Yonsei Med J.  2009 Feb;50(1):68-77. 10.3349/ymj.2009.50.1.68.

Early and Late Changes of MMP-2 and MMP-9 in Bleomycin-Induced Pulmonary Fibrosis

• Kim JY
• Choeng HC
• Ahn C
• Cho SH

• KMID: 1782970
• DOI: http://doi.org/10.3349/ymj.2009.50.1.68

Abstract

PURPOSE: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of pulmonary fibrosis. To understand the role of MMP-2 and MMP-9 in pulmonary fibrosis, we evaluated the sequential dynamic change and different cellular sources of the 2 MMPs along the time course and their differential expression in the bronchoalveolar lavage (BAL) fluid and in the lung parenchyma of the bleomycin-induced pulmonary fibrosis models in rats.
MATERIALS AND METHODS
The level of MMPs in BAL fluid of 54 bleomycin-treated rats was assessed by zymography from 1 to 28 days after intratracheal bleomycin instillation. The level of MMPs in lung parenchyma was evaluated by immunohistochemistry.
RESULTS
MMP-2 and MMP-9 were markedly increased in both the BAL fluid and in the lung parenchyma of the bleomycin-treated rats, especially in the early phase with the peak on the 4th day. The levels of both MMPs in the BAL fluid correlated generally well to those in lung parenchyma, although the level of MMP-9 in BAL fluid was higher than MMP-2. In the lung parenchyma, the 2 MMPs, in early stage, were predominantly expressed in the inflammatory cells. In late stage, type II pneumocytes and alveolar epithelial cells at the periphery of the fibrotic foci retained MMP expression, which was more prominent in the cells showing features of cellular injury and/or repair.
CONCLUSION
In bleomycin-induced pulmonary fibrosis, MMP-2 and MMP-9 may play important roles, especially in the early phase. In the late stage, the MMP-2 and MMP-9 may play a role in the process of repair.

Keyword

Pulmonary fibrosis; bleomycin; matrix metalloproteinase; matrix-metalloproteinase-2; matrix metalloproteinase-9

MeSH Terms

Animals
Antibiotics, Antineoplastic/toxicity
Bleomycin/toxicity
Bronchioles/*enzymology/pathology
Bronchoalveolar Lavage Fluid/cytology/immunology
Disease Models, Animal
Enzyme Activation
Gelatin
Immunohistochemistry
Male
Matrix Metalloproteinase 2/*metabolism
Matrix Metalloproteinase 9/*metabolism
Neutrophils/pathology
Pulmonary Fibrosis/chemically induced/*metabolism/*pathology
Rats
Rats, Sprague-Dawley

Figure

• 

  Fig. 1 Total cell count in the BAL fluid of bleomycin-treated group was significantly higher than in the control group (p < 0.05). Total cell count increased rapidly from the first day after bleomycin instillation, reaching the peak level on the 4th day. Then, it continuously decreased till the 28th day. Exp: bleomycin-treated experimental group: n = 54, Control: control group: n = 27, Average cell count from each group was used.

• 

  Fig. 2 The percentage of neutrophil in the BAL fluid in the bleomycin-treated group was significantly higher than in the control group (p < 0.05). The percentage of neutrophil was markedly increased from the first day after bleomycin instillation, reaching the peak level on the 4th day, and then decreased rapidly thereafter. Exp: bleomycin-treated experimental group: n = 54, Control: control group: n = 27. Average value from each group was used.

• 

  Fig. 3 Histological findings of bleomycin-treated group. (A) 3rd day. Swollen cuboidal epithelial cells of the terminal bronchiole and type II pneumocytes (arrows) and inflammatory cell infiltration in the peribronchiolar and perivascular area (arrowhead). (B) 5th day. A number of small tubular structures of bronchiolar epithelial cells, "bronchiolization" foci (arrow) are seen around the terminal bronchiole. (C) 14th day. Some foci of small intraalveolar fibrosis are seen (arrows). (D) 28th day. Dense mature collagen stained deep blue in Masson-trichrome stain is widening the alveolar septae and destroying the normal alveolar pattern (A, B, and C: H&E; D: Masson's trichrome, A and C: × 100; B, and D: × 40).

• 

  Fig. 4 The immunohistochemical expression of the MMP-2 and MMP-9 in bleomycin-treated group (A, C, and E: MMP-2; B, D, and F: MMP-9). (A and B) On the 4th day, the bronchiolar epithelial cells that are swollen and activated show prominent MMP positivity (arrows). MMPs are also strongly expressed in the inflammatory cells in the surrounding parenchyma. (C and D) On the 7th day, the MMP-2 and MMP-9 are expressed in the foci of bronchiolization (arrows). Some inflammatory cells, such as neutrophils and macrophages, still show expression (arrowheads). (E and F) On 14th day, the expression of MMP-2 (E) and MMP-9 (F) is decreased in most part, but still retained at the periphery of the fibrotic foci (arrows).

• 

  Fig. 5 The change in the levels of MMP-2 and MMP-9 in the lung parenchyma of bleomycin-treated group, shown by immunohistochemistry. The immunohistochemical score was evaluated semiquantitatively as described in methods. Both MMP-2 and MMP-9 rapidly increased from the first day to the 4th day, and decreased thereafter till the 28th day.

• 

  Fig. 6 Gelatin zymography of MMP-2 (72 kDa) and MMP-9 (92 kDa) in the BAL fluid of the bleomycin-treated group. The representative gelatin zymography sample shows that the band of MMP-9 is strong, particularly on the 4th days. It gets weaker from the 14th to 21st days. On the 14th day, weak band of active MMP-2 (62 kDa) is noted. 4, 14, and 21: experimental group; C: control group.

• 

  Fig. 7 The change of the gelatinolytic activity of MMP-2 and MMP-9 in the BAL fluid of the bleomycin-treated group. The activity of MMP-2 (A) and MMP-9 (B) is rapidly increased and reaches the peak level on the 4th day and then decreases thereafter. The change of the MMP-9 is more pronounced than that of the MMP-2. The MMP-2 declined less rapidly.

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