HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nishikomori, R. Articles by Strober, W. Search for Related Content PubMed PubMed Citation Articles by Nishikomori, R. Articles by Strober, W.

BALB/c Mice Bearing a Transgenic IL-12 Receptor 2 Gene Exhibit a Nonhealing Phenotype to Leishmania major Infection Despite Intact IL-12 Signaling

Ryuta Nishikomori^*, Sanjay Gurunathan, Kanako Nishikomori^* and Warren Strober^1,*

^* Mucosal Immunity Section and Clinical Immunology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In BALB/c mice infected with Leishmania major, early secretion of IL-4 leads to a Th2-type response and nonhealing. We explored the role of IL-4-induced down-regulation of the IL-12R2 chain in the establishment of this Th2 response. First, we showed that the draining lymph nodes of resistant C57BL/6 mice infected with L. major were enriched in CD4⁺/IL-12R2 chain⁺ cells producing IFN-. Next, we demonstrated that BALB/c background mice bearing an IL-12R2-chain transgene manifested a nonhealing phenotype similar to wild-type littermates despite the persistence of their ability to undergo STAT4 activation. Finally, we found that such transgenic mice display more severe infection than wild-type littermates when treated with IL-12 7 days after infection, and under this condition, the mice display increased Leishmania Ag-induced IL-4 secretion. These studies indicate that although CD4⁺/IL-12R2 chain⁺ T cells are important components of the Th1 response, maintenance of IL-12R2 chain expression is not sufficient to change a Th2 response to a Th1 response in vivo and thus to allow BALB/c mice to heal L. major infection.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The outcome of infection of mice with the intracellular protozoan parasite Leishmania major is mouse strain-dependent (1, 2). Strains that mount a Th1 response or negate a Th2 response after infection (e.g., C57BL/6, C3H mice) display a healing phenotype; this is attributable to their ability to produce high levels of IFN-, the cytokine necessary for the generation of activated macrophages that kill infecting organisms through inducible NO synthase and NO production (3, 4). In contrast, strains that mount a Th2 response after infection (e.g., BALB/c mice) display a nonhealing phenotype because of an inability to produce requisite high levels of IFN-.

The basis of the Th2 response to L. major infection in BALB/c mice is of considerable theoretical and practical interest, but is not yet completely understood. It is known that IL-4 mRNA production is up-regulated in draining lymph nodes at the early stage of infection of BALB/c mice and that such IL-4 production comes from V4/V8 CD4⁺ T cells responding to L. major Ag (5, 6, 7). It also is also known that although BALB/c mice produce IL-12 early in infection (8), they produce reduced amounts of IFN- (compared with resistant C57BL/6 mice), presumably because the early burst of IL-4 down-regulates the IL-12 receptor, especially the IL-12R2 chain, and thus precludes the IL-12 signaling necessary for Th1 development (9, 10, 11, 12, 13). The result is unopposed Th2 differentiation. However, this scenario is brought into question by the results of recent in vitro studies of CD4⁺ T cells constitutively expressing the IL-12R2 (14, 15). In these studies, it was shown that the IL-12R2 chain is not a determinative factor for Th1 cell differentiation, and that once CD4⁺ T cells differentiate to Th2 cells, IL-12 signaling cannot convert Th2 cells to Th1 cells; furthermore, it was shown that IL-12 cannot shut down IL-4-induced Th2 cell differentiation when enough IL-4 is provided during the priming in addition to IL-12. These in vitro results quite obviously call into question the notion that down-regulation of the IL-12R2 chain is a key prerequisite of nonhealing L. major infection in BALB/c mice and prompted us to determine whether in fact such infection leads to a healing phenotype in BALB/c mice bearing an IL-12R2 chain transgene. The results were surprising in that the presence of the transgene did not change the nonhealing phenotype of the mice and actually led to increased severity of infection in comparison to wild-type littermates when the mice were treated with IL-12 7 days after infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 mice expressing an IL-12R2 chain transgene under control of a CD2 promoter and CD2 locus control region was constructed as described previously (15). These mice were back-crossed to BALB/c mice five to seven times and then maintained as heterozygotes for transgene. Transgene-negative littermates served as controls. Flow cytometric analysis of lymphoid organs (thymus, spleen, and lymph nodes) showed that transgene-positive and -negative mice manifested few differences in the percentages of CD3, CD4, B220, CD11b, CD11c, DX5, and CD4⁺/V8⁺ (clone B21.14; BD PharMingen, San Diego, CA)/V4⁺ (clone KT4; BD PharMingen) cells. In contrast, with a specific mAb against mouse IL-12R2 chain (PDL-HAM10B9; Ref. 15) the transgene-positive but not the transgene-negative mice expressed IL-12R2 chain on unstimulated CD4⁺ and CD8⁺ but not on B220⁺ and CD11b^high cells. The expression of IL-12R2 chain on DX5⁺ cells was slightly positive, but an increase in staining intensity was identified in transgene-positive as compared with transgene-negative mice (data not shown). Mice aged 8–20 wk were used in all experiments.

L. major infection and parasite quantitation

L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown as described previously, and infective stage metacyclic promastigotes were isolated from growth cultures by their lack of agglutination with peanut agglutinin (16). Mice were infected with L. major by injection of metacyclic promastigotes (1 x 10⁵) into the left footpad. Infection was monitored by weekly measurement of footpad swelling with a caliper.

Parasite loads of regional popliteal lymph nodes were quantitated by serial dilution (3-fold) of cell suspensions with 50 µl of NNN medium containing 30% defibrinated rabbit blood and overlaid with 100 µl of C-M199 (199 medium supplemented with 20% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine, 25 mM HEPES, pH 7.0, 0.1 mM adenine, and 5 µg/ml hemin). The number of viable parasites were determined in duplicate from the reciprocal of the highest dilution at which promastigotes could be detected after 7 days of incubation at 26°C.

Treatment of mice with Abs and cytokines

In some studies, mice were treated with anti-mouse IL-4 mAb (clone 11B11; 3 mg on day 0 of infection) or mouse recombinant IL-12 (1.5 µg on various days as described) by i.p. injection. Anti-mouse IL-4 mAb and mouse recombinant IL-12 used in these studies were purchased from the National Cancer Institute (Bethesda, MD).

Culture of popliteal lymph nodes and analysis of culture fluids by ELISA

For assessment of cytokine production, whole regional popliteal lymph node cell populations from each mouse were cultured at 1.5 x 10⁶ cells/ml with soluble L. major Ag (SLA²; 25 µg/ml). Culture medium consisted of RPMI 1640 supplemented with 10% FCS, 15 mM HEPES pH 7.0, 100 U/ml penicillin G, 100 µg/ml streptomycin, 50 µM 2-ME, and 5% NCTC 109. Cells were cultured for 48 h, after which culture supernatants were harvested and assayed for IL-4, IFN-, and IL-10 concentration by ELISA with the Opti-EIA mouse IL-4 mini-kit, the Opti-EIA mouse IFN- kit, and the Opti-EIA IL-10 kit (BD PharMingen). Determinations were performed in duplicate as described previously, and the values obtained were averaged (15).

Flow cytometry

Flow cytometry was performed with a Becton Dickinson (Mountain View, CA) FACScan flow cytometer in conjunction with CellQuest II software (Becton Dickinson). Cells were stained with rat anti-mouse CD4-FITC (BD PharMingen), hamster anti-mouse IL-12R2 chain mAb (PDL-HAM10B9), hamster anti-TNP mAb (BD PharMingen), biotinylated goat anti-hamster IgG (H+L; Jackson ImmunoResearch Laboratories, West Grove, PA) as described previously (15). To amplify the signal intensity of mouse IL-12R2 chain expression for wild-type mouse CD4⁺ cells, streptavidin-PE (BD PharMingen) and biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA) also were also used as described by Cohen (17).

Immunoprecipitation and Western blotting

CD4⁺ cells were isolated by positive selection with mouse CD4 beads according to the manufacturer’s protocol (Miltenyi Biotech, Auburn, CA; purity of CD4⁺ population obtained: >95%). The percentage of DX5⁺/CD4⁺ cells in CD4⁺ cells isolated from draining popliteal lymph nodes was from 7 to 11% in C57BL/6 and from 4 to 5% in BALB/c. STAT4 tyrosine phosphorylation in CD4⁺ cells stimulated with mouse IL-12 (5 ng/ml) for 20 min was assessed by immunoprecipitation and Western blotting by using previously described procedures (15).

Competitive RT-PCR assay for IL-4 mRNA for draining lymph nodes

After footpad injection of 1 x 10⁵ metacyclic promastigotes, RNA was isolated from pooled popliteal draining lymph nodes for each sample. First-strand cDNA synthesis was performed with Superscriptase II (Life Technologies, Rockville, MD). The semiquantitative PCR developed by Bouaboula et al. was performed with competitor construct containing sequences for ₂-microglobulin and multiple cytokines as described by the authors (18). The first-strand cDNA was amplified by PCR in the presence of 4-fold serial dilution of the competitor construct. The PCR products were electrophoresed in agarose gel with ethidium bromide, and the ratio of the relative concentration of IL-4 cDNA to the relative concentration of ₂-microglobulin cDNA was calculated. Results are expressed as fold increase in mRNA expression with L. major-infected mice in comparison with that in noninfected wild-type littermates.

Statistics

Data were analyzed statistically by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-12R2 chain is a marker for Th1 cells in L. major infection

In initial studies, we quantified IL-12R2 chain expression on CD4⁺ cells during L. major infection in nonhealing BALB/c and healing C57BL/6 mice by flow cytometry with anti-mouse IL-12R2 chain mAb. As shown in Fig. 1A, at 6 wk of infection, 16.2 ± 2.7% (mean ± SD; isotype control 5.1 ± 0.1%) of regional lymph node (popliteal) CD4⁺ cells are IL-12R2 chain⁺ in C57BL/6 mice (noninfected: IL-12R2 chain, 1.9 ± 0.3%; isotype control, 1.6 ± 0.5%) whereas only 6.0 ± 0.6% (isotype control 3.9 ± 0.5%) of a comparable cell population were positive in BALB/c mice (noninfected: IL-12R2 chain, 2.0 ± 0.6%; isotype control, 1.3 ± 0.2%). These data confirm previous studies in which IL-12R2 chain expression was measured at the RNA level (11, 12); in addition, they are consistent with the fact that, as shown in Fig. 1B, CD4⁺ cells from C57BL/6 mice at 6 wk of infection when stimulated by IL-12 transduced STAT4 tyrosine phosphorylation to a greater extent than comparable T cells from BALB/c mice.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. A, IL-12R2 chain expression on CD4+ cells of regional lymph nodes of BALB/c mice and C57BL/6 mice at 6 wk of L. major infection. Whole lymph nodes cells were stained with rat anti-CD4 mAb and anti-mouse IL-12R2 chain mAb or hamster anti-TNP mAb (control) and analyzed by flow cytometry. Representative histograms gated on CD4+ cells are shown. In C57BL/6 mice (n = 4), 16.2 ± 2.7% (mean ± SD; isotype mAb control 5.1 ± 0.1%) of CD4+ cells were IL-12R2 chain+ cells, whereas in BALB/c mice (n = 4), 6.0 ± 0.6% (isotype mAb control 3.9 ± 0.5%) of CD4+ cells were IL-12R2 chain+ cells. B, STAT4 tyrosine phosphorylation of CD4+ cells in regional popliteal lymph node cells of C57BL/6 mice (BL/6, lanes 1 and 2), BALB/c background IL-12R2 chain-transgenic mice (BALB/c IL-12R2Tg, lanes 5 and 6), and wild-type littermates (BALB/c WT, lanes 3 and 4) at 6 wk of L. major infection. Cells (7 x 106) cells were incubated with (+, lanes 2, 4, and 6) or without (-, lanes 1, 3, and 5) IL-12 (5 ng/ml) for 20 min and subjected to immunoprecipitation Western blotting for STAT4 tyrosine phosphorylation. After stripping, the same blot was probed with anti-STAT4 Ab.

View this table: [in this window] [in a new window]   Table I. Cytokine production from sorted IL-12R2 chain-positive CD4 cells of draining lymph node of L. major-infected C57BL/6 mouse1

L. major infection in BALB/c-background IL-12R2 chain-transgenic mice

Having established the importance of the IL-12R2 chain as a marker of Th1 cells developing in response to L. major infection, we next sought to determine the relation of down-regulation of IL-12R2 chain expression to the development of a nonhealing phenotype in BALB/c mice. In initial studies, we infected mice with a BALB/c background and bearing an IL-12R2 chain transgene as well as littermate control mice with L. major by footpad injection and then recorded footpad swelling over time. We have noticed that IL-4 can suppress the IL-12R2 transgene expression on CD4⁺ cells in vitro (15), so first we checked IL-12R2 chain expression on CD4 cells of IL-12R2 chain-transgenic mice and wild-type littermates. As shown in Fig. 2A, CD4⁺ cells in the popliteal lymph nodes of the transgenic mice displayed robust expression of the IL-12R2 chain at 1, 3, and 6 wk of infection as determined by flow cytometry. In addition, as shown in the Western blots depicted in Fig. 1B, CD4⁺ cells obtained from popliteal lymph nodes of IL-12R2 chain-transgenic mice at 6 wk of infection transduced STAT4 tyrosine phosphorylation to the same extent as CD4⁺ cells from C57BL/6 mice, whereas CD4⁺ cells from wild-type littermates transduced little STAT4 tyrosine phosphorylation. These studies thus showed that the IL-12R2 chain on CD4⁺ cells was not only present but also functioning at this time during infection.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. A, IL-12R2 chain expression of CD4+ cells in regional lymph nodes of BALB/c background IL-12R2-transgenic mice and wild-type littermates during the course of L. major infection (1, 3, and 6 wk of infection). The staining was done without an amplification step by anti-streptavidin and streptavidin-PE. Histograms gated on CD4+ cells are shown. B, Footpad swelling during L. major infection in untreated BALB/c background IL-12R2 chain-transgenic mice (Tg), wild-type littermates (WT), and mice treated with 3 mg of anti-IL-4 mAb at the time of infection (Tg+aIL-4, WT+aIL-4), and C57BL/6 mice (BL/6). All untreated BALB/c-background IL-12R2 chain-transgenic mice (Tg) and wild-type littermates (WT) progressed to develop ulceration and necrosis. Each group consists of four mice. Error bars indicates SEM. Representative of three independent experiments is shown. C, Parasite number in regional popliteal lymph nodes in IL-12R2 chain-transgenic mice at 6 wk of infection. Each diamond shows parasite number in draining popliteal lymph node of an individual mouse. Horizontal bars indicate geometric mean of the parasite number in each group.

View this table: [in this window] [in a new window]   Table II. Cytokine production by draining lymph node cells of L. major-infected BALB/c background IL-12R2 chain-transgenic mice1

IL-12 treatment cannot control established L. major infection in BALB/c background IL-12R2 chain-transgenic mice

The lack of relevance of the IL-12R2 chain expression to the nonhealing phenotype of L. major-infected BALB/c mice was further explored in studies in which infected mice were subjected to IL-12 treatment at various time points of infection. These studies were based on previous reports showing that BALB/c mice develop a healing phenotype if they are administered IL-12 at the time of initiation of infection, but fail to develop a healing phenotype if they are administered IL-12 7 days after initiation of infection, presumably because at the time of initiation of infection, responding T cells express the IL-12R2 chain and respond to IL-12, whereas at 7 days of infection they do not express the IL-12R2 chain and no longer respond to IL-12 (11, 12, 20, 21). Thus, one might predict that IL-12R2 chain-transgenic mice that maintain IL-12R2 chain expression will develop a healing phenotype when treated with IL-12 both at the time of initiation of infection and later at 7 days after initiation of infection.

In fact, as shown in Fig. 3, A and B, we found that although IL-12 treatment of IL-12R2 chain-transgenic mice and wild-type littermates at the initiation of infection led to greatly decreased footpad swelling and decreased parasite load at 5 wk of infection as compared with untreated mice. IL-12 treatment of both IL-12R2 chain-transgenic mice and wild-type littermates failed to control infection when treated with IL-12 at 7 days of infection, although the footpad swelling and parasites load were less than in nontreated mice. In addition, the extent of footpad swelling and the parasite load in the IL-12R2 chain-transgenic mice treated with IL-12 7 days after infection were greater than in the wild-type littermate mice. Thus, IL-12 treatment of L. major infection in IL-12R2 chain-transgenic mice was relatively ineffective in reversing the course of infection and led to less control of infection than in wild-type littermates. The basis of this somewhat paradoxical response to IL-12 became evident from concomitant studies of cytokine secretion from popliteal lymph nodes cells stimulated with SLA. Thus, as shown in Table III, although IL-12R2-transgenic mice and wild-type littermates given IL-12 treatment at the time of initiation of infection led to an expected increase in IFN- secretion in both transgenic mice and wild-type littermates, both mice given IL-12 treatment at 7 days of infection did not produce more IFN- than nontreated mice. Furthermore, IL-12R2 chain-transgenic mice given IL-12 at 7 days of infection produced more IL-4 than nontreated IL-12R2 chain-transgenic mice and wild-type littermates given IL-12 at 7 days of infection.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. A, Footpad swelling during L. major infection in untreated IL-12R2 chain-transgenic mice and mice treated with i.p. injection of recombinant IL-12. IL-12R2 chain-transgenic mice (Tg) or wild-type littermates (WT) were treated with daily i.p. injection of 1.5 µg of mouse recombinant IL-12 on days 0–4 (Tg+IL-12 (0–4) and WT+IL-12 (0–4)), or on days 7–11 (Tg+IL-12 (7–11) and WT+IL-12 (7–11)). Mice were infected at day 0. Each group consists of four mice except Tg (n = 5) and WT+IL-12 (0–4) (n = 3). Error bars indicate SEM. B, Parasite number in regional popliteal lymph nodes of IL-12-treated IL-12R2 chain-transgenic mice at 5 wk of infection. Each diamond shows parasite number in draining popliteal lymph node of an individual mouse. Horizontal bars indicate geometric mean of parasite number in each group.

View this table: [in this window] [in a new window]   Table III. Cytokine production by draining lymph node cells of IL-12 treated L. major-infected BALB/c background IL-12R2 chain-transgenic mice1

IL-4 induction by L. major in draining popliteal lymph nodes in IL-12R2 chain-transgenic mice

In a final series of studies, we evaluated the possibility that the observed preservation of the IL-4 response in BALB/c-background IL-12R2 chain-transgenic mice after L. major infection might be artifactually attributable to the presence of a vastly increased number of CD4⁺ T cells responsive to L. major which reacted in an early and massive IL-4 response that drove Th2 differentiation despite continued IL-12 signaling. Here we injected IL-12R2 chain-transgenic mice with L. major and then serially measured IL-4 mRNA in draining popliteal lymph node cells with a semiquantitative RT-PCR technique based on the presence of an internal standard (see Materials and Methods). As shown in Fig. 4, in which the IL-4 mRNA for IL-12R2 chain-transgenic mice and wild-type littermates are expressed as fold increase after normalization with the amount of 2-microglobulin mRNA present in the sample (also measured by semiquantitative RT-PCR), the response of both groups are essentially the same with respect to both kinetics and magnitude. Thus, the IL-4 response of the IL-12R2 chain-transgenic mice is not qualitatively or quantitatively different from wild-type littermates, and the failure of cells in IL-12R2 chain-transgenic mice to undergo increased Th1 differentiation cannot be attributed to an intrinsic difference in their IL-4 response.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. IL-4 mRNA up-regulation during the early phase of L. major infection in IL-12R2 chain-transgenic mice and wild-type littermates. IL-4 mRNA and 2-microglobulin mRNA were measured by semiquantitative RT-PCR. The IL-4 mRNA level obtained was normalized by 2-microglobulin mRNA level and then expressed as fold increases at the various time after infection in comparison with that in noninfected wild-type littermates. The data shown are representative of two independent studies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

As indicated in the introduction, L. major infection in BALB/c mice has been shown to trigger an early burst of IL-4 from L. major-reactive cells expressing V4/V8, which primes the animal for a Th2-type response (5, 6, 7). This Th2 priming is reported to occur in the first week of infection when IL-12 p40 mRNA also is detected in BALB/c mice (8). Nevertheless, as shown in studies by Heath et al. (14, 15) as well as in our own previous studies, such priming occurs when enough IL-4 is present, even if IL-12 signaling also is going on, and thus it is apparent that IL-12 cannot reverse or block the IL-4 priming effect. One explanation for the overriding effect of IL-4 priming in this and other situations relates to its ability to down-regulate IL-12R2 chain expression early in the course of T cell differentiation and thereby to shut off IL-12 signaling and IFN- production (10, 11, 12, 13). However, in the present study, we showed that BALB/c-background IL-12R2 chain-transgenic mice that maintain IL-12R2 chain expression (i.e., IL-12 signaling) in the face of IL-4 stimulation nevertheless manifest a nonhealing phenotype characterized by footpad swelling and parasite loads identical with that seen in wild-type littermates not carrying the transgene. In addition, by analysis of cytokine production by isolated cells obtained from L. major-infected transgenic and wild-type littermate mice, we showed that a strong Th2 cytokine response occurred in the transgenic mice that was equivalent to that in the wild-type mice. Taken together, these data provide unequivocal evidence that down-regulation of IL-12R2 chain is not necessary for successful Th2 differentiation in vivo and that the latter can occur in the face of continued IL-12 signaling. They thus complement earlier in vitro studies showing that priming of CD4⁺ T cells expressing a competent IL-12R with IL-4 induces Th2 differentiation.

In further studies designed to probe the role of the IL-12 signaling and the IL-12R2 chain in the regulation of T cell responses, we determined whether L. major infection in IL-12R2 chain-transgenic mice would be influenced by IL-12 treatment at the initiation of infection or 1 wk later. In other words, we determined whether the continued presence of the IL-12R2 chain could revert the Th2 response to a Th1 response. We found that although IL-12-treated transgenic mice harbored fewer parasites and showed delayed footpad swelling compared with nontreated mice, late IL-12 treatment was not able to control infection in either the wild-type or the IL-12R2 chain-transgenic mouse. Even more surprisingly, late IL-12 treatment of IL-12R2 chain-transgenic mice led to a poorer outcome than observed in late IL-12-treated wild-type littermates, i.e., treated transgenic mice were less able to handle infection than littermates. This was attributable to the fact that late IL-12 treatment of IL-12R2 chain-transgenic mice led to more cells producing IL-4 in comparison to treated wild-type littermates. Thus, IL-12 abetted the Th2 response rather than caused its reversal.

One possible explanation of this paradoxical finding is that more L. major-specific IL-4-producing cells are present in IL-12R2-chain-transgenic mice than in wild-type littermates at the time of initial infection that are subsequently driven by IL-12 to further expand via the latter’s proliferative effects. However, this possibility is unlikely because draining lymph node cells from IL-12R2 chain-transgenic mice produced as much IL-4 mRNA (Fig. 4) as those from wild-type littermates during the first week of infection.

Another possible explanation is that IL-12 drives NK T cells (CD4⁺/DX5⁺ cells) to produce IL-4 because even in wild-type littermates these cells express IL-12R2 chain and can respond to IL-12. However, it recently was reported that NK T cells produce more IFN-, not more IL-4, when stimulated with anti-CD3 + IL-12 than with anti-CD3 alone (27). Thus, it is unlikely that NK T cells are the source of increased IL-4 when cells from draining lymph nodes of IL-12R2 chain-transgenic mice are stimulated with Leishmania Ag. This view is further supported by the finding that NK T cell-deficient mice have a greater parasite burden than wild-type mice after L. major infection, suggesting that NK T cells activated by IL-12 actually protect mice from L. major infection (28).

A final and most likely explanation for the increased IL-4 in mice treated with IL-12 at 7days of infection is that in transgenic mice, Th2 cells developing in response to Leishmania stimulation (as compared with the Th2 cells in wild-type mice) undergo increased expansion when exposed to the exogenous IL-12 because they maintain IL-12R2 chain expression (14, 15). More specifically, in IL-12R2 chain-transgenic mice given late IL-12 treatment, Th2 cells exhibit either increased proliferation or decreased apoptosis which leads, in turn, to an exaggerated IL-4 response; in contrast, in wild-type littermates given late IL-12 treatment, the same Th2 cells are not expanded by IL-12 because they lack IL-12R2 chain expression. This view is supported by our own cytokine studies with isolated CD4⁺ cells from draining lymph nodes, which showed that CD4⁺ T cells from IL-12R2 chain-transgenic mice treated with late IL-12 secreted more IL-4 in response to Leishmania Ag than those from wild-type mice treated with late IL-12 (3269 pg/ml vs 1701 pg/ml). In addition, it is supported by studies of human CD4⁺ T cells where the Th1/Th2 dichotomy is not as clear as in mice (29), in which it is reported that Th0/Th2 clones proliferate and produce IL-4 in response to IL-12 because of the maintenance of IL-12R2 chain on those clones (30, 31). Finally, it is supported by an in vivo study reported by Bliss et al. (32) in which it was found that in vivo immunization of mice with Ag plus IL-12 enhances Ag-specific Th2 type cytokine and Ab responses as well as Th1 responses. It should be noted that the enhanced IL-4 responses of IL-12R2 chain-transgenic mice to IL-12 lead to the somewhat unexpected conclusion that although down-regulation of IL-12R2 chain on developing Th2 cells is not necessary for Th2 differentiation, it may nevertheless be necessary for establishment of Th1 differentiation, because otherwise a nascent Th2 response greatly enhanced by IL-12 would continue and down-regulate the Th1 response.

In summary, these studies shed new light on the immunologic mechanisms that underlie healing and nonhealing responses to L. major in various mouse strains, and in so doing on the factors that regulate T cell differentiation in vivo. As noted previously, L. major infection induces a Th1 cell-dominated response via IL-12 signaling and STAT4 activation in C57BL/6 mice that results in a controlling (healing) phenotype; we show in initial studies that in these mice, CD4⁺/IL-12R2 chain⁺ T cells that are the main source of the IFN-. A different situation is obtained in BALB/c mice, where L. major infection induces a Th2/IL-4-dominated response that renders the responding T cells incapable of undergoing Th1 differentiation. We demonstrated that this effect of IL-4 does not depend on the ability of IL-4 to down-regulate the IL-12R2 chain and that continued presence of the latter in IL-12R2 chain-transgenic mice is still associated with a Th2/IL-4-dominated response. In fact, in such mice, continued IL-12 signaling further increases the Th2 response. Thus, the ability of IL-4 signaling to induce Th2 differentiation in vivo is not dependent on down-regulation of IL-12 signaling but rather on as yet poorly understood intracellular events. One possibility that requires further exploration in this regard is the recent finding that IL-4 induces GATA-3, which both facilitates Th2 cytokine gene transcription and blocks Th1 cytokine gene (IFN- gene) activation (33).

	Acknowledgments

 
We thank Dr. David Sacks (Laboratory of Parasitic Disease, National Institute of Allergy and Infectious Diseases) for providing the L. major parasite and technical advice and Dr. Rolf Ehrhardt (Protein Design Labs, Fremont, CA) for providing mouse IL-12R₂ chain mAb (PDL-HAM10B9). We also thank Dr. J. Hewitt and the staff of National Institute of Allergy and Infectious Diseases transgenic facility for creatingIL-12R2 chain-transgenic mice and maintaining its colony, S. Barbieri for FACS sorting, and S. Kaul for secretarial assistance. Finally, we would like to thank Drs. Alan Sher (Laboratory of Parasite Disease) and David Sacks for critical reading of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Warren Strober, Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1890. E-mail address: wstrober{at}niaid.nih.gov

² Abbreviation used in this paper: SLA, soluble Leishmania major Ag.

Received for publication January 30, 2001. Accepted for publication March 27, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Reiner, S. L., R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[Medline]
2. Heinzel, F. P., M. D. Sadick, B. J. Holaday, R. L. Coffman, R. M. Locksley. 1989. Reciprocal expression of interferon or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59.[Abstract/Free Full Text]
3. Green, S. J., C. A. Nacy, M. S. Meltzer. 1991. Cytokine-induced synthesis of nitrogen oxides in macrophages: a protective host response to Leishmania and other intracellular pathogens. J. Leukocyte Biol. 50:93.[Medline]
4. Stenger, S., N. Donhauser, H. Thuring, M. Rollinghoff, C. Bogdan. 1996. Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase. J. Exp. Med. 183:1501.[Abstract/Free Full Text]
5. Launois, P., T. Ohteki, K. Swihart, H. R. MacDonald, J. A. Louis. 1995. In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4⁺ T cells which are NK1.1^-. Eur. J. Immunol. 25:3298.[Medline]
6. Launois, P., K. G. Swihart, G. Milon, J. A. Louis. 1997. Early production of IL-4 in susceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness. J. Immunol. 158:3317.[Abstract]
7. Launois, P., I. Maillard, S. Pingel, K. G. Swihart, I. Xenarios, H. Acha-Orbea, H. Diggelmann, R. M. Locksley, H. R. MacDonald, J. A. Louis. 1997. IL-4 rapidly produced by V4V8 CD4⁺ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
8. 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.[Abstract]
9. 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.[Abstract]
10. Szabo, S. J., A. S. Dighe, U. Gubler, K. M. Murphy. 1997. Regulation of the interleukin (IL)-12R 2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817.[Abstract/Free Full Text]
11. 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 2-chain expression on CD4⁺ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol. 161:6156.[Abstract/Free Full Text]
12. Jones, D., M. M. Elloso, L. Showe, D. Williams, G. Trinchieri, P. Scott. 1998. Differential regulation of the interleukin-12 receptor during the innate immune response to Leishmania major. Infect. Immun. 66:3818.[Abstract/Free Full Text]
13. Himmelrich, H., P. Launois, I. Maillard, T. Biedermann, F. Tacchini-Cottier, R. M. Locksley, M. Rocken, J. A. Louis. 2000. In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. J. Immunol. 164:4819.[Abstract/Free Full Text]
14. Heath, V. L., L. Showe, C. Crain, F. J. Barrat, G. Trinchieri, A. O’Garra. 2000. Cutting edge: ectopic expression of the IL-12 receptor-2 in developing and committed Th2 cells does not affect the production of IL- 4 or induce the production of IFN-. J. Immunol. 164:2861.[Abstract/Free Full Text]
15. Nishikomori, R., R. O. Ehrhardt, W. Strober. 2000. T helper type 2 cell differentiation occurs in the presence of interleukin 12 receptor 2 chain expression and signaling. J. Exp. Med. 191:847.[Abstract/Free Full Text]
16. Belkaid, Y., S. Mendez, R. Lira, N. Kadambi, G. Milon, D. Sacks. 2000. A natural model of Leishmania major infection reveals a prolonged "silent" phase of parasite amplification in the skin before the onset of lesion formation and immunity. J. Immunol. 165:969.[Abstract/Free Full Text]
17. Cohen, J. H., J. P. Aubry, M. H. Jouvin, J. Wijdenes, J. Bancherau, M. Kazatchkine, J. P. Revillard. 1987. Enumeration of CR1 complement receptors on erythrocytes using a new method for detecting low density cell surface antigens by flow cytometry. J. Immunol. Methods 99:53.[Medline]
18. Bouaboula, M., P. Legoux, B. Pessegue, B. Delpech, X. Dumont, M. Piechaczyk, P. Casellas, D. Shire. 1992. Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control. J. Biol. Chem. 267:21830.[Abstract/Free Full Text]
19. Slifka, M. K., R. R. Pagarigan, J. L. Whitton. 2000. NK markers are expressed on a high percentage of virus-specific CD8⁺ and CD4⁺ T cells. J. Immunol. 164:2009.[Abstract/Free Full Text]
20. Sypek, J. P., C. L. Chung, S. E. Mayor, J. M. Subramanyam, S. J. Goldman, D. S. Sieburth, S. F. Wolf, R. G. Schaub. 1993. Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med. 177:1797.[Abstract/Free Full Text]
21. Heinzel, F. P., D. S. Schoenhaut, R. M. Rerko, L. E. Rosser, M. K. Gately. 1993. Recombinant interleukin 12 cures mice infected with Leishmania major. J. Exp. Med. 177:1505.[Abstract/Free Full Text]
22. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN production and type 1 cytokine responses. Immunity 4:471.[Medline]
23. Wu, C., J. Ferrante, M. K. Gately, J. Magram. 1997. Characterization of IL-12 receptor 1 chain (IL-12R1)-deficient mice: IL-12R1 is an essential component of the functional mouse IL- 12 receptor. J. Immunol. 159:1658.[Abstract]
24. Wu, C., X. Wang, M. Gadina, J. J. O’Shea, D. H. Presky, J. Magram. 2000. IL-12 receptor ss2 (IL-12Rss2)-deficient mice are defective in IL-12- mediated signaling despite the presence of high affinity IL-12 binding sites. J. Immunol. 165:6221.[Abstract/Free Full Text]
25. Carter, L. L., K. M. Murphy. 1999. Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon production from CD4⁺ versus CD8⁺ T cells. J. Exp. Med. 189:1355.[Abstract/Free Full Text]
26. Rogge, L., A. Papi, D. H. Presky, M. Biffi, L. J. Minetti, D. Miotto, C. Agostini, G. Semenzato, L. M. Fabbri, F. Sinigaglia. 1999. Antibodies to the IL-12 receptor 2 chain mark human Th1 but not Th2 cells in vitro and in vivo. J. Immunol. 162:3926.[Abstract/Free Full Text]
27. Leite-De-Moraes, M. C., A. Hameg, M. Pacilio, Y. Koezuka, M. Taniguchi, L. Van Kaer, E. Schneider, M. Dy, A. Herbelin. 2001. IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells. J. Immunol. 166:945.[Abstract/Free Full Text]
28. Ishikawa, H., H. Hisaeda, M. Taniguchi, T. Nakayama, T. Sakai, Y. Maekawa, Y. Nakano, M. Zhang, T. Zhang, M. Nishitani, M. Takashima, K. Himeno. 2000. CD4⁺ v14 NKT cells play a crucial role in an early stage of protective immunity against infection with Leishmania major. Int. Immunol. 12:1267.[Abstract/Free Full Text]
29. Romagnani, S.. 1991. Human TH1 and TH2 subsets: doubt no more. Immunol. Today 12:256.[Medline]
30. Hu, C., P. Salgame. 1999. Inability of interleukin-12 to modulate T-helper 0 effectors to T-helper 1 effectors: a possible distinct subset of T cells. Immunology 97:84.[Medline]
31. Jeannin, P., Y. Delneste, P. Life, J. F. Gauchat, D. Kaiserlian, J. Y. Bonnefoy. 1995. Interleukin-12 increases interleukin-4 production by established human Th0 and Th2-like T cell clones. Eur. J. Immunol. 25:2247.[Medline]
32. Bliss, J., V. Van Cleave, K. Murray, A. Wiencis, M. Ketchum, R. Maylor, T. Haire, C. Resmini, A. K. Abbas, S. F. Wolf. 1996. IL-12, as an adjuvant, promotes a T helper 1 cell, but does not suppress a T helper 2 cell recall response. J. Immunol. 156:887.[Abstract]
33. Ouyang, W., S. H. Ranganath, K. Weindel, D. Bhattacharya, T. L. Murphy, W. C. Sha, K. M. Murphy. 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4- independent mechanism. Immunity 9:745.[Medline]

This article has been cited by other articles:

				A. P. Nigg, S. Zahn, D. Ruckerl, C. Holscher, T. Yoshimoto, J. M. Ehrchen, F. Wolbing, M. C. Udey, and E. von Stebut Dendritic Cell-Derived IL-12p40 Homodimer Contributes to Susceptibility in Cutaneous Leishmaniasis in BALB/c Mice J. Immunol., June 1, 2007; 178(11): 7251 - 7258. [Abstract] [Full Text] [PDF]

				Y. Charoenvit, G. T. Brice, D. Bacon, V. Majam, J. Williams, E. Abot, H. Ganeshan, M. Sedegah, D. L. Doolan, D. J. Carucci, et al. A Small Peptide (CEL-1000) Derived from the {beta}-Chain of the Human Major Histocompatibility Complex Class II Molecule Induces Complete Protection against Malaria in an Antigen-Independent Manner Antimicrob. Agents Chemother., July 1, 2004; 48(7): 2455 - 2463. [Abstract] [Full Text] [PDF]

				E. von Stebut, J. M. Ehrchen, Y. Belkaid, S. L. Kostka, K. Molle, J. Knop, C. Sunderkotter, and M. C. Udey Interleukin 1{alpha} Promotes Th1 Differentiation and Inhibits Disease Progression in Leishmania major-susceptible BALB/c Mice J. Exp. Med., July 21, 2003; 198(2): 191 - 199. [Abstract] [Full Text] [PDF]

				P. Kropf, S. Herath, R. Klemenz, and I. Muller Signaling through the T1/ST2 Molecule Is Not Necessary for Th2 Differentiation but Is Important for the Regulation of Type 1 Responses in Nonhealing Leishmaniamajor Infection Infect. Immun., April 1, 2003; 71(4): 1961 - 1971. [Abstract] [Full Text]

				G.-X. Zhang, B. Gran, S. Yu, J. Li, I. Siglienti, X. Chen, M. Kamoun, and A. Rostami Induction of Experimental Autoimmune Encephalomyelitis in IL-12 Receptor-{beta}2-Deficient Mice: IL-12 Responsiveness Is Not Required in the Pathogenesis of Inflammatory Demyelination in the Central Nervous System J. Immunol., February 15, 2003; 170(4): 2153 - 2160. [Abstract] [Full Text] [PDF]

				J. Li, U. M. Padigel, P. Scott, and J. P. Farrell Combined Treatment with Interleukin-12 and Indomethacin Promotes Increased Resistance in BALB/c Mice with Established Leishmania major Infections Infect. Immun., October 1, 2002; 70(10): 5715 - 5720. [Abstract] [Full Text] [PDF]

				F. Aguilar Torrentera, J. D. Laman, M. Van Meurs, L. Adorini, E. Muraille, and Y. Carlier Endogenous Interleukin-12 Is Critical for Controlling the Late but Not the Early Stage of Leishmania mexicana Infection in C57BL/6 Mice Infect. Immun., September 1, 2002; 70(9): 5075 - 5080. [Abstract] [Full Text] [PDF]

				L. Gorelik, S. Constant, and R. A. Flavell Mechanism of Transforming Growth Factor {beta}-induced Inhibition of T Helper Type 1 Differentiation J. Exp. Med., June 3, 2002; 195(11): 1499 - 1505. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nishikomori, R. Articles by Strober, W. Search for Related Content PubMed PubMed Citation Articles by Nishikomori, R. Articles by Strober, W.