Allergy Asthma Immunol Res.  2011 Jul;3(3):205-211. 10.4168/aair.2011.3.3.205.

Mesenchymal Stem Cell Transfer Suppresses Airway Remodeling in a Toluene Diisocyanate-Induced Murine Asthma Model

• Lee SH
• Jang AS
• Kwon JH
• Park SK
• Won JH
• Park CS

• KMID: 1803327
• DOI: http://doi.org/10.4168/aair.2011.3.3.205

Abstract

PURPOSE
Severe asthma is characterized by high medication requirements to maintain good disease control or by persistent symptoms despite high medication use. The transfer of bone marrow-derived mesenchymal stem cells (BMDMSCs) to the injured lungs is a possible treatment for severe asthma. This study investigated the therapeutic effects of BMDMSCs in airway remodeling and inflammation in an experimental toluene diisocyanate (TDI)-induced asthma animal model of severe asthma.
METHODS
BMDMSCs were transferred into rats after TDI inhalation. Bronchoalveolar lavage (BAL) cell profiles, histological changes including an inflammatory index and goblet cell hyperplasia, and the airway response to methacholine using plethysmography were analyzed. Smooth muscle actin (SMA) and proliferating cell nuclear antigen (PCNA) protein expression were observed in lung tissue using immunohistochemical staining. The collagen content was measured in lung tissue sections and lung extracts using Masson's trichrome staining and an immunoassay kit.
RESULTS
The numbers of inflammatory cells in BAL fluid, histological inflammatory index, airway response to methacholine, number of goblet cells, and amount of collagen were increased in TDI-treated rats compared with sham rats (P=0.05-0.002). BMDMSC transfer significantly reduced the TDI-induced increase in the inflammatory index and numbers of eosinophils and neutrophils in BAL fluid to levels seen in sham-treated rats (P<0.05). BMDMSC transfer significantly reduced the number of goblet cells, collagen deposition, and immune staining for SMA and PCNA with concomitant normalization of the airway response to methacholine.
CONCLUSIONS
The systemic transfer of BMDMSCs effectively reduced experimental TDI-induced airway inflammation and remodeling and airway hyperreactivity.

Keyword

Airway remodeling; asthma; collagen; stem cells; TDI

MeSH Terms

Actins
Airway Remodeling
Animals
Asthma
Bronchoalveolar Lavage
Collagen
Eosinophils
Goblet Cells
Hyperplasia
Immunoassay
Inflammation
Inhalation
Lung
Mesenchymal Stromal Cells
Methacholine Chloride
Models, Animal
Muscle, Smooth
Neutrophils
Plethysmography
Proliferating Cell Nuclear Antigen
Rats
Salicylamides
Stem Cells
Toluene
Toluene 2,4-Diisocyanate
Actins
Collagen
Methacholine Chloride
Proliferating Cell Nuclear Antigen
Salicylamides
Toluene
Toluene 2,4-Diisocyanate

Figure

• 

  Fig. 1 Schematic diagram of the experimental protocol. Mice were sensitized by the intranasal administration of 3% toluene diisocyanate (TDI) once daily for 5 consecutive days. Bone marrow-derived mesenchymal stem cells (1×105 cells) were transferred via an intravenous route. The following day, mice were challenged via the airways with 1% TDI for 3 days with 1 hr ultrasonic nebulization each day. At day 12, airway hyperresponsiveness (AHR) to methacholine was measured; then, the mice were killed on day 13.

• 

  Fig. 2 Cellular profiles of bronchoalveolar lavage (BAL) fluid. Toluene diisocyanate (TDI)-sensitized/challenged mice had increased numbers of cells, macrophages, neutrophils, and eosinophils compared with sham-treated mice (*P<0.05). Bone marrow-derived mesenchymal stem cell (BMDMSC) transfer effectively reduced the increased total cell, macrophage, eosinophil, and neutrophil numbers in BAL fluid from the TDI-sensitized/challenged mice (†P<0.05).

• 

  Fig. 3 Microscopic findings of the intrapulmonary bronchi. (A) Histological examination of lung tissue. Lung sections were obtained from sham and toluene diisocyanate (TDI)-sensitized/challenged mice with or without bone marrow-derived mesenchymal stem cell transfer. Tissue sections were stained with periodic acid-Schiff (PAS) to determine the presence of goblet cells. Scale bar=200 µm. (B) Semiquantitative analysis of the severity of peribronchial inflammation. All of the randomly selected histological images were scored using the inflammatory index, and the mean is presented. (C) PAS-positive cells in epithelium and total epithelial cells were counted, and the percentage of PAS-positive cells was calculated. *P<0.0001, compared with the sham group; †P<0.0001, compared with the TDI group.

• 

  Fig. 4 Immunohistochemical staining of proteins expressed in the intrapulmonary bronchi. (A) Smooth muscle actin (SMA) protein expression increased markedly in the peribronchial muscle layer of the bronchi of toluene diisocyanate (TDI)-sensitized/challenged mice. Bone marrow-derived mesenchymal stem cell (BMDMSC) transfer reduced the expression of SMA protein. (B) Proliferating cell nuclear antigen (PCNA) protein expression increased markedly in epithelial cells in the bronchi of TDI-sensitization/challenged mice. Infusion of BMDMSCs reduced the expression of PCNA protein. A representative image from six experiments in each group is shown. Scale bar=200 µm.

• 

  Fig. 5 Histological analysis and collagen level in the lung. (A) Masson trichrome stain in lung tissue of sham, toluene diisocyanate (TDI)-sensitized/challenged, and Bone marrow-derived mesenchymal stem cell (BMDMSC) transferred mice. Scale bar=200 µm. (B) Effects of BMDMSC transfer on collagen deposition. The total soluble collagen content in the right lung was measured using a Sircol collagen kit. Differences between the BMDMSC-infusion and TDI groups were significant. *P<0.05, compared with the sham group; †P<0.05, compared with the TDI sensitized/challenged group.

• 

  Fig. 6 Effect of Bone marrow-derived mesenchymal stem cell (BMDMSC) transfer on airway hyperresponsiveness to methacholine in a toluene diisocyanate (TDI)-induced murine asthma model. TDI-sensitized/challenged mice had high enhanced pause (Penh) values at 20 and 50 mg/mL methacholine (*P<0.005 vs. sham). BMDMSC transfer significantly reduced the Penh values at 50 mg/mL methacholine in TDI-sensitized/challenged mice (†P<0.01 vs. TDI).

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