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


     
 

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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stephenson, L.
Right arrow Articles by Boothby, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stephenson, L.
Right arrow Articles by Boothby, M.
The Journal of Immunology, 2004, 173: 4523-4528.
Copyright © 2004 by The American Association of Immunologists

An IL-4R{alpha} Allelic Variant, I50, Acts as a Gain-of-Function Variant Relative to V50 for Stat6, But Not Th2 Differentiation1

Linda Stephenson, Mary H. Johns, Emily Woodward, Ana L. Mora2 and Mark Boothby3

Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Signaling through the IL-4R {alpha}-chain (IL-4R{alpha}) is crucial for the development of Th2 cells, central effectors in atopic disease. Alleles of the IL-4R{alpha} have been identified that have been variably associated with increased incidence of allergic disease, but there is little direct evidence that any variant is sufficient to alter a target that determines allergic pathophysiology or susceptibility. Variants of IL-4R{alpha} encoding isoleucine instead of valine at position 50 (I50 vs V50, respectively) can signal increased Stat6-dependent transcriptional activity, whether in an I50, Q551 or I50, R551 haplotype. Strikingly, signaling through these receptors did not increase the efficiency of Th2 development or the IL-4 mediated repression of Th1 development or a target gene, IL-18R{alpha}. Further, IL-4-induced proliferation was similar for Th2 cells independent of the variant expressed. Together these findings indicate that IL-4R{alpha} variants that exhibit gain-of-function with respect to Stat6 do not act directly through alterations in Th2/Th1 induction after Ag exposure. The data further suggest that for such variants, any mechanistic involvement is based on a role in cellular targets of Th2 cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Atopy, an immune disorder characterized by the generation of inflammatory responses to environmental Ags, underlies the clinical manifestations of allergic rhinitis and the majority of asthma cases. Although triggered by environmental exposure, the propensity to develop atopy is heritable (1). Linkage studies have identified several regions associated with an increased risk of disease development, including genes encoding the Th2 cytokine cluster (including IL-4, IL-5, and IL-13) and the IL-4R {alpha}-chain (IL-4R{alpha}) (2, 3, 4, 5). IL-4 and the ligand binding chain of its receptor have been particularly attractive as candidate genes for atopic disease susceptibility because of their central roles in promoting the immune responses responsible for atopy.

IL-4 is a pleiotropic cytokine that acts at multiple stages in the pathophysiology of allergic disease. IL-4 initiates class switching to the IgE subtype in B cells and directs the development of Th2 cells, the primary effectors of atopy (6, 7). In response to IL-4, endothelial cells increase their expression of adhesion molecules, resulting in an increased egress of eosinophils into sites of inflammation (7). To accomplish these actions, IL-4 signals through heterodimeric receptor complexes containing the IL-4R{alpha} and an accessory chain, either {gamma} common or IL-13R{alpha} (8, 9, 10). Binding of IL-4 to its receptor results in the activation of resident Jak kinases and the subsequent activation of downstream effector proteins such as Stat6 (9). Induction of Stat6 is essential for the efficient initiation of IL-4-mediated developmental programs. Targeted gene disruption studies demonstrate the requirement for Stat6 in the IL-4-initiated development programs in both T and B cells. Stat6-deficient mice exhibit severe impairment of Th2 development, Ab class switching to IgE, and up-regulation of the low affinity IgE receptor (CD23) (11, 12, 13). Gene ablation studies demonstrate that Stat6-deficient mice fail to develop allergic lung inflammation and airway hyper-responsiveness, highlighting the importance of this pathway in disease development (14). Together these findings suggest that any dysregulation in the IL-4R/Stat6 signaling axis that leads to enhanced Stat6 activity might cause an increase in Th2 cells and an increased predisposition to developing atopic disease.

Alleles of IL-4R{alpha} have been identified that correlate with an increased risk of developing atopic disease, hyperIgE syndrome, or atopic asthma in multiple populations (15, 16). One such variant, in which isoleucine replaces valine at position 50 (I50 and V50, respectively), has been associated with increased IgE levels and asthma in a study of a Japanese cohort (17). Studies performed in immortalized cell lines indicate that the I50 variant mediates a modest increase in the induction of a Stat6-responsive reporter and proliferation in response to IL-4 stimulation. A variant of IL-4R in which an arginine replaces a glutamine at position 551 (R551 and Q551, respectively) was identified as being enriched in patients with allergic inflammatory disorders (18). Additional work has suggested that the pertinent susceptibility trait is, in fact, a haplotype in which V50 is linked to R551 (19). However, it remains unclear whether any of these allelic variants could increase the strength of a Th2 response or influence target genes critical to the onset of allergic inflammation. Considering the central roles of Th2 cells in initiating atopic disease and IL-4 signaling in Th2 development, we have investigated whether IL-4Rs containing allelic variant substitutions can increase proliferation or Th2 development in CD4+ T cells.


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

IL-4R{alpha}-deficient mice on a BALB/c background (gift from N. Noben-Trauth, Department of Immunology, George Washington University Medical Center, Washington, DC) were bred with DO11.10 TCR transgenic BALB/c mice. Resultant IL-4R{alpha}–/– and DO11.10 transgenic mice were genotyped by PCR (10). Mice were maintained in microisolator cages under specific pathogen-free conditions and used with institutional approval in accordance with applicable regulations.

Plasmids and mutagenesis

Full-length huStat6 cDNA was cloned into the MSCV-IRES-Thy1.1 retrovector and mouse (m)4 IL-4R{alpha} cDNA (C57BL/6) was cloned into MSCV2.2-IRES-GFP (GFP-RV). Using a C57BL/6-derived cDNA (I50, Q551 allelotype) (8, 20) as a template, amino acid substitutions were introduced into the mIL-4R {alpha}-chain by PCR using Pfu turbo polymerase (Stratagene, La Jolla, CA) and were selected for using DpnI cleavage of the products. For consistency with the existing literature, mutations made in the mIL-4R{alpha} are referred to by the amino acid position of the highly homologous human (hu) IL-4R{alpha} sequence (9) in place of the actual amino acid residue number in the primary mIL-4R{alpha} sequence. PCR primers used for IL-4R{alpha} mutagenesis were (listing one strand only): I50V (mouse I51) 5'-GAAAACCTCACATGCGTCCCGAGGAACAGTGC-3'; and R551Q (mouse Q552) 5'-CTGCCGGTGGCTACCGGGAGTTTGTGCAGGC. Underlined sequences are the sites of mutated residues.

Reagents and generation of cell lines

Purified recombinant mIL-4 as well as purified and fluorochrome-conjugated Abs against IL-4 (allophycocyanin), Thy 1.1 (PE), IFN-{gamma} (PE), rat IgG (biotin), and streptavidin (PerCP and rPE) were obtained from BD Pharmingen (San Diego, CA). IL-18R{alpha} Ab and biotinylated anti-goat IgG were obtained from R&D Systems (Minneapolis, MN) and Jackson ImmunoResearch Laboratories (West Grove, PA), respectively. Alexa 647-BrdU was obtained from Molecular Probes (Eugene, OR), M1 anti-IL-4R{alpha} was a gift from Immunex, and HuIL-2 was a gift from Biologic Response Modifiers Program (Frederick, MD). Human T leukemic Jurkat cells were cultured in complete IMDM supplemented with 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, and 50 µM 2-ME (referred to hereafter as IMDM-10). 293T cells were cultured in complete DMEM supplemented as described for IMDM-10 and with MEM amino acids. To create the Stat6-expressing cell line S6J, vesicular stomatitis virus-G-pseudotyped retrovirions were produced by cotransfecting 293T cells with MSCV-IRES-Thy1.1-Stat6, pSV-gp and pHCMV-g using calcium phosphate-mediated transfection (21). Virus-containing supernatants were harvested 48 h after transfection. Jurkat T cells were transduced by overnight incubation with virus-containing supernatant and polybrene (8 µg/ml). Thy1.1+ progeny were isolated by FACS 4 days after infection. These cells were divided and reinfected with vesicular stomatitis virus-G pseudotyped virions containing MSCV-GFP-mIL-4R constructs. After preparative sorting for equivalent GFP and Thy1.1 expression, mIL-4R{alpha} expression was determined using the M1 anti-mIL-4R.

Flow cytometric analysis and intracellular staining

Mouse IL-4R{alpha} was detected by three-step indirect immunofluorescent staining with M1 anti-receptor Ab, followed by biotinylated anti-rat IgG and streptavidin-PerCP. For intracellular cytokine staining, polarized cells were stimulated for 6 h with PMA (50 ng/ml) plus ionomycin (1 µg/ml). Monesin (2 µM) was added to the cell culture for the final 4 h. Cells were stained with Abs against mIL-4R{alpha}, fixed with 4% paraformaldehyde, and permeabilized in PBS/0.5% saponin/1% BSA before staining with anti-IL-4 and anti-IFN-{gamma}. BrdU was detected by permeabilizing 4% paraformaldehyde fixed cells with PBS/0.5% saponin/1% BSA. After DNase treatment to expose BrdU epitopes, cells were stained with anti-BrdU Abs. Indirect immunofluorescent staining for surface IL-18R was performed by a three-step procedure using anti-IL-18R Abs, followed by biotinylated anti-goat IgG and then streptavidin-PE. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences, San Diego, CA) and were analyzed using CellQuest (BD Biosciences) or Flo-Jo (Tree Star, Palo Alto, CA) software.

Retroviral transduction and Th2 polarization

Retrovirus-containing supernatants were collected 48 h after transfection of the {Phi}NX ecotropic packaging cell line with retrovector plasmids, mixed with OVA323–339 peptide-activated splenocytes from IL-4R{alpha}–/– DO11.10 TCR transgenic mice, and centrifuged (1 h at 10,000 x g) as previously described (22, 23). Single-cell suspensions were prepared from spleen and lymph nodes as previously described (24). Cells were cultured in the presence of purified recombinant mIL-4, huIL-2 (10 ng/ml), anti-IFN-{gamma} (1 µg/ml), and anti-IL-12 (1 µg/ml). The expression of IL-4R{alpha} was determined by flow cytometric analysis 3 days post-transduction. Five days after transduction, cells were washed, sorted for GFP+ cells, and restimulated with peptide-pulsed BALB/c APCs (1 µg/ml with a 1:1 APC:T cell ratio). Supernatants were collected 48 h after stimulation, and cytokine production was determined by ELISA. The frequency of cytokine-producing cells was determined by intracellular staining 5 days after transduction.

Proliferation and reporter gene assays

Three days after transduction, cells were washed and plated in serum-free IMDM for 8 h. Cells were washed again and restimulated with the indicated cytokines for 15 h, then pulsed with BrdU for an additional 6 h in the continual presence of cytokine. Samples were collected and processed for intracellular detection of BrdU by flow cytometry. Jurkat T cells stably transduced with Stat6 and mIL-4R-encoding retrovectors were transiently transfected with the Stat6-responsive luciferase reporter N4-C/EBP using Superfect (Stratagene) according to the manufacturer’s instructions. Cells were plated in IMDM-10 for 16 h, divided into equal aliquots, and treated for 24 h with medium alone, mIL-4, or huIL-4 (5 ng/ml). Extracts of these cells were subjected to luciferase assays as described previously (25). In all experiments, fold induction was normalized to the endogenous huIL-4R [normalized fold induction = fold induction x [huIL-4 RLU(wt)]/[huIL-4 RLU(mutant)].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased transcriptional activity mediated by I50 variants

Stat6 induction by IL-4 activates transcriptional pathways essential for Th2 development (11, 12, 13). Studies performed in B cells suggest that the presence of isoleucine at position 50 in the IL-4R promotes increased Stat6 activation after IL-4 stimulation in receptors that contained either arginine or glutamine at position 551 (17). However, it is not clear in allergic pathophysiology what are the functional implications of a modest increase in Stat6 induction. To determine whether receptors containing I50 would act as gain-of-function variants in T cells, we generated a panel of mouse IL-4R{alpha} constructs with either isoleucine or valine at position 50 (I50 and V50, respectively) and arginine or glutamine at position 551 (R551 and Q551, respectively). Retroviral constructs encoding these cDNAs were transduced into a Stat6-expressing T cell line to create polyclonal populations expressing each variant of the IL-4R. As determined by FACS analysis, cell surface expression of mIL-4R{alpha} on each of the lines was equivalent (Fig. 1A).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 1. The I50 variant of IL-4R{alpha} promotes increased Stat6 transcriptional activity. A, Similar mIL-4R{alpha} expression on Jurkat T cells stably transduced with the panel of mIL-4R variants. The expression of mIL-4R{alpha} on polyclonal populations of Jurkat human T cells generated using retrovectors encoding the indicated IL-4R{alpha} variant was determined by FACS analyses using the M1 mAb against the mIL-4R{alpha} ectodomain. The data are presented as a histogram overlay of the receptor indicated (bold line) and the M1 staining from Jurkat cells transduced with vector only (dotted line). B, IL-4 dose response of Stat6-dependent transcription. Jurkat T cells expressing the allelic variants were transiently transfected with a Stat6-dependent reporter, stimulated with the indicated concentration of IL-4 for 24 h, and assayed for luciferase activity. In each of four independent experiments, IL-4 concentrations spanned the entire dose range (0.25–10 ng/ml). The two graph panels represent the mean (±SEM) dose-response curve, but have been split to allow better resolution of the data at the low IL-4 doses. C, Jurkat T cells expressing the allelic variant mIL-4Rs were transiently transfected with a Stat6-dependent reporter and stimulated with mIL-4 at 0.25 or 5.0 ng/ml for 24 h, after which luciferase activities were measured. Shown are mean (±SEM) data from four independent experiments.

 
In considering the origins of atopy, it is likely that the levels of IL-4 initiating Th2 development are quite low. Although the cells producing the initial IL-4 have not been completely elucidated, we hypothesized that the IL-4 produced may be at concentrations that are limiting for IL-4-induced signaling or regulation of genes downstream from the receptor. To determine the ability of the panel of allelic variant receptors to mediate the induction of a Stat6-responsive luciferase reporter over a range of concentrations, a dose-response experiment was performed in Jurkat T cells. The cell lines were transfected with a Stat6-dependent luciferase reporter, stimulated with increasing doses of IL-4, and analyzed. As expected, cell lines not expressing mIL-4R exhibited no inducible reporter activity after IL-4 stimulation. As shown in Fig. 1, B and C, it was determined that receptors containing isoleucine at position 50 promoted an increased induction of the Stat6-responsive reporter when IL-4 was present at concentrations less than 1 ng/ml. Induction of luciferase activity was similar for all variants when IL-4 concentrations were saturating (5 ng/ml). In contrast, receptors containing I50 (IQ and IR) promoted an ~2-fold increase in transcription compared with receptors containing V50 when stimulated by IL-4 at a limiting concentration (0.25 ng/ml). The presence of the R551 mutation did not augment induction of the reporter when present on either the I50 or V50 receptor. Together these results indicate that under limiting IL-4 conditions, the atopy-associated I50 mIL-4R variants transduced a more potent activation of Stat6 in T cells than the V50 IL-4R{alpha} variants. These data suggest that I50 variants of the IL-4R can act as a gain-of-function receptor with regard to transcriptional activity mediated by Stat6. However, when IL-4 is present at saturating levels, all receptor variants promote Stat6 induction with similar efficiency.

Equivalent Th2 development in cells expressing allelic variant IL-4R{alpha} chains

Having determined that the presence of isoleucine at position 50 increases Stat6 transcriptional activity under conditions of limiting IL-4 availability, we next investigated whether this increased Stat6 transcriptional activity would translate into an increase in IL-4-mediated Th2 development. Although the activation of Stat6 is critical for efficient Th2 development, it is not known whether an increase in receptor-mediated Stat6 activation would promote an increase in Th2 development, or if receptor-mediated Stat6 induction is already optimized (at a saturating level on a dose-response curve for differentiation efficiency) in T cells. To determine whether the increased Stat6 activation mediated by the I50 variant increased the efficiency of Th2 development, we developed an IL-4R reconstitution system based on the use of lymphoid cells from IL-4R{alpha}-deficient mice. IL-4R{alpha}-deficient DO11.10 TCR transgenic cells were activated in vitro with antigenic peptide and transduced with replication-defective retroviruses encoding the allelic variant IL-4Rs. After transduction, cells were cultured under Th2-polarizing conditions with increasing concentrations of IL-4. Of note, cell surface staining performed 2 days after transduction showed that each allelic variant was expressed at similar levels on transduced (GFP+) cells (Fig. 2A). Six days after transduction, cells were restimulated and assayed for markers of Th2 development. Importantly, only cells that were GFP+ and expressed an IL-4R developed into Th2 cells (Fig. 2B).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. IL-4R{alpha} reconstitution on IL-4R{alpha}-deficient T cells promotes efficient Th2 development. A, Similar mIL-4R{alpha} expression on cells transduced with the indicated retrovectors. Three days after transduction of IL-4R{alpha}-deficient T cells with the indicated constructs (empty, vector only, with no IL-4R{alpha} cDNA), the expression of IL-4R{alpha} on primary IL-4R{alpha}–/– T cells was determined using the M1 mAb as described in Fig. 1. B, Only GFP+ (IL-4R{alpha}+) cells differentiate into Th2 cells. After restimulation with APCs, cells transduced with the GFP-RV (Empty) and mIL-4R{alpha}(I50)-GFP-RV (mIL-4R{alpha}(I50)) were processed for intracellular staining of IL-4 and IFN-{gamma}. The GFP– and GFP+ populations were separately analyzed as indicated. IL-4R{alpha}+/+, IL-4R{alpha} expressing T cells included as a positive control.

 
As demonstrated in Fig. 2B, although receptor expression is delayed in this system, robust Th2 development was observed. To measure Th2 development in retrovirally transduced cells, IL-4 production was detected by intracellular staining after restimulation. The percentage of cells that produced IL-4 was similar for all receptor variants at all IL-4 concentrations (Fig. 3, A and B). In cells polarized in the presence of the limiting concentration of IL-4, the quantities of IL-4 and IL-5 produced by cells expressing any of the receptor variants were comparable (Fig. 3C). These data indicate that signaling through receptors containing the I50 variant did not significantly enhance Th2 development, even at a limiting concentration of IL-4.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. IL-4R{alpha} allelic variants promote Th2 development with similar efficiency. Shown in each panel are mean (±SEM) data from four independent experiments. A and B, Th2 effectors expressing allelic variants produce similar levels of IL-4 restimulation. Activated DO11.10+ IL-4R{alpha}–/– splenocytes were transduced with retrovectors encoding the indicated IL-4R{alpha}. Transduced splenocytes were polarized for 5 days in Th2 conditions with the indicated concentrations of IL-4, restimulated with PMA and ionomycin for 6 h, then processed for intracellular staining of IL-4. Shown are the mean frequencies of Th2 cells detected in the GFP+ subset of cells from three independent experiments. Note that no pairwise comparison of two receptor variants at any concentration of IL-4 revealed any statistically significant difference. Even pooling the data for I50 vs V50 variants, regardless of sequence at the 551 position, did not uncover any statistically significant difference. C, Th2 effectors expressing allelic variant receptors produce similar levels of IL-4 and IL-5 after antigenic restimulation. DO11.10+ IL-4R{alpha}–/– splenocytes were transduced with retrovectors as described above. Transduced cells were cultured under Th2 conditions using a limiting concentration of IL-4 (0.25 ng/ml) for 5 days, then restimulated with antigenic peptide-pulsed APCs for 48 h. IL-5 and IL-4 production was measured by ELISA.

 
Allelic variant receptors mediate equivalent IL-4 induced proliferation

Several studies suggest that Stat6 plays a key role in IL-4-mediated proliferation (26, 27). Although no increase in Th2 effector frequency was observed, it is possible that an allelic variant receptor increases the IL-4-mediated proliferative response via enhanced activation of Stat6. Such an increased proliferative response would mediate the growth of a larger population of Th2 effectors in vivo, providing a mechanism by which an allelic variant receptor could promote an increased risk of atopic disease. Accordingly, we measured whether the variants increase the IL-4-mediated proliferative response secondary to increased Stat6 activation. Transduced cells expressing the allelic variant receptors were grown for 3 days in culture, rested, then treated with increasing amounts of IL-4 for 21 h. Low levels of basal proliferation were observed in cells grown in medium alone or in cells not expressing an IL-4R (empty). In response to cytokine, samples expressing IL-4Rs were induced to proliferate in a dose-responsive manner that was similar for each member of the panel of variants (Fig. 4). Additional studies indicated that IL-4-induced increases in cell number were similar in comparisons among the receptor variants (data not shown). We conclude that the presence of isoleucine at position 50 does not increase the magnitude of the IL-4-induced proliferative response in T cells.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4. IL-4R{alpha} allelic variants promote similar levels of IL-4-induced S phase entry. DO11.10+ IL-4R{alpha}–/– splenocytes were transduced with retrovectors encoding the indicated IL-4R{alpha} and grown under Th2 conditions for 3 days. Cells were rinsed and rested in serum-free medium before being treated with the indicated concentrations of IL-4. After 15 h, the cells were pulsed with BrdU and cultured for an additional 6 h. The IL-4-induced proliferation (percentage of BrdU positive) is defined as the percentage of BrdU incorporated after IL-4 stimulation. Shown are the mean (±SEM) results from two independent experiments.

 
Repression of IL-18R expression and Th1 development in cells expressing allelic variants of the mIL-4R

Naive CD4+ T cells express an IL-18R whose expression promotes Th1 differentiation and function, but is repressed by IL-4 signaling through Stat6. T cells increase the expression of this receptor during differentiation into Th1 effectors, a process requiring both IFN-{gamma} and IL-12 (28, 29). Therefore, we used this target gene to determine whether allelic variants of the IL-4R{alpha} increase the efficiency of IL-18R down-regulation. The expression of the IL-18R on activated cells transduced with the allelic variant receptors was analyzed 5 days after transduction. Transduced cells were grown in Th2 conditions with increasing concentrations of IL-4, then analyzed for IL-18R expression. As expected, cells that did not express an IL-4R (empty) did not down-regulate the expression of IL-18R in response to IL-4. IL-18R expression was suppressed to a similar degree on all IL-4R-bearing samples (Fig. 5A). This down-regulation was maximally achieved by an IL-4 concentration of 0.5 ng/ml. These data indicate that IL-4 signals to suppress IL-18R expression efficiently, and that the level of Stat6 induction mediated by receptors containing valine at position 50 is already sufficient to achieve maximal effect.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 5. IL-4R{alpha} allelic variants promote equivalent inhibition of Th1 development. A, DO11.10+ IL-4R{alpha}–/– splenocytes were transduced with retrovectors encoding the indicated IL-4R{alpha} variants. Transduced cells were polarized under Th2 conditions with the indicated concentration of IL-4 for 5 days, then stained with Abs against the mIL-18R or with an isotype control. The change in mean fluorescence intensity ({Delta}MFI) is defined as: MFI of IL-18R stain on cells transduced with the indicated mIL-4R{alpha} – the MFI of the isotype control. B, Activated DO11.10+ IL-4R{alpha}–/– splenocytes were transduced with retrovectors encoding the indicated IL-4R{alpha}, cultured for 5 days in Th2 conditions (0.25 ng/ml IL-4), restimulated with PMA and ionomycin for 6 h, then processed for intracellular staining of IFN-{gamma}. Shown are the mean (±SEM) results from three independent experiments.

 
IFN-{gamma} released by Th1 cells blocks the ability of Th2 cytokines to elicit allergic inflammation and clinical manifestations (30, 31, 32). In addition to promoting Th2 differentiation, IL-4 signaling suppresses the emergence of the Th1 subset of effector cells (9). We therefore investigated the possibility that the increased efficiency of Stat6 induction by an allelic variant would promote allergic disease by repression of the Th1 program, independent from effects on the Th2 response. To determine whether augmented Stat6 activation by the I50-expressing variants enhanced inhibition of Th1 development, we analyzed the production of IFN-{gamma} by transduced cells polarized under IL-4-limiting Th2 conditions. Each IL-4R{alpha} variant mediated suppression of IFN-{gamma} production to a similar degree. The percentage of cells producing IFN-{gamma} was equivalent for cells transduced with allelic variant receptors (Fig. 5B), whereas cells lacking the mIL-4R (i.e., transduced with empty vector) became IFN-{gamma} producers at approximately twice the frequency of IL-4R{alpha}-transduced cells. In a dose-response assay, the emergence of Th1 cells, as measured by IFN-{gamma} production, was maximally achieved at the lowest dose of IL-4 tested (0.1 ng/ml; data not shown). These data indicate that suppression of Th1 development is mediated at similar efficiencies by all receptor variants tested and that this inhibition is efficiently achieved at low levels of IL-4.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The gene encoding IL-4R{alpha} is a candidate susceptibility gene for atopic diseases. Numerous linkage studies demonstrate that the risk of allergic disease maps genetically to this chromosomal region, and specific sequence variants have been reported to be associated with an increased risk of disease development (5, 15, 33). However, these associations appear to be variable, and in no case has a mechanism been established by which variants of the IL-4R{alpha} impact allergic pathophysiology. Although studies of B cells suggest that variants containing isoleucine at position 50 may independently enhance IL-4 signaling, the effect on function in B or T lymphocytes remains unclear. In this study we measured the efficiency with which four allelic variant IL-4Rs induce Th2 development. When linked to Q551 and R551, I50-containing variants signals promoted higher Stat6 transcriptional activity in a T cell line than the V50 receptors stimulated with limiting concentrations of IL-4. Interestingly, although this substitution (I for V) is located in an extracellular domain of the IL-4R, two independent groups have demonstrated that there is no alteration in IL-4 binding affinity for the receptor (17, 19, 34). However, this increase in Stat6 activation did not enhance the ability of these variants to promote Th2 development, as measured by cytokine production or frequency of IL-4-producing cells. IL-4-mediated proliferation was similar for Th2 effectors expressing all receptors. In addition, inhibition of IL-18 receptor expression and suppression of IFN-{gamma}-producing cells were mediated to an equivalent degree by cells expressing all receptor variants. We conclude that in T cells, the presence of neither the I50 nor the R551 substitution significantly influenced the ability of the receptor to induce Th2 development or the Th1/Th2 balance.

Atopy and asthma are complex human diseases resulting from the moderate contributions of many low-penetrant alleles (35, 36). As a result, genome-wide screens for susceptibility genes have demonstrated inconsistencies in the strength of linkage at any one site (36). Specifically, markers flanking the IL-4R{alpha} have shown a strong linkage with atopy, whereas genome-wide screens have failed to show a consistent linkage to this region (36). In addition, no common variant of the IL-4R {alpha}-chain associates with atopic disease across multiple populations (15). Based on these observations, it has been suggested that the true susceptibility allele is not IL-4R{alpha}, but, rather, a closely linked sequence or gene. Alternatively, a given haplotype may confer susceptibility in some populations, but not others (36). The data reported in this study indicate that a role for IL-4R{alpha} variants acting to increase the Th2 effector response or decrease the Th1 response is unlikely. If the IL-4R{alpha} gene is a true susceptibility locus, then gain-of-function variants act either on other IL-4-responsive cells or through its role as an accessory chain for the IL-13R complex. In one study of Japanese asthmatic patients, homozygosity for the I50 variant was significantly associated with atopic, but not nonatopic, asthma, whereas Arg551 did not significantly associate with either condition (17, 34). These results were contradicted by studies of Japanese and Dutch populations that failed to confirm overtransmission of the I50 variant to atopic asthmatics (16, 37). The study that demonstrated overrepresentation of I50 among atopic asthmatics analyzed I50 IL-4R{alpha} signaling in PBMCs. In PBMCs, IL-4 treatment caused increased production of IgE and an increase in up-regulation of the low affinity IgE receptor, CD23 (34). However, this study analyzed a limited sample size of volunteers with heterogeneous genetic backgrounds, making it difficult to determine whether the effect was due to enhanced signaling through the I50 IL-4R{alpha} or included the contributions of other genetic factors.

Our data also suggest that the activation of Stat6 in T cells is already at a saturating level on the dose-response curve in the context of Th2 development. This would mean that for Th2 induction, variants that alter the magnitude of the Stat6 response would not increase the efficiency of Th2 development. It remains an interesting possibility that the threshold of activation for downstream events is different in T and B cells. Although the results generated in T cells suggest that Stat6 induction is optimized, such that the levels mediated by a weaker receptor promote full Th2 development, in B cells the induction of CD23 might still be within the linear region of the dose-response curve and thus susceptible to enhancement of Stat6 activity. In this scenario, individuals expressing the I50 or V50 variant would have similar magnitudes of Th2 responses, but downstream, IL-4-mediated, B cell-mediated responses would be enhanced in I50-expressing individuals. Such a possibility is consistent with recent work in which a transcriptionally attenuated Stat6 mutant, which lacked the ability to interact with the nuclear coactivator-1 transcriptional coactivator, could promote normal IL-4 production from Th2 cells, but was crippled in its ability to transduce CD23 induction (38). This model would suggest that for some haplotypes, IL-4R{alpha} would be a susceptibility gene because of its ability to increase allergy-related processes in the targets of Th2 cytokines rather than by directly causing a change in the Th1 or Th2 response.


   Acknowledgments
 
We thank J. Price and C. Alford for preparative sorting; Immunex Corp., P. Marrack, W. Sha, and C. Aiken for plasmids and reagents; A. Keegan for critical review of the manuscript; G. Nolan for {Phi}NX cells; and N. Noben-Trauth for BALB/c-IL-4R{alpha}–/– mice.


   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 National Institutes of Health Grant GM42550, the Sandler Family Supporting Foundation through a Senior Investigator Award of the Sandler Program for Asthma Research (to M.R.B.), and Molecular Endocrinology Training Program Training Grant T32DK07563 (to L.M.S.). Core facilities essential to this work were supported by the Diabetes Research and Training Center (DK20593) and Vanderbilt Ingram Cancer Center (68485). Submitted in partial fulfillment of the requirements of the Ph.D. degree (L.M.S.) at Vanderbilt University. Back

2 Current address: Department of Medicine, Emory University Medical School, Atlanta, GA 30322. Back

3 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232-3463. E-mail address: mark.boothby{at}vanderbilt.edu Back

4 Abbreviations used in this paper: m, mouse; hu, human. Back

Received for publication July 16, 2004. Accepted for publication August 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ono, S. J.. 2000. Molecular genetics of allergic diseases. Annu. Rev. Immunol. 18:347.[Medline]
  2. Collaborative Study on the Genetics of Asthma (CSGA). 1997. A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat. Genet. 15:389.[Medline]
  3. Deichmann, K., J. Bardutzky, J. Forster, A. Heinzmann, J. Kuehr. 1997. Common polymorphisms in the coding part of the IL4-receptor gene. Biochem. Biophys. Res. Commun. 231:696.[Medline]
  4. Marsh, D. G., J. D. Neely, D. R. Breazeale, B. Ghosh, L. R. Freidhoff, E. Ehrlich-Kautzky, C. Schou, G. Krishnaswamy, T. H. Beaty, E. Marshall. 1994. Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations: strong medicine for NIH. Science 264:1152.[Abstract/Free Full Text]
  5. Ober, C., N. J. Cox, M. Abney, A. Di Rienzo, E. S. Lander, B. Changyaleket, H. Gidley, B. Kurtz, J. Lee, M. Nance, et al 1998. Genome-wide search for asthma susceptibility loci in a founder population: the Collaborative Study on the Genetics of Asthma. Hum. Mol. Genet. 7:1393.[Abstract/Free Full Text]
  6. Maggi, E.. 1998. The TH1/TH2 paradigm in allergy. Immunotechnology 3:233.[Medline]
  7. Ryan, J. J.. 1997. Interleukin-4 and its receptor: essential mediators of the allergic response. J. Allergy Clin. Immunol. 99:1.[Medline]
  8. Mosley, B., M. P. Beckmann, C. J. March, R. L. Idzerda, S. D. Gimpel, T. VandenBos, D. Friend, A. Alpert, D. Anderson, J. Jackson. 1989. The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 59:335.[Medline]
  9. Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, W. E. Paul. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17:701.[Medline]
  10. Noben-Trauth, N., L. D. Shultz, F. Brombacher, J. F. Urban, Jr, H. Gu, W. E. Paul. 1997. An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. Natl. Acad. Sci. USA 94:10838.[Abstract/Free Full Text]
  11. Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313.[Medline]
  12. Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, et al 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.[Medline]
  13. Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.[Medline]
  14. Kuperman, D., B. Schofield, M. Wills-Karp, M. J. Grusby. 1998. Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J. Exp. Med. 187:939.[Abstract/Free Full Text]
  15. Ober, C., S. A. Leavitt, A. Tsalenko, T. D. Howard, D. M. Hoki, R. Daniel, D. L. Newman, X. Wu, R. Parry, L. A. Lester, et al 2000. Variation in the interleukin 4-receptor {alpha} gene confers susceptibility to asthma and atopy in ethnically diverse populations. Am. J. Hum. Genet. 66:517.[Medline]
  16. Howard, T. D., G. H. Koppelman, J. Xu, S. L. Zheng, D. S. Postma, D. A. Meyers, E. R. Bleecker. 2002. Gene-gene interaction in asthma: IL4RA and IL13 in a Dutch population with asthma. Am. J. Hum. Genet. 70:230.[Medline]
  17. Mitsuyasu, H., K. Izuhara, X. Q. Mao, P.S. Gao, Y. Arinobu, T. Enomoto, M. Kawai, S. Sasaki, Y. Dake, N. Hamasaki, et al 1998. Ile50Val variant of the IL-4R{alpha} upregulates IgE synthesis and associates with atopic asthma. Nat. Genet. 19:119.[Medline]
  18. Hershey, G. K., F. F. Michal, L. A. Esswein, M. L. Thomas, T. A. Chatila. 1997. The association of atopy with a gain of function mutation in the {alpha} subunit of the interluekin-4 receptor. N. Engl. J. Med. 337:1720.[Abstract/Free Full Text]
  19. Risma, K. A., N. Wang, R. P. Andrews, C. M. Cunningham, M. B. Ericksen, J. A. Bernstein, R. Chakraborty, G. K. Hershey. 2002. V75R576 IL-4 receptor {alpha} is associated with allergic asthma and enhanced IL-4 receptor function. J. Immunol. 169:1604.[Abstract/Free Full Text]
  20. Galizzi, J. P., N. Harada, I. Durand, J. Banchereau, C. E. Zuber. 1990. Interleukin 4 receptors on normal human B lymphocytes: characterization and regulation. Cell. Immunol. 129:329.[Medline]
  21. Pear, W. S., G. P. Nolan, M. L. Scott, D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8392.[Abstract/Free Full Text]
  22. Bahnson, A. B., J. T. Dunigan, B. E. Baysal, T. Mohney, R. W. Atchison, M. T. Nimgaonkar, E. D. Ball, J. A. Barranger. 1995. Centrifugal enhancement of retroviral mediated gene transfer. J. Virol. Methods 54:131.[Medline]
  23. 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]
  24. Mora, A. L., D. Chen, M. Boothby, D. H. Rubin. 1999. Lineage-specific differences among CD8+ T cells in their dependence of NF-{kappa}B/Rel signaling. Eur. J. Immunol. 29:2968.[Medline]
  25. Goenka, S., J. Youn, L. M. Dzurek, U. Schindler, L.-y. Yu-Lee, M. Boothby. 1999. Paired Stat6 C-terminal transcription activation domains required both for inhibition of an IFN-responsive promoter and trans-activation. J. Immunol. 163:4663.[Abstract/Free Full Text]
  26. Wurster, A. L., D. J. Withers, T. Uchida, M. F. White, M. J. Grusby. 2002. Stat6 and IRS-2 cooperate in interleukin 4 (IL-4)-induced proliferation and differentiation but are dispensable for IL-4-dependent rescue from apoptosis. Mol. Cell. Biol. 22:117.[Abstract/Free Full Text]
  27. Zhu, J., L. Guo, B. Min, C. J. Watson, J. Hu-Li, H. A. Young, P. N. Tsichlis, W. E. Paul. 2002. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 16:733.[Medline]
  28. Smeltz, R. B., J. Chen, J. Hu-Li, E. M. Shevach. 2001. Regulation of interleukin (IL)-18 receptor {alpha} chain expression on CD4+ T cells during T helper (Th)1/Th2 differentiation: critical downregulatory role of IL-4. J. Exp. Med. 194:143.[Abstract/Free Full Text]
  29. Smeltz, R. B., J. Chen, R. Ehrhardt, E. M. Shevach. 2002. Role of IFN-{gamma} in Th1 differentiation: IFN-{gamma} regulates IL-18R{alpha} expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor {beta}2 expression. J. Immunol. 168:6165.[Abstract/Free Full Text]
  30. Brusselle, G. G., J. C. Kips, R. A. Peleman, G. F. Joos, R. R. Devos, J. H. Tavernier, R. A. Pauwels. 1997. Role of IFN-{gamma} in the inhibition of the allergic airway inflammation caused by IL-12. Am. J. Respir. Cell Mol. Biol. 17:767.[Abstract/Free Full Text]
  31. Cohn, L., C. Herrick, N. Niu, R. J. Homer, K. Bottomly. 2001. IL-4 promotes airway eosinophilia by suppressing IFN-{gamma} production: defining a novel role for IFN-{gamma} in the regulation of allergic airway inflammation. J. Immunol. 166:2760.[Abstract/Free Full Text]
  32. Cohn, L., R. J. Homer, N. Niu, K. Bottomly. 1999. T helper 1 cells and IFN-{gamma} regulate allergic airway inflammation and mucus production. J. Exp. Med. 190:1309.[Abstract/Free Full Text]
  33. Daniels, S. E., S. Bhattacharrya, A. James, N. I. Leaves, A. Young, M. R. Hill, J. A. Faux, G. F. Ryan, P. N. le Souef, G. M. Lathrop, et al 1996. A genome-wide search for quantitative trait loci underlying asthma. Nature 383:247.[Medline]
  34. Mitsuyasu, H., Y. Yanagihara, X. Q. Mao, P. S. Gao, Y. Arinobu, K. Ihara, A. Takabayashi, T. Hara, T. Enomoto, S. Sasaki, et al 1999. Cutting edge: dominant effect of Ile50Val variant of the human IL-4 receptor {alpha} chain in IgE synthesis. J. Immunol. 162:1227.[Abstract/Free Full Text]
  35. Hoffjan, S., C. Ober. 2002. Present status on the genetic studies of asthma. Curr. Opin. Immunol. 14:709.[Medline]
  36. Hall, I. P.. 2000. Interleukin-4 receptor {alpha} gene variants and allergic disease. Respir. Res. 1:6.[Medline]
  37. Noguchi, E., M. Shibasaki, T. Arinami, K. Takeda, Y. Yokouchi, K. Kobayashi, N. Imoto, S. Nakahara, A. Matsui, H. Hamaguchi. 1999. No association between atopy/asthma and the Ile50Val polymorphism of IL-4 receptor. Am. J. Respir. Crit. Care Med. 160:342.[Abstract/Free Full Text]
  38. Goenka, S., C. Marlar, U. Schindler, M. Boothby. 2003. Differential roles of C-terminal activation motifs in the establishment of Stat6 transcriptional specificity. J. Biol. Chem. 278:50362.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
A. Acacia de Sa Pinheiro, A. Morrot, S. Chakravarty, M. Overstreet, J. H. Bream, P. M. Irusta, and F. Zavala
IL-4 induces a wide-spectrum intracellular signaling cascade in CD8+ T cells
J. Leukoc. Biol., April 1, 2007; 81(4): 1102 - 1110.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
F. Zhang and M. Boothby
T helper type 1-specific Brg1 recruitment and remodeling of nucleosomes positioned at the IFN-{gamma} promoter are Stat4 dependent
J. Exp. Med., June 12, 2006; 203(6): 1493 - 1505.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. M. Stephenson, D.-S. Park, A. L. Mora, S. Goenka, and M. Boothby
Sequence Motifs in IL-4R{alpha} Mediating Cell-Cycle Progression of Primary Lymphocytes
J. Immunol., October 15, 2005; 175(8): 5178 - 5185.
[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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stephenson, L.
Right arrow Articles by Boothby, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stephenson, L.
Right arrow Articles by Boothby, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS