Experimentally, periostin promotes myofibroblast differentiation and plays a part in bleomycin-induced lung fibrosis (39)

Experimentally, periostin promotes myofibroblast differentiation and plays a part in bleomycin-induced lung fibrosis (39). passaging, and purity was assessed using morphological and immunohistochemical staining as previously described (46). Fibroblasts were grown and assayed in DMEM media, normal bronchial epithelial cells (NHBECs) were grown, and assays were performed using bronchial epithelial growth media (Cambrex, Lonza, Slough, UK). Fibroblasts or NHBECs were plated into 96-well plates (Costar, Corning, NY) at 1 105 cells/well and allowed to adhere for 8 hours. The cells were washed with PBS and cultured overnight in serum-free media containing 1% penicillin and 1% streptomycin at 37C in 5% CO2/air. Cells were stimulated with or without recombinant human IL-13 (R&D Systems, Minneapolis, MN). At the designated time points, supernatants were removed, and gene expression levels were quantitated using branched DNA technology (Panomics) as per the manufacturers instructions. Gene expression fold induction was determined after calibration of IL13R2 expression with GAPDH and normalized to corresponding unstimulated cells. Histologic Analysis Formalin-fixed and paraffin-embedded lung sections were stained with hematoxylin and eosin to assess gross morphology or Mallory’s trichrome stains to visualize collagen deposition. A modified Ashcroft histopathology scoring scale was used to quantify fibrotic alterations by a reviewer unaware of the treatment groups (32). Briefly, the entire left lung lobe from each animal was scanned and reviewed for fibrotic alterations in the alveolar Rabbit Polyclonal to OR2L5 septa. The alterations varied from none (i.e., normal) to confluent fibrotic masses in at least 50% of the visible lung structure (Grade 5). Five to eight whole lung sections were scanned from each group of mice. Formalin-fixed and paraffin-embedded lung sections were analyzed for immunohistochemical localization of IL-13R2. These assessments are described in the online supplement. Statistics Normally distributed data were expressed as means SEM and assessed for significance by Student’s test or ANOVA as appropriate. Data that were not normally distributed were assessed for significance using the Wilcoxon rank sum test or Mann-Whitney U test. Patient demographics were compared using Students test or Mann-Whitney analysis. Categorical variables were compared using Fishers exact test. values were determined for multiple comparisons using the Bonferroni correction. Data were clustered using the absolute value of correlation coefficients (distance measure) with hierarchical clustering, thereby identifying the transcripts that were strongly related to each other using the R version 2.13.0 program (47). The Ward method, MHY1485 which derives spherical clusters, was used as the agglomeration algorithm for forming clusters. Values of 0.05, 0.01, and 0.005 were considered significant. Results Overexpression of IL-13 and Not IL-4 in IPF IPF is characterized by excess ECM deposition in the MHY1485 lung due in part to an inappropriate IL-13Cdriven fibrotic process (3). Analysis of biopsied lung tissue from patients with IPF (= 8) and patients with a histologically fibrotic-dominant form of NSIP (= 4) or a histologically cellular-dominant form MHY1485 of NSIP (= 3) indicated that gene expression was more highly expressed in IPF lung tissue (Figure 1A) and that was most up-regulated in fibrotic NSIP (Figure 1B), in comparison to nonfibrotic control tissue. To further extend this observation, we confirmed the expression of IL-13 at the protein level in lung sections using immunohistochemical approaches. Biopsies from the following histologically verified lung diseases were assembled on a tissue microarray and examined concomitantly for the presence of IL-13 protein: respiratory bronchiolitis interstitial lung disease (Figures 2A and 2C), IPF MHY1485 with acute exacerbation (Figure 2B), normal lung tissue (Figure 2D), fibrotic NSIP (Figure 2E), and IPF (Figure 2F). Under higher magnification (200), it was apparent that IL-13 was most abundantly expressed in the interstitial areas of the biopsies obtained from patients with IPF who exhibited an acute.

---