TRAIL-Dependent Resolution of Pulmonary Fibrosis

Figure 4

Gr-1⁺ myeloid cells decreased collagen, fibronectin, and TGF-β expression in cultured primary lung fibroblasts. Mice were treated with Clod Lipo or Lipo one day prior to bleomycin challenge. Five days after bleomycin administration, Gr-1⁺ myeloid cells were MACS-purified from the lungs of challenged mice, and 1 × 10⁵ Gr-1⁺ cells were cocultured with 5 × 10⁵ primary mouse lung fibroblasts for 24 h. Lung fibroblasts were either untreated or exposed to IL-13 at 10 ng/ml for 24 h and washed prior to coculture. After coculture, the Gr-1⁺ myeloid cells were subsequently washed away prior to analysis of the fibroblasts. (a) Fibroblasts were fixed, permeabilized, and stained with anticollagen mAb prior to confocal microscopy. Shown are representative images taken at 1000x magnification. (b) The expression of fibronectin 1, procollagen 1, procollagen 3, and TGF-β was determined using quantitative PCR analysis of adherent primary lung fibroblasts. (c) Quantitative PCR analysis of RNA was used to determine the expression of granzyme, perforin, and TRAIL in purified Gr-1⁺ myeloid cells. (d) Purified Gr-1⁺ myeloid cells were stained for CD11b, and TRAIL and analyzed by flow cytometry. (e) Purified mouse lung fibroblasts were treated with increasing concentrations of sTRAIL for 24 h, washed, and quantitative PCR analysis was used to determine the expression of TGF-β, procollagen 1, and procollagen 3. Data are mean ± SEM, n = 3–5 independent experiments. All statistics were performed using unpaired parametric t-tests; .

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