Interleukin (IL)-13 is a T helper 2–derived cytokine that has recently been implicated in allergic airway responses. We hypothesized that IL-13 may regulate expression of eotaxin in airway epithelium. We found that IL-13 upregulated eotaxin messenger RNA and protein synthesis in the airway epithelial cell line BEAS-2B; this effect showed synergy with tumor necrosis factor (TNF)- α and also was inhibited by the glucocorticoid budesonide. To establish the mechanisms of eotaxin upregulation by IL-13, cells were transfected with an eotaxin promoter–luciferase reporter plasmid and transcription was activated by IL-13 (1.7-fold) and TNF- α (2.8-fold). The combination of IL-13 and TNF- α additively activated the promoter constructs (4.1-fold). Activation of signal transducer and activator of transcription (STAT) 6 by IL-13 was confirmed by nuclear protein binding to a DNA probe derived from the eotaxin promoter. Activation of eotaxin transcription by IL-13 and the additive effect with TNF- α were lost in plasmids mutated at a putative STAT6 binding site. Cotransfection with a wild-type STAT6 expression vector significantly enhanced activation of the eotaxin promoter after IL-13 stimulation (6-fold induction). A significant increase of eotaxin protein secretion in the supernatant of STAT6 wild-type–transfected cells was observed after IL-13 stimulation. Cotransfection with a dominant negative STAT6 mutant expression vector inhibited activation of the eotaxin promoter by IL-13. These results indicate that IL-13 stimulates eotaxin expression in airway epithelial cells and that STAT6 plays a pivotal role in this response.
Asthma is a disease characterized by the infiltration of eosinophils and lymphocytes into airway epithelium and subsequent epithelial damage and tissue remodeling (1-4). T helper (Th) 2 cells and their cytokine products play a crucial role in this process. Interleukin (IL)-13 is one of the important Th2-type cytokines which have been implicated and are upregulated in asthma (5-7). Recent studies indicate that IL-13 can induce pathologic changes reminiscent of asthma in animals, including infiltration of eosinophils and mononuclear cells, epithelial damage, hyperplasia of goblet cells, and subepithelial fibrosis (8-10).
IL-13 probably plays important roles as a mucus-stimulating cytokine (8, 11) as well as in the recruitment of eosinophils. Endothelial cell expression of vascular cell adhesion molecule (VCAM)-1, an adhesion molecule involved in eosinophil recruitment, has been shown to be induced by IL-13 (12, 13).
Eotaxin is a C-C chemokine that binds with high affinity and specificity to the chemokine receptor CCR3 (14– 17). CCR3 is expressed on important cells in allergic disease, such as eosinophils, basophils, a subset of Th2 cells, and mast cells (16, 18-20). Because eotaxin has been reported to be highly expressed in the epithelium of asthmatics (21-23), it may play a role in the movement of these cells in the airways.
Recent studies suggest that IL-13 may be an important inducer of eotaxin in the airways. Zhu and colleagues reported eosinophilic inflammation and eotaxin induction in airway epithelium in IL-13 transgenic mice (8), and Li and associates reported that IL-13 induces eotaxin in the airway epithelium of mice more effectively than does IL-4 (24). These data suggest that IL-13 may play an important role in the pathogenesis of asthma and that upregulation of epithelial expression of eotaxin may contribute to the eosinophilia caused by IL-13.
Airway epithelial cells are known to be one of the important sources of eotaxin in vivo, and eotaxin expression has been demonstrated in vitro using cultured epithelial cells (25, 26). We have recently analyzed the regulation of eotaxin expression in airway epithelial cells by tumor necrosis factor (TNF)-α, IL-4, and glucocorticoids (27), and demonstrated important roles for nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT) 6 (28).
STAT6 is a member of the family of signal transducers and activators of transcription (29). STAT6 is known to be activated through IL-4 signaling and to activate transcription of several genes, including CD23 (the low-affinity immunoglobulin [Ig] E receptor), major histocompatibility complex (MHC) class II, and IgE (30, 31). We have recently reported that STAT6 binds to the proximal region of the eotaxin promoter and contributes to the regulation of eotaxin transcription by IL-4 (28). IL-4 and IL-13 share receptor components and activate similar signal transduction pathways (32, 33). Takeda and coworkers have reported that IL-13 induces MHC class II expression via STAT6 in macrophages (34). Although data suggesting similarities between IL-4 and IL-13 signaling via STAT6 is accumulating, little is known regarding regulation of STAT6 function by IL-13 in airway epithelial cells. We report here that IL-13 upregulates eotaxin expression in airway epithelial cells by a mechanism involving activation of STAT6.
BEAS-2B is a human airway epithelial cell line transformed with adenovirus 12–simian virus 40 hybrid virus (a kind gift from Dr. Curtis Harris) (35). BEAS-2B cells were cultured in Dulbecco's modified Eagle's medium (Biofluids, Inc., Rockville, MD) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ng/ml streptomycin (GIBCO BRL, Gaithersburg, MD) at 37°C with 5% CO2 in humidified air.
Total RNA was extracted from BEAS-2B cells 18 h after the incubation with or without indicated cytokines using RNAzol B reagent (36). Northern blot analysis was performed as previously described (17, 27). Aliquots of 20 μg of RNA were run on 1% agarose/formaldehyde gels and then blotted onto a nylon membrane (DuPont-NEN, Boston, MA) with a positive pressure blotter (Stratagene, La Jolla, CA). The membrane was hybridized with 32P-labeled complementary DNA (cDNA) probes for eotaxin and exposed to X-ray film after washing with saline sodium citrate and sodium dodecyl sulfate (SDS) buffer. Autoradiographs were quantified by video densitometry and the results are shown as the ratio of eotaxin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (housekeeping gene) densitometric units. The probes used were a BamHI fragment spanning 260 base pairs (bp) from the coding region for eotaxin and a cDNA probe (1,100 bp) for GAPDH.
The concentration of eotaxin protein in culture supernatants was assayed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The limit of detection in the assay was 5 pg/ml.
Methods of eotaxin promoter–reporter plasmid (pEotx) construction have been described previously (28). A 1,363-bp fragment of the promoter region of the eotaxin gene (site −1,363 to −1) was amplified by polymerase chain reaction and ligated into Mlu I and Bgl II sites of a luciferase reporter pGL3-Basic vector (Promega, Madison, WI) and the construct is referred to as pEotx.1363. The pEotx.478 and pEotx.300 were constructed by deleting lengths of the 5′-end of the eotaxin promoter sequence of pEotx.1363. The construct pEotx.M-1 was synthesized by mutating the putative STAT6 binding sites in pEotx.1363 (see Figure 3). We previously determined that this sequence is important for STAT6 binding using electrophoresis mobility shift assays (28). The wild-type STAT6 expression vector (referred to as STAT6 WT) and the tyrosine 641 mutant of STAT6, which acts as a dominant negative (referred to as STAT6 Y641), are described elsewhere (37).
BEAS-2B cells were seeded into six-well plates and allowed to grow to 50 to 70% confluence. Cells were transfected with 3 or 6 μl of Fugene 6 transfection reagent (Boehringer-Mannheim, Indianapolis, IN) and 1 μg luciferase reporter plasmids with or without 1 μg STAT6 expression vector, as indicated later, and incubated for 24 h in 2 ml medium. Cytokine was then added, and 6 h later cells were washed twice with Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) (GIBCO-BRL), solubilized by incubation in 300 μl of lysis buffer for 20 min (Promega), transferred to microtubes, and centrifuged to pellet cellular debris. The supernatants were stored at −70°C until luciferase activity was measured using the Luciferase Assay System (Promega) and a luminometer (Analytic Luminescence Laboratories, Sparks, MD). The protein concentration of the samples was measured using the Bradford protein dye reagent (Bio-Rad, Hercules, CA) and the relative luciferase activity was normalized to protein concentration.
Whole-cell extracts were prepared by modification of a method previously reported (38, 39). Cells were harvested and resuspended in lysis buffer (20 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1 mM dithiothreitol [DTT], 1% Nonidet P-40 [NP-40], 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.2 mg/ml leupeptin, 1 mM Na3VO4, and 50 mM NaF) (GIBCO BRL). Cells were sonicated for 10 s with an ultrasonic cell disrupter (Heat System Ultrasonics, Inc., Farmingdale, NY) and then incubated for 5 min. All procedures were performed on ice. Lysates were centrifuged and the supernatant was collected and stored at −70°C.
Nuclear extracts were prepared using a modification of the method of Schreiber and colleagues (40). BEAS-2B cells were treated as indicated and then harvested with a scraper after washing twice with HBSS. The cells were washed with buffer A (10 mM N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid [Hepes], 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml leupeptin, and 1 mM Na3VO4) (GIBCO BRL). The cell pellets were resuspended in buffer B (buffer A containing 0.2% NP-40) and incubated for 2 min. Nuclei were pelleted by centrifugation and resuspended in buffer C (buffer A containing 0.25 M sucrose). Nuclei were again pelleted, then resuspended in buffer D (50 mM Hepes, 400 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml leupeptin, and 1 mM Na3VO4) and incubated with shaking for 30 min. All procedures were performed on ice. The mixture was centrifuged and the supernatant was stored at −70°C.
The pull-down of nuclear protein was based on the methods previously reported (37). Aliquots of 200 μg of nuclear extracts were incubated for 40 min at 4°C with gentle rotation in 400 μl of 10 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 0.1% NP-40, 2% glycerol, 50 μg/ml poly(dI-dC), 10 mM KCl, 1 mM PMSF, 0.2 mg/ml leupeptin, 1 mM Na3VO4, 1 mM NaF (GIBCO BRL), and streptavidin beads (Pierce, Rockford, IL) carrying a biotinylated oligonucleotide derived from the eotaxin promoter (−77 to −56) which contains a STAT6 binding site described previously (28) (see Figure 3). After washing three times with buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, 10% glycerol, 1 mM PMSF, 0.2 mg/ml leupeptin, 1 mM Na3VO4, and 1 mM NaF), the precipitated proteins were eluted by boiling for 5 min in the sample buffer containing 0.25 M Tris-HCl, 10% glycerol, 5% SDS, and 0.025% 2-ME (Sigma, St. Louis, MO).
The whole-cell extracts or precipitated nuclear extracts were subjected to 10% Tris–glycine gradient gel electrophoresis (Novex, San Diego, CA) and transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). The membrane was blocked with 5% nonfat milk powder in 50 mM Tris, 0.15 M NaCl, and 0.05% Tween 20 (TBST), incubated with 1 μg/ml rabbit anti-STAT6 antibody (Ab) (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST for 3 h, washed with TBST, and then incubated with antirabbit Ig Ab (Amersham) for 1 h. After extensive washing with TBST, chemiluminescent substrate was added (enhanced chemiluminescence Western blot detection system; Amersham) and the membrane was subjected to autoradiography.
Analysis of data was performed using Stat-View II (Abacus Concept, Inc., Berkeley, CA). Data are expressed as means ± standard error of the mean (SEM). Statistical differences were determined by analysis of variance with Fisher PLSD.
Initial experiments were performed to determine whether IL-13 stimulates expression of eotaxin messenger RNA (mRNA) and protein in BEAS-2B epithelial cells. BEAS-2B expressed small amounts of eotaxin mRNA (Figure 1). Alone, IL-13 (50 ng/ml) induced eotaxin expression to a lesser extent than did stimulation with TNF-α (100 ng/ml). A large amount of induction was observed with the combination of IL-13 and TNF-α. We also examined the effect of the potent topical glucocorticoid budesonide (BUD) on the expression of eotaxin mRNA. Pretreatment with 10−7 M BUD (a generous gift of Dr. Ralph Brattsand, Astra Zeneca, Lund, Sweden) 24 h before cytokine stimulation inhibited expression of eotaxin mRNA, induced by the combination of IL-13 and TNF-α.
A small amount of eotaxin protein was detected in the medium of unstimulated BEAS-2B (0.1 ± 0.02 ng/ml) (Figure 2A). The quantity of 50 ng/ml IL-13 stimulated eotaxin production to a lesser degree (0.57 ± 0.09 ng/ml) than did stimulation with 100 ng/ml of TNF-α (1 ± 0.23 ng/ml). The combination of IL-13 and TNF-α synergistically stimulated eotaxin production (6.8 ± 2.3 ng/ml), and this maximal induction was significantly inhibited with pretreatment of 10−7 M BUD (0.58 ± 0.09 ng/ml, P < 0.05 compared with dimethyl sulfoxide [DMSO]).
We compared the efficacy of IL-13 as an inducer of eotaxin protein with that of IL-4 in BEAS-2B epithelial cells (Figure 2B). A minimal level of eotaxin (0.08 ± 0.01 ng/ml) was detected in the supernatant of unstimulated cells. IL-13 increased eotaxin production (0.81 ± 0.07 ng/ml, P < 0.05 compared with control). This magnitude of response was similar to that induced by IL-4 (50 ng/ml) (0.78 ± 0.07 ng/ml, P < 0.05 compared with control). TNF-α (100 ng/ml) increased eotaxin levels to 0.9 ± 0.14 ng/ml (P < 0.05 compared with control). The combination of IL-13 and TNF-α synergistically increased eotaxin production (3.2 ± 0.3 ng/ml, P < 0.05 compared with TNF-α). This synergistic effect of IL-13 with TNF-α was similar to that observed with IL-4 (3.1 ± 0.44 ng/ml, P < 0.05 compared with TNF-α). There is a slight difference between the data in Figures 2A and 2B. We confirmed that the concentration of DMSO used did not influence the expression of eotaxin (data not shown). We think it may be due to the difference of cell line passage, the lot of FBS, and the lot of the cytokines.
We next focused experiments on the mechanisms of eotaxin transcription induced by IL-13. The sequences of the binding sites for STAT6 and NF-κB in the eotaxin promoter and mutant plasmid are indicated in Figure 3. IL-13 dose-dependently increased luciferase activity in BEAS-2B cells transfected with pEotx.1363 (Figure 4). Maximal activation of the eotaxin promoter was observed at 50 ng/ml of IL-13 treatment (2.6 ± 0.4-fold, P < 0.05 compared with control).
Although IL-13 has been shown to activate STAT6 in several cell types (41, 42), no information is available in epithelial cells, to our knowledge. To determine whether IL-13 activates STAT6 and induces subsequent binding to the eotaxin promoter, we performed Western blot analysis of nuclear protein precipitated by binding to an eotaxin promoter–derived DNA probe containing the STAT6 binding site (see Figure 3). STAT6 was not detected in the nuclear extract of unstimulated control BEAS-2B cells (Figure 5). A significant level of STAT6 was detected after treatment with 10 ng/ml of IL-13 by 20 and 30 min after treatment. A comparable response was observed with 10 ng/ml IL-4 used as a control. These results suggest that IL-13 activates STAT6 and induces its translocation into the nucleus of airway epithelial cells. These data also indicate that the STAT6 activated by IL-13 is capable of binding to the proximal region of the eotaxin promoter at site −74 to −65. Compared with TNF-α, IL-13 was a weaker stimulus, inducing an increase of 1.7 ± 0.1-fold (P < 0.05) compared with 2.8 ± 0.2-fold (P < 0.05) for TNF-α (Figure 6A). The combination of TNF-α and IL-13 induced a slightly more than additive activation of the promoter (4.1 ± 0.2-fold, P < 0.05 compared with TNF-α). We used Fugene 6 transfection reagent to transfect plasmids in BEAS-2B cells because the transfection efficiency is stable. The protein concentration of the samples was measured using the Bradford protein dye reagent, and the relative luciferase activity was normalized to protein concentration instead of normalizing with transfectional efficiency.
To define which region of the promoter is responsible for transcriptional activation by IL-13, we transfected cells with reporter plasmids containing 5′-deleted promoter fragments and plasmids that were mutated at the STAT6 binding site (Figures 6B, 6C, and 6D). Although IL-13 activation was retained after transfection using pEotx.478 (1.5 ± 0.2-fold, compared with control) and pEotx.300 (1.4 ± 0.1-fold, compared with control), it was diminished and not statistically significantly different from control. It is interesting to note that the more than additive effects of IL-13 combined with TNF-α were well preserved using all of these constructs (pEotx.478, 4.8 ± 0.2-fold; pEotx.300, 5.1 ± 0.3-fold; P < 0.05 compared with TNF-α alone). These data indicate that the IL-13–responsive region exists on the promoter sequence within 300 bp from the transcription start site. In contrast, IL-13 activation and the interaction with TNF-α were totally lost using transfection with pEotx.M1, a plasmid mutated at the STAT6 binding site spanning from −74 to −65. Taken together, these data suggest that the STAT6 binding site is necessary for activation of the eotaxin promoter by IL-13.
Overexpression of wild-type STAT6 was used to further assess the role of STAT6 in activation of the eotaxin promoter. We first confirmed that transfection of STAT6 WT vector induced a significant level of STAT6 protein in BEAS-2B cells (Figure 7A). We also determined that primary bronchial epithelial cells (PBEC) express STAT6 protein constitutively. Cotransfection of the reporter plasmid pEotx.1363 and STAT6 WT vector resulted in a significant increase of induction by IL-4 (6.2 ± 1.0-fold, P < 0.05 versus control) and IL-13 (6.0 ± 1.0-fold, P < 0.05 versus control) compared with the induction without cotransfection of the STAT6 expression vector (1.9 ± 0.2-fold with IL-4 and 2.0 ± 0.1-fold with IL-13) (Figure 7B). Cotransfection with a dominant negative–mutant STAT6 expression vector, STAT6 Y641, almost completely inhibited the response to IL-4 and IL-13 exposure (1.2 ± 0.03-fold and 1.1 ± 0.02-fold, respectively). Cotransfection with the same amount of pcDNA3, which is the basic plasmid of each expression vector, did not influence the promoter activity significantly with IL-13 stimulation (1.8 ± 0.1-fold induction, P < 0.05 compared with control), although IL-4 stimulation was retained but relatively diminished (1.6 ± 0.3-fold induction, P = not significant compared with control). We have titrated the vectors in the eotaxin promoter assay (referred to as STAT6 WT or STAT6 Y641 and pEotx.1363). We cotransfected BEAS-2B cells with 1 μg of pEotx.1363 and different doses of expression vector. The quantity of 2 μg of expression vector was too much and significantly inhibited the transcription efficiency of pEotx.1363. STAT6 expression using less than 1 μg of the STAT6 expression vector was not sufficient to induce much STAT6 in BEAS-2B cells (data not shown). We also confirmed that transfection with STAT6 WT induced a considerable level of eotaxin protein production in BEAS-2B cells 18 h after stimulation with IL-4 (2.82 ± 0.61 ng/ml, P < 0.05 compared with control) and IL-13 (2.7 ± 0.71 ng/ ml, P < 0.05 compared with control) (Figure 7C).
We demonstrate here that the Th2 cytokine IL-13 induces eotaxin mRNA and protein expression in airway epithelial cells in vitro. Alone, IL-13 was less effective than TNF-α. The combination of IL-13 and TNF-α synergistically stimulated eotaxin protein expression, but stimulated transcription only additively or slightly more than additively. Synergy between IL-13 and TNF-α has previously been demonstrated for the induction of VCAM-1 on endothelial cells (43). These findings support and extend recent findings indicating that IL-13 causes severe eosinophilic inflammation and induces eotaxin expression in vivo (8, 9, 24).
To analyze the mechanisms of eotaxin regulation by IL-13, we studied transcriptional activation. IL-13 activated a reporter containing 1,363 bp of the eotaxin promoter and acted more than additively with TNF-α. These transcriptional studies parallel studies of eotaxin mRNA but do not adequately explain the synergistic increase of protein induced by IL-13 and TNF-α. This divergence suggests a possible role of post-transcriptional regulation of eotaxin expression by TNF-α and IL-13, which may amplify the response. Further investigation of the mechanism of this synergy is clearly required.
Activation of STAT6 by IL-13 and subsequent binding activity for the proximal region of the eotaxin promoter were confirmed by Western blotting after pull-down of nuclear protein using a DNA probe that contains the STAT6 binding site of the eotaxin promoter at position −74 to −65 as previously described (28). This result indicates that activation of the cells by IL-13 led to activation and nuclear localization of STAT6. On the basis of studies by others, we speculate that this may have occurred via STAT6 phosphorylation via Jak 1 (33, 44).
To determine whether STAT6 was responsible for the activation of the eotaxin promoter by IL-13, a pEotx mutated at the STAT6 binding site was used in conjunction with STAT6 wild-type and mutant overexpressing plasmids. Promoter assays using the mutant reporter plasmid showed that the binding site of STAT6 is necessary for both activation of the eotaxin promoter by IL-13 and synergy with TNF-α. Cotransfection of the STAT6 WT expression vector considerably enhanced the IL-13 response, confirming a role of STAT6 in activation of eotaxin transcription by IL-13. Because IL-13–induced secretion of eotaxin protein was greatly increased when STAT6 WT was overexpressed, we conclude that IL-13 is driving the endogenous eotaxin promoter in parallel with the reporter construct. We have previously reported that IL-4 activates STAT6 and TNF-α activates NF-κB in airway epithelial cells, and that both of these transcription factors bind to an overlapping binding site on the eotaxin promoter proximal region (28). Due to the similarity of signaling between IL-13 and IL-4, IL-13 may also cooperate with TNF-α in the activation of the eotaxin promoter through activation of STAT6 and its binding to the proximal promoter region partly shared by NF-κB.
Li and colleagues reported that IL-13 more potently induced eotaxin than did IL-4 in mouse bronchial epithelium in vivo (24). In the present study, IL-13 and IL-4 activated eotaxin transcription and stimulated protein release with similar efficacy. Although some reports have indicated the possibility of different signaling between IL-4 and IL-13 (45, 46), several investigators have shown that STAT6 is responsible for the actions of both IL-4 and IL-13. Takeda and associates reported that macrophage activation in response to IL-13 was impaired in STAT6-deficient mice (34). IL-4 and IL-13 receptors share the IL-4 receptor α chain, which is believed to be sufficient to activate STAT6 through the tyrosine phosphorylation of Jak 1 (44, 47). The similar efficacy of eotaxin induction by IL-4 and IL-13 may be due to a central role of STAT6 as a regulator of expression of the eotaxin gene.
Taken together, our results indicate the importance of IL-13 in the induction of eotaxin expression in airway epithelial cells and suggest that STAT6 may play a crucial role in the activation of the eotaxin gene.
The authors thank Drs. Bruce S. Bochner, Lisa A. Beck, David Proud, Jean Kim, and Shau-Ku Huang for their excellent advice and helpful discussions; and Carol Bickel, John E. Cumberland, John W. Schmidt, Eva Ehrlich-Kautzky, Eri Matsukura, and Bonnie Hebden for their skillful assistance. This work was supported by National Institutes of Health grants RO1AR31891 and AI44885.
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*Current address: First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-0064, Japan. E-mail: smatsuku@post.
Abbreviations: antibody, Ab; base pairs, bp; budesonide, BUD; dimethyl sulfoxide, DMSO; dithiothreitol, DTT; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immunosorbent assay, ELISA; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; interleukin, IL; messenger RNA, mRNA; nuclear factor, NF; Nonidet P-40, NP-40; eotaxin promoter– luciferase reporter plasmid, pEotx; phenylmethylsulfonyl fluoride, PMSF; standard error of the mean, SEM; signal transducer and activator of transcription, STAT; the wild-type STAT6 expression vector, STAT6 WT; the tyrosine 641 mutant of STAT6, which acts as a dominant negative, STAT6 Y641; T helper, Th; tumor necrosis factor, TNF.