IκB kinase–driven nuclear factor-κB activation in patients with asthma and chronic obstructive pulmonary disease Rosalia Gagliardo, PhD, Pascal Chanez,

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Presentation transcript:

IκB kinase–driven nuclear factor-κB activation in patients with asthma and chronic obstructive pulmonary disease Rosalia Gagliardo, PhD, Pascal Chanez, MD, PhD, Mirella Profita, PhD, Anna Bonanno, PhD, Giusy Daniela Albano, PhD, Angela Marina Montalbano, PhD, Flora Pompeo, PhD, Cesare Gagliardo, MD, Anna Maria Merendino, PhD, Mark Gjomarkaj, MD Journal of Allergy and Clinical Immunology Volume 128, Issue 3, Pages 635-645.e2 (September 2011) DOI: 10.1016/j.jaci.2011.03.045 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IKKα and IKKβ protein expression in PBMCs of control subjects (C), asthmatic patients (A), HSs, and patients with COPD. A and C, Representative Western blot analysis of p-IKKα, IKKα, p-IKKβ, and IKKβ in PBMCs isolated from control subjects, asthmatic patients, HSs, and patients with COPD. B and D, Expression of p-IKKα/IKKα and p-IKKβ/IKKβ in PBMCs of control subjects, asthmatic patients, HSs, and patients with COPD. Signals corresponding to p-IKKα, IKKα, p-IKKβ, and IKKβ were semiquantified by using a densitometric scanner and expressed as the ratio of the band intensity of the phosphorylated protein versus the band intensity of the total protein (National Institutes of Health Image/Gel Plotting analysis program). Open circles represent patients with moderate asthma, and solid circles represent patients with severe asthma. Open triangles represent patients with mild-to-moderate COPD (GOLD stages I and II), and solid triangles represent patients with severe COPD (GOLD stage III). Inverted triangles represent HSs. Horizontal lines represent medians. E, Specificity of sequential immunoprecipitation for IKKα and IKKβ protein analysis. After comigration on SDS gels, immunoprecipitates were analyzed through immunoblotting by using anti-phosphorylated serine/threon/phen murine antibody to show the different p-IKKα and p-IKKβ molecular weights. ∗P < .01, ∗∗P < .001. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IKKα and IKKβ functional activity. A and B, Luminescent IKK assay. Signals corresponding to IKKα and IKKβ activity in PBMCs of control subjects (C), asthmatic patients (A), HSs, and patients with COPD were measured by using the luminescent kinase assay. Individual data are shown. Open circles represent patients with moderate asthma, and solid circles represent patients with severe asthma. Open triangles represent patients with mild-to-moderate COPD (GOLD stages I and II), and solid triangles represent patients with severe COPD (GOLD stages III). Inverted triangles represent HSs. Horizontal lines represent medians. C, Representative autoradiography of IKKα and IKKβ functional activity detected by adding radiolabeled ATP and recombinant GST-IκBα to immunoprecipitated IKKα and IKKβ obtained from PBMCs of control subjects, asthmatic patients, HSs, and patients with COPD, as described in the Methods section. IKKα and IKKβ activity is demonstrated by means of detection of GST-IκBα substrate phosphorylation. D, Correlation between IKKα activity and FEV1/FVC percent predicted in patients with COPD. Statistical analysis was performed by using the Spearman test. ∗P < .01, ∗∗P < .001. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 A and B, Correlations between IKKα activity and p-IKKα/IKKα ratio (Fig 3, A) and between IKKβ activity and p-IKKβ/IKKβ ratio (Fig 3, B). C, Correlation between IKKα and IKKβ activity in patients with COPD. Individual data are shown. Open circles represent patients with moderate asthma, and solid circles represent patients with severe asthma. Open triangles represent patients with mild-to-moderate COPD (GOLD stages I and II), and solid triangles represent patients with severe COPD (GOLD stages III). Inverted triangles represent HSs. Statistical analysis was performed by using the Spearman test. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Representative Western blotting of IKKα and IKKβ subcellular protein distribution. Nuclear and cytoplasmic fractions of PBMCs obtained from control subjects (C), asthmatic patients (A), HSs, and patients with COPD were separated and immunoblotted with anti-IKKα and anti-IKKβ. Signals corresponding to IKKα and IKKβ in nuclear and cytoplasmic compartments were semiquantified by means of densitometric scanning and normalized versus the band intensity of HDAC-1 for the nuclear protein fraction and versus α-tubulin for the cytoplasmic protein fraction. ∗P < .01, significantly different from control subjects and asthmatic patients. Open bars, Nucleus; solid bars, cytosol. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Modulation of IKK activity by FP in PBMCs from patients. Modulation of IKKα and IKKβ activity by FP treatment in PBMCs from control subjects, asthmatic patients, HSs, and patients with COPD. Results obtained by using the luminescent kinase assay are expressed as percentages of IKKα (A) and IKKβ (B) inhibition by means of FP treatment (means ± SDs) in comparison with the respective basal values. ∗∗P < .001 and ∗P < .01, significantly different from control subjects. #P < .01 for comparisons indicated. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 In vitro modulation of IKKα and IKKβ activity in PBMCs from healthy donors. Modulation of IKKα and IKKβ in PBMCs isolated from healthy donors in response to TNF-α, CSE, GSH, and FP by using the kinase activity assay is shown. Data are expressed as percentages of IKKα (A) and IKKβ (B) activity modulation in comparison with that seen in unstimulated PBMCs. The results are representative of 3 independent experiments (means ± SDs). ∗∗P < .001 and ∗P < .01, significantly different from baseline values. ##P < .001 and #P < .01 for comparisons indicated. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Release of IL-8 by PBMCs. A, Assessment of the concentrations of IL-8 released by PBMCs isolated from control subjects, asthmatic patients, HSs, and patients with COPD. Individual data are shown. Horizontal lines represent the median. ∗∗P < .001 and ∗P < .01, significant differences between the study groups. B-D, In vitro effects of FP treatment on IL-8 release by PBMCs from asthmatic patients (Fig 7, B), HSs (Fig 7, C), patients with COPD (Fig 7, D). ∗∗P < .001 and ∗P < .01 versus FP. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 In vitro modulation of IL-8 release by PBMCs. Assessment of the concentrations of IL-8 released by PBMCs isolated from healthy donors stimulated with TNF-α and 10% CSE for 24 hours in the presence or absence of FP and GSH is shown. The results are representative of 3 independent experiments (means ± SDs). ∗∗P < .001 and ∗P < .01, significantly different from baseline values. ##P < .001 and #P < .01 for comparisons indicated. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 9 Release of IL-8 by PBMCs after siIKKα transfection. A, Assessment of the concentrations of IL-8 released by PBMCs isolated from patients with COPD, HSs, and control subjects temporarily transfected with siRNA for IKKα or with control siRNA containing a scrambled sequence. ∗P < .01, significantly different from baseline values. B, Expression of IKKα protein in PBMCs not transfected (baseline), transfected with scrambled siRNA, and transfected with siRNA for IKKα was analyzed by means of Western blot analysis. Journal of Allergy and Clinical Immunology 2011 128, 635-645.e2DOI: (10.1016/j.jaci.2011.03.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions