Signal Transducer And Activator Of Transcription 6 Expression Research Articles

Th2 Cytokines Increase Staphylococcus aureus Alpha Toxin–Induced Keratinocyte Death through the Signal Transducer and Activator of Transcription 6 (STAT6)

Atopic dermatitis (AD) is an inflammatory skin disease characterized by increased Th2 cytokine expression. AD skin lesions are often exacerbated by Staphylococcus aureus mediated secretion of the lytic virulence factor, alpha toxin. In the current study, we report that alpha toxin induced cell death is greater in the skin from patients with AD compared to controls. Furthermore, we find that keratinocyte differentiation and Th2 cytokine exposure influence sensitivity to S. aureus alpha toxin induced cell death. Differentiated keratinocytes are protected from cell death, while cells treated with Th2 cytokines have increased sensitivity to alpha toxin induced lethality. Our data demonstrates that downstream effects mediated by Th2 cytokines are dependent upon host expression of STAT6. We determine that Th2 cytokines induce biochemical changes that decrease levels of acid sphingomyelinase, an enzyme that cleaves sphingomyelin, an alpha toxin receptor. Furthermore, Th2 cytokines inhibit production of lamellar bodies, organelles critical for epidermal barrier formation. Finally, we determine that sphingomyelinase and its enzymatic product, phosphocholine, prevent Th2 mediated increases in alpha toxin induced cell death. Therefore, our studies may help explain the increased propensity for Th2 cytokines to exacerbate S. aureus induced skin disease, and provide a potential therapeutic target for treatment of AD.

STAT6 expression in T cells, alveolar macrophages and bronchial biopsies of normal and asthmatic subjects

BackgroundAsthma is characterised by increased numbers of Th2-like cells in the airways and IgE secretion. Generation of Th2 cells requires interleukin (IL)-4 and IL-13 acting through their specific receptors and activating the transcription factor, signal transducer and activator of transcription 6 (STAT6). STAT6 knockout mice fail to produce IgE, airway hyperresponsiveness and bronchoalveolar lavage eosinophilia after allergen sensitisation, suggesting a critical role for STAT6 in allergic responses.MethodsWe have investigated the expression of STAT6 in peripheral blood T-lymphocytes, alveolar macrophages and bronchial biopsies from 17 normal subjects and 18 mild-moderate steroid-naïve stable asthmatic patients.ResultsSTAT6 expression was variable and was detected in T-lymphocytes, macrophages and bronchial epithelial cells from all subjects with no difference between normal and stable asthmatic subjects.ConclusionsSTAT6 expression in different cells suggests that it may be important in regulating the expression of not only Th2-like cytokines in T cells of man, but may also regulate STAT-inducible genes in alveolar macrophages and airway epithelial cells.

Expression of STAT6 and NF-κB p65 in the colon mucosa of patients with ulcerative colitis

The expression of signal transducer and activator of transcription 6 (STAT6) and nuclear factor-κB (NF-κB) in the colonic mucosa of patients with ulcerative colitis (UC) was examined. Real-time polymerase chain reaction and immunohistochemistry were used to detect the expression of STAT6 and NF-κB p65 at both mRNA and protein levels in the colonic mucosa of patients with UC and healthy volunteers. The results showed that the expression levels of STAT6 and NFκB p65 in the colonic mucosa of patients with UC were significantly higher than in normal controls at both mRNA and protein levels. These data suggest that STAT6 and NFκB p65 perhaps play an important role in the pathogenesis of UC and underscore the potential value of anti-UC strategies in the clinical management of this disease.

Effect of compound Sophorae Flavescentis Jiechangrong capsule on expression of NF-κB p65 and STAT6 in the intestinal mucosa of patients with ulcerative colitis

The effects of compound Sophorae Flavescentis Jiechangrong capsule (CSFJC) on the expression of nuclear factor-κB p65 (NF-κB p65) and signal transducer and activator of transcription 6 (STAT6) in the intestinal mucosa of patients with ulcerative colitis and the possible mechanism were investigated. Eighteen patients with ulcerative colitis were randomly divided into a traditional Chinese medicine (TCM) group (n = 11) treated by CSFJC and a western medicine (WM) group (n = 7) treated by Sulfasalazine tablets. The treatment duration lasted eight weeks. Before and after the treatment, the symptoms and the physical signs were observed, and the routine stool test, the colonoscopy, and pathological examination were performed in the two groups. The expression levels of NF-κB p65 and STAT6 were detected by using immunohistochemistry. The results showed that the total effective rate of the curative effectiveness in TCM and WM groups was 100% and 71.4%, respectively, and the total effective rate of colonic mucosa lesion in TCM and WM groups was 90.9% and 71.4%, respectively, with the differences being significant (all P<0.05). The total effective rate of syndromes of damp-heat blocking according to the TCM in TCM and WM groups was 90.9% and 71.4%, respectively. After the treatment, the expression of NF-κB p65 and STAT6 in the two groups was decreased, and the decrease of NF-κB p65 and STAT6 expression in TCM group was more significant than in WM group (P<0.05). It was concluded that CSFJC can inhibit the activation and expression of NF-κB p65 and STAT6 in the intestinal mucosa of patients with ulcerative colitis, which is a possible mechanism for CSFJC treating ulcerative colitis.

Modulation by aspirin of nuclear phospho–signal transducer and activator of transcription 6 expression: Possible role in therapeutic benefit associated with aspirin desensitization

Aspirin-exacerbated respiratory disease is characterized by asthma, nasal polyps, and intolerance to aspirin with overexpression of leukotriene (LT) C(4) synthase and cysteinyl leukotriene receptors. Through an unknown mechanism, aspirin desensitization is an effective treatment. We hypothesized that aspirin desensitization blocks IL-4-induced expression of these LT activities through inhibition of signal transducer and activator of transcription 6 (STAT6)-mediated transcription. Nuclear extracts were derived from THP-1 and normal human monocytes resting and cocultured with aspirin before IL-4 stimulation. Quantitative PCRs were conducted. Electrophoretic mobility shift assays were performed by using oligomers for STAT6 sites within the LT receptor and synthase promoters. Western blots of nuclear extracts were probed by using anti-phospho-STAT6 antibodies. Upregulation of LT receptor mRNA by IL-4 was negated by aspirin and ketorolac but not by sodium salicylate. The STAT6 site in the LT receptor and synthase promoters was confirmed by using mobility shift assays by competing with unlabeled STAT6 consensus probes and supershifts with anti-STAT6 antibodies. Aspirin and ketorolac decreased the IL-4-inducible expression of nuclear STAT6 observed in mobility shift assays and Western hybridization. The LT receptor and synthase promoters have STAT6-binding sites that are engaged by IL-4-induced nuclear extracts and inhibited by aspirin. Assuming that normal monocytes behave like monocytes from patients with aspirin-exacerbated respiratory disease, inhibition of IL-4-STAT6 might explain a mechanism in aspirin desensitization daily treatment, resulting in downregulation of production and responsiveness to cysteinyl leukotrienes. This biologic activity of aspirin likely reflects COX inhibition because it was shared with ketorolac but not sodium salicylate.

RNA interference of STAT6 rapidly attenuates ongoing interleukin‐13‐mediated events in lung epithelial cells

Signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a central role in asthma pathogenesis, with its activation driving the development of airway hyper-reactivity and local inflammation. Therefore, inhibition of local STAT6 expression provides a rationale for therapeutic intervention in bronchial asthma. Given the absence of specific inhibitory drugs, we tested the ability of small interfering RNAs (siRNAs) to target STAT6 gene expression through the molecular process of RNA interference (RNAi). At pico-molar concentrations, STAT6-specific siRNAs potently inhibited STAT6 mRNA expression in lung epithelial cells (50% inhibitory concentration range = 134-861 pm) without inducing cellular interferon responses. Detectable STAT6 protein expression was rapidly abolished within 48 hr of treatment (t(1/2) range = or < 12-37 hr) and this was unaffected by pretreatment with STAT6-activating cytokines. Furthermore, STAT6 suppression by RNAi produced downstream functional inhibitory effects in that interleukin (IL)-13- or IL-4-driven eotaxin chemokine family [chemokine (C-C motif) ligand 11 (CCL11), CCL24 and CCL26] mRNA expression was markedly inhibited. Induction of detectable CCL26 protein synthesis was completely ablated by pretreating cells with STAT6-specific siRNA. The therapeutic potential of this approach is further demonstrated by novel findings that cells pre-exposed to IL-13 or IL-4 and subsequently treated with STAT6-targeting siRNA exhibited a rapid and significant attenuation of ongoing CCL26 protein expression, suggesting that chronic asthma-associated lung inflammation will be responsive to this approach.

Effect of the Chinese Herb Extract Osthol on IL-4-induced Eotaxin Expression in BEAS-2B Cells

Asthma is an allergic inflammatory disease of the airways. The interaction between bronchial epithelial cells and eosinophils is an important feature of an asthma attack. Eotaxin, an eosinophil-specific C-C chemokine, is a potent chemoattractant involved in the mobilization of eosinophils into the airway after allergic stimulation. Cnidii monnieri fructus, the dried fruit of Cnidium monnieri Cusson, has been used as an antipruritogenic agent in ancient China. OsthoL is the major component of Cnidii monnieri fructus extract. We investigated the ability of osthol to regulate cytokine-induced eotaxin expression in the human bronchial epithelial cell line BEAS-2B. BEAS-2B cells were pretreated with osthol at different concentrations (0.1-10 microM), and then stimulated with interleukin (IL)-4 alone, or in combination with tumor necrosis factor (TNF)-alpha. Eotaxin levels were determined by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. STAT6 (signal transducer and activator of transcription 6) and MAPK (mitogen-activated protein kinase) expressions were evaluated by Western blotting, to detect possible intracellular signal transduction. IL-4 and TNF-alpha significantly induced eotaxin expression in BEAS-2B cells. Expression of eotaxin was suppressed by osthol (0.1-10 microM) in a dose-dependent manner. Osthol did not suppress IL-4-induced p38, ERK or JNK expression. Osthol did suppress IL-4-induced STAT6 in a dose-dependent manner. Osthol suppressed IL-4-induced eotaxin in BEAS-2B cells via inhibition of STAT6 expression. This data suggest that osthol might have potential for treating allergic airway inflammation.

Signal transducer and activator of transcription‐6 (STAT6) is a constitutively expressed survival factor in human prostate cancer

Signal transducer and activator of transcription (STAT)-6 is a member of the STAT family of latent transcription factors. In this investigation, we examined STAT6 expression in clinical prostate cancer tissue specimen and determined its role in prostate cell proliferation and migration. STAT6 expression in cell lines and tissues was analyzed by RT-PCR, IHC and/or immunoblot analyses. Down-regulation of STAT6 expression was achieved by STAT6 siRNA and its effect on cell migration and apoptosis was measured. STAT6 is highly expressed in the fibromuscular stroma of prostate cancer specimens. STAT6 is also expressed in the malignant epithelial layer and prostate intraepithelial neoplasia (PIN). STAT6 expression was significantly correlated with high histological grades of prostate cancer as well as with tumor size. Our data indicate deregulated STAT6 mRNA and protein expression in prostate cancer cells with high levels in the non-cancerous HPV 18C-1 and cancerous DU145 cell lines and low levels in PC3 and LNCaP cells. Phosphorylated STAT6 was expressed in all three cancer cell lines DU145, PC3, and LNCaP. Down-regulation of STAT6 using siRNA leads to the induction of early apoptosis in DU145 cells and inhibits migration of these cells. Significant reduction in cell viability and transcriptional down-regulation of the anti-apoptotic protein Bcl-X(L) was observed followed by STAT6 down-regulation in DU145 cells. Interestingly STAT6 also regulates transcription of 15-lipoxygenase-1 gene in DU145 cells. Our data suggest that STAT6 is a survival factor in prostate cancer and regulates the genetic transcriptional program that is responsible for prostate cancer progression.

Budesonide attenuates airway remodeling and modules the expression of Janus protein tyrosine kinase 1, and signal transducers and activators of transcription 6 in asthma: an experiment with mice

To investigate the effects of budesonide (BUD) on the airway remodeling and the expression of Janus protein tyrosine kinases 1 (JAK1) and signal transducer and activator of transcription 6 (STAT6) in asthma. Thirty female Balb/c mice were randomly divided into 3 equal groups: control group; asthma group, sensitized on day 1, 8, and 15 and challenged from day 21 to 52 with periodically repeated intranasal drip of ovalbumin (OVA); and BUD treated group, undergoing intranasal drip of OVA as mentioned above and intranasal administration of BUD 2 hours before each OVA challenge. 24 h after the final OVA inhalation an invasive single-chamber whole body plethysmograph was used to assess the airway responsiveness. Then bronchoalveolar lavage fluid (BALF) was obtained and ELISA was used to measure the contents of interleukin (IL)-4 and IL-13. The mice were killed and their lungs taken out. HE staining and periodic acid Schiff (PAS) staining were used to observe the airway score of goblet cells. Peribronchiolar collagen deposition was imaged in Masson-stained lung sections. Biochemical assay was used to determine the total lung tissue level of collagen. Potass hydrolyse method was used to examine the content of hydroxyproline in the lung tissue. Western blotting was used to detect the protein expression of alpha-smooth muscle actin (SMA), JAK1, and STAT6. RT-PCR was used to detect the mRNA expression of alpha-SMA. The value of LogPC100 of the asthma group was 1.88 +/- 0.34, significantly higher than those of the BUD and control groups (1.79 +/- 0.18 and 0.82 +/- 0.78 respectively, both P = 0.000). The airway score of goblet cells of the asthma group was 3.05 +/- 0.23, significantly higher than those of the BUD and control groups (1.35 +/- 0.26 and 0.40 +/- 0.13 respectively, both P < 0.01). The hydroxyproline content of the asthma group was (459 +/- 47) microg/100 mg tissue, significantly higher than those of the BUD and control groups [(284 +/- 16) and (181 +/- 22) microg/100 mg tissue respectively, both P < 0.01]. The level of IL-4 of the asthma group was (14.4 +/- 1.12) ng/L, significantly higher than those of the BUD and control groups [(7.3 +/- 0.6) and (5.6 +/- 0.8) ng/L respectively, both P < 0.01]. The IL-13 level of the asthma group was (16.8 +/- 0.9) ng/L, significantly higher than those of the BUD and control groups [(10.6 +/- 0.9) and (5.6 +/- 0.8) ng/L respectively, both P < 0.01]. Treatment of BUD attenuated the allergen-induced airway hyperresponsiveness (AHR) and structural changes in airway, and decreased the values of the airway scores of goblet cells, and levels of hydroxyproline, IL-4, and IL-13 in comparison with the asthma group (all P < 0.01). Repeated OVA challenge resulted in an upregulation of the expression levels of alpha-SMA, JAK1 and STAT6 protein and alpha-SMA mRNA, while use of BUD suppressed these changes. The changes of JAK1 and STAT6 expression were correlated significantly with the changes in the airway score of goblet cells, hydroxyproline content, expression level of alpha-SMA, and levels of IL-4 and IL-13 in BALF (all P < 0.05). BUD ameliorates the progression of airway remodeling following prolonged allergen challenge via regulation of JAK1/STAT6 signal pathway.

Inhibition of Signal Transducer and Activator of Transcription 6 Activity in the Hodgkin Lymphoma Cell Line L1236 Induces Growth Inhibition and Apoptosis.

Signal transducer and activator of transcription 6 (STAT6) plays a central role in interleukin (IL)-4 and -13 signaling. Upon binding of the cognate receptors by these cytokines, STAT6 becomes phosphorylated by Jak family kinases and subsequently translocates to the nucleus where transcription of its target genes is regulated. Expression of IL-13 and its receptor are common features of Hodgkin lymphoma (HL) tumor cells, the so-called Hodgkin Reed-Sternberg (H-RS) cells, in which this cytokine has been shown to act as an autocrine growth factor. Consequently, constitutively phosphorylated STAT6 with a nuclear localization is a common and distinctive feature of H-RS cells in classical HL. We knocked down STAT6 expression in the HL cell line L1236 with small interfering RNA (siRNA) and found that inhibition of STAT6 activity results in cell growth inhibition, decreased viability and increased apoptosis. The results depict a central role of STAT6 in the growth of H-RS cells and indicate that STAT6 could be a potential target for therapeutic intervention in HL. Moreover, in order to identify the target genes of this transcription factor in H-RS cells, a combined approach of RNA interference and microarray is performed and the results will be presented.

DNA vaccine encoding Der p2 allergen down-regulates STAT6 expression in mouse model of allergen-induced allergic airway inflammation

Activation of signal transducer and activator of transcription 6 (STAT6) plays a critical role in the late phase of Th2-dependent allergy induction. STAT6 is essential to Th2 cell differentiation, recruitment, and effector function. Our previous study confirmed that DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen. In the present study, we determined whether DNA vaccine encoding Dermatophagoides pteronyssinus group 2 (Der p2) could down-regulate the expression and activation of STAT6 in lung tissue from asthmatic mice. After DNA vaccine immunization, BALB/c mice were sensitized by intraperitoneal injection and challenged by intranasal instillation of rDer p2. The levels of the cytokines IL-4 and IL-13 in BAL fluid were measured by enzyme-linked immunosorbent assay. The lung tissue was assessed by immunohistochemical staining with anti-STAT6. The protein expression of STAT6 was determined by Western blot. The activation of STAT6 binding ability was analyzed with electrophoretic mobility shift assay. DNA vaccine encoding Der p2 allergen effectively decreased the levels of IL-4 and IL-13 in the asthmatic mice. Histological evidence and Western blot showed that the expression of STAT6 in the DNA treated mice was markedly attenuated. STAT6 binding to specific DNA motif in lung tissue from the gene vaccinated mice was inhibited. DNA vaccine encoding Der p2 prevents allergic pulmonary inflammation probably by inhibiting the STAT6 signaling pathway in mice with Der p2 allergen-induced allergic airway inflammation.

Increased numbers of committed myeloid progenitors but not primitive hematopoietic stem/progenitors in mice lacking STAT6 expression.

Signal transducer and activator of transcription-6 (STAT6) plays important roles in cytokine signaling via interleukin-4 and -13 receptors (IL-4R and IL-13R). Mice in which STAT6 has been disrupted by homologous recombination show defects in T helper cell type 2 (Th2) lymphocyte production, resulting in an accumulation of Th1 cells. In addition to defects in differentiation and proliferation of T lymphocytes, STAT6-deficient mice show increased cell-cycle activation and frequency of myeloid progenitors. Although this has been shown to be mediated through Oncostatin M production by T cells, IL-4Ralpha and STAT6 have also recently been found to be enriched for expression in primitive hematopoietic stem cells (HSCs) in gene expression-profiling studies. Therefore, we have investigated whether defects in hematopoietic function in mice lacking STAT6 expression extended into the primitive hematopoietic compartments of the bone marrow. Here, we report that STAT6 deficiency increased bone marrow-committed myeloid progenitors but did not alter the number of cells enriched for HSC/multipotent progenitors, primitive cobblestone area-forming cells assayed in vitro, or bone marrow short-term or long-term repopulating cells assayed in vivo. Therefore, the requirement for STAT6 activation during hematopoiesis is limited, and primitive hematopoietic cell types are insulated against possible effects of cytokine stimulation by Th1 cells.

Differential activation of signal transducer and activator of transcription 6 in B cells from allergic children and their non‐allergic siblings

The germline (GL) epsilon promoter is regulated by IL-4 and is essential for class switching to IgE. IL-4-induced gene expression is largely mediated through activation of latent transcription factor STAT6 (signal transducer and activator of transcription 6). We investigated whether increased levels of IgE in allergic individuals may be associated with alteration in the level or activation of STAT6 and subsequent increase in GL epsilon promoter activity. Electrophoretic mobility shift assay and Western blotting assays were used to investigate the level of expression and activation of STAT6 in Epstein-Barr virus (EBV)-transformed B cell lines from children with birch pollen allergy and their non-allergic siblings. The activity of the GL epsilon promoter was tested in a transient transfection assay. STAT6 was expressed at the same level in all B cell lines tested. In two out of five sibling pairs STAT6 was activated by IL-4 more efficiently in the allergic individuals but in the three other pairs the opposite was found. In transient transfections, no difference in IL-4-induced GL epsilon promoter function was detected, although basal promoter activity varied between allergic and healthy siblings in two out of five pairs. We demonstrate for the first time that upon IL-4 signalling STAT6 transcription factor activation differs in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude a possibility that stronger activation of this transcription factor is associated with an expression of allergic phenotype.

Molecular mechanisms of interleukin-4-induced up-regulation of type I collagen gene expression in murine fibroblasts.

There is evidence that interleukin-4 (IL-4) plays a major role in the induction of extracellular matrix protein synthesis in fibrotic disease. We therefore examined the effect of IL-4 on collagen synthesis in primary fibroblasts isolated from normal and TSK/+ mice, which spontaneously develop a scleroderma-like syndrome characterized by diffuse cutaneous hyperplasia. Expression of the IL-4 receptor was determined by flow cytometry and Western blotting. The IL-4 signal transduction cascade was analyzed by Western blotting. We assessed the role of signal transducer and activator of transcription 6 (STAT-6) in IL-4 induction of alpha2(I) collagen promoter activity and message levels via luciferase reporter assay and real-time polymerase chain reaction. The activation status of the transcription factors activator protein 1 (AP-1) and Sp-1 upon stimulation with IL-4 in normal and TSK/+ fibroblasts was examined by electrophoretic mobility shift assay. Flow cytometry and Western blotting showed that IL-4 receptor alpha expression was elevated in TSK/+ fibroblasts compared with normal fibroblasts. After IL-4 stimulation, janus-activated kinase 1 (JAK-1) and JAK-2 were phosphorylated to a greater degree in TSK/+ fibroblasts than in C57BL/6 fibroblasts. TSK/+ fibroblasts appeared to be hyperresponsive to IL-4, displaying increased synthesis of alpha1(I) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine alpha2(I) collagen promoter. Overexpression of STAT-6 enhanced this effect, whereas expression of a dominant-negative STAT-6 abrogated the ability of IL-4 to induce alpha1(I) collagen mRNA in TSK/+ fibroblasts. Moreover, IL-4 induced increased DNA binding activity of transcription factors that are important for collagen synthesis. Our observations indicate that IL-4 has a profound effect on several factors that have been identified as playing major roles in the regulation of collagen synthesis and suggest that IL-4 increases the expression of type I collagen through a mechanism involving the activation of transcription factors that bind to and activate collagen promoter.

STAT6 Mediates Interleukin-4 Growth Inhibition in Human Breast Cancer Cells

In addition to acting as a hematopoietic growth factor, interleukin-4 (IL-4) inhibits growth of some transformed cells in vitro and in vivo. In this study, we show that insulin receptor substrate (IRS)-1, IRS-2, and signal transducer and activator of transcription 6 (STAT6) are phosphorylated following IL-4 treatment in MCF-7 breast cancer cells. STAT6 DNA binding is enhanced by IL-4 treatment. STAT6 activation occurs even after IRS-1 depletion, suggesting the two pathways are independent. To examine the role of STAT6 in IL-4-mediated growth inhibition and apoptosis, a fulllength STAT6 cDNA was transfected into MCF-7 cells. Transient overexpression of STAT6 resulted in both cytoplasmic and nuclear expression of the protein, increased DNA binding in response to IL-4, and increased transactivation of an IL-4 responsive promoter. In STAT6-transfected cells, basal proliferation was reduced whereas apoptosis was increased. Finally, stable expression of STAT6 resulted in reduced foci formation compared to vector-transfected cells alone. These results suggest STAT6 is required for IL-4mediated growth inhibition and induction of apoptosis in human breast cancer cells.

Signal transducer and activator of transcription 6 (STAT-6) expression and function in asthmatic bronchial epithelium

Background: Asthma is associated with increased production of IL-4 and IL-13. Objective: Because many of the effects of these cytokines are mediated by activation of signal transducer and activator of transcription 6 (STAT-6), we investigated expression and function of this transcription factor in the airways. Methods: STAT-6 expression was investigated through use of immunohistochemistry or RT-PCR applied to bronchial biopsy specimens or brushings from normal control or asthmatic subjects. STAT-6 function was investigated by means of Western blotting and ELISA applied to primary epithelial cell cultures. Results: Immunohistochemistry revealed that the bronchial epithelium was the major site of STAT-6 expression, both cytoplasmic and nuclear staining being observed. The level of STAT-6 expression in subjects with mild asthma (median [range] percent epithelial staining, 3.4% [0% to 16.0%]; n = 14) did not differ significantly from that in normal controls (4.7% [0.0% to 20.0%]; n = 11); however, in subjects with severe asthma, epithelial STAT-6 expression (13.7% [4.8% to 25.7%]; n = 9) was increased in comparison with subjects with mild asthma and normal controls (P < .05). RT-PCR analysis showed that epithelial STAT-6 expression was heterogeneous and comprised both full-length STAT-6 and the dominant-negative variant that lacks the SH2 domain. Treatment of primary cultures of bronchial epithelial cells with IL-4 resulted in STAT-6 phosphorylation and stimulation of IL-8 secretion; however, no difference in the responses of epithelial cells was observed between normal (n = 12) and asthmatic (n = 14) donors. Conclusion: These data demonstrate expression and activation of STAT-6 in normal and asthmatic bronchial epithelium. The activity of this transcription factor is likely to play a key role in mediating the responses of the bronchial epithelium to TH2 cytokines that are characteristic of the asthmatic phenotype. (J Allergy Clin Immunol 2001;108:832-8.)

TH2 cytokine–associated transcription factors in atopic and nonatopic asthma: Evidence for differential signal transducer and activator of transcription 6 expression

Background: The expression of IL-4 and IL-5 is increased in patients with atopic asthma compared with control subjects and correlates with indices of pulmonary function. In nonatopic asthma the expression of IL-4, unlike IL-5, fails to correlate with pulmonary function, and compared with their atopic counterparts, these patients have fewer cells expressing IL-4 receptor (IL-4R). As such, a deficiency in the IL-4 signaling pathway may be implicated in nonatopic asthma. The transcription factors GATA-3 and cMAF mediate IL-4 and IL-5 synthesis, whereas signal transducer and activator of transcription 6 (STAT-6) is critical for IL-4R signaling. Objective: This study examines the expression profile of these transcription factors in asthma, according to atopic status. Methods: With immunocytochemistry, the expression of GATA-3, cMAF, and STAT-6 protein was determined in sections of bronchial biopsy specimens from patients with atopic asthma (n = 7), patients with nonatopic asthma (n = 8), and control subjects (n = 8). Results: Higher numbers of cells expressing GATA-3 and cMAF were observed in patients with atopic and those with nonatopic asthma than in control subjects and patients with tuberculosis (P < .001). There were also more STAT-6–immunoreactive cells in patients with atopic and those with nonatopic asthma than in control subjects (P < .0001, P < .05). Notably, however, fewer cells expressing STAT-6 protein were observed in nonatopic versus atopic asthma (P < .0001). Conclusions: These results demonstrate the upregulation of GATA-3 and cMAF in both variants of asthma and indicate that reduced IL-4R signaling, because of lower STAT-6 expression, may be a feature of nonatopic asthma. (J Allergy Clin Immunol 2001;107:586-91.)

Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression.

Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, the mechanism by which IFNs mediate this inhibition has not been defined. IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-beta or IFN-gamma, but not IL-1, IL-2, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IL-6, or transforming growth factor beta suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was not evident unless the cells were preincubated with IFN for at least 1 hr before IL-4 stimulation. Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-gamma-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1.