Steven Mutsaers, Asthma and Allergy Research Institute, Ground Floor E Block, Sir Charles Gairdner Hospital, Verdun Street, Nedlands, WA 6009, Australia. Email: email@example.com
The mesothelium is composed of an extensive monolayer of specialized cells (mesothelial cells) that line the body's serous cavities and internal organs. Traditionally, this layer was thought to be a simple tissue with the sole function of providing a slippery, non-adhesive and protective surface to facilitate intracoelomic movement. However, with the gradual accumulation of information about serosal tissues over the years, the mesothelium is now recognized as a dynamic cellular membrane with many important functions. These include transport and movement of fluid and particulate matter across the serosal cavities, leucocyte migration in response to inflammatory mediators, synthesis of pro-inflammatory cytokines, growth factors and extracellular matrix proteins to aid in serosal repair, release of factors to promote both the deposition and clearance of fibrin, and antigen presentation. Furthermore, the secretion of molecules, such as glycosaminoglycans and lubricants, not only protects tissues from abrasion, but also from infection and possibly tumour dissemination. Mesothelium is also unlike other epithelial-like surfaces because healing appears diffusely across the denuded surface, whereas in true epithelia, healing occurs solely at the wound edges as sheets of cells. Although controversial, recent studies have begun to shed light on the mechanisms involved in mesothelial regeneration. In the present review, the current understanding of the structure and function of the mesothelium and the biology of mesothelial cells is discussed, together with recent insights into the mechanisms regulating its repair.
The mesothelium was first described by Bichat in 1827.1 Using histological techniques, he observed that the serous cavities were lined by a single layer of flattened cells similar to those of the lymphatics. In 1880, Minot,2 in a detailed study of the embryological origins of the mesothelium, initially referred to this layer as the ‘epithelial lining of mammalian mesodermic cavities’ and subsequently proposed the term ‘mesothelium’.3 Embryologically, the mesothelium develops from the mesodermal tissue between 8 and 18 days of gestation, depending on the species.4–6 In humans, this occurs around day 14, with cells gradually differentiating from round or cuboidal cells to elongated flattened cells that line the coelomic cavities.4,6
Despite the early discovery and description of the mesothelium, it has only been in recent years that its importance, both in health and disease, has been realized. It is not just a limiting protective layer that stops the organs from sticking together, but a dynamic structure regulating serosal responses to injury, infection and disease. The present review focuses on the recent advances in our understanding of the structure and function of mesothelial cells and the mesothelium and the mechanisms regulating its repair.
STRUCTURE OF MESOTHELIAL CELLS AND THE MESOTHELIUM
The mesothelium extends as a monolayer over the entire surface of the three serosal cavities (pleural, pericardial and peritoneal) and, in the male, it also lines the sac that surrounds the testes. Mesothelium that covers the internal organs is referred to as the visceral mesothelium, whereas the parietal mesothelium lines the body wall. Morphological studies of the mesothelium of several mammalian species, including rat, mouse, dog, hamster, rabbit, horse and humans,1,7–23 have demonstrated, with minor exceptions, that mammalian mesothelium is essentially similar, irrespective of species or anatomical site. In addition, histochemical studies have supported these structural findings.24,25
The mesothelium forms a monolayer of predominantly elongated, flattened, squamous-like mesothelial cells, approximately 25 μm in diameter, with the cytoplasm raised over a central round or oval nucleus.26 The cells rest on a thin basement membrane supported by connective tissue stroma, which varies in quantity and quality between the three serous cavities, between visceral and parietal serosa and between species.20,22,27 Surface specialization, in the form of characteristic microvilli and occasional cilia, is also seen (Fig. 1).
Although the mesothelium is composed predominantly of squamous-like cells, cuboidal mesothelial cells can be found in various areas, including the septal folds of the mediastinal pleura, the parenchymal organs (liver, spleen), the ‘milky spots’ of the omentum and the peritoneal side of the diaphragm overlying the lymphatic lacunae.13,18,19,28 Cells that are morphologically similar to these cuboidal mesothelial cells can also be identified after injury or stimulation of the serosal surfaces29–31 (Fig. 2).
Ultrastructural analysis of squamous-like and cuboidal mesothelial cells show differences between their nuclei and cell organelles. Squamous-like mesothelial cells have a round or ovoid nucleus, whereas the nuclei of cuboidal cells are larger and contain a prominent nucleolus.8,17,18,32 Organelles of the squamous-like cells are located centrally, close to the nucleus. These cells contain microtubules and microfilaments, glycogen, few mitochondria, a poorly developed Golgi apparatus and little rough endoplastic reticulum (RER).9,17,18,32 The microfilaments are located parallel to the cell surface and are situated deep in the cytoplasm.9 Cuboidal mesothelial cells have abundant mitochondria and RER, a well-developed Golgi apparatus, microtubules and a comparatively greater number of microfilaments,9,18,32 suggesting a more metabolically active state.
The luminal surface of the mesothelial cell has a well-developed microvillous border, with microvilli varying in length, shape and density (Fig. 3). Variations in the concentration of microvilli have been reported on mesothelial cells of different organs, between adjacent cells and on the surface of individual cells.11,18,19,28 The entire microvilli population is also labile, with the number of microvilli expressed on each cell changing under different physiological conditions.15 Differences in the number of microvilli on regenerating mesothelial cells have also been reported and it has been suggested that their concentration reflects functional adaptation.15,18,33,34 These changes are also associated with an alteration in surface charge.34 This may reflect changes in the composition of the negatively charged glycocalyx that covers the mesothelial cell surface35 or may be due to trapping by microvilli of proteins and other molecules from the serosal fluid, protecting the mesothelium from surface friction and ensuring its integrity.9,11,14,18,34,36
Mesothelial cells also have a well-developed system of vesicles and vacuoles; most are micropinocytic, but multivesicular bodies and large vacuoles can be found.37 Like the microvilli, the concentration of these structures varies between cells and sites. Generally, the visceral mesothelium has more vesicles than the parietal mesothelium and the vesicles are more numerous along the luminal cell surface, often in contact with the plasmalemma.8,9,22,32,36,38 These vesicles are involved in transport of fluids and particulates across the mesothelium, because experimental studies have shown tracer particles up to 100 nm can traverse the cell by pinocytic transport.8,36,39
The boundaries between mesothelial cells are tortuous, with adjacent cells often overlapping. They have well-developed cell–cell junctional complexes, including tight junctions, adherens junctions, gap junctions and desmosomes.10,37,40 Tight junctions are crucial for the development of cell surface polarity and the establishment and maintenance of a semipermeable diffusion barrier. Adherens junctions are thought to form the structural and adhesive support of the cell layer and gap junctions are aqueous intercellular channels. Extensive studies in recent years have begun to elucidate the molecular mechanisms regulating junction formation. A number of protein components associated with these junctional complexes has been identified in a variety of cell types.41–44 Mesothelium expresses E-, N- and P-cadherins, although, unlike the epithelium, N-cadherin predominates.45 Furthermore, membrane-associated expression of zonula occludens (ZO)-1 has been demonstrated in intact sheets of normal mesothelium.46 Expression of other junctional proteins, such as catenins and occludin, has not been studied.
Von Recklinghausen, in 1863,47 described the presence of ‘stomata’, cavities at the junction of two or more mesothelial cells, which he postulated allowed movement of fluid and particulate matter to and from the serous cavities. Scanning and transmission electron microscopy studies combined with tracer studies have clearly demonstrated their existence, although there is still much debate about their location, density and function.12–14,19,28,39,48–56 Stomatal openings, 3– 12 μm in diameter, are generally found in those regions, where cuboidal mesothelial cells are present, such as on the peritoneal side of the diaphragm or over the omental milky spots. These openings provide a direct access to the underlying submesothelial lymphatic system, allowing rapid removal of fluid, cells, bacteria and particles from the serosal cavities.52,54,56–58 It has also been proposed that stomata provide a direct link between the pleural and peritoneal cavities.54,59 This may explain how inhaled materials, such as asbestos, can enter the peritoneal cavity and cause asbestos-induced fibrosis or malignant mesothelioma.
FUNCTION OF THE MESOTHELIUM
Classically, the two main functions attributed to the mesothelium are to provide a protective barrier and a frictionless interface for the free movement of apposing organs and tissues. In recent years, it has been discovered that this tissue also plays important roles in fluid and cell transport, initiation and resolution of inflammation, tissue repair, lysis of fibrin deposits preventing adhesion formation and protection against invading microorganisms and, possibly, tumour dissemination.
The mesothelium is involved in the transport and movement of fluid and particulate matter across the serosal cavities. Odor, in 1954,7 proposed that the mesothelial cell surface is important in transport across the serosal wall because the many microvilli increase the surface area of the luminal aspect of the cell, thereby facilitating absorption. The glycocalyx covering microvilli also contains glycosaminoglycans, which bind fluids, aiding adsorption.18 Studies involving various tracers have shown that particulates and solutes can be actively transported across the mesothelium by pinocytic vesicles.8,36,39 If required, for example when there is an elevation in extracellular fluid pressures, mesothelial cells can increase the number of plasmalemmal vesicles.60 Using similar tracers, transmesothelial transport has also been shown to occur through intracellular junctions and stomata25,36,38,61–63 rather than occurring by passive diffusion. However, others have found no evidence to support this hypothesis.38,64,65
Studies of the enzymes associated with flattened resting mesothelial cells and cuboidal ‘activated’ mesothelial cells have demonstrated a difference in the enzymatic profile of each mesothelial cell type.62,66–72 The studies indicated that resting mesothelium is mainly concerned with membrane transport, whereas cuboidal mesothelial cells have a wider spectrum of functional activities. These observations also support the concept that flat resting mesothelial cells can become metabolically active cuboidal cells.
SECRETION OF GLYCOSAMINOGLYCANS AND SURFACTANT
The mesothelial cell secretes surface glycosaminoglycans, predominantly hyaluronan, which have been demonstrated ultrastructurally both on the surface and within pinocytic vesicles of mesothelial cells.73 From this observation, it was unclear whether the glycosaminoglycans are produced by or merely transported through the mesothelium. However, several observations support the hypothesis that mesothelial cells produce glycosaminoglycans in vivo. Hyaluronan is not derived from the circulation but is ‘locally’ produced.74 An increase in the concentration of hyaluronan is associated with the presence of activated ‘cuboidal’ mesothelial cells in pleural effusions.75,76 Mesothelial cells in culture synthesize hyaluronan77–81 which is upregulated following injury,81,82 and is assembled into hyaluronan-containing pericellular matrix ‘coats’.83 The function of the hyaluronan-containing coat is not known, but it has been proposed that it may protect the cells from viral infections and the cytotoxic effects of lymphocytes and may be important in differentiation.83 Furthermore, it may also play a role in protecting serosal membranes from adhesion formation84,85 and tumour cell dissemination and growth.86,87
Dobbie and Lloyd88 reported that following tannic-glutaraldehyde fixation, lamella bodies, similar to those described in type II pneumocytes, could be visualized at the ultrastructural level within and on the surface of mesothelial cells. In vitro, rat and human mesothelial cells are capable of synthesizing large amounts of phosphatidylcholine, the major constituent of lamella bodies and pulmonary surfactant.89,90 These findings provide evidence to suggest that mesothelial cells also produce and secrete a lubricant surfactant similar to type II pneumocytes.91
Mesothelial cells are metabolically active cells that participate in serosal inflammation by secreting various pro-, anti- and immunomodulatory mediators. These include prostaglandins and prostacyclin, chemokines, nitric oxide (NO) and reactive nitrogen and oxygen species, anti-oxidant enzymes, cytokines and growth factors, extracellular matrix (ECM) molecules and products of the coagulation cascade (Table 1). These mediators are released in response to bacterial endotoxins and cytokines,92 asbestos,93 or instilled agents94 with the purpose of restoring normal serosal architecture and function.
It is likely that serosal inflammation is activated on the surface of the mesothelial cell, although this has not been proven. For example, bacteria have been shown to attach to and be phagocytosed by mesothelial cells,95,96 which can result in mesothelial cell activation and release of interleukin (IL)-8.95 Interleukin-8 release is also associated with engulfment of asbestos fibres by cells.94 Furthermore, hyaluronan and mediators released from activated macrophages, such as IL-1β, tumour necrosis factor (TNF)-α and interferon (IFN)-γ, are also potent inducers of neutrophil and monocyte chemokines, including IL-8, growth-related oncogene-α (GRO-α), IFN-γ-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1) and RANTES by mesothelial cells.94,97–101 Mesothelial cells also release GRO-α in response to IL-17, a cytokine secreted by T cells.102 Combining IL-17 with TNF-α further potentiated GRO-α release and increased the stability of GRO-α mRNA.102 Particulates, such as talc and asbestos, have been shown to stimulate mesothelial cells to secrete IL-8 and MCP-1.103,104 Levels of these chemokines can be further increased if the cells are exposed to particulates in the presence of IL-1β or TNF-α.103,104 Neutralizing studies with MCP-1- and IL-1-specific antibodies demonstrated significant decreases in bioactivity for MCP-1 and IL-8, indicating that mesothelial cell-derived MCP-1 and IL-8 play a significant role in the chemotactic activity seen in stimulated mesothelial cell supernatants.94
Recent studies have shown that mesothelial cells also secrete stromal cell-derived factor-1 (SDF-1)105–107 and eotaxin.108 Stromal cell-derived factor-1 stimulates the growth of B lymphocyte precursors (B1a) in vitro and SDF-1 production by mesothelial cells may account for the selective accumulation of B1 lymphocytes in body cavities.107 Mesothelial cells also produce eotaxin in response to TNF-α and IL-4, a T helper (Th)-2 cytokine. Eotaxin is a chemokine for eosinophils and may explain why eosinophilic pleural effusions occur in many diseases.109,110
Studies have also demonstrated that human mesothelial cells constitutively express mRNA transcripts for macrophage colony stimulating factor (M-CSF) and can secrete IL-6 and M-CSF.111–117 Interleukin-6 levels are also elevated in response to IL-1β and TNF-α.113,116,117 Interleukin-6 is often induced together with the pro-inflammatory cytokines IL-1 and TNF-α and circulating IL-6 plays an important role in the induction of acute-phase reactions. Endogenous IL-6 plays a crucial anti-inflammatory role in both local and systemic acute inflammatory responses by controlling the levels of pro-inflammatory, but not anti-inflammatory, cytokines.118 Interleukin-1β and TNF-α also stimulate mesothelial cells to secrete heat shock proteins (HSP)-72/73, which may have a cell-protective function lessening mesothelial cell damage during inflammation.119 In addition, mRNA transcripts for other cytokines, such as granulocyte stimulating factor, granulocyte–macrophage colony stimulating factor and IL-1, can be induced by bacterial lipopolysaccharide (LPS), TNF-α and epidermal growth factor (EGF).111,115
Mesothelial cells also produce NO and reactive nitrogen (NO· radical) and oxygen (O2–· radical, H2O2 and OH· radical) species in vitro in response to cytokines, bacterial products and asbestos.120–123 To counteract the effect of these reactive species, mesothelial cells contain significant quantities of anti-oxidants. When subjected to mild oxidant stress, the cells are protected mainly by the glutathione redox cycle, whereas during severe oxidant exposure they require catalase-mediated protection.124 Asbestos fibres have been shown to upregulate the expression of anti-oxidant genes (manganese containing superoxide dismutase and haem oxygenase) in human pleural mesothelial cells;125 however, it is likely that the endogenous anti-oxidant synthesis may be insufficient to counteract the injurious effects of asbestos.
Resolution of inflammation and repair of the serosa without fibrosis requires a downregulation of the inflammatory response, including inhibition of fibroblast proliferation and collagen production. Mesothelial cells are likely to contribute to controlling inflammation both in normal and inflamed tissue because they have cyclo-oxygenase (COX) activity,126 expressing COX-1 and COX-2 mRNA,127,128 and metabolize arachidonic acid to release prostaglandins and prostacyclin.127,129–132 Exposure of mesothelial cells to macrophage-conditioned medium or the cytokines IL-1β and TNF-α resulted in an increase in the levels of both COX-1 and COX-2 mRNA, with the greatest increase being seen for COX-2.127
The onset of pleural, pericardial and peritoneal infections is characterized by a massive influx of leucocytes from the vascular compartment into the serosal space.133,134 As outlined by Topley et al.,135 in order for leucocytes to be attracted to the site of inflammation four basic events have to occur: (i) there needs to be a site of activation; (ii) resident cells need to be activated and signals generated to initiate the response; (iii) there must be a chemotactic gradient to direct leucocytes to the site of inflammation; and (iv) adhesion molecule expression must be upregulated to allow leucocyte margination and transmigration into the serosal space.
As discussed previously, it is likely that serosal inflammation is activated on the surface of the mesothelial cell with subsequent release of chemokines. Secretion of chemokines is polarized towards the cell apical surface, creating a chemotactic gradient from the basolateral to the apical side of the mesothelial cell.99,100,136 Using transmigration studies, neutrophils and monocytes have been shown to follow this gradient and traverse mesothelial cell monolayers. Blocking this gradient with antibodies abolished transmigration.99 These findings suggest that, by secreting chemokines in a polarized manner, mesothelial cells promote directed transmesothelial migration of both neutrophils and monocytes.
Movement of leucocytes from the circulation to the site of inflammation is facilitated by the expression of integrins and adhesion molecules. Mesothelial cells express several cell-adhesion molecules, including intercellular adhesion molecule (ICAM-1), vascular cellular adhesion molecule (VCAM-1), E-cadherin, N-cadherin, CD49a, CD49b and CD29,40,112,137–140 which can be induced by IL-1β, TNF-α and IFN-γin vitro.138,139,141 Leucocytes express the β2 integrin family members, lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) and Mac-1 (CD11b/CD18) on their surface, which are counter-receptors for ICAM-1. Interaction between LFA-1/Mac-1 and ICAM-1 leads to cell–cell adherence and results in transmigration of leucocytes across mesothelial cell monolayers. However, neutrophils showed minimal adherence to and migration across unactivated mesothelial cell monolayers, despite an extensive amount of ICAM-1 on the mesothelial membrane.139 Pretreatment of the monolayers with IL-1β induced enhanced neutrophil adherence and migration across the mesothelial monolayer, together with a further increase in ICAM-1 expression on the mesothelial membrane.139 Therefore, neutrophil migration not only required ICAM-1 expression, but also activation and release of cytokines from the mesothelial cells. Interleukin-8 appeared to be the major cytokine involved, with platelet-activating factor (PAF) and transforming growth factor (TGF)-β playing lesser roles. The importance of ICAM-1 and LFA-1 in leucocyte transmigration across the mesothelium was clearly demonstrated both in pleural and peritoneal cells because the incubation of cells with the soluble form of ICAM-1 and neutralizing antibodies to ICAM-1 and LFA-1 significantly reduced neutrophil transmigration.100,112,138
Recently, it was shown that ICAM-1 and VCAM-1 were only expressed on the microvilli of mesothelial cells, with VCAM-1 less numerous and on less microvilli (24%) than ICAM-1 (90%).142 Anti-VLA-4 (a counter-receptor for VCAM-1) does not inhibit leucocyte binding to mesothelium, whereas both anti-LFA-1 and anti VLA-4 do.112 It is interesting that adhesion molecules are only expressed on microvilli, suggesting that leucocytes may not ‘crawl’ on the cell surface, but to and from microvilli.
It has been proposed that the main function of microvilli is to trap proteins from the serosal fluid to help ensure mesothelial cell integrity. However, it seems more likely that, by modulating microvilli density together with adhesion molecule expression, mesothelial cells can regulate trafficking of leucocytes into and out of the serosal cavities. This is supported by the observation that the greatest concentration of microvilli on mouse testicular mesothelial cells occurred at mesothelial cell junctions 6 days after injury, when regeneration of the mesothelium was almost complete and inflammatory cells were being cleared.34
A great deal is known about leucocyte influx into an inflamed site, but the subsequent events are less clear. It is likely that leucocyte clearance from serosal cavities is via stomata and the draining lymphatics,143 in contrast with influx directly across the mesothelium from the vasculature. Furthermore, kinetic studies on macrophages within the peritoneum suggest that resident and inflammatory macrophages are cleared at different rates.143,144 The role of the mesothelial cell in regulating clearance of leucocytes during resolution of inflammation is yet to be elucidated.
Several studies have demonstrated that mesothelial cells have the capacity to secrete a variety of growth factors and ECM molecules that are likely to be involved in inflammation and tissue repair, analogous to that of epithelial cells. Growth factors initiate cell proliferation and migration at the edge of a lesion and repair cells migrate over ECM molecules that are exposed or deposited at the wound site to cover the injury.145,146 Growth factors, including TGF-β, platelet-derived growth factors (PDGF), fibroblast growth factors (FGF) and members of the EGF family (EGF, heparin-binding EGF (HB-EGF) and vascular-EGF (VEGF)) are likely to regulate this process.145,146
Cultured peritoneal and pleural mesothelial cells have been shown to synthesize and secrete TGF-β1 and TGF-β2, with TGF-β1 levels 60-fold higher than those of TGF-β2.147,148 Cultured mesothelial cells also express TGF-β1 and TGF-β2 mRNA147,149,150 and TGF-β1 expression has been shown by in situ hybridization in the visceral pleura of mice exposed to intratracheal instillation of bleomycin.151 Treatment of cells with IL-1 significantly increases protein and mRNA expression of both TGF-β isoforms147,148 and the steady state levels of the mRNA.147 Hypoxia also increases TGF-β1 and TGF-β2 mRNA levels, which is enhanced for TGF-β2 in the presence of exogenous TGF-β1.149 From this finding, it was proposed that changes in the TGF-β1/TGF-β2 ratio may reduce scarring and fibrosis in serosal tissues.149 Transforming growth factor-β3 mRNA and protein were not detected in any of the studies.147–149
Platelet-derived growth factor-A and PDGF-B chain mRNA have been detected in cultured pleural and peritoneal mesothelial cells,152–154 although the level of B chain mRNA was much lower than for the A chain.152 Langerak et al.153 found that the B chain was only expressed following spontaneous transformation in vitro of a normal mesothelial cell line, consistent with high levels of PDGF-B chain found in malignant mesothelioma cell lines.152,155 Mesothelial cells also constitutively express mRNA for the heparin-binding growth factors, basic FGF (bFGF), HB-FGF and VEGF50,156–158 and, upon stimulation with IL-1 and TNF-α, increase their mRNA and protein levels for HB-EGF and VEGF, but not bFGF.156 However, IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF, mRNA expression.156 Cronauer et al.159 showed constitutive bFGF protein in cultured human peritoneal mesothelial cells, predominantly intracellular, which increased following IL-1 stimulation. This increase in intracellular bFGF concentration was associated with an induction of the release of bFGF and an increase in the steady state levels of bFGF mRNA. Mesothelial VEGF production also increased following exposure to TGF-β2 and was inhibited in the presence of a TGF-β-blocking antibody.158 Recently, mesothelial cells have been shown to synthesize other growth mediators, such as endothelin-1 and PAF following stimulation160 with thrombin, hepatocyte growth factor (HGF)161,162 and keratinocyte growth factor (KGF).161 Interestingly, HGF and KGF are generally considered as epithelial cell-derived growth factors, stimulating mesenchymal cell proliferation and migration. The finding that mesothelial cells also express the receptors for these factors confirms the dual epithelial/mesenchymal properties characteristic of this cell.161,162
Mesothelial cells also respond to a host of other cytokines and growth factors. Epidermal growth factor, TGF-β1 and TGF-β2, PDGF, FGF, IL-1, TNF-α and IFN-α,β,γ are mitogenic for human mesothelial cells in vitro and in vivo.163–167 In addition, mesothelial cells are capable of proliferation and chemotaxis in response to products of the coagulation cascade, including thrombin168 and fibrinopeptides.169
Mesothelial cells also have the capacity to synthesize a variety of ECM macromolecules in vitro. Cultured rat mesothelial cells produce collagen types I, III and IV, elastin, fibronectin and laminin.170–175 Furthermore, electron microscopy studies reveal that pleural mesothelial cells can organise these components into complex structures that resemble components of the ECM in vivo (thick collagen fibres, the amorphous components of elastic fibres and basement membrane-like structures), which were restricted to the basal region below the cells in culture.170 Several studies have also demonstrated increased production of these molecules following incubation of cells with various cytokines and growth factors, such as TNF-α, EGF and PDGF.173,174,176 Interestingly, NO suppressed collagen production by mesothelial cells, suggesting that NO may compromise the ability of the mesothelial cell to repair the serosal tissue during conditions associated with significant pleural inflammation. Alternatively, NO may prevent the excessive formation of fibrotic tissue.175
Cultured human pleural mesothelial cells synthesize large amounts of collagen types I and III, but no evidence has been reported for collagen type IV.111,177 Incubation of human peritoneal mesothelial cells with TGF-β increased mRNA expression for collagen type III, but reduced expression for collagen type I. The same results were obtained when cells were cultured under hypoxic conditions.178 Mesothelial cells also increase collagen synthesis following IL-1β treatment179 and exposure to peritoneal effluents obtained from patients with acute peritonitis.180 Evidence from these studies suggests that upregulation of ECM molecules by IL-1, hypoxia and factors present in infected serosal effluents are likely to occur, at least in part, through a TGF-β-dependent mechanism. It is likely that overexpression of TGF-β in serosal tissues can lead to fibrosis and adhesion formation. It is possible that mesothelial cells regulate TGF-β activity by secreting the TGF-β inhibitors, decorin and biglycan.181
Mesothelial cells are also likely to play an important role in regulating the ECM turnover that follows serosal injury by secreting metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP).182–184 It has been proposed that the state of differentiation of these cells has a marked influence on MMP and TIMP expression, such that cells with an epitheloid morphology adopt a more ECM-degradative phenotype.182 Regulating the balance of these molecules, together with the level of ECM production, is important for the outcome of injury, leading either to tissue regeneration and re-establishment of normal function or fibrosis and adhesion formation.
Cell-to-cell and cell-to-ECM adhesion has roles in embryonic development, maintenance of tissue architecture, inflammatory response, tumour metastases and tissue repair.146,185 The role of integrins in the adhesion of mesothelial cells to ECM in response to injury and their effects on cell function have not been widely studied. Integrins are a family of heterodimeric molecules, composed of an α-subunit non-covalently associated with a β-subunit186 that bind to ECM molecules. To date, at least 23 structurally distinct integrins have been described. Human primary pleural mesothelial cells demonstrated high expression of α2, α3, α5, β1, β3 and αvβ3 integrins, with less expression of α1, α4 and α6.187 Functionally, mediators, such as EGF, have been shown to stimulate a reversible change in mesothelial cells to a fibroblastic phenotype that is accompanied by an increased expression of β1 integrins, in particular α2β1, facilitating enhanced adhesion to and migration on collagen type I.188 In addition, in response to asbestos, integrins on rabbit pleural mesothelial cells interact with specific ECM molecules causing cells to change their shape and ability to internalize asbestos.189 Plating human peritoneal mesothelial cells on different ECM proteins also had a profound effect on cell proliferation.190 These examples clearly demonstrate that integrin expression on mesothelial cells influences cell shape and behaviour. The role of integrins in mesothelial cell function and repair demands further study.
Antigen presentation and T cell activation are the first steps in the generation of a specific immune response. Recognition of foreign antigens by Th cells is dependent on their presentation by major histocompatibility complex (MHC) class II mole-cules, which are expressed on professional antigen-presenting cells (APC), such as dendritic cells, macrophages, monocytes and B cells.191 Other cells normally do not express class II molecules, although non-professional APC, such as endothelial cells and keratinocytes, can express these molecules and present antigen following cytokine stimulation.191,192 For cells to present antigen, there must be interaction between the T cell receptor, class II molecules and specific accessory molecules on the presenting cells, such as B7-1 and B7-2. Intercellular adhesion molecule-1 can also act as an accessory molecule, usually on non-professional APC.191,192
Valle et al.193 demonstrated that human peritoneal mesothelial cells are able to present tetanus toxoid and Candida albicans bodies to mononuclear cells depleted of adherent cells and to a T cell clone. In a similar study, Hausmann et al.141 generated highly purified Th cells, depleted of B cells, dendritic cells and monocytes. They demonstrated that, in the absence of professional APC, human peritoneal mesothelial cells stimulated by IFN-γ effectively induced the proliferation of CD4+ Th cells when the antigen tetanus toxoid or the super antigen Staphylococcus aureusα toxin were present. High levels of ICAM-1 were also detected on the mesothelial cell surface following IFN-γ treatment, whereas B7-1 and B7-2 molecules were not, suggesting that ICAM-1 is the major accessory molecule for antigen presentation by mesothelial cells.141,193
Activation of mesothelial cells with IFN-γ also induced IL-15 production by mesothelial cells.141 Interleukin-15 is a pleotrophic cytokine with many activities related to cell-mediated immunity, such as stimulation of T cell growth.194 In addition, it has been proposed that IL-15 cooperates with IL-2 in the initiation of an immune response and, subsequently, enhances IL-2 responsiveness.195 Elevated levels of IL-15141 and IFN-γ196 were also found in the effluent of patients suffering from peritonitis, suggesting that IFN-γ release from activated Th cells during peritonitis could induce peritoneal cells to produce IL-15.
TUMOUR GROWTH AND DISSEMINATION
There have been very few studies examining the role of the mesothelial cell in tumour growth and dissemination within serosal cavities. Most studies have concentrated on examining tumour recurrence following surgery, clearly showing that traumatized mesothe-lial surfaces are privileged sites for tumour cell adhesion.197 Van der Wal et al.198 suggested that this was due to upregulation of adhesion molecules on mesothelial cells in response to inflammatory mediators, thus promoting the anchoring of tumour cells followed by growth promotion of the adhered tumour cells through the action of locally produced growth factors. Although mesothelial cells may play a role, it is more likely that entrapment of tumour cells in the fibrinous exudate deposited following trauma and binding of tumour cells via integrins to exposed submesothelial connective tissue are the main mechanisms of attachment.199 However, as discussed, mesothelial cells synthesize a host of growth factors in response to inflammatory stimuli and, therefore, may play a role in stimulating tumour growth.
Several experimental studies have demonstrated that, following surgical trauma, tumour growth is also enhanced at sites distal to the injury.200–202 In addition, increased tumour growth was observed in animals exposed to surgical wound fluid or a combination of the growth factors TGF-β and bFGF, suggesting that mediators produced after surgical trauma enhance local and distant tumour growth.202 It is likely that these mediators induce upregulation of cell adhesion molecules on mesothelial cells, promoting tumour cell attachment. Once the tumour cells adhere to mesothelial cells, they can migrate through the mesothelium, invade local organs and move to distant sites. Interleukin-1β, TNF-α and IFN-γ upregulate adhesion molecule expression on mesothelial cells138,139,141 and IL-1β and EGF increase tumour cell adhesion to cultured mesothelial cells.140 Interestingly, incubation of mesothelial cells with TNF-α had no effect on tumour cell attachment.140 In the absence of surgical trauma, it is possible that the tumours themselves secrete mediators that upregulate adhesion molecule expression on mesothelial cells.
Numerous studies have demonstrated that adhesion of tumour cells to the hyaluronan pericellular coat of mesothelial cells is an important step in the peritoneal spread of ovarian and colorectal cancer.86,203–208 A wide variety of malignancies of epithelial and mesenchymal origin express high levels of the hyaluronan receptor CD44,209 although the degree of adhesion does not necessarily relate to the amount of CD44 expressed.204,205 This may be due to differences in CD44 glycosylation205 or variant forms of CD44.209 Blocking interaction of CD44 with hyaluronan using antisense CD44 cDNA208 monoclonal antibodies that block the hyaluronan-binding site of CD44,86,203,205,207 intact hyaluronan and hyaluronan oligomers,86 reduced cell adhesion and inhibited cell migration. However, because blocking CD44 did not totally inhibit mesothelial binding in all studies, it is likely that other surface molecules are involved.
The role of integrins in the interaction of tumour cells with mesothelial cells has also been explored.86,206,207 As well as synthesis of ECM molecules by mesothelial cells, damage to the mesothelium or rounding of mesothelial cells after stimulation can expose underlying connective tissue. Hepatocyte growth factor and TGF-β can both induce rounding and separation of mesothelial cells, exposing submesothelial connective tissue.210,211 Lessan et al.207 demonstrated that they could reduce adhesion of an ovarian carcinoma cell line to mesothelial cells by using a monoclonal antibody against the β1 integrin subunit, which is common to many integrin molecules and can bind a variety of ECM proteins. Migration of ovarian carcinoma cell lines towards fibronectin, type IV collagen and laminin can be blocked by antibodies against α5β1, α2β1 and α6β1, respectively.86 Equally, antibodies against CD44 reduced cell adhesion and migration, suggesting that tumour migration is regulated by both integrin-dependent and -independent mechanisms.
Although mesothelial cells appear to mainly promote tumour dissemination and growth, intact hyaluronan inhibits the adhesion of tumour cells to mesothelium.86 Similarly, conditioned medium from a confluent mesothelial cell culture containing high amounts of hyaluronan prevented tumour cell attachment to mesothelial cells, but hyaluronidase treatment increased tumour cell adhesion.87 Free hyaluronan in the conditioned medium would have bound to the CD44 molecules on the tumour cells and blocked their interaction with the hyaluronan present on the surface of the mesothelial cells. Removal of free hyaluronan may explain why tumour cells adhered to mesothelial cells in other studies. Therefore, under normal physiological conditions, secretion of hyaluronan by mesothelial cells into the serosal fluid may protect the serosal surface from tumour implantation. Following resection of peritoneal tumours, many surgeons lavage the peritoneal cavity to remove blood and other biological material. It is possible that this practice is detrimental to the patient because it actually provides residual tumour cells with a favourable environment to attach and reseed onto the serosal surface.
COAGULATION AND FIBRINOLYSIS
Mesothelial cells play an important role in local fibrin deposition and clearance within serosal cavities. Their fibrinolytic activity is a key factor in the prevention and removal of fibrin deposits that form following mechanical injury, hemothoraces and infection (Fig. 4). If the fibrinolytic capacity is insufficient and fibrin accumulation is not resolved, fibrous adhesions form between opposing serosal surfaces. In the peritoneum, adhesions occur in 93–100% of patients who have undergone abdominal surgery.212 These adhesions are the major cause of intestinal obstruction,213 are responsible for up to 20% of infertility cases in women214 and are thought to cause abdominal pain.215–217 In the thorax, pleural loculation and adhesions can impair lung function and cardiac adhesions may have a deleterious effect on cardiac function218 and/or result in decreased coronary artery bypass graft patency.219 In addition, adhesions make the growing number of repeat surgical procedures difficult for the surgeon and hazardous for the patient.
Mesothelial cells have both procoagulant and fibrinolytic activity. The procoagulant activity is due to tissue factor, the main cellular initiator of the extrinsic coagulation cascade,220 and assembly of the prothrombinase complex at the mesothelial cell surface.221–223 Fibrin deposition is also aided by the secretion of the plasminogen activator inhibitors (PAI) PAI-1 and PAI-2.221,224–228 The main plasminogen activators (PA) are tissue PA (tPA) and urokinase PA (uPA). The PA convert the inactive zymogen plasminogen into active plasmin, which, in turn, enzymatically breaks down fibrin.221,226,229 Mesothelial cells are the main source of tPA in serosal cavities but secrete lower levels of uPA.230
There is a fine balance between fibrin deposition and breakdown in serosal cavities, which, if inappropriately regulated, can cause reduced fibrin clearance and result in adhesion formation.231 Both pro- and antifibrinolytic mediators are regulated by inflammatory factors, including LPS, TNF-α and IL-1,232–235 and fibrogenic mediators, such as TGF-β and thrombin.227,236,237 These mediators reduce production of tPA by mesothelial cells while increasing synthesis of PAI-1, causing a significant delay in fibrinolysis. Transforming growth factor-β is also a potent inducer of collagen production and has been used experimentally to induce pleurodesis in animal models.238,239 Redressing this imbalance by blocking PAI or the mediators upregulating their synthesis may be a way of preventing adhesions. This proposal is supported by experimental studies in rodents, where it was shown that blocking the activity of PAI-1240 or TGF-β241 with neutralizing antibodies had a significant effect on reducing adhesion formation.240
There have been many reports describing materials, primarily barrier membranes and gels, that reduce adhesion formation by separating serosal surfaces.242 However, these materials are not widely used because of problems associated with their clinical application and their limited success. It is widely accepted that the future direction in preventing adhesions is through the application of growth factors and mediators designed to increase the rate of serosal repair and to break down fibrin. However, despite the clinical importance of adhesions, there is little information available regarding the basic physiology of serosal wound repair and adhesion formation and the mediators regulating these processes.
The exact mechanisms involved in mesothelial regeneration are controversial. Hertzler, in 1919,243 was the first to note that both large and small peritoneal injuries healed at the same rate. He concluded from this observation that mesothelium could not heal solely by the centripetal migration of proliferating cells at the wound edge into the injured area, as occurs for healing of squamous epithelium. Since Hertzler's early observations, many studies using different types of serosal injury have been performed to elucidate the mechanisms regulating mesothelial regeneration, following deep lacerations,67,244,245 broad abrasions,46,246 minor linear scarifications,247 drying,248 chemical treatment249 and heat injury.29–31,34,46,250 Based on these studies, it is now generally accepted that the healing process begins, within 24 h of injury, upon the arrival at the wound surface of a population of round cells and is completed 7–10 days later when the area is covered by cells displaying all the characteristics of mesothelial cells.29–31,245,246 The origin of these new mesothelial cells has not yet been confirmed, but a number of possibilities have been proposed (Fig. 5), including centripetal migration of mesothelial cells, exfoliation of mature or proliferating mesothelial cells from adjacent or opposing surfaces, which settle on the wound surface and replicate, pre-existing free-floating serosal reserve cells that settle on the wound surface and gradually differentiate into new mesothelium, macrophage transformation, submesothelial mesenchymal precursors and bone marrow-derived circulating precursors.
LOCAL CELL PROLIFERATION AND CENTRIPETAL MIGRATION
The mesothelium is a slowly renewing tissue that can be stimulated by a variety of agents and direct physical damage to increase its turnover rate. Watters and Buck251 reported that apposing serosal surfaces undergo maximal division 2 days after injury and further demonstrated that both surfaces are triggered simultaneously, suggesting that a stimulus may traverse the space between them. Subsequent kinetic studies using [3H]-thymidine uptake as a marker for DNA synthesis have demonstrated that 60–80% of mesothelial cells at the wound edge and on the apposing surface are dividing 24–48 h after injury29,250 (Fig. 6). There is no doubt that proliferating cells surrounding the wound play an important role in the repair process, many migrating across the injured serosa from the edge of the wound to the centre (Fig. 7). However, this does not explain how large and small wounds can heal at the same time. It is clear that simple migration of cells is not the only mechanism of regeneration.
Of the remaining proposals, irradiation29,30 and cell labelling studies30,46,67,244–246,252 have clearly shown that macrophage transformation and a circulating bone marrow-derived mesothelial precursor are unlikely. Therefore, as well as migration of cells into the wound from the edge of the lesion, regenerating mesothelium is likely to originate either from a submesothelial mesenchymal precursor or free-floating mesothelial cells.
SUBMESOTHELIAL MESENCHYMAL PRECURSOR
It was proposed that, upon appropriate stimulation, subserosal connective tissue ‘fibroblast-like’ cells migrated to the serosal surface and differentiated into mature mesothelial cells.244,245,252 This proposal was supported by embryology and cell culture studies that demonstrated that mesothelial cells can modulate their morphology under different conditions and display many of the characteristics associated with fibroblasts, such as ECM production.174,175,177,179,207,253
Furthermore, Bolen et al.,254,255 examining intermediate filament expression in serosal tissues from human pathologies, demonstrated that some submesothelial fibroblast-like cells expressed cytokeratins, a feature characteristic of epithelial and mesothelial cells, but not normally expressed by fibroblasts. These results were interpreted to suggest that submesothelial spindle cells are multipotential and have the capacity to differentiate into surface mesothelial cells. Whitaker et al.,256 in a similar study, were unable to reproduce these findings and suggested that the staining pattern seen by Bolen et al. may not be a result of submesothelial cells differentiating into surface mesothelial cells but, instead, may be due to mature mesothelial cells migrating into the subserosal connective tissue.
Although the proposal of a multipotential submesothelial precursor for regenerating mesothelial cells has generally been favoured, substantial evidence against such a theory exists.29,250,257–259 Implantation of polyethylene sheeting onto denuded serosa did not prevent remesothelialization.257 A mesothelial membrane was grown from free serosal cells by using a diffusion chamber placed in the peritoneal cavity of rats, ensuring no possible contact with fibroblast-like cells.258 Irradiation studies have demonstrated impaired local mesothelial regeneration, which was recoverable by addition of peritoneal lavage cells.29 In addition, a kinetic study of serosal repair demonstrated that subserosal cells were not essential for mesothelial healing and that the regenerating cells were likely to originate from the surrounding uninjured serosal surface.250
FREE-FLOATING MESOTHELIAL CELLS
In 1957, Cameron et al.247 proposed that mesothelial healing involved attachment of free-floating mesothelial cells to the injured surface. Peritoneal lavage fluid recovered from experimental animals following injury to the mesothelium was found to contain a significantly increased number of viable free-floating mesothelial cells, 2 days post-injury compared with controls.29 The increased free-floating cell population was thought to be due to proliferation of mesothelial cells adjacent to250,257 and opposing30,260 the serosal injury. Further support for this proposal came from implantation of polyethylene sheets and diffusion chamber studies, as described previously, demonstrating that reconstitution of the mesothelium could occur without contact with submesothelial fibroblast-like cells.257,258 Furthermore, Cleaver et al.261 showed that the healing rate of the mesothelium was retarded following postoperative peritoneal lavages, possibly due to the removal of the free-floating serosal cells. Replacement of these cells in irradiated animals resulted in a marked increase in the healing rate of the testicular mesothelium.29
The most compelling evidence to support a free-floating origin for regenerating mesothelium is from recent in vivo cell-tracking studies.46 Cultured mesothelial cells, peritoneal lavage cells and control fibroblasts were labelled with a tracking dye (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbacyanine perchlorate; DiI) and injected into rats immediately following mesothelial injury. Labelled cultured mesothelial and peritoneal lavage cells, but not cultured fibroblasts, implanted onto the wound surface and began to proliferate. By examining ZO-1 expression in the regenerating mesothelium, it was also shown that the DiI-labelled cells began to form junctional complexes, clearly indicating that implanted cells were incorporated into the regenerated mesothelium.
From these findings and those of others, a model of mesothelial repair was proposed.46 Mesothelial regeneration requires recruitment of inflammatory cells to the wound surface and release of mediators to activate and stimulate mesothelial cell proliferation surrounding the wound.30,31 Activated mesothelial cells break their cell-to-cell contacts and migrate onto the wound surface,29 possibly through the action of HGF, which is secreted by mesothelial cells161,162,262 and surrounding fibroblasts.210 A population of mesothelial cells adjacent to the wound and on the apposing surface detach and become free floating,29 move along chemotactic gradients and attach to ECM components exposed beneath the mesothelium or deposited from the serosal fluid, proliferate and reconstitute an intact mesothelial monolayer.
SUMMARY AND CONCLUSIONS
The mesothelium was first described 175 years ago, but it has only been in the past 20 years that we have started to appreciate the roles that mesothelial cells and the mesothelium play in maintaining normal serosal membrane integrity and function. Mesothelial cells are biologically active cells that can sense and respond to signals within their microenvironment. They secrete glycosaminoglycans and surfactant to provide a frictionless free surface between parietal and visceral serosa. They actively transport fluids and particulates across the serosal membrane and form openings or stomata, directing movement of cells to and from the serosal cavities. Mesothelial cells can synthesize and secrete a diverse array of mediators in response to external signals, initiating and regulating an inflammatory response, recruiting cells into the serosal cavities and presenting antigen to T cells. They also play an active role in tissue repair through the release of growth factors and ECM molecules and their protease and fibrinolytic properties are of major importance in preventing fibrosis and the formation of adhesions. In addition, secretion of hyaluronan into serosal fluids may play an important role in preventing the dissemination and growth of tumours.
Despite the realization that the mesothelium is not merely a protective slippery barrier there are still many questions that remain unanswered. Mesothelial cells can clearly regulate the influx of inflammatory cells into serosal cavities upon stimulation, but their role in cell clearance during the resolution of inflammation has not been examined. Stomata have been identified in the mesothelial membrane, but nothing is known about how mesothelial cells and lymphatic endothelium communicate to form these openings. It is possible that mesothelial cells act in concert with underlying fibroblast-like cells to ensure serosal integrity, but the relationship between these cells is unclear. It has been proposed that adhesion formation between serosal tissues occurs because damage to the mesothelium reduces the fibrinolytic capacity of the serosal membranes. No one has started to examine the role mesothelial cells may actively play in the formation of adhesions. Mesothelial cells are able to change between epithelial and fibroblastic phenotypes. Identifying the genes regulating this transformation may provide some insight into the development of the different histogenic forms of malignant mesothelioma.
Regeneration of the mesothelium is unlike other epithelial-like surfaces because healing does not occur solely by centripetal migration of cells from the wound edge. The mechanism of repair is controversial, but recent evidence suggests that free-floating mesothelial cells implant, proliferate and incorporate into the regenerating mesothelium. Before this proposal is fully accepted, many questions must first be answered. For example, the mechanisms by which these cells break cell–cell contacts and become detached from the basement membrane are not known, although regulation of integrins or proteolytic dissociation of ECM may be involved. We do not understand what signalling mechanisms allow the free-floating cells to remain viable in the serosal fluid and not undergo apoptosis. It is not clear what directs the free-floating cells to the injured site and allows them to attach and migrate on the wound surface. In addition, and possibly most importantly, we need to understand more about the nature of the free-floating cell population and determine whether these are just desquamated mesothelial cells or a dedicated precursor cell population.
The present review has highlighted some of our current knowledge on the structure and function of mesothelial cells in maintaining tissue homeostasis and their role in repair. However, many questions still need to be answered before we can begin to understand the pathogenesis of serosal pathologies, such as pleural loculations, adhesion formation and malignant mesothelioma.