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Published ahead of print on December 11, 2003, doi:10.1164/rccm.200307-888OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 596-603, (2004)
© 2004 American Thoracic Society

Signaling Pathways Regulating Interleukin-13–stimulated Chemokine Release from Airway Smooth Muscle

Qi Peng, Takeshi Matsuda and Stuart J. Hirst

Department of Asthma, Allergy, and Respiratory Science, Guy's, King's and St. Thomas' School of Medicine, King's College London, Guy's Hospital Campus, London, United Kingdom

Correspondence and requests for reprints should be addressed to Stuart J. Hirst, Ph.D., Department of Asthma, Allergy, and Respiratory Science, Guy's, King's and St Thomas' School of Medicine, Thomas Guy House, Guy's Hospital Campus, London SE1 9RT, UK. E-mail: stuart.hirst{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13–stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 ± 4 and 75 ± 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1ß was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4–dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 µM) and p38 (SB202190, 10 µM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase–dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Key Words: airway smooth muscle • antisense oligodeoxynucleotide • eotaxin • mitogen-activated protein kinases • STAT6

Accumulating evidence supports interleukin (IL)-13 as a central regulator of the pathogenic mechanism underlying allergic disease. Exaggerated levels of IL-13 are observed in atopic and nonatopic asthma and in other allergic disorders (1–4). Furthermore, transgenic pulmonary overexpression of either IL-13 or IL-4 in mice, and administration of IL-13 into the airways, induces key features of the airways inflammation and remodeling process seen in asthma, including lymphocyte and eosinophil accumulation, mucous cell metaplasia, subepithelial fibrosis, and airway hyperresponsiveness in experimental models (5, 6). Although the key cell types mediating the in vivo activity of IL-13 are uncertain, one report suggests that the effects of IL-13 do not require participation of T or B cells (7), and conditioned supernatants from activated T helper Type 2-polarized CD4+ cells when applied intranasally to the airways of naïve mice can induce airway hyperresponsiveness to acetylcholine within 6 hours in the absence of inflammatory cell recruitment (8). Such studies suggest that the effects of IL-13 and other T helper cell Type 2 cytokines may be more dependent on resident airway cells. Of these, emerging evidence suggests that airway smooth muscle (ASM) cells may fulfill an immunomodulatory role in airway wall inflammatory events by expressing cell adhesion receptors and costimulatory molecules, and by synthesizing various cytokines and chemokines including the eosinophil-activating chemokines regulated on activation, normal T cell expressed and secreted (RANTES), and eotaxin (9–14). Local induction of these chemokines may prime tissues in the lung to produce an exaggerated eosinophilic response in asthma (6, 9–12).

IL-13 and IL-4 appear to act relatively selectively to release eotaxin from human ASM cells, with no effect on the induction of granulocyte-macrophage colony-stimulating factor, RANTES, or IL-8 release in the same culture supernatants (12), which is consistent with similar findings in other systems (6, 15). Moreover, IL-13 or IL-4 in combination with tumor necrosis factor-{alpha} (TNF-{alpha}) or IL-1ß induces a marked synergy in eotaxin levels released from human ASM cells (12, 14). The mechanisms regulating eotaxin production and release in ASM cells are presently unclear. Our own studies have shown that induction of eotaxin release from human ASM cells by IL-13 or IL-4 is mediated by cell surface IL-13 receptors (IL-13Rs), which comprise the common IL-4 receptor (IL-4R) {alpha} subunit and IL-13R{alpha}1 (12), but not the common {gamma} chain that is shared by the IL-2R as well as other cytokines (16). Although these cells also express the IL-13R{alpha}2 subunit (17), available evidence suggests that only receptors containing the IL-13R{alpha}1 subunit are capable of inducing subsequent signals (5). Indeed, ligation of IL-13R{alpha}1/IL-4R{alpha} heterodimers with IL-13 or IL-4 induces tyrosine phosphorylation and activation, via JAK1 (Janus kinase-1), of the transcription factor STAT6, a member of the family of signal transducers and activators of transcription (STAT) (18, 19). Recruitment of STAT6 to tyrosine-phosphorylated IL-4R{alpha} initiates C-terminal tyrosine phosphorylation on STAT6 residues by JAKs and causes homodimer formation through reciprocal SH2 domain interactions and enables nuclear translocation and binding to specific DNA recognition sequences within the promoter region of several target genes, including eotaxin (20, 21). Although other signaling pathways including insulin receptor substrate (IRS)-1 and IRS-2 and phosphatidylinositol 3'-kinase are activated by IL-13 (22, 23), STAT6 is generally regarded to be critical in mediating intracellular signals elicited by IL-4 and IL-13, and most studies in allergic models of asthma have focused on the role of STAT6. Indeed, in STAT6-deficient mice there is abrogation of IL-13–stimulated airway eosinophilia, antigen-induced airway hyperresponsiveness, and mucus production (24, 25). Likewise, IL-13–induced airway hyperreactivity is STAT6 dependent (26), and STAT6 levels are reported to be elevated in the airway epithelium of patients with severe asthma compared with mild asthma or normal control subjects (27).

In keeping with reports that IL-13 activates multiple intracellular signaling pathways in target cells, a study from our laboratory using cultured human ASM cells showed in addition to STAT6 phosphorylation that IL-13 and IL-4 activate key elements of the mitogen-activated protein (MAP) kinase signaling cascade such as p42/p44 extracellular signal-regulated kinase (ERK) and p38 MAP kinase (12). By using inhibitors of p42/p44 ERK and p38 MAP kinase activation we showed a partial requirement for these pathways in IL-13R activation and subsequent eotaxin release (12). However, a causative role for STAT6 was not examined. Here, using human ASM cells transfected with antisense oligodeoxynucleotides (AS-ODNs) to downregulate STAT6 protein, we report that release of eotaxin from human ASM cells by IL-13 or IL-4 is also STAT6 dependent. In addition, STAT6 contributes to synergistic increases in eotaxin by IL-13 or IL-4 in combination with IL-1ß. Likewise, the mechanism by which IL-13 or IL-4 can suppress IL-1ß–stimulated RANTES release is STAT6 dependent. Some of the results of these studies have been previously reported in the form of an abstract (28).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Culture of Human Airway Smooth Muscle Cells
In accordance with procedures approved by the Research Ethics Committee of Guy's and St Thomas' Hospitals, macroscopically normal human bronchial smooth muscle was obtained from the lobar or main bronchus of 33 nonasthmatic patients of either sex (mean age, 61 ± 6 years; range, 25–77 years; 19 male, 14 female) undergoing lung resection for carcinoma of the bronchus. Smooth muscle cells were isolated and placed into culture as previously described (12). Using fluorescence immunocytochemistry or flow cytometric techniques, near-confluent, serum-deprived human ASM cells (Passage 2) stained (greater than 95%) for smooth muscle-specific {alpha}-actin and calponin. When examined by light and electron microscopy, these cells displayed all the reported characteristics of viable smooth muscle cells in culture (29). Cells at Passages 3–5 were used in all experiments.

ODN Design and Transfection into Human Airway Smooth Muscle Cells
Three antisense 5'- and 3'-end phosphorothioate (PS)-modified ODNs (AS-ODN1, 5'-ccC CAC AGA GAC ATG Atc tg-3'; AS-ODN2, 5'-cgG TCC ATC TCA GAG Aag gc-3'; AS-ODN3, 5'-gtG AGG TCC TGT TCA Gtg gg-3' [lower case denotes PS-modified bases]) that targeted different regions of human STAT6 mRNA (bp 157–176, 1456–1475, and 2554–2573, respectively) were chosen on the basis of previously described sequences (30). A sense ODN3 (Sense, 5'-ccC ACT GAA CAG GAC Ctc ac-3') and a scrambled ODN3 (SCR, 5'-caC TCC AGG ACA AGT Cac cc-3') served as controls. All ODNs (ODN3 labeled with fluorescein isothiocyanate [FITC] at the 5' end) were purchased from MWG Biotech (Ebersberg, Germany). Transfection of ODNs, using cationic lipid-mediated transfection with FuGENE 6, was performed according to the manufacturer's protocol (Roche, Indianapolis, IN) with some modifications. Briefly, a transfection cocktail ratio of 2.5 ODN (0.05 to 3 µM) to 1 FuGENE 6 (10 µl of ODN to 4 µl of FuGENE 6) was added to subconfluent (about 60 to 70%) cells during growth arrest in RPMI 1640 containing 25 mM HEPES, 2 mM L-glutamine, penicillin (100 U/ml)–streptomycin (100 µg/ml), and bovine serum albumin (1 mg/ml) ("supplemented RPMI") with the addition of 1 µM insulin, transferrin (5 µg/ml), and 100 µM ascorbate. Untransfected cells were incubated with supplemented RPMI containing FuGENE 6 only. After 72 hours, cells were retransfected by treatment with the same concentration of AS-ODN or sense/SCR-ODNs and then incubated for a further 24 hours in the absence or presence of human recombinant cytokines (R&D Systems, Abingdon, UK). In experiments examining phosphorylation of STAT6, cells were stimulated with cytokines for 15 minutes only, and were not retransfected during the stimulation. Elsewhere, cells were pretreated with 10 µM inhibitors of MAP kinase [SB202190: 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; U0126: 1,4-diamino-2,3-dicyano-1,4-bis-(2-aminophenylthio)butadiene; Calbiochem, Nottingham, UK) 30 minutes before and throughout stimulation with recombinant cytokines.

Measurement of Cytokine Levels
Cytokine levels in cell-conditioned medium were determined in duplicate by specific sandwich enzyme-linked immunosorbent assays (ELISAs) using matched monoclonal capture and biotinylated, or polyclonal (RANTES, eotaxin) detection antibody pairs (R&D Systems) as described previously (12).

Fluorescence Microscopy and Flow Cytometry
To confirm transfection and to determine efficiency an FITC-labeled ODN (ODN3) was used. In localization studies, FITC-ODN3-transfected cells on Lab-Tek four-chamber microscope glass slides (Nunc; Invitrogen, Paisley, UK) were fixed for 10 minutes in 4% paraformaldehyde (methanol free, EM grade; Polysciences, Warrington, PA) before application of the nuclear counterstain Hoechst 33342 (1 µg/ml; Molecular Probes, Leiden, The Netherlands). Overall transfection efficiency was confirmed by flow cytometry. FITC-ODN-tranfected cells were removed from 25-cm2 flasks using 0.02% trypsin–EDTA in phosphate-buffered saline and fixed for 10 minutes in ice-cold 4% paraformaldehyde. Numbers of FITC-positive cells were determined on a FACSCalibur flow cytometer (Becton Dickinson, Oxford, UK) using the CellQuest Pro analysis program (Becton Dickinson) (12). After analysis, transfected cells were confirmed microscopically to be intact. See the online supplement for a full description of fluorescence microscopy and flow cytometric techniques.

Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was isolated from cytokine-stimulated human ASM cells after transfection with or without AS-ODNs, using an RNeasy minikit (Qiagen, Hilden, Germany), and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described in the online supplement. RT-PCR primers were as follows: eotaxin, 5'-CCC AAC CAC CTC CTG CTT TAA C-3' as sense and 5'-CCA GAT ACT TCA TGG AAT CCT GCA C-3' as antisense; 18S rRNA, 5'-TGA CTC AAC ACG GGA AAC CTC AC-3' as sense and 5'-GGA CAT CTA AGG GCS TCA CAG ACC-3' as antisense; and STAT6, 5'-AGA TCA TGT CTC TGT GGG GTC T-3' as sense and 5'-TAG AAG AGC TGT CTC TTT GGG TTC-3' as antisense. STAT6 primers were designed to amplify a PCR product containing all three regions (157–2573 bp) targeted by the AS-ODNs. All primers were from MWG Biotech.

Western Immunoblot Analyses
Total cell protein extracts were prepared and separated (7.5 µg/lane) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (14, 31). Total STAT3 and STAT5 were detected with mouse monoclonal antibodies (610189 and 610191, respectively; BD-Pharmingen, Oxford, UK). Total STAT6 (sc 4220; Santa Cruz Biotechnology, Santa Cruz, CA) and phospho-STAT6 (9361; New England BioLabs, Beverly, MA) were detected with rabbit polyclonal antibodies as previously described (12). Primary antibodies were detected with goat anti–rabbit or anti–mouse IgG horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence (Amersham Biosciences, Amersham, UK). Blots were stripped and reprobed with a mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6G5; Biogenesis, Poole, UK) to control for differences in loading.

Statistical Analysis
Data are expressed as means ± SEM of observations obtained from cells cultured from patient donors (n). IC50 values were estimated for individual concentration–response curves by nonlinear least-squares regression analysis (SigmaPlot; SPSS, Chicago, IL). Data were compared by one- or two-way analysis of variance (ANOVA), where appropriate, followed by Bonferroni's t-test post hoc to evaluate statistical differences between treatment groups (SigmaStat; SPSS). A probability value (p) of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selective Inhibition of STAT6 Protein Expression by AS-ODNs
To determine optimal conditions for ODN uptake, an FITC-conjugated AS-ODN (AS-ODN3) was titrated at concentrations of 0.05 to 3 µM together with the cationic lipid-mediated transfection reagent, FuGENE 6, at volume ratios of 2.5:1 FuGENE 6:ODN. Positive cells were identified by flow cytometry and the uptake was found to be concentration dependent, with maximal transfection (84.6 ± 3%) occurring at 1 µM AS-ODN3 (n = 4; Figure 1A) and 50% of cells being labeled at 0.21 µM. FITC-positive cells were also confirmed by fluorescence microscopy, by which intense nuclear and cytoplasmic FITC labeling was observed in IL-13–stimulated cells transfected with 1 µM FITC-conjugated AS-ODN3, compared with treatment with FuGENE 6 alone (Figure 1B). A similar pattern of FITC localization, although less intense, was observed in unstimulated cells (not shown).



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  		Figure 1. Transfection and localization of signal transducer and activator of transcription (STAT) 6 antisense oligodeoxynucleotides (AS-ODN) in cultured human airway smooth muscle (ASM) cells. Cells were incubated with the transfection reagent FuGENE 6 in the absence or presence of fluorescein isothiocyanate (FITC)-conjugated AS-ODN3 during growth arrest for 72 hours and during stimulation for 24 hours with interleukin (IL)-13 (10 ng/ml). In (A), typical flow cytometry data are shown. Numbers in gates represent mean ± SEM of the proportion of FITC-positive cells (n = 3). In (B), localization of FITC-conjugated AS-ODN is shown compared with cells treated with FuGENE 6 only (top). Cell nuclei are also shown using the Hoechst 33342 nuclear stain (bottom). Scale bar: 50 µm (for all panels). Photomicrographs are representative of independent experiments using cells cultured from three or four different donors.

 
To examine the effect of AS-ODN on STAT6 mRNA and protein levels, human ASM cells were transfected during growth arrest, and subsequently during stimulation with IL-13 (10 ng/ml) for a further 24 hours, with a panel of AS-ODNs against the human STAT6-coding sequence. Transfection with a complementary (sense) or scrambled ODN3 (SCR) served as controls. In three of four similar experiments, unstimulated cells expressed low levels of STAT6 mRNA but high levels of STAT6 protein, possibly indicating a low basal turnover of STAT6 protein. In support of this, IL-13 upregulated STAT6 mRNA, but appeared to reduce corresponding protein levels, perhaps by increasing STAT6 mRNA stability or by increasing mRNA translation and protein turnover. All three STAT6 AS-ODNs (1.0 µM) appeared to reduce the abundance of STAT6 mRNA and STAT6 protein, whereas sense or SCR-ODN3 (1.0 µM) had no effect (Figures 2A and 2B) . Of note, preincubation of AS-ODN3 with its complementary (sense) sequence at 37°C for 20 minutes before transfection also appeared to downregulate STAT6 mRNA and STAT6 protein. The mixture was used to reverse the effects of AS-ODN3. It is possible that the unexpected efficacy of the mixture against STAT6 is because the sense sequence did not neutralize the effects of the AS-ODN3 strand, and so the significance of these findings must be interpreted with caution.



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  		Figure 2. Selective downregulation of STAT6 messenger RNA (mRNA) and protein by AS-ODN. Human ASM cells were transfected with a panel of STAT6 AS-ODNs (1.0 µM) during growth arrest (72 hours) and stimulation (24 hours) with IL-13 (10 ng/ml). Untransfected cells received FuGENE 6 only and were left either unstimulated (Unstim) or treated with IL-13 at 10 ng/ml (IL-13). Sequence specificity of AS-ODN was investigated on (A) STAT6 mRNA by RT-PCR and on (B) total STAT6 protein by Western immunoblot analysis using antibodies to STAT6, STAT3, and STAT5. A complementary ODN3 (Sense) and a scrambled AS-ODN3 (SCR), as well as reblotting membranes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), served as controls. Data are representative of independent experiments using cells cultured from three or four different donors. In (C), supernatants derived from experiments in (B) were assayed by ELISA for eotaxin. Columns and error bars represent means ± SEM of duplicate values from independent experiments (n = 4). *p < 0.05, ***p < 0.001 compared with IL-13–stimulated cells in the absence of AS-ODN treatment.

 
Selectivity of the effect of the STAT6 AS-ODNs was demonstrated by reblotting with antibodies to other STAT family members, STAT3 and STAT5. In each case, transfection with AS-ODNs appeared not to affect levels of these proteins (Figure 2B), although a small reduction in GAPDH levels was noted, but this was not a consistent finding. The presence of a doublet band for total STAT6, but not STAT3 or STAT5, likely reflects detection of both unphosphorylated and phosphorylated STAT6 after IL-13 stimulation.

STAT6 AS-ODNs Prevent IL-13- and IL-4–dependent, But Not IL-1ß–dependent, Eotaxin Release
The selective reduction in STAT6 levels in human ASM cell lysates by STAT6 AS-ODN1, -2, or -3 (1.0 µM) was paralleled by attenuation of IL-13 (10 ng/ml)–stimulated eotaxin levels in supernatants, as determined by ELISA (p < 0.05–0.01 by one-way ANOVA, n = 3) (Figure 2C). AS-ODN1 and AS-ODN3 appeared to be the most effective of the three tested in suppressing both STAT6 mRNA and protein abundance, and in reducing IL-13–stimulated eotaxin release (Figure 2). Attenuation with AS-ODN3 was concentration dependent with 50% inhibition (IC50) of IL-13–dependent eotaxin release occurring at 0.33 µM, and almost complete suppression (81.4 ± 4.4%) present at 1 µM ODN (p < 0.001 by one-way ANOVA, n = 3) (Figure 3) . No attenuation was found with the corresponding sense sequence (p > 0.05, n = 3). Transfection with STAT6 AS-ODN3 also attenuated IL-4–dependent eotaxin release by 75.1 ± 6.7% compared with treatment with FuGENE 6 alone (p < 0.001 by one-way ANOVA, n = 13), but no effect was found (p > 0.05, n = 7) on IL-4–dependent release with the sense or SCR sequence (Figure 4A) . In contrast, in parallel experiments transfection with AS-ODN3 at 1 µM had only a minimal reduction (16.4 ± 5.3%) on eotaxin release induced by IL-1ß (10 ng/ml), which was not significant (p > 0.05 by one-way ANOVA, n = 7) (Figure 4A). Likewise, whereas eotaxin mRNA content appeared to be reduced in AS-ODN3–tranfected cells after stimulation with IL-13 or IL-4, no attenuation of eotaxin mRNA was found in transfected cells after stimulation with IL-1ß (Figure 4B).



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  		Figure 3. STAT6 AS-ODN concentration–response relationship for attenuation of IL-13–dependent eotaxin release from human ASM cells in culture. Cells were either left untransfected and incubated in medium alone (Unstim, open column) or stimulated for 24 hours with IL-13 at 10 ng/ml (solid columns) in the absence or presence of transfection with AS-ODN3 (0.1–3 µM). A complementary ODN3 sequence (Sense) served as a control in IL-13–stimulated cells. Columns and error bars represent means ± SEM of duplicate values from independent ELISA experiments using cells cultured from four different donors. *p < 0.05, ***p < 0.001 compared with IL-13–stimulated cells in the absence of AS-ODN treatment.

 


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  		Figure 4. STAT6 AS-ODN (1 µM AS-ODN3) attenuates eotaxin production from human ASM cells by IL-13 (open columns) and IL-4 (solid columns), but not IL-1ß (hatched columns). Untransfected cells were either left unstimulated (Unstim) or treated for 24 hours with cytokine at 10 ng/ml. Transfection of cytokine-treated cells with a complementary ODN3 (Sense) or a scrambled AS-ODN3 (SCR) served as AS-ODN controls. In (A), columns and error bars represent means ± SEM of duplicate values from independent ELISA experiments using cells cultured from 7–13 different donors. In (B), effects of transfection with (+) or without (–) AS-ODN3 on cytokine–stimulated eotaxin mRNA levels are shown by RT-PCR. The effect of sense and SCR on IL-13–stimulated cells is also shown. Data are representative of two experiments using cells cultured from different donors. ***p < 0.01 compared with cytokine-stimulated cells in the absence of AS-ODN treatment.

 
STAT6 AS-ODN Prevents Synergistic Eotaxin Release
In keeping with our previous finding of synergy between IL-13 or IL-4 and IL-1ß (12), levels of eotaxin in supernatants from cells treated with IL-13 or IL-4 (10 ng/ml) in combination with IL-1ß were more than 50-fold above those of unstimulated cells (10.3 ± 2.7 ng/ml per million cells, n = 8), compared with a 7- to 8-fold increase in cells stimulated by the cytokines alone (Figure 5) . In cells transfected with STAT6 AS-ODN3 (1 µM) this synergy was significantly attenuated (p < 0.05; n = 6–8). However, the extent of the reduction was only 32.9 ± 5.6 or 40.1 ± 3.7% in cells stimulated IL-13 and IL-1ß (Figure 5A) or IL-4 and IL-1ß (Figure 5B), respectively. This was about 50% less (p < 0.05) than the attenuation found when AS-ODN-tranfected cells were stimulated with either IL-13 or IL-4 alone (compare Figures 4A and 5).



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  		Figure 5. Effect of STAT6 AS-ODN3 on synergistic release of eotaxin from cultured human ASM cells after stimulation (10 ng/ml) for 24 hours with (A) IL-13 or (B) IL-4 in combination with IL-1ß. Cells were either left untransfected and incubated in medium alone (Unstim) or stimulated for 24 hours with cytokine at 10 ng/ml in the absence (FuGENE reagent only) or presence of transfection with 1 µM AS-ODN3. Transfection of cytokine-stimulated cells with a complementary ODN3 (Sense) or scrambled AS-ODN3 (SCR) served as AS-ODN controls. Columns and error bars represent means ± SEM of duplicate values from independent ELISA experiments using cells cultured from six to eight different donors. *p < 0.05, compared with synergistic increases in eotaxin in the absence of AS-ODN treatment.

 
STAT6 AS-ODN Prevents Suppression of IL-1ß–stimulated RANTES by IL-13 or IL-4
We have reported that neither IL-13 nor IL-4 alone induce RANTES release from human ASM cells (12). However, both IL-13 and IL-4 reduce IL-1ß– or TNF-{alpha}–dependent RANTES by about 50% (9, 12). The mechanism underlying this attenuation has not previously been investigated. IL-13 (10 ng/ml) suppressed IL-1ß–stimulated RANTES release by 42.8 ± 6.3% (p < 0.05, n = 11) (Figure 6A) and IL-4 attenuated the response by 49.8 ± 9.2% (p < 0.01, n = 9) (Figure 6B). This reduction was reversed in cells transfected with AS-ODN3 (1 µM), but not by transfection with sense-ODN or SCR-ODN (Figure 6).



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  		Figure 6. Effect of STAT6 AS-ODN3 on attenuation of IL-1ß–stimulated RANTES release from cultured human ASM cells by (A) IL-13 or (B) IL-4. Cells were either left untransfected and incubated in medium alone (Unstim, open column) or stimulated for 24 hours with cytokine at 10 ng/ml (solid columns) in the absence (FuGENE reagent only) or presence of transfection with 1 µM AS-ODN3 or a control sense ODN3 or scrambled AS-ODN3 (SCR). Columns and error bars represent means ± SEM of duplicate values from independent ELISA experiments using cells cultured from 9–11 different donors. *p < 0.05, **p < 0.01 compared with IL-1ß–stimulated cells in the absence of AS-ODN treatment.

 
STAT6 AS-ODN Prevents STAT6 Activation by IL-13 or IL-4
Consistent with our initial findings in cultures of human ASM cells (12) and those reported by others (14, 32), incubation of cells with IL-13 (10 ng/ml) for 15 minutes increased tyrosine phosphorylation of STAT6 protein compared with unstimulated cells, whereas IL-1ß (10 ng/ml) caused comparatively little detectable increase in STAT6 activation (Figure 7) . Phosphotyrosine levels of STAT6 appeared to be marginally further increased when IL-13 was combined with IL-1ß, but this was no more than additive (Figure 7 and Hirst and coworkers [12]). In all cases, levels of STAT6 phosphorylation induced by either IL-13 or IL-4 alone or in combination with IL-1ß were reduced in cells transfected with AS-ODN3 (1 µM), consistent with the parallel reduction in total STAT6 protein levels (Figure 7). No reduction in STAT6 phosphorylation or total STAT6 protein was observed in IL-13–stimulated cells transfected with SCR-ODN. A similar profile was obtained with a 10-ng/ml concentration of IL-4 in place of IL-13 (not shown).



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  		Figure 7. Effect of STAT6 AS-ODN3 on cytokine-stimulated activation of STAT6 by IL-13 alone or in combination with IL-1ß in human ASM. Cells, untransfected (–) or transfected (+) with 1 µM AS-ODN3 during growth arrest for 72 hours, were treated with cytokines (10 ng/ml for 15 minutes). Transfection with a scrambled AS-ODN3 (SCR) served as a control in IL-13–stimulated cells. Unstimulated cells (Unstim) were untransfected and were left untreated. Levels of phosphorylated STAT6 (p-STAT6) and total STAT6 in cell lysates were detected with antibodies that recognize total STAT6 and tyrosine-phosphorylated forms of STAT6. Membranes were reprobed with a monoclonal anti-GAPDH antibody to demonstrate equal gel loading.

 
MAP Kinase Inhibitors Attenuate Eotaxin Release by IL-13 or IL-4
Initial studies from our laboratory have demonstrated in human ASM that, in addition to STAT6 activation, IL-13 and IL-4 activate p42/p44 ERK and p38 MAP kinase pathways (12). In addition, it was demonstrated that U0126 and SB202190, potent and specific inhibitors of p42/p44 ERK and p38 MAP kinases, respectively, each reduced IL-13– or IL-4–dependent eotaxin release by as much as 60 to 70% (12, 14) raising the possibility that the residual component was mediated by STAT6, as suggested by Moore and colleagues (14). We tested this hypothesis by examining the effects of maximally effective concentrations of SB202190 (p38 MAP kinase inhibitor) or U0126 (MEK inhibitor) on IL-13– or IL-4–stimulated eotaxin release from cells transfected with a maximally effective concentration of STAT6 AS-ODN3 (1 µM [Figure 3]). In untransfected cells SB202190 (10 µM) reduced IL-13– or IL-4–induced eotaxin release by 67.5 ± 6.3 and 54.5 ± 7.1%, respectively (p < 0.01–0.001, n = 6) and U0126 reduced the response by 76.7 ± 4.1% (IL-13) and 72.3 ± 5.5% (IL-4) (p < 0.001, n = 6) (Figure 8) . When SB202190 and U0126 were combined the extent of inhibition to constitutive levels was further increased to 90%. In cells treated with STAT6 AS-ODN, attenuation of IL-13– or IL-4–stimulated eotaxin release by SB202190 or U0126 alone was not different from that in untransfected cells (p > 0.05, n = 6). However, when SB202190 and U0126 were combined in STAT6 AS-ODN–treated cells, IL-13– and IL-4–stimulated eotaxin release could be abolished, suggesting a requirement for each of the three pathways in the regulation of eotaxin production by IL-13 or IL-4 in human ASM (Figure 8).



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  		Figure 8. Comparison of the effects of inhibitors (10 µM) of p42/p44 ERK (U0126) or p38 MAP kinase (SB202190) activation on IL-13–dependent (open columns) or IL-4–dependent (solid columns) eotaxin release from untransfected (Control) human ASM cells or after transfection with AS-ODN3 (1 µM). Maximally effective concentrations of inhibitors and AS-ODN3 were selected. MAP kinase inhibitors were examined alone or in combination (SB/U). Transfection of cytokine-stimulated cells with a complementary ODN3 (Sense) or scrambled AS-ODN3 (SCR) served as AS-ODN controls. Data represent means ± SEM of independent ELISA experiments using cells cultured from six different donors. Excepting sense or SCR-ODN, all treatments resulted in significant reductions (p < 0.01–0.001) of control IL-13– or IL-4–dependent eotaxin release (statistical annotations omitted for clarity). However, differences were not found in the extent of reduction between untransfected control cells and cells treated with AS-ODN3 (two-way analysis of variance).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we examined a requirement for STAT6 in IL-13– and IL-4–dependent chemokine release from human ASM cells, using AS-ODNs directed against human STAT6 mRNA to reduce total STAT6 protein levels (30). We report that STAT6-specific AS-ODNs selectively attenuated STAT6 protein expression in cultured human ASM cells and were similarly effective in preventing eotaxin mRNA expression and protein release induced by either IL-13 or IL-4 alone and when in combination with IL-1ß. We also demonstrate a requirement for STAT6 protein in mediating suppression of IL-1ß–stimulated RANTES release by IL-13 or IL-4. Finally, we report that complete abolition of IL-13– or IL-4–stimulated eotaxin release occurred only when inhibitors of both p42/p44 ERK and p38 MAP kinase activation were present in STAT6 AS-ODN–tranfected cells, suggesting distinct MAP kinase pathways are required in addition to STAT6.

Lack of selective small chemical inhibitors of STAT6 activation has hindered understanding of STAT6 function in many cell types including smooth muscle (33). Here, we used a panel of three 5'- and 3'-end PS-modified AS-ODNs targeted to human STAT6 mRNA. With an FITC-tagged STAT6 AS-ODN we demonstrated concentration-dependent uptake by flow cytometry and fluorescence microscopy, which was maximal at 1 µM AS-ODN with about 85% of human ASM cells labeled. Using Western immunoblot detection, AS-ODN specificity was confirmed by selective downregulation of total STAT6 protein by the AS-ODN panel, but not by sense or SCR-ODNs, and the absence of downregulation of the highly related STAT family proteins, STAT3 and STAT5, or of GAPDH. Moreover, STAT6 AS-ODN, but not SCR-ODN, reduced levels of STAT6 phosphorylation induced by IL-13 or IL-4. The AS-ODN panel we used was based on sequences identified by Hill and colleagues by a STAT6 limited gene walk approach (30). The efficacy and selectivity we observed against total STAT6 protein and STAT6 phosphotyrosine levels in human ASM agree with the reduction in baseline levels of STAT6 protein by these AS-ODNs in A549 human lung epithelial cells and in L929 murine lung fibroblasts (30).

On the basis of selective downregulation of STAT6 we employed this antisense approach to examine a direct role for STAT6 protein in mediating IL-13– or IL-4–dependent eotaxin release, either alone or during synergy with IL-1ß. Consistent with a requirement for STAT6, transfection of cells with AS-ODN1, -2, or -3, but not sense or SCR-ODN3, markedly reduced eotaxin release by IL-13 or IL-4. The reduction in IL-13- or IL-4–stimulated eotaxin release with AS-ODN3 was about 75–80% of the response in untransfected cells, consistent with the observed marked reduction in total and phosphotyrosine STAT6, and in keeping with the overall number of cells shown to take up the FITC-labeled AS-ODN3 (about 85%). The IC50 obtained for attenuated IL-13–dependent eotaxin release (0.33 µM) was remarkably close to the value of 0.22 µM reported with this AS-ODN sequence for total STAT6 downregulation in A549 human airway epithelial cells, despite having a 2'-O-(2-methoxy)-ethyl modification in place of the 5'- and 3'-end PS modifications used in the present study (30). Overall, this accords with STAT6 activation being a major effector pathway for IL-13– or IL-4–stimulated eotaxin release from human ASM, and is consistent with other reports examining eotaxin regulation by IL-13 or IL-4. For example, in BEAS-2B human airway epithelial cells, IL-4–induced eotaxin gene reporter activity is blocked after transfection with a reporter plasmid mutated at the putative STAT6-binding site (34), and in human dermal fibroblasts eotaxin release by IL-13 is prevented after transfection with a dominant-negative STAT6 construct (21). In contrast, eotaxin gene activation by TNF-{alpha} was unaffected in BEAS-2B cells transfected with the STAT6 mutant eotaxin promoter reporter plasmid, consistent with TNF-{alpha} inducing eotaxin gene expression by a non-STAT6 mechanism (34). Likewise, we found that IL-1ß–dependent eotaxin release was largely unaffected after downregulation of STAT6, suggesting in human ASM cells that STAT6 is not required for the majority of eotaxin release induced by IL-1ß. Nevertheless, transfection with AS-STAT6 did cause a small (about 16%) and consistent reduction in IL-1ß–dependent eotaxin. This could involve abrogation of the effects of low levels of IL-1ß-induced autocrine IL-13 production, as occurs in human ASM after IgE sensitization (35), although it is unlikely this also accounts for the low levels of STAT6 phosphorylation by IL-1ß, seen here and in our previous study (12), given that stimulation was only for 15 minutes.

In general, transfection with STAT6 AS-ODN reduced but did not abolish release of eotaxin from human ASM cells. This may reflect transfection efficiencies of less than 100% or limitations in AS-ODN efficacy once inside the cells. However, STAT6 AS-ODN was more effective in preventing eotaxin release by IL-13 or IL-4 alone (about 70–80%) than under conditions of synergy with IL-1ß (about 35%), which may indicate that additional signaling pathways contribute to eotaxin release, particularly during synergy. In keeping with this possibility, we have previously reported that in addition to IL-4R{alpha}–dependent STAT6 phosphorylation, there is IL-4R{alpha}–dependent activation of p42/p44 ERK and p38 MAP kinases in human ASM cells (12). A requirement for MAP kinase activation in the release of eotaxin by IL-13 and IL-4 alone (or when combined with IL-1ß) was identified in human ASM, using maximally effective concentrations of the p38 inhibitor SB202190, or of the MEK-1/2 inhibitor U0126, with each attenuating eotaxin release by about 40–50% (12). The latter finding confirmed a report that appeared at the same time and that also hypothesized that the residual component of the response was mediated by STAT6 (14). Here, we found the inhibition by either SB202190 or U0126 could not be further increased in cells transfected with STAT6 AS-ODN, suggesting this hypothesis was incorrect. Abolition of eotaxin release was found, but only after abrogation of all three pathways when SB202190 and U0126 were combined in AS-ODN–treated cells. Thus, it appears that both p42/p44 ERK- and p38-dependent mechanisms are cooperatively involved in modulating a STAT6-dependent signaling pathway to eotaxin expression. The molecular mechanisms by which this occurs remain unclear, but are currently being addressed in other cell systems (21, 34). In ASM, it was reported that U0126 inhibited IL-13-induced eotaxin mRNA expression, suggesting that p42/p44 ERK may act at the level of gene transcription (14). On the basis of the presence within the eotaxin gene promoter of putative consensus transcription factor-binding sites for activator protein-1 and for nuclear factor for IL-6, which can be activated or directly phosphorylated by p42/p44 ERK in some cell systems (36, 37), it was speculated that ERK induces activation of transcription factors important for induction of eotaxin expression (14). Optimal STAT6 transcriptional activity at the eotaxin promoter may require posttranslational modification involving serine phosphorylation of STAT6 by p42p/44 ERK- or p38-dependent mechanisms. Indeed, other STATs (STAT-1, -3, -4, and -5a/b) contain serine phosphorylation sites within their C-terminal transactivation domains, and p42 ERK directly phosphorylates several of these STAT proteins (38). Likewise, a report of work performed in HepG2 cells indicates that p38 MAP kinase provides a costimulatory signal for IL-4–induced gene responses and stimulates the STAT6 transcriptional activity via an unknown intermediate kinase (39).

As an additional complication in the synergy studies reported here, IL-1ß–dependent eotaxin release from human ASM also involves activation of both p42p/44 ERK- and p38-dependent mechanisms (31), which on the basis of the present findings involves a non-STAT6 mechanism. Thus, under conditions of synergy, MAP kinase activation likely involves both STAT6-dependent and STAT6-independent pathways leading to eotaxin release. The latter may also involve NF-{kappa}B–dependent pathways, because its activation in human ASM is also increased by IL-1ß (40), and putative binding consensus response elements for NF-{kappa}B and STAT6 are found overlapping at a unique site in the eotaxin gene promoter (20). Functional analysis of the eotaxin promoter, using mutant promoter reporter plasmids, suggests STAT6 and NF-{kappa}B are directly involved in the synergy elicited by IL-13 or IL-4 and IL-1ß in human airway epithelial cells and in dermal fibroblasts (20, 21, 41). However, the detailed molecular mechanism by which STAT6 connects to the basal transcriptional machinery is currently poorly understood in ASM cells and in other cell types (39).

In contrast to synergistic increases in eotaxin release, both IL-13 and IL-4 suppress release of RANTES by IL-1ß (12). Attenuation of RANTES release under these conditions was completely abolished in cells transfected with a STAT6 AS-ODN, but not in those treated with sense or SCR-ODNs, suggesting STAT6 is required for the suppression. No putative consensus STAT6-binding response elements have been reported within the RANTES gene promoter, although supershift analysis suggests STAT1 may bind a novel IFN-responsive element in the promoter region of RANTES gene in hematopoietic cells (42). The mechanism by which STAT6 acts on the RANTES promoter or interacts with other transcription factors is unknown. Nevertheless, our findings provide the first evidence in ASM that suppression of RANTES release by IL-13 or IL-4 is STAT6 dependent.

In conclusion, we have demonstrated a functional requirement for STAT6 by the finding that IL-13– or IL-4–dependent eotaxin release was prevented in cells transfected with STAT6 AS-ODN, but not sense or SCR-ODN. A requirement was also demonstrated for STAT6 in mediating suppression of IL-1ß–stimulated RANTES release by IL-13 or IL-4. Finally, inhibition of p42/p44 ERK and p38 MAP kinase activation suggests that eotaxin release induced by IL-13 or IL-4 also requires activation of distinct MAP kinase pathways. Our present study emphasizes the complexities of chemokine release from ASM cells, particularly for eotaxin, through activation of multiple cellular signaling pathways. The precise relationship between these pathways in eliciting IL-13– or IL-4–dependent eotaxin release from ASM requires further investigation. Nevertheless, the data support the importance of STAT6 as a key player in some features of the pathogenesis of asthma including eosinophilic inflammation, mucus hypersecretion, and airway hyperreactivity as suggested by animal models of asthma (24–26).


   Acknowledgments
 
The authors thank the thoracic surgeons, operating theater staff, and pathologists of Guy's and St Thomas' Hospitals, London, for supply of human lung tissue, and Dr. David Cousins and Dr. Paul Lavender for critical reading of the manuscript.


   FOOTNOTES
 
Funded by a grant from the National Asthma Campaign, UK (no. 00/44).

This article has an online supplement, which is accessible from this issue's table of content online at www.atsjournals.org

Conflict of Interest Statement: Q.P. has no declared conflict of interest; T.M. has no declared conflict of interest; S.J.H. received $3,000 for speaking at a conference (October 2003) sponsored by GlaxoSmithKline and has participated as a speaker in scientific meetings or courses organized and financed by GlaxoSmithKline and AstraZeneca in 2001–2003.

Received in original form July 2, 2003; accepted in final form December 10, 2003


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