Previous studies have shown that the in vitro ligation of FcγRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common γ-chain of Fc receptors (FcγR−/−) or mice genetically altered to lack circulating Ab (JHD) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcγR−/− mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcγR−/− mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-γ-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-β within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcγRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.

The receptors for Igs (FcRs) are a crucial component of the immune system and are present on most effector cells. The FcRs for IgG (FcγRs) comprise the largest family and mediate many of the biological functions such as phagocytosis, Ab-dependent cell-mediated cytotoxicity, induction of inflammatory cascades, and modulation of immune responses (reviewed in Refs. 1, 2, 3, 4). FcγRs are found on many cells of hemopoietic lineage and mediate both high and low affinity binding to IgG (5). The high affinity FcγRI is expressed on macrophages and is unique among these receptors in its inducibility by IFN-γ (6, 7, 8). FcγRI is capable of mediating Ab-dependent cell-mediated cytotoxicity and phagocytosis in response to cross-linking by Ab and is thought to be principally involved in augmenting Ab-mediated effector responses by macrophages and neutrophils at the site of a local inflammatory response. The low affinity receptor for IgG, FcγRIII, is thought to be responsible for effector cell responses to immune complexes and appears to selectively bind IgG1. In contrast, FcγRII is an inhibitory receptor and down-regulates cellular responses following coligation with activating receptors (9, 10).

The majority of studies examining the role of FcγR in immunity to parasites have made use of FcR γ-chain-deficient (FcγR−/−) mice. The γ-chain, a subunit common to FcγRI, FcγRIII, FcεRI, and FcαRI, is required for efficient cell surface expression and signal transduction. Mice deficient in γ-chain (FcγR−/−) show a loss of macrophages’ surface expression of FcγRIII, FcεRI, and FcαRI, and have markedly reduced FcγRI expression (10, 11, 12). Despite the continued low level expression of high affinity FcγRI (approximately one-fifth the level of receptor expressed on normal macrophages), macrophages from FcγR−/− mice have been shown to lack the ability to phagocytize IgG-opsonized particles (12).

Earlier studies have shown the importance of FcγRs in the invasion process by Leishmania parasites and in regulation of the inflammatory response during infection. Leishmania are intracellular parasites that reside primarily within host tissue macrophages and are killed via production of NO by activated macrophages. With respect to the development of resistance, there is a general consensus that the activation of an IL-12-dependent Th1 response is required for optimum production of the macrophage-activating cytokine, IFN-γ, and cure of infection. A recent study has shown that ligation of the FcγRs by IgG-opsonized Leishmania amastigotes plays a crucial role during development of the immune response by inducing synthesis and secretion of IL-10, which inhibits macrophage activation and contributes to parasite growth in lesions (13). Studies using FcγR−/− mice have shown the importance of FcγRs in the invasion process by Leishmania pifanoi (14). These studies demonstrated that γ-chain-deficient mice are refractory to infection with L. pifanoi and have markedly reduced numbers of monocytes and lymphocytes recruited or retained at the site of infection in comparison with wild-type mice. Furthermore, genetically altered mice, possessing no Ab and with or without functional B cells, are similarly unable to maintain an infection with L. pifanoi, suggesting a crucial role for Ab and FcγRs in L. pifanoi invasion (14). However, unlike infection with L. pifanoi, B cell-deficient JHD mice infected with Leishmania major develop lesions and have parasite burdens similar to those of wild-type mice, possibly suggesting a different role for B cells and/or Ab in the pathogenesis of L. major infection (15, 16). Colmenares et al. (16) have further shown that infection with L. pifanoi parasites induces comparable T cell responses (proliferative and cytokine production) in wild-type control (susceptible) or Ab-deficient (nonsusceptible) mice. Interestingly, the levels of IL-10 produced by lymph node (LN) 4-derived CD4+ T cells from wild-type or Ab-deficient mice were similar. This study suggests that Ag-specific T cells producing IL-10 may be necessary, but not sufficient for maintenance of infection.

Given the differences in patterns of disease in inbred strains of mice infected with the Old World parasite, L. major, and New World Leishmania species and the relative importance of different cytokines in resistance or susceptibility to different Leishmania species, we have explored how a deficiency in FcγRs influences infection with L. major in susceptible BALB/c mice. We investigated the role of FcγRs during L. major infection using FcγR−/− mice and show that while FcγRs appear to have little or no influence during the acute stage of infection with L. major, the absence of FcγRs, required for FcR-mediated uptake of IgG-opsonized parasites, ultimately results in enhanced resistance and resolution of infection. Healing in FcγR−/− mice appears to correlate with reduced levels of IL-10- and TGF-β in infected lesions, particularly within parasitized cells.

Female BALB/c mice were purchased from The Jackson Laboratory. BALB/cFcγR−/− and BALB/cIL-10−/− mice were originally obtained from D. Mosser (University of Maryland, College Park, MD) and from R. Coffman (DNAX, Palo Alto, CA), respectively, and were bred and maintained within the animal facility at the University of Pennsylvania. All mice were 6–10 wk old at the time of infection. L. major (WHO MHOM/IL/80/Friedlin) parasites were maintained in Grace’s insect cell culture medium (Invitrogen Life Technologies) containing 20% FBS, 2 mM l-glutamine, 100 μg of streptomycin, and 100 U/ml penicillin G sodium.

Mice were inoculated into one hind footpad with 5 × 104 (high dose) or 5 × 103 (low dose) L. major metacyclic promastigotes isolated from stationary-phase cultures by negative selection using peanut agglutinin (Sigma-Aldrich), as described previously (17). Lesion size was measured with Vernier caliper and expressed as the difference in thickness between the infected and the uninfected contralateral footpads. Parasites were enumerated by a limiting dilution assay, as described previously (18). In brief, the homogenates of infected lesions were serially diluted in Grace’s insect culture medium plus 20% FBS and observed 5–7 days later for growth of promastigotes. Parasite numbers are expressed as the negative log10 dilution at which promastigotes’ growth was observed.

Single cell suspensions of LNs draining infected lesions were cultured at 5 × 106 cells/ml in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 5 × 10−5 M 2-ME in the presence of soluble leishmanial Ag (SLA, prepared as described previously (19)) at 50 μg/ml. Supernatants were collected at 72 h and assayed for IFN-γ ELISA, as previously described (20). rIFN-γ (PeproTech) was used as standard for ELISA. IL-10 production was measured using ELISA kit (BD Pharmingen), as directed by the manufacturer.

The number of IL-12p40- and IL-4-secreting cells in LN suspensions was determined using an ELISPOT assay, as previously described (21, 22). The mAbs C17.8 and biotinylated C15.6, generously provided by C. Hunter at the University of Pennsylvania (Philadelphia, PA), were used for IL-12 ELISPOT assay. IL-4-producing cells were detected using mAbs 11B11 and biotinylated BVD6-24G2. IL-12- and IL-4-secreting cells were determined in cell cultures following overnight stimulation with SLA (50 μg/ml).

For intracellular detection of IFN-γ and IL-10, purified LN cells were plated in a 96-well plate (Costar) at a density of 4 × 105 cells/well in a final volume of 200 μl. Cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 5 h. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added to cultures during the last 2 h of stimulation. Cells were harvested, washed, resuspended in FACS buffer (1× PBS, 0.2% BSA fraction V, 4 mM sodium azide), and then preincubated with saturating concentrations of Fc block for 20 min on ice and stained with FITC-conjugated anti-CD4 (BD Pharmingen) for 30 min on ice. Cells were then washed with FACS buffer, fixed with 1% (w/v) formaldehyde overnight at 4°C, washed again, and permeabilized with 0.1% (w/v) saponin in FACS buffer. After permeabilization, cells were stained with allophycocyanin-conjugated anti-IFN-γ and PE-conjugated anti-IL-10 (BD Pharmingen) for 30 min on ice. Cells were washed once with 0.1% saponin buffer and then with FACS buffer. Analysis of the cells was performed using a FACSCalibur flow cytometer (BD Biosciences). Results were analyzed using CellQuest software (BD Biosciences). The recommended Ab concentrations were used to give optimal staining for flow cytometric analyses.

Infected footpads were fixed in 10% phosphate-buffered Formalin and embedded in paraffin, and tissue sections on slides were used for immunostaining. Slides were heated in a 60°C oven for 1 h to help adherence of paraffin-embedded tissue sections to slides, and deparaffinized and rehydrated with several changes of PRO PAR (clearant) and grades of ethanol. After breaking the cross-linkages, created by Formalin fixative in citrate buffer (pH 6.0) for 7 min, slides were treated with H2O2 to quench endogenous peroxidase. Slides were incubated with rabbit anti-mouse IL-10 (PeproTech) or rabbit anti-mouse TGF-β Abs (Abcam) for 30 min at room temperature and detection, followed by incubation with peroxidase-conjugated goat anti-rabbit Ab (Jackson ImmunoResearch Laboratories) for 15 min, and visualized with 3,3′-diaminobenzidine solution (diaminobenzidine chromogen). Slides were counterstained with hematoxylin for 1 min.

Leishmania-specific Ab was determined by ELISA based on a previously described assay (23). Briefly, polystyrene microtiter plates (Dynatech Laboratories) were coated with 1.0 μg of L. major Ag (SLA) per ml in PBS (pH 7.4) overnight at 4°C. The plates were washed, as described previously, and blocked with 0.5% newborn calf serum in PBS for 2 h at 37°C. Serum samples were then serially diluted with 0.5% FCS in PBS starting at a dilution of 1/1010. The samples were incubated overnight at 4°C. The plates were washed in PBS-0.05% Tween 20 five times, and bound Abs were detected with goat anti-mouse IgG1 or IgG2a directly conjugated to HRP (The Jackson Laboratory). After 2 h of incubation at 37°C, the plates were washed with PBS-0.05% Tween 20, and the ABTS peroxidase substrate (Kirkegaard & Perry Laboratories) was then added. The plates were read in a spectrophotometer at OD405.

Statistically significant differences between groups were determined using unpaired Student’s t test. Significance was assumed if p < 0.05.

To determine whether FcγRs play a role in susceptibility to L. major, we examined patterns of infection in FcγR−/− BALB/c mice and wild-type BALB/c mice inoculated with two different doses of parasites. At the higher dose (5 × 104 promastigotes), BALB/c and FcγR−/− mice developed a similar pattern of infection. Although lesions in FcγR−/− mice were slightly smaller from weeks 8–12 of infection (Fig. 1), the differences were not significant. However, when lesion parasite numbers were enumerated at week 12 of infection, we observed ∼5 log fewer parasites in the lesions of FcγR−/− mice, compared with wild-type mice (Fig. 1). In marked contrast, when inoculated with a lower dose of 5 × 103 promastigotes, FcγR−/− mice were able to heal their lesions (Fig. 1) and almost totally eliminate parasites from their lesions. Healing in FcγR−/− mice inoculated with 5 × 103 promastigotes was associated with total resistance to reinfection with a challenge dose of 2 × 105 promastigotes delivered into the contralateral footpad (data not shown).

FIGURE 1.

Mice lacking the γ-chain for FcR exhibit increased control of infection with L. major following inoculation of high numbers of parasites. FcγR−/− and BALB/c mice were inoculated with 5 × 104 (A) or 5 × 103 (B) metacyclic promastigotes, and course of infection was followed for 12–15 wk. Lesion size (left) was expressed as the difference in thickness between the infected and the uninfected contralateral footpads. Numbers of lesion parasites (right) at the termination of infection were determined by limiting dilution assay and expressed as the negative log10 dilution at which promastigote growth was observed. Error bars represent the mean ± SE of five mice per group, and are representative of results of two separate experiments. ∗, Indicates statistically different at p < 0.05.

FIGURE 1.

Mice lacking the γ-chain for FcR exhibit increased control of infection with L. major following inoculation of high numbers of parasites. FcγR−/− and BALB/c mice were inoculated with 5 × 104 (A) or 5 × 103 (B) metacyclic promastigotes, and course of infection was followed for 12–15 wk. Lesion size (left) was expressed as the difference in thickness between the infected and the uninfected contralateral footpads. Numbers of lesion parasites (right) at the termination of infection were determined by limiting dilution assay and expressed as the negative log10 dilution at which promastigote growth was observed. Error bars represent the mean ± SE of five mice per group, and are representative of results of two separate experiments. ∗, Indicates statistically different at p < 0.05.

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To determine whether a deficiency in FcγR expression altered the in vivo invasion of parasites into host macrophages, we also assessed infection levels during an earlier stage of infection. Mice infected with 5 × 103L. major were sacrificed at peak lesion size (week 6 postinfection) and lesion parasite numbers were enumerated. Interestingly, the levels of infection in both FcγR−/− and BALB/c mice were comparable, suggesting that a lack of FcRs did not impair macrophage uptake of parasites (Fig. 2). We also examined lesion parasite numbers at day 14 of infection and observed no statistical difference between parasites in the two strains of mice (log 2.9 ± 0.3 lesion parasites in BALB/c mice vs log 2.3 ± 0.6 in FcγR−/− mice).

FIGURE 2.

FcγR−/− and BALB/c harbor similar numbers of lesion parasites at week 6 following infection with 5 × 103 promastigotes. Numbers of lesion parasites were determined as in Fig. 1. Error bars represent the mean ± SE of five mice per group and are representative of results of two separate experiments. Infection levels were not statistically different in the two groups of mice.

FIGURE 2.

FcγR−/− and BALB/c harbor similar numbers of lesion parasites at week 6 following infection with 5 × 103 promastigotes. Numbers of lesion parasites were determined as in Fig. 1. Error bars represent the mean ± SE of five mice per group and are representative of results of two separate experiments. Infection levels were not statistically different in the two groups of mice.

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Next, we examined immune responses in infected mice to determine whether absence of the Fc γ-chain had any impact on the cytokine production patterns. LNs draining to lesion sites were collected from mice at week 6 of infection and cultured in vitro for 72 h following stimulation with SLA. The supernatants of the in vitro cultures were used to analyze IL-10 and IFN-γ production by ELISA. The numbers of IL-12p40- and IL-4-secreting cells in LN suspensions were determined using an ELISPOT assay following overnight stimulation with SLA. Cells from FcγR−/− mice produced significantly lower levels of IL-10 protein than cells from BALB/c mice (Fig. 3), but production of IFN-γ was comparable in both strains of mice (Fig. 3). In addition, IFN-γ production was similar in wild-type and FcγR−/− mice at week 15 of infection (data not shown). LNs from FcγR−/− had fewer IL-4-secreting cells (Fig. 4) compared with those in wild-type control mice; however, numbers of IL-12-secreting cells were comparable in both groups (Fig. 4). Together, these results suggest a reduction in the production of Th2-associated cytokines such as IL-4 and IL-10, but no concomitant increase in Th1-associated cytokines such as IFN-γ and IL-12.

FIGURE 3.

Cytokine production in BALB/c and FcγR−/− mice at week 6 of infection. LN cells from the mice in Fig. 3 were stimulated in vitro with SLA (50 μg/ml) for 72 h, and cell supernatants were assayed for IL-10 (left) and IFN-γ (right) by ELISA. Values are the mean ± SE of five mice per group. ∗, Indicates statistically different at p < 0.05.

FIGURE 3.

Cytokine production in BALB/c and FcγR−/− mice at week 6 of infection. LN cells from the mice in Fig. 3 were stimulated in vitro with SLA (50 μg/ml) for 72 h, and cell supernatants were assayed for IL-10 (left) and IFN-γ (right) by ELISA. Values are the mean ± SE of five mice per group. ∗, Indicates statistically different at p < 0.05.

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FIGURE 4.

Healing FcγR−/− mice have reduced numbers of IL-4-producing cells, but similar numbers of IL-12p40-producing cells at week 6 of infection. LN cells from infected mice inoculated with 5 × 103 parasites were assayed for IL-4- and IL-12p40-producing cells by ELISPOT assay. The data are expressed as mean frequency per 106 LN cells ± SE of four to five mice per group and are representative of results of two separate experiments. ∗, Indicates statistically different at p < 0.05.

FIGURE 4.

Healing FcγR−/− mice have reduced numbers of IL-4-producing cells, but similar numbers of IL-12p40-producing cells at week 6 of infection. LN cells from infected mice inoculated with 5 × 103 parasites were assayed for IL-4- and IL-12p40-producing cells by ELISPOT assay. The data are expressed as mean frequency per 106 LN cells ± SE of four to five mice per group and are representative of results of two separate experiments. ∗, Indicates statistically different at p < 0.05.

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To further assess the in vivo immune response against L. major, we compared levels of Leishmania-specific IgG1 and IgG2a Abs in sera from both groups of mice. Fig. 5 shows that at 6 wk after infection, there was a significant increase in the level of Leishmania-specific IgG1 Abs in the wild-type mice compared with the level in the FcγR−/− mice. In contrast, FcγR−/− mice had significantly higher levels of Leishmania-specific IgG2a Ab (Fig. 5). The higher IgG2a Ab levels and reduced IgG1 titers suggest that FcγR−/− mice developed a more dominant Th1-type response during infection.

FIGURE 5.

Ab production in infected mice. Serum levels of parasite-specific IgG1 and IgG2a in BALB/c and FcγR−/− mice at week 6 of infection. Values represent mean ± SE of four mice per group.

FIGURE 5.

Ab production in infected mice. Serum levels of parasite-specific IgG1 and IgG2a in BALB/c and FcγR−/− mice at week 6 of infection. Values represent mean ± SE of four mice per group.

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We extended our analysis of the immune response and assessed the numbers of IL-10- and IFN-γ-secreting CD4+ T cells in LNs using flow cytometric analysis. Cells from LNs of mice at week 6 postinfection were stimulated with PMA and ionomycin and analyzed for IL-10 and IFN-γ production. As can be seen in Fig. 6, FcγR−/− mice have significantly decreased numbers of IL-10-secreting CD4+ T cells compared with wild-type mice, whereas the numbers of IFN-γ-secreting CD4+ T cells were comparable. Thus, it appears that a deficiency in the FcR γ-chain results in decrease in the numbers of IL-10-secreting CD4+ T cells, a result that is compatible with ELISA results from 6-wk-infected mice.

FIGURE 6.

Healing FcγR−/− mice have reduced numbers of IL-10-producing CD4+ T cells, but similar numbers of IFN-γ-producing cells at week 6 of infection. LN cells from mice were stimulated with PMA and ionomycin for 5 h. Brefeldin A was added to cell cultures during the last 2 h of stimulation and analyzed for CD4 expression and intracellular cytokine secretion by flow cytometry. Gated CD4+ T cells were examined for IL-10 and IFN-γ production. Numbers represent percentage of IFN-γ- and IL-10-secreting CD4+ T cells in each quadrant and are representative of results from three mice.

FIGURE 6.

Healing FcγR−/− mice have reduced numbers of IL-10-producing CD4+ T cells, but similar numbers of IFN-γ-producing cells at week 6 of infection. LN cells from mice were stimulated with PMA and ionomycin for 5 h. Brefeldin A was added to cell cultures during the last 2 h of stimulation and analyzed for CD4 expression and intracellular cytokine secretion by flow cytometry. Gated CD4+ T cells were examined for IL-10 and IFN-γ production. Numbers represent percentage of IFN-γ- and IL-10-secreting CD4+ T cells in each quadrant and are representative of results from three mice.

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H&E-stained sections from FcγR−/− mice and wild-type control mice infected with L. major revealed similar lesion morphologies and similar numbers of parasites at 6 wk postinfection (data not shown). However, immunohistochemistry analysis using Abs that detected IL-10 and TGF-β (Fig. 7) indicated that both IL-10 and TGF-β production was significantly higher in wild-type control mice compared with FcγR−/− mice. These results suggest that FcγR signaling through γ-chain may play an important in vivo role in promoting the production of IL-10 and TGF-β at the site of infection, and provide an explanation as to why FcγR−/− mice go on to resolve infection even though we could not observe an increase in Th1-type cytokines in these mice.

FIGURE 7.

Reduced levels of IL-10 and TGF-β in lesions of FcγR−/− mice. Lesions from BALB/c mice (A and C) and FcγR−/− mice (B and D) infected for 6 wk were stained for IL-10 (A and B) or TGF-β Abs. Inserts in A and C focus on parasitized cells from lesions of BALB/c mice that are positive for IL-10 and TGF-β, respectively. Results are representative of studies from three mice. ×400 magnification.

FIGURE 7.

Reduced levels of IL-10 and TGF-β in lesions of FcγR−/− mice. Lesions from BALB/c mice (A and C) and FcγR−/− mice (B and D) infected for 6 wk were stained for IL-10 (A and B) or TGF-β Abs. Inserts in A and C focus on parasitized cells from lesions of BALB/c mice that are positive for IL-10 and TGF-β, respectively. Results are representative of studies from three mice. ×400 magnification.

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Because the central hypothesis of this study is that uptake of opsonized parasites is a stimulus for IL-10 (and TGF-β) production, we also compared L. major infection in BALB/c mice genetically deficient in gene for IL-10 with those lacking the gene for the FcR γ-chain. As can be seen in Fig. 8, IL-10−/− and FcγR−/− mice inoculated with 5 × 103 metacyclic promastigotes developed a similar pattern of infection, and both groups of mice healed their lesions. However, when inoculated with 5 × 104 promastigotes, IL-10−/− mice controlled infection, while FcγR−/− mice developed progressive disease (Fig. 8). Analysis of parasite numbers at week 8 of infection in mice infected with the higher parasite dose revealed a 10 log difference in lesion parasites between IL-10-deficient and FcγR-deficient mice (data not shown). Production of IFN-γ by LN cells from IL-10−/− mice was significantly higher compared with FcγR−/− (data not shown). Together, these results point to important differences in the ability of FcγR−/− and IL-10−/− mice to control infection, and suggest that production of IL-10 by CD4+ T cells in FcγR−/− mice may be critical to their inability to control infection at higher parasite doses.

FIGURE 8.

BALB/c IL-10−/− mice are more resistant to infection than FcγR−/− mice following inoculation of a high dose of parasites. Mice were inoculated with either 5 × 103 or 5 × 104 promastigotes, and the course of infection was followed for 12 wk, as described in Fig. 1. Error bars represent the mean ± SE of five mice per group.

FIGURE 8.

BALB/c IL-10−/− mice are more resistant to infection than FcγR−/− mice following inoculation of a high dose of parasites. Mice were inoculated with either 5 × 103 or 5 × 104 promastigotes, and the course of infection was followed for 12 wk, as described in Fig. 1. Error bars represent the mean ± SE of five mice per group.

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FcγRs are known to regulate accessory and effector cell functions that are required for the induction and expression of both humoral and cell-mediated immunity. Because opsonized Leishmania amastigotes have been shown to enter macrophages via FcRs and Leishmania amastigotes are known to be coated with Ab in vivo (24, 25, 26), it is logical to assume that FcR-mediated events can influence the response to infection. Indeed, a previous study has shown that the in vivo invasion process of Leishmania mexicana complex parasites may be influenced by the absence of FcγR expression on macrophages because Fc γ-chain-deficient mice exhibit delayed and reduced lesion development following infection with L. pifanoi (14). Although parasite numbers were not quantified and the immune response was not analyzed in the L. pifanoi study, it was suggested that a lack of FcγRs could influence either the efficiency of parasite uptake or the production of cytokines by infected macrophages. Either mechanism might result in reduced inflammation and lesion development.

We show in this study that a deficiency in FcγR expression can result in a dramatic alteration in the course of infection by L. major in highly susceptible BALB/c mice. When inoculated with a modest dose of 5,000 metacyclic promastigotes, FcγR−/− mice are able to resolve their infections and are totally resistant to a challenge infection with a high dose of parasites. Even following infection with a higher dose (50,000) of promastigotes, FcγR−/− mice exhibit enhanced resistance, as evidenced by a dramatic reduction in numbers of lesion parasites, although overall lesion size is only modestly reduced. The reduction in parasite numbers within lesions of healing mice cannot be attributed to a reduced ability of parasites to efficiently infect macrophages because lesion parasite numbers are similar in BALB/c and FcγR−/− mice through week 6 of infection. As might be expected, control of infection in FcγR−/− mice is accompanied by reduction in the production of Th2-type cytokines that predominate in BALB/c control mice. We observed that higher numbers of LN cells draining lesions in BALB/c mice produce IL-4 and IL-10 compared with those in FcγR−/− mice, and detected little parasite-specific IgG2a Ab in sera from BALB/c mice compared with significant levels of this IFN-γ-dependent isotype in sera from FcγR−/− mice. Interestingly, healing in FcγR−/− mice was not accompanied by a significant increase in IFN-γ production by LN cells, as assessed by ELISA or by intracellular staining of CD4+ T cells. The absence of increased levels of IFN-γ production in mice lacking the FcR γ-chain suggests that although healing was associated with decreased production of Th2-type cytokines, there was not a dramatic shift to a dominant Th1-type response.

A number of recent studies have shown that uptake of opsonized particles by macrophages can be a strong stimulus for IL-10 production, and FcγR signaling through γ-chain has also been associated with increased IL-10 production by Leishmania infection of bone marrow-derived macrophages (13). Several studies have also demonstrated the critical role of IL-10 in mediating susceptibility and pathogenesis to both cutaneous and visceral leishmaniasis. IL-10-transgenic mice are highly susceptible to progressive L. major disease despite producing IFN-γ (27). BALB/c mice deficient in IL-10 control L. major infection and harbor reduced parasite numbers (13), and IL-10-deficient C57BL/6 mice or C57BL/6 mice treated with anti-IL-10R Ab achieve sterile cure of L. major infection (28). In addition, we have shown that IL-10-deficient mice exhibit a dramatic increase in resistance to infection with Leishmania donovani and L. mexicana complex parasites (29, 30). As is the case with IL-10, a number of studies have shown that neutralization of TGF-β promotes enhanced resistance to both cutaneous and visceral leishmaniasis (31, 32). Although we are unaware of evidence that uptake of opsonized particles is a direct stimulus for macrophages to produce TGF-β, ligation of FcRs has been shown to enhance the production of TGF-β following phagocytosis of apoptotic cells (33). It is also possible that TGF-β and IL-10 influence each other’s production, and it has been shown that TGF-β can enhance IL-10 production in tumor-bearing mice (34). If in vivo uptake of opsonized amastigotes via FcγRs provides a stimulus for macrophages to produce IL-10 and/or TGF-β, we would expect levels of IL-10 and TGF-β to be decreased within parasitized lesions of FcγR−/− mice. Such appears to be the case because levels of staining for IL-10 and TGF-β are clearly reduced in FcγR−/− mice at week 6 of infection. Our observation that parasitized cells stain positively for IL-10 and TGF-β in lesions of BALB/c, but not FcγR−/− mice is consistent with our hypothesis that macrophage uptake of opsonized amastigotes via FcRs provides a stimulus for the production of these anti-inflammatory cytokines.

Although reduced numbers of CD4+ T cells from FcγR−/− mice produce IL-10 and levels of IL-10 are reduced in parasitized lesions, FcγR−/− mice are still more susceptible to infection than IL-10−/− mice, especially when inoculated with a higher dose of parasites. It is possible that IL-10 produced by T cells is sufficient to suppress healing in FcγR−/− mice. Such an idea would be consistent with recent reports showing a clear role for IL-10-producing T regulatory cells in susceptibility to L. major infection (35). It is also possible that at higher doses of parasites, production of elevated levels of Th2-type cytokines such as IL-4 and IL-13 provides a stimulus for macrophages to produce IL-10 because both cytokines have been shown to promote IL-10 production in vitro. We tried to address this question by treating FcγR−/− with Ab to IL-4 during the first week of infection. Although anti-IL-4 treatment promoted healing in FcγR−/− mice inoculated with 50,000 parasites, similar treatment promoted healing in control BALB/c mice. Thus, the question as to why FcγR−/− mice are able to resolve infection with 5,000 parasites, but only partially control infection with 50,000 parasites, remains open.

Although induction of IL-10 (and TGF-β) production by lesion macrophages following uptake of Ab-coated amastigotes is consistent with our data, it is possible that alternative mechanisms could also influence the development of resistance. Studies of L. pifanoi have shown that Ab-deficient JHD mice develop a course of disease similar to that in FcγR−/− mice (14). Although the level of in vivo T cell activation was similar in JHD and BALB/c mice, numbers of monocytes and lymphocytes recruited into lesions were markedly reduced in JHD mice (16). In addition, in vitro studies showed that macrophages from FcγR−/− mice were defective in their ability to activate Leishmania-specific T cells following uptake of opsonized amastigotes. Although it is still possible that uptake of parasites via FcγRs alters the Ag presentation function of macrophages, we did not observe any defect in recruitment of cells into lesions of L. major-infected mice, as evidenced by our observation that lesion development was similar in both wild-type and FcγR−/− mice through week 6 of infection. In addition, examination of histological sections did not reveal differences in the architecture of lesions in the two strains of mice. Curiously, JHD mice infected with L. major, in contrast to L. pifanoi, developed similar levels of infection and a similar course of disease to wild-type BALB/c mice (16). However, these studies used a high dose of parasites (2 million), and it is possible that JHD mice would have proven more resistant at a lower dose of parasites.

Together, our demonstration that FcγR−/− mice can control L. major infection combined with our observation that levels of IL-10 and TGF-β, produced by parasitized cells, are dramatically reduced in lesions of FcγR−/− mice lend credence to in vitro studies that show that uptake of opsonized parasites is a strong stimulus for production of anti-inflammatory cytokines. By implication, our results suggest that Ab production may negatively regulate the development of resistance to leishmaniasis, and suggest that strategies for vaccine development must take into account not only the induction of a strong Th1-type response, but should also minimize the induction of a strong humoral response.

We thank Jacqueline Farracone for performing immunohistochemical staining.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI-27828.

4

Abbreviations used in this paper: LN, lymph node; SLA, soluble leishmanial Ag.

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