Destiny and Intracellular Survival of Leishmania amazonensis in Control and Dexamethasone-treated Glial Cultures : Protozoa-specific Glycoconjugate Tagging and TUNEL Staining
Departmento de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (WB-d-C,AH-P,SC-R); Laboratório de Protozoologia, Escola Nacional de Saúde Pública, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (RMM-S,MFM); and Laboratório de Neurobiologia do Desenvolvimento, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (AS-S,LAC)
Correspondence to: Dr Leny A. Cavalcante, Laboratório de Neurobiologia do Desenvolvimento, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil. E-mail: Lacav{at}abc.org.br
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Summary |
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Key Words: microgliaprotozoa interactions lipophosphoglycan microglial cytotoxicity lipopolysaccharide
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Introduction |
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Leishmania are obligatory intracellular pathogenic parasites that must gain entrance into mononuclear phagocytes to successfully complete their cell cycle and parasitize various species of mammalian hosts (for reviews, see Cunningham 2002; Sacks and Sher 2002
; Vannier-Santos et al. 2002
). However, it is not known how the "macrophages" of the central nervous system (the microglia) interact with this obligatory intracellular parasite in their physiological environment, i.e., in the presence of other major components of the neural tissue, such as astrocytes. Thus, we have chosen to study the interactions of Leishmania amazonensis with microglia in mixed glial cultures from neonatal tissue and compare these interactions with known features of Leishmaniamacrophage interactions. We have also used treatment of microglia in mixed glial cultures with immune modulators [lipopolysaccharide (LPS) or dexamethasone (DM)] before interaction with L. amazonensis and have followed the destiny of the parasites by their specific lipophosphoglycan (LPG) membrane labeling plus terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL) staining.
We have found that, in contrast to macrophages (Kane and Mosser 2000; for reviews, see Cunningham 2002
; Vannier-Santos et al. 2002
), microglia eliminate intracellular parasites very rapidly and that this process seems to be slowed by DM treatment. Our results indicate that microglia represent a highly effective barrier to the invasion of the brain by L. amazonensis and suggest that an extensive investigation of the mechanisms involved may provide additional clues to microglial physiological functions vis-a-vis intracellular parasites.
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Materials and Methods |
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Macrophages
Murine macrophages were obtained from adult Swiss mice by harvesting of the peritoneal cavity, followed by plating of the cell suspension in 24-well culture dishes and incubation in DMEM in a humidified 95% air/5% CO2 atmosphere at 37C for 1 hr. Afterward, nonadherent cells were removed by washing and the adherent cells were cultured in complete medium. Macrophages were preincubated with 0.1 µg/ml DM or 1 µg/ml LPS at 37C for 24 hr and used for comparisons of adhesion to and/or internalization of L. amazonensis by microglial cells in mixed glial cultures after 2 hr of interaction of either mammalian cell with parasites (Table 1).
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For identification of the parasites, the cultures infected as described above or the promastigotes attached onto poly-L-lysine-coated microscope slides were fixed in 4% paraformaldehyde (Sigma) at room temperature for 5 min. Afterward, the samples were washed three times in PBS and incubated in blocking solution (10% normal rabbit serum diluted in PBS plus 1% BSA) plus 0.1% saponin at 37C for 1 hr. Later, the cover slips were incubated with 45D3 monoclonal (IgG1 anti-LPG) antibody (kindly donated by Dr David Sacks, National Institutes of Health, Bethesda, MD) diluted 1:50 at 37C for 1 hr. After incubation with the primary antibody (Lang et al. 1991), the samples were washed three times in PBS and incubated with a Cy3-tagged rabbit anti-mouse IgG secondary antibody (Caltag) diluted 1:200 (Figure 1B). Controls were treated by incubating the samples in the absence of the primary antibody (Figure 1C). The nuclei and kinetoplasts of the parasites were stained with DAPI (Sigma) (Figures 1B and 1C; see Figure 5) at a concentration of 0.1 µg/ml. The samples on cover slips were mounted on slides with DABCO (Sigma) and analyzed with a Zeiss epifluorescence photomicroscope.
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Kinetic studies of MicrogliaLeishmania Interaction
Mixed glial cultures were preincubated in DMEM/FCS with 0.1 µg/ml DM or 1 µg/ml LPS or maintained without treatment (control) at 37C for 24 hr. After that, the cultures were washed several times with serum-free DMEM and the total number of cells was estimated by counting several fields. Promastigote forms of L. amazonensis, previously centrifuged and suspended in serum-free DMEM, were added to the cultures to achieve a ratio of 10:1 parasites/glial cells, and the cell/parasite contact was maintained for 2 hr. After this period, cultures were rinsed with PBS to remove extracellular parasites, DMEM with 2% BSA was added, and the infection was followed at 37C for up to 72 hr, with fixation of infected cells at 0, 2, 4, and 10 hr after PBS rinsing (2, 4, 6, and 12 hr of interaction). Infected cells were fixed with Bouin's solution and stained with Giemsa. The percentage of microglial cells containing parasites attached or internalized was then determined by examining, in bright-field optics, randomly selected cells in at least 300 fields at 1000x magnification with a Zeiss photomicroscope (see Figures 3 and 4). To avoid missing internalized parasites that might have undergone shape or size changes, every microglial cell was analyzed in a through-focus mode.
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For the TUNEL labeling, the samples on cover slips, previously fixed in 4% paraformaldehyde at room temperature, were permeabilized with 0.1% saponin (Sigma) on ice for 30 min. After washing, the samples were incubated with the TUNEL reaction mixture containing Terminal deoxyribonucleotide Transferase (enzyme solution) and fluorescein-dUTP (labeling solution) at 37C for 1 hr. The positive controls were treated by incubating the infected cells in the presence of DNase I (Sigma) and applying the TUNEL procedure. The cover slips were mounted on slides with DABCO and analyzed under bright-field and epifluorescence conditions.
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Results |
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Surface Morphology of Infected Microglial Cells
To demonstrate the in vitro influence of immune function mediators on the morphology of microglial cells, we used the macrophage function modulators DM and LPS during the interaction of microglia with promastigote forms of L. amazonensis. After pretreatment of the host cell for 24 hr, followed by washing and interaction with the parasite at 4C for 1 hr, scanning electron microscopy analysis showed numerous filopodia (Figure 2A) in DM-treated cells, whereas these projections were rarely observed in LPS-treated (data not shown) or control (Figure 2B) cultures, even in the presence of adhered parasites.
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After DM treatment, there was a significant increase in the percentage of microglial cells with adhered parasites, increasing from 1% in the untreated cultures to 60% in the DM-treated cultures (Table 1). On the other hand, this increase was not observed in macrophages after the same treatment with DM, so that the percentage of cells with adhered parasites remained nearly identical to half of the cells. There were no major changes in the number of adhered parasites per microglial cell (Figure 3) or per peritoneal macrophage (Table 1) vis-à-vis the treatment with DM.
With respect to the internalization of parasites, similar to what was observed in adhesion assays, a very low number of untreated microglial cells presented intracellular parasites compared with untreated peritoneal macrophages (1% vs 80%; Table 1). No intracellular parasites were observed in microglial cells after treatment with LPS, whereas a very large percentage (90%) of macrophages contained intracellular parasites. The number of intracellular parasites per LPS-treated infected macrophage was similar to that in control cells.
The preincubation of microglial cells with DM favored parasitecell interactions. Thus, the percentage of cells with intracellular parasites increased from 1% in the control to 39% after DM treatment (Table 1). However, the number of intracellular parasites per cell tended to remain unaltered in this short-term experiment. In the case of macrophages, we observed that 80% of the untreated cells already showed an average of 3.1 parasites per cell, whereas the percentage observed after treatment with DM was 75% with 3.0 parasites per cell (Table 1).
Kinetics of Microglia Infection by L. amazonensis
In preparations stained by the Giemsa procedure, we evaluated the kinetics of endocytosis of the promastigote forms of L. amazonensis in microglial cells maintained only in medium or preincubated with LPS or DM for 24 hr. The intracellular destiny of the parasites was analyzed after interaction with microglia for 2 hr, after which the cultures were washed and maintained in serum-free DMEM (2% BSA added), and the infection was followed for periods of 2, 4, 6, and 12 hr. In the cultures preincubated with LPS, we observed no internalized parasites or their residues in the host cell at any of the time intervals used (data not shown).
A significant number of internalized parasites was seen in DM-treated cells at 2 hr after infection (Figure 4B) compared with the untreated cells (Figure 4A). At 4 hr, stained profiles that could not be identified as viable parasites were observed in untreated cultures. In these untreated cells, there were structures in vacuoles distributed in different regions of the cytoplasm, but such structures were not clearly identified as parasites (Figure 4C). At the same time interval, in DM-treated cultures, several parasitophorous vacuoles with a large amount of cellular remains similar to killed parasites were observed in the microglial cytoplasm (Figure 4D), indicating a delay in the elimination of parasites in DM-treated cultures compared with untreated cultures. At 12 hr after infection, only vestiges of presumptive parasites were found in any of the cultures (Figures 4E and 4F).
DNA Degradation of Internalized Parasites
To validate the findings of our study of the kinetics of L. amazonensis endocytosis by microglia, we used the TUNEL technique to evaluate the intracellular survival of the parasites. That was done through the identification of fragments of nuclear and kinetoplast DNA generated during the degradation and/or death process by digestion of the parasite by cells identified as microglia via BSI-B4 isolectin staining. We also used the (anti-LPG) 45D3 monoclonal antibody, which recognizes a characteristic LPG on the surface of Leishmania promastigotes (Lang et al. 1991) that is absent on amastigotes (Pimenta et al. 1991
), for the localization of intracellular parasites or their residues during the infection of microglia. The promastigote forms of L. amazonensis, used for infection of the cultures, showed binding of the anti-LPG antibody throughout their surfaces (compare Figure 1B).
The LPG antigen and fragmented DNA of the parasite were detected at the 2-hr interval within both control cells and DM-treated microglia (data not shown). TUNEL+ nuclei and kinetoplasts of the presumptive promastigotes colocalized with LPG+ membrane structures of the parasite. There was a larger number of these LPG-labeled profiles together with nuclei of dead parasites in cells treated with DM compared with control cells (data not shown). At 4 hr, LPG+ profiles and nuclei of the tagged parasites were numerous, with apparent aggregation of LPG+ profiles in the control cells (Figures 5A and 5B) and their punctual distribution close to the nucleus of the DM-treated cells (Figures 5C and 5D). At 6 hr, the amounts of parasite membrane profiles and fragmented DNA were noticeably reduced in relation to the previous times, but the distribution of both membrane remains and DNA fragments was not altered largely in either group (Figures 5E5H). Similarly, there was a common tendency for the aggregation of the LPG+ structures in both control and DM-treated cells (Figures 5E5H). At 12 hr after infection, no LPG+ profiles or TUNEL-labeled particles were detected in control microglial cells (Figures 5I and 5J), whereas rare LPG+ profiles (Figure 5K) but no TUNEL label (Figure 5L) were found in DM-treated cells.
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Discussion |
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Both LPS and DM treatments resulted in larger percentages of microglial cells with adhered parasites than for controls, possibly attributable to different mechanisms. Because LPS downregulates the mannose fucose receptor (MFR) (Marzolo et al. 1999), which is required for the establishment of intracellular parasitism in mononuclear phagocytes (Wilson and Pearson 1986
), enhanced parasite adhesion in LPS-treated microglia may depend on the upregulation of molecules that do not trigger the internalization of parasites (Table 1).
In spite of the verification of a larger number of adhered parasites and increased percentage of cells that internalized parasites among those previously treated by DM compared with control cells, it was observed that apparently viable or intact parasites were no longer stained by the Giemsa method as soon as after 4 hr of interaction. This observation was reinforced by the TUNEL procedure, showing that in both untreated and DM-treated cultures there was fragmentation of the parasite nucleus and kinetoplast DNA as early as after 24 hr of interaction. The larger percentage of cells that internalized parasites in DM-treated cultures may derive from either or both of the following effects. First, the MFR could be upregulated by DM treatment, and this upregulation could lead to increased adhesion of parasites to the microglia surface and consequent internalization. This effect would be in agreement with the DM-induced MFR upregulation demonstrated in purified microglial cultures (Marzolo et al. 1999). Furthermore, preliminary studies of ours have shown that the addition of D-mannose to DM-treated mixed glial cultures impairs both microglial infection and adhesion as well as the internalization of the neoglycoprotein mannosyl-BSA (W. Baetas-da-Cruz, unpublished master's thesis). However, the simple alternative that a heavier load of parasites could lead to an apparent delay in the killing and disposal of the parasite can probably be ruled out because the number of internalized parasites per cell at 2 hr of interaction is not different in DM-treated and control cells (Table 1).
A second effect would be DM treatment causing the downmodulation of inducible nitric oxide synthase and, thus, the reduction of nitric oxide synthesis and release. Again, it is known that cortisol (and possibly DM) inhibits the activation of microglia and decreases the production and release of nitric oxide and tumor necrosis factor- (Drew and Chavis 2000
), important cytotoxic and cytotoxicity-inducer molecules, respectively, for Leishmania (Roach et al. 1991
). At present, we have no evidence that the apparently longer survival of L. amazonensis after DM treatment involves either the upregulation of the MFR receptor or the reduction of nitric oxide production by microglia in mixed cultures. The major astrocyte population of these cultures may express the MFR (Burudi et al. 1999
) and the endothelial type of nitric oxide synthase (Wiencken and Casagrande 1999
), making the identification of the source and/or the quantitative assessment of either MFR or nitric oxide ambiguous or unreliable. Alternative culture models are being tested in our laboratories to approach the questions of the mechanisms involved in phagocytosis and disposal of the parasite by microglia.
It cannot be ruled out that, as in monocytes (Ma et al. 2004), DM acts through the suppression of interleukin-12 production by microglia. In other words, DM would further decrease any remaining interleukin-12 production by microglia, which is already depressed by astrocytes (Aloisi et al. 1997
), in mixed glial cultures. Clearly, additional work is necessary to investigate this issue.
The results obtained with the TUNEL procedure are compatible with the notion that DM treatment delays the killing and eventual disposal of Leishmania by microglia. More importantly, they emphasize that the microglial response to the parasite differs from that of peritoneal macrophages. Thus, there is an almost total absence of parasite residues in both DM-treated and untreated microglia after exposure of the cultures to promastigote forms for 12 hr.
In summary, microglial cells are highly effective in the elimination of Leishmania, at least in the particular case of mixed glial cultures. This cytotoxicity is apparently slowed by DM, indicating that microglia and other mononuclear phagocytes present both common and atypical functional features vis-à-vis intracellular parasites that must gain entrance into host cells to successfully complete their cell cycles. The mechanisms responsible for the microglial cytotoxicity of Leishmania remain to be determined.
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Acknowledgments |
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We thank Dr Carlos Alves for helpful suggestions on the detection of the LPG antigen. The excellent technical assistance of Bruno Avila and Sergio L. Carvalho is gratefully acknowledged. We also thank two unknown referees for their criticisms of and suggestions on a first version of this article.
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Footnotes |
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Received for publication December 23, 2003; accepted April 2, 2004
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