The anti-inflammatory effect of glucocorticoids is mediated by glucocorticoid-induced leucine zipper in epithelial cells  Jane Eddleston, PhD, Jack Herschbach, BS, Amy L. Wagelie-Steffen, MD, Sandra C. Christiansen, MD, Bruce L. Zuraw, MD  Journal of Allergy and Clinical Immunology  Volume 119, Issue 1, Pages 115-122 (January 2007) DOI: 10.1016/j.jaci.2006.08.027 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Detection of GILZ expression in airway epithelial cells. A, cDNA from BEAS-2B cells, A549 cells, human peripheral blood monocytes, and blood monocyte–derived macrophages was analyzed by means of quantitative real-time PCR for the expression of GILZ mRNA and the mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression was calculated as the quantity of GILZ mRNA over the quantity of GAPDH mRNA detected in each sample. B, Cell lysate from A549 cells overexpressing FLAG-tagged GILZ were immunoblotted with either the rabbit anti-GILZ sera (lane 1) or the anti-FLAG antibody (lane 2). The cell lysate from A549 cells overexpressing FLAG-GILZ protein was used for immunoprecipitation with the rabbit anti-GILZ sera (lane 3) or the anti-FLAG antibody (lane 5), followed by immunoblotting with either the anti-FLAG antibody (lane 3) or the anti-GILZ sera (lane 5). Lanes 4 and 6 show cell lysates from mock-transfected A549 cells immunoprecipitated with either anti-GILZ sera or anti-FLAG antibody followed by immunoblotting with anti-FLAG antibody or anti-GILZ sera, respectively. C, Cell lysates from BEAS-2B cells were immunoblotted for GILZ protein with the rabbit anti-GILZ sera. aFLAG, Anti-FLAG; aGILZ, anti-GILZ. Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Dexamethasone increases the expression of GILZ in BEAS-2B cells. A, BEAS-2B cells were stimulated with dexamethasone (DEX; 10−7 mol/L) or media alone (control) for 30 minutes and 2 hours in triplicate, and then total RNA was harvested and analyzed by means of quantitative real-time PCR in triplicate for the expression of GILZ and β-actin mRNA. B, BEAS-2B cells were treated with dexamethasone (DEX; 10−7 mol/L) alone or in combination with RU486 (10−5 mol/L) for 2 hours in triplicate, and GILZ mRNA levels were assessed (∗P < .05, Mann-Whitney U test). C, Cell lysates from BEAS-2B cells overexpressing FLAG-GILZ (F-GILZ) protein and lysates from BEAS-2B cells stimulated with media alone or dexamethasone (DEX; 10−7 mol/L) for 16 hours were immunoblotted with the rabbit anti-GILZ sera (∗P < .05, Mann-Whitney U test). D, BEAS-2B cells were stimulated for 1, 2 and 4 hours with dexamethasone (DEX; 10−7 mol/L) or media alone, and cell lysates were isolated. The level of GILZ protein was assessed by immunoblotting with the rabbit anti-GILZ sera. Blots were stripped and rehybridized with an antibody to β-actin. Results are representative of 3 separate experiments. Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 GILZ suppresses the activation of NF-κB by inflammatory mediators. A, BEAS-2B cells were transiently transfected with either pcDNA3.1(+)-GILZ (GILZ) or pcDNA3.1(+) (Mock) along with TransLucent NF-κB vector and pCMV-β-galactosidase vector. After 48 hours, cells were placed on supplement-free media for 2 hours, followed by treatment with either media alone, LPS (1 mg/mL), IL-1β (10 ng/mL), or Poly I:C (1 mg/mL). After 4 hours, cell lysates were harvested, and luciferase and β-galactosidase activity was detected. Luciferase activity was normalized to β-galactosidase activity for each sample and expressed as relative luciferase activity. B, LPS-, IL-1β–, and Poly I:C–induced NF-κB activity from 3 separate experiments is presented as the percentage of luciferase activity induced in GILZ-transfected cells compared with that seen in mock-transfected cells (∗P < .05 compared with mock-transfected cells, Mann-Whitney U test). Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Knockdown of GILZ blocks the effects of dexamethasone. A, HEK293 cells were transfected with either control siRNA (siControl) or GILZ siRNA (siGILZ), left untreated (Untreated), or treated with transfection reagent alone (Mock) for 24 hours. Total protein was isolated and assessed by means of immunoblotting for GILZ and β-actin protein. B, Total RNA was isolated from siControl and siGILZ HEK293 cells 24 hours after siRNA transfection and assessed by means of quantitative real-time PCR for IL-8 and β-actin mRNA levels (∗P < .05, Mann-Whitney U test). Data shown are of a representative experiment performed in triplicate. C, HEK293 cells were treated simultaneously with either siControl or siGILZ RNA along with 10−7 mol/L dexamethasone (DEX) or media alone (Untreated) for 24 hours, and cell lysates were isolated and assessed by means of immunoblotting for GILZ protein and β-actin. D, HEK293 cells were treated simultaneously with either siControl or siGILZ RNA along with 10−7 mol/L dexamethasone for 24 hours, followed by IL-1β (1 ng/mL) for 2 hours. Total RNA was isolated and assessed by means of quantitative real-time PCR for levels of IL-8 and β-actin mRNA (∗P < .05, Mann-Whitney U test). Data shown are from 3 separate experiments, each performed in triplicate. Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Cytokines decrease GILZ expression in BEAS-2B cells. A, BEAS-2B cells were stimulated with either media alone, IL-1β (10 mg/mL), TNF-α (10 mg/mL), IFN-γ (10 mg/mL), or CytoMix (10 mg/mL of each cytokine) for 2, 4, and 24 hours in triplicate. Total RNA was isolated and assessed for GILZ and β-actin gene expression by means of quantitative real-time PCR in triplicate. Expression was calculated (from 3 separate experiments) as the mean percentage of GILZ mRNA compared with that seen in control cells. B, BEAS-2B cells were stimulated with either media alone, IL-1β (10 mg/mL), TNF-α (10 mg/mL), IFN-γ (10 mg/mL), or CytoMix (10 mg/mL of each cytokine) for 16 hours. Cell lysates were harvested and immunoblotted for GILZ protein. The blots were stripped and rehybridized for β-actin (∗P < .05, Mann-Whitney U test). NS, No stimulus/media alone. Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 GILZ expression and regulation in NHBE cells. A, NHBE cells were treated for 30 minutes, 2 hours, and 4 hours with either media alone or dexamethasone (DEX; 10−7 mol/L). Total RNA was isolated and assessed by using quantitative real-time PCR for the levels of GILZ and β-actin mRNA. B, NHBE cells were treated for 16 hours with media alone or dexamethasone (DEX; 10−7 mol/L). Cell lysates were analyzed by means of immunoblotting for GILZ protein expression with a nitrocellulose membrane (∗P < .05, Mann-Whitney U test). C, NHBE cells were stimulated with CytoMix (10 mg/mL of IL-1β, TNF-α, and IFN-γ) for 0.5, 2, or 4 hours in triplicate. Total RNA was isolated and assessed by means of quantitative real-time PCR in triplicate for GILZ and β-actin mRNA levels. Expression was calculated as the mean percentage of GILZ mRNA compared with that seen in control cells. D, NHBE cells were stimulated for 16 hours with CytoMix. Cell lysates were harvested and analyzed by means of immunoblotting for GILZ protein. The blot was stripped and rehybridized for β-actin (∗P < .05, Mann-Whitney U test). Journal of Allergy and Clinical Immunology 2007 119, 115-122DOI: (10.1016/j.jaci.2006.08.027) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions