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Originally published online as doi:10.1189/jlb.0706440 on April 20, 2007

Published online before print April 20, 2007
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(Journal of Leukocyte Biology. 2007;82:288-299.)
© 2007 by Society for Leukocyte Biology

Leishmania major induces distinct neutrophil phenotypes in mice that are resistant or susceptible to infection

Mélanie Charmoy1, Rosette Megnekou1,2, Cindy Allenbach, Christine Zweifel, Cynthia Perez, Katia Monnat, Mélanie Breton, Catherine Ronet, Pascal Launois and Fabienne Tacchini-Cottier3

WHO Immunology Research and Training Center, Department of Biochemistry, Epalinges, Switzerland

3 Correspondence: WHO Immunology Research and Training Center, Department of Biochemistry, University of Lausanne, 155, ch. des Boveresses, CH-1066 Epalinges, Switzerland. E-mail: fabienne.tacchini-cottier{at}unil.ch

ABSTRACT

Polymorphonuclear neutrophils (PMN) are key components of the inflammatory response contributing to the development of pathogen-specific immune responses. Following infection with Leishmania major, neutrophils are recruited within hours to the site of parasite inoculation. C57BL/6 mice are resistant to infection, and BALB/c mice are susceptible to infection, developing unhealing, inflammatory lesions. In this report, we investigated the expression of cell surface integrins, TLRs, and the secretion of immunomodulatory cytokines by PMN of both strains of mice, in response to infection with L. major. The parasite was shown to induce CD49d expression in BALB/c-inflammatory PMN, and expression of CD49d remained at basal levels in C57BL/6 PMN. Equally high levels of CD11b were expressed on PMN from both strains. In response to L. major infection, the levels of TLR2, TLR7, and TLR9 mRNA were significantly higher in C57BL/6 than in BALB/c PMN. C57BL/6 PMN secreted biologically active IL-12p70 and IL-10. In contrast, L. major-infected BALB/c PMN transcribed and secreted high levels of IL-12p40 but did not secrete biologically active IL-12p70. Furthermore, IL-12p40 was shown not to associate with IL-23 p19 but formed IL-12p40 homodimers with inhibitory activity. No IL-10 was secreted by BALB/c PMN. Thus, following infection with L. major, in C57BL/6 mice, PMN could constitute one of the earliest sources of IL-12, and in BALB/c mice, secretion of IL-12p40 could contribute to impaired, early IL-12 signaling. These distinct PMN phenotypes may thus influence the development of L. major-specific immune response.

Key Words: IL-12 • Toll-like receptors • IL-10

INTRODUCTION

The intracellular protozoan Leishmania major is an obligate parasite of macrophages, which can infect various mammalian hosts such as rodents, dogs, and humans. Cutaneous leishmaniasis is mainly a result of L. major in the Old World. It is self-limiting and self-curing. Spontaneous cure and long-standing immunity are the most common outcomes.

s.c. injections of 3 x 106 L. major stationary-phase promastigotes in mice of most inbred strains such as C57BL/6 lead to control of parasite replication and the development of self-healing, cutaneous lesions. On the contrary, in BALB/c mice, infection induces uncontrolled parasite proliferation associated with the development of a lesion that does not heal. Resistance to infection has been shown to correlate with the development of CD4+ Th1 cells, and susceptibility was correlated with the development of CD4+ Th2 cells (reviewed in ref. [1 ]).

As most of the events, which have been reported to affect CD4+ Th differentiation and thus resistance or susceptibility to infection, take place during the first days after parasite inoculation, the first cells recruited to the site of parasite inoculation may influence the subsequent events leading to CD4+ Th cell differentiation. It is interesting that qualitative and quantitative differences in the onset of the inflammatory response to L. major have been reported in mice susceptible or resistant to infection. In BALB/c mice, polymorphonuclear leukocytes (PMN) constitute the major population of cells recruited the first day after parasite inoculation, and their number still contributes to 50% of the cellular infiltrate 10 days after infection. In contrast, in mice of the resistant phenotype, PMN contributed to 60% of the cellular infiltrate the first day after infection; however, 3 days after infection, they represented only 1–2% of the cellular infiltrate, and monocytes were the predominant cells [2 , 3 ].

We have reported previously that depletion of neutrophils in mice of genetically susceptible BALB/c strains, prior to the s.c. injection of 3 x 106 L. major, prevented the development of unhealing lesions and rendered these otherwise susceptible mice partially resistant to infection, with a significant reduction of the CD4+ Th2 cytokine IL-4 and of parasite burden within the site of parasite injection [4 ].

It was also reported that in C57BL/6 mice transiently depleted of neutrophils, parasite number within the footpad increased 35 days after parasite inoculation, but the mice could develop a Th1 type of immune response and finally healed their lesions, suggesting that in resistant strains of mice, neutrophils contribute to the destruction of L. major parasites but have no major effect on Th differentiation [4 , 5 ].

Thus, neutrophils appear to play a protective and/or immunoregulatory role in the early response to infection with L. major. Recently, the composition of the cellular infiltrate recruited following infection with L. major was shown to be regulated by cellular interactions between macrophages and PMN. Macrophages were reported to induce PMN apoptosis through a cell-cell contact involving membrane TNF. The susceptibility to macrophage-induced apoptosis differed between BALB/c and C57BL/6 PMN, a process attributed to potential differences in autocrine secretion of cytokines such as TGF-β [6 ]. Indeed, PMN have been reported to synthesize, in response to microorganism-derived stimuli, numerous proteins including chemokines and cytokines (reviewed in ref. [7 ]). The diversity of the cytokines produced by PMN is large, but the magnitude of cytokine production by PMN is generally lower than that of mononuclear cells (10–20x less mRNA/cell) [7 ]. Nevertheless, following infection with L. major, PMN constitute the majority of infiltrating cells in inflammatory tissues and thus, may be an important source of cytokines contributing to the initiation of the immune responses.

We therefore investigated if in L. major-susceptible or -resistant mice, infection of PMN with L. major could result in the differentiation of distinct types of PMN. Here, we report that following exposure to L. major, BALB/c and C57BL/6 neutrophils acquire a distinct phenotype including the expression of different cell surface integrins, TLRs, and the transcription and secretion of distinct cytokines.

MATERIALS AND METHODS

Mice and parasites
Female BALB/c and C57BL/6 mice were purchased from Harlan Olac Ltd. (Bicester, UK). All mice were used at 5–6 weeks of age. L. major LV 39 (MRHO/Sv/59/P strain) was maintained in vivo and grown in vitro as described previously [8 ]. Groups of two to three mice were infected i.p. with 107 stationary-phase L. major promastigotes in a final volume of 1 ml or injected with 0.25 ml, 2% starch, in PBS. Inflammatory peritoneal neutrophils were obtained 4 h after injection.

Isolation of neutrophils
Bone marrow neutrophils were isolated from the femur of mice, labeled with the neutrophil-specific mAb NIMP-R14-FITC [9 ] or 1A8-PE from BD PharMingen (San Diego, CA, USA), incubated on ice for 30 min, and purified further using MACS-positive selection using anti-FITC or anti-PE magnetic beads (Miltenyi Biotech, France). Purity of neutrophils was >98%. Inflammatory neutrophils were isolated by washing the peritoneal cavity of mice 4 h after the injection of 250 µl, 2% starch, or of 1 ml RPMI-1640 media containing 107 stationary-phase L. major.

Cell cultures
Mature PMN (1A8+) were purified by MACS, and 106 cells/ml were cultured in RPMI-1640 media supplemented with 10% FCS and antibiotics in the presence or absence of metacyclic L. major (at a 5:1 parasite:cell ratio) and/or LPS (40 ng/ml, Sigma, Buchs, CH, Switzerland) and/or IFN-{gamma} (50 U/ml, BD PharMingen) during 16, 24, or 48 h. Culture supernatants were harvested, filtered, and frozen at –80°C. The protease inhibitor aprotinin (0.4 µg/ml, Sigma Chemical Co., St. Louis, MO, USA) was added to the culture to facilitate cytokine detection.

FACS analysis
For cell surface staining, cells were incubated first with the mAb 24G2 to block FcRs and then with the mAb 1A8-PE (anti-Ly6G), anti-CD11b-FITC, isotype controls IgG2bk-FITC, streptavidin-Cy (all from BD PharMingen), and the anti-CD49d-biotinylated mAb (from eBiosciences, San Diego, CA, USA). Cells were analyzed with a FACScan (BD Biosciences, Mountain View, CA, USA) and analyzed with the program Flow Jo (Tree Star. Inc., Ashland, OR, USA).

RNA preparation and RT-PCR of cytokine genes
mRNA was isolated with Trizol (Invitrogen, Basel, Switzerland) and cDNA obtained using a kit (Pharmacia, Uppsala, Sweden). Semiquantitative PCR was performed as described previously. First-strand cDNA synthesis was performed on total RNA using a first-strand cDNA synthesis kit (Pharmacia). The semiquantitative PCR developed by Reiner et al. [10 ] was performed using the competitor construct containing sequences for multiple cytokines, the primers for hypoxanthine guanine phosphoribosyl transferase (HPRT) at the conditions described by the authors. The first-strand cDNA was used directly as a template in the presence of fivefold serial dilutions of the competitor. After separation of the PCR products by electrophoresis in agarose gel containing ethidium bromide, the ratio of the relative concentration of the gene of interest to the relative concentration of HPRT was calculated. Results are expressed as the fold increases in mRNA expression in neutrophils cultured in RPMI compared with those incubated with L. major, IFN-{gamma}, LPS, or L. major + IFN-{gamma}. For quantitative real-time RT-PCR, SYBR green was used, and PCR was done on a LightCycler (Roche, Rotkreuz, Switzerland) as described previously [11 ]. The primers for the real-time PCR were the following: p40 forward (F) 5' GGA AGC ACG GCA GCA GAA TAA 3', reverse (R) 5' CTT GAG GGA GAA GTA GGA ATG 3'; IL-12p35 F 5' AGG ACT TGA AGA TGT ACC AG 3', R 5' CTA TCT GTG TGA GGA GGG 3'; IL-23p19 F 5' AAT GTG CCC CGT ATC C 3', R 5' GGA GGT GTG AAG TTG CT 3'; HPRT F 5' GTT GGA TAT GCC CTT GAC 3', R 5' AGG ACT AGA ACA CCT GCT 3'. Results were expressed as fold increase compared with unstimulated PMN.

Cytokine detection
The level of IL-12p40 in culture supernatant was measured by an ELISA using the mAb C17.15 and biotinylated C15.6 mAb (gift of Giorgio Trinchieri, National Cancer Institute, Frederick, MD, USA). For IL-12p70, a capture bioassay was used [12 ]. Briefly, IL-12 biological activity was measured by the induction of splenocytes to produce IFN-{gamma}. Spleen cells were cultured in the presence or absence of recombinant (r)IL-12p70 or PMN supernatants and cultured for 72 h. IFN-{gamma} was measured by ELISA, and a standard curve was established with the splenocytes that received rIL-12p70. The IL-12-induced IFN-{gamma} secretion was specific, as checked by the addition of an anti-IL-12 mAb to the standard and the supernatants, blocking IFN-{gamma} secretion by splenocytes. In selected experiments, the mAb antibody against murine TGF-β1–3 (1D11.16.8; ATCC HB-9849) was also included.

IL-12p70 was also measured using an ELISA kit (OptEIATM, BD Biosciences). The level of IFN-{gamma} was measured by ELISA using the mAb O1E70 3B2 and the biotinylated AN-18.14.24 mAb. TGF-β was measured by a bioassay for TGF-β activity in PMN cell culture, using the cells transfected with a plasminogen activator inhibitor-1 (PAI-1) promoter construct [13 ], as described previously (ref. [6 ]; gift of Dr. Daniel Rifkin, New York University Medical Center, New York, NY, USA). This quantitative bioassay is based on the ability of TGF-β to up-regulate PAI-1 expression. To measure the release of latent TGF-β, PMN supernatant was acidified and added to the PAI-1-transfected cells. The luciferase assay was performed, and luciferase activity was determined using the luciferase assay substrate (Promega, Madison, WI, USA). The levels of IL-10 cytokines were measured using ELISA kits from OptEIATM (BD Biosciences).

TLR mRNA detection
Bone marrow-derived and inflammatory neutrophils recruited following injection of L. major or PBS-2% starch were purified by MACS.

Total RNA was isolated from PMN cells using the Qiagen RNA extraction kit according to the manufacturer’s instruction, including DNase treatment. First-strand cDNA synthesis was performed on total RNA using random nonamers and SuperScriptIITM (Invitrogen), according to the manufacturer’s instruction. Real-time, quantitative PCR was performed for HPRT and the TLRs (1–9) with specific primers, which were designed to measure only target transcripts, avoiding the interference with the genomic DNA. Intron-spanning primers used for the real-time PCR: HPRT F 5'-GTTGGATATGCCCTTGAC-3', HPRT R 5'-AGGACTAGAACACCTGCT-3'; TLR1 F 5'-TCTTGCTGGCACCCATTC-3', TLR1 R 5'-CATGAGAGTTTTGAGCTTGTGG-3'; TLR2 F 5'-ACCGAAACCTCAGACAAAGC-3', TLR2 R 5'-AGCGTTTGCTGAAGAGGACT-3'; TLR3 F 5'-TTGTCTTCTGCACGAACCTG-3', TLR3 R 5'-CGCAACGCAAGGATTTTATT-3'; TLR4 F 5'- GGACTCTGATCATGGCACTG-3', TLR4 R 5'-ACTACCTCTATGCAGGGA-3'; TLR5 F 5'-CTGCAACTGTGAACTTAGCA-3', TLR5 R 5'-ACTTTAGGGACCGCAT-3'; TLR6 F 5'-CAGAACTCACCAGAGGTCCAA-3', TLR6 R 5'-CGAGTATAGCGCCTCCTTTG-3'; TLR7 F 5'-ACCAGACCTCTTGATTCC-3', TLR7 R 5'-CTGTGCAGTCCACGAT-3'; TLR8 F 5'-ATGGAAGATGGCACTGGTTC-3', TLR8 R 5'-CAAACGTTTTACCTTCCTTTGTCT-3'; TLR9 F 5'-ATCCTCCATCTCCCAAC-3', TLR9 R 5'-ACGGGGTACAGACTTC-3'.

PCR amplification was prepared using LightCycler Faststart DNA Master SYBR Green I kit (Roche) according to the manufacturer’s instructions. The amplification was undertaken using the LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany). Analysis was carried out with the LightCycler 3.5 software (Roche). The efficiency of each real-time PCR was calculated (>98%). The results were analyzed using the comparative threshold cycle method ({Delta}CT) for relative quantitation of gene expression normalized to the housekeeping gene HPRT.

Leishmanicidal assay and NO detection
Intracellular survival assay of L. major in macrophages
Inflammatory PMN were isolated from the peritoneal cavity 4 h after i.p. infection with 107 L. major stationary-phase promastigotes, washed 3x, and purified by MACS. The purified neutrophils were then lysed with 0.01% SDS in serum-free medium and incubated for 72 h in a L. major growth medium (M199+10% FCS) in a 6% CO2 atmosphere at 26°C. [3H]Thymidine was added for the last 16 h, and incorporation of radioactivity by viable parasites was measured using a LKB Wallac harvester and a 1205 Beta plateTM Wallac liquid scintillation counter. The culture was performed in five to six replicates. Proliferation of the parasites, as measured by the incorporation of the [3H]thymidine, is relative to the quantity of viable parasites within the neutrophils at the time of lysis.

Analysis of nitrites
Neutrophils isolated 4 h after i.p. injection of 107 L. major were purified by MACS and cultured for 48 h in the absence or presence of L. major, IFN-{gamma}, or both as described above. Culture supernatants were analyzed for their content of nitrites (NO2–) by the Griess reaction as described previously [14 ] with a detection limit of 1 µM.

Statistics
Statistical analysis was done using the two-tailed t-test for unpaired data.

RESULTS

Expression of CD11b and CD49d on the surface of PMN
The leukocyte β2 integrin CD11b (membrane-activated complex-1 or {alpha}mβ2) at the surface of PMN plays a critical role in PMN migration [15 ] and has been shown to interact with L. major [16 ]. In addition, expression of the integrins CD11b as well as that of CD49d ({alpha}4β1, VLA-4) was shown to correlate with the differentiation of a distinct population of PMN [17 ]. We therefore analyzed CD11b and CD49d expression ex vivo, 4 h after the i.p. injection of stationary-phase L. major. BALB/c and C57BL/6 PMN expressed high levels of CD11b, but basal expression of CD49d was low (data not shown). Starch-recruited PMN expressed similar levels of CD11b, suggesting that the regulation of CD11b expression is not specific to L. major-recruited PMN, which were purified and cultured for 16 h, alone or in the presence of metacyclic L. major at a 5:1 parasite:cell ratio. BALB/c and C57BL/6 PMN retained high levels of CD11b expression under all conditions tested with a slight decrease in L. major-infected PMN (Fig. 1 ).


Figure 1
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  		Figure 1. Expression of CD11b and CD49d by PMN. L. major-recruited, inflammatory BALB/c and C57BL/6 PMN were isolated from the peritoneal cavity and cultured in the absence (gray histogram) or presence (bold line) of metacyclic L. major promastigotes at a 1:5 PMN:parasite ratio. Sixteen hours later, cells were stained with mAb against CD11b and CD49d and analyzed by FACS. This is a representative experiment of four.

The expression of CD49d/CD29, which is a receptor for VCAM-1 on the inflammatory endothelium, was also analyzed. Infection of purified inflammatory PMN with L. major induced an increase in the expression of CD49d in BALB/c but not in C57BL/6 PMN (Fig. 1) . This increase was observed consistently in four independent experiments. The association of CD49d with VCAM-1 in numerous animal models of chronic inflammatory diseases has been well documented (reviewed in ref. [18 ]), and the expression of CD49d in PMN of BALB/c mice, which develop chronic, inflammatory lesions, but not in C57BL/6 PMN, which resolve their lesion, fits with these views. A similar increase in CD49d expression was observed in starch-recruited BALB/c PMN infected with L. major, and no increase was measured in L. major-infected C57BL/6 PMN (data not shown).

As CD11b and CD49d are also involved in PMN extravasation, the kinetic of expression of these cell surface molecules was also assessed in the blood of mice subsequent to the s.c. infection of 3 x 106 L. major stationary-phase promastigotes. CD11b was expressed at elevated levels on circulating PMN, and expression did not vary within the period analyzed. In contrast, expression of CD49d was low in C57BL/6 and BALB/c blood PMN during the first days following infection; this low level of CD49d expression did not change in C57BL/6 PMN, and the level of CD49d expression increased in BALB/c PMN after 3 days of infection and remained similar and distinct from that of C57BL/6 PMN up to 35 days after infection (data not shown).

Thus, following infection with L. major, blood PMN expressed high levels of CD11b but low levels of CD49d. However, following exposure to L. major, inflammatory PMN expressed a distinct pattern of surface integrins. CD11b was expressed at high levels on PMN from both strains of mice, and L. major induced the expression of CD49d selectively on BALB/c but not on C57BL/6 PMN.

Expression of TLR mRNAs in C57BL/6 and BALB/c PMN following infection with L. major
Pathogen-recognition receptors are key components of the immune system, involved in innate effector mechanisms and induction of adaptive immunity [19 ]. TLRs are able to recognize microorganisms and transduce specific signals involved in the host defense. TLR2, TLR4, and TLR9 have been shown to play a role in the control of infection with L. major [20 , 21 ], and the role of other TLRs in this infection remains to be established. Humans and mouse PMN express TLRs [17 , 22 23 24 25 ]. We therefore investigated by quantitative real-time PCR if the transcription of the different TLRs was regulated differentially by L. major in BALB/c versus C57BL/6 PMN. Basal levels of transcription for the different TLRs (TLR1–9) were first measured in bone marrow-isolated neutrophils. TLR mRNA levels did not differ in BALB/c versus C57BL/6 neutrophils (Fig. 2 ), and no transcription was detected for TLR3 (data not shown). Expression of TLRs was then measured in inflammatory neutrophils recruited following i.p. injection of stationary-phase L. major promastigotes and compared with that of neutrophils recruited following injection of PBS-2% starch to determine L. major-specific induction. No expression of TLR3 mRNA was detected, and only low levels of TLR1, TLR5, TLR6, and TLR8 mRNA were measurable in inflammatory neutrophils (data not shown). In contrast, the level of TLR4 mRNA was increased significantly in C57BL/6 and BALB/c inflammatory PMN to similar levels in both strains of mice, and with both types of stimuli (Fig. 2) , TLR2, TLR7, and TLR9 mRNA levels were also increased in inflammatory PMN, but L. major induced significantly higher levels in C57BL/6 compared with BALB/c PMN (Fig. 2) , as measured in three independent experiments. Thus, L. major induced distinct levels of TLR mRNA expression in C57BL/6 versus BALB/c neutrophils, mainly for TLR2, TLR7, and TLR9.


Figure 2
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  		Figure 2. Expression of TLR mRNA in response to L. major in C57BL/6 and BALB/c neutrophils. Quantitative real-time PCR was performed on bone marrow (BM) neutrophils or inflammatory peritoneal neutrophils induced by infection with L. major (LM) or 2% starch in PBS. Neutrophils were isolated directly (bone marrow) or 4 h after i.p. injection (inflammatory PMN) and purified by MACS, as described in Materials and Methods. Values of the different TLR mRNAs were normalized to endogenous levels of HPRT mRNA and are represented as arbitrary units. mRNA levels were compared between BALB/c and C57BL/6 PMN. Data are the mean ± SD of neutrophil mRNA values from one experiment representative of three.

BALB/c and C57BL/6 L. major-recruited PMN secrete distinct levels of p40 following stimulation in vitro
As the expression of distinct TLRs on neutrophils may affect their cytokine secretion, we have examined the transcription and secretion of immunomodulatory cytokines in C57BL/6 and BALB/c neutrophils. IL-12 is a heterodimer of 70 kD formed by two covalently linked chains of 40 and 35 kD. Two of the main biological functions of IL-12 are the induction of IFN-{gamma} production and the polarization of CD4+ Th differentiation toward Th1. Production of IL-12 as a p40-free chain and as the biologically active IL-12p70 heterodimer has been reported in human PMN following in vitro stimulation with LPS and IFN-{gamma} [7 , 26 ] and is also observed in murine neutrophils. Mature PMN were isolated from the bone marrow and infected with L. major at a 5:1 parasite:PMN ratio and/or activated with IFN-{gamma}. Sixteen hours (mRNA) or 24 h (cytokines) later, cells were collected for mRNA analysis, and cell-free supernatant was analyzed for the production of the p40 chain. The levels of p40 mRNA were low in bone marrow neutrophils from C57BL/6 and BALB/c mice. L. major induced a small increase in p40 transcription, which was significantly higher in BALB/c PMN compared with C57BL/6 PMN, an effect increased in the presence of IFN-{gamma} (Fig. 3A , left panel). To assess the quantity of cytokine released upon stimulation in vitro, the levels of p40 were measured in culture supernatant. Unstimulated bone marrow neutrophils produced only low levels of p40 (approximately 100 pg/ml), a quantity within the limit of detection of the assay (Fig. 3B) . In vitro stimulation with L. major, IFN-{gamma} alone, or combined with L. major or LPS induced a small but significant production of p40 (between 200 and 600 pg/ml). However, no significant difference was observed between C57BL/6 or BALB/c PMN nor between the different modes of stimulation (Fig. 3B , left panel). Thus, in mature bone marrow PMN, only a low level of p40 is transcribed and secreted, and this low level is minimally inducible by activation in vitro.


Figure 3
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  		Figure 3. Expression of p40 in PMN stimulated in vitro. (A) Bone marrow PMN or L. major-recruited peritoneal PMN were isolated as described in Materials and Methods and stimulated in the presence of L. major (5:1 ratio) or IFN-{gamma} (alone, with L. major, or with LPS). Sixteen hours later, cells were collected and processed for mRNA and RT-PCR. Results are expressed as fold increase compared with untreated PMN. (B) Supernatant of the culture was collected 24 h after infection and analyzed for the p40 presence by ELISA. Data represent the mean ± SD of five experiments. *, P < 0.05, between C57BL/6 and BALB/c values.

As neutrophils are recruited rapidly to the site of parasite inoculation following infection with L. major, it was relevant to analyze the secretion of p40 in L. major-infected mice. As it is technically not feasible to obtain PMN from the infected footpads, L. major peritoneally recruited neutrophils were used and purified by MACS. Incubation of these inflammatory neutrophils with L. major, IFN-{gamma}, or both induced a significant increase in p40 mRNA transcription in BALB/c PMN but not in C57BL/6 PMN (Fig. 3A , right panel).

Activation of bone marrow-isolated BALB/c PMN with IFN-{gamma} alone induced only low levels of p40 transcription and secretion. In contrast, in L. major-recruited, inflammatory BALB/c PMN, IFN-{gamma} induced high levels of p40 transcription and secretion (Fig. 3A) . These results suggested that L. major-recruited PMN were primed to respond to IFN-{gamma} by L. major. To confirm this hypothesis, PMN were recruited with 2% starch and stimulated with IFN-{gamma}. No induction of p40 transcription and secretion was observed, and incubation with L. major induced a marked increase in p40 transcription in BALB/c PMN (data not shown). Thus, L. major plays a significant role in the induction of p40 secretion by PMN.

On the contrary, neither L. major nor any other mode of stimulation induced a significant increase in p40 transcription in inflammatory C57BL/6 neutrophils (Fig. 3A , right panel). The basal level of p40 transcription was not statistically different in BALB/c and C57BL/6 PMN (data not shown).

These mRNA data correlated with the secretion of p40 cytokine by PMN, as measured by ELISA. Inflammatory C57BL/6 PMN secreted low levels of p40 on all types of stimulation. In contrast, BALB/c PMN stimulated with L. major produced a high level of IL-12p40, which was increased further in the presence of IFN-{gamma}. The quantity of p40 secreted by BALB/c neutrophils was elevated and significantly higher than that produced by C57BL/6 PMN (Fig. 3B , right panel).

C57BL/6 but not BALB/c PMN secrete significant levels of IL-12p70
As biologically active IL-12 is formed by the association of p35 and p40, the production of bioactive IL-12p70 by PMN was measured using a bioassay, as described in Materials and Methods. Low levels of bioactive IL12-p70 were detected in bone marrow-derived PMN of C57BL/6 mice, and the levels in BALB/c PMN were within the limit of detection of the assay (Fig. 4A , left panel). In C57BL/6 inflammatory PMN recruited with L. major, low levels (between 100 and 150 pg/ml) of bioactive IL-12p70 were detected in the absence of stimulation or following stimulation with L. major (Fig. 4B , right panel). Significantly higher levels (500 pg/ml) were measured when PMN were stimulated with IFN-{gamma} (alone, with L. major, and with LPS). In contrast, no IL-12p70 was detectable in BALB/c PMN (Fig. 4A , right panel). These results were confirmed by ELISA in four additional independent experiments in which IL-12p70 levels were high in supernatants of C57BL/6 PMN but not in those of BALB/c PMN (Fig. 4B , left panel).


Figure 4
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  		Figure 4. Secretion of bioactive IL-12 by PMN. (A) Bone marrow-derived and L. major-recruited PMN C57BL/6 and BALB/c PMN were treated as described in Figure 2 and analyzed for IL-12p70 biologic activity by bioassay. Data represent the mean ± SD of five experiments. *, P < 0.05, compared with none. (B) Secretion of IL-12p70 by L. major-recruited, inflammatory PMN in the absence or presence of L. major, IFN-{gamma}, or both, as detected by ELISA (left panel). Inhibitory activity of BALB/c supernatant on IL-12 p70 secretion. Supernatant from C57BL/6 PMN treated with L. major and IFN-{gamma} was collected 24 h later and mixed at the indicated ratio, with supernatant of BALB/c PMN similarly treated and tested in the IL-12 bioassay. The results are the mean ± SD of triplicates, representative of two experiments (right panel). *, P < 0.05, compared with 50:50. (C) Induction of IL-12 p35 and IL-23 p19 mRNA by L. major in BALB/c and C57BL/6 PMN. L. major-recruited PMN were purified and cultured in the presence of L. major, IFN-{gamma}, or both for 16 h. Cells were collected and PMN purified by MACS and processed for real-time PCR analysis. Results are represented as the fold increase in mRNA, and compared with PMN not stimulated in vitro, results are the mean of triplicate measurement representative of three experiments.

Taken together, these results show that incubation with L. major and/or IFN-{gamma} in vitro induces a significant increase of transcription and secretion of p40 in inflammatory neutrophils from BALB/c mice. However, this release does not contribute to the formation of significant levels of biologically active IL-12p70. In contrast, the low levels of p40 secreted by C57BL/6 PMN contribute to the formation of biologically active IL-12p70.

The secreted p40 chain can also form monomers or homodimers, which have been reported in vitro and in vivo to bind to the IL-12 receptor (IL-12R) and thus act as an inhibitor of IL-12, preventing IL-12 binding. To test if the IL-12p40 secreted by L. major-stimulated BALB/c PMN could compete with the binding of IL-12p70, C57BL/6 splenocytes were cultured with supernatant of PMN treated for 24 h with L. major + IFN-{gamma}, as described in Materials and Methods. In these supernatants, no trace of IFN-{gamma} remained, as assessed by ELISA. BALB/c PMN supernatant was added to C57BL/6 PMN supernatant to evaluate its inhibitory potential. The presence of 50% of BALB/c supernatant decreased by 1.4-fold the IL-12-dependent, IFN-{gamma} secretion induced by the C57BL/6 PMN supernatant (Fig. 4B) . Adding 75% of BALB/c supernatant to 25% of C57BL/6 supernatant also decreased the IL-12-dependent IFN-{gamma} by 1.8x (Fig. 4B , right panel). This experiment was also performed with the supernatant of LPS + IFN-{gamma}-treated PMN with similar results (data not shown). The inhibition of IL-12 bioactivity by BALB/c supernatants indicates that one part of the p40 secreted by BALB/c PMN is composed of homodimers displaying an inhibitory function. Inflammatory neutrophils secrete many factors, which could also contribute to the inhibitory effect conferred by the BALB/c inflammatory neutrophil supernatant. One of them could be TGF-β. Therefore, anti-TGF-β was included with the neutrophil supernatants. It slightly (<0.5x) decreased the production of bioactive IL-12, but addition of a mAb against IL-12 decreased the IL-12-induced secretion of IFN-{gamma} by activated splenocytes to background levels. Similar low levels were also obtained following addition of anti-TGF-β and anti-IL-12 mAb (data not shown).

L. major does not induce the secretion of a significant level of IL-23 by PMN
The p40 chain associates with the IL-12p35 chain to form biologically active IL-12p70. To assess if the lack of IL-12p70 secreted by BALB/c PMN could result from defective transcription of p35, analysis of IL-12p35 mRNA was performed in inflammatory PMN. IL-12p35 mRNA levels increased in the presence of L. major in BALB/c PMN but to a lower level than that in C57BL/6 PMN (Fig. 4C) . Thus, in BALB/c PMN, IL-12p35 is transcribed, but nevertheless, no detectable IL-12p70 is secreted, revealing that the p35 transcribed may not be sufficient to associate with p40 to form IL-12p70.

In addition to its association with IL-12p35, the p40 subunit has been shown recently to associate with the IL-23p19 chain, forming the functional IL-23 heterodimer (reviewed in ref. [27 ]). We thus investigated if the high levels of p40 secreted by BALB/c PMN, in addition to the formation of homodimers, could associate with p19 to form IL-23. As significantly high levels of IL-12p40 are produced by L. major-recruited PMN, the levels of IL-23p19 transcription were measured in BALB/c- and C57BL/6-inflammatory PMN, stimulated or not with L. major, IFN-{gamma}, or both, as described above. Only low levels of p19 transcription were measured in BALB/c and C57BL/6 PMN, and no increase of transcription was detected in each of the conditions tested (data not shown). In line with these results, no IL-23 proteins could be detected by ELISA (data not shown). Potential release of preformed IL-23 was investigated in PMN supernatant several time-points after stimulation (1, 6, 12, and 24 h), but in four independent experiments, no IL-23 was detectable. It cannot be excluded that low levels of IL-23 (below the detection limit of the ELISA, 30 pg/ml) could be secreted by inflammatory PMN; however, these results strongly suggest that most of the IL-12p40 secreted by BALB/c and C57BL/6 inflammatory PMN does not associate with IL-23p19 to form IL-23.

As bone marrow-derived BALB/c and C57BL/6 PMN secreted equivalent, low levels of p40 (Fig. 3A) , we also evaluated IL-23p19 transcription in L. major-stimulated bone marrow PMN. Basal transcription levels of IL-23 were equally low in C57BL/6 and BALB/c PMN. However, L. major induced a significant increase in IL-23p19 mRNA levels in PMN from both strains of mice. This increase was prevented significantly in the presence of IFN-{gamma} (Fig. 4C , right panel). Accordingly, low levels of IL-23 (between 60 and 30 pg/ml) were measured in supernatants of BALB/c and C57BL/6 PMN stimulated with L. major and L. major + IFN-{gamma}, respectively, and no IL-23 was detectable in PMN not stimulated or stimulated with IFN-{gamma} alone (data not shown). Thus, low levels of IL-23 are released by bone marrow PMN in response to L. major at similar levels between BALB/c and C57BL/6 PMN, and no detectable IL-23 was released by inflammatory PMN.

C57BL/6 but not BALB/c PMN secrete IL-10 following stimulation with L. major and IFN-{gamma}
IL-10 plays a critical role in the control of inflammation through inhibition of the production of inflammatory cytokines such as IL-1, IL-12, and TNF (reviewed in ref. [28 ]). IL-10 has also been reported to inhibit the release of proinflammatory cytokines by human PMN [29 ]. To evaluate if the production of IL-10 could influence the secretion of IL-12 by PMN, transcription of IL-10 was measured in BALB/c and C57BL/6 PMN stimulated as above.

The basal levels of IL-10 mRNA in bone marrow PMN was low and equivalent in C57BL/6 and BALB/c PMN. IFN-{gamma} induced a low increase in IL-10 mRNA levels in the presence of LPS, and L. major induced a more significant increase of IL-10 mRNA in C57BL/6 PMN in the presence or absence of IFN-{gamma}. In contrast, no modulation of IL-10 mRNA levels was measured in BALB/c PMN (Fig. 5A ). In line with these results, small levels of IL-10 (between 30 and 100 pg/ml) were detected in C57BL/6 bone marrow PMN stimulated with L. major and IFN-{gamma}, but no detectable IL-10 was released by BALB/c PMN (data not shown).


Figure 5
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  		Figure 5. Distinct expression of IL-10 in C57BL/6 versus BALB/c PMN. (A) Bone marrow PMN or L. major-recruited, inflammatory PMN were isolated as described in Figure 2 and RT-PCR performed. Data are the mean ± SD of three experiments performed in triplicate. Results are expressed as fold increase of mRNA compared with unstimulated PMN. *, P < 0.05, between C57BL/6 and BALB/c values. (B) Secretion of IL-10 in the supernatant of C57BL/6 and BALB/c L. major-recruited, inflammatory PMN cultured for 24 h in the presence of L. major, IFN-{gamma}, or both, as detected by ELISA. Results are the mean ± SD of four experiments. ND, Not detectable.

In purified BALB/c-inflammatory PMN, cultured in the presence of L. major ± IFN-{gamma}, no modulation of IL-10 mRNA was detected (Fig. 5A , right panel). In contrast, C57BL/6-inflammatory PMN cultured with L. major and to a lesser extent, with IFN-{gamma}, showed an increase in IL-10 mRNA. Similar to what was observed with bone marrow PMN, no modulation of IL-10 mRNA was detectable in BALB/c-inflammatory PMN (Fig. 5A , right panel). Significant levels of IL-10 were secreted in the presence of L. major, and higher levels of IL-10 were secreted following stimulation of C57BL/6-inflammatory PMN with IFN-{gamma} (Fig. 5B) . In contrast, no release of IL-10 was measurable in inflammatory BALB/c PMN under all the conditions tested, as performed in five independent experiments (Fig. 5B and data not shown). Thus, L. major and IFN-{gamma} can induce the transcription and the release of IL-10 selectively in C57BL/6 PMN.

Production of TGF-β is induced selectively in BALB/c bone marrow PMN
TGF-β is another cytokine with an anti-inflammatory role. We thus measured the transcription and release of TGF-β by PMN activated with L. major or LPS in the presence or not of IFN-{gamma}. We first measured TGF-β transcription in bone marrow-derived and L. major-recruited PMN. Stimulation of PMN with L. major, LPS + IFN-{gamma}, as well as IFN-{gamma} alone resulted in TGF-β transcription levels, which were significantly higher in BALB/c than in C57BL/6 PMN (Fig. 6A ). It is remarkable that L. major induced the most significant increase in TGF-β mRNA, and the combination of LPS-IFN-{gamma} resulted in a lower increase in TGF-β mRNA. As the TGF-β is known to be regulated at the post-transcriptional level, the bioactive TGF-β was measured in neutrophil supernatants.


Figure 6
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  		Figure 6. Distinct transcription of TGF-β by BALB/c PMN in response to L. major. (A) Bone marrow-derived and L. major-recruited PMN were isolated and cultured as described in Figure 2 . Cells were collected and processed for RT-PCR. (B) Secretion of TGF-β, as analyzed on culture supernatant, using the PAI/luciferase bioassay (PAI/L) as described in Materials and Methods. *, P < 0.05, between C57BL/6 and BALB/c. Results are represented as the mean ± SD of four experiments.

The PAI/L assay was used to measure the release of activated, latent, bioactive TGF-β. Only low levels of TGF-β were secreted by bone marrow PMN not stimulated in vitro. These levels may correspond to the release of prestored TGF-β. L. major induced a significant release of TGF-β in BALB/c PMN but not in C57BL/6 PMN. IFN-{gamma} induced the release of TGF-β by C57BL/6 and BALB/c PMN, but higher levels were released by BALB/c PMN. However, in response to the combination of IFN-{gamma} plus L. major or plus LPS, C57BL/6 and BALB/c PMN released comparable levels of latent TGF-β (Fig. 6B) . The present results confirm the difference in active TGF-β release previously reported between BALB/c and C57BL/6 and reveal that C57BL/6 PMN stimulated with L. major or LPS in the presence of IFN-{gamma} have potentially equivalent levels of latent TGF-β compared with BALB/c PMN (as detected upon acid activation), but L. major and LPS are not sufficient to induce their release (ref. [6 ] and data not shown). Thus, L. major induces preferentially the release of TGF-β in BALB/c bone marrow PMN.

In contrast, in inflammatory PMN (recruited with L. major), 24 h after in vitro stimulation, the production of TGF-β did not differ significantly at the mRNA level (Fig. 6A , right panel) or at the protein level (data not shown).

Distinct NO production and increased number of L. major in BALB/c versus C57BL/6 neutrophils
Following infection with L. major, the phenotype of BALB/c and C57BL/6 neutrophils was distinct; we therefore investigated if this could have functional consequences on the survival of parasites within neutrophils. Four hours after L. major infection, i.p.-inflammatory PMN were isolated and purified by MACS as described in Materials and Methods. Neutrophils were lysed with 0.01% SDS and incubated for 72 h in a L. major growth medium at 26°C. Higher proliferation, as measured by the incorporation of the [3H]thymidine, was measured in BALB/c PMN than in C57BL/6 PMN (Fig. 7A ), suggesting a greater number of viable parasites within BALB/c neutrophils at the time of lysis. This difference could be a result of distinct parasite uptake and/or killing. During L. major infection, one of the protective effects of IFN-{gamma} occurs through its induction of NO synthase and the secretion of NO, which is involved in the killing of L. major. We therefore measured NO secretion by L. major-recruited neutrophils cultured in the absence or presence of L. major, IFN-{gamma}, or both. L. major induced NO secretion in C57BL/6 but not in BALB/c neutrophils (Fig. 7B) . In contrast, IFN-{gamma} induced the release of NO in C57BL/6 and BALB/c neutrophils, but the amounts of NO produced were significantly higher in C57BL/6 neutrophils (Fig. 7B) , as observed in four independent experiments.


Figure 7
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  		Figure 7. Secretion of NO and measure of L. major presence in BALB/c versus C57BL/6 neutrophils. (A) Neutrophils were recruited 4 h after i.p. infection with L. major promastigotes and purified by MACS. The cells were then lysed and the parasites cultured for 72 h at 26°C. For the last 16 h, [3H]thymidine was added. Proliferation of the parasites, as measured by the incorporation of the [3H]thymidine, reflects the quantity of viable parasites within the neutrophils. This is a representative experiment of two. (B) L. major-recruited neutrophils were cultured for 48 h in the absence or presence of L. major, IFN-{gamma}, or both. NO production was measured in cell-free supernatants of C57BL/6 and BALB/c neutrophils using the Griess reagent. This is a representative experiment of three. *, P < 0.05, compared with C57BL/6.

DISCUSSION

Neutrophils are key components of the inflammatory response and not only contribute to the primary protective response as effector cells, but they also contribute to the recruitment, activation, and programming of the APC (reviewed in ref. [30 ]). We report here that following infection with L. major, inflammatory neutrophils from C57BL/6 and BALB/c mice expressed equally high levels of CD11b. In contrast, the expression of CD49d was induced by L. major only on BALB/c PMN. Leukocyte integrins are important in migration but also in events that follow transendothelial migration such as activation and survival of leukocytes. In mice, blockade of CD49d on intrapulmonary leukocytes was reported to decrease allergen-induced lung inflammation [31 ]. The presence of CD49d on BALB/c PMN may thus contribute to their reported persistence at the site of inoculation following infection with L. major.

Tsuda et al. [17 ] characterized PMN-I or PMN-II as PMN involved in resistance or susceptibility to infection by methicillin-resistant Staphylococcus aureus, respectively, and PMN-I expressed high levels of CD49d, and PMN-II did not express CD49d. Our results show that CD49d is expressed by inflammatory PMN from L. major-susceptible mice; however, no difference in CD11b expression was noted in inflammatory or in blood PMN. Thus, the criteria for defining PMN-I or PMN-II may vary according to the infectious agent.

Expression of TLRs on neutrophils has been reported to affect neutrophil migration, apoptosis, and secretion of cytokines (reviewed in ref. [32 ]). Human neutrophils express mRNA for all the TLRs except TLR3 [33 ]. It is interesting that this was also true in mouse PMN, as we could not detect any TLR3 mRNA in inflammatory or in bone marrow-isolated neutrophils. TLR4 mRNA was induced and highly expressed in BALB/c and C57BL/6 inflammatory neutrophils with no difference between PBS-starch and L. major. TLR4 was reported previously to be of importance during L. major infections, as lack of TLR4 resulted in increased parasite growth in the early phase of infection and delayed healing in mice of a resistant L. major genetic background [20 ]. Our results suggest that L. major stimulates TLR4 mRNA to similar levels in BALB/c and C57BL/6 mice. Following exposure to L. major, a higher induction of TLR2, TLR7, and TLR9 mRNA was measured in L. major-recruited C57BL/6 as compared with BALB/c neutrophils. Lipophosphoglycan (LPG), one of the TLR2 ligands, contains long carbohydrate branches with repeated phosphoglycan units. LPG is prominent at the surface of Leishmania promastigotes. L. major has been reported to activate human macrophages as well as human NK cells through TLR2 [21 , 34 ]. In addition, activation of human macrophages by Leishmania donovani was reported to depend on TLR2, as demonstrated by RNA interference experiments [35 ]. Stimulation of TLRs in neutrophils has been reported to trigger the production of cytokines, and IL-10 production was induced in response to lipoteichoic acid, another TLR2 ligand [24 ]. It is interesting that in the present study, we measured IL-10 production only in L. major-stimulated C57BL/6 but not in BALB/c neutrophils, suggesting that the higher induction of TLR2 mRNA in C57BL/6 PMN may be linked to their selected secretion of IL-10, a mechanism currently under investigation. TLR9 is a receptor for unmethylated, bacterial CpG DNA motifs. Protozoan DNA from Trypanosoma cruzi and Trypanosoma brucei has been reported to induce macrophage and dendritic cell (DC) activation putatively through TLR9 [36 , 37 ]. In addition IL-18 gene therapy (injection of IL-18 in a CpG-including vector) induced a Th1 response in L. major-infected BALB/c mice, which was thought to occur via the CpG-TLR9 signaling. In our study, fragments of parasite genomic DNA could be responsible for the increase in TLR9 expression on L. major-recruited neutrophils. It is interesting that L. major parasites were reported to be more damaged in C57BL/6 than in BALB/c neutrophils, where they remained almost intact [2 ], a process in line with the higher levels of NO measured in L. major-infected C57BL/6 neutrophils (Fig. 7) . The higher TLR9 mRNA expression measured consistently in C57BL/6 versus BALB/c L. major-recruited neutrophils may go in this sense. TLR7 is another endosomal TLR, where mRNA levels were higher in C57BL/6 than in BALB/c L. major-recruited neutrophils. Thus, higher expression of TLR2, TLR7, and TLR9 is preferentially associated with C57BL/6 neutrophils, which are secreting bioactive IL-12 and IL-10, demonstrating that multiple and distinct TLRs are involved in the early neutrophil response to L. major in mice susceptible or resistant to infection with L. major.

We report here that highly purified, L. major-recruited neutrophils from C57BL/6 or BALB/c mice, respectively, resistant and susceptible to infection with this parasite, secrete a distinct pattern of cytokines following in vitro activation with infectious L. major promastigotes. In a first-step, secretion of IL-12 was investigated. It is striking that L. major recruited BALB/c PMN transcribed and secreted significantly higher amounts of p40 mRNA and protein in response to in vitro stimulation with L. major, an increase amplified by stimulation with IFN-{gamma}. IL-12p40 is secreted as monomers and homodimers five to 90 times as much as IL-12p70 in vitro and in vivo (reviewed in ref. [38 ]). The low levels of IL-12p40 secreted by C57BL/6 PMN associated with IL-12p35 to form the bioactive IL-12p70. However, in BALB/c PMN, no secretion of IL-12p70 was observed.

We showed by real-time PCR that the p35 mRNA level was induced by L. major in BALB/c PMN but to a lower extent than in C57BL/6 PMN. In addition, we showed that part of the high levels of IL-12p40 secreted by BALB/c PMN was inhibiting IL-12 signaling in activated splenocytes. We previously reported the synthesis of active TGF-β selectively by bone marrow BALB/c PMN [6 ]. Here, we showed further that TGF-β is also transcribed more actively in response to stimuli in BALB/c bone marrow PMN. It is interesting that TGF-β has been reported to inhibit IL-12p40 production and also to reduce IL-12p40 mRNA stability [39 ]; thus, there is a possibility that part of the IL-12p40 mRNA produced by BALB/c PMN may not be stable. However, the levels of TGF-β transcribed and secreted by inflammatory PMN were comparable between BALB/c and C57BL/6 PMN.

The production of biologically active IL-12 by PMN from C57BL/6 mice may be one of the earliest sources of IL-12, a cytokine required for the polarization of a CD4+ Th1 response.

IL-12p40 homodimers have been shown to be antagonists of IL-12 p70 by binding competitively to the IL-12R, suppressing immune responses to several antigens in vitro [40 , 41 ] and in vivo [12 , 42 , 43 ]. An excess of IL-12p40 homodimers has been reported to promote a Th2 predominant response [43 ]. Thus, the production of IL-12p40 monomers and/or homodimers by BALB/c PMN observed in vitro could also be inhibitory in vivo, preventing the development of CD4+ Th1 differentiation. In this line, in BALB/c mice, transient depletion of PMN prior to infection led to a significant decrease in the production of Th2 cytokines, as well as the development of lesions, which were significantly smaller than those of untreated, L. major-infected BALB/c mice [4 ]. However, when the production of IL-12 by all types of cells was inhibited in BALB/c mice, by transient injection of a mAb-inactivating IL-12 [4 ] or by using genetically deficient IL-12p40 [44 ], BALB/c mice remained susceptible to infection with L. major, suggesting that the production of IL-12p40 homodimers by BALB/c PMN could be one of the factors preventing the polarization of CD4+ Th1 cells but that other factors released by PMN or other cells of the innate immune response also contribute to this polarization.

It has been reported that the p40 chain can also associate with p19 to form IL-23 [45 ]. Here, we show that the excess of p40 measured in L. major-recruited BALB/c PMN did not associate with p19, as no IL-23 was detectable in inflammatory PMN following activation. IL-23 was secreted only at low levels in L. major-stimulated bone marrow-derived PMN but at equal levels between BALB/c and C57BL/6 PMN. Thus, the production of IL-23 by PMN does not appear to be of significance following L. major infection.

IL-10 plays a critical role in the control of inflammation through inhibition of the production of inflammatory cytokines such as IL-1, IL-12, and TNF by macrophages, DC, and CD4+ Th1 cells (reviewed in ref. [28 ]). IL-10 has also been reported to inhibit the release of proinflammatory cytokines by human PMN [29 ]. The production of IL-10 by C57BL/6 PMN but not BALB/c PMN could therefore contribute to the suppression of the inflammatory response observed at the site of L. major inoculation in infected C57BL/6 mice. IL-10 has also been reported to inhibit IL-12 production by blocking transcription of IL-12p40 [46 ]. This may explain the low levels of IL-12p40 transcription measured in C57BL/6 PMN but not in BALB/c PMN, which do not secrete IL-10. The role of IL-10 in resistance to infection with L. major is not well defined. Mice on a L. major-resistant genetic background, expressing an IL-10 transgene under a MHC II E{alpha} promoter, were more susceptible to infection than C57BL/6 mice [47 ]. In contrast, transgenic mice expressing IL-10 under the control of the IL-2 promoter on a C57BL/6-healing, genetic background retained resistance to L. major infection and Th1 response comparable with that of wild-type C57BL/6 mice [48 ]. These results imply that the cellular source of IL-10 may be of importance in the development of resistance or susceptibility to infection.

Several groups have reported the importance of neutrophils early in L. major infection. Transient depletion of neutrophils during the first days of infection with L. major had a consequence on the number of parasites surviving at the site of parasite inoculation in resistant C57BL/6 and in susceptible BALB/c mice. In resistant strains of mice (C57BL/6, C3H/HeJ), absence of PMN caused by the injection of mAb-depleting neutrophils (NIMPR14) or neutrophils and eosinophils (RB6-8C5) at the time of infection and/or during the first week of infection led to a significant increase in the parasite load, suggesting a protective role for neutrophils in these strains of mice [4 , 5 , 49 , 50 ]. In sharp contrast, the absence of PMN in BALB/c mice during the first week of infection had the opposite effect, reducing significantly the parasite number within the draining lymph nodes and at the site of infection [4 , 50 ]. Using different strains of L. major parasites, an increase in parasite number in BALB/c mice depleted with the RB6 mAb was observed [5 , 49 ], suggesting that different strains of L. major may induce different PMN responses. It is interesting that following restimulation with this strain of L. major, Chen et al. [49 ] did not detect any IL-12p40 secretion in BALB/c neutrophils. Whether this results from the strain of parasite used or the use of parasite antigens for restimulation in vitro, rather than live parasites used in the present study, remains to be determined.

We report here that BALB/c neutrophils harbored more parasites than C57BL/6 neutrophils 4 h after i.p. infection. In addition, C57BL/6 neutrophils secreted significantly higher levels of NO than BALB/c neutrophils in response to L. major and both L. major and IFN-{gamma}. These results are in line with a previous study reporting difference in the survival of parasites within neutrophils in both strains of mice following s.c. infection with L. major [2 ] and suggest that the distinct neutrophil phenotypes observed in C57BL/6 and BALB/c neutrophils may act directly or indirectly (through cytokine secretion) on parasite survival within the neutrophils.

In conclusion, the present study shows that in response to L. major, IFN-{gamma}, or both, distinct up-regulation of selective TLR mRNAs is observed in neutrophils from L. major-resistant or susceptible strains of mice. The production of cytokines also differs significantly in PMN derived from C57BL/6 or BALB/c mice. IL-12 and IL-10 are produced only by PMN of the resistant C57BL/6 strain, and BALB/c PMN produce IL-p40 homodimers and TGF-β, two cytokines preventing and inhibiting the differentiation of CD4+ Th1 immune responses.

Altogether, these results reveal a potentially important role for PMN in the regulation of the early events leading to the development of a protective response against the protozoan L. major parasite.

ACKNOWLEDGEMENTS

The Swiss National Foundation for Scientific Research (SNF) supported this work. We thank Prof. J. Mauël for critical reading of the manuscript and Olivier Fröhlicher for technical help with some of the experiments.

FOOTNOTES

1 These authors contributed equally to this work. Back

2 Current address: Biotechnology Centre and Faculty of Sciences, University of Yaoundé I, Yaoundé, Cameroon. Back

Received July 11, 2006; revised March 15, 2007; accepted April 2, 2007.

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