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Id Subject Object Predicate Lexical cue
T1 0-57 Sentence denotes Suppression of GATA-3 Nuclear Import and Phosphorylation:
T135 0-120 Sentence denotes Suppression of GATA-3 Nuclear Import and Phosphorylation: A Novel Mechanism of Corticosteroid Action in Allergic Disease
T2 58-159 Sentence denotes A Novel Mechanism of Corticosteroid Action in Allergic Disease Steroid Suppression of GATA-3 Function
T3 162-327 Sentence denotes Peter Barnes and colleagues show that corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that suppress Th2 cytokine expression.
T4 328-444 Sentence denotes This novel mechanism of corticosteroid action may help explain the efficacy of corticosteroids in allergic diseases.
T5 446-454 Sentence denotes Abstract
T136 455-465 Sentence denotes Background
T6 455-465 Sentence denotes Background
T137 466-658 Sentence denotes GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases.
T7 466-658 Sentence denotes GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases.
T138 659-774 Sentence denotes Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown.
T8 659-774 Sentence denotes Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown.
T139 775-912 Sentence denotes We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.
T9 775-912 Sentence denotes We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.
T140 914-934 Sentence denotes Methods and Findings
T10 914-934 Sentence denotes Methods and Findings
T141 935-1374 Sentence denotes In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-κB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-α.
T11 935-1374 Sentence denotes In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-κB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-α.
T142 1375-1579 Sentence denotes In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation.
T12 1375-1579 Sentence denotes In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation.
T143 1580-1653 Sentence denotes These inhibitory effects of fluticasone are rapid, potent, and prolonged.
T13 1580-1653 Sentence denotes These inhibitory effects of fluticasone are rapid, potent, and prolonged.
T144 1654-1802 Sentence denotes We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.
T14 1654-1802 Sentence denotes We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.
T145 1804-1815 Sentence denotes Conclusions
T15 1804-1815 Sentence denotes Conclusions
T146 1816-1952 Sentence denotes Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression.
T16 1816-1952 Sentence denotes Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression.
T147 1953-2107 Sentence denotes This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.
T17 1953-2107 Sentence denotes This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.
T148 2109-2161 Sentence denotes Please see later in the article for Editors' Summary
T18 2109-2161 Sentence denotes Please see later in the article for Editors' Summary
T19 2163-2179 Sentence denotes Editors' Summary
T20 2181-2191 Sentence denotes Background
T21 2192-2279 Sentence denotes The immune system protects the human body from viruses, bacteria, parasites, and fungi.
T22 2280-2555 Sentence denotes When one of these foreign invaders enters the body, immune system cells called T lymphocytes recognize specific molecules on the invader's surface and release chemical messengers (cytokines) that recruit and activate other types of immune cell, which then attack the invader.
T23 2556-2787 Sentence denotes Sometimes, however, the immune system responds to a normally harmless material (for example, house-dust mites or grass pollen; scientists call these materials allergens) and triggers an allergic disease such as asthma or hay fever.
T24 2788-2974 Sentence denotes Contact with an allergen activates a type of T lymphocyte called a T helper-2 (Th2) cell that subsequently makes (expresses) three cytokines called interleukin-4 (IL-4), IL-5, and IL-13.
T25 2975-3080 Sentence denotes These cytokines ultimately cause inflammation (swelling) of the part of the body exposed to the allergen.
T26 3081-3213 Sentence denotes Corticosteroids, which suppress the expression of cytokines by Th2 cells, are often used to treat inflammation in allergic diseases.
T27 3214-3455 Sentence denotes Other treatments for these common conditions—about 50 million people in the US have an allergic disease—include minimizing exposure to allergens and diminishing the response of the immune system to allergens by using various immunotherapies.
T28 3457-3481 Sentence denotes Why Was This Study Done?
T29 3482-3626 Sentence denotes Scientists know that corticosteroids reduce allergic inflammation by binding to proteins in immune system cells called glucocorticoid receptors.
T30 3627-3828 Sentence denotes After binding to a corticosteroid, these receptors move into the nucleus of the cell (the part of the cell that contains its genes), where they suppress the expression of certain proinflammatory genes.
T31 3829-3920 Sentence denotes However, it is still not known how corticosteroids inhibit the expression of Th2 cytokines.
T32 3921-4043 Sentence denotes A key regulator of the expression of these cytokines and of allergic inflammation is a transcription factor called GATA-3.
T33 4044-4188 Sentence denotes Transcription factors are proteins that control the expression of other proteins by binding to specific sequences in the genes that encode them.
T34 4189-4413 Sentence denotes In this study, the researchers try to discover more about how corticosteroids reduce allergic inflammation by investigating the effects of the corticosteroid fluticasone on the regulation of GATA-3 activity in T lymphocytes.
T35 4415-4452 Sentence denotes What Did the Researchers Do and Find?
T36 4453-4692 Sentence denotes Transcription factors have to move into the nucleus of cells (so-called nuclear translocation) to control the expression of their target genes, so the researchers first asked whether fluticasone affects the cellular localization of GATA-3.
T37 4693-4856 Sentence denotes Fluticasone treatment of activated T lymphocytes growing in dishes, they report, inhibited the nuclear translocation of GATA-3 and reduced Th2 cytokine expression.
T38 4857-5108 Sentence denotes Other experiments showed that the inhibition of GATA-3 nuclear translocation was partly caused by competition between the glucocorticoid receptor bound to fluticasone and GATA-3 for binding to importin-α, a protein that is required for nuclear import.
T39 5109-5197 Sentence denotes However, fluticasone also prevented the nuclear translocation of GATA-3 in a second way.
T40 5198-5298 Sentence denotes Before GATA-3 can bind to importin-α, phosphate groups have to be added to specific sites in GATA-3.
T41 5299-5522 Sentence denotes This “phosphorylation” requires an enzyme called p38 MAP kinase, and the researchers found that fluticasone treatment of activated T lymphocytes induced the expression of MAP kinase phophatase-1, a p38 MAP kinase inhibitor.
T42 5523-5745 Sentence denotes Finally, when the researchers treated seven patients with mild asthma with inhaled fluticasone, they found that fluticasone also inhibited GATA-3 nuclear translocation in the lymphocytes circulating in the patients' blood.
T43 5747-5775 Sentence denotes What Do These Findings Mean?
T44 5776-5983 Sentence denotes These findings, obtained both in the laboratory and in patients, suggest that corticosteroids inhibit the expression of Th2 cytokines and thus reduce allergic inflammation through two interacting mechanisms.
T45 5984-6263 Sentence denotes They suggest that corticosteroids prevent the nuclear translocation of GATA-3, a key regulator of Th2 cytokine expression, by competing with GATA-3 for binding to importin-α and by preventing the phosphorylation of GATA-3, a modification that allows GATA-3 to bind to importin-α.
T46 6264-6584 Sentence denotes This dual mechanism of corticosteroid action may help to explain why these drugs are so effective in the treatment of allergic diseases, although further experiments are needed to show that the lymphocytes resident at sites of allergic inflammation respond to corticosteroids in the same way as lymphocytes in the blood.
T47 6585-6752 Sentence denotes Finally, these findings suggest that the interaction between phosphorylated GATA-3 and importin-α might be a potential target for new treatments for allergic diseases.
T48 6754-6776 Sentence denotes Additional Information
T49 6777-6896 Sentence denotes Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000076.
T50 6897-7228 Sentence denotes The US National Institute of Allergy and Infectious Diseases provides information on allergic diseases and a simple description of the immune system The UK National Health Service Choices service provides information about allergies Links to other information about allergies are available from MedlinePlus (in English and Spanish)
T51 7233-7245 Sentence denotes Introduction
T1295 7246-7436 Sentence denotes Inflammation in allergic diseases such as asthma, rhinitis, and atopic dermatitis is mediated via expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells.
T52 7246-7436 Sentence denotes Inflammation in allergic diseases such as asthma, rhinitis, and atopic dermatitis is mediated via expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells.
T1296 7437-7566 Sentence denotes IL-4 and IL-13 regulate the expression of IgE from B lymphocytes, whereas IL-5 plays a key role in eosinophilic inflammation [1].
T53 7437-7566 Sentence denotes IL-4 and IL-13 regulate the expression of IgE from B lymphocytes, whereas IL-5 plays a key role in eosinophilic inflammation [1].
T1297 7567-7697 Sentence denotes Th2 cytokines are regulated by the zinc finger transcription factor GATA-3, which is predominantly expressed in Th2 cells [2],[3].
T54 7567-7697 Sentence denotes Th2 cytokines are regulated by the zinc finger transcription factor GATA-3, which is predominantly expressed in Th2 cells [2],[3].
T1298 7698-7844 Sentence denotes GATA-3 determines Th2 cell differentiation and selectively activates the promoters of IL-4, IL-5, and IL-13 through chromatin remodelling [4]–[7].
T55 7698-7844 Sentence denotes GATA-3 determines Th2 cell differentiation and selectively activates the promoters of IL-4, IL-5, and IL-13 through chromatin remodelling [4]–[7].
T1299 7845-8103 Sentence denotes The key role of GATA-3 in allergic airway inflammation has been demonstrated in mice by the reduced release of Th2 cytokines in animals treated with dominant-negative mutants of GATA-3 and by local application of antisense oligonucleotides to GATA-3 [8],[9].
T56 7845-8103 Sentence denotes The key role of GATA-3 in allergic airway inflammation has been demonstrated in mice by the reduced release of Th2 cytokines in animals treated with dominant-negative mutants of GATA-3 and by local application of antisense oligonucleotides to GATA-3 [8],[9].
T1300 8104-8308 Sentence denotes Furthermore, conditional knock-out of the Gata3 gene in mice reduces expression of Th2 cytokines in vitro and in vivo [10], and similar results have been reported in isolated murine CD4+ lymphocytes [11].
T57 8104-8308 Sentence denotes Furthermore, conditional knock-out of the Gata3 gene in mice reduces expression of Th2 cytokines in vitro and in vivo [10], and similar results have been reported in isolated murine CD4+ lymphocytes [11].
T1301 8309-8449 Sentence denotes Finally, knockdown of GATA-3 expression using siRNA in human T cells results in loss of anti-CD3/CD28-mediated Th2 cytokine expression [12].
T58 8309-8449 Sentence denotes Finally, knockdown of GATA-3 expression using siRNA in human T cells results in loss of anti-CD3/CD28-mediated Th2 cytokine expression [12].
T1302 8450-8582 Sentence denotes In order for GATA-3 to regulate gene expression, it must translocate from the cytoplasm into the nucleus to access its target genes.
T59 8450-8582 Sentence denotes In order for GATA-3 to regulate gene expression, it must translocate from the cytoplasm into the nucleus to access its target genes.
T1303 8583-8807 Sentence denotes Enhanced nuclear expression of GATA-3 following T cell receptor activation was first demonstrated in murine T cells [13] and was recently confirmed in human T cells following T cell receptor and co-receptor stimulation [12].
T60 8583-8807 Sentence denotes Enhanced nuclear expression of GATA-3 following T cell receptor activation was first demonstrated in murine T cells [13] and was recently confirmed in human T cells following T cell receptor and co-receptor stimulation [12].
T1304 8808-8975 Sentence denotes GATA-3 contains a classical nuclear import signal [14] and is transported into the nucleus by the nuclear import protein importin-α (also known as karyopherin-α) [12].
T61 8808-8975 Sentence denotes GATA-3 contains a classical nuclear import signal [14] and is transported into the nucleus by the nuclear import protein importin-α (also known as karyopherin-α) [12].
T1305 8976-9134 Sentence denotes Deletion of a region encompassing the GATA-3 nuclear localisation sequence (NLS) region in murine and human cells prevents its nuclear localisation [12],[14].
T62 8976-9134 Sentence denotes Deletion of a region encompassing the GATA-3 nuclear localisation sequence (NLS) region in murine and human cells prevents its nuclear localisation [12],[14].
T1306 9135-9425 Sentence denotes The affinity of the importin-α–NLS interaction is regulated by phosphorylation [15], and we have shown that p38 mitogen-activated protein kinase (MAPK) plays a critical role in phosphorylating GATA-3 to enhance its interaction with importin-α and subsequent transport into the nucleus [12].
T63 9135-9425 Sentence denotes The affinity of the importin-α–NLS interaction is regulated by phosphorylation [15], and we have shown that p38 mitogen-activated protein kinase (MAPK) plays a critical role in phosphorylating GATA-3 to enhance its interaction with importin-α and subsequent transport into the nucleus [12].
T1307 9426-9587 Sentence denotes Corticosteroids are highly effective in the treatment of allergic inflammation, with marked suppression of Th2 cytokines in airways of patients with asthma [16].
T64 9426-9587 Sentence denotes Corticosteroids are highly effective in the treatment of allergic inflammation, with marked suppression of Th2 cytokines in airways of patients with asthma [16].
T1308 9588-9849 Sentence denotes Corticosteroids mediate their anti-inflammatory effects through binding to glucocorticoid receptors (GRs), which then translocate to the nucleus where they interact with glucocorticoid response elements (GREs) in the promoter regions of steroid-sensitive genes.
T65 9588-9849 Sentence denotes Corticosteroids mediate their anti-inflammatory effects through binding to glucocorticoid receptors (GRs), which then translocate to the nucleus where they interact with glucocorticoid response elements (GREs) in the promoter regions of steroid-sensitive genes.
T1309 9850-10154 Sentence denotes Alternatively, activated GR interacts with coactivator molecules to suppress the expression of inflammatory genes by inhibiting the action of proinflammatory transcription factors such as nuclear factor-κB (NF-κB) through the recruitment of co-repressor molecules such as histone deacetylase-2 [17],[18].
T66 9850-10154 Sentence denotes Alternatively, activated GR interacts with coactivator molecules to suppress the expression of inflammatory genes by inhibiting the action of proinflammatory transcription factors such as nuclear factor-κB (NF-κB) through the recruitment of co-repressor molecules such as histone deacetylase-2 [17],[18].
T1310 10155-10407 Sentence denotes Nuclear localisation and retention of GR is mediated through the nuclear localisation sequences NL1 and NL2 [19], by nuclear retention signals [20], and by control of nuclear export via a chromosomal region maintenance 1 (CRM-1) dependent pathway [21].
T67 10155-10407 Sentence denotes Nuclear localisation and retention of GR is mediated through the nuclear localisation sequences NL1 and NL2 [19], by nuclear retention signals [20], and by control of nuclear export via a chromosomal region maintenance 1 (CRM-1) dependent pathway [21].
T1311 10408-10472 Sentence denotes NL1, which is similar to the SV40 NLS, binds to importin-α [22].
T68 10408-10472 Sentence denotes NL1, which is similar to the SV40 NLS, binds to importin-α [22].
T1312 10473-10644 Sentence denotes NL1 is activated both by glucocorticoid agonists such as dexamethasone and fluticasone propionate (FP) and by glucocorticoid antagonists such as mifepristone (RU486) [23].
T69 10473-10644 Sentence denotes NL1 is activated both by glucocorticoid agonists such as dexamethasone and fluticasone propionate (FP) and by glucocorticoid antagonists such as mifepristone (RU486) [23].
T1313 10645-10837 Sentence denotes NL1 can be mutated, and the resulting GR still translocates to the nucleus in response to ligands, but via interaction with importin 7, an event that requires an as-yet unknown component [23].
T70 10645-10837 Sentence denotes NL1 can be mutated, and the resulting GR still translocates to the nucleus in response to ligands, but via interaction with importin 7, an event that requires an as-yet unknown component [23].
T1314 10838-10961 Sentence denotes NL2 is poorly defined, residing in the ligand-binding domain, and much less is known about its mechanism of GR import [24].
T71 10838-10961 Sentence denotes NL2 is poorly defined, residing in the ligand-binding domain, and much less is known about its mechanism of GR import [24].
T1315 10962-11230 Sentence denotes A variety of other factors are also important for the regulation of GR activation and nuclear import including chaperones such as Hsp90 and other immunophilins [24]–[26] and FK506-binding proteins that may be linked to dynein and/or peptidylprolyl isomerase [27],[28].
T72 10962-11230 Sentence denotes A variety of other factors are also important for the regulation of GR activation and nuclear import including chaperones such as Hsp90 and other immunophilins [24]–[26] and FK506-binding proteins that may be linked to dynein and/or peptidylprolyl isomerase [27],[28].
T1316 11231-11499 Sentence denotes However, the molecular basis for the inhibition of Th2 cytokines by corticosteroids is not well understood, because the genes encoding IL-4, IL-5, and IL-13 do not have any recognisable GRE sequence [29] and are only partly regulated by NF-κB in human cells [30]–[32].
T73 11231-11499 Sentence denotes However, the molecular basis for the inhibition of Th2 cytokines by corticosteroids is not well understood, because the genes encoding IL-4, IL-5, and IL-13 do not have any recognisable GRE sequence [29] and are only partly regulated by NF-κB in human cells [30]–[32].
T1317 11500-11670 Sentence denotes Using overexpression and CAT-reporter genes, Lavender and colleagues [33] have shown that GR reduced GATA-3-mediated IL-5 and -13 promoter activity in human CD4+ T cells.
T74 11500-11670 Sentence denotes Using overexpression and CAT-reporter genes, Lavender and colleagues [33] have shown that GR reduced GATA-3-mediated IL-5 and -13 promoter activity in human CD4+ T cells.
T1318 11671-11897 Sentence denotes The authors postulated that local recruitment of GR may alter the ability of GATA-3 either to bind to its target site, to cause transcriptional up-regulation, or to maintain an environment that is permissive for transcription.
T75 11671-11897 Sentence denotes The authors postulated that local recruitment of GR may alter the ability of GATA-3 either to bind to its target site, to cause transcriptional up-regulation, or to maintain an environment that is permissive for transcription.
T1319 11898-12158 Sentence denotes We therefore investigated the effects of a synthetic corticosteroid, FP, on GATA-3 phosphorylation and nuclear translocation in a T lymphocyte cell line (HuT-78) and in peripheral blood mononuclear cells activated by anti-CD3 and anti-CD28 antibodies in vitro.
T76 11898-12158 Sentence denotes We therefore investigated the effects of a synthetic corticosteroid, FP, on GATA-3 phosphorylation and nuclear translocation in a T lymphocyte cell line (HuT-78) and in peripheral blood mononuclear cells activated by anti-CD3 and anti-CD28 antibodies in vitro.
T1320 12159-12325 Sentence denotes We also studied the effects of inhaled fluticasone therapy on GATA-3 subcellular localization in peripheral blood mononuclear cells (PBMCs) from patients with asthma.
T77 12159-12325 Sentence denotes We also studied the effects of inhaled fluticasone therapy on GATA-3 subcellular localization in peripheral blood mononuclear cells (PBMCs) from patients with asthma.
T78 12327-12348 Sentence denotes Materials and Methods
T3670 12350-12379 Sentence denotes Participants and Study Design
T79 12350-12379 Sentence denotes Participants and Study Design
T3671 12380-12577 Sentence denotes We studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34].
T80 12380-12577 Sentence denotes We studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34].
T3672 12578-12827 Sentence denotes Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d.
T81 12578-12827 Sentence denotes Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d.
T3673 12828-12908 Sentence denotes Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration.
T82 12828-12908 Sentence denotes Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration.
T83 12909-13051 Sentence denotes All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust.
T3674 12909-13140 Sentence denotes All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.
T84 13052-13140 Sentence denotes The clinical study was conducted before the requirement for Clinical Trial Registration.
T3976 13142-13165 Sentence denotes Antibodies and Reagents
T85 13142-13165 Sentence denotes Antibodies and Reagents
T3977 13166-13296 Sentence denotes The monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom).
T86 13166-13296 Sentence denotes The monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom).
T3978 13297-13785 Sentence denotes Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK).
T87 13297-13785 Sentence denotes Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK).
T3979 13786-13900 Sentence denotes The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis.
T88 13786-13900 Sentence denotes The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis.
T3980 13901-13958 Sentence denotes All other reagents were purchased from Sigma (Poole, UK).
T89 13901-13958 Sentence denotes All other reagents were purchased from Sigma (Poole, UK).
T4349 13960-13991 Sentence denotes Cell Culture and PBMC Isolation
T90 13960-13991 Sentence denotes Cell Culture and PBMC Isolation
T4350 13992-14141 Sentence denotes A human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12].
T91 13992-14141 Sentence denotes A human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12].
T4351 14142-14295 Sentence denotes PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34].
T92 14142-14295 Sentence denotes PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34].
T4352 14296-14452 Sentence denotes Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M).
T93 14296-14452 Sentence denotes Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M).
T4353 14453-14564 Sentence denotes Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].
T94 14453-14564 Sentence denotes Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].
T4615 14566-14591 Sentence denotes Reverse Transcription PCR
T95 14566-14591 Sentence denotes Reverse Transcription PCR
T4616 14592-14669 Sentence denotes Total RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK).
T96 14592-14669 Sentence denotes Total RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK).
T4617 14670-14765 Sentence denotes During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences).
T97 14670-14765 Sentence denotes During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences).
T4618 14766-14884 Sentence denotes Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK).
T98 14766-14884 Sentence denotes Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK).
T4619 14885-14955 Sentence denotes For relative quantification, RT-PCR was carried out using cDNA probes.
T99 14885-14955 Sentence denotes For relative quantification, RT-PCR was carried out using cDNA probes.
T4620 14956-15013 Sentence denotes Primers for IL-4 were from Sigma-Genosys (Cambridge, UK).
T100 14956-15013 Sentence denotes Primers for IL-4 were from Sigma-Genosys (Cambridge, UK).
T4621 15014-15125 Sentence denotes Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.
T101 15014-15125 Sentence denotes Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.
T4846 15127-15193 Sentence denotes Cell Fractionation, Immunoprecipitation, and Western Blot Analysis
T102 15127-15193 Sentence denotes Cell Fractionation, Immunoprecipitation, and Western Blot Analysis
T4847 15194-15271 Sentence denotes Nuclear and cytoplasmic fractions were prepared as previously described [35].
T103 15194-15271 Sentence denotes Nuclear and cytoplasmic fractions were prepared as previously described [35].
T4848 15272-15441 Sentence denotes Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor.
T104 15272-15441 Sentence denotes Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor.
T4849 15442-15557 Sentence denotes Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris.
T105 15442-15557 Sentence denotes Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris.
T4850 15558-15809 Sentence denotes Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA).
T106 15558-15809 Sentence denotes Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA).
T4851 15810-15946 Sentence denotes Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine.
T107 15810-15946 Sentence denotes Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine.
T4852 15947-16052 Sentence denotes Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).
T108 15947-16052 Sentence denotes Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).
T5202 16054-16083 Sentence denotes Chromatin Immunoprecipitation
T109 16054-16083 Sentence denotes Chromatin Immunoprecipitation
T110 16084-16332 Sentence denotes Chromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C.
T5203 16084-16467 Sentence denotes Chromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C. Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse:
T111 16333-16467 Sentence denotes Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse:
T5204 16468-16496 Sentence denotes 5′-TCATGGCTCTGAAACGTTCTG-3′.
T112 16468-16496 Sentence denotes 5′-TCATGGCTCTGAAACGTTCTG-3′.
T5205 16497-16677 Sentence denotes PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.
T113 16497-16677 Sentence denotes PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.
T5489 16679-16750 Sentence denotes GR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)
T114 16679-16750 Sentence denotes GR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)
T5490 16751-16926 Sentence denotes Immunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36].
T115 16751-16926 Sentence denotes Immunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36].
T5491 16927-17109 Sentence denotes Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells.
T116 16927-17109 Sentence denotes Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells.
T5492 17110-17172 Sentence denotes These proteins were subsequently refolded in glycine solution.
T117 17110-17172 Sentence denotes These proteins were subsequently refolded in glycine solution.
T5493 17173-17292 Sentence denotes 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody.
T118 17173-17292 Sentence denotes 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody.
T5494 17293-17421 Sentence denotes After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells.
T119 17293-17421 Sentence denotes After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells.
T5495 17422-17513 Sentence denotes GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells.
T120 17422-17513 Sentence denotes GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells.
T5496 17514-17739 Sentence denotes After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies.
T121 17514-17739 Sentence denotes After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies.
T5497 17740-17930 Sentence denotes FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR.
T122 17740-17930 Sentence denotes FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR.
T5498 17931-18031 Sentence denotes FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).
T123 17931-18031 Sentence denotes FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).
T6074 18033-18049 Sentence denotes NF-κB Activation
T124 18033-18049 Sentence denotes NF-κB Activation
T6075 18050-18182 Sentence denotes NF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium).
T125 18050-18182 Sentence denotes NF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium).
T6076 18183-18293 Sentence denotes In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide.
T126 18183-18293 Sentence denotes In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide.
T6077 18294-18353 Sentence denotes Plates were washed before addition of an anti-p65 antibody.
T127 18294-18353 Sentence denotes Plates were washed before addition of an anti-p65 antibody.
T6078 18354-18458 Sentence denotes Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate.
T128 18354-18458 Sentence denotes Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate.
T6079 18459-18512 Sentence denotes The intensity of the reaction was measured at 450 nm.
T129 18459-18512 Sentence denotes The intensity of the reaction was measured at 450 nm.
T6273 18514-18541 Sentence denotes Immunofluorescence Staining
T130 18514-18541 Sentence denotes Immunofluorescence Staining
T6274 18542-18613 Sentence denotes Immunofluorescence staining was performed as previously described [34].
T131 18542-18613 Sentence denotes Immunofluorescence staining was performed as previously described [34].
T6275 18614-18686 Sentence denotes All staining was performed at room temperature and under humidification.
T132 18614-18686 Sentence denotes All staining was performed at room temperature and under humidification.
T6276 18687-18786 Sentence denotes Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK).
T133 18687-18786 Sentence denotes Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK).
T6277 18787-18971 Sentence denotes Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature.
T134 18787-18971 Sentence denotes Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature.
T6278 18972-19127 Sentence denotes After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako).
T135 18972-19127 Sentence denotes After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako).
T6279 19128-19304 Sentence denotes Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50).
T136 19128-19304 Sentence denotes Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50).
T6280 19305-19508 Sentence denotes The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany).
T137 19305-19508 Sentence denotes The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany).
T6281 19509-19659 Sentence denotes To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls.
T138 19509-19659 Sentence denotes To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls.
T6282 19660-19778 Sentence denotes Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.
T139 19660-19778 Sentence denotes Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.
T6774 19780-19800 Sentence denotes GATA-3-GFP Construct
T140 19780-19800 Sentence denotes GATA-3-GFP Construct
T6775 19801-19950 Sentence denotes The GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector.
T141 19801-19950 Sentence denotes The GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector.
T6776 19951-20081 Sentence denotes GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI.
T142 19951-20081 Sentence denotes GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI.
T143 20082-20278 Sentence denotes DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C.
T6777 20082-20606 Sentence denotes DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C. Klenow was inactivated by incubation for 10 min at 75°C. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector.
T144 20279-20335 Sentence denotes Klenow was inactivated by incubation for 10 min at 75°C.
T145 20336-20606 Sentence denotes DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector.
T6778 20607-20721 Sentence denotes Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.
T146 20607-20721 Sentence denotes Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.
T7169 20723-20735 Sentence denotes Transfection
T147 20723-20735 Sentence denotes Transfection
T7170 20736-21007 Sentence denotes HuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection.
T148 20736-21007 Sentence denotes HuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection.
T7171 21008-21171 Sentence denotes The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.
T149 21008-21171 Sentence denotes The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.
T7353 21173-21194 Sentence denotes Time-Lapse Microscopy
T150 21173-21194 Sentence denotes Time-Lapse Microscopy
T7354 21195-21463 Sentence denotes HuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA).
T151 21195-21463 Sentence denotes HuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA).
T7355 21464-21729 Sentence denotes Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK).
T152 21464-21729 Sentence denotes Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK).
T7356 21730-21784 Sentence denotes Photographs were taken at 0, 30, 60, 120, and 240 min.
T153 21730-21784 Sentence denotes Photographs were taken at 0, 30, 60, 120, and 240 min.
T7605 21786-21808 Sentence denotes Densitometric Analysis
T154 21786-21808 Sentence denotes Densitometric Analysis
T7606 21809-21938 Sentence denotes Densitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK).
T155 21809-21938 Sentence denotes Densitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK).
T7607 21939-22019 Sentence denotes Briefly, immunoblots were scanned and gates were drawn tightly around each band.
T156 21939-22019 Sentence denotes Briefly, immunoblots were scanned and gates were drawn tightly around each band.
T7608 22020-22099 Sentence denotes Background values from each lane were subtracted to normalize each measurement.
T157 22020-22099 Sentence denotes Background values from each lane were subtracted to normalize each measurement.
T7609 22100-22154 Sentence denotes The bands were quantified using the Gelworks software.
T158 22100-22154 Sentence denotes The bands were quantified using the Gelworks software.
T7610 22155-22326 Sentence denotes All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.
T159 22155-22326 Sentence denotes All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.
T7781 22328-22348 Sentence denotes Statistical Analysis
T160 22328-22348 Sentence denotes Statistical Analysis
T7782 22349-22563 Sentence denotes Data from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com).
T161 22349-22563 Sentence denotes Data from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com).
T7783 22564-22787 Sentence denotes Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test.
T162 22564-22787 Sentence denotes Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test.
T7784 22788-22853 Sentence denotes Data from this analysis are presented as a box-and-whiskers plot.
T163 22788-22853 Sentence denotes Data from this analysis are presented as a box-and-whiskers plot.
T7785 22854-23003 Sentence denotes Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels.
T164 22854-23003 Sentence denotes Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels.
T7786 23004-23161 Sentence denotes We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p<0.05.
T165 23004-23161 Sentence denotes We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p<0.05.
T166 23163-23170 Sentence denotes Results
T8238 23172-23247 Sentence denotes The Effect of Corticosteroids on GATA-3 Nuclear Translocation and IL-4 mRNA
T167 23172-23247 Sentence denotes The Effect of Corticosteroids on GATA-3 Nuclear Translocation and IL-4 mRNA
T8239 23248-23356 Sentence denotes Corticosteroids are effective in inhibiting GATA-3-regulated IL-4 gene expression in vitro and in vivo [32].
T168 23248-23356 Sentence denotes Corticosteroids are effective in inhibiting GATA-3-regulated IL-4 gene expression in vitro and in vivo [32].
T8240 23357-23464 Sentence denotes We therefore investigated whether corticosteroids affect anti-CD3/CD28–stimulated nuclear import of GATA-3.
T169 23357-23464 Sentence denotes We therefore investigated whether corticosteroids affect anti-CD3/CD28–stimulated nuclear import of GATA-3.
T8241 23465-23616 Sentence denotes Stimulation of cells with anti-CD3/CD28 resulted in a rapid cytoplasmic/nuclear GATA-3 translocation (Figure 1A), confirming our previous results [12].
T170 23465-23616 Sentence denotes Stimulation of cells with anti-CD3/CD28 resulted in a rapid cytoplasmic/nuclear GATA-3 translocation (Figure 1A), confirming our previous results [12].
T8242 23617-23746 Sentence denotes We also confirmed a clear separation of nuclear and cytosolic fractions as indicated by histone H1 and MEK-1 markers (Figure 1B).
T171 23617-23746 Sentence denotes We also confirmed a clear separation of nuclear and cytosolic fractions as indicated by histone H1 and MEK-1 markers (Figure 1B).
T8243 23747-23958 Sentence denotes The potent topical corticosteroid FP caused sustained loss of nuclear GATA-3 expression and cytoplasmic retention of GATA-3 at concentrations ranging from 10−12 to 10−8 M, which cover the therapeutic range [37].
T172 23747-23958 Sentence denotes The potent topical corticosteroid FP caused sustained loss of nuclear GATA-3 expression and cytoplasmic retention of GATA-3 at concentrations ranging from 10−12 to 10−8 M, which cover the therapeutic range [37].
T8244 23959-24274 Sentence denotes This effect was concentration- and time-dependent, with a peak effect of 11.6-fold at 30 min at a concentration of 10−8 M (Figure 1C) and was associated with marked reductions in anti-CD3/CD28–stimulated IL-4 and IL-5 mRNA expression (Figure 1D) and a loss of GATA-3 binding to the native IL-5 promoter (Figure 1E).
T173 23959-24274 Sentence denotes This effect was concentration- and time-dependent, with a peak effect of 11.6-fold at 30 min at a concentration of 10−8 M (Figure 1C) and was associated with marked reductions in anti-CD3/CD28–stimulated IL-4 and IL-5 mRNA expression (Figure 1D) and a loss of GATA-3 binding to the native IL-5 promoter (Figure 1E).
T174 24275-24421 Sentence denotes 10.1371/journal.pmed.1000076.g001 Figure 1 Fluticasone propionate down-regulates Th2 cytokine gene expression and inhibits GATA-3 nuclear import.
T16029 24319-24421 Sentence denotes Fluticasone propionate down-regulates Th2 cytokine gene expression and inhibits GATA-3 nuclear import.
T16030 24422-24921 Sentence denotes (A) Anti-CD3/CD28 treatment of HuT-78 cells results in translocation of GATA-3 from the cytoplasm to the nucleus within 30 min. (B) Histone H1 and MEK-1 were used to confirm distinct separation of cytoplasmic and nuclear extracts in three separate experiments. (C) Western blot analysis of FP-treated HuT-78 cells demonstrated impaired nuclear localization of GATA-3 induced by anti-CD3/CD28 co-stimulation in a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent manner.
T175 24422-24921 Sentence denotes (A) Anti-CD3/CD28 treatment of HuT-78 cells results in translocation of GATA-3 from the cytoplasm to the nucleus within 30 min. (B) Histone H1 and MEK-1 were used to confirm distinct separation of cytoplasmic and nuclear extracts in three separate experiments. (C) Western blot analysis of FP-treated HuT-78 cells demonstrated impaired nuclear localization of GATA-3 induced by anti-CD3/CD28 co-stimulation in a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent manner.
T16031 24922-24984 Sentence denotes Cells were pretreated with FP for 30 min prior to stimulation.
T176 24922-24984 Sentence denotes Cells were pretreated with FP for 30 min prior to stimulation.
T16032 24985-25081 Sentence denotes MEK1 and histone H1 were used to demonstrate equal cytoplasmic and nuclear loading respectively.
T177 24985-25081 Sentence denotes MEK1 and histone H1 were used to demonstrate equal cytoplasmic and nuclear loading respectively.
T16033 25082-25305 Sentence denotes Results are presented graphically below as mean±SEM of at least three independent experiments. *** p<0.001 compared to t = 0. (D) RT-PCR showing that FP inhibits IL-4 and IL-5 mRNA expression in CD3/CD28-costimulated cells.
T178 25082-25305 Sentence denotes Results are presented graphically below as mean±SEM of at least three independent experiments. *** p<0.001 compared to t = 0. (D) RT-PCR showing that FP inhibits IL-4 and IL-5 mRNA expression in CD3/CD28-costimulated cells.
T16034 25306-25342 Sentence denotes GAPDH was used as a loading control.
T179 25306-25342 Sentence denotes GAPDH was used as a loading control.
T16035 25343-25676 Sentence denotes Lower panels show graphical analysis of results presented as mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to anti-CD3/CD28–stimulated. (E) FP (10 nM) reduces the ability of anti-CD3/CD28-stimulated GATA-3 to associate with the native IL-5 promoter 60 min after stimulation.
T180 25343-25676 Sentence denotes Lower panels show graphical analysis of results presented as mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to anti-CD3/CD28–stimulated. (E) FP (10 nM) reduces the ability of anti-CD3/CD28-stimulated GATA-3 to associate with the native IL-5 promoter 60 min after stimulation.
T16036 25677-25754 Sentence denotes Data are also shown graphically as mean±SEM of three independent experiments.
T181 25677-25754 Sentence denotes Data are also shown graphically as mean±SEM of three independent experiments.
T16037 25755-25822 Sentence denotes All data were analysed by ANOVA followed by Newman-Keuls post-test.
T182 25755-25822 Sentence denotes All data were analysed by ANOVA followed by Newman-Keuls post-test.
T8940 25824-25879 Sentence denotes Ligand-Activated GR Competes with GATA-3 for Importin-α
T183 25824-25879 Sentence denotes Ligand-Activated GR Competes with GATA-3 for Importin-α
T8941 25880-26034 Sentence denotes We confirmed and extended previous data [20] to show that ligand-activated GR as well as GATA-3 uses importin-α for its nuclear import (Figure 2A and 2B).
T184 25880-26034 Sentence denotes We confirmed and extended previous data [20] to show that ligand-activated GR as well as GATA-3 uses importin-α for its nuclear import (Figure 2A and 2B).
T8942 26035-26161 Sentence denotes This interaction between GR and importin-α was significant at concentrations as low as 10−12 M and was maximal with 10−8 M FP.
T185 26035-26161 Sentence denotes This interaction between GR and importin-α was significant at concentrations as low as 10−12 M and was maximal with 10−8 M FP.
T8943 26162-26274 Sentence denotes Subsequent GR nuclear translocation was rapid and sustained at significant levels for at least 14 h (Figure 2B).
T186 26162-26274 Sentence denotes Subsequent GR nuclear translocation was rapid and sustained at significant levels for at least 14 h (Figure 2B).
T8944 26275-26476 Sentence denotes Using IP-Western blotting we showed that FP at 10−12–10−8 M decreased the association between GATA-3 and importin-α induced by anti-CD3/CD28 stimulation in a concentration-dependent manner (Figure 2C).
T187 26275-26476 Sentence denotes Using IP-Western blotting we showed that FP at 10−12–10−8 M decreased the association between GATA-3 and importin-α induced by anti-CD3/CD28 stimulation in a concentration-dependent manner (Figure 2C).
T8945 26477-26690 Sentence denotes In addition, using GFP-labelled GATA-3 and confocal microscopy we demonstrated that GATA-3 nuclear import following anti-CD3/CD28 stimulation for 30 min was attenuated by pretreatment with FP (10−8 M) (Figure 2D).
T188 26477-26690 Sentence denotes In addition, using GFP-labelled GATA-3 and confocal microscopy we demonstrated that GATA-3 nuclear import following anti-CD3/CD28 stimulation for 30 min was attenuated by pretreatment with FP (10−8 M) (Figure 2D).
T189 26691-26827 Sentence denotes 10.1371/journal.pmed.1000076.g002 Figure 2 Fluticasone propionate reduces GATA-3 association with importin-α and GATA-3 nuclear import.
T16815 26735-26827 Sentence denotes Fluticasone propionate reduces GATA-3 association with importin-α and GATA-3 nuclear import.
T16816 26828-27014 Sentence denotes (A) Western blot analysis demonstrates a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR interaction with importin-α (Imp-α).
T190 26828-27014 Sentence denotes (A) Western blot analysis demonstrates a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR interaction with importin-α (Imp-α).
T16817 27015-27076 Sentence denotes A positive control for GR association with importin is shown.
T191 27015-27076 Sentence denotes A positive control for GR association with importin is shown.
T16818 27077-27132 Sentence denotes Quantification of the densitometry data is shown below.
T192 27077-27132 Sentence denotes Quantification of the densitometry data is shown below.
T16819 27133-27438 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. *** p<0.001 compared to control, ### p<0.001. (B) Western blot analysis demonstrated a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR nuclear translocation measured by IP.
T193 27133-27438 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. *** p<0.001 compared to control, ### p<0.001. (B) Western blot analysis demonstrated a time- (at 10−8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR nuclear translocation measured by IP.
T16820 27439-27494 Sentence denotes Quantification of the densitometry data is shown below.
T194 27439-27494 Sentence denotes Quantification of the densitometry data is shown below.
T16821 27495-27781 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ***p<0.001 compared to control. (C) Western blot analysis of HuT-78 cells treated with FP and anti-CD3/CD28 co-stimulation demonstrated a concentration-dependent decrease in GATA-3–importin-α association at 20 min.
T195 27495-27781 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ***p<0.001 compared to control. (C) Western blot analysis of HuT-78 cells treated with FP and anti-CD3/CD28 co-stimulation demonstrated a concentration-dependent decrease in GATA-3–importin-α association at 20 min.
T16822 27782-27837 Sentence denotes Quantification of the densitometry data is shown below.
T196 27782-27837 Sentence denotes Quantification of the densitometry data is shown below.
T16823 27838-28103 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to αCD3/CD28-stimulated cells. (D) GFP-tagged GATA-3 was overexpressed and cells stimulated (b, c) or not (a) for 30 min with anti-CD3/CD28.
T197 27838-28103 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to αCD3/CD28-stimulated cells. (D) GFP-tagged GATA-3 was overexpressed and cells stimulated (b, c) or not (a) for 30 min with anti-CD3/CD28.
T16824 28104-28181 Sentence denotes The effect of 30 min pretreatment of cells with FP (10−8 M, c) is also shown.
T198 28104-28181 Sentence denotes The effect of 30 min pretreatment of cells with FP (10−8 M, c) is also shown.
T16825 28182-28249 Sentence denotes All data were analysed by ANOVA followed by Newman-Keuls post-test.
T199 28182-28249 Sentence denotes All data were analysed by ANOVA followed by Newman-Keuls post-test.
T9685 28251-28266 Sentence denotes Effect on MKP-1
T200 28251-28266 Sentence denotes Effect on MKP-1
T9686 28267-28468 Sentence denotes Dexamethasone inhibits p38 MAPK function in a cell type–specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28].
T201 28267-28468 Sentence denotes Dexamethasone inhibits p38 MAPK function in a cell type–specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28].
T9687 28469-28688 Sentence denotes FP (10−8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B).
T202 28469-28688 Sentence denotes FP (10−8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B).
T9688 28689-28765 Sentence denotes This effect was detected at 30 min and lasted for at least 14 h (Figure 3B).
T203 28689-28765 Sentence denotes This effect was detected at 30 min and lasted for at least 14 h (Figure 3B).
T9689 28766-28935 Sentence denotes FP (10−8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C).
T204 28766-28935 Sentence denotes FP (10−8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C).
T9690 28936-29023 Sentence denotes This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP.
T205 28936-29023 Sentence denotes This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP.
T9691 29024-29187 Sentence denotes We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10−8 M after 10 min (Figure 3D and 3E).
T206 29024-29187 Sentence denotes We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10−8 M after 10 min (Figure 3D and 3E).
T9692 29188-29358 Sentence denotes However, the effects of FP on GATA-3 nuclear import, importin-α association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10−12 M, see Figure 2).
T207 29188-29358 Sentence denotes However, the effects of FP on GATA-3 nuclear import, importin-α association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10−12 M, see Figure 2).
T208 29359-29572 Sentence denotes 10.1371/journal.pmed.1000076.g003 Figure 3 Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.
T17603 29403-29572 Sentence denotes Fluticasone propionate–mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.
T17604 29573-30060 Sentence denotes (A) Western blot analysis shows that FP (10−8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10−8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3.
T209 29573-30060 Sentence denotes (A) Western blot analysis shows that FP (10−8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28–co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10−8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation–induced serine phosphorylation (P-Ser) of GATA-3.
T17605 30061-30126 Sentence denotes For (A–C), quantification of the densitometry data is also shown.
T210 30061-30126 Sentence denotes For (A–C), quantification of the densitometry data is also shown.
T17606 30127-30345 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to αCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner.
T211 30127-30345 Sentence denotes Each bar represents mean±SEM of at least three independent experiments. ### p<0.001 compared to control, ***p<0.001 compared to αCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner.
T17607 30346-30527 Sentence denotes All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *p<0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner.
T212 30346-30527 Sentence denotes All results are representative of at least three independent experiments and where appropriate expressed as means±SEM, *p<0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner.
T17608 30528-30586 Sentence denotes Results are representative of two independent experiments.
T213 30528-30586 Sentence denotes Results are representative of two independent experiments.
T17609 30587-30665 Sentence denotes All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.
T214 30587-30665 Sentence denotes All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.
T9693 30668-30927 Sentence denotes Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-α, and activated GR, we demonstrated that activated GR significantly increased GR-importin-α association in the presence and absence of activated GATA-3 (Figure 4B).
T215 30668-30927 Sentence denotes Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-α, and activated GR, we demonstrated that activated GR significantly increased GR-importin-α association in the presence and absence of activated GATA-3 (Figure 4B).
T9694 30928-31030 Sentence denotes This effect is not mutual, since activated GATA-3 did not block GR–importin-α association (Figure 4C).
T216 30928-31030 Sentence denotes This effect is not mutual, since activated GATA-3 did not block GR–importin-α association (Figure 4C).
T9695 31031-31271 Sentence denotes These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-α (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-α interaction in a concentration-dependent manner (Figure 4E).
T217 31031-31271 Sentence denotes These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-α (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-α interaction in a concentration-dependent manner (Figure 4E).
T9696 31272-31408 Sentence denotes Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-α and thereby limit GATA-3 nuclear import.
T218 31272-31408 Sentence denotes Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-α and thereby limit GATA-3 nuclear import.
T219 31409-31520 Sentence denotes 10.1371/journal.pmed.1000076.g004 Figure 4 Fluticasone propionate competes with phospho-GATA-3 for importin-α.
T18308 31453-31520 Sentence denotes Fluticasone propionate competes with phospho-GATA-3 for importin-α.
T18309 31521-32365 Sentence denotes (A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10−8 M) stimulated cells enhances GR–importin-α binding in the presence (•) and absence (▪) of activated GATA-3. * p<0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28–stimulated cells does not attenuate GR–importin-α association. *p<0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28–stimulated cells interacting with immobilised importin-α in an in vitro binding assay. *p<0.05 compared to GATA-3 isolated from unstimulated cells. # p<0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (•) versus unstimulated (○) GR on attenuation of GATA-3–importin-α association was concentration-dependent. *p<0.05, **p<0.01 between groups.
T220 31521-32365 Sentence denotes (A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10−8 M) stimulated cells enhances GR–importin-α binding in the presence (•) and absence (▪) of activated GATA-3. * p<0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28–stimulated cells does not attenuate GR–importin-α association. *p<0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28–stimulated cells interacting with immobilised importin-α in an in vitro binding assay. *p<0.05 compared to GATA-3 isolated from unstimulated cells. # p<0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (•) versus unstimulated (○) GR on attenuation of GATA-3–importin-α association was concentration-dependent. *p<0.05, **p<0.01 between groups.
T18310 32366-32494 Sentence denotes All results are expressed as mean±SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test.
T221 32366-32494 Sentence denotes All results are expressed as mean±SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test.
T9697 32497-32617 Sentence denotes Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation.
T222 32497-32617 Sentence denotes Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation.
T9698 32618-32745 Sentence denotes Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A).
T223 32618-32745 Sentence denotes Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A).
T9699 32746-32865 Sentence denotes Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B).
T224 32746-32865 Sentence denotes Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B).
T9700 32866-33052 Sentence denotes Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export.
T225 32866-33052 Sentence denotes Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export.
T9701 33053-33217 Sentence denotes Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10−8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).
T226 33053-33217 Sentence denotes Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10−8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).
T227 33218-33323 Sentence denotes 10.1371/journal.pmed.1000076.g005 Figure 5 Fluticasone propionate does not affect GATA-3 nuclear export.
T18713 33262-33323 Sentence denotes Fluticasone propionate does not affect GATA-3 nuclear export.
T18714 33324-33842 Sentence denotes (A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10−8 M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***p<0.001 compared to unstimulated cells, ### p<0.001 compared to anti-CD3/CD28–stimulated cells. (B) Western blot analysis showing that FP (10−8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b–j) or not (a) with anti-CD3/CD28.
T228 33324-33842 Sentence denotes (A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10−8 M) to prevent anti-CD3/CD28–stimulated GATA-3 nuclear localization measured at 60 min. ***p<0.001 compared to unstimulated cells, ### p<0.001 compared to anti-CD3/CD28–stimulated cells. (B) Western blot analysis showing that FP (10−8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b–j) or not (a) with anti-CD3/CD28.
T18715 33843-34106 Sentence denotes The effect of treating cells with FP (10−8 M, f–j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10−8 M) does not prevent anti-CD3/CD28–stimulated p65 nuclear translocation at 60 min after stimulation. **p<0.01 compared to unstimulated cells.
T229 33843-34106 Sentence denotes The effect of treating cells with FP (10−8 M, f–j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10−8 M) does not prevent anti-CD3/CD28–stimulated p65 nuclear translocation at 60 min after stimulation. **p<0.01 compared to unstimulated cells.
T18716 34107-34205 Sentence denotes All results are representative of at least four independent experiments and are shown as mean±SEM.
T230 34107-34205 Sentence denotes All results are representative of at least four independent experiments and are shown as mean±SEM.
T18717 34206-34267 Sentence denotes Results were analysed by ANOVA followed by Newman-Keuls test.
T231 34206-34267 Sentence denotes Results were analysed by ANOVA followed by Newman-Keuls test.
T11291 34269-34385 Sentence denotes The Inhibitory Effect of Corticosteroids on GATA-3 Nuclear Localization in Primary T Lymphocytes Ex Vivo and In Vivo
T232 34269-34385 Sentence denotes The Inhibitory Effect of Corticosteroids on GATA-3 Nuclear Localization in Primary T Lymphocytes Ex Vivo and In Vivo
T11292 34386-34744 Sentence denotes Treatment with FP ex vivo demonstrated a concentration-dependent decrease in the direct interaction between phospho-GATA-3 and importin-α in PBMCs from patients with asthma (Figure 6A and 6B), which was significantly inhibited at 10−12 M FP (p<0.001, ANOVA and Newman-Keuls test) and completely attenuated by 10−8 M FP (p<0.001, ANOVA and Newman-Keuls test).
T233 34386-34744 Sentence denotes Treatment with FP ex vivo demonstrated a concentration-dependent decrease in the direct interaction between phospho-GATA-3 and importin-α in PBMCs from patients with asthma (Figure 6A and 6B), which was significantly inhibited at 10−12 M FP (p<0.001, ANOVA and Newman-Keuls test) and completely attenuated by 10−8 M FP (p<0.001, ANOVA and Newman-Keuls test).
T234 34745-34907 Sentence denotes 10.1371/journal.pmed.1000076.g006 Figure 6 Fluticasone propionate impairs GATA-3 interaction with importin-α and GATA-3 nuclear localization in vivo and ex vivo.
T19286 34789-34907 Sentence denotes Fluticasone propionate impairs GATA-3 interaction with importin-α and GATA-3 nuclear localization in vivo and ex vivo.
T19287 34908-35102 Sentence denotes (A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naïve asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-α measured at 60 min.
T235 34908-35102 Sentence denotes (A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naïve asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-α measured at 60 min.
T19288 35103-35464 Sentence denotes Each bar represents the mean±SEM of at least three independent experiments; *** p<0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naïve asthma patients treated with inhaled FP (500 µg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-α.
T236 35103-35464 Sentence denotes Each bar represents the mean±SEM of at least three independent experiments; *** p<0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naïve asthma patients treated with inhaled FP (500 µg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-α.
T19289 35465-35635 Sentence denotes The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-α expression was unaffected by inhalation of FP.
T237 35465-35635 Sentence denotes The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-α expression was unaffected by inhalation of FP.
T19290 35636-35691 Sentence denotes Blot is representative of gels from three participants.
T238 35636-35691 Sentence denotes Blot is representative of gels from three participants.
T11293 35694-35872 Sentence denotes Our previous T cell line studies indicated that 10−12 M FP suppresses IL-4 and -5 gene expression and attenuated the interaction of GATA-3 with importin-α (see Figures 1D and 2).
T239 35694-35872 Sentence denotes Our previous T cell line studies indicated that 10−12 M FP suppresses IL-4 and -5 gene expression and attenuated the interaction of GATA-3 with importin-α (see Figures 1D and 2).
T11294 35873-35994 Sentence denotes This concentration is close to peak plasma levels obtained from asthmatic patients treated with inhaled FP (500 µg) [27].
T240 35873-35994 Sentence denotes This concentration is close to peak plasma levels obtained from asthmatic patients treated with inhaled FP (500 µg) [27].
T11295 35995-36151 Sentence denotes Inhaled FP (500 µg) treatment of seven steroid-naive asthma patients significantly reduced GATA-3–importin-α interaction in vivo in a time-dependent manner.
T241 35995-36151 Sentence denotes Inhaled FP (500 µg) treatment of seven steroid-naive asthma patients significantly reduced GATA-3–importin-α interaction in vivo in a time-dependent manner.
T11296 36152-36314 Sentence denotes This produced a >90% decrease in GATA-3–importin-α association at 2 h (median [95% CI], 13,494 [6,828–17,829] versus 879 [597–1,165]; p<0.05 Friedman's analysis).
T242 36152-36314 Sentence denotes This produced a >90% decrease in GATA-3–importin-α association at 2 h (median [95% CI], 13,494 [6,828–17,829] versus 879 [597–1,165]; p<0.05 Friedman's analysis).
T11297 36315-36447 Sentence denotes However, this did not reach significance using Wilcoxon's post-test analysis (W = 6.00) probably due to low numbers of participants.
T243 36315-36447 Sentence denotes However, this did not reach significance using Wilcoxon's post-test analysis (W = 6.00) probably due to low numbers of participants.
T11298 36448-36545 Sentence denotes Similar results were observed when GATA-3–importin-α association was measured (Figure 6C and 6D).
T244 36448-36545 Sentence denotes Similar results were observed when GATA-3–importin-α association was measured (Figure 6C and 6D).
T11299 36546-36594 Sentence denotes The lower dose of FP (100 µg) was not effective.
T245 36546-36594 Sentence denotes The lower dose of FP (100 µg) was not effective.
T11300 36595-36801 Sentence denotes The attenuated interaction of GATA-3 did not result from the defective recycling of importin-α, as a significant decrease in the abundance of importin-α in the cytoplasmic pool was not detected (Figure 6E).
T246 36595-36801 Sentence denotes The attenuated interaction of GATA-3 did not result from the defective recycling of importin-α, as a significant decrease in the abundance of importin-α in the cytoplasmic pool was not detected (Figure 6E).
T11301 36802-36914 Sentence denotes We further examined whether inhaled FP could affect cellular localization of GATA-3 in peripheral blood T cells.
T247 36802-36914 Sentence denotes We further examined whether inhaled FP could affect cellular localization of GATA-3 in peripheral blood T cells.
T11302 36915-37265 Sentence denotes Treatment with inhaled FP (500 µg) for 2 h significantly increased GR nuclear translocation (Figure 7A) and concomitantly decreased the number of nuclear GATA-3 immunoreactive peripheral blood T cells (37%±4.2% versus 58.2%±4.95%, p = 0.016, W = 28.0, Wilcoxon's rank test) compared with placebo as measured by immunocytochemistry (Figure 7A and 7B).
T248 36915-37265 Sentence denotes Treatment with inhaled FP (500 µg) for 2 h significantly increased GR nuclear translocation (Figure 7A) and concomitantly decreased the number of nuclear GATA-3 immunoreactive peripheral blood T cells (37%±4.2% versus 58.2%±4.95%, p = 0.016, W = 28.0, Wilcoxon's rank test) compared with placebo as measured by immunocytochemistry (Figure 7A and 7B).
T11303 37266-37397 Sentence denotes This was confirmed by Western blotting, which also indicated that this effect was both time- and dose-dependent (Figure 7C and 7D).
T249 37266-37397 Sentence denotes This was confirmed by Western blotting, which also indicated that this effect was both time- and dose-dependent (Figure 7C and 7D).
T11304 37398-37795 Sentence denotes Thus, inhaled FP (500 µg) induced significant loss in nuclear GATA-3 at 2 h (median [95% CI], 0.40 [0.27–0.53] versus 0.14 [0.11–0.19], p<0.05, W = 21.00, Wilcoxon's rank test) (Figure 7C) and cytoplasmic GATA-3 levels were enhanced by inhaled FP in a dose-dependent manner (median [95% CI], 0.0032 [0.0026–0.0039] versus 0.658 [0.592–0.720], p<0.05, W = −21.00, Wilcoxon's rank test) (Figure 7D).
T250 37398-37795 Sentence denotes Thus, inhaled FP (500 µg) induced significant loss in nuclear GATA-3 at 2 h (median [95% CI], 0.40 [0.27–0.53] versus 0.14 [0.11–0.19], p<0.05, W = 21.00, Wilcoxon's rank test) (Figure 7C) and cytoplasmic GATA-3 levels were enhanced by inhaled FP in a dose-dependent manner (median [95% CI], 0.0032 [0.0026–0.0039] versus 0.658 [0.592–0.720], p<0.05, W = −21.00, Wilcoxon's rank test) (Figure 7D).
T11305 37796-37931 Sentence denotes In addition, FP (500 µg) inhibited p38 MAPK phosphorylation in primary T cells in vivo at 2 h in samples from two patients (Figure 7E).
T251 37796-37931 Sentence denotes In addition, FP (500 µg) inhibited p38 MAPK phosphorylation in primary T cells in vivo at 2 h in samples from two patients (Figure 7E).
T252 37932-38052 Sentence denotes 10.1371/journal.pmed.1000076.g007 Figure 7 Inhaled fluticasone propionate impairs GATA-3 nuclear localization in PBMCs.
T19726 37976-38052 Sentence denotes Inhaled fluticasone propionate impairs GATA-3 nuclear localization in PBMCs.
T19727 38053-38324 Sentence denotes (A) Representative immunocytochemistry of showing the effect of inhaled FP (500 µg) on GR and GATA-3 nuclear localisation. (B) Nuclear GATA-3 immunoreactivity in PBMCs from seven steroid-naïve asthma patients 2 h following inhaled FP treatment (100 or 500 µg via spacer).
T253 38053-38324 Sentence denotes (A) Representative immunocytochemistry of showing the effect of inhaled FP (500 µg) on GR and GATA-3 nuclear localisation. (B) Nuclear GATA-3 immunoreactivity in PBMCs from seven steroid-naïve asthma patients 2 h following inhaled FP treatment (100 or 500 µg via spacer).
T19728 38325-38840 Sentence denotes The median and interquartile ranges for each treatment are presented as a box-and-whiskers plot (n = 7); * p<0.05 Wilcoxon's rank test compared with placebo. (C) Immunoblotting analyses of PBMCs demonstrated a time-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression after inhalation of FP. (D) Immunoblotting analyses of PBMCs demonstrated a dose-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression 2 h after inhalation of FP.
T254 38325-38840 Sentence denotes The median and interquartile ranges for each treatment are presented as a box-and-whiskers plot (n = 7); * p<0.05 Wilcoxon's rank test compared with placebo. (C) Immunoblotting analyses of PBMCs demonstrated a time-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression after inhalation of FP. (D) Immunoblotting analyses of PBMCs demonstrated a dose-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression 2 h after inhalation of FP.
T19729 38841-38975 Sentence denotes Histone H1 and MEK-1 immunoblotting confirmed equivalent total protein loading for the nuclear and cytoplasmic fractions respectively.
T255 38841-38975 Sentence denotes Histone H1 and MEK-1 immunoblotting confirmed equivalent total protein loading for the nuclear and cytoplasmic fractions respectively.
T19730 38976-39337 Sentence denotes Quantification of the densitometry data in (C) and (D) is shown as a box-and-whiskers plot of results from n = 6 participants for which data were available. *p<0.05 compared to control. (E) Western blot analyses of PBMCs demonstrated a time-dependent decrease in dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK after inhalation of FP (500 µg).
T256 38976-39337 Sentence denotes Quantification of the densitometry data in (C) and (D) is shown as a box-and-whiskers plot of results from n = 6 participants for which data were available. *p<0.05 compared to control. (E) Western blot analyses of PBMCs demonstrated a time-dependent decrease in dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK after inhalation of FP (500 µg).
T19731 39338-39415 Sentence denotes The results shown in (E) are representative of samples from two participants.
T257 39338-39415 Sentence denotes The results shown in (E) are representative of samples from two participants.
T11306 39418-39572 Sentence denotes Taken together, our data suggest that inhaled FP reduces nuclear localization of GATA-3 in vivo by acutely inhibiting phospho-GATA-3–importin association.
T258 39418-39572 Sentence denotes Taken together, our data suggest that inhaled FP reduces nuclear localization of GATA-3 in vivo by acutely inhibiting phospho-GATA-3–importin association.
T11307 39573-39759 Sentence denotes This effect may be direct, through competition for importin-α or associated molecules, or secondary to an effect on p38 MAPK-mediated GATA-3 phosphorylation via rapid induction of MKP-1.
T259 39573-39759 Sentence denotes This effect may be direct, through competition for importin-α or associated molecules, or secondary to an effect on p38 MAPK-mediated GATA-3 phosphorylation via rapid induction of MKP-1.
T11308 39760-39907 Sentence denotes The combination of these two interacting effects can result in complete suppression of GATA-3 nuclear import and thus Th2 cytokine gene expression.
T260 39760-39907 Sentence denotes The combination of these two interacting effects can result in complete suppression of GATA-3 nuclear import and thus Th2 cytokine gene expression.
T261 39909-39919 Sentence denotes Discussion
T13323 39920-40132 Sentence denotes Here we have demonstrated, to our knowledge for the first time, that corticosteroids may inhibit GATA-3 function and therefore the transcription of Th2 genes via two distinct but interacting molecular mechanisms.
T262 39920-40132 Sentence denotes Here we have demonstrated, to our knowledge for the first time, that corticosteroids may inhibit GATA-3 function and therefore the transcription of Th2 genes via two distinct but interacting molecular mechanisms.
T13324 40133-40312 Sentence denotes Firstly, corticosteroid-activated GR appears to compete with activated GATA-3 for nuclear import via importin-α, which is required for the nuclear transport of both GATA-3 and GR.
T263 40133-40312 Sentence denotes Firstly, corticosteroid-activated GR appears to compete with activated GATA-3 for nuclear import via importin-α, which is required for the nuclear transport of both GATA-3 and GR.
T13325 40313-40630 Sentence denotes Secondly, corticosteroids at higher concentrations increase the expression of MKP-1, a potent inhibitor of p38 MAPK activity and thereby prevent T cell receptor/co-receptor activation of p38 MAPK to prevent the phosphorylation of GATA-3 that is necessary for interaction with importin-α and subsequent nuclear import.
T264 40313-40630 Sentence denotes Secondly, corticosteroids at higher concentrations increase the expression of MKP-1, a potent inhibitor of p38 MAPK activity and thereby prevent T cell receptor/co-receptor activation of p38 MAPK to prevent the phosphorylation of GATA-3 that is necessary for interaction with importin-α and subsequent nuclear import.
T13326 40631-40820 Sentence denotes We have previously shown that translocation of GATA-3 from the cytoplasm to the nucleus involves the nuclear transporter protein importin-α, which interacts with phosphorylated GATA-3 [12].
T265 40631-40820 Sentence denotes We have previously shown that translocation of GATA-3 from the cytoplasm to the nucleus involves the nuclear transporter protein importin-α, which interacts with phosphorylated GATA-3 [12].
T13327 40821-41052 Sentence denotes We have also previously reported that GATA-3 knockdown using siRNA results in suppression of anti-CD3/CD28–stimulated IL-4/IL-5 mRNA induction, thus implicating an essential role for GATA-3 in the transcription of these genes [12].
T266 40821-41052 Sentence denotes We have also previously reported that GATA-3 knockdown using siRNA results in suppression of anti-CD3/CD28–stimulated IL-4/IL-5 mRNA induction, thus implicating an essential role for GATA-3 in the transcription of these genes [12].
T13328 41053-41154 Sentence denotes We now confirm, in human T cells, that GR also uses the same nuclear import mechanism as GATA-3 [22].
T267 41053-41154 Sentence denotes We now confirm, in human T cells, that GR also uses the same nuclear import mechanism as GATA-3 [22].
T13329 41155-41268 Sentence denotes We therefore propose that there is competition between ligand-activated GR and phospho-GATA-3 for nuclear import.
T268 41155-41268 Sentence denotes We therefore propose that there is competition between ligand-activated GR and phospho-GATA-3 for nuclear import.
T13330 41269-41542 Sentence denotes Furthermore, we have shown that there is preferential binding of importin-α to activated GR over phospho-GATA-3, so that corticosteroids would preferentially reduce GATA-3 entry and thus rapidly switch off Th2 gene transcription without any need for any intermediate steps.
T269 41269-41542 Sentence denotes Furthermore, we have shown that there is preferential binding of importin-α to activated GR over phospho-GATA-3, so that corticosteroids would preferentially reduce GATA-3 entry and thus rapidly switch off Th2 gene transcription without any need for any intermediate steps.
T13331 41543-41702 Sentence denotes Furthermore, there was some degree of specificity for GATA-3, as nuclear translocation of the p65 subunit of NF-κB was not affected by corticosteroid exposure.
T270 41543-41702 Sentence denotes Furthermore, there was some degree of specificity for GATA-3, as nuclear translocation of the p65 subunit of NF-κB was not affected by corticosteroid exposure.
T13332 41703-41897 Sentence denotes We tested some alternative explanations for this effect of FP on GATA-3 nuclear exclusion and failed to show that FP either enhances GATA-3 nuclear export directly or induces GATA-3 degradation.
T271 41703-41897 Sentence denotes We tested some alternative explanations for this effect of FP on GATA-3 nuclear exclusion and failed to show that FP either enhances GATA-3 nuclear export directly or induces GATA-3 degradation.
T13333 41898-42104 Sentence denotes The evidence from the in vitro competition assays does, however, suggest that purified activated GR can clearly attenuate purified phospho-GATA-3–importin-α association and that the converse does not occur.
T272 41898-42104 Sentence denotes The evidence from the in vitro competition assays does, however, suggest that purified activated GR can clearly attenuate purified phospho-GATA-3–importin-α association and that the converse does not occur.
T13334 42105-42187 Sentence denotes Furthermore, we have shown that only phospho-GATA-3 can associate with importin-α.
T273 42105-42187 Sentence denotes Furthermore, we have shown that only phospho-GATA-3 can associate with importin-α.
T13335 42188-42337 Sentence denotes This mechanism is sensitive to very low concentrations of corticosteroid and would be rapid in onset as no changes in protein synthesis are required.
T274 42188-42337 Sentence denotes This mechanism is sensitive to very low concentrations of corticosteroid and would be rapid in onset as no changes in protein synthesis are required.
T13336 42338-42537 Sentence denotes This acute mechanism may also contribute to the reduction in GATA-3 nuclear import and may play a major role at low corticosteroid concentrations and/or at early time points prior to MKP-1 induction.
T275 42338-42537 Sentence denotes This acute mechanism may also contribute to the reduction in GATA-3 nuclear import and may play a major role at low corticosteroid concentrations and/or at early time points prior to MKP-1 induction.
T13337 42538-42674 Sentence denotes Corticosteroids can modulate p38 MAPK activity through the induction of MKP-1, a potent endogenous inhibitor of MAPK function [38],[39].
T276 42538-42674 Sentence denotes Corticosteroids can modulate p38 MAPK activity through the induction of MKP-1, a potent endogenous inhibitor of MAPK function [38],[39].
T13338 42675-42795 Sentence denotes We report here a rapid induction of MKP-1 mRNA following stimulation of cells with relatively high concentrations of FP.
T277 42675-42795 Sentence denotes We report here a rapid induction of MKP-1 mRNA following stimulation of cells with relatively high concentrations of FP.
T13339 42796-42989 Sentence denotes We hypothesize that this rapid induction of MKP-1 can reduce GATA-3 nuclear import by attenuating p38 MAPK activity and subsequent GATA-3 phosphorylation, thus preventing nuclear translocation.
T278 42796-42989 Sentence denotes We hypothesize that this rapid induction of MKP-1 can reduce GATA-3 nuclear import by attenuating p38 MAPK activity and subsequent GATA-3 phosphorylation, thus preventing nuclear translocation.
T13340 42990-43256 Sentence denotes The location of the serine residue(s) of GATA-3 that are phosphorylated by p38 MAPK are currently unknown, but a bioinformatics search (Motif Scanner, http://scansite.mit.edu/motifscan_seq.phtml) indicates at least three potential p38 MAPK-sensitive serine residues.
T279 42990-43256 Sentence denotes The location of the serine residue(s) of GATA-3 that are phosphorylated by p38 MAPK are currently unknown, but a bioinformatics search (Motif Scanner, http://scansite.mit.edu/motifscan_seq.phtml) indicates at least three potential p38 MAPK-sensitive serine residues.
T13341 43257-43439 Sentence denotes As predicted from these in vitro data, impairment of GATA-3 nuclear import by FP may, at least in part, underlie the efficacy of corticosteroids in suppressing allergic inflammation.
T280 43257-43439 Sentence denotes As predicted from these in vitro data, impairment of GATA-3 nuclear import by FP may, at least in part, underlie the efficacy of corticosteroids in suppressing allergic inflammation.
T13342 43440-43740 Sentence denotes Although we did not assess the acute inhibitory effect of FP on the expression of IL-4 mRNA in vivo, a single inhalation of FP (500 µg) may have comparable effects, as it provides plasma levels within a relevant range of concentrations used to suppress IL-4 transcription in our in vitro system [37].
T281 43440-43740 Sentence denotes Although we did not assess the acute inhibitory effect of FP on the expression of IL-4 mRNA in vivo, a single inhalation of FP (500 µg) may have comparable effects, as it provides plasma levels within a relevant range of concentrations used to suppress IL-4 transcription in our in vitro system [37].
T13343 43741-43872 Sentence denotes A lower dose of inhaled FP (100 µg) was not effective, but plasma concentrations may be below those required for GATA-3 inhibition.
T282 43741-43872 Sentence denotes A lower dose of inhaled FP (100 µg) was not effective, but plasma concentrations may be below those required for GATA-3 inhibition.
T13344 43873-44053 Sentence denotes However, it is likely that the higher concentrations of FP in the airways after inhaled administration would be effective in inhibiting GATA-3 in airway T cells of asthma patients.
T283 43873-44053 Sentence denotes However, it is likely that the higher concentrations of FP in the airways after inhaled administration would be effective in inhibiting GATA-3 in airway T cells of asthma patients.
T13345 44054-44276 Sentence denotes The study of PBMCs from asthma patients treated with inhaled corticosteroid therapy clearly demonstrates that these molecular mechanisms are likely to also occur in patients at therapeutic doses of inhaled corticosteroids.
T284 44054-44276 Sentence denotes The study of PBMCs from asthma patients treated with inhaled corticosteroid therapy clearly demonstrates that these molecular mechanisms are likely to also occur in patients at therapeutic doses of inhaled corticosteroids.
T13346 44277-44452 Sentence denotes In addition, previous studies have shown that corticosteroids can suppress IL-4 and IL-5 release from peripheral blood cells of asthma patients in vitro and in vivo [40]–[42].
T285 44277-44452 Sentence denotes In addition, previous studies have shown that corticosteroids can suppress IL-4 and IL-5 release from peripheral blood cells of asthma patients in vitro and in vivo [40]–[42].
T13347 44453-44896 Sentence denotes In summary, our data provide evidence for a novel action of corticosteroids: suppression of allergic inflammation through a rapid inhibitory effect on GATA-3 nuclear translocation by preferential binding to the shared nuclear import protein importin-α and by a second mechanism involving increased synthesis of MKP-1, which inhibits p38 MAPK, thus preventing the phosphorylation of GATA-3 that is necessary for nuclear translocation of GATA-3.
T286 44453-44896 Sentence denotes In summary, our data provide evidence for a novel action of corticosteroids: suppression of allergic inflammation through a rapid inhibitory effect on GATA-3 nuclear translocation by preferential binding to the shared nuclear import protein importin-α and by a second mechanism involving increased synthesis of MKP-1, which inhibits p38 MAPK, thus preventing the phosphorylation of GATA-3 that is necessary for nuclear translocation of GATA-3.
T13348 44897-45035 Sentence denotes These two mechanisms are likely to be synergistic, accounting for the rapid and potent effect of corticosteroids on allergic inflammation.
T287 44897-45035 Sentence denotes These two mechanisms are likely to be synergistic, accounting for the rapid and potent effect of corticosteroids on allergic inflammation.
T13349 45036-45235 Sentence denotes This is exemplified by the rapid inhibitory effect of topical corticosteroids on nasal Th2 cytokine release after allergen provocation in individuals with seasonal allergic rhinitis (hay fever) [43].
T288 45036-45235 Sentence denotes This is exemplified by the rapid inhibitory effect of topical corticosteroids on nasal Th2 cytokine release after allergen provocation in individuals with seasonal allergic rhinitis (hay fever) [43].
T13350 45236-45398 Sentence denotes Prevention of phospho-GATA-3 interaction with importin-α may provide a new approach for the development of novel therapies for the treatment of allergic diseases.
T289 45236-45398 Sentence denotes Prevention of phospho-GATA-3 interaction with importin-α may provide a new approach for the development of novel therapies for the treatment of allergic diseases.