In the present study MMP2 and MMP9 decreased the transmesothelial resistance of parietal and visceral sheep pleura in in vitro experiments. This decrease supports that MMPs augment pleural mesothelial permeability. The paracellular pathway, through which mesothelial permeability may increase, was studied. After incubation of primary mesothelial cells with MMP2 or MMP9, mesothelial monolayer integrity was not disrupted and the expression of TJ proteins occludin and claudin-1 in the cytoplasmic membrane remained intact.
MMP2 and MMP9 were selected for our study because they have been found elevated in exudative pleural effusions of different origin [3
]. MMP2 is expressed and secreted constitutively in the pleural cavity by mesothelial cells [12
]. In exudates high levels of MMP2 may be due to increased expression as a result of stimulation of mesothelial cells by cytokines or other cells, such as mononuclear cells, might collaborate in MMP2 release [10
]. MMPs can degrade almost all components of the extracellular matrix (ECM) and to date it is widely known that MMPs can cleave not only ECM components, such as collagen and elastin, but also non-ECM protein substrates, such as cell surface molecules, ECM-bound growth factors and cytokines released on the ECM [13
In the present study the decrease in transmesothelial electrical resistance of sheep pleura that occurs after incubation with MMP2 or MMP9 suggests for an increase in the permeability of the pleura. As expected for an enzyme, the decrease in resistance of the pleura was time-dependent with the maximum decline in resistance occurring on the 40 min. Regarding the dose-dependent effect, a remarkable difference between MMP2 and MMP9 occurred: for MMP2 the greatest increase in permeability occurred at the highest concentration studied, which is 20 ng/ml. On the contrary, for MMP9 the greatest increase in permeability occurred at the lowest concentration studied, which is 0.1 ng/ml. The reason for this discrepancy is not elucidated but we should take into account differences between enzyme kinetics and substrate specificity.
TIMP2 is a tissue inhibitor of metalloproteinases and inhibits MMPs activity by binding noncovalently to their active site [15
]. TIMP2 is found in exudates whereas in transudates its levels are usually no detectable [3
]. The application of TIMP2 on the pleura inhibited the decrease in RTM
which was induced by MMPs on both the parietal and visceral pleura. This finding enhances our previous finding and suggests that the decline in RTM
is an effect caused by MMPs and not a non-pecific result. TIMP2 was selected at the present study because it is 2-9 times more effective than TIMP1 for the inhibition of MMP2 and MMP9 activity [16
]. Moreover, TIMP2 tended to increase pleural permeability particularly at visceral pleura and at its basolateral side, when it was applied alone at the pleura. This finding can be interpreted considering the growth-promoting-activity that has been attributed to TIMP2 [17
]. A wide range of human, bovine and mouse cells proliferate when incubated with TIMP2. Indeed, receptors of TIMP2 have been identified at the surface of the above cells [18
]. One possible explanation is that TIMP2 acts on mesothelial cells via receptor-binding and influences mesothelial permeability, as is the case for numerous growth factors.
We next looked for a possible mechanism explaining the impact of MMPs on the transmesothelial resistance and asked whether TJ proteins, occludin and claudin-1, may be hydrolyzed by MMPs. TJs are a specific type of cell-cell contact which are located in the most apical region of the lateral plasma membrane. The paracellular passage of small molecules, such as water and solutes, is highly regulated by the TJ proteins, including occludin and claudin-1 [9
]. Indirect immunofluorescence experiments for occludin and claudin-1 showed a continuous staining at cell periphery which was not weakened and remained at the cell borders after incubation with MMPs. Similarly, western blot revealed that these proteins are expressed at pleural mesothelium under normal conditions and are not proteolytically disrupted by MMPs. More specifically for occludin, western blot analysis revealed three distinct bands: at 60 KDa, 85 KDa and a broad band at about > 60 KDa. These bands may be due to occurrence of splice variants or post-transational modification, i.e phosphorylation. Previous studies have shown that occludin is widely phosphorylated on serine and threonine resides and modulation of occludin phosphorylation regulates cellular localization and paracellular permeability [19
]. Some degree of phosphorylation may also be the case in the present study. As far as the 80 KDa band is concerned, we cannot rule out the possibility that this band corresponds to a protein complex between occludin and claudin (MWs 60 and 28 KDa respectively). Occludin and claudin do not interact directly to each other, but they crosslink by integral TJ proteins, such as ZO-1, ZO-2 and ZO-3 [9
]. The intensity of the above band increased after MMPs incubation, implying that MMPs interfere with occludin and claudin interactions. Because the 80 KDa band appeared enhanced to all three western blot experiments that were performed, it is less likely to represent an artifact. The mesothelial cells used for immunofluorescence and western blotting derived from upper-, middle- and lower-heighted visceral pleura and no difference between them occurred.
Our data is contradictory to previous results which have revealed MMPs as major contributors to the control of paracellular permeability by proteolytic degradation of TJ proteins. More specifically, MMPs have been correlated with an increase in capillary permeability that follows ischemia-reperfusion injury in brain [8
], in myocardium [4
], in lung [21
] and in kidney [22
]. These studies attributed to MMPs an elementary role in the inflammatory process and the breakdown of the paracellular capillary permeability during inflammation. In some studies a selective cleavage of TJ proteins occurred [7
] but the results of our study clearly showed that TJ integrity is not disrupted at mesothelial cells by MMPs. It is possible that other substrates, different from occludin and claudin-1, are a molecular target for MMPs at pleural mesothelium. For example, MMPs have been found to disrupt or reorganize the basement membrane of endothelial cells and thus result to increased permeability [24
]. Moreover, adherent junctions are also degraded by MMPs and their hydrolysis leads to TJ disassembly and to increased permeability [22
The limitations of the present study are the followings: Firstly, the Ussing chamber technique investigates permeability alterations provoked on mesothelial membrane, which consists a confluent membrane between the apical and basolateral compartment of a chamber. However, under in vivo conditions it is possible that MMPs act not only on mesothelial cells but also on vascular capillaries lying beneath the basement membrane. Secondly, the precise role of the mesothelial layer at pleural fluid turnover is not fully established. Although we used to believe that mesothelial cells are leaky and display no resistance at pleural fluid passage, more recent investigations indicate that the permeability to solutes of mesothelium is of the same order of magnitude as that of the capillary endothelium [27
]. This means that pleural fluid is a filtrate of pleural capillaries and mesothelium too. Moreover, damage of pleural mesothelial monolayer by lipopolysaccharide (LPS), thrombin and bacteria increase pleural permeability to proteins and demonstrate to play a central role in the formation of effusions [29
]. Finally, sheep pleura resembles to human pleura as far as morphology and function is concerned. On both pleurae the blood supply comes from the systemic circulation and microscopically two different types of mesothelial cells are found: the cyboidal cells with less developed TJs and flattened cells with more TJs [31
]. However, further studies should be performed at human pleura in order to confirm the results of the present study.
Our findings imply an important role for MMPs in the pathogenesis of pleural effusions. Under normal conditions, MMPs and especially MMP2 are found only in small amounts in the pleural fluid. The balance of MMPs in pleural fluid may serve the degradation and turnover of ECM that underlies mesothelial cells and which normally occurs in low rates. However in pleural exudates MMP2 and MMP9 levels increase, as shown from previous studies, and a role for MMP2 and MMP9 in pleural fluid formation is proposed by our study. Tight junctions do not apparently loosen in mesothelial cells after the addition of MMPs. It is possible that other mechanisms exist through which MMPs increase mesothelial permeability. The revelation of the mechanims by which MMPs compromise the mesothelial barrier will provide a better understanding of the pathogenesis of pleural fluid formation.