Modulation of cytokine and nitric oxide by mesenchymal stem cell transfer in lung injury/fibrosis

Abstract

Background: No effective treatment for acute lung injury and fibrosis currently exists. Aim of this study was to investigate the time-dependent effect of bone marrow-derived mesenchymal stem cells (BMDMSCs) on bleomycin (BLM)-induced acute lung injury and fibrosis and nitric oxide metabolites and inflammatory cytokine production.

Methods: BMDMSCs were transferred 4 days after BLM inhalation. Wet/dry ratio, bronchoalveolar lavage cell profiles, histologic changes and deposition of collagen were analyzed.

Results: Nitrite, nitrate and cytokines were measured weekly through day 28. At day 7, the wet/dry ratio, neutrophilic inflammation, and amount of collagen were elevated in BLM-treated rats compared to sham rats (p = 0.05-0.002). Levels nitrite, nitrate, IL-1beta, IL-6, TNF-alpha, TGF-beta and VEGF were also higher at day 7 (p < 0.05). Degree of lymphocyte and macrophage infiltration increased steadily over time. BMDMSC transfer significantly reduced the BLM-induced increase in wet/dry ratio, degree of neutrophilic infiltration, collagen deposition, and levels of the cytokines, nitrite, and nitrate to those in sham-treated rats (p < 0.05). Fluorescence in situ hybridization localized the engrafted cells to areas of lung injury.

Conclusion: Systemic transfer of BMDMSCs effectively reduced the BLM-induced lung injury and fibrosis through the down-regulation of nitric oxide metabolites, and proinflammatory and angiogenic cytokines.

Figure 1

Flowcytometric Analysis of rat mesenchymal stem cells wased in the study. The cell surface marker phenotype of these MSCs was shown to be for CD45-, CD106-, CD44H+, CD90+, and CD11b+.

Figure 2

Variable differential potential of rat mesenchymal stem cells depending on culture method. (A) Culture-expanded mesenchymal stem cells showed a fibroblast-like morphology following culture expansion in vitro (×100). (B) Presence of adipocytes was demonstrated by oil red O staining of cytoplasmic inclusions of neutral lipids (×200). (C) Chondrocytes were demonstrated by toluidine blue staining (×100). (D) Calcium in the differentiated osteocytes was showed by Von Kossa staining (×100).

Figure 3

Schematic diagram of the experimental protocol. Specific pathogen-free, 6-week-old female SD rats (200-250 g body weight) were treated with 3 mg/kg bleomycin (BLM) dissolved in 5 ml of endotoxin-free water via inhalation. Bone marrow-derived mesenchymal stem cells (1 × 10⁶) were administered via a tail vein on day 4 after BLM treatment. The rats were killed on days 0, 7, 14, 21, and 28. Lung specimens were obtained before and after bronchoalveolar lavage as described in the Materials and Methods.

Figure 4

Effect of BMDMSC transfer on body weight and survival rate. (A) BMDMSC transfer increased body weight gain in the bleomycin - treated rats. (B) Bleomycin - treated rats showed higher mortality rate compared to the sham - treated rats while BMDMSC transfer attenuated the mortality of the bleomycin - treated rats.

Figure 5

Effect of bone marrow-derived mesenchymal stem cell (BMDMSC) transfer on the bleomycin (BLM)-induced increase in the lung wet/dry ratio. The transfer of BMDMSCs significantly reduced the BLM-induced increase in the wet/dry ratio on day 7. * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 6

(A) Time-dependent changes in the inflammatory cell profile in bronchoalveolar lavage (BAL) fluid. Bleomycin (BLM)-induced increases in the number of neutrophils, lymphocytes, and macrophages in the BAL fluid were significantly reduced between days 7 and 28 by the transfer of BMDMSCs. * and ** indicate p < 0.05 and p < 0.01, respectively. (B) Effect of BMDMSC transfer on BLM-induced histologic changes in the lungs. Lung tissues were stained with hematoxylin & eosin on days 7, 14, 21, and 28. BLM treatment induced the intra-alveolar and interstitial infiltration of inflammatory cells. The extent of inflammation significantly decreased from day 7 to day 28 following BMDMSC transfer. Magnification = × 200.

Figure 7

Effect of BMDMSC transfer on collagen deposition in lung tissues and the total amount of soluble collagen in lung extracts. (A) Collagen deposition in the lungs was analyzed using Masson's Trichrome stain. (B) The Sircol collagen assay was used to measure the total amount of soluble collagen in lung tissue lysates. * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 8

Effect of bone marrow-derived mesenchymal stem cell (BMDMSC) transfer on the levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and IL-1β, vascular endothelial growth factor (VEGF), and TGF-β in lung lysates. Bleomycin treatment significantly increased the levels of IL-1β (A), VEGF (B), IL-6 (C), TNF-α (D), and TGF-β (E) between days 7 and 28. The increases in IL-1β, VEGF, IL-6, TNF-α and TGF-β were significantly reduced by the transfer of BMDMSCs. * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 9

The effect of bone marrow-derived mesenchymal stem cell (BMDMSC) transfer on the bleomycin (BLM)-induced increases in nitrate and nitrite in lung extracts. BLM (n = 6) increased the nitrate (A) and nitrite levels (B) in lung extracts from day 7 to day 21; however, the effect was significantly reduced by BMDMSC transfer (n = 6). * and ** indicate p < 0.05 and p < 0.01, respectively.

Figure 10

Localization of the Y chromosome in bleomycin (BLM)-treated rats following bone marrow-derived mesenchymal stem cell transfer. A 2-μm-thick section of lung tissue from a BLM-treated rat hybridized with a fluorescein isothiocyanate-conjugated Y chromosome paint probe and counterstained with ethidium bromide is shown. (A) Bleomycin treated rats (n = 3). (B) Bone marrow-derived mesenchymal stem cell in recipient lungs under fluorescent microscope two weeks after BMDMSC injection in BMDMSC transfer rats (n = 3). The arrows indicate nuclei containing a Y chromosome. Magnification = 1000×. (C) Donor cells containing the Y chromosome zoomed out pictures of the lung. Magnification = 400×. (D) Localization of the cells containing the Y chromosome in serial section of lung tissue of BMDMSC transfer rats. H&E stain, magnification = 400×.