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The Journal of Immunology, 2006, 176: 1115-1121.
Copyright © 2006 by The American Association of Immunologists

Impairment of Alternative Macrophage Activation Delays Cutaneous Leishmaniasis in Nonhealing BALB/c Mice1

Christoph Hölscher*,{dagger}, Berenice Arendse*, Anita Schwegmann*, Elmarie Myburgh* and Frank Brombacher2,*

* Institute for Infectious Diseases and Molecular Medicine and Division of Immunology, Health Science Faculty, University of Cape Town, Cape Town, South Africa; and {dagger} Junior Research Group Molecular Infection Biology, Research Center, Borstel, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expressed on various cell types, the IL-4R{alpha} is a component of both receptors for IL-4 and IL-13. Susceptibility of BALB/c mice to Leishmania major is believed to be dependent on the development of IL-4- and IL-13-producing Th2 cells, while IFN-{gamma} secretion by Th1 cells is related to resistance. Despite a sustained development of Th2 cells, IL-4R{alpha}-deficient BALB/c mice are able to control acute cutaneous leishmaniasis, suggesting that IL-4R{alpha}-bearing cells other than Th2 cells contribute to susceptibility. To analyze the contribution of the IL-4R{alpha} on macrophages, recently generated macrophage/neutrophil-specific IL-4R{alpha}-deficient mice on a susceptible BALB/c genetic background were infected with L. major. Strikingly, macrophage/neutrophil-specific IL-4R{alpha}-deficient mice showed a significantly delayed disease progression with normal Th2 and type 2 Ab responses but improved macrophage leishmanicidal effector functions and reduced arginase activity. Together, these results suggest that alternative macrophage activation contributes to susceptibility in cutaneous leishmaniasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leishmania major is the organism that has been most intensely studied and from which the original Th1/Th2 paradigm of immunological development during infectious disease was determined (1). Protective immunity against L. major is dependent on an IL-12-driven cell-mediated immune response mainly characterized by the production of IFN-{gamma} in CD4+ Th1 cells and type 1 Ab isotypes in B cells. IFN-{gamma} in turn mediates protection by inducing NO synthase-2 (NOS-2)3 expression and NO production in classically activated macrophages. In contrast, an IL-4-driven Th2 response and associated cytokines such as IL-13 counterregulate Th1 responses, and consequently, it would be expected that a Th2 response would be detrimental to the outcome of disease. Because susceptible BALB/c mice deficient for IL-4 (2), IL-13 (3), the IL-4R{alpha} (4), or STAT6 (5) are able to contain infection with L. major downstream, IL-4R{alpha}-mediated mechanisms became the center of interest. The IL-4R{alpha} is a common component of the receptor complexes for IL-4 and IL-13. Accordingly, IL-4 and IL-13 have many functional properties in common, including the modulation of Th2 cell development, type 2 Ig class switching in B cells, and inflammatory responses due to the regulation of macrophage functions (6). However, although IL-4R{alpha}–/– BALB/c mice control acute cutaneous leishmaniasis, these mice still develop a Th2 response following infection (7). Thus, IL-4R{alpha}-mediated mechanisms other than inducing Th2 responses must account for disease progression in L. major-infected BALB/c mice. One likely IL-4R{alpha}-mediated mechanism could be the suppression of classical macrophage activation (caM{phi}) after L. major infection.

To distinguish the role of IL-4R{alpha} signaling in specific cellular populations in vivo, we have generated macrophage/neutrophil-specific IL-4R{alpha}–/– (LysMcreIL-4R{alpha}flox/–) mice to be in a position to differentiate IL-4R{alpha}-dependent functions on the cellular level in vivo (8). Macrophages can be activated by different stimuli, with IFN-{gamma} leading to caM{phi} and IL-4/IL-13 resulting in alternative macrophage activation (aaM{phi}) (reviewed in Ref.9). Accordingly, we have recently demonstrated that LysMcreIL-4R{alpha}flox/– mice showed impaired aaM{phi}, which is essential for down-modulation of cell-mediated immune responses, immunopathology, and survival during schistosomiasis (8).

In this study, LysMcreIL-4R{alpha}flox/– mice on a susceptible BALB/c genetic background were infected with L. major to explore a possible role of aaM{phi} in cutaneous leishmaniasis. In contrast to susceptible BALB/c mice, LysMcreIL-4R{alpha}flox/– BALB/c mice showed a significantly delayed disease progression after infection with L. major concomitant with normal Th2 and type 2 Ab immune responses but improved macrophage leishmanicidal activities. These results suggest that alternatively activated macrophages are contributing to the susceptible phenotype in nonhealer BALB/c mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All experimental animals were bred under specific-pathogen-free conditions at the University of Cape Town (Cape Town, South Africa). IL-4R{alpha}–/– (4) and conditional IL-4R{alpha}flox/flox (8) mice were on a pure BALB/c genetic background. To obtain cell-specific IL-4R{alpha}-deficient BALB/c mice, LysMcre mice (10) on an F9 BALB/c genetic background were intercrossed with conditional IL-4R{alpha}flox/flox BALBL/c mice and further mated with complete IL-4R{alpha}–/– BALB/c mice to generate hemizygous LysMcreIL-4R{alpha}flox/– BALB/c mice. LysMcre-negative IL-4R{alpha}flox/– BALB/c mice (referred to as IL-4R{alpha}flox/– BALB/c mice) and C57BL/6 mice were used as wild-type controls. For infection experiments, mice were matched for age and sex and maintained under barrier conditions in the biosafety level 2 facility in individually ventilated cages.

Infection with L. major and histopathology

L. major LV 39 (MRHO/Sv/59/P-strain) was maintained and prepared for infection as described previously (4). Anesthetized mice were infected s.c. into one hind footpad with 2 x 106 stationary phase metacyclic L. major promastigotes. Swelling of infected footpads was measured weekly using a Mitutoyo micrometer caliper. To calculate the parasite burden in the draining popliteal lymph nodes (pLN) from individual mice at different time points, pLN cells were resuspended in 6.4 ml of medium. One hundred microliters of 24, 2-fold serial dilutions were cultured at 26–28°C in microtiter plates containing 50 µl of a solid layer of rabbit blood agar. After 10 days, each individual well was scored microscopically for parasite growth, and the fraction of negative wells per eight wells was determined for each dilution and subjected to statistical analysis for the calculation of minimal estimates of the number of viable L. major per lymph node by applying Poisson statistics and the {chi}2 minimization method as described (11). For histopathological analysis, the infected footpad and a sample of liver were taken at different time points after infection and processed as described elsewhere (4).

Determination of Ab isotypes

Blood was collected and serum was prepared using serum separator tubes (BD Biosciences). Ag-specific IgG1, IgG2a, and IgG2b were quantified by ELISA, as previously described (7). Detection limits were 5 ng/ml for IgG1 and IgG2b and 0.1 ng/ml for IgG2a and IgG3. Total IgE was determined as described (4). The detection limits were 5 ng/ml for IgG1 and IgG2b, 0.1 ng/ml for IgG2a and IgG3, and 8 ng/ml for IgE.

Restimulation assays

Single-cell suspensions from draining pLNs were isolated by straining through a metal sieve. Without any additional stimulus, peritoneal exudate cells were harvested by flushing the peritoneal cavity of infected mice with medium. After depletion of erythrocytes, cells were resuspended in complete IMDM (Invitrogen Life Technologies) supplemented with 10% FCS (Invitrogen Life Technologies), 0.05 mM 2-ME (Sigma-Aldrich), and penicillin and streptomycin (100 U/ml and 100 µg/ml; Invitrogen Life Technologies), and cultured in triplicates at 2 x 106/ml in 48-well plates. Restimulation was performed with L. major Ag (106/ml inactivated parasites) or LPS (Sigma-Aldrich; 15 ng/ml). After 48 h, the content of IFN-{gamma}, IL-4, and NO was quantified in culture supernatants (8). Arginase activity was measured in cell lysates as described previously (12). Briefly, cells were lysed with 50 µl of 0.1% Triton X-100 (Sigma-Aldrich). After 30 min on a shaker, 50 µl of 10 mM MnCl2 (Merck), 50 mM Tris-HCl (Merck) were added, and the enzyme was activated by heating for 10 min at 55°C. Arginine hydrolysis was conducted by incubating 25 µl of the activated lysate with 25 µl of 0.5 M L-arginine (Merck; pH 9.7) at 37°C for 60 min. The reaction was stopped with 400 µl of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). As a degree of arginase activity, the urea concentration was measured at 540 nm after addition of 25 µl of {alpha}-isonitrosopropiophenone (Sigma-Aldrich; dissolved in 100% ethanol) followed by heating at 95°C for 45 min. One unit of arginase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol urea/min.

Infection of bone marrow-derived macrophages (BMM{phi})

BMM{phi} were generated and triplicate cultures were infected with L. major promastigotes in a parasite-to-cell ratio of 10:1 as published (13). After 24 h, extracellular parasites were removed and cells were incubated for 16 h with medium or IL-4 (BD Biosciences; 1000 U/ml). Macrophages were subsequently stimulated with IFN-{gamma} (BD Biosciences; 100 U/ml) and LPS (15 ng/ml). After 48 h, supernatants were collected for quantification of NO, and the amount of parasites was determined as described (13).

Statistics

Data are expressed as means of individual determinations and SDs. Statistical analysis was performed using unpaired Student’s t test defining differences to IL-4R{alpha}flox/– mice as significant (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To understand IL-4R{alpha}-mediated mechanisms in distinct cell types leading to suppression of protective immune responses during cutaneous leishmaniasis, macrophage/neutrophil-specific IL-4R{alpha}-deficient (LysMcreIL-4R{alpha}flox/–) BALB/c mice were infected with 2 x 106 L. major metacyclic promastigotes (Fig. 1). Healing C57BL/6 mice initially showed a transient and moderate footpad swelling but generated resistance to L. major infection (Fig. 1a) with low amounts of parasites in draining pLN (Fig. 1b). Susceptible nonhealing IL-4R{alpha}flox/– BALB/c mice developed progressive footpad swelling (Fig. 1a) and showed an enhanced parasite load in draining pLN (Fig. 1b) accompanied by ulceration and necrosis requiring termination of the experiment 11 wk after infection (Fig. 1c). In contrast, IL-4R{alpha}–/– BALB/c mice controlled acute L. major infection as previously shown (4) with moderate footpad swelling (Fig. 1a) and significantly reduced parasite burden in draining pLN (Fig. 1b) in the absence of ulceration and necrosis (Fig. 1c). Significantly, LysMcreIL-4R{alpha}flox/– BALB/c mice showed an increased resistance, compared with susceptible IL-4R{alpha}flox/– BALB/c mice (and BALB/c mice, data not shown) with a similar moderate footpad swelling as observed in IL-4R{alpha}–/– BALB/c mice for up to 13 wk after infection (Fig. 1a). Importantly, the parasite load in draining pLN from LysMcreIL-4R{alpha}flox/– BALB/c mice was significantly reduced 9 wk after infection with L. major, compared with susceptible IL-4R{alpha}flox/– BALB/c control mice (Fig. 1b). Similar results were found in the footpad at week 6 postinfection (p.i.; data not shown). Thereafter, LysMcreIL-4R{alpha}flox/– BALB/c mice developed disease progression with increasing footpad swelling accompanied by ulceration and necrosis (Fig. 1c) as well as elevated parasite burden in draining pLN (Fig. 1b) requiring termination of the experiment only at week 18. Histopathological analysis at this late time point demonstrated a similar inflammatory response in infected tissue accompanied with necrosis, bone destruction and inflammatory foci around disseminated pathogens in other organs as observed in IL-4R{alpha}flox/– BALB/c mice 9 wk earlier (Fig. 1c). These data reveal that IL-4R{alpha}-mediated mechanisms in macrophages are involved in the development of early disease progression after L. major infection.



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  		FIGURE 1. L. major infection. Experimental mice were s.c. infected into one hind footpad with L. major. a, Footpad swelling was calculated as the difference between the infected and the uninfected footpad. Data represent means and SDs of eight mice per group. b, After 9 and 18 wk p.i., the parasite burden in draining pLN was determined by 2-fold limiting dilutions of single-cell suspensions. c, At the indicated time points (#), mice were killed and samples from infected footpads and liver were processed for histopathology (insets, parasites; arrows, inflammatory foci). One experiment representative of three performed is shown. Statistical analysis was performed using unpaired Student’s t test defining differences to IL-4R{alpha}flox/– (*, p ≤ 0.05; ***, p ≤ 0.001) or IL-4R{alpha}–/– (#, p ≤ 0.05) BALB/c mice as significant.

 
As Th2 and type 2 Ab immune responses were originally implicated with the fatal outcome of cutaneous leishmaniasis (2), we addressed whether delayed disease progression in LysMcreIL-4R{alpha}flox/– BALB/c mice was accompanied by an altered Th1/Th2 and type1/type2 Ab immune response. Therefore, we analyzed humoral (Fig. 2) and cellular (Fig. 3) immune responses after infection with L. major. As determined by ELISA, 9 wk after infection with L. major, Leishmania-specific Ab isotypes detected in sera of infected LysMcreIL-4R{alpha}flox/– BALB/c mice were similar to those observed in susceptible control IL-4R{alpha}flox/– BALB/c mice (Fig. 2), showing dominant type 2 Ab responses (IgG1 and IgE). In contrast, IL-4R{alpha}–/– BALB/c mice developed a dominant type 1 isotype response (IgG2a, IgG2b). After Ag-specific restimulation, lymphocytes from IL-4R{alpha}flox/–, IL-4R{alpha}–/–, and LysMcreIL-4R{alpha}flox/– BALB/c mice expressed comparable amounts of IL-4 (Fig. 3). LysMcreIL-4R{alpha}flox/– BALB/c mice, however, produced a marked IFN-{gamma} response, which was also observed in healing C57BL/6 mice but not in susceptible IL-4R{alpha}flox/– or IL-4R{alpha}–/– BALB/c mice (Fig. 3). This may suggest that proinflammatory macrophages may promote Th1 responses mediating protection in experimental cutaneous leishmaniasis.



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  		FIGURE 2. Ab response after infection with L. major. Experimental mice were s.c. infected with L. major, and specific IgGs and total IgE Abs were quantified 9 wk p.i. Data represent means and SDs of eight mice per group. One experiment representative of three performed is shown.

 


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  		FIGURE 3. Th cytokine response in L. major-infected mice. Experimental mice were s.c. infected with L. major, and cells from draining pLN were incubated with medium or L. major Ag 9 wk p.i. The production of IFN-{gamma} and IL-4 was determined after 48 h. Data represent means and SDs of triplicate cultures. One experiment representative of two performed is shown. Statistical analysis was performed using unpaired Student’s t test defining differences to IL-4R{alpha}flox/– BALB/c mice as significant (**, p ≤ 0.01; ***, p ≤ 0.001).

 
caM{phi} plays an important role in combating infection with L. major through IFN-{gamma}-induced expression of effector molecules such as NOS-2-dependent NO and other reactive nitrogen intermediates (RNI) (14, 15, 16). This induction is counterregulated by IL-4R{alpha}-dependent mechanisms, leading to aaM{phi} and the induction of arginase I. Because IL-4R{alpha}-induced arginase I hydrolyzes L-arginine to urea and L-ornithine, this IL-4R{alpha}-mediated pathway regulates NO production in macrophages through depletion of L-arginine as the substrate for NOS-2 (17, 18). Therefore, we speculated that, in the absence of IL-4R{alpha}-mediated arginase I activity, the production of NO was increased in macrophages from L. major-infected LysMcreIL-4R{alpha}flox/– BALB/c mice. Indeed, 9 wk after infection C57BL/6, IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BALB/c macrophages showed a significantly reduced arginase activity (Fig. 4a) after restimulation with LPS, compared with susceptible IL-4R{alpha}flox/– BALB/c mice. Due to the absence of IL-4R{alpha}-dependent competition with arginase I, our results suggest a reduced aaM{phi} in IL-4R{alpha} mutant mouse strains. Vice versa, macrophages from C57BL/6, IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BALB/c mice produced significantly increased amounts of NO in response to LPS, compared with susceptible IL-4R{alpha}flox/– BALB/c mice (Fig. 4b), suggesting that classical macrophage effector mechanisms were enhanced in the absence of IL-4R{alpha} responsiveness in macrophages.



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  		FIGURE 4. aaM{phi} vs caM{phi} in L. major-infected mice. Experimental mice were s.c. infected with L. major, and cells from the peritoneal cavity or draining pLN were isolated 9 wk p.i. Adherent peritoneal cells and draining pLN were restimulated with LPS, and the (a) arginase activity and (b) production of NO were determined after 48 h. Data represent means and SDs of four mice per group, respectively. One experiment representative of two performed is shown. Statistical analysis was performed using unpaired Student’s t test defining differences to IL-4R{alpha}flox/– BALB/c mice as significant (**, p ≤ 0.01; ***, p ≤ 0.001).

 
In accordance with biased caM{phi} in the absence of the IL-4R{alpha} on macrophages, LysMcreIL-4R{alpha}flox/– BALB/c mice should be able to better control L. major infection. Together, IL-4R{alpha}-mediated mechanisms appear to directly suppress leishmanicidal effector functions in macrophages and may therefore contribute to the enhanced susceptibility in nonhealer BALB/c mice. To evaluate this IL-4R{alpha}-dependent suppression of macrophage effector mechanisms, we conducted a leishmanicidal assay in BMM{phi} from IL-4R{alpha}flox/–, IL-4R{alpha}–/–, and LysMcreIL-4R{alpha}flox/– BALB/c mice (Fig. 5). After in vitro infection with L. major promastigotes, BMM{phi} were preincubated with medium or IL-4 and subsequently stimulated with IFN-{gamma}/LPS. Whereas BMM{phi} from all groups were able to eliminate intracellular Leishmania in response to IFN-{gamma}/LPS, parasite elimination was strikingly inhibited by IL-4 in IL-4R{alpha}flox/– BMM{phi}. In contrast, in vitro IFN-{gamma}/LPS-mediated leishmanicidal activity was unaffected by the addition of IL-4 in infected IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BMM{phi} (Fig. 5). Moreover, IL-4R{alpha}-mediated suppression of leishmanicidal effector functions in BMM{phi} from IL-4R{alpha}flox/– mice correlated with a decreased production of IFN-{gamma}/LPS-induced NO. In contrast, NO production by IFN-{gamma}/LPS-stimulated and L. major-infected IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– macrophages could not be inhibited by IL-4 (Fig. 5). Together, our data provide conclusive evidence that alternatively activated macrophages were not able to express a leishmanicidal effector and may therefore accelerate the development of cutaneous leishmaniasis in nonhealer BALB/c mice.



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  		FIGURE 5. Leishmanicidal macrophage effector functions. To assess IL-4R{alpha}-mediated suppression of leishmanicidal macrophage effector functions, BMM{phi} from uninfected mice were incubated with medium or IL-4, infected with L. major and stimulated with IFN-{gamma}/LPS. After 48 h, NO production and the leishmanicidal activity in macrophages were determined. Data represent means and SDs of triplicate cultures. One experiment representative of two performed is shown. Statistical analysis was performed using unpaired Student’s t test defining differences to IL-4R{alpha}flox/– mice as significant (**, p ≤ 0.01; ***, p ≤ 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The differentiation of naive Th cells to cytokine-expressing effector cells is important for the orchestration of immunity. Th1 and Th2 cells, which express the signatory (or archetypal) cytokines IFN-{gamma} and IL-4, respectively, mediate the release of type 1 or type 2 Ab isotypes by B cells and promote the defense against distinct types of antigenic challenges, with Th1 cells more typically involved in systemic, and Th2 cells involved in mucosal, immunity. Murine L. major infection remains the primary model for investigation of Th subset development because resistance and susceptibility have been definitively linked to Th cell differentiation; resistant healing mice generate a protective Th1 response, whereas susceptible mice develop a nonprotective Th2 response (19). Nevertheless, despite the usefulness of this model, the paradigm of Th1-mediated protection vs Th2-dependent susceptibility remains elusive because a number of alternative mechanisms to account for nonhealing disease have been postulated. Particularly, studies using neutralizing anti-IL-4 Abs or IL-4–/– mice revealed that the role of IL-4 for susceptibility to L. major infection is far more complex. Although neutralization of IL-4 promotes resistance in BALB/c mice, the development of a healing phenotype in these mice appears not to be dependent on IFN-{gamma}, whereas resistance in C57BL/6 mice strictly depends on IFN-{gamma} (20). Therefore, mechanisms mediating a nonhealing phenotype may be different in BALB/c and C57BL/6 mice. The outcome of L. major infection in IL-4–/– BALB/c mice has also been a controversial subject. Whereas Noben-Trauth et al. (21) have shown that IL-4–/– BALB/c mice develop progressive lesions and could not contain parasites, Kopf et al. (2) reported that IL-4–/– mice are able to resist infection. The use of parasite strains of different virulence may have partially accounted for this contrasting outcome of infection in IL-4–/– mice (22). Importantly, studies in IL-4R{alpha}–/– BALB/c mice which are, despite a profound Th2 and type 2 Ab response, resistant to L. major infection, suggest that IL-4R{alpha}-bearing cells other than lymphocytes are involved in IL-4R{alpha}-dependent susceptibility (7). Moreover, comparative infection studies using IL-4–/– and IL-4R{alpha}–/– or IL-13–/– and IL-4/IL-13–/– mice demonstrated that IL-13 contributes to disease progression in BALB/c mice during L. major (3, 4) and Leishmania mexicana infection (23). As murine lymphocytes are not responsive to IL-13, other cell types would appear to be involved in these functions. To understand IL-4R{alpha}-mediated mechanisms in distinct cell types leading to suppression of protective immune responses during cutaneous leishmaniasis, macrophage/neutrophil-specific IL-4R{alpha}-deficient (LysMcreIL-4R{alpha}flox/–) BALB/c mice were infected with L. major. Significantly, LysMcreIL-4R{alpha}flox/– BALB/c mice showed an increased resistance, compared with susceptible IL-4R{alpha}flox/– BALB/c mice for up to 13 wk after infection. However, although disease progression was significantly delayed in LysMcreIL-4R{alpha}flox/– BALB/c mice, macrophage/neutrophil-specific IL-4R{alpha}-deficient BALB/c mice became eventually susceptible, whereas complete IL-4R{alpha}-deficient BALB/c mice remained resistant to infection with L. major. Therefore, our data reveal that IL-4R{alpha}-mediated mechanisms in macrophages are involved in the development of early disease progression after L. major infection. In contrast, IL-4/IL-13 responsiveness in cells different from macrophages/neutrophils appear to promote the outcome of disease in susceptible BALB/c mice during later stages of experimental leishmaniasis. Because the expression of IL-10 is impaired in CD4+ T cells from L. major-infected IL-4–/– mice (2), one explanation for the transient effect of IL-4R{alpha} deficiency in neutrophils/macrophages after L. major infection could be that the IL-4-driven production of immunosuppressive cytokines by CD4+ T cells abolished the initial protective immune responses in LysMcreIL-4R{alpha}flox/– BALB/c mice. However, we could not find significant differences between infected IL-4R{alpha}flox/–, IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BALB/c (data not shown).

IL-4- and IL-4R{alpha}-mediated signaling have been implicated in a variety of experimental systems in the development of Th2 and type 2 Ab immune responses indicated by the development of IL-4-producing CD4+ Th2 cells and a distinct profile of Ab isotype secretion by B cells (IgG1 and IgE) (24, 25). In contrast, Th1 and type 1 Ab responses are characterized by IFN-{gamma}-producing CD4+ Th1 cells and a different Ab isotype profile (IgG2a and IgG2b). As Th2 and type 2 Ab immune responses were originally implicated in the fatal outcome of cutaneous leishmaniasis (2), we addressed whether delayed disease progression in LysMcreIL-4R{alpha}flox/– BALB/c mice was accompanied by an altered Th1/Th2 and type1/type2 Ab immune response. Interestingly, L. major-infected LysMcreIL-4R{alpha}flox/– BALB/c mice displayed a mixed Th1/Th2 immune response. Whereas the type 2 Ab isotype profile was similar to IL-4R{alpha}flox/– BALB/c mice, lymphocytes from LysMcreIL-4R{alpha}flox/– BALB/c mice apparently developed a Th1/Th2 phenotype. A profound IL-4 response to L. major Ag was present in all mutant strains but not in C57BL/6 mice, confirming our previous data that Th2 development and the production of type 2 Abs in cutaneous leishmaniasis is independent of the IL-4R{alpha} (7). Other factors, like IL-10 or TGF-{beta} could also have been involved in the observed immune modulation (26, 27), even so, we did not find obvious differences during the analysis (data not shown). Increased Th1-mediated IFN-{gamma} production in LysMcreIL-4R{alpha}flox/– BALB/c mice compared with complete IL-4R{alpha}–/– BALB/c mice may indicate that IL-4R{alpha} responsiveness is necessary for optimal IFN-{gamma} production. This suggests that caM{phi} may promote Th1 responses as previously found in Schistosoma mansoni-infected LysMcre IL-4R{alpha}flox/– BALB/c mice (16). Because IL-4R{alpha} engagement suppresses IL-12 production in macrophages (8), macrophage-specific IL-4R{alpha} deficiency may uncover IL-4R{alpha}-dependent proinflammatory mechanisms such as the IL-4-mediated instruction of dendritic cells to produce IL-12 which has been shown to promote Th1 cell development and resistance in L. major-infected BALB/c mice (28). However, we did not consistently find increased IL-12 in infected LysMcreIL-4R{alpha}flox/– BALB/c mice (data not shown). Factors produced by alternatively activated macrophages may also be directly involved in the regulation of Th1 immune responses. Accordingly, we recently identified reactive oxygen intermediates and 12/15 lipoxygenase to be suppressive molecules produced by alternatively activated myeloid cells capable of inhibiting T cell proliferation (29).

caM{phi} plays an important role in combating infection with L. major through IFN-{gamma}-induced expression of effector molecules such as NOS-2-dependent production of RNI (14, 15, 16). Importantly, inhibition of endogenous NOS-2 reactivates latent leishmaniasis in resistant mouse strains during the whole course of infection, indicating that NOS-2-dependent mechanisms in macrophages are crucial for the control of L. major persisting in immunocompetent hosts after resolution of the primary infection (30). The production of RNI is counterregulated by IL-4R{alpha}-dependent mechanisms, leading to arginase I-expressing alternatively activated macrophages. Because NOS-2 shares L-arginine as a substrate with arginase I, substrate depletion by either enzyme is a key regulatory mechanism in macrophages and differential expression of NOS-2 and arginase 1 is important for regulating macrophage effector functions (17, 18). Accordingly, in contrast to macrophages from L. major-infected susceptible IL-4R{alpha}flox/– BALB/c mice, macrophages from infected resistant C57BL/6, IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BALB/c mice showed biased classical activation phenotypes, suggesting that classical macrophage effector mechanisms were enhanced in the absence of IL-4R{alpha} responsiveness. This was directly shown after LPS restimulation as arginase activity was reduced and NO production was increased in macrophages from infected C57BL/6, IL-4R{alpha}–/–, and LysMcreIL-4R{alpha}flox/– BALB/c mice, compared with macrophages from infected IL-4R{alpha}flox/– BALB/c mice. Although we have not directly addressed the expression of NOS-2 and arginase I, our results indicate that caM{phi} from IL-4R{alpha}–/– and LysMcreIL-4R{alpha}flox/– BALB/c mice were able to efficiently express NOS-2-dependent leishmanicidal effector mechanisms in the presence of IL-4 possibly due to impaired IL-4R{alpha}-mediated arginase I activities. In line with our findings are recent published results which postulated a role of aaM{phi} and arginase I expression for susceptibility to experimental L. major infection (31, 32). In the absence of endogenous IL-9, increased resistance of BALB/c mice to infection with L. major was accompanied with a switch from alternative to caM{phi} (32). Vice versa, arginase I activity in alternatively activated macrophages from TLR 4-deficient C57BL/10 ScSn mice was coincident with an increased susceptibility to L. major infection (31). The most striking evidence that arginase does play an essential role in mediating susceptibility to infection with L. major came from a recent report in which neutralization of endogenous arginase with N-hydroxy-nor-L-arginine ameliorated disease progression in BALB/c mice (33). Importantly, IL-4 was shown to mediate arginase I expression and to promote elevated parasite growth in macrophages dependent on arginase activity and the arginase-dependent production of polyamines. Therefore, in our study, IL-4-mediated suppression of leishmanicidal effector mechanisms in alternatively activated macrophages from IL-4R{alpha}flox/–BALB/c mice was likely due to the induction of arginase I as originally described by Kropf et al. (33). This suppressive effect of IL-4-induced arginase activity could on the one hand lead to the reduced RNI synthesis by substrate depletion. On the other hand, IL-4-dependent production of polyamines through the enzymatic activity of arginase could directly promote parasite growth. In contrast, other studies have shown that, in the absence of LPS, IL-4 can synergize with IFN-{gamma} for the intracellular elimination of L. major in a TNF- and RNI-dependent manner (34, 35). Different incubation protocols could have been responsible for these discrepancies. Whereas coincubation of IL-4 and IFN-{gamma} results in a synergistic induction of RNI (34, 35), preincubation of IL-4 down-regulates NO production (8, 36). In contrast to the mentioned in vitro studies, LysMcreIL-4R{alpha}flox/– BALB/c mice enabled us to analyze the impact of IL-4R{alpha}-mediated signaling in macrophages on the outcome of experimental leishmaniasis in vivo. Our studies revealed that, after infection with L. major, a switch from aaM{phi} to caM{phi} results in an increased production of RNI followed by a delayed disease progression in LysMcreIL-4R{alpha}flox/– BALB/c mice. A similar increased production of RNI accompanied with an impairment in aaM{phi} was recently shown by us in S. mansoni-infected LysMcreIL-4R{alpha}flox/– BALB/c mice (8). However, as NOS-2-dependent production of RNI is crucial for controlling latent L. major infection during later stages of the disease (30) and because suppression by IL-4 appears to be effective only at the initial stages of RNI induction (36), an only transiently increased RNI production in LysMcreIL-4R{alpha}flox/– BALB/c mice could have delayed leishmaniasis in these mice. In contrast, increasing arginase I production in IL-4R{alpha}-responsive cells different from macrophages/neutrophils such as fibroblast and dendritic cells (37) may also have generated more favorable conditions for the parasite and have contributed to the delayed disease progression in L. major-infected LysMcreIL-4R{alpha}flox/– BALB/c mice. In addition to macrophages, neutrophils in LysMcreIL-4R{alpha}flox/– BALB/c mice also display a deficient IL-4R{alpha} expression (8). Although the protective role of macrophage effector functions during cutaneous leishmaniasis is well established (14, 15, 16), only conflicting data on the impact of neutrophils on resistance to L. major infection has been published (38, 39). Together, it seems unlikely that IL-4R{alpha} responsiveness on neutrophils does have a decisive role in the late outcome of L. major infection.

From our data, we conclude that impairment of IL-4R{alpha}-mediated signaling in BALB/c macrophages is responsible for a delayed disease progression in LysMcreIL-4R{alpha}flox/– BALB/c mice by promoting caM{phi} and their leishmanicidal effector functions. aaM{phi} and subsequent arginase I activity is, as our study showed, rather of advantage for the parasite and may be part of its evolved evasion mechanisms. This hypothesis is also substantiated by the fact that Leishmania itself expresses endogenous arginase I, which plays a pivotal role in polyamine precursor metabolism and is essential for parasite survival as recently shown using arginase I-deficient parasites (40). Second, it has been demonstrated that the parasite is able to exploit the host’s IL-4R{alpha}-dependent arginase I-mediated polyamine metabolism (33). Overall, our data show evidence that aaM{phi} is disadvantageous for host protection against leishmaniasis, as it prevents efficient Th1 and type 1 Ab responses and suppresses macrophage effector functions. However, aaM{phi} can also modulate Th1/type1 responses to prevent excessive inflammation to ensure host survival during infection such as acute schistosomiasis (8). In conclusion, aaM{phi} limits the pathological sequelae of excessive inflammation but may also prevent optimal antimicrobial protection.


   Acknowledgments
 
We thank A. Hölscher, R. Peterson, M. Simpson, W. Dwyer, E. Smith, L. Fick, and all the animal facility staff for excellent technical assistance. We thank Drs. J. Alexander, R. Guler, T. Cutler, D. Herbert, and E. von Stebut for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Medical Research Council and National Research Foundation of South Africa. F.B. is the holder of a Wellcome Trust Research Senior Fellowship for Medical Science in South Africa (Grant No. 056708/Z/99). Back

2 Address correspondence and reprint requests to Dr. Frank Brombacher, Institute for Infectious Diseases and Molecular Medicine and Division of Immunology, Health Science Faculty, University of Cape Town, Werner Beith South Building, Anzioroad, Observatory 7925, Cape Town, South Africa. E-mail address: fbrombac{at}uctgsh1.uct.ac.za Back

3 Abbreviations used in this paper: NOS-2, NO synthase-2; caM{phi}, classical macrophage activation; aaM{phi}, alternative macrophage activation; pLN, popliteal lymph node; BMM{phi}, bone marrow-derived macrophage; p.i., postinfection; RNI, reactive nitrogen intermediate. Back

Received for publication March 30, 2005. Accepted for publication November 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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