IL-13 Augments Compressive Stress-induced Tissue Factor Expression in Human Airway Epithelial Cells

Transcription

1 IL-13 Augments Compressive Stress-induced Tissue Factor Expression in Human Airway Epithelial Cells Jennifer A. Mitchel, Silvio Antoniak, Joo-Hyeon Lee, Sae-Hoon Kim, Maureen McGill, David I. Kasahara, Scott H. Randell, Elliot Israel, Stephanie A. Shore, Nigel Mackman, and Jin-Ah Park ONLINE DATA SUPPLEMENT

2 E2 SUPPLEMENTARY MATERIALS AND METHODS Isolation of Extracellular vesicles and immunogold labeling To determine the subcellular location of TF in extracellular vesicles isolated from CM, we performed immunogold labeling of TF. Extracellular vesicles were isolated as previously described 1. Immunogold labeling was performed in the Harvard Medical School (HMS) EM core facility 2 Western blot analysis To detect TF protein in the cell lysates and CM, we performed Western blot analysis, as previously described 1,3. Immunohistochemistry Paraffin-sectioned lung tissues were immunostained with antibodies against the basal cell marker p63 (1:200, Santa Cruz Biotechnology) and TF (1:100, R&D Systems). Secondary antibodies against goat or rabbit were conjugated with Alexa 488 or 594 (1:400, Invitrogen). Vectastain with DAPI was used for nuclear staining. Fluorescence microscopy and imaging were performed with a Nikon 90i and a Roper Scientific CCD camera (Nikon) and NIS-Elements software. Quantitative RT-PCR To detect mrna expression of TF, RNA was isolated from cell lysates, cdna was generated, and qrt-pcr was performed, all as described previously 1. The expression of TF was normalized to GAPDH and the fold changes were calculated using the delta-delta Ct method 4.

3 E3 Isolation of mouse primary lung epithelial cells Mouse primary lung epithelial cells were isolated as previously described 5,6. Distal lung tissues and tracheas were minced and digested, and sorted using FACS (Becton Dickinson, New Jersey). Type II epithelial cells were isolated from Sftpc-H2B-GFP mice 7. Club cells were isolated from Scgb1a1-CreER TM ; Rosa26R-eYFP mice 8. Basal cells were isolated by labeling with NGFR antibody 6 (Abcam). Staining with DAPI (Sigma) was used to eliminate dead cells. Allergen sensitized and challenged mice Four-week old mice were sensitized by i.p. injection with a mixture of ovalbumin (OVA) (0.1 mg/ml) and alum (20 mg/ml) on days 0 and 14. Beginning on day 28, mice were challenged for 30 minutes daily, for three or six consecutive days, with either aerosolized PBS or 1% OVA in PBS 9.

4 E4 SUPPLEMENTAL TABLE ID # Age Sex ICS Ethnicity % Eosinophils 1 29 F N Caucasian F N Hispanic M N Caucasian F N Caucasian F N Caucasian M N Caucasian 82.1 Supplemental Table 1: Patient information for materials collected before and after allergen challenge. ICS = inhaled corticosteroid. For all patients, <0.5% of total cells were eosinophils before allergen challenge; % Eosinophils values are reported at 48 hours post-allergen challenge.

5 E5 References 1 Park, J. A. et al. Tissue factor-bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo. The Journal of allergy and clinical immunology 130, , doi: /j.jaci (2012). 2 Griffiths, G., Burke, B. & Lucocq, J. Fine structure immunocytochemistry. (Springer- Verlag, 1993). 3 Park, J. A., Drazen, J. M. & Tschumperlin, D. J. The chitinase-like protein YKL-40 is secreted by airway epithelial cells at base line and in response to compressive mechanical stress. The Journal of biological chemistry 285, , doi: /jbc.m (2010). 4 Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, , doi: /meth (2001). 5 Kim, C. F. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, , doi: /j.cell (2005). 6 Rock, J. R. et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 106, , doi: /pnas (2009). 7 Lee, J. H. et al. Surfactant protein-c chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells. Am J Respir Cell Mol Biol 48, , doi: /rcmb oc (2013). 8 Rawlins, E. L. et al. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4, , doi: /j.stem (2009). 9 Williams, A. S. et al. Role of the adiponectin binding protein, T-cadherin (cdh13), in allergic airways responses in mice. PloS one 7, e41088, doi: /journal.pone (2012).