Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

Cell culture and transfections. L. major MHOM/IL/67/JERICHO II is a WRAIR/WHO reference strain obtained from the American Type Culture Collection. This strain was used to transfect the episomal GM-CSF-expressing vectors. L. major LV39 and L. donovani 1S2D are described elsewhere (35, 41). The LV39 strain was used to integrate the mGM-CSF gene into the ribosomal DNA locus by homologous recombination and to infect mice. All strains were grown in SDM-79 medium supplemented with 10% fetal bovine serum (Multicell, Wisent Inc.) and 5 μg of hemin per ml. Approximately 15 μg of DNA from the episomal expression vector and 2 to 3 μg of the linearized targeting construct for genomic integration were used for transfections, as described previously (36). All transfectants were selected with 40 μg of G418 (Geneticin; Gibco-BRL) per ml. The murine macrophage cell line J774, originally obtained from the American Type Culture Collection was cultured in Dulbecco's modified Eagle's medium (Gibco-BRL) supplemented with 10% fetal bovine serum and incubated at 37°C in a 5% CO₂ atmosphere. Human peripheral blood monocytes were isolated from heparinized venous blood of normal donors by the Canadian Red Cross. The cells were centrifuged over a Ficoll-Paque gradient (Pharmacia) as previously described (34). After several washes, the cells were resuspended in RPMI 1640 medium (Gibco-BRL) containing 10% human serum (Gibco-BRL). To differentiate monocytes into macrophages, 3 × 10⁶ peripheral blood leukocytes, counted using trypan blue, were adhered to tissue culture plates and cultured for 5 days at 37°C in 5% CO₂-95% air in a humidified atmosphere to allow cell transformation.

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 pSPαNEOα (constructed by subcloning the SmaI-BamHI-digested αNEOα expression cassette from the vector PGEM7-αNEOα [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 pSPαNEOα 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 pSPαNEOα downstream of the α-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 × 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 × 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 × 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 × 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 60×1.4NA 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.

Generation of transgenic L. major producing and secreting GM-CSF.The goal of this study was to engineer “suicide” Leishmania expressing cytokine genes known to activate the host immune responses in better controlling parasite infection. This type of live vectors may constitute an interesting approach to the design of a Leishmania vaccine. For these studies, we selected GM-CSF, a cytokine shown to be important in the control of Leishmania infections (see Discussion). First, we engineered L. major transgenic strains expressing either the mGM-CSF or the hGM-CSF genes as part of an episomal vector under the constitutive control of the 800-bp intergenic region of the L. enriettii α-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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FIG. 1.

Recombinant L. major expressing GM-CSF as part of an episomal vector. (A) Expression vectors pNEO-mGM-CSF and pNEO-hGM-CSF were made by inserting the murine mGM-CSF and hGM-CSF genes, respectively, downstream of the neomycin phosphotransferase (NEO) expression cassette as described in Materials and Methods. Arrows indicate the orientation of GM-CSF transcript maturation. In pNEO-hGM-CSF/R, the hGM-CSF gene is cloned in the opposite orientation relative to mRNA maturation signals. Bg, BglII. (B) Southern blot of L. major genomic DNA digested with BglII and hybridized to the murine and human GM-CSF-specific probes. Lanes: 1, L. major pNEO control; 2, L. major expressing either the mGM-CSF or the hGM-CSF gene. (C) GM-CSF expression in L. major GM-CSF recombinant cells. A Northern blot of total Leishmania RNA hybridized to the same probes as above. Lanes 1 and 2 are as in panel B. Lane 3, L. Major expressing the hGM-CSF gene.

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FIG. 2.

Stable integration of the GM-CSF gene into the L. major LV39 genome. (A) Schematic representation of the L. major rRNA locus with the RNA pol I promoter indicated by a flag. The GM-CSF expression cassette was inserted downstream of the rRNA promoter for stable and high expression. The EcoRV (EV) and BamHI (B) restriction sites and the size of the restriction fragments are indicated. (B) Southern blot hybridization of the L. major LV39 wild-type strain (lane 1) and of L. major mGM-CSF recombinant strain made by the integration of the mGM-CSF gene into the ribosomal locus (lane 2) with probes specific to the rRNA promoter sequence and the mGM-CSF gene, respectively. The EcoRV restriction fragments confirming the correct integration of the GM-CSF gene into the parasite genome are indicated by arrows. (C) Clamped homogeneous electric field electrophoresis and Southern blot hybridization of L. major chromosomes (lanes and probes are as indicated in panel B). The ribosomal locus is part of chromosome 27. (D) Northern blot hybridization of L. major total RNA (lanes are as indicated in panel B) with the GM-CSF gene probe. RNA loading was monitored by ethidium bromide staining (lower panel).

Because the GM-CSF clone used for these studies harbors the amino-terminal signal sequence necessary for targeting secreted proteins into the lumen of the endoplasmic reticulum in eukaryotes, it is expected that Leishmania should secrete GM-CSF into the medium. It has been reported that Leishmania utilizes similar signal sequences for targeting proteins to the endoplasmic reticulum to that observed in yeast and higher eukaryotes (51). Using an ELISA, we first measured the release into the culture medium of GM-CSF by GM-CSF-expressing L. major promastigotes. Our results indicated that 2 × 10⁶L. major promastigotes expressing the integrated mGM-CSF gene secreted 550 pg of GM-CSF per ml while 2 × 10⁶L. major promastigotes expressing the episomal GM-CSF secreted 450 pg/ml. We similarly evaluated whether GM-CSF was released by intracellular parasites during the course of an in vitro infection. The release of GM-CSF was measured in both the intracellular (the cytosol of infected macrophages including phagosomes) and extracellular (culture supernatants of infected macrophages) milieu. More than 850 pg of GM-CSF per ml was produced by 10⁶ parasites inside macrophages 4 h following infection, and this amount remained relatively constant at later stages of macrophage infection (Fig. 3A). In contrast, macrophages infected with the wild-type parasites did not produce or secrete detectable levels of GM-CSF (Fig. 3). Approximately 25% of the intracellularly produced GM-CSF (Fig. 3A) was secreted in the extracellular milieu during the course of infection (Fig. 3B). The amount of GM-CSF released in the supernatants of infected macrophages (∼425 pg/ml for 2 × 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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FIG. 3.

GM-CSF release by intracellular Leishmania expressing the GM-CSF gene. (A) CM-CSF production and release within macrophages at different time points following infection of J774 macrophages with L. major expressing the episomal form of the mGM-CSF gene (□) 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.

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FIG. 4.

GM-CSF detection by immunofluorescence within macrophages infected with GM-CSF-producing parasites. After overnight infection of J774 murine macrophages with either L. major LV39 expressing the integrated mGM-CSF gene or L. major expressing the episomal GM-CSF vector, macrophages were washed, fixed, and then incubated with the monoclonal anti-mouse GM-CSF antibody and Alexa Fluor 546, as indicated in Materials and Methods. Coverslips were then mounted and sealed on the microscope slide for confocal observation. Color images were created with Fluoview 300 version 3.3 software, and color contrasts were adjusted using Photoshop version 6.0 software. (A) macrophages infected with L. major wild type (dilutions are at 1/250 for the first antibody and 1/1,000 for the second antibody), (B) macrophages infected with L. major expressing the episomal form of the mGM-CSF gene (dilutions are at 1/250 for the first antibody and 1/1,000 for the second antibody), (C) uninfected macrophages (dilutions are at 1/1,000 for the first antibody and 1/2,000 for the second antibody), and (D) macrophages infected with L. major LV39 expressing the integrated form of the mGM-CSF gene (dilutions are at 1/1,000 for the first antibody and 1/2,000 for the second antibody).

Increased intracellular killing of recombinant Leishmania producing GM-CSF.GM-CSF plays a protective role in Leishmania infections (15, 19, 29, 44, 54). Indeed, pretreatment of macrophages with recombinant GM-CSF enhances the intracellular killing of L. tropica (19), L. donovani (54), and L. mexicana amazonensis (21) in vitro. We therefore tested whether parasites expressing biologically active GM-CSF were more susceptible to elimination by macrophages. The intracellular survival of Leishmania amastigotes expressing either the mGM-CSF or the hGM-CSF gene was first evaluated using an in vitro system to infect macrophages/monocytes (31). The murine J774 cell line or human peripheral blood monocytes were infected with late-stationary-phase Leishmania promastigotes expressing GM-CSF at a parasite-to-cell ratio of 20:1, and infection rates were compared to those of the Leishmania NEO control strain. The outcome of the infection was determined at 6, 24, 48, and 72 h by microscopic examination, as indicated under Materials and Methods. Our results demonstrated that the intracellular survival of L. major amastigotes expressing either the mGM-CSF or the hGM-CSF gene was decreased by more than 80% 48 h following the infection compared to that of the L. major NEO control (Fig. 5). Similar results were obtained with recombinant L. donovani expressing the mGM-CSF gene (data not shown). Moreover, infection of peritoneal mouse macrophages with GM-CSF-expressing L. major resulted in a similar decrease in the survival of amastigotes (data not shown). Parasite infectivity levels were further confirmed by measuring the luciferase (LUC) activity in macrophages infected with recombinant GM-CSF parasites expressing the LUC reporter gene at several time points following infection (data not shown). To evaluate whether the decreased intracellular survival of GM-CSF-expressing parasites was specifically related to the effect of GM-CSF, we incubated the parasites with an anti-GM-CSF neutralizing antibody few hours prior to macrophage infection. Our data showed that in this case, the anti-leishmanial effect of GM-CSF was almost completely blocked (Fig. 5A). Also, when the human GM-CSF gene was cloned in the reverse orientation relative to mRNA maturation signals, we saw no effect in the intramacrophage survival of the parasites (Fig. 5B).

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FIG. 5.

Intracellular killing of L. major amastigotes expressing the mGM-CSF and/or the hGM-CSF gene. J774 murine macrophages and/or human monocytes differentiated to macrophages (5 × 10⁴ cells/well) were incubated for 6, 48, or 72 h with late-stationary-phase L. major promastigotes (parasite-to-cell ratio, 20:1) expressing either the mGM-CSF or the hGM-CSF gene as described in Materials and Methods. At these fixed time points, cell cultures were dried and stained with Diff Quik to determine the level of infection. (A) Infection of murine macrophages with the L. major pNEO control, L. major mGM-CSF expressing the episomal mGM-CSF gene, and L. major mGM-CSF incubated with an anti-GM-CSF neutralizing antibody prior to macrophage infection. (B) Infection of human monocytes with L. major pNEO, L. major hGM-CSF, and L. major hGM-CSF/R (with the human GM-CSF gene cloned in the reverse orientation). The results shown here are the mean of three independent experiments with duplicate samples and the standard errors of the mean.

The antileishmanial effect of GM-CSF was also evaluated in susceptible BALB/c mice infected with GM-CSF-expressing L. major. For these studies, both recombinant L. major strains expressing either the episomal (Fig. 1A) or the integrated (Fig. 2A) mGM-CSF gene were used. The outcome of Leishmania infection was monitored by weekly measurements of the mouse footpad lesions. Our data showed a significant delay in the development of cutaneous lesions in mice infected with GM-CSF-expressing parasites compared to the NEO control strain (Fig. 6). Indeed, mice infected with Leishmania carrying the episomal GM-CSF vector start developing the first signs of cutaneous infection after 6 weeks, compared to 2 weeks for the control L. major NEO strain. However, mice infected with parasites carrying the integrated mGM-CSF gene, where GM-CSF expression is expected to be higher due to the promoter activity, start to develop cutaneous lesions after 7 to 8 weeks, 1 to 2 weeks later than the time noted for mice infected with the GM-CSF-episomal construct (Fig. 6). In this experiment, mice infected with the L. major NEO control strain (Fig. 6) or with L. major wild type (data not shown) were sacrificed after 7 weeks due to the large size of the lesions. Overall, these data support the notion that GM-CSF production and release by intracellular L. major can remarkably reduce parasite viability in vitro and also delay lesion development in mice in vivo.

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FIG. 6.

Outcome of cutaneous disease in susceptible BALB/c mice infected with late-stationary-phase L. major promastigotes expressing the murine GM-CSF gene. The infection was monitored for up to 7 weeks postinoculation as described in Materials and Methods. Results obtained with mice infected with the L. major NEO control, mice infected with the L. major LV39 recombinant strain expressing the mGM-CSF gene integrated into the 18S ribosomal locus under the control of the rRNA promoter, and mice infected with L. major GM-CSF expressing the episomal form of the mGM-CSF gene are shown. The result shown here is the average mean of three independent experiments.

GM-CSF released by GM-CSF-expressing Leishmania activates macrophage functions.To better understand how exposure of macrophages to GM-CSF could initiate parasite killing, we tested whether parasites expressing GM-CSF could modify the cytokine response of the macrophage. Murine macrophage cell lines were infected in vitro either with L. major LV39 expressing the integrated GM-CSF gene or with L. major wild type, and the expression profile of several cytokines was measured by an RNase protection assay. LPS was used as a control for macrophage stimulation. As shown in Fig. 7, several proinflammatory cytokines such as IL-1β, IL-18, and IL-6 were transiently induced from 2.5- to 4.5-fold in macrophages infected with GM-CSF-expressing parasites. Maximal induction for IL-18 was seen at 24 h postinfection, and that for IL-1β and IL-6 was seen at 4 h postinfection (Fig. 7). Moreover, our data showed an increase in IL-1Ra gene expression 24 h following infection with GM-CSF-expressing parasites, suggesting a certain balance control in the inflammatory response. It is known that early IL-1 release and enhanced inflammatory response could also exert negative effects on cell function (13). IL-1Ra can competitively block the binding of IL-1α and IL-1β to type I and type II IL-1 receptors modulating thus IL-1 expression (17).

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FIG. 7.

A time course pattern of cytokine expression by J774 macrophages infected with L. major LV39 expressing the integrated mGM-CSF gene. Graphs represent densitometric quantification of bands from cytokine mRNA (IL-1Ra, IL-1β, IL-18, and IL-6) normalized to the GAPDH signal. LPS was used as a control. The relative increase in the levels of the various cytokines is in comparison to macrophages infected with the wild-type parasites. The data presented here are the mean of three independent experiments. Error bars, ± 0.1 to 0.5.

Given that induction of proinflammatory cytokines is known to modulate chemokine expression and that L. major can induce chemokine expression in vitro and in vivo (26, 27), we tested whether CXC and CC chemokine mRNA expression was further enhanced in macrophages infected with GM-CSF-expressing Leishmania strains. As shown in Fig. 8, chemokine gene expression of RANTES, MIP-1α, MIP-1β, 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-1α, MIP-1β, and MIP-2. Lymphotactin, IP-10, TCA-3, and TNF-α gene expression was not altered in GM-CSF-expressing parasites (data not shown).

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FIG. 8.

Patterns of chemokine expression by J774 macrophages infected with L. major LV39 expressing the integrated mGM-CSF gene. Graphs represent densitometric quantification of bands from chemokine mRNA (RANTES, MCP-1, MIP-2, MIP-1α, and MIP-1β) normalized to the GAPDH signal. The relative increase in the levels of the various chemokines is in comparison to macrophages infected with the wild-type parasites is shown. The data presented here are the mean of three independent experiments. Error bars, ± 0.03 to 0.4.

It was reported recently that MCP-1 and MIP-1α 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-γ 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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FIG. 9.

No inhibition in mice infected with GM-CSF-expressing recombinant parasites. BALB/c mice infected either with L. major LV39 expressing the integrated mGM-CSF gene (L.m-GM-CSF) or with the L. major NEO strain (L.m-NEO) were treated with aminoguanidine, a selective inhibitor of inducible iNOS synthesis, and the outcome of the cutaneous infection in BALB/c mice was monitored for up to 4 weeks and compared to that for the untreated animals. The data presented here are the mean of two independent experiments.