Engraftment of bone marrow progenitor cells in a rat model of asbestos-induced pulmonary fibrosis

Abstract

Rationale: Bone marrow-derived cells have been shown to engraft during lung fibrosis. However, it is not known if similar cells engraft consequent to inhalation of asbestos fibers that cause pulmonary fibrosis, or if the cells proliferate and differentiate at sites of injury.

Objectives: We examined whether bone marrow-derived cells participate in the pulmonary fibrosis that is produced by exposure to chrysotile asbestos fibers.

Methods: Adult female rats were lethally irradiated and rescued by bone marrow transplant from male transgenic rats ubiquitously expressing green fluorescent protein (GFP). Three weeks later, the rats were exposed to an asbestos aerosol for 5 hours on three consecutive days. Controls were bone marrow-transplanted but not exposed to asbestos.

Measurements and main results: One day and 2.5 weeks after exposure, significant numbers of GFP-labeled male cells had preferentially migrated to the bronchiolar-alveolar duct bifurcations, the specific anatomic site at which asbestos produces the initial fibrogenic lesions. GFP-positive cells were present at the lesions as monocytes and macrophages, fibroblasts, and myofibroblasts or smooth muscle cells. Staining with antibodies to PCNA demonstrated that some of the engrafted cells were proliferating in the lesions and along the bronchioles. Negative results for TUNEL at the lesions confirmed that both PCNA-positive endogenous pulmonary cells and bone marrow-derived cells were proliferating rather than undergoing apoptosis, necrosis, or DNA repair.

Conclusions: Bone marrow-derived cells migrated into developing fibrogenic lesions, differentiated into multiple cell types, and persisted for at least 2.5 weeks after the animals were exposed to aerosolized chrysotile asbestos fibers.

Figures

Figure 1.

Pulmonary fibrosis model based on bone marrow transplantation and subsequent asbestos exposure. (A) Female Sprague-Dawley rats were irradiated at a lethal level (11 Gy) and immediately rescued by tail vein injection of 5 × 106 bone marrow mononuclear cells from male GFP-transgenic rats. After 3 wk of hematopoietic reconstitution, chimeric rats were exposed to asbestos for three consecutive days (5 h per day). Animals were killed 1 day or 2.5 weeks after the last asbestos exposure. (B) Control rat lung has normal morphology and exhibits normal bronchiolar–alveolar duct bifurcations (arrow). (C) Lung of chimeric rat 6 weeks after bone marrow transplant. There is slight bronchiolar inflammation but in general, lung architecture is normal and bronchiolar–alveolar duct morphology is unaffected (arrow). (D) Control (no bone marrow transplant) rat 1 day after asbestos exposure. (E) Chimeric (transplanted) rat 1 day after the last asbestos exposure. Arrows indicate asbestos lesions. Bone marrow transplantation does not alter the development of focal asbestos lesions at the first bronchiolar–alveolar ducts. (F) Gomori Trichrome stain of lung section from a chimeric asbestos-exposed rat (2.5 wk after exposure, ×10). Parenchymal lung tissue outside of areas exposed to asbestos does not display fibrosis (6 wk after bone marrow transplantation). (G) Asbestos lesion at the alveolar duct bifurcation from F. Note blue staining of the lesion that is indicative of interstitial cell fibrosis (×100). (H) Immunohistochemistry for GFP on lung section from transgenic GFP rat (control). Arrow indicates first alveolar duct bifurcation. B indicates bronchial space. The majority of pulmonary cells are positive for GFP. Note that many cells exhibit perinuclear and nuclear, as well as cytoplasmic staining. (I) Epifluorescent microscopy (FITC channel) of live adherent bone marrow cells from transgenic GFP rat. Bone marrow–derived cells also exhibit perinuclear and nuclear, as well as cytoplasmic staining.

Figure 2.

Bone marrow–derived cells engraft at asbestos lesions. (A) Immunohistochemistry for GFP (Alexa 594, red) demonstrating bone marrow–derived cell engraftment 1 day after asbestos exposure. (B) Bone marrow–derived cell engraftment persists 2.5 weeks after asbestos exposure. (C) Fluorescent in situ hybridization (FISH) for the Y chromosome confirming male bone marrow–derived cell engraftment at first alveolar duct bifurcation 1 day after the last asbestos exposure (arrow). (D) Y chromosome FISH demonstrating bone marrow–derived cell engraftment at the second alveolar duct bifurcation 1 day after the last asbestos exposure. A typical small fibrogenic lesion is identified at a duct junction (arrow). (E) Quantification of bone marrow–derived cell engraftment in the first bronchiolar–alveolar duct in chimeric control animals (no asbestos) and chimeric animals 1 day and 2.5 weeks after asbestos exposure. *P ⩽ 0.001 for comparison of control and asbestos exposed first bronchiolar–alveolar ducts. **P ⩽ 0.001 for comparison of first bronchiolar–alveolar ducts to surrounding lung parenchyma within the same animals (1 day, n = 3 [5 lesions per rat]; 2.5 weeks, n = 4 [5 lesions per rat]).

Figure 3.

Double immunohistochemistry for differentiation markers. (A–C) Staining for monocytes/macrophages (green) demonstrates that many of the bone marrow–derived cells (red) in the developing asbestos lesions 1 day after exposure are monocytes or macrophages (arrows). However, many bone marrow–derived cells are identified that are negative for blood cell markers (arrowheads). (D) Positive control (PC) showing bone marrow–derived collagen 1–positive fibrocytes in healing skin wound of chimeric rat. (E) Rabbit nonspecific IgG-negative control for lung 2.5 weeks after asbestos exposure (NC, 10 μg/ml). (F) Deconvolution microscopy of bone marrow–derived collagen 1–positive cell in asbestos lesion 2.5 wk after exposure (arrow). Collagen 1–negative/GFP-positive cells are also present (arrowheads).

Figure 4.

Deconvolution microscopy of bone marrow–derived cells in asbestos lesions. (A) Immunostaining for pro–surfactant protein C (pro–SP-C, red) combined with FISH for the Y chromosome (green) identifies bone marrow–derived type II pneumocytes in the asbestos-exposed lung. We did not observe bone marrow–derived type II cells in asbestos lesions. (B, C) Double fluorescent in situ hybridization (FISH) for the rat Y chromosome (red dots) and chromosome 4 (green dots) in 1 day and 2.5 week asbestos lesions. By FISH analysis of multiple asbestos lesions at the first bronchiolar–alveolar duct junctions, we did not find evidence of cell fusion. If present, cell fusion would be demonstrated by extra chromosome 4 signals (more than two) within a single cell nucleus. Insets show male bone marrow–derived cells (Y chromosome positive) that also stain for two chromosome 4s (indicative of a normal cell). These data do not exclude cell fusion from occurring at low levels, since the process of sectioning may occasionally remove chromosomes from the nucleus.

Figure 5.

Double immunohistochemistry for GFP and proliferating cell nuclear antigen (PCNA) to identify dividing bone marrow–derived cells in the lung. (A, B) Positive control (PC) showing PCNA-positive keratinocytes (green nuclei) in healing skin wound of chimeric rat. (C) Mouse nonspecific IgG negative control (NC, 10 μg/ml). (D–F) Lung of chimeric rat 1 day after the last asbestos exposure. Rare GFP bone marrow–derived cells are positive for PCNA (arrows). The majority of the PCNA-positive cells are resident epithelial cells. (G) Deconvolution microscopy of asbestos-exposed lung 1 day after exposures. Large arrows: PCNA- positive host cells in asbestos lesion. Arrowheads: PCNA-negative bone marrow–derived cells. Circle: PCNA-positive bone marrow–derived cell. Small arrows: PCNA-positive bone marrow–derived cell from the xz and yz planes, showing nuclear localization of PCNA. (H) Deconvolution microscopy of asbestos-exposed lung 2.5 weeks after exposures. Proliferation has resolved in 2.5 week lesion. Large arrows: PCNA-positive host cells in bronchial lining. Arrowheads: PCNA-negative bone marrow–derived cells that remain engrafted in lesion after 2.5 weeks. Small arrows: PCNA-positive bronchial host cell from the xz and yz planes, showing nuclear localization of PCNA. (I) Relative proportions of PCNA-postive GFP and host (Non-GFP) cells in asbestos lesions 1 day and 2.5 weeks after exposures. Cell counts from two different animals are shown for both time points (animals A and B, 1 d; animals C and D, 2.5 wk)

Figure 6.

TUNEL to identify apoptotic or necrotic cells following asbestos exposure. (A) One day after three consecutive 5-hour daily exposures to airborne asbestos fibers, there were no TUNEL-positive cell nuclei in cells at the alveolar duct junction lesions (arrow) or in cells that line the walls of the bronchus. A macrophage is stained nonspecifically in the cytoplasm, but not in the nucleus (arrowhead). (B) Rare TUNEL-positive cell nuclei were observed in the bronchial walls 2.5 weeks after the asbestos exposures. However, there were not TUNEL-positive cells in the proliferative lesion at the alveolar duct junction (arrow). Inset magnification: ×100. (C) We also observed rare randomly distributed parenchymal cells that were TUNEL-positive 2.5 weeks after asbestos exposure, perhaps due to irradiation. (D) TUNEL-positive cell from C. DAPI signal without blue pseudocolor. Nuclear disorganization was clearly visible in TUNEL-postitive cells (inset).

Figure 7.

Double immunostaining demonstrates mesenchymal cell engraftment from bone marrow progenitors after asbestos exposure. (A–C) Vimentin (VIM) staining shows fibroblast engraftment from cells that migrate from the bone marrow (arrow). (D–F) Vimentin staining of GFP-positive cells cultured from dispase-digested lung tissues after asbestos exposure confirms in vivo immunohistochemistry (arrows). (G–I) α smooth muscle actin (SMA) staining demonstrates myofibroblast or smooth muscle cell differentiation from bone marrow progenitors. (J–L) SMA immunocytochemistry of GFP-positive bone marrow–derived cells isolated and cultured from the lungs of asbestos-exposed chimeric rats also confirms in vivo stains.