Thierry Lang,* Nathalie Courret, Jean-Hervé Colle, Geneviève Milon, and Jean-Claude Antoine

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1 INFECTION AND IMMUNITY, May 2003, p Vol. 71, No /03/$ DOI: /IAI Copyright 2003, American Society for Microbiology. All Rights Reserved. The Levels and Patterns of Cytokines Produced by CD4 T Lymphocytes of BALB/c Mice Infected with Leishmania major by Inoculation into the Ear Dermis Depend on the Infectiousness and Size of the Inoculum Thierry Lang,* Nathalie Courret, Jean-Hervé Colle, Geneviève Milon, and Jean-Claude Antoine Unité d Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, Paris, France Received 16 July 2002/Returned for modification 11 September 2002/Accepted 12 February 2003 The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (10 6 parasites) or low (10 3 parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN- ) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN- and later on IL-4; the level of IFN- produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN- and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN- does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 10 6 LP, but in this case CD4 T lymphocytes did not release IFN- or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation. Leishmania spp. are protozoan parasites transmitted from the sandfly during a blood meal to a variety of mammalian hosts. Inbred mice inoculated with these parasites have been widely used as experimental hosts for elucidating and characterizing the various processes involved in leishmaniases (38). The infection of mouse strains such as C57BL/6 with Leishmania major results in a disease that gradually resolves following the development of a dominant Th1 immune response, whereas the infection of BALB/c mice results in a progressive disease that does not resolve following the development of a dominant Th2 immune response. An explanation for these different outcomes of L. major infection in mice is that the gamma interferon (IFN- ) produced by Th1 cells is required for the induction in macrophages of nitric oxide synthase and the production of the leishmanicidal molecule nitric oxide (7, 27, 48). In contrast, Th2 cytokines such as interleukin 4 (IL-4), IL-10, and IL-13 may prevent or abolish macrophage activation (35), making intracellular parasite growth possible (16, 34). * Corresponding author. Mailing address: Unité d Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25 rue du Docteur Roux, Paris Cedex 15, France. Phone: Fax: address: tlang@pasteur.fr. Present address: Département des Maladies Infectieuses, Institut Cochin, Paris, France. Because IL-4 is required for the differentiation of naive CD4 T lymphocytes into Th2 effector cells (42), the role of this cytokine in susceptibility to L. major has been studied (15). The use of anti-il-4 antibodies or gene disruption to block IL-4 production in BALB/c mice converts the L. major-immune reactivity to a healing response in most but not all cases (22, 32 34, 40). Other studies have shown that the burst of IL-4 mrna production observed in the draining lymph nodes (LN) of infected BALB/c mice in the first 16 to 20 h after parasite delivery plays a key role in determination of the susceptible phenotype lesion (18, 24, 25). These early IL-4 transcripts are transiently produced by a restricted population of V 8 V 4 CD4 T lymphocytes (24) recognizing a single epitope of the parasite protein LACK (Leishmania homolog of receptors for activated C kinase), located between amino acids 158 and 173 (19, 30). It has also been shown that L. major-infected BALB/c mice rendered tolerant to this antigen by its transgenic expression in the thymus or rendered deficient in V 4 T lymphocytes developed a Th1 response and were eventually cured of the disease (17, 19, 24). In these models, the role of LACK-reactive T cells in susceptibility to L. major probably results from the rapid production of IL-4 mrna and the secretion of IL-4 into the LN of infected mice. However, it should be borne in mind that, to date, this phenomenon has been described only after experi- 2674

2 VOL. 71, 2003 CYTOKINES INDUCED BY L. MAJOR INOCULATION 2675 mental infections involving millions of stationary-phase promastigotes (SP), which are known to be highly heterogeneous and to include both infectious and noninfectious parasites. The noninfectious parasites generally predominate and are much more sensitive to microbicidal activity than the infectious stage, the metacyclic promastigotes (MP). The vast majority of these noninfectious parasites die and are degraded at the injection site, but they may release a large number of proteins, the processing of which may result in rapid and early activation of parasite-reactive T cells able to shape the immune response. In the present study, we examined the early cytokines produced by CD4 LN T lymphocytes recovered from BALB/c mice inoculated with high (10 6 ) or low (10 3 ) doses of L. major MP. In addition, we investigated whether the presence of noninfectious but viable parasites in the inoculum affected the pattern of cytokines released by CD4 T lymphocytes of infected mice. To address this issue, mice were inoculated with L. major promastigote populations exhibiting various degrees of infectiousness, log-phase promastigotes (LP) or SP, into the ear dermis (4, 6, 31) because this injection route is closer to the natural route of metacyclic delivery by the sandfly than the subcutaneous inoculation currently performed in most laboratories. The outcome of infection was evaluated by measuring ear swelling and parasite burden at the inoculation site and in the draining LN. Here, we show that the levels and profiles of cytokines (IFN- and IL-4) secreted by the LN cells clearly depend on the infectiousness and the dose of parasites used for inoculation. MATERIALS AND METHODS Mice. We obtained 2- to 4-month-old female BALB/c and Swiss nu/nu mice from the Pasteur Institute (Paris, France), Iffa Credo (St Germain-sur-l Arbresle, France), or Harlan (Gannat, France). Parasite preparation and intradermal inoculation. The L. major NIH 173 strain (MHOM/IR/ /173) was maintained by passage in Swiss nu/nu mice. Amastigotes were prepared from infected mice as previously described (2). Promastigotes, freshly derived from amastigotes, were cultured at 26 C in HOS- MEM-II medium (8) supplemented with 20% fetal bovine serum (Dutscher, Brumath, France), 100 U of penicillin per ml, and 100 g of streptomycin per ml (Seromed, Berlin, Germany). LP were obtained from cultures passaged every 2 days. Infectious MP were isolated from 6-day stationary-phase cultures by negative selection with peanut agglutinin (Vector Laboratories, Burlingame, Calif.) (39). Parasites were suspended in Dulbecco s phosphate-buffered saline (PBS), and 10 3 MP or 10 6 LP, SP, or MP in a volume of 10 l were injected intradermally into the mice with a 30-gauge needle. Inoculations were done into the inner face of the ear. The development of the lesions was monitored by measuring the thickness of the inoculated ear and comparing it to that of the uninfected contralateral ear with a direct-reading Vernier caliper (Thomas, Swedesboro, N.J). Antibodies. The monoclonal antibody M5/114, an anti-i-a b,d,q and I-E d,k rat immunoglobulin G2b (IgG2b) (9), was purified by adsorption chromatography as previously described (1) from ascites prepared in nude mice. The monoclonal antibody SFR8-B6, a rat IgG2b directed against HLA-Bw6, was used as a control (37). The fluorescein-conjugated rat anti-mouse CD4 monoclonal antibody CT- CD4 (IgG2a) and the R-phycoerythrin-conjugated rat anti-mouse V 4 TCR monoclonal antibody CTVBA (IgG2a) were purchased from Caltag (San Francisco, Calif.). Irrelevant labeled rat IgG2a (Caltag) was used as a control. The levels of cytokines produced by the cultured cells were determined with the following pairs of antibody, the second antibody of each pair being biotinylated: R4-6A2 (rat IgG1) and AN (rat IgG1) for IFN- and 1D11 (rat IgG2b) and 24G2 (rat IgG1) (Endogen, Woburn, Mass.) for IL-4. Quantification of parasites. We estimated the number of parasites present in parasite-loaded ears and the draining LN as previously described (26). The two sheets of the infected ears were separated, cut into small pieces, and ground in HOSMEM-II culture medium with a glass tissue homogenizer. LN were removed and mechanically dissociated, and the number of cells they contained was determined. The tissue and organ homogenates were serially diluted in HOS- MEM-II culture medium and then dispensed into 96-well plates containing semisolid agar (Bacto-Agar; Difco, Detroit, Mich.) supplemented with 10% sterile heparin-treated rabbit blood. The plates were incubated for 10 days, and then each well was examined and classified as positive or negative according to whether or not viable promastigotes were present. Limiting-dilution analysis was then applied to the data to estimate the number of viable parasites, expressed in limiting-dilution assay units (LDAU). Statistical analysis of the results was based on the maximum-likelihood method (44, 46). Preparation of mouse cell suspensions and ex vivo reactivation. Retromaxillar draining LN were recovered and mechanically dissociated. Tissue homogenates were filtered through a cell strainer with 70- m pores (Falcon Products, St. Louis, Mo.). The cells were then resuspended in ACK lysis buffer to remove red blood cells (23). Cell viability was assessed by trypan blue exclusion. For the measurement of in vitro cytokine production, single LN cell suspensions obtained from 5 to 15 mice were pooled, and CD4 T lymphocytes were purified by magnetic cell sorting on a VarioMacs apparatus (Miltenyi Biotech, Bergish- Gladbach, Germany). For this purpose, cells were labeled with anti-cd4 antibody-coated magnetic microbeads and positively selected on VS columns. Enrichment efficiency ( 90% purity) was checked by cytofluorimetric analysis as described below. CD4 T cells were dispensed into 96-well plates ( cells/well) in the presence of 3,000-rad-irradiated spleen cells ( cells/well) from normal mice. They were cultured in a final volume of 200 l of RPMI 1640 supplemented with 10% fetal bovine serum, 50 U of penicillin per ml, 50 g of streptomycin per ml, and M 2-mercaptoethanol, with or without 5 to 15 M LACK( ) (Chiron Technologies, Suresnes, France) (19), or 2 g of the recombinant LACK- 1 protein per ml (a truncated 24-kDa form of LACK kindly provided by N. Glaichenhaus, Université de Nice, Sophia Antipolis, France) (30) or 0.08 to 12.5 g ofl. major antigens per ml (freeze-thawed preparation of promastigotes). Cultures were incubated at 37 C in an atmosphere of 5% CO 2 and 95% air. Cell culture supernatants were harvested at 92 h and stored at 20 C until used for IL-4 and IFN- determinations. Quantitative RT-PCR analysis of IL-4 transcripts. At designated time points after injection of PBS or parasites into ear dermis, retromaxillar LN were collected. The transcripts of IL-4 and -actin were quantified either from total LN cells or from CD4 T cells by competitive reverse transcription (RT)-PCR as described previously (11). Briefly, total RNA was isolated with the RNeasy kit (Qiagen, Hilden, Germany) and reverse transcribed. Reverse transcripts (cdna) were quantified with a PCR method involving coamplification of cdna with an internal standard. To eliminate the variations due to RNA extraction and cdna synthesis steps, quantification of IL-4 transcripts in a given sample was expressed with respect to a constant number (10 6 )of -actin mrna copies. Variations between replicates of transcript quantitation in the same sample were less than 25%. FACS analysis of LN-derived leukocytes. Cells were prepared from the LN of uninfected or infected mice and stained for fluorescence-activated cell sorting (FACS) analysis. To detect plasma membrane molecules, cells were dispensed into a 96-well plate ( cells/well). They were first incubated on ice for 30 min with PBS supplemented with 1% bovine serum albumin and 0.1% sodium azide to block nonspecific binding sites. Cells were then incubated for 30 min at 4 C with saturating concentrations of labeled monoclonal antibody or with the same concentration of labeled isotype-matched irrelevant antibody. The cells were fixed in 1% paraformaldehyde in PBS. For each sample, 10,000 cells were examined on a FACScan fluorescence-activated cell sorter (Becton Dickinson, Mountain View, Calif.). Detection of cytokines in cell culture supernatants. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the amounts of IL-4 and IFN- present in cell cultures according to classical protocols (36). For each assay, a standard curve was generated with known amounts of the corresponding cytokine. A supernatant from the IL-4-producing X63Ag8-653 cell line (20) was used as the IL-4 standard. Mouse recombinant IFN-, used as a standard for IFN-, was purchased from Genentech (San Francisco, Calif.). Data were standardized and processed with KC4 software (Bio-Tek Instruments Inc., Winooski, Vt.). The sensitivities of the ELISA were 55 pg/ml for IFN- and 5.5 pg/ml for IL-4. Statistical analysis. Comparisons between experimental groups were carried out by the two-tailed t test, and P values were calculated with SigmaPlot software (SPSS, Chicago, Ill.). A difference in mean values was considered statistically significant when P was 0.05 or very significant when P was 0.01.

3 2676 LANG ET AL. INFECT. IMMUN. RESULTS FIG. 1. Development of ear lesions in BALB/c mice following inoculation with a high or low dose of L. major promastigotes at various stages of differentiation. Either 10 6 MP ( ), 10 3 MP ( ), 10 6 SP ( ), or 10 6 LP (F) were injected intradermally into the ears of mice. Lesion sizes correspond to the difference in thickness between the inoculated ear and the contralateral noninoculated ear. These data are expressed as mean values 1 standard deviation (15 mice per group). At weeks 3 and 4, lesion sizes of mice inoculated with 10 6 MP ( )orsp( ) were significantly (P 0.01) different from those of mice injected with 10 6 LP (F) or10 3 MP ( ). Onset and development of ear lesions in BALB/c mice infected with L. major promastigotes at various stages of development. MP were injected into the ear dermis of BALB/c mice. The development of lesions was assessed by measuring the thickness of the ear over a period of 7 weeks (Fig. 1) and compared with that of lesions induced by the inoculation of SP or LP. In BALB/c mice infected with 10 6 MP or SP, the onset of cutaneous clinical signs was first detectable around week 1 or 2. Lesions then rapidly increased in size (Fig. 1). In both cases, this increase was accompanied by ulceration (Fig. 2A and B), tissue necrosis, and dermal erosion after 4 to 5 weeks. Following inoculation with 10 6 LP, BALB/c mice developed lesions that first became detectable around week 3 (Fig. 1; Fig. 2C). The thickness of the ear then increased very slowly but continuously, and ulceration and necrosis were noted by weeks 10 to 12 (data not shown). Inoculations with large numbers of parasites are commonly carried out in experimental infections but poorly reflect what happens in natural infections. Indeed, only around 10 to 1,000 infectious promastigotes are transmitted by the bite of a parasite-carrying sandfly (47; M. E. Rogers and P. A. Bates, com- FIG. 2. Photomicrographs of ear lesions (white arrows in C and D) developed in BALB/c mice inoculated intradermally 21 days previously with (A) 10 6 MP, (B) 10 6 SP, (C) 10 6 LP, or (D) 10 3 MP of L. major.

4 VOL. 71, 2003 CYTOKINES INDUCED BY L. MAJOR INOCULATION 2677 FIG. 3. Relationship between parasite burden of BALB/c mice inoculated with L. major 3 days previously and amount of IFN- produced by LACK-reactive CD4 LN T cells recovered from these mice. Either 10 6 MP, SP, or LP or PBS was injected into the dermis of both ears of BALB/c mice. (A) The parasite loads of ears and LN were determined by limiting-dilution assays and are expressed in LDAU. For each time point, we analyzed at least three animals per group, and the results are expressed as means 1 standard deviation. ND, not detected. Ear parasite loads of mice inoculated with MP or SP were significantly different from those of mice injected with LP (P 0.05). (B) IFN- produced by LACK-reactive CD4 T cells present in the draining LN. LN-derived CD4 T cells were purified and then reactivated ex vivo ( cells/well) in the presence of LACK- 1 (2 g/ml) or LACK( ) (15 M). Syngeneic irradiated splenocytes were used as antigen-presenting cells ( APC cells/well). In some wells, the anti I-A d monoclonal antibody M5/114 (3 g/ml) or the irrelevant monoclonal antibody SFR8-B6 (3 g/ml) was added. After 92 h of incubation at 37 C, supernatants were harvested and assayed for IFN- by ELISA. The data shown are from a single experiment representative of three. Each bar represents the mean 1 standard deviation of triplicate determinations (10 to 15 mice per group). Levels of IFN- released by CD4 T cells recovered from MP-infected mice were significantly different from the amounts of IFN- produced by CD4 T cells recovered from SP- or LP-infected mice (P 0.05). munication at the Worldleish 2 Congress, Crete, Greece, May 2001). Therefore, we also investigated the outcome of infection after intradermal inoculation of a small number (10 3 )ofmpin the ear. Inoculations with 10 3 MP or 10 6 LP induced cutaneous lesions displaying similar patterns of development (Fig. 1; Fig. 2C and D). Early cytokine production by LACK-reactive CD4 T cells recovered from mice inoculated with 10 6 promastigotes at various developmental stages. BALB/c mice were inoculated into the ear dermis with 10 6 MP, SP, or LP. The amounts of cytokines (IFN-, IL-4) produced by LACK-reactive CD4 T cells recovered from the draining retromaxillar LN were determined on day 3 postinoculation. We also investigated the relationship between the stimulation of LACK-reactive CD4 T cells and parasite burden by determining the number of parasites present in infected ears and draining LN and expressing them in LDAU (Fig. 3A). We found that 1,300 to 2,000 viable parasites ( 0.2% of the initial parasite dose) were present in the ears of mice inoculated with 10 6 MP or SP. In these animals, we also detected less than 100 parasites in the draining LN (Fig. 3A). Otherwise, no more than 100 viable parasites (less than 0.01% of the initial parasite inoculum) were present in the ears of mice inoculated with 10 6 LP and no viable parasites were detected in the LN (Fig. 3A). It must be stressed, however, that all these estimations are valid only if we assume that 1 LDAU corresponds to a single parasite. Overall, these results seem to indicate that, whatever the parasite stage used, a large proportion of the promastigotes died after intradermal injection. Nonetheless, we found a correlation between the developmental stage of the promastigotes used and the number of parasites surviving at the inoculation site and in the LN, the most infectious being the more numerous. We then investigated whether this difference was correlated with the activation or expansion of the LACK-reactive CD4 T-cell population in the draining LN. Reactivation of the CD4 T lymphocytes with the LACK( ) peptide or the LACK- 1 protein clearly led to the production of significantly higher levels of IFN- than those detected if these cells were not reactivated (control) or if CD4 T cells from mice inoculated with PBS alone were incubated with LACK- 1 or LACK( ) (Fig. 3B). Interestingly, CD4 T lymphocytes from mice inoculated with MP were much less reactive to LACK- 1 or LACK( ) than were those from mice which had received SP or LP. Thus, shortly after inoculation, the amount of IFN- produced by LACK-reactive T cells clearly depended on the infectiousness of the injected parasites. Furthermore, in our experimental conditions, no IL-4 secretion was detected at this time point, whatever the promastigote stage used for inoculation (data not shown). We also checked, in control experiments, that the stimulation of LACK-reactive cells was major histocompatibility complex (MHC) class II restricted, as shown by the significant inhibition of IFN- production observed in the presence of the anti-ia d M5/114 monoclonal antibody (Fig. 3B). Otherwise, we investigated whether the activation of LACK-reactive T cells was linked to the expansion or recruitment of V 4 CD4 T lymphocytes by analyzing cells recovered from the draining LN by flow cytometry after V 4 labeling. The percentages of V 4 CD4 T cells were similar in the LN of uninfected mice (PBS) and those of mice inoculated with promastigotes at various developmental stages. For instance, despite the large amounts of IFN- produced by LACK-reactive T cells present in the LN of mice inoculated with 10 6 LP, no significant increase in the proportion of V 4 cells was detected in these LN. Thus, the percentage of V 4 cells reached % of total LN cells and % of CD4 T cells in uninfected mice and % of total LN cells and % of CD4 T cells in mice infected with 10 6 LP 3 days previously. Reactivity of CD4 T lymphocytes to parasite antigens following low- or high-dose inoculations of L. major promastigotes: kinetic study over a 3-week period. The experiments described above clearly indicated that 3 days after inoculation of L. major promastigotes into the ear dermis of BALB/c mice, LACK-reactive CD4 T lymphocytes were present in the draining LN and that they secreted IFN- but no IL-4. These find-

5 2678 LANG ET AL. INFECT. IMMUN. FIG. 4. Monitoring of parasite load and total number of retromaxillar LN cells in BALB/c mice inoculated intradermally in each ear with 10 6 MP, 10 3 MP, or 10 6 LP. (A and B) At various time points after inoculation, parasite loads were estimated in the ears (A) and in the draining LN (B) by limiting-dilution assays. Results are expressed in LDAU per ear or LN. Histograms show the mean values 1 standard deviation for three individual ears or the mean values for 10 to 15 pooled LN. ND, not detected. At all time points, ear parasite loads of mice inoculated with 10 6 MP were significantly different from those of mice injected with 10 6 LP or 10 3 MP (P 0.01). (C) Total number of cells in the retromaxillar LN of mice inoculated in each ear with 10 6 MP ( ), 10 3 MP ( ), or 10 6 LP (F) (mean values of 10 to 15 pooled LN). Similar results were obtained in two independent experiments. ings were apparently contrary to published data showing that, after inoculation of L. major promastigotes into the footpads of BALB/c mice, rapid production of IL-4 mrna or IL-4 by LACK-reactive T cells is detectable (18, 24, 25). It was, however, possible that the time chosen in our preceding experiments was too late to detect the early production of IL-4 by these cells. To examine this point, a kinetic study of CD4 lymphocyte reactivity was undertaken, starting at 16 h postinoculation. Furthermore, to see whether some of the results obtained with the antigen LACK, namely the early production of IFN- by specific CD4 T cells and the inverse relationship between the level of IFN- secretion and the infectiousness of the parasites injected, could be extended to other parasite antigens, we assessed the reactivity of CD4 T cells to parasite lysates. In this series of experiments, we also examined the effect of the parasite size inoculum on the type and levels of cytokines released by parasite-specific CD4 T lymphocytes. Mice were inoculated with 10 6 MP, SP, or LP or with 10 3 MP. Sixteen hours to 19 days later, CD4 T lymphocytes were purified from retromaxillar LN and reactivated in vitro with LACK or parasite lysates, later designated L. major antigens. In parallel, we monitored changes in the parasite burden in the ears and draining LN over time. Sixteen hours after the injection of 10 6 MP into the ear dermis, viable parasites were detected in the ear (about 0.08% of the inoculum) and the draining LN (less than 0.01% of the initial parasite dose). The number of parasites then increased substantially at both sites, reaching and parasites, respectively, by day 19 (Fig. 4A and B). The parasite burden at day 19 was much lower following inoculation with 10 3 MP or 10 6 LP, reaching only 10 4 in the ears and 300 to 1,500 parasites in the LN (Fig. 4A and B). Thus, in all cases, the occurrence of lesion development (see Fig. 1) was correlated with the multiplication of parasites at the inoculation site and in the draining LN. We also estimated the total number of LN cells at the different time points examined. In mice inoculated with 10 6 MP, the very large increase in the number of parasites observed in the LN on day 19 was associated with a 10-fold increase in the number of LN cells (Fig. 4C). At the same time, the number of cells present in the LN of mice inoculated with 10 3 MP or 10 6 LP was similar to the number of cells present in the LN of uninfected mice (day 0; Fig. 4C). After their purification, CD4 T lymphocytes from the retromaxillar LN of these mice were stimulated with the LACK- 1 protein or the LACK( ) peptide. Shortly after infection (16 h), no IL-4 was produced by CD4 T cells from mice infected with 10 6 MP, SP, or LP (Fig. 5B; data not shown for SP). At day 3, CD4 T cells from mice infected with 10 6 LP secreted a small amount of IL-4 (Fig. 5D; LP-infected mice versus MP-infected mice, P 0.05). In contrast, during the same period of time, CD4 T cells from mice infected with 10 6 MP secreted significant amounts of IFN- (Fig. 5A and C). The level of IFN- production was low at 16 h (Fig. 5A; MPinfected mice versus control mice, P 0.05) and peaked on day 3 (Fig. 5C; MP-infected mice versus control mice, P 0.05). At this time point, the IFN- concentration was higher in the supernatants of cells recovered from mice infected with 10 6 LP than in those of cells taken from mice infected with 10 6 MP (Fig. 5C; LP-infected mice versus MP-infected mice, P 0.05). On days 8 and 19, LACK-reactive T cells were detected in the LN of both groups of mice, but they produced much smaller amounts of IFN- than on day 3 (Fig. 5E and G). Even at these time points, only moderate levels of IL-4 secretion were induced by LACK- 1 or LACK( ) in both groups of LN cells (Fig. 5F and H; LP- or MP-infected mice versus control mice, P 0.05). On the other hand, no secretion of IFN- or IL-4 was detected in the supernatants of cells prepared from mice inoculated with 10 3 MP regardless of the time point considered (Fig. 5A to H). Finally, no IFN- and little if any IL-4 was detected in the supernatants of LN CD4 T lymphocytes from uninfected mice (PBS) cultured in the presence of LACK- 1 or LACK( ) and of CD4 LN T lymphocytes derived from infected mice but not reactivated in vitro (Fig. 5A to H). We next assessed overall CD4 T-cell reactivity to L. major by monitoring the presence of L. major antigen-reactive CD4 T

6 VOL. 71, 2003 CYTOKINES INDUCED BY L. MAJOR INOCULATION 2679 FIG. 5. Cytokine production by LACK-reactive CD4 T cells present in the retromaxillar LN of mice inoculated intradermally in each ear with 10 6 MP, 10 3 MP, or 10 6 LP. LN CD4 T cells from PBS-inoculated mice were used as negative controls. At various time points after injection, ex vivo restimulation of CD4 T cells ( cells/well) was carried out with medium alone (control) or with LACK- 1 (2 g/ml) or LACK( ) (15 M) in the presence of syngeneic irradiated splenocytes, used as antigen-presenting cells ( cells/well). After 92 h of incubation, supernatants were collected and assayed for IFN- and IL-4 by ELISA. Each bar indicates the mean cytokine concentration of triplicates 1 standard deviation (10 to 15 mice per group). Similar results were obtained in two independent experiments. cells in the retromaxillar LN. The reactivation of CD4 T cells from mice inoculated with large numbers of parasites (10 6 MP, 10 6 LP) led to the production of significant amounts of IFN- as early as 16 h (LP-infected mice) or 3 days (MP-infected mice) postinjection (Fig. 6A and C; LP- or MP-infected mice versus control mice, P 0.05). In contrast, only very low levels of IL-4 were detected (Fig. 6B and D; LP- or MP-infected mice versus control mice, P 0.05). Both cytokines were secreted in larger amounts into the supernatants of LN cells derived from LP-inoculated mice than into those of LN cells isolated from MP-inoculated mice if suboptimal concentrations of L. major antigens (3.1 and 6.2 g/ml) were used for restimulations (Fig. 6C and D; LP-infected mice versus MP-infected mice, P 0.05). We observed a significant but low level of IFN- secretion by CD4 T cells without restimulation with L. major antigens 16 h and 3 days after inoculation with 10 6 LP (Fig. 6A and C; LP-infected mice versus control mice, P 0.05). As for cells incubated with L. major antigens (see below), this weak secretion of IFN- was MHC class II restricted (data not shown). On days 8 and 19, production of IFN- by cells derived from 10 6 MP- or 10 6 LP-infected mice fell sharply (Fig. 6E and G), but an increase in the amount of IL-4 produced by CD4 T cells was noted during this period (Fig. 6F and H; LP- or MPinfected mice versus control mice, P 0.01). However, similar amounts of this cytokine were found in the supernatants of CD4 LN T cells derived from mice inoculated with either 10 6 MP or 10 6 LP. Under the same conditions, no IFN- or IL-4 was produced by the LN cells of mice inoculated with PBS alone. In contrast to the data obtained with high-dose inoculations, the injection of 10 3 MP led to the production of significant amounts of both IFN- and IL-4 only on day 19 (Fig. 6G and H; mice inoculated with 10 3 MP versus control mice, P 0.01), a time point corresponding to the onset of lesion development (see Fig. 1) and to an increase in parasite burden in the ears and in the LN (see Fig. 4A and B). Regardless of the experimental group, the stimulation of L. major antigen-specific CD4 T cells was MHC class II restricted because it was specifically inhibited by the anti-ia d M5/114 monoclonal antibody (data not shown). Under our experimental conditions, we failed to detect an early production of IL-4 by CD4 T cells as described in others models of experimental leishmaniasis. As the possible consumption of IL-4 by cultured LN cells could account for this lack of detection, we also examined by quantitative RT-PCR the ability of parasites to stimulate IL-4 expression in vivo at the mrna level. Mice were inoculated with 10 6 MP, SP, or LP or with PBS, and 3 days to 21 days later, quantitation of IL-4 transcripts present in total LN cells or in CD4 LN T cells was carried out after extraction of total RNA. Shortly after inoculation (day 3), no IL-4 transcript was detected in total LN cells or in purified CD4 LN T cells (Fig. 7A). An 8- to 12-fold increase in IL-4 transcripts was observed at days 8 and 21 of infection with either parasite population. Thus, these findings are quite consistent with the results described above for the ex

7 2680 LANG ET AL. INFECT. IMMUN. FIG. 6. Cytokine production by L. major antigen-reactive CD4 T lymphocytes present in the retromaxillar LN of mice inoculated in each ear with 10 6 MP, 10 3 MP, or 10 6 LP. LN-derived CD4 T cells from PBS-inoculated mice were used as negative controls. At various time points after inoculation, ex vivo restimulation of CD4 T cells ( cells/well) was carried out in the absence (point 0) or presence of various concentrations of L. major antigens (LmAg), with syngeneic irradiated splenocytes used as antigen-presenting cells ( cells/well). After 92 h of incubation, supernatants were collected and assayed for IFN- (A, C, E, and G) and IL-4 (B, D, F, and H) production by ELISA. For each time point, we determined the mean cytokine concentration of triplicates 1 standard deviation (10 to 15 mice per group). Similar results were obtained in two independent experiments. vivo secretion of IL-4 by CD4 LN T cells (compare Fig. 7 with Fig. 5 and 6). Additionally, in a further experiment with SPinfected mice, it was shown that IL-4 transcripts in the LN cells were not detected before day 3 (Fig. 7B). The absence of IL-4 transcripts very early (16 h) after inoculation of MP or LP was also noted (data not shown). DISCUSSION The subcutaneous delivery of high doses of L. major SP to mice of various inbred strains is a widely used model for studying the pathogenesis of cutaneous leishmaniasis and the relationships between the phenotype of the mice inoculated (resistant or susceptible to Leishmania) and the type of immune response developed. The use of these experimental conditions has made it possible to demonstrate that the fate of infectious parasites is associated with T helper cell development and the balance between the Th1 and Th2 lymphocyte subsets that expand after parasite delivery (for a review, see reference 38). However, both the dose and the developmental stage of the parasites used in these experiments were expected to influence the kinetics of the events following inoculation, especially the early activation of T lymphocytes, as assessed by their cytokine profiles. Using a model which takes into account a natural site of infectious parasite delivery, the ear dermis, we investigated the early response of Leishmania-specific CD4 T lymphocytes by injecting low and high doses of L. major metacyclic promastigotes into BALB/c mice. To determine the influence of the infectiousness of the parasite inoculum and the developmental stage of the parasites, in some experiments we also injected viable parasite populations containing various percentages of infective parasites. In vitro stimulation of retromaxillar LNderived CD4 T cells was carried out with either L. major antigens or the antigen LACK, which has been shown to play a major role in expression of the susceptible phenotype of BALB/c mice to L. major (19, 24). Shortly after the delivery (16 h, day 3) of 10 6 MP, low levels of IFN- production by LACKreactive CD4 T lymphocytes were detected. In contrast, much higher amounts of IFN- were produced by LACK-reactive T cells derived from mice inoculated with a high dose of LP, although more viable parasites were observed in the ears of mice infected with high doses of MP than in those of mice infected with high doses of LP. CD4 LN T cells derived from mice inoculated with SP produced intermediate levels of IFN-. Although the data were less clear than those obtained with the LACK antigen, similar observations were made when L. major antigens were used to stimulate CD4 T lymphocytes. Thus, the reactivation of LN-derived CD4 T cells by L. major

8 VOL. 71, 2003 CYTOKINES INDUCED BY L. MAJOR INOCULATION 2681 FIG. 7. Quantitative analysis of IL-4 mrna transcripts present in retromaxillar LN cells of mice inoculated with PBS alone (negative controls) or with 10 6 promastigotes in the ear dermis. Total RNA was extracted from the LN at various time points after parasite injection, and quantitative reverse transcription-pcr was carried out as described in Materials and Methods. Results are expressed as the number of IL-4 transcripts per 10 6 copies of actin mrna. (A) Results obtained with mice inoculated 3 days to 21 days before with promastigotes at various developmental stages are compared. The amounts of IL-4 mrna contained by both total LN cells and purified CD4 LN T lymphocytes are shown. (B) Kinetics of IL-4 transcript production at very early times following inoculation of mice with SP. antigens led to the secretion of both IFN- and IL-4 in amounts that depended on the developmental stage of the parasites used for inoculation, with the highest levels recorded for mice inoculated with LP, at least at early time points after infection (16 h and 3 days). These data are consistent with the observation that, 3 days after inoculation of 10 6 MP into the ear dermis of BALB/c mice, the amount of IFN- transcripts associated with the LN-derived CD4 T cells was similar to that in the CD4 LN T cells of mice inoculated with PBS, whereas at the same time point, the amount of IFN- transcripts contained by the CD4 LN T cells of LP-infected mice increased by a factor of 5 to 7 (J.-H. Colle, N. Courret, and T. Lang, unpublished results). Together, our findings indicate that, until day 3, the CD4 component of the LACK- and L. major antigen-induced immune responses that developed in the draining LN of BALB/c mice into which 10 6 MP had been injected into the ear dermis clearly displayed a Th1 cytokine profile, with IFN- secretion and the absence of IL-4 production or the release of only very small amounts of IL-4. Furthermore, it has not been possible to detect a peak of IL-4 mrna in the retromaxillar LN early after infection of BALB/c mice with 10 6 parasites in the ear dermis, whatever the stage of the promastigotes inoculated. From days 3 to 8 postinfection, moderate to large amounts of IL-4 were secreted by CD4 T cells in response to L. major antigens or LACK. This higher level of IL-4 production was accompanied by a decrease in the IFN- secretion induced by these antigens. These findings are apparently at variance with previous data showing that inoculation of the footpads of BALB/c or B10.D2 mice with L. major SP rapidly induces the production of IL-4 mrna or IL-4 by CD4 popliteal LN T cells reactive to the LACK antigen (18, 24, 25). Although these experimental results are not easily comparable to ours because the strain of L. major used, the virulence of the parasites, the dose of parasites injected, and the inoculation site were not identical, it must be stressed that under our experimental conditions, the subcutaneous inoculation of 10 6 NIH173 SP into the footpads of BALB/c mice did not induce IL-4 mrna transcripts in the draining LN cells before day 3 postinfection (data not shown), and a recent study demonstrated that IL-4 expression by LACK-specific CD4 LN T cells of BALB/c mice infected into the footpads with 10 6 metacyclic promastigotes (strain NIH173) is not detectable before day 3 postinoculation (43). Therefore, factors other than the L. major strain and the inoculation site of the parasites seem to be at the origin of the differences described above. Even if the kinetics of the appearance of IL-4 transcripts is similar in ears and footpads, recent data from our laboratory suggest that the mechanisms involved in the expression of the susceptible phenotype of BALB/c mice to L. major could vary with the inoculation site. Indeed, injection of an IL-4-neutralizing monoclonal antibody (11B11) shortly before intradermal inoculation of a high dose of L. major SP (LV39 strain) into the ear dermis was unable to prevent the disease process, which nevertheless started with a delay of 1 or 2 weeks compared to that observed in BALB/c mice treated with an irrelevant monoclonal antibody used as an isotype control before parasite inoculation. In contrast, the same treatment with the IL-4-neutralizing monoclonal antibody completely prevented lesion formation when the same high dose of LV39 SP was injected subcutaneously into the footpads (J.-H. Colle and G. Milon, personal communication). Could the differences observed with the various parasite developmental stages be due to their sensitivity to leishmanicidal processes and thus, possibly, to the amount of released antigen processed by antigen-presenting cells? At first sight, our in vivo/ex vivo data are reminiscent of the in vitro results that showed that stimulation of CD4 T-cell hybridomas or of CD4 T-cell lines reactive to various Leishmania antigens is much stronger if the macrophages used as antigen-presenting cells are loaded with parasite stages very sensitive to the microbicidal mechanisms of the host cells (13, 21, 36). In particular, it has been shown that the presentation of LACK by promastigote-loaded macrophages is correlated with the infectiousness of the parasites taken up by phagocytosis, with the least virulent, LP, being the best source of LACK( ) I-A d complexes (13). Very likely, this differential presentation of LACK is linked, at least partially, to the destruction of the parasites within macrophages. Indeed, MP remain viable after internalization, most differentiating into amastigotes (12), whereas most LP and a large part of SP are destroyed after phagocytosis (13). However, this experimental in vitro model is clearly much too simple to provide a complete explanation for all the findings reported here because most MP, SP, and LP were rapidly destroyed following their injection into the ear dermis. Nevertheless, the two sets of data could be reconciled if we assume that, in vivo, LP and MP follow very different pathways leading to various degrees of exposure of the Leish-

9 2682 LANG ET AL. INFECT. IMMUN. mania antigens to the antigen presentation machinery of antigen-presenting cells. For instance, LP have been shown to be much more sensitive than MP to the complement system (for a review, see reference 29), and it is likely that after their injection, these parasites are rapidly killed, even before being taken up by phagocytic cells (14). In contrast, viable MP may be taken up by phagocytosis and then destroyed in intracellular compartments. As previously demonstrated, the sites at which the parasites are killed may be very important for the subsequent presentation of parasite antigens (13, 36). Of course, there may be other reasons for the lower level of activation of Leishmania-reactive T lymphocytes observed shortly after infection with MP. Various cell types (polymorphonuclear leukocytes, macrophages, dendritic leukocytes) may be involved in the internalization of the parasites depending on their stage of differentiation. Stage-dependent regulation of the costimulatory molecules produced by the antigenpresenting cells involved in the uptake of LP or MP could also explain our results, as could the level and/or type of cytokines produced after LP or MP inoculation. In this respect, L. major LP have been shown to be much more active than L. major MP in inducing the production of IL-12 p40, IL-12 p70, IFN-, tumor necrosis factor alpha, and IL-10 in cultures of human peripheral blood mononuclear cells (41). Other studies have shown that the infection of murine macrophages with MP results in selective inhibition of IL-12 induction (5, 10). We also studied the effect of decreasing the dose of infective promastigotes (MP) on the parasitic process. Thus, following inoculation with very small numbers of MP, the infection process involved a discreet phase which lasted more than 8 days and during which the number of parasites in the ears increased by a factor of about 10. No cytokine production (IFN-, IL-4) by Leishmania-specific LN-derived CD4 T lymphocytes was detected during this period. Lesions were first observed 3 weeks after inoculation, and their formation coincided with a further increase in the number of parasites in the ears, the presence of parasites in the draining LN, and the presence in the LN of Leishmania-reactive CD4 T lymphocytes secreting IFN- and IL-4. Rather similar data concerning the silent phase were recently obtained with other experimental models based on inoculation of the footpads or ears of genetically resistant C57BL/6 mice with 10 2 L. major metacyclic promastigotes (6, 28). However, the results of these studies differed from those of our study on two points, namely, a much larger increase in the number of parasites at the inoculation sites during the silent phase and the development of the lesion that was concomitant with the killing of the parasites. It is also important to note that after inoculation with a low dose of MP, LACK-reactive T cells were not detected during the 3 weeks preceding the onset of ear swelling. These results thus demonstrate that the kinetics, level, and reactivity of the CD4 T-cell response are at least partly dictated by the size of the parasite inoculum. The differences could be due to the rapid induction, after inoculation with a high dose of parasites, of intense inflammatory processes at the site of parasite delivery. In this respect, it has been shown that large numbers of neutrophils are rapidly recruited to the site of infection after the inoculation of BALB/c mice with to SP and that these cells persist for more than 11 days (3, 45; Y. Belkaïd and G. Milon, unpublished results). These cells recruited early after parasite inoculation have also been shown to play a role in shaping the L. major-specific T-cell response (45). The lineages of cells encountered by the parasites and their reactivity to them may also depends heavily upon the initial number of parasites and their infectiousness. For instance, a large number of Leishmania parasites can target a larger number of host cells or host cell lineages. Overall, these data show that, under our experimental conditions, there is no causal relationship between early production of IL-4 by CD4 T cells and susceptibility to L. major in BALB/c mice and that other, as yet unidentified factors may be responsible for the nonhealing cutaneous lesions observed in these mice (32 34). Second, the presence of early LACKspecific, IFN- -producing CD4 T lymphocytes in the LN of mice inoculated with high doses of parasites does not prevent lesion development. Thus, in this experimental model, the two cytokines analyzed are not predictive of the outcome of infection. Our results also demonstrate that inoculation with a very small number of parasites leads to the generation of IL-4- producing cells without affecting the generation of IFN- -producing cells. Thus, under certain conditions, IFN- and IL-4 cytokine production during L. major infection is not mutually exclusive. ACKNOWLEDGMENTS We thank Nicolas Glaichenhaus (Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France) for supplying LACK- 1, Fritz Melchers (Basel Institute, Basel, Switzerland) for providing the IL-4- producing cell line, and Karim Sebastien for his expertise on mice. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique, the Direction des Systèmes de Forces et de la Prospective (DSP/STTC, grant 97/2506A), and the Ministère de l Enseignement Supérieur, de la Recherche et de la Technologie (Programme de Recherche Fondamentale Microbiologie, Maladies Infectieuses et Parasitaires ). 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