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Table of Contents
Vol. 75, No. 2, 2008
Issue release date: March 2008
Respiration 2008;75:121–133
(DOI:10.1159/000113629)

Pathophysiology of the Pleura

Jantz M.A. · Antony V.B.
Division of Pulmonary, Critical Care, and Sleep Medicine, University of Florida, Gainesville, Fla., USA
email Corresponding Author

Abstract

The pleural mesothelial cell is an essential cell in maintaining the normal homeostasis of the pleural space and it is also a central component of the pathophysiologic processes affecting the pleural space. In this review, we will review the defense mechanisms of the pleural mesothelium and changes in pleural physiology as a result of inflammatory, infectious, and malignant conditions with a focus on cytokine and chemokine networks. We will also review the processes involved in the pathogenesis of pleural fibrosis.


 Outline


 goto top of outline Key Words

  • Empyema
  • Malignant pleural effusion
  • Mesothelium
  • Pleura
  • Pleural fibrosis
  • Pleurodesis

 goto top of outline Abstract

The pleural mesothelial cell is an essential cell in maintaining the normal homeostasis of the pleural space and it is also a central component of the pathophysiologic processes affecting the pleural space. In this review, we will review the defense mechanisms of the pleural mesothelium and changes in pleural physiology as a result of inflammatory, infectious, and malignant conditions with a focus on cytokine and chemokine networks. We will also review the processes involved in the pathogenesis of pleural fibrosis.

Copyright © 2008 S. Karger AG, Basel


goto top of outline Pathophysiology of the Pleura

The pleural mesothelium is a monolayer of cells that may vary from a flattened ovoid shape to columnar or cuboidal cells that lie loosely over the underlying substructure. A unique characteristic of the pleural space is that it is a potential space within a closed environment. The connective tissue of the pleural basement membrane is a complex structure that underlies the surface layer of mesothelial cells and is involved in inflammation of the pleural space. A large meshwork of capillaries originates from the bronchial arterial vessels present in the subpleural layer. Lymphatic connections are also present in the subpleural layer and drain into the mediastinal, intercostal, and sternal lymph nodes. The volume of fluid in the pleural space is small, in the range of 0.2–0.5 ml. Normally, the protein content (and the cellular content) is low with an absence of inflammatory cells.

 

goto top of outline Defense Mechanisms of the Pleura

The pleura is intricately connected with the underlying lung parenchyma through a network of balancing cellular and humoral factors that participate in host defense of the pleural space. The pleural membrane not only serves a barrier function, but also has multiple other defense mechanisms to maintain the homeostatic balance of the pleural space. In contrast to the lung, the pleura does not interface with the external environment since the pleura encircles a closed, potential space [1]. Inflammation and changes in the delicate homeostatic balance of the pleural space can be initiated by introduction of foreign cells, proteins, microbes, blood, or air as well as by mechanical disruption of the mesothelial monolayer (fig. 1).

FIG01
Fig. 1. Illustration of the various pathophysiologic mechanisms in the pleural space. Neutrophils are recruited to the pleural space by mesothelial cell release of IL-8, while mononuclear cells (MN) are recruited through their response to mesothelial cell-derived MCP-1. Both mononuclear cells and neutrophils interdigitate with ICAM-1 expressed on the mesothelial cell through their CD11 receptors. GM-CSF, an anti-apoptotic protein released by mesothelial cells, prolongs the life span of the recruited inflammatory cells. White blood cells (WBC) may enter the pleural space through gap junctions between cells following downregulation through phosphorylation of β-catenin (β-Cat) and n-cadherins (N-Cad). VEGF and nitric oxide (NO) are released in response to bacterial and mycobacterial presence, and permeability of the pleura is modulated via activation of hypoxia-inducible factor-1α (HIF-1α). Bacteria such as Staphylococcus aureus and mycobacteria (Mycobacterium tuberculosis and Bacillus Calmette-Guérin) interact with TLR2 via MyD88 to increase production of inflammatory gene regulators such as NF-κB and activator protein-1 (AP-1). Cancer cells can invade the pleura via changes in gap junctions mediated by alterations in β-catenin and n-cadherin with growth supported by elucidation of VEGF. Sialomucins provide mechanical repulsion to pleural invasion by microbes and malignant cells. Endostatin (Ends) is released by the mesothelial cell as a defense mechanism against invading malignant cells. PMN = Polymorphonuclear neutrophils.

The innate immunity of the pleura is seen early during inflammation, within the first few hours following an insult to the pleural space [2]. Much of the innate immunity of the pleura is provided by the multipotent pleural mesothelial cell that completely lines the pleural space. The pleural mesothelial cell not only recognizes the offending organism, but subsequently initiates the process of inflammatory response and coordinates the perpetuation of the inflammatory changes. These inflammatory responses may differ depending on the invading microbe or cell. Malignant cells must be recognized as foreign in spite of the development of multiple factors that allow malignant cells to present themselves as innocuous agents which can freely enter the pleural space.

The free surface of the mesothelium is covered by glycoconjugates, which consist of pleural mesothelial cell-associated sialomucins [3]. These mesothelial cell-associated sialomucins are strong anionic sites that, in essence, coat the pleural surface with a negative charge and act to repulse the presence of abnormal cells, organisms, and particles. Not only do these glycoproteins electrostatically repel the opposing pleural membrane because of the strong negative charges, they also provide a second level of mechanical repulsion to invading cells, microbes, and particulates [4, 5]. The presence of these sialomucins on the surface of the mesothelial cells allow the mesothelium to function mechanically as a ‘Teflon’-coated surface rather than a ‘Velcro-like’ surface. Removal of the sialomucins from the surface of the mesothelium increases the adherence of bacteria and inflammatory cells. The pleural membrane is bathed in secretions with antimicrobial properties such as lysozyme. Normal pleural fluid contains immunoglobulins, principally IgG and IgA. The pleural fluid also contains complement. Complement activation can lead to microbial lysis and may also play a role in the amplification of inflammation with cytokine production and increased phagocytosis of cells. In addition, mesothelial cells produce fibronectin, i.e. a large glycoprotein that prevents adherence of organisms such as Pseudomonas aeruginosa.

One of the innate responses of the pleural mesothelium is the release of reactive oxygen species and reactive nitrogen intermediates. The nitric oxide radical is a diatomic molecule containing an unpaired electron that permits it to react with other molecules. The reaction of the nitric oxide radical with superoxide anion leads to the formation of peroxynitrite anion and peroxynitrous acid. The inducible form of nitric oxide synthase is capable of producing micromolar quantities of nitric oxide radicals over a prolonged period of time. Pleural mesothelial cells produce large quantities of nitric oxide radicals in response to the stimulation by cytokines, lipopolysaccharide (LPS), and particulates [6, 7]. Inducible nitric oxide synthase may thus contribute to the control of infections in the pleural space and may be involved in pleural inflammation from other insults.

The mesothelial cell is pivotal in recruiting additional inflammatory cells as a second line of defense as well as maintaining its own response against inflammatory changes in the pleural space. Mesothelial cells have multiple pattern recognition receptors, which recognize the carbohydrate residua of microbial metabolism and LPS on the surface of some microbial pathogens. The innate immune system of the mesothelial cell also recognizes pathogen-associated molecular patterns, which can then initiate multiple levels of defense mechanisms [8]. Some of these pattern recognition receptors include CD14, integrins, and the mannose receptor. Inflammatory responses initiated by the pleural mesothelial cell include release of chemokines to recruit neutrophils, mononuclear cells, and lymphocytes as well as the release of cytokines such as interleukin (IL)-1, IL-6, and interferons (IFNs), which function as costimulators of T cells. T-cell-independent mesothelial responses are initiated following phagocytosis of microbes and particulate material, e.g. asbestos fibers, with subsequent release of IL-12 and tumor necrosis factor (TNF)-α. These cytokines are involved in the proinflammatory response.

Acquired immunity involves the T- and B-cell lymphocyte components of the immune system with expression of distinct antigenic receptors [9, 10]. Activated T lymphocytes participate in the orchestration of specific immune responses in the pleural space. Mesothelial cells contribute to the cytokine networks that allow for undifferentiated T lymphocytes to become T-helper (Th)-1 or Th2-type cells that subsequently direct different inflammatory responses in the pleural space [7]. Thus, the defense mechanisms of the pleura include functions starting from providing a mechanical barrier to invasion as well as a sophisticated, multilayered, and coordinated system of cytokine and inflammatory cell recruitment.

 

goto top of outline Cytokine Networks in the Pleural Space

Cytokines are polypeptide structures with multiple biological functions and are key effectors for the process of initiation, perpetuation, and resolution of the inflammatory responses of the pleura. Cytokines do not act alone, but form a multitiered, interconnected network that establishes communication between cells and allows for an orchestrated inflammatory cascade of events critical to the inflammatory response [11, 12]. Accumulation of inflammatory cells and fluid in the pleural space is a classic response of pleural inflammation. The mesothelial cell is known to produce multiple chemokines [13]. The chemokine family is named according to the location of the amino-terminal cysteine residue with a C, C-C, C-X-C, or CCXXXC motif. Pleural mesothelial cells release chemokines from all of the member family groups. The C-X-C chemokines, such as IL-8, have been well characterized. These chemokines are critical in neutrophil chemotaxis and activation. The C-X-C chemokines include those that contain an amino acid residue that precedes the first N-terminal cysteine residue. For the majority of the C-X-C chemokines, the amino acids are GLU-LEU-ARG, otherwise known as the ELR motif. The presence or absence of the ELR motif appears to be critical in determining the function of the C-X-C chemokines. The chemokines that lack the ELR motif are not potent neutrophil chemotaxins and demonstrate angiostatic properties, whereas those containing an ELR motif, such as IL-8, are chemotactic for neutrophils and are also angiogenic [14]. The C-C group of chemokines is defined by the position of the first two end-terminal cysteines. These include macrophage inflammatory protein (MIP)-1α, -1β, and -1γ; macrophage chemoattractant protein (MCP)-1, -2, -3, and -4; and RANTES (regulated upon activation in normal T cells, expressed, and secreted protein). The C-C chemokine lymphotaxin is important for the recruitment of lymphocytes to the pleural space. Mesothelial cells have also been described to produce fractalkine. Fractalkine mediates chemotaxis, cell adhesion in the absence of substrates for other adhesion molecules, and activates natural killer (NK) cells, leading to increased cytotoxicity and IFN-γ production.

 

goto top of outline Cytokine Networks during Acute Infections

As noted, infectious pathogens express a set of molecules called pathogen-associated molecular patterns (PAMPs). These microbial molecular markers may be composed of proteins, carbohydrates, lipids, or nucleic acids and may be intracellular or surface bound [15]. Such PAMP markers include LPS, bacterial lipoproteins, lipoteichoic acids of gram-positive bacteria, bacterial cell wall peptidoglycans, cell wall components of fungi and mycobacteria, bacterial DNA, and viral RNA [16]. The innate immune system takes advantage of the expression of PAMPs to recognize the pathogen with the help of pattern recognition receptors such as the Toll-like receptors (TLRs) [17]. Eleven TLRs, TLRs 1–11, have been identified in mammals to date. Binding of the PAMP with the respective TLR initiates downstream signaling with production of various cytokines, chemokines, and peptides with antimicrobial activity. TLR binding generates a signal through an adaptor molecule (MyD88) that leads to intracellular association with IL-1 receptor-associated kinase which then causes activation of TNF receptor-associated factor 6. This results in nuclear translocation of nuclear factor κB (NF-κB). NF-κB is an important transcription factor that activates the promoters of the genes for cytokines and other proinflammatory products such as TNF-α, IL-1, IL-6, and IL-8 [16]. TLRs also induce the production of cytokines such as IL-12 and IL-18 from antigen-presenting cells. Our group has recently demonstrated that mouse pleural mesothelial cells constitutively express TLR1–9 messenger RNA, particularly TLR4, and, after exposure to staphylococcal peptidoglycan, the pleural mesothelial cells upregulated expression of TLR2 as well as expression of the antimicrobial peptide β-defensin-2 [unpubl. data]. This was associated with an increase in p38 mitogen-activated protein kinase activity. Defensins are small cationic peptides with antimicrobial function and are part of the innate immunity system. In addition to its role in innate immunity, human β-defensin-2 promotes adaptive immune responses by recruiting dendritic cells and T lymphocytes to sites of microbial invasion and as a chemoattractant for neutrophils [18, 19]. Elevated human β-defensin-2 levels have been noted in pleural fluids from patients with empyema [20]. These results suggest that pleural mesothelial cells are able to recognize and mount an immune response against a wide variety of infectious agents.

Accumulation of neutrophils and mononuclear phagocytes is a characteristic feature of parapneumonic effusions. IL-8 is found in significant quantities in pleural fluids obtained from patients with parapneumonic effusions. Pleural fluid from patients with uncomplicated parapneumonic effusions as well as empyemas is chemotactic for neutrophils and contains higher levels of IL-8 than pleural effusions from patients with malignancy, tuberculosis, or heart failure [21]. In patients with uncomplicated parapneumonic effusions, levels of epithelial neutrophil-activating protein-78 (ENA-78) correlated with neutrophil counts and the correlation of neutrophil counts with ENA-78 levels was greater than that observed with IL-8 levels. This chemokine is also known to have specific chemotactic activity for neutrophils. Broaddus et al. [22], using an endotoxin model of pleurisy, demonstrated that inhibition of neutrophil entry into the pleural space was mediated by antibodies to rabbit IL-8. Other early inflammatory mediators released by mesothelial cells include IL-1β and TNF-α. In acute inflammation, the early response of the pleural mesothelial cell leads to a widening of the inflammatory changes with recruitment of several phagocytic cells that release additional cytokines which communicate back to the resident mesothelial cell. In the exudative stage of parapneumonic effusions, the pleural fluid is still free flowing. During the fibrinopurulent stage, there is further movement of inflammatory cells and bacteria into the pleural space and other cytokine networks appear to be initiated.

Pleural mesothelial cells release growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, and fibroblast growth factor (FGF). These factors are known to be mitogenic for fibroblasts and are also angiogenic. These growth factors are putatively thought to induce walling off of the pleural space in a fibrous peel with development of new capillaries and neovascularization of the injured mesothelium. This allows for an increased influx of inflammatory cells into the area to prevent further spread of the infection to other areas. In effect, the pleural space is transformed into an abscess cavity.

 

goto top of outline Cytokine Networks in Granulomatous Disease of the Pleura

Tuberculosis is a classic example of a pleural granulomatous response. The pleural granulomatous response includes the development of cytokine networks that drive the Th1/Th2 responses. Early during the course of granulomatous disease there is a neutrophil-predominant response [23], but the more persistent response is that of mononuclear phagocytes that engulf mycobacteria, resulting in a coalescence of mononuclear cells into granulomas. Mesothelial cells release members of the C-C chemokine family, including MCP-1. MCP-1 is a member of the supergene family that has been demonstrated in tuberculous pleural fluids. Tuberculous pleural fluids have also been described to contain MIP-1α, a specific chemokine for monocytes.

IFN-γ, a critical cytokine for the recruitment of mononuclear cells, is also present in pleural fluids of patients with granulomatous inflammation [24]. Mesothelial cells produce IL-12, which drives the Th lymphocyte response towards the Th1 cytokines including IL-4. Neutralization of the IFN-γ response in the pleural space causes abrogation of the development of granulomas. IFN-γ augments cytokine and chemokine production by local cells and allows for a significant increase in MCP-1 and MIP-1 production by mesothelial cells.

IFN-γ upregulates antimicrobial, phagocytic, and T-cell-activating functions as well as nitric oxide release by mesothelial cells [6]. Nitric oxide is part of the antimicrobial mechanism of mesothelial cells that results in the killing of mycobacteria as well as increased production of other oxidants such as hydrogen peroxide and superoxide anion. IFN-γ itself is regulated by other cytokines, such as TNF-α, which act synergistically with IFN-γ in macrophage activation. IL-12 also functions with TNF-α, IL-1β, IL-15, and IL-18 in producing optimal IFN-γ expression. In patients with human immunodeficiency virus infection, there is a disruption of the cytokine network in the pleural space with disastrous results for the patients. Cytokines such as IL-10 are present in significant quantities in the pleural fluid of patients with disseminated tuberculosis and can prevent critical Th1-type responses from functioning. This leads to poor granuloma formation and dissemination of the disease.

 

goto top of outline Changes in Pleural Cell Populations

The pleural mesothelial cell is the most common cell of the pleural space. It is also the primary cell that initiates responses to noxious stimuli [7]. The mesothelial cell is a metabolically active cell that maintains a dynamic state of homeostasis in the pleural space until provoked. It is actively phagocytic and is capable of producing several cytokines, as mentioned above [13]. Mesothelial cells are ciliated and have multiple tight intercellular adherens junctions as well as focal adhesions that anchor the mesothelial cell onto the extracellular membrane via integrins. Mesothelial cell expression of adhesion molecules such as the intercellular adhesion molecules (ICAMs) and selectins (e.g. L-, P-, and E-selectin) as well as CD44 come into play during the movement of cells into the pleural space. ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), and VCAM-1 (CD106) are upregulated on mesothelial cells during the transfer of neutrophils, mononuclear cells, and lymphocytes into the pleural space [13].

When injured, mesothelial cells respond via proliferation and chemotaxis to cover areas of denuded extracellular matrix. This proliferative and chemotactic response is mediated in part by an autocrine response to the production of chemokines in the local area of injury. Mesothelial cells also maintain both juxtacrine and paracrine communications between cells to allow for a rapid response during inflammation.

goto top of outline Neutrophils

Neutrophils are the first cells to respond during inflammation [25]. During inflammation, these cells move from the vascular compartment into the pleural space and form the first line of inflammatory cell defense against invading organisms or particulates such as asbestos [26]. A significantly large number of neutrophils are found in the lung vasculature. During inflammation, neutrophils move out of the vascular compartment and into the pleural space using the adhesion molecule ICAM-1 on mesothelial surfaces to interdigitate with the CD11/CD18 ligands on their surfaces [27]. The primary function of neutrophils in the pleural space is phagocytosis and bacterial killing [28]. They have potent antibacterial defense mechanisms such as release of oxidants and proteases [29]. Neutropenic animals with empyema are unable to clear bacteria and develop disseminated disease. The eventual fate of neutrophils in the pleural space during acute inflammatory events such as empyema is unclear. Neutrophils are cleared from the pleural space by macrophages that engulf apoptotic cells. Mesothelial cells regulate the process of apoptosis of neutrophils via production of granulocyte-macrophage colony stimulating factor (GM-CSF), and thus manipulate the life span of the neutrophil in the pleural space [30,31,32].

goto top of outline Lymphocytes

The mesothelial cell releases several chemokines which are directed at lymphocyte recruitment into the pleural space. These chemokines include MCP-1, MCP-2, MCP-3, and RANTES. Both B- and T-cell lymphocytes are found during inflammatory disease. T-cell lymphocytes are common in granulomatous disease, while both T- and B-cell lymphocytes are found in malignant pleural effusions and in effusions caused by inflammatory processes such as systemic lupus erythematosus and rheumatoid disease [33]. Pleural fluids from patients with tuberculosis contain NK cells as well as γ/δ T cells which are critical for responses against mycobacteria. The T lymphocytes are divided into CD4 and CD8 lymphocytes. The CD4 lymphocytes predominate in diseases such as tuberculosis while CD8 lymphocytes predominate in diseases such as lymphoma. Activated T lymphocytes in the pleural space release multiple cytokines [34]. Th1 cells produce IFN-γ and IL-12 while Th2 cells produce IL-4, IL-5, IL-10, and IL-13. CD8 T lymphocytes can function as specific cytotoxic cells, while NK cells can regulate B-cell function. NK cells also play a role in host defense against viral infections and malignant cells. Activated NK cells are an important source of INF-γ, which limits tumor angiogenesis and promotes the development of specific protective immune responses [35].

goto top of outline Fibroblasts

Pleural fibroblasts, though not present in large numbers under normal conditions, are often recruited to the pleural space when there is denudation of the mesothelium. Fibroblasts are known to produce collagen and can release several cytokines and chemokines which can then perpetuate the inflammatory process [36]. The proliferation of fibroblasts is determined by the environment present in the pleural milieu. Potent mitogens for fibroblasts include PDGF, FGF, and epithelial growth factor (EGF). Inhibitors of fibroblast growth include prostaglandin E2, which can be produced by pleural mesothelial cells [37]. IFN-γ may have either an inhibitory or a stimulatory effect on the growth of pleural fibroblasts. Fibroblasts derived from different tissue sites display distinct morphological, structural, and functional characteristics. Pleural fibroblasts appear to have specific functions and demonstrate specific behavior in response to injury of the overlying extracellular matrix and cells [38].

 

goto top of outline Malignant Pleural Effusions

Certain malignant cells demonstrate a greater predilection for the pleural space than elsewhere. Metastases from cancers of the lung, breast, stomach, and ovary are seen in greater frequency in the pleural space than metastases from other malignancies. The presence of a malignant cell in the pleural space indicates that the malignant cell has overcome the pleural defense mechanisms to localize in the pleural space. Malignant cells have a large armamentarium of mechanisms whereby they can present themselves as innocuous cells to the mesothelial cellular environment [39]. One such mechanism is the use of receptors for CD44, one ligand for which is hyaluronan [40]. Hyaluronan is produced in significant quantities by the pleural mesothelial cell and interdigitates with the mesothelial cell [41]. The CD44-hyaluronan complex is internalized by malignant cells and broken down by hydrolysis to several low-molecular-weight oligosaccharides. The oligosaccharides are chemotactic for malignant cells as well as angiogenic and increase the permeability of the mesothelial monolayer. Low-molecular-weight hyaluronan also induces malignant mesothelioma cell proliferation and haptotaxis via interaction of the CD44 receptor [42].

Angiogenesis is critical for the ability of the malignant cells to develop an environment surrounded by blood vessels through which the malignant cells can be nourished. Malignant cells can produce multiple cytokines including vascular endothelial growth factor (VEGF) and basic FGF (bFGF), which are angiogenic and increase the permeability of the tissues around the malignant cells to allow for growth of new capillaries and neovascularization of the pleural surface. In addition, the cancer cells can induce pleural mesothelial cells to release VEGF [43]. This leads to establishment of tumor implants on the pleural surface and independent growth of the tumor by eluding the defense mechanisms of the pleura. Malignant cells can also produce autocrine growth factors.

Malignant mesothelioma cells exhibit dysregulated growth and produce a variety of growth factors. Malignant mesothelioma cells express a number of receptor tyrosine kinases including epidermal growth factor receptor, c-MET, PDGF receptor, and VEGF receptor. The expression of Eph receptors on malignant mesothelioma cells, specifically EphA2, has recently been described by our group [44]. The Eph transmembrane tyrosine kinases constitute the largest family of receptor tyrosine kinases. The Eph receptors are capable of recognizing signals from the cell microenvironment and influencing cell-cell interaction and migration. The Eph receptors are divided into two classes, A and B, based on structure and binding affinity. Currently, 14 Eph receptors and 8 ephrin ligands are recognized in humans. EphA2 receptor overexpression has been implicated in tumor growth, angiogenesis, and metastasis, and EphA2 overexpression has been noted in aggressive malignancies [45,46,47,48]. In our initial study of malignant mesothelioma cells, high expression of EphA2 was noted in all three malignant mesothelioma cell lines tested while low-level expression was observed in pleural mesothelial cells. Overexpression of EphA2 by transfecting the malignant mesothelial cell lines with pcDNA/EphA2 plasmids significantly increased the haptotactic migration of the malignant mesothelioma cells compared to nontransfected cells. Transfection of the malignant mesothelioma cells with small interfering RNA produced downregulation of EphA2 expression, inhibition of cell proliferation and haptotactic migration, and induction of apoptosis through caspase-9 activation [44]. In a subsequent study, we observed that activation of the EphA2 receptor by its ligand ephrinA1 downregulates total EphA2 expression via phosphorylation and suppressed growth of malignant mesothelioma cells via ERK1/2 signaling [49].

Endostatin, an inhibitor of angiogenesis, is released by normal cells and tissues. Endostatin inhibits endothelial cell migration, induces cell cycle arrest and apoptosis, and reduces tumor growth [50]. Endostatin is one potential defense mechanism of the pleural mesothelium against invading malignant cells. We evaluated endostatin levels in pleural fluid samples from patients with effusions due to malignancy and congestive heart failure [51]. Endostatin expression by pleural mesothelial cells was also demonstrated by Western analysis and confocal microscopy. The pleural fluids from patients with congestive heart failure contained significantly higher levels of endostatin when compared with fluids from patients with malignant pleural effusions. Pleural mesothelial cells alone released a significantly greater amount of endostatin when compared with ovarian cancer cells. When the pleural mesothelial cells were cocultured with ovarian cancer cells without contact, there was an increase in the endostatin production. However, when the pleural mesothelial cells were cocultured in direct contact with ovarian cancer cells, the endostatin levels significantly decreased. Endostatin production was upregulated in the presence of tumor cells but not when ovarian cancer cells were adherent to the underlying pleural mesothelial cell monolayer. These findings suggest that pleural mesothelial cells play a key role in the anti-angiogenic process by producing endostatin in the pleural space, and thus attempting to prevent tumor spread and metastasis in the pleural space.

Talc insufflation has been noted to induce pleural mesothelial cells to release endostatin [52]. The biological effects of pleural fluids and conditioned media from talc-activated pleural mesothelial cells on endothelial cells were evaluated by performing proliferation, invasion, tube formation and apoptosis assays. Pleural fluids from patients with malignant pleural effusions who received thoracoscopic talc insufflation contained significantly higher levels of endostatin compared with pre-talc instillation. Talc-activated pleural mesothelial cells released significantly greater amounts of endostatin when compared with a malignant mesothelioma cell line treated with talc. Pleural fluid from patients who received talc insufflation as well as culture supernatants from talc-activated pleural mesothelial cells significantly inhibited the proliferation of human umbilical vein endothelial cells and, in addition, inhibited endothelial cell invasion and endothelial cell tube formation as compared to pleural mesothelial cell conditioned media. Pleural fluids collected after talc insufflation and conditioned media from talc-activated pleural mesothelial cells were also noted to induce apoptosis in human umbilical vein endothelial cells. Thus, talc appears to alter the angiogenic balance in the pleural space from a biologically active and angiogenic environment to a more angiostatic milieu.

 

goto top of outline Pleural Effusion Formation during Inflammation

One of the hallmarks of an inflammatory process in the pleural space is the development of an exudative pleural effusion. Pleural inflammation is not only associated with an influx of a large number of inflammatory cells, but also with a transfer of proteins and a change in pleural permeability. Individual pleural mesothelial cells are linked together into a tight membrane by connecting intracellular proteins at key areas called adherens junctions. Activation of the pleural mesothelial monolayer by malignant cells, bacteria, or cytokines causes a breach in the integrity of the pleura and results in altered shape and gap formation between mesothelial cells, leakage of protein and fluids, and movement of phagocytic cells into the pleural space. Vascular permeability factor, also commonly known as VEGF, is upregulated in mesothelial cells when they are activated [53]. VEGF has been found in large quantities in parapneumonic effusions as well as malignant pleural effusions [54,55,56]. VEGF is a 35- to 45-kDa dimeric polypeptide expressed in several isoforms resulting from alternative mRNA splicing of a single gene and is now recognized to be a pivotal permeability and angiogenic factor mediating neovascularization under many conditions [54].

Adherens junction proteins, namely cadherins and catenins, are transmembrane proteins that function as a zipper between cells, allowing a change in permeability to occur via signaling mechanisms that lead to contraction of the intracellular actin cytoskeletal filaments and to gap formation between mesothelial cells [57]. A major cadherin in pleural mesothelial cells is neural cadherin (n-cadherin). When adherens junctions are stabilized as in tightly confluent cells, the majority of n-cadherin loses tyrosine phosphorylation and combines with plakoglobin and actin [58]. However, when cells have weakened junctions, n-cadherin is heavily phosphorylated in tyrosine, and there is decreased expression of β-catenin, too [59].

Thus, n-cadherin and β-catenin are critical determinants of mesothelial paracellular permeability. This interaction is a dynamic one since this permeability is also reversible. VEGF induces tyrosine phosphorylation of adherens junction proteins to increase paracellular permeability. During the formation of a pleural effusion, not only can cells migrate via the interaction of surface ligands for intercellular molecules expressed on mesothelial cells, but this also allows proteins of high molecular weight to leak across the pleural membrane. Exposure of pleural mesothelial monolayers to malignant cells or organisms leads to mesothelial barrier dysfunction.

 

goto top of outline Resolution of Pleural Inflammation

The resolution of a pleural inflammatory process is dependent on multiple factors, but primarily on the neutralization of the inciting agent. In infections secondary to bacteria, mycobacteria, or viruses, for example, death of the organism and eventual clearance of the microbial products from the pleural space is associated with resolution. However, in malignant pleural effusions, this may not occur on its own without the use of chemotherapeutic options. For example, in diseases such as metastatic small-cell carcinoma, chemotherapy can cause a rapid decrease and resolution of malignant pleural effusions, while in mesothelioma or non-small-cell lung cancer effective resolution of the pleural effusion is much more difficult. Interestingly, the pleural mesothelial cell plays a role during the process of resolution of acute inflammatory diseases such as empyema. GM-CSF is known to prolong the life span of leukocytes by inhibition of apoptosis [30]. High levels of GM-CSF are found in parapneumonic effusions and in empyema [31]. Mesothelial cells are also shown to undergo apoptosis when stimulated with live bacteria, but not dead organisms. Inflammation of the pleural surface may resolve with fibrosis or without fibrosis. The cytokine networks that move the resolution of inflammation towards fibrosis are not clear. However, it is apparent that resolution without fibrosis requires regeneration of a normal mesothelial surface following injury and denudation, while repair with fibrosis involves the production and proliferation of fibroblasts.

 

goto top of outline Pleural Fibrosis

As noted, the mesothelial cell plays a critical role in the initiation of inflammatory responses in the pleural space because it is the first cell to recognize a perturbation in the pleural space. Pleural inflammation is not only associated with an influx of a large number of inflammatory cells but also with a transfer of proteins and a change in the permeability of the pleura. The pleural mesothelial cells release cytokines in a polar fashion, with a high concentration being released on the apical surface, which leads to directed migration of leukocytes into the pleural space. In addition to the release from mesothelial cells, a number of cytokines are also released from the inflammatory cells recruited to the pleural space. We will examine some of these cytokines that may play a part in the pathogenesis of pleural fibrosis. We will also discuss the role of disordered fibrin turnover in the development of pleural fibrosis.

goto top of outline Transforming Growth Factor-β

Transforming growth factor-β (TGF-β) is a family of multifunctional growth-modulating cytokines. Virtually all cells, including mesothelial cells, can produce and have receptors for TGF-β. Overproduction of TGF-β is the principal abnormality in most fibrotic diseases and elevated levels of TGF-β have been found in pleural effusions [60]. TGF-β regulates a number of cellular processes, including cell proliferation, cell migration, cell differentiation, and extracellular matrix production. It is a potent chemoattractant for fibroblasts which are important in collagen synthesis and pleural fibrosis [61]. Mesothelial cells also participate in extracellular matrix turnover. Following stimulation by TGF-β, mesothelial cells can synthesize collagen, matrix proteins, matrix metalloproteinase-1 and -9, and tissue inhibitor of matrix metalloproteinases-2 [62, 63]. TGF-β suppresses fibrinolysis by reducing tissue plasminogen activators as well as increasing the mesothelial cell production of plasminogen activator inhibitor (PAI)-1 and PAI-2 [64, 65]. TGF-β has been demonstrated to be present in high levels in empyema, tuberculous pleuritis, and asbestos-related pleural effusions, suggesting a role in the pleural fibrosis associated with these conditions [66,67,68]. In addition, intrapleural administration of TGF-β has been demonstrated to induce pleurodesis, i.e. pleural fibrosis, in animal models [69, 70].

goto top of outline Basic Fibroblast Growth Factor

bFGF, also known as FGF-2, is one of the FGF families. bFGF is known to stimulate mesothelial cell proliferation in vitro and in vivo [71]. It is mitogenic for fibroblasts, smooth muscle cells, and endothelial cells, and, in addition, is also a known angiogenic factor [72,73,74]. bFGF is present in pleural effusions of various etiologies [75, 76]. In a recent study, bFGF levels were higher in the pleural fluid of patients who underwent successful talc pleurodesis compared to those who failed talc pleurodesis or had thoracoscopy alone without talc pleurodesis [77]. In this study, the addition of bFGF antibody to the pleural fluids obtained from these patients caused a significant decrease in fibroblast growth activity. Mesothelial cells stimulated with talc were noted to release higher amounts of bFGF when compared to controls [77].

goto top of outline Platelet-Derived Growth Factor

PDGF is a mitogenic cytokine for mesothelial cells [78]. Mesothelial cells are known to produce PDGF [79]. PDGF can also promote the growth of fibroblasts and stimulates hyaluronan production in fibroblasts and mesothelial cells [80, 81]. In addition, PDGF can stimulate collagen production by mesothelial cells. PDGF has been demonstrated to be an important mediator of fibroblast proliferation in the pleura in response to inhaled crocidolite asbestos fibers in rodent models. Antibodies against PDGF inhibit fibroblast proliferation in these models [82]. Lastly, PDGF also induces the expression of TGF-β, further potentiating the fibrotic response [83].

goto top of outline Disordered Fibrin Turnover

During the process of wound healing, formation of a transitional fibrin neomatrix contributes to tissue organization and fibrotic repair. It has been proposed that disordered fibrin turnover plays a central role in the pathogenesis of pleural fibrosis [84]. The extravascular deposition of fibrin that occurs along the parietal and visceral pleural surfaces is a marker of early pleural injury. As a result of pleural injury and increased microvascular permeability, plasma is extravasated into the tissue or body compartment. Coagulation at the site of injury is initiated by tissue factor forming a complex with activated factor VII and resultant formation of transitional fibrin. Remodeling of the transitional fibrin occurs through the release of proteases from inflammatory cells that invade the neomatrix. Continued formation and resorption of extravascular fibrin is facilitated by cytokines such as TNF-α and TGF-β. The mesothelial cells and recruited inflammatory cells can produce components of both the fibrinolytic system and inhibitors of the fibrinolytic system including tissue plasminogen activator, urokinase, urokinase receptor, and PAI-1. The relative expression of urokinase, which is thought to be the major plasminogen activator of extravascular fibrin in the lung, versus that of PAIs and antiplasmins, is a key determinant of local fibrinolytic activity. With ongoing remodeling rather than clearance of transitional fibrin, collagen deposition occurs which ultimately leads to progressive scarring and fibrotic repair [84, 85].

Tissue factor is locally secreted in the pleural compartment and is detectable in the pleural fluid [86]. In addition, tissue factor is also expressed by cells in the pleural compartment including mesothelial cells, macrophages, and fibroblasts [64, 87, 88]. The process of coagulation in the pleural space is regulated by concurrent expression of tissue factor pathway inhibitor (TFPI) [86]. Pleural mesothelial cells elaborate both tissue factor as well as TFPI [89]. In the setting of pleural injury, the intrapleural elaboration of tissue factor appears to exceed that of TFPI given the intrapleural fibrin deposition that is observed with pleural inflammation. Intrapleural coagulation has been demonstrated to be upregulated in patients with exudative effusions compared to patients with effusions due to congestive heart failure [86].

The urokinase, urokinase receptor, and PAI-1 are also hypothesized to be involved in the pathogenesis of pleural injury and fibrosis. These components of the fibrinolysis system have been identified in the pleural fluid [86]. Plasminogen is present in pleural fluids and can be activated by urokinase or tissue plasminogen activator with the subsequent generation of plasmin. Both urokinase and tissue plasminogen activator are secreted by cultured human pleural mesothelial cells, and both of these molecules are detectable in pleural effusions in a free form and complexed to PAI-1 and PAI-2 [64, 86]. Tissue plasminogen activator is mainly responsible for intravascular thrombolysis while urokinase is mainly involved in extravascular proteolysis and tissue remodeling [90]. Localized generation of plasmin by urokinase, either in free form or via interaction with urokinase receptors on the cell surface, allows mesothelial cells and other cells to degrade extracellular matrix [90]. Urokinase receptors are expressed on the surface of pleural mesothelial cells, macrophages, and lung fibroblasts [91,92,93]. Both urokinase and urokinase receptor are involved in the regulation of cytokine-mediated cellular signaling and cell trafficking [94]. In addition, urokinase is a chemotaxin and a mitogen for mesothelial cells and lung fibroblasts [91, 95]. PAI-1 and PAI-2 are the major inhibitors of urokinase. They are both produced by mesothelial cells and lung fibroblasts [64]. The expression of urokinase-mediated fibrinolytic activity in pleural fluids is inhibited by PAI-1 as well as by antiplasmins [86]. Levels of PAI-1 have been noted to be markedly increased in exudative effusions compared to transudative effusions due to congestive heart failure. By inhibiting intrapleural fibrin clearance, these PAIs produce a fibrinolytic defect that leads to accelerated pleural connective tissue matrix organization and pleural fibrosis. Thus, the interplay of urokinase, urokinase receptor, and PAI responses appear to influence the processes of pleural inflammation and repair versus development of pleural fibrosis.

 

goto top of outline Pleurodesis

Pleurodesis implicates the obliteration of the pleural space and absence of defining surfaces between the parietal and visceral pleura. All pleurodesis agents aim at initiating an inflammatory response that eventually results in the development of pleural fibrosis. Talc has been demonstrated to cause release of several FGFs by pleural mesothelial cells. These include bFGF and PDGF [77]. Initiation of an inflammatory response that allows for the directed movement of the inflammation to resolve with fibrosis has been the goal (fig. 2). Interestingly, if malignant disease is advanced to the point where the pleural mesothelial surface is covered by malignant deposits so that the talc or another sclerosing agent has little interaction with the normal pleural mesothelial surface, the fibrotic response has been found to be attenuated with decreases in the amount of FGFs in the pleural fluid. This finding emphasizes the important role played by the mesothelial cell in the process of pleural fibrosis.

FIG02
Fig. 2. Mechanisms of action of talc pleurodesis. Introduction of talc particles into the pleural space produces elaboration of TGF-β and bFGF and subsequent production of connective tissue growth factor (CTGF) via the SMAD3/4 pathway. These growth factors lead to recruitment and proliferation of fibroblasts which produce pleural fibrosis and obliteration of the pleural space.


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 goto top of outline Author Contacts

Veena B. Antony, MD
Division of Pulmonary, Critical Care, and Sleep Medicine
University of Florida, 1600 SW Archer Road-Box 100225
Gainesville, FL 32610-0225 (USA)
Tel. +1 352 392 2666, Fax +1 352 392 0821, E-Mail Veena.Antony@medicine.ufl.edu


 goto top of outline Article Information

Previous article in this series: 1. Froudarakis ME: Diagnostic work-up of pleural effusions. Respiration 2008;75:4–13.

Number of Print Pages : 13
Number of Figures : 2, Number of Tables : 0, Number of References : 95


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 75, No. 2, Year 2008 (Cover Date: March 2008)

Journal Editor: Bolliger C.T. (Cape Town)
ISSN: 0025–7931 (Print), eISSN: 1423–0356 (Online)

For additional information: http://www.karger.com/RES


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

The pleural mesothelial cell is an essential cell in maintaining the normal homeostasis of the pleural space and it is also a central component of the pathophysiologic processes affecting the pleural space. In this review, we will review the defense mechanisms of the pleural mesothelium and changes in pleural physiology as a result of inflammatory, infectious, and malignant conditions with a focus on cytokine and chemokine networks. We will also review the processes involved in the pathogenesis of pleural fibrosis.



 goto top of outline Author Contacts

Veena B. Antony, MD
Division of Pulmonary, Critical Care, and Sleep Medicine
University of Florida, 1600 SW Archer Road-Box 100225
Gainesville, FL 32610-0225 (USA)
Tel. +1 352 392 2666, Fax +1 352 392 0821, E-Mail Veena.Antony@medicine.ufl.edu


 goto top of outline Article Information

Previous article in this series: 1. Froudarakis ME: Diagnostic work-up of pleural effusions. Respiration 2008;75:4–13.

Number of Print Pages : 13
Number of Figures : 2, Number of Tables : 0, Number of References : 95


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 75, No. 2, Year 2008 (Cover Date: March 2008)

Journal Editor: Bolliger C.T. (Cape Town)
ISSN: 0025–7931 (Print), eISSN: 1423–0356 (Online)

For additional information: http://www.karger.com/RES


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Wang N: Mesothelial cells in situ; in Chretien J, Bignon J, Hirsch A (eds): The Pleura in Health and Disease. New York, Dekker, 1985, pp 23–42.
  2. Medzhitov R, Janeway CA Jr: Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997;9:4–9.
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