Leishmania major Modulates Chemokine and Chemokine Receptor Expression by Dendritic Cells and Affects Their Migratory Capacity
http://www.100md.com
感染与免疫杂志 2005年第4期
Institute for Molecular Biology of Infectious Diseases, University of Würzburg, Würzburg, Germany
ABSTRACT
Dendritic cells (DC) both produce and respond to chemokines. We examined the profiles of chemokines and chemokine receptors expressed by DC and their chemotactic response after interaction with Leishmania major. Expression of the chemokine receptors CCR2 and CCR5 by DC and their responsiveness to the respective ligands, CCL2 and CCL3, were downregulated, while the level of CCR7 and the DC response to its ligand CCL21 were enhanced. These parasite-induced alterations were observed with DC from L. major-resistant and -susceptible mice. In contrast, expression of the chemokine CXCL10 was elicited only in DC from L. major-resistant mice.
TEXT
Immunity against infectious agents requires the coordinated action of components of the innate and adaptive immune systems. Dendritic cells (DC) guard the sites of pathogen entry to the host, such as skin and mucosae, and fulfill the critical task of conveying microbial signals to lymphocytes, thus instructing the development of an appropriate specific immune response. A fundamental aspect of this function is the recruitment of DC into peripheral tissues and their ability to migrate via tissue-draining lymphatic vessels to the T-cell areas of regional lymph nodes. The migratory capacity of DC is tightly regulated by their responsiveness to chemokines (5, 11). In the present study, we analyzed the profiles of chemokine receptors and chemokines expressed by DC after exposure to the protozoan parasite Leishmania major. Moreover, L. major-treated DC were tested for their chemotactic response.
Parasites and generation of DC. Promastigotes of the L. major isolate MHOM/IL/81/FE/BNI were grown in vitro in blood agar cultures. For the preparation of lysate, stationary-phase promastigotes were subjected to three cycles of rapid freezing and thawing. To generate DC (4), freshly prepared bone marrow cells from 4- to 6-week-old C57BL/6 or BALB/c mice were cultured in Click RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM HEPES buffer, 17 mM NaHCO3, 0.05 mM 2-mercaptoethanol, 60 μg of penicillin per ml, and 20 μg of gentamicin per ml in the presence of 200 U of granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech, London, United Kingdom) per ml. Cultures were fed with GM-CSF at days 3, 6, and 8. After 10 days, the nonadherent cells were collected and shown to have a typical DC morphology with a myeloid DC phenotype (7). Cells were resuspended at 106/ml in 24-well plates in culture medium containing 200 U of GM-CSF per ml and exposed for 24 h to stationary-phase promastigotes (10 parasites per DC), L. major lysate (equivalent to 30 parasites per DC), lipopolysaccharide (LPS) (Sigma-Aldrich, Taufkirchen, Germany) at 1 μg/ml, or LPS plus gamma interferon (IFN-) (BD Biosciences, Heidelberg, Germany) at 200 U/ml. The cells were then collected for RNA extraction or resuspended in culture medium for analysis of their chemotactic activity. Upon DC exposure to live parasites, more than 98% of the cells were infected with L. major, as determined by fluorescence microscopy after staining with acridine orange and ethidium bromide.
RNA extraction, RPA, and RT-PCR. Total RNA was extracted from DC using the RNeasy Midi kit (QIAGEN Operon, Cologne, Germany) according to the manufacturer's instructions. The expression of chemokine and chemokine receptor genes was analyzed using the RiboQuant RNase protection assay (RPA) system kit (BD Biosciences) following the instructions of the supplier. The quantity of protected RNA was determined using ImageMaster software (Amersham Biosciences Europe, Freiburg, Germany). The values are expressed as units relative to the L32 rRNA gene.
For reverse transcription-PCR (RT-PCR), 2 μg of RNA was reverse transcribed (One-Step RT-PCR kit; BD Biosciences), and the relative amounts of the respective mRNA were determined using the following primers: CXCL10 antisense, 5'-GAG CCT GAG CTA GGG AGG AC-3'; CXCL10 sense, 5'-CGG GCC AGT GAG AAT GAG GG-3'. -Actin primers were used to normalize the amount of template RNA (-actin antisense, 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'; -actin sense, 5'-GTG GGC CGC TCT AGG CAC CAA-3'). One-Step RT-PCR conditions were as follows: (i) reverse transcription (1 h at 50°C); (ii) an initial denaturation step (5 min at 94°C); (iii) 25 cycles of amplification, with 1 cycle consisting of denaturation (30 s at 94°C), annealing (30 s at 64°C), and elongation (1 min at 68°C); and (iv) a final extension step (2 min at 68°C).
Chemotaxis assay. The chemotactic activity of DC was examined using 24-well Transwell chamber plates (8-μm pore size; Corning Costar, Bodenheim, Germany). The upper compartments were supplemented with 106 cells in 0.5 ml of culture medium, and 1.5 ml of culture medium alone or culture medium containing 50 ng of the chemokine CCL2, CCL3, or CCL21 (PeproTech) per ml was added to the lower chambers. After overnight incubation, the cells were harvested from the bottom chambers and labeled with biotin-conjugated anti-I-Ad or anti-I-Ab monoclonal antibodies (BD Biosciences) and streptavidin-conjugated fluorescein isothiocyanate (Serotec, Düsseldorf, Germany), followed by enumeration of major histocompatibility complex class II-positive cells using a fluorescence microscope.
Exposure to L. major modulates the expression of chemokine receptors and chemokines in DC. Differences in the pattern of chemokine receptor expression by DC regulate their responsiveness to chemoattractants and, consequently, their migratory activities (5, 11). Therefore, we examined whether the interaction of DC with L. major parasites leads to a modulation of chemokine receptor gene expression. LPS, a well-characterized stimulator of DC maturation (11), was used as a positive control to validate the extent of the signals induced by L. major. RPA of RNA isolated from DC that had been infected with L. major or had been treated with parasite lysate showed that expression of chemokine receptors CCR1 and CCR6 was not altered (data not shown). In contrast, the expression of CCR2 and CCR5 was significantly downregulated by L. major, whereas that of CCR7 was upregulated compared with DC of the same maturation stage that had not been exposed to antigen (Fig. 1A and B). The parasite-induced changes in chemokine receptor gene expression were observed with DC from both L. major-susceptible BALB/c mice and L. major-resistant C57BL/6 mice. Flow cytometric analyses and confocal microscopy demonstrated that exposure to L. major also modulated the expression of these chemokine receptors on the surfaces of DC from both strains of mice (data not shown). The modulation of CCR2, CCR5, and CCR7 expression did not require infection of DC with L. major, because DC exposure to both live parasites and lysed organisms altered the profile of chemokine receptor expression.
DC are both a target and a source of chemokines. Therefore, we also examined whether mRNA expression of chemokines is modulated by L. major. DC were found to express a range of chemokines, such as CCL3, CCL4, and CCL5, that were not regulated by the parasites (Fig. 1 and data not shown). In contrast, expression of chemokines CCL2 and CXCL10, which could not be detected in unstimulated DC, was induced by exposure of DC to L. major (Fig. 1A and B). The modulation of DC chemokine expression by L. major was also observed at the protein level, as determined by an enzyme-linked immunosorbent assay (data not shown). While a CCL2 response was elicited in DC from both L. major-susceptible and -resistant mice, parasite-induced expression of CXCL10 was observed only in DC from L. major-resistant C57BL/6 mice but not in those from L. major-susceptible BALB/c mice. This is not due to a general failure of BALB/c DC to express CXCL10, since control experiments with various DC stimuli demonstrated that, although the well-established DC activator LPS was also unable to trigger significant CXCL10 levels in these DC, the chemokine was expressed in response to BALB/c DC stimulation with the combination of LPS and IFN- (Fig. 1C).
DC infection with L. major or treatment with parasite lysate modulates their migratory activity. To evaluate whether the parasite-induced modulations in chemokine receptor expression correlate with changes in the responsiveness to the corresponding ligands, DC from L. major-susceptible BALB/c and L. major-resistant C57BL/6 mice were tested for their migratory activity in response to the respective chemokines. The results of the chemotaxis assay showed that exposure of DC to L. major caused a reduction of about 50% in the chemotactic response to CCL2 and CCL3, the ligands of CCR2 and CCR5, respectively (Fig. 2). This is in accordance with the parasite-induced downregulation of CCR2 and CCR5 expression in DC (Fig. 1). On the other hand, the increased expression of CCR7 by L. major-treated DC (Fig. 1) was associated with a 2.3- to 3-fold enhancement of the migration in response to CCL21, the ligand of CCR7 (Fig. 3). Consistent with the findings obtained by RPA and immunofluorescence analysis of chemokine receptor expression, the chemotactic capacities of DC from L. major-susceptible BALB/c mice and L. major-resistant C57BL/6 mice were very similar.
Conclusions. DC play a central role in the initiation and regulation of the immune response to Leishmania parasites (6, 15). The results of this study show that DC respond to L. major by altering their migratory properties through the expression of various chemokine receptors and by releasing inflammatory chemokines. Interaction with L. major downmodulated CCR2 and CCR5 expression in DC and their responsiveness to the respective ligands, CCL2 and CCL3, while CCR7 expression and the DC response to its ligand CCL21 were enhanced. Such a change in the profile of chemokine receptors expressed by DC has been demonstrated to be associated with their recruitment from peripheral tissues to secondary lymphoid organs (10, 11, 13, 17), a requirement for the encounter of antigen-bearing DC and responder T cells. Interestingly, the L. major-induced modulations in chemokine receptor expression and responsiveness to their ligands were comparable for DC from L. major-susceptible and -resistant mice, suggesting that the differential ability of these mice to control leishmaniasis is not due to host-dependent effects of the parasite on the expression of chemokine receptors directing DC migration. In contrast, the selective modulation of chemokine receptor expression appears to depend upon the Leishmania species, since DC from mice infected with Leishmania donovani were reported to have reduced expression of CCR7 and decreased responsiveness to CCR7 ligands (1).
We observed considerable levels of L. major-induced CCL2 expression in DC from both L. major-resistant and -susceptible mice. Contrasting functions of this chemokine in leishmaniasis have been reported. CCL2-deficient mice do not mount a Th2 response, and upon L. major infection of CCL2–/– mice on an L. major-susceptible BALB/c background, their lesions are significantly smaller than those of infected wild-type mice (3). On the other hand, deficiency in the expression of CCR2 (the receptor for CCL2) of mice on an L. major-resistant C57BL/6 x 129/J background shifts the L. major resistance to susceptibility that is dominated by Th2 cytokines and correlates with an impairment of Langerhans cell migration to the draining lymph nodes (14). CCL2 is preferentially expressed in draining lymph nodes of L. major-resistant mice (18, 20), and we previously demonstrated that self-healing localized cutaneous leishmaniasis in humans is associated with strong CCL2 expression in the skin lesions (9). Moreover, we showed that dermal macrophages are the source of CCL2 in lesions of patients and that CCL2 triggers leishmanicidal activity in human monocytes (8). Together, our findings suggest that CCL2 expression by macrophages correlates with parasite elimination, while the L. major-induced CCL2 response of DC appears to have different functions with host-independent significance, such as the recruitment of immature DC, which is important to sustain antigen sampling in peripheral tissues, and of other cell types that are involved in the effector phase of the immune response (11, 12).
Notably, L. major parasites elicited a CXCL10 response in DC from L. major-resistant mice, while they failed to induce expression of this chemokine in DC from L. major-susceptible mice. Previous studies (18, 20) demonstrated that L. major infection upregulates CXCL10 expression in the draining lymph nodes of L. major-resistant mice, but not in those of L. major-susceptible mice, and showed that the cytokines interleukin 12 and IFN- are required for increased levels of CXCL10 expression (20). The present study extends these findings by documenting for the first time that CXCL10 derived from L. major-stimulated DC is relevant for the higher level of this chemokine in L. major-resistant mice and, therefore, underlines the significance of DC functions for cure versus susceptibility to experimental leishmaniasis. A particular feature of CXCL10 is its ability to recruit and activate NK cells (16, 18). This is of considerable interest in light of the recent observation that the cross talk between DC and NK cells is essential for their reciprocal activation (2). Moreover, DC-derived CXCL10 has been demonstrated to attract and retain T helper cells within the DC-T-cell clusters in the draining lymph nodes (19) and may thus contribute to Th1 cell polarization in L. major-resistant mice.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft of Germany.
REFERENCES
1. Ato, M., S. Stger, C. R. Engwerda, and P. M. Kaye. 2002. Defective CCR7 expression on dendritic cells contributes to the development of visceral leishmaniasis. Nat. Immunol. 3:1185-1191.
2. Gerosa, F., B. Baldani-Guerra, C. Nisii, V. Marchesini, G. Carra, and G. Trinchieri. 2002. Reciprocal activating interaction between natural killer cells and dendritic cells. J. Exp. Med. 195:327-333.
3. Gu, L., S. Tseng, R. M. Horner, C. Tam, M. Loda, and B. J. Rollins. 2000. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404:407-411.
4. Lutz, M., N. Kukutsch, A. Ogilvie, S. Rner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77-92.
5. McColl, S. R. 2002. Chemokines and dendritic cells: a crucial alliance. Immunol. Cell Biol. 80:489-496.
6. Moll, H., and C. Berberich. 2001. Dendritic cell-based vaccination strategies: induction of protective immunity against leishmaniasis. Immunobiology 204:659-666.
7. Ramírez-Pineda, J. R., A. Frhlich, C. Berberich, and H. Moll. 2004. Dendritic cells (DC) activated by CpG DNA ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC. J. Immunol. 172:6281-6289.
8. Ritter, U., and H. Moll. 2000. Monocyte chemotactic protein-1 stimulates the killing of Leishmania major by human monocytes, acts synergistically with IFN- and is antagonized by IL-4. Eur. J. Immunol. 30:3111-3120.
9. Ritter, U., H. Moll, T. Laskay, E. B. Brcker, O. Velazco, I. Becker, and R. Gillitzer. 1996. Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis. J. Infect. Dis. 173:699-709.
10. Saeki, H., A. M. Moore, M. J. Brown, and S. T. Hwang. 1999. Secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J. Immunol. 162:2472-2475.
11. Sallusto, F., and A. Lanzavecchia. 1999. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J. Exp. Med. 189:611-614.
12. Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I. Julkunen, R. Forster, R. Burgstahler, M. Lipp, and A. Lanzavecchia. 1999. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Immunol. 29:1617-1625.
13. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:2760-2769.
14. Sato, N., S. K. Ahuja, M. Quinones, V. Kostecki, R. L. Reddick, P. C. Melby, W. A. Kuziel, and S. S. Ahuja. 2000. CC chemokine receptor (CCR)2 is required for Langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells: absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, B cell outgrowth, and sustained neutrophilic inflammation. J. Exp. Med. 192:205-218.
15. Scott, P., and C. A. Hunter. 2002. Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr. Opin. Immunol. 14:466-470.
16. Taub, D. D., T. J. Sayers, C. R. D. Carter, and J. R. Ortaldo. 1995. and chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155:3877-3888.
17. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Bonecchi, P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Differential responsiveness to constitutive vs. inducible chemokines of immature and mature mouse dendritic cells. J. Leukoc. Biol. 66:489-494.
18. Vester, B., K. Müller, W. Solbach, and T. Laskay. 1999. Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major. Infect. Immun. 67:3155-3159.
19. Yoneyama, H., S. Narumi, Y. Zhang, M. Murai, M. Baggiolini, A. Lanzavecchia, T. Ichida, H. Asakura, and K. Matsushima. 2002. Pivotal role of dendritic cell-derived CXCL10 in the retention of T helper cell 1 lymphocytes in secondary lymph nodes. J. Exp. Med. 195:1257-1266.
20. Zaph, C., and P. Scott. 2003. Interleukin-12 regulates chemokine gene expression during the early immune response to Leishmania major. Infect. Immun. 71:1587-1589.(Mario Steigerwald and Hei)
ABSTRACT
Dendritic cells (DC) both produce and respond to chemokines. We examined the profiles of chemokines and chemokine receptors expressed by DC and their chemotactic response after interaction with Leishmania major. Expression of the chemokine receptors CCR2 and CCR5 by DC and their responsiveness to the respective ligands, CCL2 and CCL3, were downregulated, while the level of CCR7 and the DC response to its ligand CCL21 were enhanced. These parasite-induced alterations were observed with DC from L. major-resistant and -susceptible mice. In contrast, expression of the chemokine CXCL10 was elicited only in DC from L. major-resistant mice.
TEXT
Immunity against infectious agents requires the coordinated action of components of the innate and adaptive immune systems. Dendritic cells (DC) guard the sites of pathogen entry to the host, such as skin and mucosae, and fulfill the critical task of conveying microbial signals to lymphocytes, thus instructing the development of an appropriate specific immune response. A fundamental aspect of this function is the recruitment of DC into peripheral tissues and their ability to migrate via tissue-draining lymphatic vessels to the T-cell areas of regional lymph nodes. The migratory capacity of DC is tightly regulated by their responsiveness to chemokines (5, 11). In the present study, we analyzed the profiles of chemokine receptors and chemokines expressed by DC after exposure to the protozoan parasite Leishmania major. Moreover, L. major-treated DC were tested for their chemotactic response.
Parasites and generation of DC. Promastigotes of the L. major isolate MHOM/IL/81/FE/BNI were grown in vitro in blood agar cultures. For the preparation of lysate, stationary-phase promastigotes were subjected to three cycles of rapid freezing and thawing. To generate DC (4), freshly prepared bone marrow cells from 4- to 6-week-old C57BL/6 or BALB/c mice were cultured in Click RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM HEPES buffer, 17 mM NaHCO3, 0.05 mM 2-mercaptoethanol, 60 μg of penicillin per ml, and 20 μg of gentamicin per ml in the presence of 200 U of granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech, London, United Kingdom) per ml. Cultures were fed with GM-CSF at days 3, 6, and 8. After 10 days, the nonadherent cells were collected and shown to have a typical DC morphology with a myeloid DC phenotype (7). Cells were resuspended at 106/ml in 24-well plates in culture medium containing 200 U of GM-CSF per ml and exposed for 24 h to stationary-phase promastigotes (10 parasites per DC), L. major lysate (equivalent to 30 parasites per DC), lipopolysaccharide (LPS) (Sigma-Aldrich, Taufkirchen, Germany) at 1 μg/ml, or LPS plus gamma interferon (IFN-) (BD Biosciences, Heidelberg, Germany) at 200 U/ml. The cells were then collected for RNA extraction or resuspended in culture medium for analysis of their chemotactic activity. Upon DC exposure to live parasites, more than 98% of the cells were infected with L. major, as determined by fluorescence microscopy after staining with acridine orange and ethidium bromide.
RNA extraction, RPA, and RT-PCR. Total RNA was extracted from DC using the RNeasy Midi kit (QIAGEN Operon, Cologne, Germany) according to the manufacturer's instructions. The expression of chemokine and chemokine receptor genes was analyzed using the RiboQuant RNase protection assay (RPA) system kit (BD Biosciences) following the instructions of the supplier. The quantity of protected RNA was determined using ImageMaster software (Amersham Biosciences Europe, Freiburg, Germany). The values are expressed as units relative to the L32 rRNA gene.
For reverse transcription-PCR (RT-PCR), 2 μg of RNA was reverse transcribed (One-Step RT-PCR kit; BD Biosciences), and the relative amounts of the respective mRNA were determined using the following primers: CXCL10 antisense, 5'-GAG CCT GAG CTA GGG AGG AC-3'; CXCL10 sense, 5'-CGG GCC AGT GAG AAT GAG GG-3'. -Actin primers were used to normalize the amount of template RNA (-actin antisense, 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3'; -actin sense, 5'-GTG GGC CGC TCT AGG CAC CAA-3'). One-Step RT-PCR conditions were as follows: (i) reverse transcription (1 h at 50°C); (ii) an initial denaturation step (5 min at 94°C); (iii) 25 cycles of amplification, with 1 cycle consisting of denaturation (30 s at 94°C), annealing (30 s at 64°C), and elongation (1 min at 68°C); and (iv) a final extension step (2 min at 68°C).
Chemotaxis assay. The chemotactic activity of DC was examined using 24-well Transwell chamber plates (8-μm pore size; Corning Costar, Bodenheim, Germany). The upper compartments were supplemented with 106 cells in 0.5 ml of culture medium, and 1.5 ml of culture medium alone or culture medium containing 50 ng of the chemokine CCL2, CCL3, or CCL21 (PeproTech) per ml was added to the lower chambers. After overnight incubation, the cells were harvested from the bottom chambers and labeled with biotin-conjugated anti-I-Ad or anti-I-Ab monoclonal antibodies (BD Biosciences) and streptavidin-conjugated fluorescein isothiocyanate (Serotec, Düsseldorf, Germany), followed by enumeration of major histocompatibility complex class II-positive cells using a fluorescence microscope.
Exposure to L. major modulates the expression of chemokine receptors and chemokines in DC. Differences in the pattern of chemokine receptor expression by DC regulate their responsiveness to chemoattractants and, consequently, their migratory activities (5, 11). Therefore, we examined whether the interaction of DC with L. major parasites leads to a modulation of chemokine receptor gene expression. LPS, a well-characterized stimulator of DC maturation (11), was used as a positive control to validate the extent of the signals induced by L. major. RPA of RNA isolated from DC that had been infected with L. major or had been treated with parasite lysate showed that expression of chemokine receptors CCR1 and CCR6 was not altered (data not shown). In contrast, the expression of CCR2 and CCR5 was significantly downregulated by L. major, whereas that of CCR7 was upregulated compared with DC of the same maturation stage that had not been exposed to antigen (Fig. 1A and B). The parasite-induced changes in chemokine receptor gene expression were observed with DC from both L. major-susceptible BALB/c mice and L. major-resistant C57BL/6 mice. Flow cytometric analyses and confocal microscopy demonstrated that exposure to L. major also modulated the expression of these chemokine receptors on the surfaces of DC from both strains of mice (data not shown). The modulation of CCR2, CCR5, and CCR7 expression did not require infection of DC with L. major, because DC exposure to both live parasites and lysed organisms altered the profile of chemokine receptor expression.
DC are both a target and a source of chemokines. Therefore, we also examined whether mRNA expression of chemokines is modulated by L. major. DC were found to express a range of chemokines, such as CCL3, CCL4, and CCL5, that were not regulated by the parasites (Fig. 1 and data not shown). In contrast, expression of chemokines CCL2 and CXCL10, which could not be detected in unstimulated DC, was induced by exposure of DC to L. major (Fig. 1A and B). The modulation of DC chemokine expression by L. major was also observed at the protein level, as determined by an enzyme-linked immunosorbent assay (data not shown). While a CCL2 response was elicited in DC from both L. major-susceptible and -resistant mice, parasite-induced expression of CXCL10 was observed only in DC from L. major-resistant C57BL/6 mice but not in those from L. major-susceptible BALB/c mice. This is not due to a general failure of BALB/c DC to express CXCL10, since control experiments with various DC stimuli demonstrated that, although the well-established DC activator LPS was also unable to trigger significant CXCL10 levels in these DC, the chemokine was expressed in response to BALB/c DC stimulation with the combination of LPS and IFN- (Fig. 1C).
DC infection with L. major or treatment with parasite lysate modulates their migratory activity. To evaluate whether the parasite-induced modulations in chemokine receptor expression correlate with changes in the responsiveness to the corresponding ligands, DC from L. major-susceptible BALB/c and L. major-resistant C57BL/6 mice were tested for their migratory activity in response to the respective chemokines. The results of the chemotaxis assay showed that exposure of DC to L. major caused a reduction of about 50% in the chemotactic response to CCL2 and CCL3, the ligands of CCR2 and CCR5, respectively (Fig. 2). This is in accordance with the parasite-induced downregulation of CCR2 and CCR5 expression in DC (Fig. 1). On the other hand, the increased expression of CCR7 by L. major-treated DC (Fig. 1) was associated with a 2.3- to 3-fold enhancement of the migration in response to CCL21, the ligand of CCR7 (Fig. 3). Consistent with the findings obtained by RPA and immunofluorescence analysis of chemokine receptor expression, the chemotactic capacities of DC from L. major-susceptible BALB/c mice and L. major-resistant C57BL/6 mice were very similar.
Conclusions. DC play a central role in the initiation and regulation of the immune response to Leishmania parasites (6, 15). The results of this study show that DC respond to L. major by altering their migratory properties through the expression of various chemokine receptors and by releasing inflammatory chemokines. Interaction with L. major downmodulated CCR2 and CCR5 expression in DC and their responsiveness to the respective ligands, CCL2 and CCL3, while CCR7 expression and the DC response to its ligand CCL21 were enhanced. Such a change in the profile of chemokine receptors expressed by DC has been demonstrated to be associated with their recruitment from peripheral tissues to secondary lymphoid organs (10, 11, 13, 17), a requirement for the encounter of antigen-bearing DC and responder T cells. Interestingly, the L. major-induced modulations in chemokine receptor expression and responsiveness to their ligands were comparable for DC from L. major-susceptible and -resistant mice, suggesting that the differential ability of these mice to control leishmaniasis is not due to host-dependent effects of the parasite on the expression of chemokine receptors directing DC migration. In contrast, the selective modulation of chemokine receptor expression appears to depend upon the Leishmania species, since DC from mice infected with Leishmania donovani were reported to have reduced expression of CCR7 and decreased responsiveness to CCR7 ligands (1).
We observed considerable levels of L. major-induced CCL2 expression in DC from both L. major-resistant and -susceptible mice. Contrasting functions of this chemokine in leishmaniasis have been reported. CCL2-deficient mice do not mount a Th2 response, and upon L. major infection of CCL2–/– mice on an L. major-susceptible BALB/c background, their lesions are significantly smaller than those of infected wild-type mice (3). On the other hand, deficiency in the expression of CCR2 (the receptor for CCL2) of mice on an L. major-resistant C57BL/6 x 129/J background shifts the L. major resistance to susceptibility that is dominated by Th2 cytokines and correlates with an impairment of Langerhans cell migration to the draining lymph nodes (14). CCL2 is preferentially expressed in draining lymph nodes of L. major-resistant mice (18, 20), and we previously demonstrated that self-healing localized cutaneous leishmaniasis in humans is associated with strong CCL2 expression in the skin lesions (9). Moreover, we showed that dermal macrophages are the source of CCL2 in lesions of patients and that CCL2 triggers leishmanicidal activity in human monocytes (8). Together, our findings suggest that CCL2 expression by macrophages correlates with parasite elimination, while the L. major-induced CCL2 response of DC appears to have different functions with host-independent significance, such as the recruitment of immature DC, which is important to sustain antigen sampling in peripheral tissues, and of other cell types that are involved in the effector phase of the immune response (11, 12).
Notably, L. major parasites elicited a CXCL10 response in DC from L. major-resistant mice, while they failed to induce expression of this chemokine in DC from L. major-susceptible mice. Previous studies (18, 20) demonstrated that L. major infection upregulates CXCL10 expression in the draining lymph nodes of L. major-resistant mice, but not in those of L. major-susceptible mice, and showed that the cytokines interleukin 12 and IFN- are required for increased levels of CXCL10 expression (20). The present study extends these findings by documenting for the first time that CXCL10 derived from L. major-stimulated DC is relevant for the higher level of this chemokine in L. major-resistant mice and, therefore, underlines the significance of DC functions for cure versus susceptibility to experimental leishmaniasis. A particular feature of CXCL10 is its ability to recruit and activate NK cells (16, 18). This is of considerable interest in light of the recent observation that the cross talk between DC and NK cells is essential for their reciprocal activation (2). Moreover, DC-derived CXCL10 has been demonstrated to attract and retain T helper cells within the DC-T-cell clusters in the draining lymph nodes (19) and may thus contribute to Th1 cell polarization in L. major-resistant mice.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft of Germany.
REFERENCES
1. Ato, M., S. Stger, C. R. Engwerda, and P. M. Kaye. 2002. Defective CCR7 expression on dendritic cells contributes to the development of visceral leishmaniasis. Nat. Immunol. 3:1185-1191.
2. Gerosa, F., B. Baldani-Guerra, C. Nisii, V. Marchesini, G. Carra, and G. Trinchieri. 2002. Reciprocal activating interaction between natural killer cells and dendritic cells. J. Exp. Med. 195:327-333.
3. Gu, L., S. Tseng, R. M. Horner, C. Tam, M. Loda, and B. J. Rollins. 2000. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404:407-411.
4. Lutz, M., N. Kukutsch, A. Ogilvie, S. Rner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77-92.
5. McColl, S. R. 2002. Chemokines and dendritic cells: a crucial alliance. Immunol. Cell Biol. 80:489-496.
6. Moll, H., and C. Berberich. 2001. Dendritic cell-based vaccination strategies: induction of protective immunity against leishmaniasis. Immunobiology 204:659-666.
7. Ramírez-Pineda, J. R., A. Frhlich, C. Berberich, and H. Moll. 2004. Dendritic cells (DC) activated by CpG DNA ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC. J. Immunol. 172:6281-6289.
8. Ritter, U., and H. Moll. 2000. Monocyte chemotactic protein-1 stimulates the killing of Leishmania major by human monocytes, acts synergistically with IFN- and is antagonized by IL-4. Eur. J. Immunol. 30:3111-3120.
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