Impaired Expression of Inflammatory Cytokines and Chemokines at Early Stages of Infection with Leishmania amazonensis

Mice.Female C57BL/6 and BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, Ind.). Female IL-10^−/− and IL-4^−/− mice generated with a C57BL/6 background and wild-type controls were purchased from Jackson Laboratory (Bar Harbor, Maine). All mice were maintained under specific-pathogen-free conditions and used for experiments when they were 6 to 8 weeks old with protocols approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, Tex.). Age- and sex-matched mice (five to eight mice/group) were infected subcutaneously in the hind foot with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis or L. major. Lesion size was monitored with a digital micrometer (Control Company, Friendswood, Tex.) and was expressed as the difference between the thickness of infected feet and the thickness of uninfected feet. Tissue parasite burdens were measured at different times by using a limiting dilution assay as previously described (49).

Parasite culture and antigen preparation.The infectivity of L. amazonensis (MHOM/BR/77/LTB0016) and the infectivity of L. major (MRHO/SU/59/P/LV39; obtained from Richard Titus, Colorado State University, Fort Collins) were maintained by regular passage through BALB/c mice. Promastigotes were cultured at 23°C in Schneider's Drosophila medium (pH 7.0 to 7.3) (Life Technologies, Rockville, Md.) supplemented with 20% fetal bovine serum (Sigma, St. Louis, Mo.), 2 mM l-glutamine, and 50 μg of gentamicin per ml. To prepare amastigote lysates for the generation of T-cell lines and other experiments, lesion-derived amastigotes were recovered from foot tissues of BALB/c mice and cultured at 32°C for 5 to 7 days in the same Schneider's Drosophila medium except that the pH was adjusted to 5.0 to 5.3 and the gentamicin concentration was reduced to 25 μg/ml. Parasites (1 × 10⁸ cells/ml in phosphate-buffered saline) were subjected to three freeze-thaw cycles and sonication for 45 min in an ice bath and then were stored at −70°C.

Detection of cytokines and chemokines and their receptor mRNAs by RPA.Total RNAs were extracted from draining LNs, isolated CD4⁺ LN T cells, or soft tissue of the infected foot at different times during infection by using a Micro RNA isolation kit (Stratagene, La Jolla, Calif.). An RNase protection assay (RPA) was conducted by using RiboQuant multiprobe RPA kits together with the following four template sets (all obtained from BD Biosciences): (i) set mCK-2b for the cytokines IL-4, IL-12p40, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-18/IGIF, and IFN-γ; (ii) set mCK-5b for the chemokines RANTES, eotaxin, MIP-1β, MIP-1α, MIP-2, and MCP-1; (iii) set mCR-3 for the cytokine receptors IL-12β1, IL-12β2, IL-6R, IFN-γRα, and IFN-γRβ; and (iv) set mCR-5 for the chemokine receptors CCR1, CCR3, CCR4, CCR5, and CCR2. Briefly, antisense cRNA probes were transcribed via T7 RNA polymerase in the presence of [α-³²P]UTP (3,000 Ci/mmol; ICN Biomedical, Inc., Irvine, Calif.). After heating the preparations briefly at 90°C to denature the RNA, we incubated the labeled probes with 5-μg portions of total RNA samples in hybridization buffer at 45°C for 16 h. Annealed products were digested with an RNase A-RNase T₁ mixture for 1 h at 30°C. The protected fragments were precipitated, dried, resuspended in loading buffer, and then denatured for 3 min at 90°C before electrophoresis on QuickPoint gels (NOVEX, San Diego, Calif.). The gels were dried for 1 h at 80°C before exposure to Kodak X-AR film at −70°C. The identity of each protected fragment was established by analyzing its migration distance by using a standard curve of migration distance versus the log nucleotide length for each undigested probe. The quantity of a given mRNA species in the original RNA sample was determined based on the signal densities as determined with an AlphaImager 2200 optical densitometer (Alpha Innotech Corp., San Leandro, Calif.) for the appropriately sized, protected probe fragment bands. Sample loading was normalized to the amount of the housekeeping gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Measurement of cytokines and chemokines by ELISA.LN cells (5 × 10⁶ cells) were prepared at different times during infection and cultured in 24-well plates in the presence or absence of parasite lysates (5 × 10⁶ parasite equivalents). Culture supernatants were collected at 72 h. Specific ELISAs were performed by using pairs of monoclonal antibodies and recombinant mouse IL-4, IL-10, and IFN-γ (BD Biosciences). To directly assess the levels of cytokines and chemokines at the inflamed sites, foot tissues were homogenized in lysis buffer (15 mM Tris-HCl, 0.5% Triton X-100, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂). Homogenized tissue samples were kept on ice for 30 min before centrifugation (850 × g for 30 min). After filtration through 0.2-μm-pore-size syringe filters, supernatants were tested by using ELISA kits specific for IL-1β, MIP-1α (R & D System, Minneapolis, Minn.), and MCP-1 (BD Biosciences).

Proliferation of draining LN cells and purified CD4⁺ LN T cells.CD4⁺ T cells were purified from draining LNs by positive selection by using magnetic beads (Dynal, Lake Success, N.Y.). Briefly, LN cells (pooled from five mice/group) were incubated with appropriate amounts of anti-CD4 monoclonal antibody-coated beads for 20 min at 4°C with gentle shaking. The rosetted cells were collected by using a magnetic separator, and bound cells were separated from beads by using DETACHaBEAD mouse CD4 (Dynal). Purified cell preparations usually contained >90% CD4⁺ T cells, as analyzed by fluorescence-activated cell sorting. CD4⁺ LN T cells (10⁵ cells) were cultured with 5 × 10⁵ irradiated syngeneic splenocytes of naive C57BL/6 mice together with promastigote lysates (equivalent to 0, 10³, 10⁴, 10⁵, and 10⁶ parasites/well) for 4 days in 96-well plates. One microcurie of [³H]thymidine was added 18 h before harvest, and the radioactivity was counted. Proliferative responses of LN cells were similarly tested in the presence or absence of parasite lysates (29).

Generation of L. amazonensis-reactive Th1-cell line and adoptive cell transfer.The procedures used for generating CD4⁺ T-cell lines were similar to those of Holaday et al. (28). Briefly, splenocytes were recovered from L. amazonensis-infected C57BL/6 mice (age, 4 months) and depleted CD8⁺ cells by using anti-CD8-coated beads (Dynal). CD8-depleted cells (5 × 10⁶ cells) were cultured with L. amazonensis amastigote lysates (equivalent to 2 × 10⁶ parasites) in complete IMDM containing 5 ng of IFN-γ per ml and a 1:8 dilution of culture supernatant of an anti-IL-4 hybridoma (11B11; American Type Culture Collection) for 7 days in 24-well plates. After centrifugation with Lympholyte-M separation medium (Cedarlane, Westbury, N.Y.) at 2,000 rpm (model BR 4i centrifuge; Jouan, Inc., Winchester, Va.) for 20 min, viable cells enriched at the interphase were collected, the concentration was adjusted to 2 × 10⁵ cells/ml, and the preparation was allowed to rest in the absence of antigen for 7 days. After this, 2 × 10⁵ viable cells were restimulated every 12 to 14 days with 5 × 10⁶ syngeneic splenocytes pulsed with amastigote lysates in the presence of IFN-γ and anti-IL-4. Viable cells harvested after a 7-day rest were injected intravenously into the tail vein at different concentrations (1 × 10⁶ to 5 × 10⁶ or 10⁷ cells/mouse) 1 day prior to infection with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis.

Statistical analysis.The differences between experimental groups were examined by using the Student's t test. A difference in mean values was considered significant when the P value was <0.05 or very significant when the P value was <0.01.

Delayed production of inflammatory cytokines and CC chemokines during L. amazonensis infection.Following infection with L. amazonensis, C57BL/6 mice developed progressive disease, with the lesion sizes increasing from 0.5 mm at 2 weeks to 2.1 mm at 8 weeks, while the lesions in L. major-infected mice reached the peak values (1.1 mm) at 4 weeks and then declined to baseline values (0.1 mm) by 8 weeks (Fig. 1). At 10 weeks, the parasite loads in foot tissues of L. amazonensis-infected mice were about 4 logs higher than the parasite loads in foot tissues of L. major-infected mice (log₁₀ 6.2 ± 0.49 versus log₁₀ 2.1 ± 0.18). However, it is unclear why L. amazonensis-infected mice failed to mount a strong Th1 response, despite the absence of Th2 dominance (29). To test the hypothesis that the failure of these mice to mount a Th1 response was due to a change in the production of proinflammatory cytokines and Th1-favored CC chemokines at an early stage of infection, we examined the expression profiles of cytokines and chemokines during the course of infection with these two parasite species. Total RNAs were isolated from draining LNs and foot tissues after 0, 1, 2, 4, or 8 weeks of infection (three to five mice/group), and the levels of specific transcripts were measured by the RPA. The quantity of each mRNA species was normalized and expressed relative to the level of the housekeeping GAPDH gene. Consistent with previous reports (30, 31), we found that LNs from L. amazonensis-infected mice expressed almost no IL-12 or IFN-γ and a low level of IL-4 mRNA during the first 2 weeks of infection (Fig. 2A and B). Compared to the 2-week data, a very similar profile of gene expression was observed in LNs of L. amazonensis-infected mice at 4 weeks (data not shown). Expression of IFN-γ and IL-1β became detectable after 8 weeks of infection with L. amazonensis. In sharp contrast, the transcripts for IL-12, IFN-γ, IL-1α, and IL-1β were readily detectable even after 1 week of infection with L. major. Expression of these genes reached their peak levels after 2 weeks, and the levels remained relatively high at 8 weeks. After 2 weeks, the levels of IFN-γ and IL-1β in L. major-infected mice were approximately 20- and 6-fold higher, respectively, than the levels in L. amazonensis-infected mice.

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FIG. 1.

Lesion development during the course of infection with L. amazonensis and L. major. C57BL/6 mice (five mice/group) were infected subcutaneously with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis (La) or L. major. Lesion sizes were measured with a micrometer. The parasite load in foot tissues was assessed after 10 weeks of infection, and the numbers in parentheses are means ± standard deviations for the groups (in log scale). Two asterisks indicate that there is a statistically significant difference (P < 0.01) between the two infection groups.

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FIG. 2.

Deficient expression of proinflammatory cytokines during infection with L. amazonensis. C57BL/6 mice were infected subcutaneously with 2 × 10⁶ stationary-phase promastigotes of L. amazonensis (La) or L. major. At 0, 1, 2, and 8 weeks after infection, draining LNs were harvested and pooled (three to five LNs/group) for total RNA extraction. RPA analyses were performed for the genes indicated, as described in Materials and Methods, and the results of a representative assay are shown (A). (B and C) Quantities of each mRNA species detected in LNs (B) and foot tissues (C), normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. wk, week(s).

A more striking difference was observed for the foot tissues of L. amazonensis- and L. major-infected mice (Fig. 2C). In this case, expression of IL-1α and IL-1β was barely detectable throughout the 4-week infection period with L. amazonensis. However, the levels of expression peaked after 2 weeks of infection with L. major and were approximately 35- to 70-fold greater than the levels in L. amazonensis-infected mice, and then they decreased by 4 weeks. These results indicate that there was prompt and regulated expression of IL-1-type cytokines in self-healing mice; however, expression of these genes in the nonhealing mice was delayed for about 2 to 4 weeks and significantly reduced after onset. Interestingly, expression of IL-1Ra, a specific IL-1 receptor antagonist that functions as an anti-inflammatory cytokine by competing with IL-1α and IL-1β for binding to the IL-1 receptor (3), was induced like expression of IL-1α and IL-1β was induced (Fig. 2), suggesting that there is a balance between IL-1 and IL-1Ra in local tissues of L. major-infected hosts.

Chemokines in the CC family, especially the ligands for CCR5 and CCR2, are involved in differentiation of T-cell subsets, recruitment of leukocytes to the site of infection (37, 38), and activation of the leishmanicidal activity of macrophages (9, 45). We next examined whether a deficient Th1-type response in L. amazonensis-infected mice correlated with alterations in expression of CC chemokines. Foot tissues of L. amazonensis-infected mice showed no sign of chemokine expression after 1 and 2 weeks but exhibited low levels of chemokine expression after 4 weeks (Fig. 3). In contrast, significantly higher levels of CCL5/RANTES, CCL3/MIP-1α, CCL4/MIP-1β, and MIP-2 were detected after 2 weeks of infection with L. major, and the levels were about 6- to 20-fold higher than those in L. amazonensis-infected mice. Except for expression of CCL5/RANTES and CCL2/MCP-1, chemokine expression declined after 4 weeks of infection with L. major (Fig. 3B). As in the foot tissues, deficient expression of these chemokines was also observed in draining LNs of L. amazonensis-infected mice (data not shown).

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FIG. 3.

Deficient expression of CC chemokines during infection with L. amazonensis. C57BL/6 mice (five mice/group) were infected with 2 × 10⁶ promastigotes of L. amazonensis (La) or L. major. At 0, 1, 2, and 4 weeks after infection, total RNA was extracted from foot tissues. RPA analyses were conducted for the genes indicated, and the results of a representative assay are shown (A). (B) Quantity of each mRNA species normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. wk, week(s).

To determine whether the change in cytokines and chemokines at the mRNA level correlated with protein expression, we collected LN cells and foot tissue extracts 1, 2, and 4 weeks after infection. The levels of IL-4, IL-10, and IFN-γ in culture supernatants of in vitro restimulated LN cells, as well as the levels of IL-1β, MIP-1α, and MCP-1 in foot tissue extracts, were determined by specific ELISAs. Compared with infection in L. major controls, L. amazonensis infection resulted in significant reductions in protein levels for IFN-γ, IL-1β, MIP-1, and MCP-1 (Fig. 4). Although expression of the IL-10 gene was not always easy to document in an RPA (data not shown), the levels of IL-4 and IL-10 secretion were consistently higher in LN cells derived from L. amazonensis-infected mice than in cells from L. major-infected mice (Fig. 4). Once again, differences between the two infection models were most apparent after 2 and 4 weeks. Consistent with the RPA data (Fig. 2), IL-4 production was not detectable after 2 weeks of infection with L. major, while the levels in L. amazonensis-infected mice decreased from 1.6 ng/ml after 1 week to 0.05 ng/ml by 4 weeks. It should be mentioned that although the levels of IL-4 production and IL-10 production at early stages of L. amazonensis infection were significantly higher than those in L. major-infected C57BL/6 mice, they were much lower than those reported for L. major-infected BALB/c mice (26, 29).

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FIG. 4.

Differential production of cytokines and chemokines following infection with L. amazonensis or L. major. C57BL/6 mice (six to eight mice/group) were infected with 2 × 10⁶ promastigotes of L. amazonensis (open bars) or L. major (solid bars). LN cells were cultured in vitro in the presence of parasite lysates (equivalent to 5 × 10⁶ parasites/well), and the supernatant was collected 3 days later. Foot tissue homogenates were prepared and pooled from three naive and infected mice as described in Materials and Methods. The levels of IL-4, IL-10, and IFN-γ in the supernatants of LN cell cultures and of IL-1β, MIP-1α, and MCP-1 in foot tissue homogenates were determined by ELISAs. The data are means ± standard deviations for three animals per group in three independent experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

Deficient expression of cytokine and chemokine receptors in L. amazonensis-infected mice.The functions of inflammatory and chemotactant cytokines are often regulated at the level of their corresponding receptors. To examine whether L. amazonensis-infected mice were also deficient in expression of cytokine receptors, CD4⁺ T cells were purified from draining LNs after 2 weeks of infection (five to eight mice/group). Total RNA was immediately extracted for RPA. Consistent with a previous report (31), we found a five- to sevenfold reduction in expression for the IL-12 receptor β2 subunit (IL-12Rβ2) in L. amazonensis-infected mice compared to the expression in L. major-infected controls (Fig. 5A and B) (P < 0.01). Furthermore, L. amazonensis-infected mice had significantly lower levels of CCR1, CCR2, and CCR5 mRNAs than L. major-infected controls (Fig. 5C and D) (P < 0.05). No apparent changes were detected in the levels of expression of IL-12Rβ1, IL-6R, gp130, IFN-γRα, CCR3, or CCR4, suggesting that there was selective, rather than global, impairment in expression of cytokine and chemokine receptors in CD4⁺ T cells of L. amazonensis-infected mice.

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FIG. 5.

Reduced expression of cytokine and chemokine receptors in L. amazonensis-infected mice. C57BL/6 mice (five to eight mice/group) were not treated or were infected with 2 × 10⁶ promastigotes of L. amazonensis (La) or L. major. At 2 weeks after infection, draining LNs were harvested and pooled for isolation of CD4⁺ T cells by positive selection. Total RNA was extracted from CD4⁺ T cells, and RPA analyses were conducted for cytokine receptors (A) and chemokine receptors (B). (C and D) Quantities of mRNA species for cytokine receptors (C) and chemokine receptors (D) normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

Reduced T-cell responses to L. amazonensis antigens.To test whether the decreased cytokine and cytokine receptor levels have a major effect on T-cell responsiveness to L. amazonensis antigens, we collected draining LN cells from two groups of mice after 2 weeks of infection and stimulated these cells (5 × 10⁵ cells/well/200 μl) with lysates of L. amazonensis or L. major promastigotes. At concentrations equivalent to 10³ to 10⁶ parasites/well, there was a significant reduction in thymidine incorporation in LN cells of L. amazonensis-infected mice (Fig. 6A) compared with the incorporation in L. major-infected controls (P < 0.01). The differences were not due to a reduction in the levels of CD4⁺ T cells in the LNs of L. amazonensis-infected mice because the percentages of CD4⁺ T cells per LN were 22.97% ± 0.82% and 17.71% ± 0.52% in L. amazonensis- and L. major-infected mice, respectively. Moreover, LN cells from both groups of mice responded similarly to concanavalin A (data not shown). These data suggest that there was selective impairment in parasite-specific immune responses during L. amazonensis infection. To further define the function of CD4⁺ cells, we isolated CD4⁺ LN T cells after 2 weeks of infection and stimulated the cells with the corresponding parasite lysates together with irradiated, syngeneic splenocytes of naive C57BL/6 mice. A reduction in the proliferation of CD4⁺ LN T cells of L. amazonensis-infected mice was still evident, but the reduction was less (Fig. 6B) (P < 0.05). Therefore, it appears that CD4⁺ T cells in L. amazonensis-infected mice are intrinsically deficient in responsiveness to L. amazonensis antigen, probably due to impaired priming and optimal activation through their interaction with dendritic cells, which are known to shape antigen-specific T-cell responses in L. amazonensis-infected mice (42). In agreement with this view, we found significantly smaller LNs in L. amazonensis-infected mice than in L. major-infected controls after 5 and 9 days of infection (data not shown). After 2 weeks, the total numbers of cells in L. amazonensis- and L. major-infected mice were 7.6 × 10⁶ and 21.0 × 10⁶ cells per LN, respectively (P < 0.01). These in vitro and in vivo observations suggest that there was impairment in CD4⁺ T-cell priming and activation during L. amazonensis infection.

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FIG. 6.

Parasite-specific proliferation of draining LN cells and purified CD4⁺ LN T cells. Draining LN cells were collected from C57BL/6 mice at 2 weeks after infection with L. amazonensis (□) or L. major (▪), and CD4⁺ LN T cells were isolated by positive selection. Draining LN cells (5 × 10⁵ cells) (A) and CD4⁺ LN T cells (10⁵ cells) (B) were cultured without and with 5 × 10⁵ irradiated syngeneic splenocytes in the presence of different concentrations of promastigote lysates for 4 days in 96-well plates. One microcurie of [³H]thymidine was added 18 h before harvest, and the radioactivity was counted. The data are means ± standard deviations for two separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the two infection groups.

Reversal of L. amazonensis-specific immune deficiency via adoptive transfer of Th1 cells.Given that there is insufficient priming of antigen-specific CD4⁺ T cells in L. amazonensis-infected mice and that treatment with recombinant IFN-γ or IL-12 alone does not result in parasite resolution (7, 31), we next examined whether bypassing T-cell priming via adoptive transfer of fully primed Th1 cells would restore gene expression and reverse the outcome of infection. We generated a Th1 cell line, which contained about 40% TNF-α-producing cells and 95% IFN-γ-producing cells and expressed predominantly Vβ4 (14%) and Vβ6 (58%) T-cell receptors. C57BL/6 mice were transferred intravenously with increasing doses of Th1 cells (2 × 10⁵, 1 × 10⁶, 5 × 10⁶, or 1 × 10⁷ cells per mouse) 1 day prior to infection. As shown in Fig. 7A, there were dose-dependent reductions in lesion size and tissue parasite burdens in mice undergoing cell transfer. Mice receiving 5 × 10⁶ or 1 × 10⁷ cells were completely protected against promastigote challenge, with no sign of lesions after 12 weeks and a 3- to 4-log reduction in the parasite load. This protection correlated with an average fivefold increase in IFN-γ production in LN cells and a 10-fold increase in splenocytes (data not shown).

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FIG. 7.

Reversal of immunodeficiency and control of L. amazonensis infection following Th1-cell transfer. C57BL/6 mice (five to eight mice/group) were not treated or received different doses of Th1 cells and then were infected with 2 × 10⁶ promastigotes of L. amazonensis 1 day later. (A) Lesion sizes, as determined biweekly with a micrometer. The parasite load in foot tissues was assessed after 14 weeks of infection, and the numbers in parentheses are means ± standard deviations for the groups (in log scale). The parasite loads in mice that received 5 × 10⁶ or 1 × 10⁷ Th1 cells were lower than those in nontransferred controls (P < 0.01). Mice that received 5 × 10⁶ Th1 cells were selected for subsequent gene expression analyses. At zero time and 2 weeks after infection, draining LNs (B), foot tissues (C), and purified CD4⁺ LN T cells (D and E) were collected and used for total RNA extraction. RPA analyses were performed for different genes. The quantity of each mRNA species was normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments. One asterisk (P < 0.05) and two asterisks (P < 0.01) indicate that there are statistically significant differences between the transferred and nontransferred groups.

Mice that received 5 × 10⁶ Th1 cells were selected for detailed gene expression analyses. Compared to control animals, cell-transferred mice had significantly higher levels of IFN-γ, IL-1β, and IL-1Ra in LN cells after 2 weeks (Fig. 7B), higher levels of RANTES, MIP-1α, MIP-1β, and MIP-2 in foot tissues (Fig. 7C), and increased levels of IL-12Rβ2, CCR2, and CCR5 in CD4⁺ LN T cells (Fig. 7D and E) (P < 0.01). Enhanced levels of IL-12, IL-1α, IL-12β1, and CCR1 were also detected in mice that received cells (P < 0.05). Overall, the levels of expression of these genes in cell-transferred, L. amazonensis-infected mice closely resembled the levels observed in L. major-infected mice (compare Fig. 7 with Fig. 2 to 4). These studies suggest that an early, vigorous Th1 response to the parasite contributes to the induction of inflammatory mediator production in the skin.

Partial role of IL-10 in immune down-regulation.It is known that IL-4 and IL-10 can down-regulate inflammatory responses in many infection models (25, 55) and that both cytokines play critical roles in disease progression and parasite persistence in L. major infection (32). Given the enhanced production of IL-4 and IL-10 in the first few weeks of L. amazonensis infection (Fig. 4), it was important to examine whether endogenous production of these two cytokines contributed to impaired expression of inflammatory mediators. IL-10^−/− and wild-type C57BL/6 mice were infected with L. amazonensis promastigotes, and total RNA was extracted from LNs or foot tissues at zero time and after 2 and 4 weeks for RPA analysis. As shown in Fig. 8B, approximately 3- to 10-fold increases in the levels of IL-12, IFN-γ, and IL-1β were detected in LNs of IL-10^−/− mice compared with the levels in wild-type controls. Foot tissues of IL-10^−/− mice exhibited two- to threefold increases in the levels of CCL5/RANTES, CCL3/MIP-1α, CCL4/MIP-1β, and MIP-2 compared to the levels in wild-type controls. This enhancement was most evident after 4 weeks of infection with L. amazonensis; however, the overall levels of gene expression in L. amazonensis-infected mice remained much lower than those in resistant mice (compare the data for L. amazonensis-infected mice to the data for L. major-infected mice in Fig. 2 and 3). Consistent with a previous report (30), IL-10^−/− mice remained susceptible to L. amazonensis infection and developed slightly smaller, but progressive lesions that contained about 10-fold fewer parasites than the wild-type control lesions contained (Fig. 8A). Similar studies were conducted with IL-4^−/− mice; however, we did not observe a significant increase in gene expression (data not shown). As reported by Jones et al. (31), L. amazonensis-infected IL-4^−/− mice developed progressive lesions with a high number of parasites in foot tissue (log₁₀ 4.55 ± 0.37 parasites at 2 weeks and log₁₀ 5.32 ± 0.51 parasites at 4 weeks), compared to the numbers in the wild-type controls (log₁₀ 4.32 ± 0.28 parasites at 2 weeks and log₁₀ 5.37 ± 0.45 parasites at 4 weeks). There were no statistical differences between the two infection models at the 2- and 4-week time points (P > 0.05). Collectively, these data suggest that IL-10, but not IL-4, contributes in part to immune down-regulation in L. amazonensis infection.

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FIG. 8.

Partial reversal of immunodeficiency in L. amazonensis-infected IL-10^−/− mice. IL-10^−/− and control (WT) C57BL/6 mice were infected with 2 × 10⁶ promastigotes of L. amazonensis. (A) Lesion sizes, as determined with a micrometer. (Inset) Parasite loads in foot tissues were assessed (log₁₀ scale) after 4 and 10 weeks of infection. One asterisk indicates that there is a statistically significant difference (P < 0.05) between the two infection groups at 4 and 10 weeks after infection. (B) Total RNAs were extracted from LNs or infected foot tissues at zero time and 2 and 4 weeks after infection and used for RPA analyses of different genes. The quantity of each mRNA species detected in LNs and foot tissues was normalized and expressed relative to the amount of GAPDH. The data are means ± standard deviations for three separate experiments.