeneity. Iowa State College. J. Sci. 8, 75-81 (1933) Received September 5, 1978
Z. Parasitenkd. 59, 31-41 (] 979)
Parasitenkunde ParasitDIc~gY Research
9 by Springer-Verlag 1979
In vitro Light and Electron Microscope Studies on Different Virulent Promastigotes of Leishmania donovani in Hamster Peritoneal Macrophages F. Ebert*, E. Buse, and H. Mfihlpfordt Bernhard-Nocht-[ast/tut f~.r Scbiffs- und Tropenkrankheicen, Hamburg, Abteilung ftir ProIozoologie: Bernhard-Nocht-Strage 74, D-2000 Hamburg 4, Federal Republic of Germany
Summary. Hamster peritoneal macrophages were infected with avirulent and virulent promastigotes of a L. donovani strain using various ratios (1:1; I :10) of parasites and peritoneal ceils. Light microscope studies have shown that there was a significant difference in the number of parasites taken up by phagocylic cells between the macrophage cultures infected with avirulent and virulent promastigotes at 4 h as well as during the following 14 days of infection. In both virulent groups the number of anaastigotes were sharply increased. However, the surviving parasites were eliminated continuously when the macrophage cultures were infected with avirulent parasites. Electron microscope examinations of the different infected macrophage cultures did not show any difference in the localization of the surviving parasites. At oae and 24 h post-refection, parasites have beet~ observed in typical parasitophorous vacuoles. However, by day 4, 7, and 14 post-infections, the majority of intact parasites were surrounded by a four-laminar membrane without a space between parasite and vacuole membrane. Besides, some amastigotes were seen in large parasitophorous vacuoles. It seemed as if some of these amastigotes were trying to leave the parasitophorous vacuoles. In all cases acid phosphatase could be demonstrated in the parasitophorous vacuoles and around the parasites indicating that the lysosomes of the host ce~l have been fosed with t'ne parasitophorous vacuole. It is indicated that the virulent L e i s h m a n i a parasites are mare resistant to the digestive system of the macrophages. Introduction The host-parasite relationship of visceral leishmaniasis has been investigated by infecting peritoneal maeraphages in viva and in vitro with L e i s h m a n i a dana*
Present address." Centro de Microscopia Electronica, Funda~gto Oswatdo Cruz, Rio de Janeiro,
Brasil
0044-3255/79/0059/0031/$02.20
32
F. Ebert et al.
vani (Miller and Twonhy, 1967; Akiyama and McQuillen, 1972; Ebert et al.,
1976; Chang and Dwyer, 1978). The results of these investigations are contradictory and the factors explaining the resistance of L. donovani and other species of Leishmania to the digestive system of macrophages remained unclear. Macrophage cultures which have been infected with amastigotes of L. donovani showed that the parasitophorous vacuoles containing Leishmania fuse with secondary lysosomes (Chang and Dwyer, 1978). Similar findings have been observed in vivo with avirulent promastigotes in hamster peritoneal macrophages (Ebert et al., 1976), However, Akiyama and McQuillen (1972) demonstrated that some of the engulfed promastigotes were enclosed within vacuoles of the macrophages before being degraded, whereas other promastigotes escaped vacuolization and were transformed into amastigotes. Moreover, they found that macrophages were not very susceptible host cells in vitro for studying the parasite host interaction of L. donovani, although Leishmania are obligate intracellular parasites of the mononuclear phagocytes. The purpose of the present study was to examine the influence of different virulent Leishmania parasites on their specific host cells and to study the fate of the intracellular parasites at the ultrastructural level.
Materials and Methods Source o f the Parasites P romastigotes of an Indian Le&hmania donovani strain (LRC-L51)I used in the present experiments were collected from two sources. (I) F r o m a modified N a k a m u r a culture continuously maintained in the laboratory since 1973 (Ebert, 1973) and (2) hamster-spleen-derived amastigotes cultured in N N N - m e d i u m for four subcultures at weekly intervals. The cultures were maintained at 26 ~ C. The parasites were harvested during the log-phase, washed in medium 199 containing 20% heat inactivated foetal bovine serum (M199 + 2 0 % F B S ) , and adjusted to the desired concentration. The virulence of promastigotes was examined by intraperitoneal inoculation of 3 x 107 parasites per hamster. Smears of spleen and liver were taken not earlier than four weeks and not later than 10 m o n t h s after infection. Only the hamsters infected by the freshly isolated parasites showed amasfigotes in spleen and liver after 4-6 weeks. Experimental infections with the promastigotes of the N a k a m u r a m e d i u m were without success over a ten-months observation period.
Macrophage Cultures The peritoneal cavity of normal hamsters was washed with 10 ml physiological NaC1 solution. The peritoneal cells were centrifuged and resuspended in M 1 9 9 + 2 0 % FBS and antibiotics (200 gg/ ml Streptomycin, 200 U/ml Penicillin). The ceils were counted in a Neubauer counting chamber and adjusted to 4 • 106 cells/ml. One ml of the cell suspension was plated in a 10 x 35 m m Leighton tube for 4 h at 37 ~ C and then washed with M 1 9 9 + 2 0 % FBS to remove non-adherent cells. The adherent cells were incubated overnight (16-18 h) until used in experiments. 1 The strain was sent by Prof~ A. Zuckermann, Leishmania Reference Centre, Jerusalem, in t971 and was maintained by hamster to hamster passage and in cultures since that time
L. donovani in Macrophages
33
Infection of Macrophages Macrophage monolayers were rinsed with M 199 + 2 0 % FBS before being infected with promastigotes at ratios of 1:1 and 1:10 (peritoneal ceils:parasites). After 30 min the infected macrophage cultures were rinsed again to wash out extracellular parasites.
Preparation oJ"the Cells for Microscopy a) Light Microscopy. The coverslips were harvested at intervals of two days, fixed in 2% OsO, in cacodylate buffer (0.1 M, pH 7.2) for a few seconds, and stained with Giemsa solution. b) Electron Microscopy. Macrophages were cultured in 250 ml flasks. At intervals of one and 24 h and 4, 7, and 14 days after infection, the monolayers were fixed in 2.5% glutaraldehyde in 0.I M cacodyla~e buffer (pH 7.2), washed several times in the same buffer, subjected to the Gomori reaction, and then postfixed in 1% OsO4, T1ae complete process was done in situ. Subseql~ent to postfixation, the cells were gently scraped off the surface of the flasks with a rubber policeman. The procedure of embedding was as described previously (Ebert et al., 1976), The statistical significance of the results was tested by the variance test on homogeneity of the binorninal distribution after Snedecor and Irwin (1933).
Results
Two experiments were performed in which normal hamster peritoneal macrophages were challenged with promastigotes of L. donovani from the two sources using different ratios of parasites and peritoneal cells. There was no prefered position of the first contact of Leishmania to the macrophages. In slides harvested five up to 20 min after inoculation promastigotes were seen to attach themselves to the surface of macr0phages either by means of the flagellum or body of the parasite. The process of engulfment of parasites into macrophages was very rapid. After 5 min of the infection some of the parasites had been phagocytized completely. After 4 h the majority of Leishmania were engulfed by their posterior end and only the flagellum could be seen outside the phagocytic cells (Fig. 1A). At 24 h the surviving parasites had the appearance of amastigotes (Fig. 1 B). The quantitative study is presented in the growth curve of Fig. 2. There was a significant difference in the number of parasites taken up by the phagocytic cells between the macrophage cultures infected with Leishmania of both sources at 4 h as well as during the following 14 days of infection. The figure illustrates that Group C (infected with avirulent parasites, 1:10) had phagocytized a significantly higher percentage of Leishmania parasites (66%) than Group B (46%), infected with virulent parasites, l : l . However, only in Group C was the number of surviving parasites decreasing continuously. Likewise it can be seen in Group D that a strong reduction of the number of amastigotes had taken place during the experiment. In this group parasites were found in less than 1% of macrophages from 7 to 14 days post-infection. It is further seen that in the virulent groups (A and B) the amastigotes were increased as evidenced by the increase of infected macrophages after 14 days of infection, However,
Fig. 1 A Light micrograph of hamster peritoneal macrophages infected with promastigotes of L. donovani (1 : 10) four hours after infection, x 680. B Light micrograph of hamster peritoneal macrophages infected with L. donovani (arrowed) after 7 days of infection, x 710
lO0-
A
8o
o o '~ 60 40 z_
g 20
ll;
~ 4h 1
3
5
7
9
11
13
15
DAYS
Fig. 2. The course of L. donovani infection in hamster peritoneal macrophages with different doses (peritoneal cells:parasites). 9 1 : 10, virulent (A); 9 1 : 1, virulent (B); r 1 : 10, avirulent (C); [] 1:1, avirulent (D)
~60I
VIRULENT ./~
t DAYS
ul uJ
VIRULENT B so
40
I,,2o u.
z
4h 1
3
5
7 DAYS
9
11
13
15
-
-" ~--___,~__.._.~
AVIRULENT C 60,
O 40. uJ cJ M. 20. z
/, h 1
3
5
7 DAYS
g
11
13
15
AVIRULENT I ~ so
120 u. Z
i
.
4h 1
3
-,
5
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7 DAYS
9
9
11
13
15
Fig. 3 A - D . N u m b e r of L. donovani per hamster peritoneal macrophage after infection with virulent and avirulent promastigotes at rates 1:10 (A and C) and 1:1 (B and D). 9 1 parasite per cell, [] 2 parasites per cell, ~ 3 parasites per cell, [] 5 parasites per cell, 9 more than 5 parasites per cell
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Fig. 4. A Leishmania parasites (P) in hamster peritoneal macrophage, 4 h after addition of the parasites. The parasites are located in typical parasitophorous vacuoles (PV). x 20,000. B Histochemical localization of acid phosphatase, 4 h after infection. Black deposits of lead phosphate indicate sites of activity, x22,400. F/, flagellum; Ki, kinetoplast; Nm, nucleus of macrophage; Np, nucleus of parasite; P, parasite; P V, parasitophorous vacuole
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Fig. 5. A Infected macrophage 7 days after infection. Note the absence of the parasitophorous vacuoles, x 13,200. B Higher magnification showing the typical four-laminar structure of parasitehost-membranes. Note that some areas show only a three-laminar membrane (arrowed). • 103,200. C Intact Leishmania parasite 7 days after infection. A lysosome (L) is located near the parasite. x 28,800. D Phosphatase activity around the intact parasite 7 days after addition of the parasites. x 36,720. Fl, flagellum; Ki, kinetoplast; L, lysosome; Mi, mitochondrium; Nm, nucleus of macrophage; Np, nucleus of parasite; P, parasite
Group A did not show a significant increase in the number of infected macrophages, since its rate of infection was as high as 92% from the beginning. The multiplication of amastigotes found only in the virulent groups can be clearly seen in Fig. 3. This figure represents the number of parasites per macrophage during the experiment. In Group A the numbers of amastigotes representing more than five per host cell were increased from 27 to 64% and in Group B from 2 to 18% during the 14 days of infection. Electron microscopic examinations of macrophage cultures inoculated with virulent and avirulent Leishmania parasites did not show any differences in the localization of survived parasites within their host cell. In specimens prepared
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Fig. 6. A Two parasites in a parasitophorous vacuole 7 days after infection. Note the amorphous electron dense material, x 15,750. B Detail of a macrophage infected 7 days showing a parasite changing from the parasitophorous vacuole to the cytoplasm, x 13,500. C Infected macrophage 7 days after infection. One parasite leaving the parasitophorous vacuole. Another parasite is already located in the cytoplasm, x14,250. Inset: high magnification showing the typical four-laminar membrane, x40,000. D Parasitophorous vacuole shows acid phosphatase activity, x 31,500. p, parasite; PV, parasitophorous vacuole one and 24 h post-infection, parasites have been frequently observed in phagosomes which were losely surrounded by host m e m b r a n e s with a clear space between the m e m b r a n e s of the parasite and the p a r a s i t o p h o r o u s vacuole (Fig. 4A). In these p a r a s i t o p h o r o u s vacuoles heavy deposits o f lead p h o s p h a t e were observed indicating that the lysosomes have been fused with the parasitop h o r o u s vacuole (Fig. 4B). By day 4, 7, and 14 post-infection, the majority of intact parasites was surrounded by a four-laminar m e m b r a n e w i t h o u t a
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space between the membrane of the parasitophorous vacuole and the membrane of the parasite (Fig. 5A and B). Using the Gomori technique, a positive reaction could be shown around the parasites in most cases (Fig. 5 C and D). Moreover, the deposit of lead phosphate was less than that observed one and 24 h postinfection. Besides, some healthy amastigotes in large parasitophorous vacuoles were associated with amorphous electron dense material (Fig. 6A) after 4 and 7 days of infection. It seemed as if some of the amastigotes were trying to leave the parasitophorous vacuole (Fig. 6B, and C). Near the Vacuole, parasites were lying within the cytoplasm of the host cell and were surrounded by a four-laminar membrane as already noticed in the majority of surviving parasites (Fig. 6 C). Acid phosphatase could be demonstrated in the vacuoles which indicate that amastigotes persist within the phagosome-lysosomal complexes of the macrophages (Fig. 6D).
Discussion
The present results show that only virulent Leishmania parasites survived in normal hamster peritoneal macrophages in vitro. The higher attachment of the virulent promastigotes to macrophages in both 1:1 and 1:10 infections at 4 h cannot explain the subsequent higher multiplication of the parasites. Only avirulent parasites were continuously eliminated during the two-weekly infection although in Group C more parasites were engulfed than in Group B at 4 h. Thus the degree of virulence of promastigotes seems to be an important factor in determining the survival of parasites. The differences of attachment between the two Leishmania cultures are not understood. The mobility of the cells was not different. However, the variation of morphology of virulent parasites was much higher than the avirulent ones (unpublished data). Whether or not these morphological different ' stages' of L. donovani promatigotes show different behaviour in macrophages requires further investigations. Our results are not identical with the findings of Akiyama and McQuillen (1972) who reported that only a small percentage of promastigotes of L. donovani survived in hamster peritoneal macrophages although Leishmania with a minimum of in vitro passages was used. The transformation of promastigotes into amastigotes does not necessarily mean that the parasites survived in macrophages because the avirulent promastigotes were transformed after 24 h but their elimination was continued by macrophages. Other investigations of promastigotes of L. donovani and hamster peritoneal macrophages have been carried out (Pulvertaft and Hoyle, 1960; Miller and Twonhy, 1967), however, the observation period was limited. Moreover, all of the strains used were not inoculated in hamsters to examine their virulence. On the other hand, a 7-fold increase of L. donovani in hamster peritoneal macrophages over the period of some days was reported by Chang and Dwyer (1978) when macrophages were infected with amastigotes. Handman and Spira (1977) reported the successful growth of L. tropica promastigotes in mouse macrophages for 26 days.
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There is no ultrastructural evidence to explain the significant higher multiplication of virulent Leishmania parasites in macrophages. At 4 and 24 h L. donovani are localized in a typical parasitophorous vacuole of the host cell. Similar findings were reported by Alexander and Vickerman (1975) and Lewis and Peters (1976) using L. mexicana and mouse macrophages. However, at 4, 7, and 14 days post-infection the morphological features of amastigotes have been changed from the vacuole to the cytoplasm, here, surrounded by a four-laminar membrane. There is no doubt that one part of the four-laminar membrane represents the unit membrane of the parasitophorous vacuole and the other one is part of the membrane of the parasite. Moreover, in peritoneal macrophages from highly infected hamsters with L. donovani we found intact amastigotes which were also surrounded by a four-laminar membrane (unpublished data). Rudzinska et al. (1964) described similar observations in their electron microscopic study of amastigotes of L. donovani within spleen ceils. The host cell lysosomes should have been fused with the parasitophorous vacuole as evident by their acid phosphatase. Our results conform with those of other Leishmania spp. (Alexander and Vikerman, 1975; Lewis and Peters, 1976). Recent studies on the intracellular protozoa, Trypanosoma cruzi and Toxoplasma gondii, have shown that other mechanisms are involved. T. cruzi (Nogueira and Cohn, 1976; Kress et al., 1977) has been reported to escape from the parasitophorous vacuole of the macrophage without being surrounded by the host membrane. The growth of T. gondii in mouse macrophages is ascribed to the non-fusion o f lysosomes with the phagocytic vacuole (Jones and Hirsch, 1972). According to our results, Chang and Dwyer (1978) described several types of parasitophorous vacuoles. Parasites in loose vacuoles and amastigotes appear to 'push into' the host cytoplasm as has also been seen in our study (Fig. 6B, and C). Another type was characterized by the close apposition of host-parasite membranes, leaving no space for thorotrast granules. This type could be identical with our one, seen in Fig. 5 A and 6 C. Contrary to Chang and Dwyer we found, however, that in this type acid phosphatase activity could be demonstrated around the amastigotes (Fig. 5 D). The presence of intact Leishmania parasites and the acid phosphatase in all types of parasitophorous vacuoles suggest that amastigotes are resistant to the digestive system of macrophages. The four-laminar membrane surrounding the amastigotes could behave as a functional physical barrier. Acknowledgements. This study was supported by BMZ, Research program-Fiocruz/Rio de Janeiro, BNI/Hamburg. The authors wish to thank Mr. H. Jabionski for his technical assistance and Mr. R. Geister for his help to elaborate statistical analysis. References
Akiyama, H.J., McQuillen, N.K. : Interaction and transformation of Leishmania donovani within in vitro cultured cells. Am. J. Trop. Med. Hyg. 21, 873 879 (1972) Alexander, J., Vickerman, K. : Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexieana infected macrophages. J. Protozool. 22, 502-508 (1975) Chang, K.P., Dwyer, D.M. : Leishmania donovani. Hamster macrophage interactions in vitro. Cell entry, intracellular survival, and multiplicationof amastigotes. J. Exp. Med. 147, 515-529 (1978)
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Ebert, F.: Charakterisierung von Leishmania donovani-Stfimmen mit der Disk-Elektrophorese. Z. Tropenmed. Parasitol. 24, 517 524 (1973) Ebert, F., Enriquez, G.L., Mfihlpfordt, H. : Electronmicroscopic studies of phagocytosis of Leishmania donovani by hamster peritoneal macrophages and its lysosomal activity in vivo. Behring Inst. Mitt. 60, 65 74 (1976) Handman, E., Spira, D.T.: Growth of Leishmania amastigotes in macrophages from normal and immune mice. Z. Parasitenkd. 53, 78 81 (1977) Jones, T.C., Hirsch, J.G.: The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 136, 1173-1193 (1972) Kress, Y., Tanowitz, H., Bloom, B., Wittner, M.: Trypanosoma cruzi.: Infection of normal and activated mouse macrophages. Exp. Parasitol. 41, 385 396 (1977) Lewis, D.H., Peters, W. : The resistance of intracellular Leishmania parasites to digestion by lysosomal enzymes. Ann. Trop. Med. Parasitol. 71,295-312 (1977) Miller, H.C., Twonhy, D.W. : Infection of macrophages in culture by leptomonads of Leishmania donovani. J. Protozool. 14, 781-789 (1967) Nogueira, N., Cohn, Z. : Trypanosoma cruzi. : Mechanism of entry and intracellular fate in mammalian ceils. J. Exp. Med. 143, 1402 1420 (1976) Pulvertaft, R.J.V., Hoyle, G.F.: Stages in the life cycle of Leishmania donovani. Trans. R. Soc. Trop. Med. Hyg. 54, 191-196 (1960) Rudzinska, N.A., D'Alessandro, P., Trager, W.: The fine structure of Leishmania donovani and the role of kinetoplast in the leishmania-leptomonad transformation. J. Protozool. 11, 166 191 (1964) Snedecor, G.W., Irwin, M.R.: On the Chi-square test for homogeneity. Iowa State College. J. Sci. 8, 75-81 (1933) Received September 5, 1978
