Pleura, peritoneum and pericardium are squamous epithelia of mesothelial origin, which line lungs, chest cavity, abdomen, and heart, respectively. Pleura can be divided in the pleura visceralis or pleura pulmonalis covering lungs, and pleura parietalis covering the ribs. Both are delimitated by a single layer of epithelial cells which attach to a lamina propria, and have different functions regarding transport and barrier properties. Between the two layers, the pleura cavity is located (Figure 1). The pleura provides a fluid layer for lung movement, and therefore is necessary for breathing This small volume of pleural liquid is a result of filtration and absorptive processes, and its volume and composition is tightly regulated (Zocchi, 2002). An imbalance of this regulation can result in an accumulation of fluid between the parietal and the visceral pleura, a condition defined as pleural effusion (Light, 1997).

The difference between hydrostatic and colloid-osmotic pressure of pleural liquid and capillary blood results in filtration of pleural liquid in the pleural cavity. In caudal parietal pleura, the elimination of liquid from a pleural cavity occurs through lymphatic vessels. In this context, further mechanisms related to mesothelial cell activity have been discussed to be more relevant, including a solute-coupled absorption of liquid from the pleural space (Zocchi, 2002). However, vectorial transport across epithelia depends on a concerted action of transcellular transport mechanisms in combination with paracellular channel and barrier functions, and several transcellular transport mechanisms have been identified to participate in the formation of pleural liquid. For analysis, electrophysiological measurements have been performed in different species including man (Hatzoglou et al., 2001; Sarkos et al., 2002; Kouritas et al., 2008; Markov et al., 2011). Moreover, the molecular correlate of paracellular channel and barrier function has been discovered in recent years, namely the tight junction (TJ) which shows a tissue specific expression of tight junction proteins determining the functional properties of the tissues (for review, see e.g., Amasheh et al., 2011; Rosenthal et al., 2012). Neighboring epithelial cells are linked together by these structures, which show a distinct strand pattern in freeze fracture electron micrographs (Staehelin, 1973). Typically tight junctions are located between the apical and the basolateral membrane of epithelial cells, providing not only a gate function describing the role for paracellular permeability, but also a fence function which is a prerequisite for polarity of the cells, as it delimits diffusion of basolateral and apically located integral membrane molecules to the opposite membrane compartment, respectively (Tsukita et al., 2001). Taken together, both transcellular and paracellular determinants of transport and barrier function can be regarded to contribute to the generation of different ionic composition of pleural fluid compared to plasma (Table 1).

Furthermore, inflammation was identified as one major mechanism perturbing barrier integrity, as shown previously in inflammatory bowel diseases (Amasheh et al., 2009a). In this context, also an effect of pleura inflammation on tight junction proteins was observed (Markov et al., 2011).

The ignition of molecular tight junction protein research occurred in 1993, when the group of Shoichiro Tsukita described the first discovery of a molecular correlate of barrier function, namely occludin (Furuse et al., 1993). Before this time point, it was even not clear if the tight junction, which had been visualized in electron microscopic images for decades before, was of protein or lipoid origin. This finding soon catalyzed the establishment of a new research field, resulting in a broad variety of novel basic physiological as well as clinically related findings. Soon after Tsukita's pioneer work in the field of molecular tight junction analysis, a second landmark paper was published, this time presenting the first members of the family of claudins, namely claudin-1 and -2. Paralleled by the finding that occludin might not be the most crucial factor for barrier integrity, as the complete knock out did not show a perturbation of barrier properties (Furuse et al., 1998; Schulzke et al., 2005), a series of further publications highlighted the functional contribution of single claudins to major epithelial functions (Amasheh et al., 2011). These studies led to a classification of single members of the claudin family in barrier building (sealing), pore-forming (paracellular channel function) and intermediate claudins, which still is valid and can be regarded as a general outcome of tight junction research. Finally, a third type of tight junction protein was described, which led to a novel classification of non-claudin tight junction proteins as members of the TAMP family, namely tricellulin, MarvelD3, and occludin (Ikenouchi et al., 2005; Steed et al., 2009).

Among the variety of tetraspan TJ proteins that have been reported to be localized within TJ strands of different organs, occludin (Furuse et al., 1993) and claudins (Furuse et al., 1993) have been identified to primarily provide a barrier against the paracellular passage of ions. Although occludin was the first tetraspan membrane protein that was detected within TJ strands (Furuse et al., 1993), its contribution to barrier properties is still discussed, though (Schulzke et al., 2005). At least, ubiquitous detection in immunofluorescent stainings, established occludin as a general marker of TJ localization, which has been evaluated in many studies (Amasheh et al., 2002, 2005, 2009a,b; Dittmann et al., 2014; Markov et al., 2014). Moreover, occludin can also be visualized in pleura (Figure 3).

In contrast, members of the claudin family have been attributed to the organ-specific properties of epithelia, as e. g. shown in intestine, mammary gland, kidney and lungs (Kaarteenaho et al., 2010; Kirk et al., 2010; Markov et al., 2010, 2012). The single contribution of claudins to barrier properties varies, though. As e.g., claudin-1, -3, and -5, decrease tight junctional ion permeability, other claudins have been demonstrated to specifically mediate paracellular permeability, as it has been shown in detail for claudin-2 which forms a paracellular channel (Amasheh et al., 2002, 2005; Furuse et al., 2002; Milatz et al., 2010).

Information on the molecular correlate of barrier function in the pleura reaches back to the pre-molecular era of tight junction research, when desmosomes and TJs have been discovered as intercellular contacts by electron microscopy (Wang, 1974), and a final breakthrough was achieved by detection of claudins and the correlation with functional barrier properties (Markov et al., 2011).

Recently, for the first time a combined analysis of pleura mesothelial barrier function and expression of TJ proteins was performed. In this study, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7 (Markov et al., 2011; Figure 4). Moreover, in inflamed pleura, claudin-2 was induced, indicating a typical pathophysiological mechanism (see paragraph 4).

According to the functional contribution to barrier properties, claudins can be divided in three groups, namely (i) sealing tight junction proteins, (ii) claudins mediating paracellular permeability and (iii) claudins with ambiguous function. Among the claudins detected in pleura mesothelium, claudin-1, -3, and -5 belong to the first group, whereas claudin-2 belongs to the second, and claudin-7 to the third group.

The expression of claudin-1, -3, -5, and -7 in human pleura indicates an important role of specific barrier properties of the mesothelial cell layers (Markov et al., 2011). These findings are in accordance with previous studies, which have underlined specific contributions of single members of the tight junction protein family of claudins to barrier function of other leaky epithelia such as small intestine and proximal tubule (Markov et al., 2010; Amasheh et al., 2011).

Expression of claudins in pleura mesothelium has been shown to be altered in several pathophysiological conditions. These changes can be found in inflammatory events and in cancer. In previous studies, the paracellular channel claudin-2 has been reported to be induced by tumor necrosis factor α (TNFα), which explains e.g., the pathomechanism of inflammatory bowel diseases (Amasheh et al., 2009a, 2010). The two main forms of inflammatory bowel diseases, namely Crohn's disease and ulcerative colitis, as well as collagenous colitis and pouchitis, all typically show a perturbation of TJ protein expression and localization. This mechanism is regarded to be an important factor for the sustained and aggravated course of the diseases mediated by TNFα (Bürgel et al., 2002; Heller et al., 2005; Zeissig et al., 2007; Amasheh et al., 2009a).

In inflamed pleura, a general reduction of tightening TJ proteins and an increase of permeability mediating TJ proteins was reported, recently (Markov et al., 2011). Claudin-2 was reported to be elevated in inflamed pleura, which has also been shown for Crohn's disease, ulcerative colitis, and pouchitis (Heller et al., 2005; Zeissig et al., 2007; Amasheh et al., 2009a). Moreover, in inflamed pleura, a decrease of occludin, claudin-1, -3, -5, and -7 was detected, which also shows parallels with mechanisms reported in inflammatory bowel diseases (Heller et al., 2005; Zeissig et al., 2007; Amasheh et al., 2009a).

In accordance, pleural exudates showed a marked increase of TNFα levels in a mouse lung inflammation model (Mazzon and Cuzzocrea, 2007). Moreover, TNFα is also elevated in other lung pathologies, e.g., tuberculosis and cancer (Qian et al., 2012).

TNFα at higher levels was reported to induce a down-regulation of the tight junction scaffolding protein zonula occludens protein 1 (ZO-1), and an opening of the paracellular barrier (Ma et al., 2004). As a prerequisite of the pathomechanism of TNFα altering tight junction protein expression in lungs, the TNFα receptor TNFR1 was identified (Mazzon and Cuzzocrea, 2007). If, apart from claudin-2, a number of tightening tight junction proteins are reduced due to a general perturbation of ZO-1, this results in a decrease of barrier properties despite reduction of the paracellular pore claudin-2 (Schneeberger and Lynch, 1992; Fink and Delude, 2005; Jacob et al., 2005), though. Taken together, whereas the increase of claudin-2 in pleural inflammation indicates a separate regulatory pathway which has been reported for inflammatory bowel diseases, the general reduction of tightening proteins in inflamed pleura also points at a mechanism reported in a mouse lung inflammation model, which is not even based on an increase of claudin-2, but a universal decrease of tight junction proteins which may less specifically perturb the epithelial barrier. Both regulatory pathways however appear to be primarily targeted by TNFα.

These alterations in claudin expression are in contrast to findings reported in analyses of pleura cancer and mesothelioma, which show an alteration of claudin-4. In this context, Claudin 4 was identified to be an important marker for carcinoma vs. mesothelioma diagnosis in pleural and peritoneal biopsies and effusions, as it is detected in metastatic tumor cells but not in benign forms of mesotheliomas (Facchetti et al., 2007; Lonardi et al., 2011).

Transport and barrier function of pleura mesothelium indicate typical epithelial characteristics. Expression of the major sealing tight junction proteins-1, -3, and -5 indicate a physiological role of the mesothelial barrier function for pleural liquid formation. Moreover, the effects observed in inflamed pleura, namely an induction of the paracellular pore claudin-2 and the decrease of tightening tight junction proteins are in accordance with regulatory mechanisms observed in inflamed epithelia, as shown e.g., for inflammatory bowel diseases. These findings may contribute to future therapeutic and preventive approaches regarding mesothelia.

Alexander G. Markov and Salah Amasheh conceived the review, conducted literature survey and wrote the review.