Steven E. Mutsaers

Mesothelial cells: Their structure, function and role in serosal repair

The mesothelium is composed of an extensive monolayer of specialized cells (mesothelial cells) that line the bodys 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 mesothelial cell

Mesothelial cells form a monolayer of specialised pavement-like cells that line the bodys serous cavities and internal organs. The primary function of this layer, termed the mesothelium, is to provide a slippery, non-adhesive and protective surface. However, mesothelial cells play other pivotal roles involving transport of fluid and cells across the serosal cavities, antigen presentation, inflammation and tissue repair, coagulation and fibrinolysis and tumour cell adhesion. Injury to the mesothelium triggers events leading to the migration of mesothelial cells from the edge of the lesion towards the wound centre and desquamation of cells into the serosal fluid which attach and incorporate into the regenerating mesothelium. If healing is impaired, fibrous serosal adhesions form between organs and the body wall which impede vital intrathoracic and abdominal movement. Neoplastic transformation of mesothelial cells gives rise to malignant mesothelioma, an aggressive tumour predominantly of the pleura. Although closely associated with exposure to asbestos, recent studies have implicated other factors including simian virus 40 (SV40) in its pathogenesis.

Presence and Distribution of Sensory Nerve Fibers in Human Peritoneal Adhesions

ObjectiveTo assess the distribution and type of nerve fibers present in human peritoneal adhesions and to relate data on location and size of nerves with estimated age and with clinical parameters such as reports of chronic pelvic pain. Summary Background DataPeritoneal adhesions are implicated in the cause of chronic abdominopelvic pain, and many patients are relieved of their symptoms after adhesiolysis. Adhesions are thought to cause pain indirectly by restricting organ motion, thus stretching and pulling smooth muscle of adjacent viscera or the abdominal wall. However, in mapping studies using microlaparoscopic techniques, 80% of patients with pelvic adhesions reported tenderness when these structures were probed, an observation suggesting that adhesions themselves are capable of generating pain stimuli. MethodsHuman peritoneal adhesions were collected from 25 patients undergoing laparotomy, 20 of whom reported chronic pelvic pain. Tissue samples were prepared for histologic, immunohistochemical, and ultrastructural analysis. Nerve fibers were characterized using antibodies against several neuronal markers, including those expressed by sensory nerve fibers. In addition, the distribution of nerve fibers, their orientation, and their association with blood vessels were investigated by acetylcholinesterase histochemistry and dual immunolocalization. ResultsNerve fibers, identified histologically, ultrastructurally, and immunohistochemically, were present in all the peritoneal adhesions examined. The location of the adhesion, its size, and its estimated age did not influence the type of nerve fibers found. Further, fibers expressing the sensory neuronal markers calcitonin gene-related protein and substance P were present in all adhesions irrespective of reports of chronic abdominopelvic pain. The nerves comprised both myelinated and nonmyelinated axons and were often, but not invariably, associated with blood vessels. ConclusionsThis study provides the first direct evidence for the presence of sensory nerve fibers in human peritoneal adhesions, suggesting that these structures may be capable of conducting pain after appropriate stimulation.

Adhesion Molecule–dependent Mechanisms Regulate the Rate of Macrophage Clearance During the Resolution of Peritoneal Inflammation

Macrophage clearance is essential for the resolution of inflammation. Much is known about how monocytes enter the inflammatory site but little is known about how resultant macro-phages are cleared. We have previously demonstrated that macrophage clearance from resolving peritonitis occurs by emigration into draining lymphatics rather than local apoptosis. We now examine mechanisms for this process, in particular by evaluating the hypothesis that modulation of adhesion interactions between macrophages and cells lining the lymphatics regulates the rate of macrophage clearance. We demonstrate in vivo that macrophages adhere specifically to mesothelium overlying draining lymphatics and that their emigration rate is regulated by the state of macrophage activation. We observed that macrophage–mesothelial adhesion is Arg-Gly-Asp (RGD) sensitive and partially mediated by very late antigen (VLA)-4 and VLA-5 but not αv or β2 integrins. Moreover, macrophage clearance into lymphatics can be blocked in vivo by RGD peptides and VLA-4 and VLA-5 but not β2 blocking antibodies. This is the first evidence that macrophage emigration from the inflamed site is controlled and demonstrates that this is exerted through specific adhesion molecule regulation of macrophage–mesothelial interactions. It highlights the importance of adhesion molecules governing entry of cells into the lymphatic circulation, thus opening a new avenue for manipulating the resolution of inflammation.

Fibroblasts Isolated from Normal Lungs and Those with Idiopathic Pulmonary Fibrosis Differ in Interleukin-6/gp130-Mediated Cell Signaling and Proliferation

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.

Pathogenesis of pleural fibrosis

Abstract:  Pleural fibrosis resembles fibrosis in other tissues and can be defined as an excessive deposition of matrix components that results in the destruction of normal pleural tissue architecture and compromised function. Pleural fibrosis may be the consequence of an organised haemorrhagic effusion, tuberculous effusion, empyema or asbestos‐related pleurisy and can manifest itself as discrete localised lesions (pleural plaques) or diffuse pleural thickening and fibrosis. Although the pathogenesis is unknown, it is likely that the complex interactions between resident and inflammatory cells, profibrotic mediators and coagulation, and fibrinolytic pathways are integral to pleural remodelling and fibrosis. It is generally considered that the primary target cell for pleural fibrosis is the subpleural fibroblast. However, increasing evidence suggests that mesothelial cells may also play a significant role in the pathogenesis of this condition, both by initiating inflammatory responses and producing matrix components. A greater understanding of the interactions between pleural and inflammatory cells, cytokines and growth factors, and blood derived proteins is required before adequate therapies can be developed to prevent pleural fibrosis from occurring.

Myeloperoxidase Deficiency Attenuates Lipopolysaccharide-Induced Acute Lung Inflammation and Subsequent Cytokine and Chemokine Production

Lung neutrophilia is common to a variety of lung diseases. The production of reactive oxygen and nitrogen species during neutrophil oxidative burst has been associated with protein and DNA damage. Myeloperoxidase (MPO) is an enzyme stored in the azurophilic granula of neutrophils. It is important in host defense because it generates the reactive oxidant hypochlorous acid and has been described to play a role in the activation of neutrophils during extravasation. We hypothesized that MPO contributes directly to the development of acute lung neutrophilia via stimulation of neutrophil extravasation and indirectly to the subsequent production of cytokines and chemokines in the lung. To test this hypothesis, wild-type (WT) and Mpo−/− mice were given a single LPS instillation, after which the development of neutrophil-dominated lung inflammation, oxidative stress, and cytokine and chemokine levels were examined. Mpo−/− mice demonstrated a decreased lung neutrophilia that peaked earlier than neutrophilia in WT mice, which can be explained by decreased neutrophil chemoattractant levels in LPS-exposed Mpo−/− compared with WT mice. However, oxidative stress levels were not different in LPS-exposed WT and Mpo−/− mice. Furthermore, in vivo findings were confirmed by in vitro studies, using isolated neutrophils. These results indicate that MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production by other cell types in the lung.

Genetic partitioning of interleukin-6 signalling in mice dissociates Stat3 from Smad3-mediated lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)‐6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)‐β signalling. We examined bleomycin‐induced inflammation and fibrosis in mice carrying a mutation in the shared IL‐6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL‐6‐mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130757F mice. The extent of fibrosis was attenuated in B lymphocyte‐deficient gp130757F;µMT−/− compound mutant mice, but fibrosis still occurred in their Smad3−/− counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF‐β/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin‐induced lung fibrosis.

Stimulation of Mesothelial Cell Proliferation by Exudate Macrophages Enhances Serosal Wound Healing in a Murine Model

Examination of thermally induced serosal lesions in mice displayed collections of inflammatory cells, predominantly macrophages, on and surrounding the wound within 48 hours of injury. Furthermore, by 2 days a large number of uninjured mesothelial cells adjacent to the wound were synthesizing DNA. From these findings, it was hypothesized that macrophages play a major role in serosal repair by stimulating mesothelial cell proliferation. Again, using a murine model of mesothelial regeneration, depletion of circulating monocytes significantly delayed serosal healing whereas addition of peritoneal exudate cells to the wound site 36 hours before injury increased the healing rate. In vivo assessment of mesothelial cell proliferation using tritiated thymidine incorporation and autoradiography demonstrated that peritoneal exudate cells stimulated mesothelial cell proliferation (12.44 +/- 1.63% labeling index, compared with controls in which medium only was used 4.48 +/- 0.71%). The mesothelial proliferation was predominantly because of macrophage-secreted products with molecular weights of 36 to 53 kd or 67 to 100 kd. These data support the hypothesis that macrophages play an important role in serosal healing by stimulating mesothelial cell proliferation.

Structure and function of mesothelial cells.

The mesothelium was first described about 180 years ago but only in the last twenty years have we begun to appreciate the roles that mesothelial cells play in maintaining normal serosal membrane integrity and function. Mesothelial cells are sentinel cells that can sense and respond to signals within their microenvironment. They secrete glycosaminoglycans and surfactant to allow the parietal and visceral serosa to slide over each other. They actively transport fluids, cells and particulates across the serosal membrane and between serosal cavities. They synthesise and secrete a diverse array of mediators in response to external signals which play important roles in regulating inflammatory, immune and tissue repair responses. In addition, they are likely to protect from peritoneal dissemination of tumours until the integrity of the mesothelium is breached. Although the importance of the mesothelial cell is being realised, we still do not understand the mechanisms regulating many of their functions. How the cells communicate with each other and surrounding cells, whether mesothelial cells differentiate into different cell types or if a mesothelial stem cell exists, the mechanisms regulating mesothelial repair and the role mesothelial cells play in serosal pathologies, all need further study. Although it has long been accepted that mesothelial cells are similar irrespective of site or species, apart from morphology, few studies have truly compared biochemical and functional characteristics of these cells between species and within different anatomical sites. In a recent study examining the effect of aging on human peritoneal mesothelial cells, there was a positive correlation between the age of the donors cells and the proinflammatory profile. Although mesothelial cells share many similarities, it is likely that functional and physiological adaptation will alter these cells. Addressing these questions are paramount if we hope to find better ways to protect serosal integrity and prevent peritoneal dissemination of tumours.