In normal condition, the homeostasis of the extracellular matrix of the lung is maintained and tightly controlled. In idiopathic pulmonary fibrosis, however, this homeostasis is severely disrupted and accumulation of ECM continues, resulting in progressive fibrosis. The pathogenesis of IPF is not yet completely understood. Inflammation after epithelial injury is believed to be the integral part of the pathophysiology of pulmonary fibrosis,16
especially in early phase, although the links between fibrosis and inflammation have been debated.17-19
Dysregulated wound repair is also believed to be important pathogenetic mechanisms.18,19
Key players, such as TGF-beta 1, TNF, and IL-1 beta have been reported to be crucial in sustaining inflammation and promoting progressive fibrosis.10,20,21
MMPs and their inhibitors, TIMPs, have been known to play important roles in pulmonary fibrosis through their function in matrix protein degradation and its control.3-5
Among many types of MMPs, MMP-2 and MMP-9 have been known to be important and to show increased activity in many pulmonary diseases including pulmonary fibrosis.4,5,22,23
Herein, we demonstrated that the activity of MMP-2 and MMP-9 in the bronchoalveolar lavage fluid and lung parenchyma increased rapidly in the early phase, reaching the peak levels on the 4th day, and then consistently decreased thereafter. This is in accordance with the previous studies,24,25
which showed that the activity of the MMP-2 and MMP-9 increases in the early stage and decreases thereafter. However, the timing of the peak level in previous studies is somewhat different, and this may be due to the difference of experimental animals used or to the different time schedules when animals were sacrificed. The changes of the MMPs in the BAL fluid correlated to the total/differential cell counts in the BAL fluid. The level of the 2 MMPs in the lung parenchyma was not significantly different from each other, however, the change of the gelatinolytic activity of the MMP-9 in the BAL fluid was more prominent than that of MMP-2. This is maybe due to that in early phase, the MMP-9 was expressed strongly in neutrophils which more frequently existed in the intraalveolar spaces rather than in the lung parenchyma per se.
There was a temporal change in the major sources of the MMPs. In early phase until the 4th day, both two MMPs were mainly expressed in the inflammatory cells. However, more specifically, MMP-2 was noted more frequently in alveolar macrophages, whereas MMP-9 in neutrophils. Since previous studies showed that these MMPs were co-localized with the type IV collagen in the basement membrane,3
the MMP-2 and MMP-9 in this early phase secreted by macrophages and neutrophils may play an important role in degrading the basement membrane, thereby facilitating the inflammatory cell migration. In midphase (the 5th to 7th day), parenchymal cells, such as bronchiolar epithelial cells and type II pneumocytes, in addition to the inflammatory cells, also comprise important cellular sources expressing these MMPs. Especially, those with the features of cellular injury, such as cellular swelling, vacuolization, and nuclear atypia, expressed MMP-2 and MMP-9 more prominently. The bronchiolization foci15
observed frequently from the 4th to 7th day also showed prominent MMP-2 and MMP-9 reactivity. In later phase (from 14th to 28th days), the main cellular sources of the MMPs were again the type II pneumocytes, however, mainly the ones surrounding the fibrotic foci. After the 14th day when fibrosis progressed, the overall expression of the MMP-2 and MMP-9 was very low. The fibroblasts, immature or mature collagen fibers at the center of the fibrotic foci, didn't show much MMP expression, while a few alveolar macrophages and type 2 pneumocytes retained the expression of the MMPs at the periphery of the fibrotic foci, presumably the advancing front of the fibrosis.
Bronchiolization, frequently observed from 4th to 7th day, is regarded as a reparative reaction to cellular injury to the alveolar epithelial cells.15,26
Also, cells surrounding the fibrotic foci are known to be derived from regenerating pneumocytes.27
The fact that the MMP-2 and MMP-9 were expressed in the cells with features of cellular injury/repair may suggest the roles of MMP-2 and MMP-9 in cellular regeneration. The roles of MMPs in relation with cellular regeneration in pulmonary fibrosis has also been suggested by several previous studies,28-30
implicating their role in facilitating the migration of epithelial cells to the injured site.
Proinflammatory cytokines such as TNF-alpha and IL-6, which were increased in the early phase of pulmonary fibrosis and are essential in progression of early pulmonary inflammation to pulmonary fibrosis31,32
have been associated with increased MMP levels33
or induction of MMP production from various cell types including alveolar epithelial cells, alveolar fibroblasts, and alveolar macrophages.34-36
They were also shown to induce other cytokines such as TGF-beta 1.32
These proinflammatory cytokines may play roles in pulmonary fibrosis by recruiting inflammatory cells and inducing MMPs production from recruited cells, thus facilitating the migration of those inflammatory cells and further promoting fibrosis through a synergistic action with profibrotic cytokine TGF-beta 1. TGF-beta 1, the most potent fibrogenic cytokine, has been reported to increase in pulmonary fibrosis and to be expressed in alveolar epithelial cells in higher levels even in later phase of pulmonary fibrosis.37,38
It was also reported that TGF-beta 1 induces transition of alveolar epithelial cells to mesenchymal/fibroblasts-like cells and MMP-2 expression in those cells,39
possibly suggesting that MMP-2 production in alveolar epithelial cells by TGF-beta 1 may promote pulmonary fibrosis. However, the mechanisms of cytokines and MMPs need to be further elucidated, since many other cytokines such as IL-1040
are also involved, and there are conflicting results, too.42
In our study, the levels of the MMP-2 and MMP-9 in the lung parenchyma corresponded well to those of BAL fluid. The antibodies used for immunohistochemistry could equally stain both latent and active forms without any preference, therefore, active forms of both MMPs were to be evaluated by the zymography. It was expected that the active forms of MMP-2 and MMP-9 might have been increased in the early phase of pulmonary fibrosis when the total MMP-2 and MMP-9 activity reached the maximum. However, the zymography of the BAL revealed no active forms of the MMPs from the 4th to 7th day when the MMP levels were the highest. Only a small amount of active MMP-2 was detected on the 14th day, and the active form of MMP-9 was not noted throughout the entire time course. It is quite possible that the active forms of MMPs were more likely present in the lung parenchyma where active protein degradation occurs and that the level of the active MMPs in the BAL fluid might be too low to be detected, or might have been degraded too quickly after their normal function. There are some studies reporting that the levels of active MMP did not necessarily correlate to total MMP levels.10,11,43
The fact that active form of only MMP-2, but not MMP-9, was detected in later phase of fibrosis is in accordance with an another study which showed similar result, suggesting different roles of MMP-2 and MMP-9 along in the time course.25
Presumably, MMP-9 is more important in early phase when active degradation of the basement membrane and inflammatory cell migration occur, whereas MMP-2 may play roles in later phase in regard to the formation of the fibrotic foci and cellular repair.
In this study, we didn't investigate the activity of TIMPs. Considering the fact that MMP activity is regulated by TIMPs, the role of MMPs in pulmonary fibrosis has to be understood in association with that of TIMPs. The activities of TIMP-1 or TIMP-23,44-46
were reported to increase in pulmonary fibrosis, especially in the acute phase of pulmonary fibrosis or acute lung injury model. It has also been suggested that TIMP is related to the process of bronchial epithelial cell regeneration.30
More detailed information on the functions of MMP in relation with TIMPs in pulmonary fibrosis needs to be clarified.