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Marc Ouellette, Martin Olivier,* and Barbara Papadopoulou*
Infectious Diseases Research Center, CHUL Research Center, Department of Medical Biology, Faculty of Medicine, Laval University, Quebec, Canada
Received 7 February 2003/ Returned for modification 8 April 2003/ Accepted 6 August 2003
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ABSTRACT |
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/CCL3, MIP-1ß/CCL4, MIP-2/CXCL2, and MCP-1/CCL2, which enhance parasite killing. Indeed, GM-CSF-expressing parasites survive poorly in macrophages in vitro and produce delayed lesion development in susceptible BALB/c mice in vivo. Selective killing of intracellular Leishmania expressing cytokine genes capable of activating cellular responses may constitute a promising strategy to control and/or prevent parasitic infections. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which is required to activate infected macrophages for parasite killing (38). The genetic predisposition for susceptibility to L. major infection in mice correlates with the early production of interleukin-10 (IL-10), IL-13 and IL-4 that drives the polarized TH2 response, hence suppressing TH1-cell development. Resistance to L. major infection in mice is associated with the ability of IL-12 to redirect the early TH2 response through IL-12/IL-12R signaling, which is essential to establish and maintain a curative TH1 response (for a review, see reference 43).
It has been reported that cytokines can modulate macrophage differentiation by causing selective changes in macrophage gene expression, hence allowing alterations of macrophage functions (15, 38). It has also been shown that macrophages preincubated in vitro with cytokines prior to infection with Leishmania acquire the capacity to kill the intracellular parasites (21, 29). Moreover, cytokines such as IFN-, tumor necrosis factor alpha (TNF-), IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as antileishmanial therapy in experimental model systems (20, 25, 29, 30, 49, 52). The role of proinflammatory cytokines in cell-mediated immunity in leishmaniasis has not been fully defined. Leishmania infections induce proinflammatory cytokines (IL-1, IL-6, and TNF-) and chemokines (11, 27, 49), but the involvement of these substances in the control of leishmaniasis is not yet clearly established.
The proinflammatory hematopoietic GM-CSF has well documented stimulatory effects on monocyte and macrophage functions. These effects include enhanced proliferation of their progenitor cells, increase in endocytosis, increased function as antigen-presenting cells, release of other proinflammatory cytokines, and increased inhibition of killing of intracellular fungi, bacteria, protozoa, and viruses (for reviews, see references 4 and 23). GM-CSF enhances host defenses against a broad spectrum of invading organisms (4), including Leishmania infections (15, 19, 29, 44, 54). As part of our interest in developing a safe and effective attenuated live-vaccine strategy against Leishmania infections, we evaluated the potential of delivering anti-leishmanial cytokines, known to induce parasite killing, using L. major as a vehicle. We engineered recombinant Leishmania organisms in which both developmental stages of the parasite express and release high levels of GM-CSF and evaluated their "suicide" potential in macrophages in vitro and in mice in vivo. We report here that GM-CSF secreted by recombinant GM-CSF-expressing L. major can activate macrophage functions to kill the parasites more efficiently in vitro and to produce delayed lesion development in vivo.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA constructs.
The vectors pXMT1 and pXMT2 expressing the cDNA of the human and murine GM-CSF genes, respectively, were generously donated by Walid Mourad (Immunology Research Unit, CHUL Research Center). The Leishmania vector pNEO-mGM-CSF was made by subcloning the 1.2-kb KpnI-KpnI fragment from pXMT2 containing the murine GM-CSF (mGM-CSF) gene (filled in with T4 DNA polymerase [New England Biolabs]) into the BamHI site (filled in with the large Klenow fragment [New England Biolabs]) of vector pSPNEO (constructed by subcloning the SmaI-BamHI-digested NEO expression cassette from the vector PGEM7-NEO [36] into the SmaI-BamHI sites of pSP72 [Promega]). The Leishmania construct expressing the human GM-CSF (hGM-CSF) gene was made by subcloning a 750-bp XhoI-XhoI fragment from the vector pXMT1 into the SalI site of pSPNEO to yield pNEO-hGM-CSF. The construct pNEO-hGM-CSF/R corresponds to the reverse orientation of the hGM-CSF gene with respect to the NEO expression cassette. For stable expression, the mGM-CSF gene was integrated downstream of the L. major rRNA promoter on chromosome 27 by homologous recombination. The targeting cassette harboring sequences necessary for homologous recombination was made by subcloning the PCR-amplified mGM-CSF gene (5'-end primer, 5'-GAGGTCTGCGATTGACGTAG-3'; 3'-end prime, 5'-CTACTGGCAGAATCAACCAG-3') into the BamHI site of vector pSPNEO downstream of the -tubulin intergenic region. Then, the NEO-mGM-CSF cassette was digested with BglII-XbaI, filled in with Klenow fragment, and subcloned into the unique BssHII site of the L. major ribosomal promoter-containing construct (41).
DNA manipulations. Total genomic DNA from Leishmania was digested with BglII resolved on 0.7% agarose gels and transferred to nylon membranes (Hybond-N; Amersham). Total RNA from L. major transfectants was prepared using Trizol (Gibco BRL). Southern and Northern blot hybridizations and washings were done by standard procedures (45). The GM-CSF-specific gene probes used in this study correspond to the 1.2-kb KpnI-KpnI fragment for the mGM-CSF gene and to the 750-bp XhoI-XhoI fragment for the hGM-CSF gene.
Leishmania infection in macrophages in vitro and in mice. The infection rates of Leishmania GM-CSF transfectants were tested in murine and human macrophages in vitro in comparison to the Leishmania NEO control cells. Briefly, macrophages were seeded (200 µl per well, 5 x 104 cells/ml) into eight-well chamber slides and infected with late-stationary-phage L. major NEO- and L. major GM-CSF-expressing promastigotes at a parasite-to-cell ratio of 20:1 for 6 h as described previously (31). Following this incubation, the nonengulfed parasites were removed by three to five washes with warm medium and the chambers were replenished with 500 µl of fresh culture medium. The level of infection was determined at 6, 24, 48, and 72 h by optical microscopy examination following Diff Quik staining of cell preparations. To neutralize GM-CSF activity, 200 µl of J774 murine macrophages was incubated for 1 h at 37°C in the presence of 2 µg of an anti-murine GM-CSF polyclonal antibody (R&D Systems) per ml prior to infection with Leishmania. Preincubated macrophages were then infected with L. major-mGM-CSF recombinant parasites as described above. Female BALB/c mice (6 to 8 weeks of age; six mice per group) were injected intradermally at the base of the footpad with 5 x 106 late-stationary-phase L. major promastigotes. The L. major strains used for infections were low-passage strains, and parasites were initially isolated from mouse lesions and then transfected to yield the recombinant parasites. Development of cutaneous lesions at the site of injection was monitored on a weekly basis for 8 weeks postinfection by measuring the thickness of the infected and the noninfected footpads. To selectively inhibit inducible nitric oxide synthase (iNOS) production by murine macrophages, BALB/c mice were treated twice a day with aminoguanidine sulfate (Sigma) at 9 mg/mouse for a 4-week period.
ELISAs. Supernatants from recombinant L. major-hGM-CSF parasites and from the L. major-NEO control were harvested by centrifugation following 5 days of culture when a density of 2 x 107 promastigotes/ml was reached. Supernatants were assayed directly using a monoclonal antibody enzyme-linked immunosorbent assay (ELISA) kit (Endogen) as recommended by the manufacturer. Standard curves for quantification and comparison were generated using a recombinant hGM-CSF protein. To detect GM-CSF inside the phagosomes and also in the supernatants of infected macrophages, a sandwich ELISA was performed. Briefly, J774 murine macrophages (2 x 105) were infected with late-stationary-phase promastigotes of either L. major expressing the episomal form of the mGM-CSF gene or L. major LV39 expressing the integrated form of the mGM-CSF gene at a parasite-to-cell ratio 20:1, and supernatants as well as macrophage lysates were collected at 4, 8, and 16 h postinfection. The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA. Dilutions of the antibodies and the assay procedure were as specified by the manufacturer (BD Biosciences).
Confocal microscopy.
After overnight infection of J774 murine macrophages with either L. major expressing the episomal GM-CSF form or L. major LV39 expressing the integrated GM-CSF gene, macrophages were washed three times with phosphate-buffered saline to remove unbound Leishmania and fixed with 100% cold acetone for 10 min. Then, infected macrophages were incubated for 30 min with the monoclonal anti-mouse GM-CSF antibody (MAB415; R&D Systems) and then for 30 min with the second antibody, Alexa Fluor 546 goat anti-rat (A-11081 immunoglobulin G (heavy plus light chains; Molecular Probes). The samples were prepared as specified by the manufacturer. Coverslips were then mounted and sealed on the microscope slide for confocal microscopy observation. Fluorescence images acquired through a 60x1.4NA objective (PlanApo; Olympus) were captured at serial optical sections (
20 to 30 sections at 0.5-µm intervals) by the Flowview FV300 confocal scanning unit. Fluorescence from each channel was imaged sequentially to eliminate cross talk between channels. Alexa 546 was excited with a helium-neon laser line. The red fluorescence was imaged using a 575- to 630-nm bandpass emission filter. Color images were created with Fluoview 300 version 3.3 software (Olympus). Then color contrasts were adjusted using Photoshop v 6.0 software.
RNase protection assay. Cytokine and chemokine gene expression induced by GM-CSF-expressing Leishmania following infection of J774 murine macrophages in vitro was monitored by an RNase protection assay using the mCK-2b and mCK-5 Riboquant kits (Pharmingen), respectively, as described previously (27). Briefly, 10 µg of RNA isolated from infected macrophages after 4, 8, 24, or 48 h of infection was hybridized separately with the 32P-labeled multiprobe template set in excess. The free probe and other single-stranded RNAs were digested with RNase. The RNase-protected probes were purified, resolved on denatured 5% polyacrylamide gels, and quantified with a PhosphorImager (ImageQuant 3.1 software) relative to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GADPH). LPS (lipopolysaccharide of Escherichia coli serotype O111:B4 [Sigma]) at 10 ng/ml was used as a control of J774 cell activation, and this action was stopped after 4 h.
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RESULTS |
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-tubulin gene (Fig. 1A), which provides the necessary signals for transcript maturation by trans splicing and polyadenylation (24). Southern and Northern blot hybridization analysis confirmed the presence of the GM-CSF-expressing plasmids in both L. major pNEO-mGM-CSF and pNEO-hGM-CSF transfectants (Fig. 1B) and showed that the corresponding RNAs were properly maturated (Fig. 1C). To further increase cytokine expression and to avoid possible loss of the Leishmania-expressing plasmids in infected mice, we generated a second series of transgenic Leishmania strains that carry the GM-CSF gene integrated into the genome. To engineer these parasites, the mGM-CSF gene was integrated into the rRNA locus of L. major LV39 downstream of the RNA polymerase I (pol I) promoter on chromosome 27. We have previously shown that foreign genes can be expressed at high levels when integrated downstream of the Leishmania ribosomal promoter and transcribed by RNA pol I (9, 41). Genomic analysis of the transfectants confirmed the proper integration of the mGM-CSF gene into the ribosomal locus. Indeed, Southern blot hybridization indicated two additional bands of 1 and 11 kb hybridizing to the rRNA promoter probe (Fig. 2A and B) and two bands of 3 and 5 kb hybridizing to the GM-CSF probe (Fig. 2B), as expected for a successful targeting. By comparing the intensity of the hybridizing bands with the ribosomal promoter probe we concluded that integration of the mGM-CSF gene occurred probably at one location within the multiple-copy ribosomal locus (Fig. 2B and data not shown). The genomic integration of the mGM-CSF gene into the rRNA locus was further confirmed by pulsed-field gel electrophoresis and Southern blot hybridization (Fig. 2C). The rRNA promoter initiates transcription by RNA pol I resulting in high levels of GM-CSF gene expression, as indicated by Northern blot hybridization (Fig. 2D). GM-CSF-expressing parasites have been maintained in culture for several months without any significant effect on growth or morphology, indicating that GM-CSF expression is not detrimental to Leishmania itself.
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425 pg/ml for 2 x 106 parasites after 16 h of infection [Fig. 3B]) was very similar to the amount of GM-CSF secreted by extracellular promastigotes (450 pg/ml). Moreover, immunofluorescence studies indicated that GM-CSF is being produced in the phagosomes of infected macrophages (Fig. 4B and D). Indeed, macrophages infected with GM-CSF-expressing parasites were highly fluorescent (Fig. 4B) compared to macrophages infected with wild-type parasites (Fig. 4A) and/or to uninfected macrophages (Fig. 4C). Taken together, these results suggest that both extracellular and intracellular GM-CSF-producing L. major organisms release GM-CSF in the extracellular milieu.
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and IL-1ß to type I and type II IL-1 receptors modulating thus IL-1 expression (17).
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, MIP-1ß, MIP-2, and MCP-1, as evaluated by an RNase protection assay, was induced by L. major, as previously reported (27). However, of utmost interest, GM-CSF-expressing L. major was capable of further inducing expression of the above chemokine genes by 2.5- to 4.5-fold (Fig. 8). Maximal induction was seen at 8 h postinfection for MCP-1 and at 24 h postinfection for RANTES, MIP-1, MIP-1ß, and MIP-2. Lymphotactin, IP-10, TCA-3, and TNF- gene expression was not altered in GM-CSF-expressing parasites (data not shown).
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could induce a nitric oxide (NO)-mediated regulatory mechanism to control the intracellular growth of L. donovani (7). We therefore tested whether nitric oxide production was increased in J774 macrophages infected with L. major LV39 expressing the integrated mGM-CSF gene compared to the control L. major NEO. Following incubation of the infected macrophages with IFN- for 24 or 48 h, the accumulation of nitrite after the addition of the Griess reagent was measured in the supernatants spectrophotometrically at 543 nm. Our results indicated a slight increase in NO production by the J774 macrophage cell line 24 h following infection with GM-CSF-expressing parasites (data not shown). To better assess the effect of GM-CSF release in inducing NO production, mice infected with either the GM-CSF-expressing parasites or the control strain were treated with aminoguanidine, a selective inhibitor for iNOS. No significant difference in the development of cutaneous lesions was detected between the two groups of mice (Fig. 9), thus excluding any contribution of NO production in delaying the development of disease in mice infected with GM-CSF-expressing parasites, as shown for Trypanosoma species (33). However, a significant increase in the size of the lesions was observed in aminoguanidine-treated mice infected with the L. major NEO strain, as expected (Fig. 9).
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DISCUSSION |
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gene (50). However, IFN- had no effect on disease progression in susceptible BALB/c mice (50). Here we have chosen to express the GM-CSF gene by the parasite, since this cytokine has often been associated with both macrophage activation and subsequent parasite killing. Indeed, it has been reported that pretreatment of macrophages with GM-CSF enhances the intracellular killing of L. tropica (19), L. donovani (54), L. major (2), and L. mexicana amazonensis (21) in vitro. Moreover, GM-CSF showed clear-cut antileishmanial activity in vivo, associated with both mobilization of neutrophils and monocytes (29), and its combination with pentavalent antimony has been used successfully for the treatment of visceral leishmaniasis (5). Interestingly, Leishmania antigens presented by GM-CSF-derived macrophages could protect susceptible mice against challenge with L. major by activating TH1 cells (15). While GM-CSF demonstrates a clear-cut leishmanicidal activity in vitro and in vivo in the L. donovani model (29, 54), the results obtained in the literature with L. major were more conflicting, suggesting that GM-CSF may play a positive (2, 15, 19, 21), neutral (12), or negative (18, 47) role in host defense. Our results indicated that recombinant GM-CSF parasites were eliminated much more rapidly inside macrophages compared to control cells and that this occurred independently of the macrophage type used for infection (Fig. 5 and 6, and data not shown). The intramacrophage parasite killing was specifically associated to GM-CSF activity since this effect was successfully caused to revert by an anti-GM-CSF antibody treatment prior to macrophage infection (Fig. 5A).
GM-CSF plays a central role in initiating a cytokine network by releasing numerous proinflammatory cytokines from the activated macrophages (42). Although several studies have reported an antileishmanial effect of GM-CSF in vitro and in vivo, little is known about how GM-CSF induces its antileishmanial activity. It is possible that GM-CSF induces parasite killing by activating macrophages to enhance H2O2 release (2, 37), to produce more nitric oxide, or to augment IFN- and IL-1 production (21). Our study has shed some light on the mechanism by which GM-CSF exerts its antiparasitic action. We showed here that GM-CSF, once released by both extracellular and intracellular forms of Leishmania, can activate phagocytic cells to express proinflammatory cytokines such as IL-1ß, IL-6, and IL-18 (Fig. 7) and a number of chemokines (RANTES, MIP-1, MIP-1ß, MIP-2, and MCP-1) (Fig. 8), known to be crucial to cellular recruitment and to facilitate the initiation of inflammatory responses. As suggested by our data (Fig. 3, 4, 5A, and data not shown), macrophage activation could be mediated both at the beginning of infection by promastigotes secreting GM-CSF and later during infection when amastigotes inside the phagosomal compartment also release GM-CSF. We showed recently that L. major is a strong inducer of early inflammatory events in vivo compared to L. donovani (27). In this study, we show that GM-CSF-expressing parasites further enhance the expression of several proinflammatory molecules in vitro (Fig. 7 and 8). An enhanced inflammatory reaction during the early steps of the Leishmania infection may result in a more rapid elimination of the parasites, which could explain the significant delay (5 to 6 weeks postinfection) in the development of cutaneous lesions in susceptible BALB/c mice (Fig. 6). This increased accumulation of a specific subset of chemokine transcripts after macrophage activation with L. major-GM-CSF-expressing parasites could result in the recruitment of a greater number of inflammatory leukocytes (i.e., neutrophils) at the inoculation site, which could kill the parasites, hence explaining the antileishmanial effect of GM-CSF seen in mice (Fig. 6). It is very likely that the larger number of neutrophils recruited in the inoculation site by, for example, MIP-2 (Fig. 8) could promote a more efficient killing of Leishmania. Experiments using the air pouch system (27) are expected to answer some of these questions.
An increase in the production of IL-1ß and IL-18 could have important consequences in controlling the parasitic load and activating a number of lymphocytic cells to kill the parasite. IL-18 and IL-1ß are related to the same family in terms of structure, processing, receptor, and signal transduction pathways (for a review, see reference 8). It has been shown that IL-1 may be required for normal regulation of the TH1 response (46). IL-18, originally identified as IFN--inducing factor, plays a pivotal role in systemic and local inflammation (14), but it is also related to IL-12 with regard to its capacity to induce the production of TH1 cytokines and to enhance cell-mediated immunity (40). IL-18 plays an important role in host defense against a variety of infectious pathogens (for a review, see reference 8) and has been proven effective as a treatment against L. major infection (32) and in the early control of cutaneous L. major lesion growth (53). The role of the inflammatory cytokine IL-6 in controlling Leishmania infection is controversial; however, recent studies demonstrated that IL-6 deficiency down regulates both TH1- and TH2-associated cytokines in BALB/c mice infected with L. major (48) but is not required for efficient immunity (28). In addition to IL-1ß induction, IL-1R antagonist (IL-1Ra) expression is induced 24 h following macrophage infection with L. major expressing GM-CSF (Fig. 7), as also reported for human monocytes (22), suggesting that the balance between IL-1Ra and IL-1 might be an important factor in host resistance to Leishmania infection. IL-1 plays a key role in inflammation, exerting its effects on a wide variety of cells and often leading to tissue destruction (13). To limit the potential for immunopathology and to suppress the inflammatory consequences of early IL-1 release, the proinflammatory response is down regulated by cytokines such as IL-1Ra (for a review, see reference 3), which competitively blocks the binding of IL-1 and IL-1ß to type I and type II IL-1 receptors (17).
It is well known that the production of IL-1ß and IL-6 plays a central role in the proinflammatory response and that in L. major infection, IL-1ß leads to a greater up regulation of RANTES, MIP-1, MIP-1ß, MIP-2, IP-10, and MCP-1, which are known to recruit monocytes/macrophages, neutrophils, and eosinophils (27). Indeed, we show here that infection of murine macrophages in vitro with GM-CSF-expressing parasites led to a rapid and transient expression of various chemokines, including RANTES, MCP-1, MIP-1, MIP-1ß, and MIP-2 (Fig. 8). Self-healing localized cutaneous leishmaniasis in humans is associated with higher levels of MCP-1 expression (6), and MCP-1 directly stimulates the elimination of intracellular L. major parasites within human monocytes (39). MCP-1 and MIP-1 orchestrate an antileishmanial activity in murine macrophages infected with L. donovani via the induction of a NO-mediated regulatory mechanism to control the intracellular growth of Leishmania (7). We observed only a slight increase in NO production at early time points following infection with GM-CSF-expressing Leishmania strains (data not shown). However, mice infected with the L. major GM-CSF strain and treated with aminoguanidine, a selective inhibitor of iNOS synthesis, showed no difference in the size of cutaneous lesions compared to the untreated mice (Fig. 9). GM-CSF has been also involved in the induction of major histocompatibility complex class II antigen on the surface of human monocytes (1) and on the induction of HLA-DR expression by human monocytes (10) in other systems. However, fluorescence-activated cell sorter analysis using the HLA-DR 2.06 mouse monoclonal antibody did not reveal any up regulation of the major histocompatibility complex class II antigen on the surface of human macrophages infected with GM-CSF-expressing Leishmania strains (data not shown).
GM-CSF-expressing Leishmania can provide a potential means of creating a favorable immune response in the host. Indeed, a combination of inactive vaccine against American cutaneous leishmaniasis together with GM-CSF as an adjuvant has been administered to 56 volunteers and has been associated with an improved immune response (16). Moreover, by activating and augmenting many of the functions of neutrophils, monocytes/macrophages, and dendritic cells (4), GM-CSF could enhance host defenses against Leishmania infection. Strategies utilizing suicide-type vectors expressing antileishmanial cytokines that can significantly decrease parasite replication combined with genetic attenuation of parasite's infectivity or with DNA vaccine immunization may be a promising step in the development of an effective vaccine against this parasite.
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ACKNOWLEDGMENTS |
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This work was supported by a Connaught Laboratories grant through its Canadian University Research Program and Medical Research Council Industry Program and by CIHR (Canadian Institutes of Health Research) group grant GR-14500 to B. Papadopoulou, M. Ouellette, and M. Olivier. B. Papadopoulou and M. Olivier hold an FRSQ (Fonds de Recherche en Santé du Québec) Senior Research scholarship and are recipients of a Burroughs Wellcome Fund New Investigator Award in Molecular Parasitology. M. Ouellette is a holder of a Canada Research Chair in Antimicrobial Resistance and a recipient of a Burroughs Wellcome Fund Senior Award in Molecular Parasitology.
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FOOTNOTES |
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Present address: Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada.
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) or with L. major wild type (•). The amount of GM-CSF in lyzed macrophages was measured by ELISA. (B) CM-CSF release in the extracellular milieu of macrophages infected with L. major expressing the episomal form of the mGM-CSF gene (
) or with L. major wild type (
). The amount of GM-CSF in the culture supernatants of infected macrophages was measured by ELISA. Dilutions of both the capture and biotinylated antibodies were at 1/250.
