The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178, 7251 -7258
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nigg, A. P.
Right arrow Articles by von Stebut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nigg, A. P.
Right arrow Articles by von Stebut, E.

Dendritic Cell-Derived IL-12p40 Homodimer Contributes to Susceptibility in Cutaneous Leishmaniasis in BALB/c Mice1

Axel P. Nigg2,*, Sabine Zahn2,*, Dominik Rückerl{dagger}, Christoph Hölscher{dagger}, Takayuki Yoshimoto{ddagger}, Jan M. Ehrchen§, Florian Wölbing*, Mark C. Udey and Esther von Stebut3,*

* Department of Dermatology, Johannes Gutenberg-University, Mainz, {dagger} Forschungszentrum Borstel, Germany; {ddagger} Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan; § Department of Dermatology, Münster, Germany; and Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1{alpha}beta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released ~2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1{alpha}beta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In murine experimental leishmaniasis, the development of genetically determined Th responses is observed (1). Protective, healing immune responses against Leishmania are dependent on effective IFN-{gamma} release mainly from Th1 CD4+ T cells (2), but also from Tc1 CD8+ T cells (3). Infected macrophages are subsequently activated by IFN-{gamma} that enables them to kill intracellular organisms. Most mouse strains (including C57BL/6 mice) develop Th1 immune responses associated with limited disease followed by lifelong immunity. In contrast, due to the inability to mount protective Th1 immunity, BALB/c mice experience progressive lesion development, dissemination of parasites, and ultimately succumb to infection. This experimental model has been used extensively to characterize factors determining Th1/Th2 development. Several differences at the level of T cells have been associated with disease outcome: e.g., increased numbers of Leishmania homologue of receptors for activated C-kinase 2-reactive, IL-4-producing CD4+ T cells as well as an early IL-4-mediated down-regulation of the IL-12R beta2 chain on Th2 cells during the course of infection in BALB/c mice (for a review see Ref. 1).

However, in addition to CD4+ T cells, myeloid cells such as macrophages and dendritic cells (DC)4 have recently been described to influence the outcome of cutaneous leishmaniasis in susceptible BALB/c mice (4, 5). DC contribute to the development of protective immunity against intracellular pathogens (e.g., Leishmania) by initiating Leishmania-specific T cell priming and by directing Th development toward a Th1 phenotype. Particularly, the production of IL-12p70 by DC appears to be a prerequisite for generating optimal Th1 responses and plays a pivotal role in promoting cell-mediated immunity against intracellular pathogens. IL-12p70 is a heterodimeric cytokine composed of a p35 and a p40 subunit. However, beside dimerization with the p35 subunit to IL-12p70, p40 also dimerizes with an IL-12p35-related subunit, p19, to form a novel cytokine IL-23 (6). Additionally, IL-12p40 subunits also form disulfide-linked homodimers among each other (IL-12(p40)2 or IL-12p80), which exhibit mainly antagonistic functions at the IL-12R (7, 8, 9).

We and others have previously shown that differential production of IL-1{alpha}beta by DC provided a partial explanation for disparate disease outcome in Leishmania infections (10, 11, 12). The present study was initiated to attempt to identify additional DC-derived cytokines responsible for directing Th differentiation in cutaneous leishmaniasis. Specifically, our previous studies revealed that susceptible BALB/c DC produce more IL-12p40 than resistant C57BL/6 cells, whereas the release of IL-12p70 was comparable in both strains. Because IL-12p40 can be released as monomeric IL-12p40, as inhibitory homodimer, or as bioactive heterodimeric IL-12p70 (7), we analyzed the differential production of IL-12p40- containing cytokines by Leishmania major-infected DC from these susceptible and resistant mouse strains. Interestingly, we found that BALB/c DC released significantly more IL-12p80 than C57BL/6 cells both in vitro and in vivo. We propose that, in BALB/c mice, the presence of increased levels of inhibitory IL-12p80 during T cell priming might inhibit protective Th1 priming. Genetically determined increased production of IL-12p80 as well as decreased IL-1{alpha} levels by infected DC may therefore contribute to progressive disease in susceptible BALB/c mice infected with L. major.


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

Six- to 8-wk-old BALB/c, C57BL/6, or IL-12p40 transgenic (Tg) mice (C57BL/6 background) (13) were obtained from the specific pathogen-free facility of the Central Animal Facility Production Program of the University of Mainz. All animals were housed in accordance with institutional and federal guidelines. All experiments were undertaken with an approved license from the Animal Care and Use Committee of the Rheinland-Pfalz Region.

Cells and parasites

Bone marrow-derived DC (BMDC) were generated as described previously (14). Bone marrow cells were plated at 2 x 106 cells/ml medium (RPMI 1640, 5% FCS) supplemented with 10 ng/ml GM-CSF (PeproTech) ±10 ng/ml IL-4 (PeproTech) in 24-well plates, and medium was partially replaced every 48 h. On day 6, nonadherent immature BMDC were harvested. Purity was determined by assessing the number of CD11c+ MHC class II+ cells by flow cytometry (10, 15). Metacyclic promastigotes or amastigotes of L. major clone VI (MHOM/IL/80/Friedlin) were prepared as described previously (12). Isolated parasites were opsonized with 5% BALB/c normal mouse serum and washed before in vitro or in vivo infections.

Quantitation of cytokine production by DC in vitro

For stimulation, BMDC were plated at 2 x 105 or 2 x 106/ml in their basal medium for 18 h and LPS (100 ng/ml; Sigma-Aldrich), IFN-{gamma} (1,000 U/ml; Peprotec/Tebu), or parasites (parasite:cell ratio of 3:1 or 10:1) were added as indicated. Cytokine accumulation in 18- or 72-h supernatants was measured using ELISA kits specific for murine IL-12p40, IL-12p70, and TNF-{alpha} (BD Pharmingen).

Detection of IL-12p40 homodimer

Aliquots of DC supernatants were stored at –20°C and assayed for the presence of IL-12p80 by Western blot and ELISA. For Western blots, supernatants were mixed with nonreducing loading buffer and resolved on 7% SDS polyacrylamide gels. Aliquots of Blue Pre-Stained Standards (Sigma-Aldrich) were included on each gel. Proteins were transferred to polyvinylidene difluoride-membranes (Millipore) by semidry blotting (Bio-Rad) in blotting buffer (Roti-Blot A, Roti-Blot K; Roth) according to the manufacturer’s instructions. Membranes were blocked with 5% BSA/0.05% Tween 20 in PBS overnight at 4°C. IL-12 polypeptides were detected by incubation with biotinylated anti-IL-12p40 mAb (clone C17.8 (BD Pharmingen), 0.25 µg/ml in 1% BSA with 0.05% Tween 20 in PBS) for 2 h at 4°C, followed by incubation with streptavidin-HRP conjugate (156 ng/ml; Zytomed) diluted in 1% BSA with 0.05% Tween 20 in PBS for 1 h at 4°C. The membranes were then developed using chemiluminescence (ECL Plus; Amersham Biosciences). Band sizes of 80 kDa are shown.

IL-12p80 protein was quantitated by ELISA. Flat-bottom 96-well plates (Nunc) were coated overnight with anti-mouse IL-12p40 (clone 17.8 (Caltag Laboratories), 0.4 µg/ml in carbonate buffer), blocked for 1 h with PBS/2% BSA/0.05% Tween 20, and incubated for 2 h with dilutions of supernatants or sera or rIL-12p80 (R&D Systems) as a standard. Subsequently, biotinylated anti-mouse IL-12p40 (clone 17.8; BD Pharmingen) was added (0.2 µg/100 µl) for 1 h at room temperature. ELISA plates were developed using commercially available ELISA kit components (BD Biosciences), and reaction products were quantified spectrophotometrically. To determine the specificity of the IL-12p80 ELISA, we used the standard for IL-12p40, IL-12p70 (both obtained from R&D Systems), and IL-23 (Natutec) as provided in their respective ELISA kits.

Western blotting for STAT-4

A total of 2 x 106 Con A-activated splenocytes from C57BL/6 mice was cultured in IMDM (PAA Laboratories) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all obtained from Biochrom) and incubated with medium, rIL-12p70 (1 ng/ml; PeproTech), and rIL-12p80 (1 ng/ml; R&D Systems). After 40 min, cells were washed with PBS, lysed in a buffer containing protease inhibitors, and resultant cell lysates were subjected to Western blot analysis with anti-STAT-4 and anti-phospho-STAT-4 Abs (Zymed Laboratories). Detection was conducted using IRDye800-conjugated goat anti-rabbit Abs (Rockland) and visualized with the Li-Cor detection system (Li-Cor Biosciences).

Effect of IL-12p80 on in vitro Th1 cell differentiation

CD4+ T cells from C57BL/6 lymph nodes (LN) were cultured at 2 x 105 cells/flat-bottom well precoated with anti-CD3 (clone 145-2C11, 10 µg/ml) and anti-CD28 (clone 37/51, 10 µg/ml) and cultured in complete IMDM supplemented with IL-2 (50 U/ml; PeproTech). Th1 development was driven by IL-12 (1 ng/ml; PeproTech) and anti-IL-4 mAb (clone 11B11, 10 µg/ml). Medium or IL-12p80 (R&D Systems) was added at various concentrations. After 72 h, cells were washed extensively, transferred to fresh wells, and cultured for 24 h in the presence of IL-2 (50 U/ml). Finally, cells were transferred to wells precoated with anti-CD3. IFN-{gamma} levels in cell-free supernatants were detected in 3-fold serial dilutions by an IFN-{gamma}-specific ELISA (BD Biosciences) after 48 h.

Infections

Groups of mice were infected intradermally into ear skin with 2 x 105 infectious stage metacyclic promastigotes (12, 15). In some experiments, mice were euthanized in wk 3, and DC from draining submandibular LNs were positively selected using anti-CD11c microbeads (Miltenyi Biotec) and plated at 1 x 106 cells/200 µl in RPMI 1640/5% FCS. Supernatants were harvested after 18 h and analyzed for the presence of IL-12p40 and IL-12p80 by ELISA as described above.

In other experiments, murine IL-12p70 or IL-12p80 (both R&D Systems) were diluted in serum-free PBS and injected intradermally on days 1–3 postinfection as indicated. Lesion sizes were measured weekly using a caliper and ellipsoid volumes were calculated. Organisms present in lesional tissue were enumerated at the indicated times using limiting dilution assays as described previously (12). For determination of cytokine profiles in infected mice, retroauricular LNs were recovered at wk 3 and wk 5 and single-cell suspensions were prepared. Cells were added to 96-well plates without Ag or with L. major lysate (106 cells/200 µl) in complete RPMI 1640 containing 2-ME (5 x 10–5 M). Culture supernatants were assayed for cytokine production after 48 h using ELISA specific for murine IFN-{gamma} (R&D Systems) and IL-4 (BD Pharmingen).

Statistics

Statistical analysis was performed using the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
L. major-infected BMDC from susceptible and resistant mouse strains release IL-12p40 containing cytokines

Previous studies have shown that DC are capable of taking up L. major amastigotes and, to a lesser extent, promastigotes (10, 15, 16, 17). Infected DC were activated and released detectable amounts of IL-12p40 and IL-12p70 (10, 11). Strain-dependent differences in the production of IL-12p70 that would explain genetic determination of disease outcome were not detected by ELISA. To extend these findings using another source of DC, we initiated studies using BMDC generated in the presence of GM-CSF and IL-4 (Ref. 15 and this study). BMDC were stimulated with L. major promastigotes or amastigotes. Coincubation with promastigotes led to infection of only 4 ± 2% of C57BL/6 DC and 8 ± 1% of BALB/c DC, whereas 28 ± 3% and 26 ± 3% of BMDC were infected when amastigotes were used (parasite:cell ratio 10:1, C57BL/6 and BALB/c, respectively; n ≥ 5). Therefore, as in previous studies using skin-DC (11), no strain-specific difference in the percentage of infected cells or the number of ingested parasites/cell was observed. BMDC derived from genetically resistant C57BL/6 or susceptible BALB/c mice both displayed an activated phenotype after infection with L. major amastigotes, as determined by up-regulation of MHC class II and costimulatory molecules (CD40, CD54 and CD86; data not shown).

Next, release of IL-12p40 and IL-12p70 from stimulated BMDC was determined. As shown previously with skin-DC (11), stimulation with LPS or infection with L. major amastigotes led to increased release of IL-12p40 from BMDC compared with unstimulated controls (Fig. 1A). In contrast, the amount of IL-12p70 was ~100-fold lower compared with IL-12p40 (Fig. 1B), which is consistent with previous reports using other sources of DC (10, 11, 18). However, the pattern of release of IL-12p70 from BMDC was comparable to IL-12p40. Strain-dependent differences in the release of IL-12p70 from activated BMDC were not evident. As in previous studies, BALB/c DC released more IL-12p40 in response to maximal stimulation with LPS than their C57BL/6-derived counterparts (11). However, these differences did not reach statistical significance.


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 1. Strain-dependent differences in the release of IL-12p40 containing cytokines from L. major-infected DC. A–F, BMDC from C57BL/6 or BALB/c mice were generated with GM-CSF/IL-4 and stimulated with LPS (100 ng/ml), L. major promastigotes (Prom.), or amastigotes (Am.) (parasite:cell ratio of 3:1 or 10:1). D–F, BMDC were generated and stimulated in the presence or absence of IL-4 as indicated. A–F, Cytokine release was determined using ELISA specific for murine IL-12p40, IL-12p70, or TNF-{alpha}. Cytokine values were expressed as mean ± SEM (*, p < 0.05, **, p < 0.005, and ***, p < 0.002 compared with unstimulated control cells; n ≥ 3). Statistically significant differences between strains were indicated as bars above columns.

 
We also assayed the supernatants of stimulated BMDC for the release of other Th1-inducing cytokines. Release of IL-18 was not found in culture supernatants of 2 x 105 or 2 x 106 cells/ml using two commercially available ELISA kits. In parallel to the release of IL-12, TNF-{alpha} production of stimulated BMDC was also detected (Fig. 1C). No strain-dependent differences in the amount released were observed. Production of Th2 cytokines (IL-4, IL-10, and TGF-beta1) by BMDC generated from C57BL/6 or BALB/c mice was also determined (data not shown). All three cytokines were found to be produced by DC at baseline levels, but the amounts were comparably low between the mouse strains analyzed and induction in activated cells was not observed.

Effect of IL-4 on IL-12 release from BMDC

IL-4 is commonly used for the generation of murine BMDC. However, IL-4 has been shown to regulate the production of IL-12p40 and IL-12p70 by DC (19, 20, 21, 22, 23). To test the effect of IL-4 on IL-12p40 containing cytokine production, DC were generated from C57BL/6 or BALB/c mice in the presence or absence of this cytokine. Cells were subsequently stimulated with either LPS or L. major amastigotes (plus or minus IL-4 during stimulation; Fig. 1, D–F). Interestingly, although BALB/c DC stimulated in the presence of IL-4 produced marginally more IL-12p40 than C57BL/6 DC (a difference that never reached statistical significance), in the absence of IL-4 BALB/c DC produced more IL-12p40 than C57BL/6 DC (Fig. 1D). This effect was most dramatic in LPS-treated, maximally stimulated cells, but was also detectable in L. major-activated DC (Fig. 1F). A similar effect was observed with regard to the release of Th1-promoting TNF-{alpha} (data not shown). Interestingly, confirming previous findings (19, 21), the effect of IL-4 on IL-12p70 production was reversed. In the absence of IL-4, the DC produced significantly less bioactive IL-12p70. With regard to IL-12p70, IL-4-activated DC from both mouse strains produced equivalent amounts of this cytokine (Fig. 1E).

To confirm that DC generated with or without IL-4 have otherwise similar phenotypes and allostimulatory properties, we determined their surface Ag profiles and performed proliferation assays with alloreactive T cells (data not shown). The expression levels of CD11c, MHC class II, and costimulatory molecules were comparable on DC generated in the presence or absence of IL-4. In addition, their ability to activate alloreactive T cells in proliferation assays was similar.

DC from susceptible BALB/c mice release more antagonistic IL-12p80 than C57BL/6 DC

IL-12p40 is released as a component of bioactive IL-12p70 (in association with p35) or IL-23 (together with p19), as inactive IL-12p40 monomer, or as antagonistic IL-12p80 (8). We therefore analyzed whether DC stimulated with LPS or infected with L. major also released IL-12p80. DC were generated in the presence or absence of IL-4 and stimulated with LPS for 18 h at 2 x 105 cells/ml. The culture supernatants were analyzed by Western blot for the presence of IL-12p40 reactivity under nonreducing conditions. In Fig. 2A, reactivity at ~80 kDa reflecting IL-12p80 production is shown. IL-12p70 production was not detected. Interestingly, under all conditions studied, supernatants of BALB/c DC contained more IL-12p80 than C57BL/6 cultures. From four independent experiments performed, semiquantitative analyses of band densities revealed that BALB/c DC cultures contained ~1.5- to 2-fold more IL-12p80 compared with supernatants of C57BL/6 cells (Fig. 2A). In contrast to a previous report (19), no dramatic effect of IL-4 on the amount of homodimer produced from the DC was observed.


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 2. Strain-dependent differences in the release of IL-12p80 from activated DC. A, The amount of IL-12p40 released as inhibitory homodimer was determined in supernatants of 2 x 105 LPS-stimulated DC/ml by Western blot under nonreducing conditions. One of four independent experiments with similar results is shown. The relative density of IL-12p80 expression was determined and is expressed as mean ± SEM. B, The specificity of the IL-12p80 ELISA for detection of homodimeric p40 as compared with IL-12p40, IL-12p70, or IL-23 was tested. Several proteins containing the IL-12p40 subunit (IL-12p80, IL-12p70, IL-12p40, and IL-23) were diluted in PBS and assayed for cross-reactivity in an IL-12p80 ELISA. Reaction products were quantified at 450 nm using a spectrophotometer and expressed as mean ± SEM. C, IL-12p80 release was quantitated by ELISA. BALB/c or C57BL/6 DC were plated at 2 x 105/ml, stimulated with LPS/IFN-{gamma} (100 ng/1,000 U/ml), or amastigotes of L. major (1:10). Supernatants were harvested after 18 h and assayed for the presence of IL-12p80 as described in Materials and Methods. IL-12p80 levels were expressed as mean ± SEM (*, p < 0.05; n ≥ 5).

 
To quantitate the amount of IL-12p80 synthesis, we developed an ELISA specific for IL-12p80 (Fig. 2B). To accomplish this process, we used the same monoclonal anti-IL-12p40 Ab recognizing a single epitope on the p40 monomer (clone 17.8) both for capturing as well as detecting the protein. Using this approach, we determined that only rIL-12p80 was detectable by this ELISA, whereas IL-12p70 or IL-23—both containing one IL-12p40 subunit—were not measurable. Small amounts of immunoreactivity were detected at high concentrations of the commercial IL-12p40 standard, suggesting that the IL-12p40 preparation contains small amounts of contaminating IL-12p80. The IL-12p80 ELISA was highly reproducible and the detection limit was 31.25 pg/ml (similar to other commercially available ELISA).

We next measured IL-12p80 release from activated DC by ELISA. DC were generated in the absence or presence of IL-4, harvested, and stimulated for an additional 18 h with LPS/IFN-{gamma} or amastigotes of L. major (2 x 105 cells/ml). Significant IL-12p80 release by both BALB/c and C57BL/6 DC was detected after stimulation (Fig. 2C). BALB/c DC produced more IL-12p80 than C57BL/6 DC after stimulation with either LPS/IFN-{gamma} or L. major. This difference was statistically significant in the absence of IL-4, confirming previous findings regarding the role of IL-4 in IL-12p80 generation (19). Interestingly, approximately one-tenth of total IL-12p40 appeared to be released from DC in the form of the inhibitory IL-12p80, an amount ~10-fold greater than stimulatory IL-12p70. In a prior study, it was postulated that up to 40% of p40 protein in the serum is present as IL-12p40 homodimer (24).

Finally, we attempted to quantitate the production of IL-12p80 by DC from C57BL/6 and BALB/c mice in a more physiologic setting. Production of IL-12p40 in draining LN cells restimulated with soluble Leishmania Ag usually ranges between 200 and 400 pg/ml if cells were plated at 106/200 µl and IL-12p70 and IL-12p80 are below the detection limit. Thus, we enriched for LN DC in an attempt to increase the sensitivity of our approach. Mice were infected by injection of 2 x 105 metacyclic promastigotes of L. major into ear skin. Three weeks later, draining LN CD11c+ cells were enriched using microbeads (Miltenyi Biotec) and plated in medium at 1 x 106/200 µl for 18 h, and supernatants were assayed for the presence of IL-12p40 containing cytokines by ELISA. As determined previously, production of total IL-12p40 was comparable between DC from the two mouse strains analyzed (BALB/c, 10.2 ± 2.0 vs C57BL/6, 12.5 ± 2.3 ng/ml; n ≥ 11). In contrast, release of inhibitory IL-12p80 by BALB/c DC in vivo was ≥2-fold increased as compared with C57BL/6 DC (BALB/c, 162 ± 49 vs C57BL/6, 76 ± 30 pg/ml; n ≥ 11).

IL-12p40 homodimer blocks IL-12p70 signaling

In prior studies it was demonstrated that IL-12p80 preferentially binds IL-12Rbeta1, but not IL-12Rbeta2 (25, 26, 27, 28, 29). Although naive T cells from BALB/c mice initially respond to IL-12, T cells from L. major-infected animals lose their IL-12 responsiveness due to down-modulation of the IL-12beta2 chain (30, 31, 32). The IL-12beta2 down-regulation of BALB/c T cells was in part dependent on IL-4. However, because BALB/c mice that express a transgene for IL-12beta2 are still susceptible to infection, the role of IL-12beta2 for disease outcome is unclear to date (33).

To confirm that IL-12p80 inhibits IL-12R-mediated signaling in T cells, we investigated the role of IL-12p40 homodimer on the phosphorylation of STAT-4 and IFN-{gamma} release by IL-12-stimulated cells (Fig. 3). Fig. 3A shows a representative Western blot in which phospho-STAT-4 levels were visualized after stimulation of splenocytes with IL-12p70 (1 ng/ml) and/or IL-12p80 (1 ng/ml). Phopho-STAT-4 was readily detectable in IL-12p70-stimulated cultures (Fig. 3A, lane 3), whereas addition of IL-12p80 abrogated this effect (Fig. 3A, lane 4). Next, BALB/c CD4+ T cells were subjected to Th1-inducing conditions (IL-12/anti-IL-4) and IL-12p80 was added in different concentrations (Fig. 3B). Resulting IFN-{gamma} levels were determined in supernatants of anti-CD3-restimulated cells. As shown in Fig. 3B, IL-12p80 inhibited the IL-12p70-induced IFN-{gamma} release in a concentration-dependent fashion. Thus, our data confirm that BALB/c T cells respond to IL-12p80 by down-modulating IL-12p70-induced events.


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 3. IL-12p80 blocks IL-12p70-mediated STAT-4 phosphorylation and IFN-{gamma} production. A, Con A-stimulated splenocytes (2 x 106/ml) were incubated with IL-12p70 and/or IL-12p80 (1 ng/ml). After 40 min, STAT-4 and phospho-STAT-4 levels were determined by Western blot as indicated in Material and Methods. One representative blot is shown. B, CD4+ T cells from naive mice stimulated with plate-bound anti-CD3/CD28 were incubated for 3 days with IL-12p70/anti-IL-4 with or without addition of IL-12p80 as indicated. After a resting period of 24 h, cells were restimulated with anti-CD3 and IFN-{gamma} levels were determined in 48-h supernatants by ELISA (mean ± SEM; n = 3).

 
IL-12p80 augments Th2 immunity in vivo

To determine whether IL-12p80 has the ability to regulate Th development in cutaneous leishmaniasis, susceptible BALB/c and resistant C57BL/6 mice were infected with 2 x 105 infectious stage promastigotes of L. major into ear skin and treated locally with recombinant cytokine during T cell priming. In previous reports demonstrating an effect of IL-12p80 in disease settings, recombinant cytokine was applied in doses ranging from 1 to 100 µg/mouse i.p. (34, 35). In addition, DC-supernatants generally contain ~100- to 1,000-fold more IL-12p40 than IL-12p70. Therefore, we chose to apply 2 µg of IL-12p80/mouse on days 1, 2, and 3 postinfection—a treatment schedule that has proven to be efficacious for IL-1{alpha} to protect BALB/c mice from progressive disease (12). For comparison, IL-12p70 was administered in a similar fashion (50 ng/mouse) (12).

In BALB/c (Fig. 4A) as well as C57BL/6 mice (Fig. 4B), transient treatment with IL-12p80 resulted in significantly increased lesion volumes that persisted for several weeks. In addition, increased lesional parasite loads as well as impaired Th1 and enhanced Th2 development were observed in BALB/c mice as determined 3 wk postinfection (Fig. 4, C and D). Interestingly, prolonged treatment with IL-12p80 for an additional 3 wk did not further promote disease progression (data not shown).


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 4. Administration of inhibitory IL-12p80 leads to increased lesion development and inhibition of sufficient Th1 development. Groups of ≥5 BALB/c (A, C, and D) or C57BL/6 (B) mice were infected with 2 x 105 metacyclic promastigotes of L. major. On day 1, 2, and 3, mice were treated locally with 2 µg of IL-12p80, 50 ng of IL-12p70, or 20 µl of PBS. A and B, Lesion development was monitored weekly over the course of 2 mo in three dimensions and lesion volumes were calculated as ellipsoid (mean ± SEM; *, p < 0.05, **, p < 0.005, and ***, p < 0.002 compared with PBS-treated control mice). C, Lesional parasite loads of different BALB/c treatment groups were compared with controls at wk 3 postinfection using limiting dilution assays (pooled data from three independent experiments are shown, n ≥ 8 mice/group; *, p < 0.05). D, LN cells were harvested from infected BALB/c mice and plated at 106 cells/200 µl. Cells were stimulated with soluble Leishmania Ag (SLA) for 48 h, and Ag-specific cytokine release was determined using ELISA specific for murine IFN-{gamma} and IL-4 (mean ± SEM). For each mouse, the ratio between IFN-{gamma} and IL-4 was calculated and statistical significance was determined (*, p < 0.05 and ***, p < 0.002; n ≥ 12).

 
Treatment with bioactive IL-12p70 during T cell priming protected BALB/c mice from progressive disease, but did not promote healing. This result was in accordance with previous findings demonstrating that sustained IL-12p70 expression is required to mediate full protection in susceptible mice (36). In contrast, C57BL/6 mice treated with IL-12p70 did not develop significant lesions (Fig. 4B). However, similar to BALB/c mice, treatment of C57BL/6 mice with IL-12p80 resulted in enhanced lesion volumes and delayed healing for several weeks.

IL-12p40 Tg mice showed enhanced disease susceptibility due to increased levels of IL-12p80

Previously, we generated Tg mice that overexpress IL-12p40 under the control of a liver-specific promoter (13). Serum levels of IL-12p40Tg mice were high and IL-12p40 circulates as monomer and homodimer. In addition, previous studies demonstrated that Ag-driven cytokine responses in these mice were skewed toward a Th2 phenotype.

We additionally investigated the relevance of IL-12p80 in L. major infections using IL-12p40Tg mice on a resistant C57BL/6 genetic background. Wild-type C57BL/6 or IL-12p40Tg mice were infected with 2 x 105 L. major metacyclic promastigotes. Compared with wild-type mice, IL-12p40Tg mice showed enhanced lesion progression and delayed involution beginning 3 wk after infection (Fig. 5A). Interestingly, lesions of female IL-12p40Tg mice were significantly larger than those of male IL-12p40Tg mice. In contrast, no sex-specific difference was found in wild-type mice. Lesion volumes of IL-12p40Tg mice were up to 3-fold larger as compared with those of control mice. Lesion involution was delayed by ~4 wk in IL-12p40Tg mice as compared with controls. Furthermore, the skin of female IL-12p40Tg mice contained greater numbers of parasites reaching a peak load of 9.8 ± 0.9 x 108 parasites/ear at wk 5 as compared with 2.4 ± 1.6 x 105 in male IL-12p40Tg mice or 7.4 ± 2.1 x 104 in wild types (p ≤ 0.005) (Fig. 5B). In addition, enhanced parasite dissemination to the spleen was found after 5 wk in female IL-12p40Tg mice.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 5. IL-12p40 Tg mice show enhanced susceptibility in infections with L. major. Groups of ≥5 IL-12p40 Tg or control C57BL/6 mice were infected with 2 x 105 metacyclic promastigotes of L. major. A, Lesion volumes were assessed weekly over the course of ~4 mo and expressed as mean ± SEM (pooled data from four independent experiments are shown; *, p < 0.05 and ***, p < 0.002 as compared with sex-matched controls; n ≥ 7). B, Parasite loads of lesions and spleens were determined in wk 3 and 5 using limiting dilution assays, and bars indicate arithmetic means. Pooled data of three experiments are shown (*, p < 0.05; n ≥ 3).

 
The IFN-{gamma}:IL-4 ratios of IL-12p40Tg mice were skewed toward Th2 predominance (Fig. 6A) as compared with cytokine profiles in control mice. Specifically, the levels of IFN-{gamma} in Tg mice were significantly decreased in both male and female mice. Interestingly, IFN-{gamma} production was almost absent in LN cultures of female IL-12p40Tg mice. In contrast, IL-4 release was increased in IL-12p40Tg mice as compared with C57BL/6 controls.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 6. Infected IL-12p40 Tg mice display impaired Th1 immunity which correlates with increased levels of serum IL-12p40 homodimer. A, LN cells of infected IL-12p40Tg or control C57BL/6 mice were harvested in wk 3 and 5, and Ag-specific cytokine release was determined after 48 h using ELISA specific for murine IFN-{gamma} and IL-4 (mean ± SEM; *, p < 0.05 and ***, p < 0.002; n ≥ 5). B, Levels of IL-12p40 and IL-12p80 were determined in serum of naive IL-12p40Tg or C57BL/6 mice by ELISA (mean ± SEM; *, p < 0.05 and ***, p < 0.002; n ≥ 9 mice).

 
Finally, we quantitated IL-12p40 levels in the serum of IL-12p40Tg mice as compared with wild-type controls (Fig. 6B). As expected, the amount of total IL-12p40 was increased ~100- to 150-fold in naive mice Tg for IL-12p40. IL-12p40 levels in the serum of naive C57BL/6 were almost absent. No increase of serum IL-12p40 was detected in wild-type mice infected with L. major over the course of several weeks (data not shown), suggesting that IL-12p40 is not a cytokine abundant in serum under physiological conditions. We next determined the proportion of IL-12p40 present in the serum of mice in the form of the inhibitory IL-12p80. The levels of IL-12p80 were ~20- to 40-fold lower as compared with total IL-12p40. Interestingly, the amount of IL-12p80 detected in the serum of naive female IL-12p40Tg mice was significantly higher as compared with IL-12p80 in male IL-12p40Tg animals or controls. The levels of IL-12p80 in serum of C57BL/6 controls were very low. As in wild-type mice, the levels of IL-12p40 or IL-12p80 were not additionally increased upon infection with L. major.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DC influence Th cell priming and differentiation in vivo by producing an array of cytokines including IL-12p70. Using the system of resistance and susceptibility in cutaneous leishmaniasis, we and others have previously identified DC-derived IL-1{alpha}beta to be an important regulator of Th1 immune responses in addition to IL-12p70 (12, 37, 38). In the present study, we investigated strain-specific differences in the release of IL-12 in more detail. We found that DC derived from susceptible BALB/c mice produced significantly more inhibitory IL-12p80 than DC from resistant C57BL/6 mice. In addition, treatment with IL-12p80 resulted in decreased Th1 development and more progressive lesions after infection. Therefore, BALB/c DC may inhibit IL-12p70 signaling in T cells by overproducing IL-12p80 that blocks the IL-12R and thus contributes to genetic determination of disease outcome.

Previous studies using Langerhans cell-like fetal skin-derived DC or freshly isolated DC have shown that DC take up L. major amastigotes, are activated and release detectable amounts of IL-12p40 and IL-12p70 (10, 11, 39). In this study, BMDC were used to confirm these initial findings. Again, no strain-specific differences in the percentages of infected cells or the number of ingested parasites per cell were detected. In addition, BMDC derived from C57BL/6 or BALB/c mice were both activated after infection with L. major amastigotes, and strain-dependent differences in the release of IL-12p40 or IL-12p70 from activated BMDC, as detected by ELISA, were not obvious.

Several groups have previously demonstrated potent effects of IL-4 on the production of IL-12p70 from DC (19, 20, 21, 22, 23). In an attempt to determine the amount of IL-4 present in L. major-infected skin during DC activation, we harvested ear skin of infected BALB/c or C57BL/6 mice at several time points postinfection (data not shown). Using either Ab-based detection methods (ELISA, cytometric bead array) or the RT-PCR technique, we found no or only minute amounts of IL-4 a few days after parasite inoculation (≤25 pg/ear). Biedermann et al. (21) suggested that BALB/c mice have a deficiency in local IL-4 production early on after infection resulting in inefficient DC activation. Administration of sufficient amounts of IL-4 during DC activation (within the first 8 h postinfection) in BALB/c mice resulted in efficient Th1 priming and resistance against Leishmania infection (21). We therefore investigated how IL-4 influences the release of IL-12p40 subunits in our system of L. major-infected or LPS-stimulated DC. We confirmed that DC stimulated in the presence of IL-4 produce more IL-12p70 (presumably by up-regulating IL-12p35 expression) compared with control treatment (19, 21). In the context of Leishmania infections, IL-4R expression was increased on infected LC from susceptible BALB/c mice, but not on those from resistant mice (20). In these studies, IL-4 treatment of infected BALB/c DC strongly decreased LPS-induced IL-12p40 responses, but IL-12p70 production was not determined. In this study, BALB/c DC produced more IL-12p40 than cells derived from C57BL/6 mice in the absence of IL-4. Under these conditions, the difference in IL-12p40 production that was observed in previous studies (11) became statistically significant. In vivo, increased IL-12p40 production was found in infected BALB/c mice (as compared with C57BL/6 mice) especially very early on after infection with 2 x 106 L. major promastigotes (40). When compared with C57BL/6 mice, ~2- to 6-fold more IL-12p40 was detected in LN cell cultures of BALB/c mice on day 1 and 7 postinfection.

Bioactive IL-12p70 is a heterodimeric cytokine composed of IL-12p40 and IL-12p35 (7). Production of IL-12p40 and IL-12p35 have been shown to be regulated independently. IL-12p35 is secreted only in association with IL-12p40 as part of the IL-12p70 heterodimer. IL-12p40, however, is released as part of the bioactive heterodimer, but also as inactive monomeric IL-12p40 and IL-12p80. Both IL-12p40 and IL-12p80 are secreted in excess of IL-12p70, but IL-12p80 has been shown to act as potent modulator of IL-12p70 signaling at its receptor. In several studies, IL-12p80 was shown to have inhibitory properties (Refs. 13, 25, 41, 42 and present study), whereas we have also demonstrated an agonistic function in IL-12p35/p40-deficient mice (8). In cutaneous leishmaniasis, the role of IL-12p80 has not been previously studied. We now confirmed that a substantial amount of IL-12p40 was released by DC in the form of the inhibitory IL-12p40 homodimer (19). In addition, BALB/c DC produced more IL-12p80 than C57BL/6 DC.

As compared with prior studies (19, 20, 21, 22, 23), the effect of IL-4 on IL-12p80 release in our study was not strong. Even though we were able to reproduce the up-regulating effect of IL-4 on IL-12p70 release, the influence of IL-4 on down-modulating IL-12p80 protein synthesis from BMDC in vitro was less striking. Thus, IL-4 may not be the only cytokine/factor responsible for the induction/regulation of IL-12p80 production.

In vivo, administration of IL-12p80 led to protection from IL-12-dependent shock (42). We therefore analyzed the role of IL-12p80 during T cell priming in cutaneous leishmaniasis in vivo. Both susceptible BALB/c and resistant C57BL/6 mice demonstrated increased susceptibility in the presence of IL-12p80 during T cell priming and this effect lasted for >6 wk, although IL-12p80 was only applied during days 1–3 postinfection. The cytokine profile in BALB/c LN cultures was altered toward a Th2 phenotype, whereas IL-12p70 treatment induced a shift toward Th1 cell differentiation as expected. Interestingly, prolonged treatment with IL-12p80 did not additionally worsen disease outcome, suggesting that physiological IL-12p80 acts early after infection.

IL-12p40Tg mice, producing increased amounts of IL-12p80, displayed reduced Th1 responses and increased susceptibility toward infection with e.g., Plasmodium berghei (13). In this study, we demonstrate that IL-12p40Tg mice on a genetically resistant C57BL/6 background are also more susceptible to infection with L. major. The IL-12p40Tg mice displayed increased lesion volumes, higher lesional parasite loads, and enhanced spreading of the parasite to visceral organs. In addition, the Tg mice showed IFN-{gamma}:IL-4 ratios that were altered toward Th2. Interestingly, the level of susceptibility (in female vs male IL-12p40Tg mice) strongly correlated with the amount of homodimer present in the serum. The fact that female IL-12p40Tg mice displayed higher levels of IL-12p80 despite similar levels of IL-12p40 as compared with male IL-12p40Tg is unexplained and requires further examination.

In summary, our data indicate that in BALB/c mice, DC may contribute to disease susceptibility not only by producing less IL-1{alpha}beta, but also by releasing more inhibitory IL-12p80 after infection with L. major amastigotes than their C57BL/6 counterparts. In comparison with C57BL/6 cells, BALB/c DC display an imbalance of factors regulating Th development. BALB/c DC produce lesser amounts of cytokines (e.g., IL-1) that favor the development of Th1 immunity and increased amounts of others (e.g., IL-12p80) that promote Th2 cell differentiation. In addition, we also demonstrated an important role of IL-12p80 for the efficient induction of protective Th1-predominant immunity in vivo. These data provide additional support for the concept that genetic differences that contribute to susceptibility to cutaneous leishmaniasis in mice are expressed at the level of DC as well as in T cells.


   Acknowledgments
 
We thank Drs. Tilo Biedermann, Martin Röcken, and Hubertus Hochrein for helpful discussions, and Drs. Kerstin Steinbrink, Christian Becker, and Helmut Jonuleit for critically reading the manuscript. We also thank Susanna Lopez Kostka for expert technical assistance.


   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 grants from the Deutsche Forschungsgemeinschaft (SFB 548 to E.v.S. and HO2145/2-1 to C.H.), the MAIFOR program of the University of Mainz (to E.v.S.), and the Intramural Program of the National Institutes of Health, Center for Cancer Research, National Cancer Institute (to M.C.U.). Back

2 A.P.N. and S.Z. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Esther von Stebut, Department of Dermatology, Johannes Gutenberg-University, Langenbeckstrasse 1, 55131 Mainz, Germany. E-mail address: vonstebu{at}mail.uni-mainz.de Back

4 Abbreviations used in this paper: DC, dendritic cell; Tg, transgenic; BMDC, bone marrow-derived DC; LN, lymph node. Back

Received for publication May 30, 2006. Accepted for publication March 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sacks, D., N. Noben-Trauth. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2: 845-858. [Medline]
  2. Reiner, S. L., R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13: 151-177. [Medline]
  3. Belkaid, Y., E. von Stebut, S. Mendez, R. Lira, C. Caler, S. Bertholet, M. C. Udey, D. Sacks. 2002. CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J. Immunol. 168: 3992-4000. [Abstract/Free Full Text]
  4. von Stebut, E., M. C. Udey. 2004. Requirements for Th1-dependent immunity against infection with Leishmania major. Microbes Infect. 6: 1102-1109. [Medline]
  5. Holscher, C., B. Arendse, A. Schwegmann, E. Myburgh, F. Brombacher. 2006. Impairment of alternative macrophage activation delays cutaneous leishmaniasis in nonhealing BALB/c mice. J. Immunol. 176: 1115-1121. [Abstract/Free Full Text]
  6. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, X. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715-725. [Medline]
  7. Holscher, C.. 2004. The power of combinatorial immunology: IL-12 and IL-12-related dimeric cytokines in infectious diseases. Med. Microbiol. Immunol. 193: 1-17. [Medline]
  8. Holscher, C., R. A. Atkinson, B. Arendse, N. Brown, E. Myburgh, G. Alber, F. Brombacher. 2001. A protective and agonistic function of IL-12p40 in mycobacterial infection. J. Immunol. 167: 6957-6966. [Abstract/Free Full Text]
  9. Picotti, J. R., S. Y. Chan, K. Li, E. J. Eichwald, D. K. Bishop. 1997. Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-{gamma}-producing cells. J. Immunol. 158: 643-648. [Abstract]
  10. von Stebut, E., Y. Belkaid, T. Jakob, D. L. Sacks, M. C. Udey. 1998. Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J. Exp. Med. 188: 1547-1552. [Abstract/Free Full Text]
  11. von Stebut, E., Y. Belkaid, B. V. Nguyen, M. Cushing, D. L. Sacks, M. C. Udey. 2000. Leishmania major-infected murine Langerhans cell-like dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis. Eur. J. Immunol. 30: 3498-3506. [Medline]
  12. von Stebut, E., J. M. Ehrchen, Y. Belkaid, S. Lopez Kostka, K. Molle, J. Knop, C. Sunderkotter, M. C. Udey. 2003. Interleukin 1{alpha} promotes Th1 differentiation and inhibits disease progression in Leishmania major-susceptible BALB/c mice. J. Exp. Med. 198: 191-199. [Abstract/Free Full Text]
  13. Yoshimoto, T., C. R. Wang, T. Yoneto, S. Waki, S. Sunaga, Y. Komagata, M. Mitsuyama, J. Miyazaki, H. Nariuchi. 1998. Reduced T helper 1 responses in IL-12p40 transgenic mice. J. Immunol. 160: 588-594. [Abstract/Free Full Text]
  14. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176: 1693-1702. [Abstract/Free Full Text]
  15. Woelbing, F., S. Lopez Kostka, K. Moelle, Y. Belkaid, C. Sunderkoetter, S. Verbeek, A. Waisman, A. P. Nigg, J. Knop, M. C. Udey, E. von Stebut. 2006. Uptake of Leishmania major by dendritic cells is mediated by Fc-{gamma} receptors and facilitates acquisition of protective immunity. J. Exp. Med. 203: 177-188. [Abstract/Free Full Text]
  16. Moll, H., H. Fuchs, C. Blank, M. Rollinghoff. 1993. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur. J. Immunol. 23: 1595-1601. [Medline]
  17. Scott, P., C. A. Hunter. 2002. Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Curr. Opin. Immunol. 14: 466-470. [Medline]
  18. Heufler, C., F. Koch, U. Stanzl, G. Topar, M. Wysocka, G. Trinchieri, A. Enk, R. M. Steinman, N. Romani, G. Schuler. 1996. Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-{gamma} production by T helper 1 cells. Eur. J. Immunol. 26: 659-668. [Medline]
  19. Hochrein, H., M. O’Keeffe, T. Luft, S. Vandenabeele, R. J. Grumont, E. Maraskovsky, K. Shortman. 2000. Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells. J. Exp. Med. 192: 823-833. [Abstract/Free Full Text]
  20. Moll, H., A. Scharner, E. Kampgen. 2002. Increased interleukin 4 (IL-4) receptor expression and IL-4-induced decrease in IL-12 production by Langerhans cells infected with Leishmania major. Infect. Immun. 70: 1627-1630. [Abstract/Free Full Text]
  21. Biedermann, T., S. Zimmermann, H. Himmelrich, A. Gumy, O. Egeter, A. K. Sakrauski, I. Seegmuller, H. Voigt, P. Launois, A. D. Levine, et al 2001. IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat. Immunol. 2: 1054-1060. [Medline]
  22. Kalinski, P., H. H. Smits, J. H. Schuitemaker, P. L. Vieira, M. van Eijk, E. C. de Jong, E. A. Wierenga, M. L. Kapsenberg. 2000. IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells. J. Immunol. 165: 1877-1881. [Abstract/Free Full Text]
  23. Yao, Y., W. Li, M. H. Kaplan, C.-H. Chang. 2005. Interleukin-4 inhibits IL-10 to promote IL-12 production by dendritic cells. J. Exp. Med. 201: 1899-1903. [Abstract/Free Full Text]
  24. Heinzel, F. P., A. M. Hujer, F. N. Ahmed, R. M. Rerko. 1997. In vivo production and function of IL-12 p40 homodimers. J. Immunol. 158: 4381-4388. [Abstract]
  25. Gillessen, S., D. Carvajal, P. Ling, F. J. Podlaski, D. L. Stremlo, P. C. Familletti, U. Gubler, D. H. Presky, A. S. Stern, M. K. Gately. 1995. Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur. J. Immunol. 25: 200-206. [Medline]
  26. Gately, M. K., D. M. Carvajal, S. E. Connaughton, S. Gillessen, R. R. Warrier, K. D. Kolinsky, V. L. Wilkinson, C. M. Dwyer, G. F. Higgins, Jr, F. J. Podlaski, et al 1996. Interleukin-12 antagonist activity of mouse interleukin-12p40 homodimer in vitro and in vivo. Ann. NY Acad. Sci. 795: 1-12. [Medline]
  27. Presky, D. H., L. J. Minetti, S. Gillessen, V. L. Wilkinson, C. Y. Wu, U. Gubler, R. Chizzonite, M. K. Gately. 1998. Analysis of the multiple interactions between IL-12 and the high affinity IL-12 receptor complex. J. Immunol. 160: 2174-2179. [Abstract/Free Full Text]
  28. Wang, X., V. L. Wilkinson, F. J. Podlaski, C. Wu, A. S. Stern, D. H. Presky, J. Magram. 1999. Characterization of mouse interleukin-12p40 homodimer binding to the interleukin-12 receptor subunits. Eur. J. Immunol. 29: 2007-2013. [Medline]
  29. Russell, T. D., Q. Yan, G. Fan, A. P. Khalifah, D. K. Bishop, S. L. Brody, M. J. Walter. 2003. IL-12p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta1. J. Immunol. 171: 6866-6874. [Abstract/Free Full Text]
  30. Guler, M. L., J. D. Gorham, C. S. Hsieh, A. J. Mackey, R. G. Steen, W. F. Dietrich, K. M. Murphy. 1996. Genetic susceptibility to Leishmania: IL-12 responsiveness in Th1 cell development. Science 271: 984-987. [Abstract]
  31. Szabo, S. J., A. S. Dighe, U. Gubler, K. M. Murphy. 1997. Regulation of the interleukin (IL)-12Rbeta2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185: 817-824. [Medline]
  32. Himmelrich, H., C. Parra-Lopez, F. Tacchini-Cottier, J. A. Louis, P. Launois. 1998. The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol. 161: 6156-6163. [Abstract/Free Full Text]
  33. Nishikomori, R., S. Gurunathan, K. Nishikomori, W. Strober. 2001. BALB/c mice bearing a transgenic IL-12 receptor beta2 gene exhibit a nonhealing phenotype to Leishmania major infection despite intact IL-12 signaling. J. Immunol. 166: 6776-6783. [Abstract/Free Full Text]
  34. Mattner, F., S. Fischer, S. Guckes, S. Jin, H. Kaulen, E. Schmitt, E. Rude, T. Germann. 1993. The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer. Eur. J. Immunol. 23: 2202-2208. [Medline]
  35. Rothe, H., R. M. O’Hara, Jr, S. Martin, H. Kolb. 1997. Suppression of cyclophosphamide induced diabetes development and pancreatic Th1 reactivity in NOD mice treated with the interleukin (IL)-12 antagonist IL-12(p40)2. Diabetologia 40: 641-646. [Medline]
  36. Stobie, L., S. Gurunathan, C. Prussin, D. L. Sacks, N. Glaichenhaus, C. Y. Wu, S. A. Seder. 2000. The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. Proc. Natl. Acad. Sci. USA 97: 8427-8432. [Abstract/Free Full Text]
  37. Filippi, C., S. Hugues, J. Cazareth, V. Julia, N. Glaichenhaus, S. Ugolini. 2003. CD4+ T cell polarization in mice is modulated by strain-specific major histocompatibility complex-independent differences within dendritic cells. J. Exp. Med. 198: 201-209. [Abstract/Free Full Text]
  38. Shibuya, K., D. Robinson, F. Zonin, S. B. Hartley, S. E. Macatonia, C. Somoza, C. A. Hunter, K. M. Murphy, A. O’Garra. 1998. IL-1{alpha} and TNF-{alpha} are required for IL-12-induced development of Th1 cells producing high levels of IFN-{gamma} in BALB/c but not C57BL/6 mice. J. Immunol. 160: 1708-1716. [Abstract/Free Full Text]
  39. Moll, H., S. Flohe. 1997. Dendritic cells induce immunity to cutaneous leishmaniasis in mice. Adv. Exp. Med. Biol. 417: 541-545. [Medline]
  40. Scharton-Kersten, T., L. C. Afonso, M. Wysocka, G. Trinchieri, P. Scott. 1995. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 154: 5320-5330. [Abstract]
  41. Mattner, F., L. Ozmen, F. J. Podlaski, V. L. Wilkinson, D. H. Presky, M. K. Gately, G. Alber. 1997. Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor {alpha}-dependent shock. Infect. Immun. 65: 4734-4737. [Abstract]
  42. Ling, P., M. K. Gately, U. Gubler, A. S. Stern, P. Lin, K. Hollfelder, C. Su, Y. C. Pan, J. Hakimi. 1995. Human IL-12p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J. Immunol. 154: 116-127. [Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Mathew, K. L. Bauer, A. Fischoeder, N. Bhardwaj, and S. J. Oliver
The Anergic State in Sarcoidosis Is Associated with Diminished Dendritic Cell Function
J. Immunol., July 1, 2008; 181(1): 746 - 755.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Soong
Modulation of Dendritic Cell Function by Leishmania Parasites
J. Immunol., April 1, 2008; 180(7): 4355 - 4360.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nigg, A. P.
Right arrow Articles by von Stebut, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nigg, A. P.
Right arrow Articles by von Stebut, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS