Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic airways  Weimin Liu, MD, Qiaoling Liang, MD, Silvana Balzar, MD, Sally Wenzel, MD, Magdalena Gorska, MD, PhD, Rafeul Alam, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 121, Issue 4, Pages 893-902.e2 (April 2008) DOI: 10.1016/j.jaci.2008.02.004 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Phospho-ERK1/2 (p-ERK1/2) immunofluorescence staining of airway biopsy samples. A, Biopsy specimens from 17 patients with severe asthma, 17 patients with mild asthma, and 15 healthy control subjects were immunostained for phospho-ERK1/2 by using the mouse monoclonal IgG2a antibody (Santa Cruz Biotechnology). An Alexa488-labeled goat anti-mouse IgG antibody detects phospho-ERK1/2–positive cells by means of green fluorescent staining. The section with the isotype control antibody (mouse IgG2a) staining was counterstained with DAPI (blue) to demonstrate the presence of nucleus. Representative images (×20 magnification, scale bar = 50 μm) from each study group are shown. B and C, Morphometric analyses of phospho-ERK1/2 staining of biopsy samples. Images from the immunostained biopsy samples obtained from patients with severe and mild asthma (n = 17) and healthy control subjects (n = 15) were analyzed by using the Metamorph software (Molecular Devices Corp, Downingtown, Pa). The mean fluorescence intensity (MFI) of phospho-ERK1/2 per image (Fig 1, B) and the mean phospho-ERK1/2–positive area per epithelial layer (Fig 1, C) for each study group was calculated by analyzing the threshold area from a representative image from each study subject. The statistical difference between the study groups was analyzed by using the Mann-Whitney U test (pairwise analyses). Fig 1, B: ∗P = .000000001, ∗∗P = .00000004, and ∗∗∗P = .001. Fig 1, C: ∗P = .000003, ∗∗P = .000006, and ∗∗∗P = .006. Each symbol indicates a study subject, and the line indicates the median value. Journal of Allergy and Clinical Immunology 2008 121, 893-902.e2DOI: (10.1016/j.jaci.2008.02.004) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Expression of phospho-p38 MAPK (p-p38) in airway tissue samples. Biopsy samples from 17 patients with severe asthma, 17 patients with mild asthma, and 15 healthy control subjects were processed for immunostaining with a rabbit phospho-specific p38 MAPK antibody (Santa Cruz Biotechnology, Inc) or control rabbit serum. The primary antibody was detected with an Alexa488-labeled goat anti-rabbit secondary antibody. The section with the control antibody staining was counterstained with DAPI (blue) to detect nucleus. Representative images from each study group are shown in A. Morphometric analyses of the phospho-p38–stained area for each study group were performed as described in the legend of Fig 1. B presents the mean fluorescence intensity (MFI) of phospho-p38 staining per image. C presents the percentage of phospho-p38–immunostained area per epithelial layer. Pairwise statistical analyses were done by using the Mann-Whitney U test. Fig 2, B: ∗P = .02, ∗∗P = .0001, and ∗∗∗P = .06. Fig 2, C: ∗P = .0004, ∗∗P = .00005, and ∗∗∗P = .1. Each symbol indicates a study subject, and the line indicates the median value. Journal of Allergy and Clinical Immunology 2008 121, 893-902.e2DOI: (10.1016/j.jaci.2008.02.004) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Expression of phospho-JNK (p-JNK) in the airway tissue. Biopsy samples from 17 patients with severe asthma, 17 patients with mild asthma, and 15 healthy control subjects were immunostained with a mouse monoclonal phospho-specific anti-JNK antibody or a mouse IgG1 isotype control antibody followed by an Alexa488-labeled goat anti-mouse secondary antibody. The sections were counterstained with DAPI (blue) to detect nucleus. Representative images from each study group are shown in A. Morphometric analyses of the phospho-JNK–stained area for each study group were performed as described in the legend of Fig 1 and presented in B. Statistical analyses were done by using the Mann-Whitney U test (pairwise analysis). MFI, Mean fluorescence intensity. ∗P = .004, ∗∗P = .7, and ∗∗∗P = .003. Each symbol indicates a study subject, and the line indicates the median value. Journal of Allergy and Clinical Immunology 2008 121, 893-902.e2DOI: (10.1016/j.jaci.2008.02.004) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Expression of ERK1/2-inducible downstream effectors sprouty-2 and JunB. Biopsy sections from patients with severe asthma and healthy control subjects (n = 6 for each group) were stained with a rabbit anti-sprouty-2 and a mouse monoclonal anti-JunB antibody. The primary antibodies were detected with Alexa488-labeled goat anti-rabbit and anti-mouse IgG antibodies. Representative images from each study group are shown. Isotype controls (rabbit IgG and mouse IgG1) for these antibodies are shown in Figs 2 and 3. The right panel shows morphometric data from 6 patients with severe asthma and 6 healthy control subjects. Statistical analyses were done with the Mann-Whitney U test. Each symbol indicates a study subject, and the line indicates the median value. Journal of Allergy and Clinical Immunology 2008 121, 893-902.e2DOI: (10.1016/j.jaci.2008.02.004) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 A, Activation of ERK1/2 in airway epithelial cells. Primary human airway epithelial cells were incubated with amphiregulin (100 ng/mL), EGF (100 ng/mL), IL-6 (100 ng/mL), IL-13 (10 ng/mL), and eotaxin (10 ng/mL) for various periods of time and then Western blotted with the anti-phospho-ERK1/2 antibody (n = 3). The membranes were reprobed with an anti-ERK1/2 antibody to determine the protein loading. B, Induction of p38 phosphorylation in epithelial cells. Primary human airway epithelial cells were stimulated with IL-4 (50 ng/mL), EGF (100 ng/mL), TNF-α (100 ng/mL), and IL-13 (10 ng/mL) for 10 minutes and Western blotted for phospho-p38. The membrane was reprobed with an anti-p38 antibody to demonstrate equal protein loading (n = 3). C and D, Effect of ERK1/2 inhibition on epithelial gene expression. Airway epithelial cells were cultured in the air-liquid interface and then preincubated with the MEK1/2 inhibitor PD98059 (PD, 50 μmol/L), the p38 inhibitor SB212190 (SB, 10 μmol/L), or the diluent (Dil) dimethyl sulfoxide for 30 minutes and then stimulated with IL-13 (20 ng/mL) and TNF-α (100 ng/mL). The secretion of chemokines in the culture supernatant was measured at 48 hours by means of ELISA. The secretion in the presence of the inhibitors was compared with that of the diluent (Fig 5, C: n = 4; ∗P = .04 and ∗∗P = .004, t test). The expression of mRNA for chemokine genes was measured after an overnight culture by means of real-time PCR, and the results were expressed as the fold increase in mRNA over the baseline (diluent-treated cells) after correcting for the difference in expression of the housekeeping gene β-actin (Fig 5, D: n = 8; ∗P < .004, t test). E, The effect of MEK1/2 and p38 inhibition on epithelial cell proliferation. Primary airway epithelial cells were cultured in the growth medium with IL-13 (10 ng/mL) or amphiregulin (100 ng/mL) in the presence or absence of diluent (Dil), PD98059 (PD, 50 μmol/L), or SB212190 (SB, 10 μmol/L). Tritiated thymidine incorporation was measured at 48 hours (n = 3; ∗P = .005 for Dil vs PD for IL-13, P = .004 for Dil vs PD for amphiregulin, P = .02 for Dil vs SB for IL-13, and P = .009 for Dil vs SB for amphiregulin, t test). The mean (± SEM) basal proliferation in the absence of the added growth factors was 2720 ± 78 (cpm). Journal of Allergy and Clinical Immunology 2008 121, 893-902.e2DOI: (10.1016/j.jaci.2008.02.004) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions