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The aging of the population in the United States and throughout the developed world has increased morbidity and mortality attributable to lung disease, while the morbidity and mortality from other prevalent diseases has declined or remained stable. Recognizing the importance of aging in the development of lung disease, the American Thoracic Society (ATS) highlighted this topic as a core theme for the 2014 annual meeting. The relationship between aging and lung disease was discussed in several oral symposiums and poster sessions at the annual ATS meeting. In this article, we used the input gathered at the conference to develop a broad framework and perspective to stimulate basic, clinical, and translational research to understand how the aging process contributes to the onset and/or progression of lung diseases. A consistent theme that emerged from the conference was the need to apply novel, systems-based approaches to integrate a growing body of genomic, epigenomic, transcriptomic, and proteomic data and elucidate the relationship between biologic hallmarks of aging, altered lung function, and increased susceptibility to lung diseases in the older population. The challenge remains to causally link the molecular and cellular changes of aging with age-related changes in lung physiology and disease susceptibility. The purpose of this review is to stimulate further research to identify new strategies to prevent or treat age-related lung disease.

The pleural mesothelium, derived from the embryonic mesoderm, is formed by a metabolically active monolayer of cells that blanket the chest wall and lungs on the parietal and visceral surfaces, respectively. The pleura and lungs are formed as a result of an intricate relationship between the mesoderm and the endoderm during development. Mesenchymal signaling pathways such as Wnt/B-catenin, Bmp4, and sonic hedgehog appear to be quintessential for lung development. Pleural Mesothelial Cells (PMCs) are known to express Wilms tumor-1 (Wt1) gene and in lineage labeling studies of the developing embryo, PMCs were found to track into the lung parenchyma and undergo mesothelial-mesenchymal transition (MMT) to form α-smooth muscle actin (α-SMA)-positive cells of the mesenchyme and vasculature. There is definite evidence that mesothelial cells can differentiate and this seems to play an important role in pleural and parenchymal pathologies. Mesothelial cells can differentiate into adipocytes, chondrocytes, and osteoblasts; and have been shown to clonally generate fibroblasts and smooth muscle cells in murine models. This supports the possibility that they may also modulate lung injury-repair by re-activation of developmental programs in the adult reflecting an altered recapitulation of development, with implications for regenerative biology of the lung. In a mouse model of lung fibrosis using lineage-tracing studies, PMCs lost their polarity and cell-cell junctional complexes, migrated into lung parenchyma, and underwent phenotypic transition into myofibroblasts in response to the pro-fibrotic mediator, transforming growth factor-β1 (TGF-β1). However, intra-pleural heme-oxygenase-1 (HO-1) induction inhibited PMC migration after intra-tracheal fibrogenic injury. Intra-pleural fluorescein isothiocyanate labeled nanoparticles decorated with a surface antibody to mesothelin, a surface marker of mesothelial cells, migrate into the lung parenchyma with PMCs supporting a potential role for pleural based therapies to modulate pleural mesothelial activation and parenchymal disease progression.

Pleural mesothelial cells (PMCs), which are derived from the mesoderm, exhibit an extraordinary capacity to undergo phenotypic changes during development and disease. PMC transformation and trafficking has a newly defined role in idiopathic pulmonary fibrosis (IPF); however, the contribution of Wilms' tumor 1 (Wt1)-positive PMCs to the generation of pathognomonic myofibroblasts remains unclear. PMCs were obtained from IPF lung explants and healthy donor lungs that were not used for transplantation. Short hairpin Wt1-knockdown PMCs (sh Wt1) were generated with Wt1 shRNA, and morphologic and functional assays were performed in vitro. Loss of Wt1 abrogated the PMC phenotype and showed evidence of mesothelial-to-mesenchymal transition (MMT), with a reduced expression of E-cadherin and an increase in the profibrotic markers α-smooth muscle actin (α-SMA) and fibronectin, along with increased migration and contractility, compared with that of the control. Migration of PMCs in response to active transforming growth factor (TGF)-β1 was assessed by live-cell imaging with 2-photon microscopy and 3D imaging, of Wt1-EGFP transgenic mice. Lineage-tracing experiments to map the fate of Wt1(+) PMCs in mouse lung in response to TGF-β1 were also performed by using a Cre-loxP system. Our results, for the first time, demonstrate that Wt1 is necessary for the morphologic integrity of pleural membrane and that loss of Wt1 contributes to IPF via MMT of PMCs into a myofibroblast phenotype.

The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung myofibroblast differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung myofibroblast differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-β1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-β1 induced SMA-α expression. miR-145 did not affect the activity of TGF-β1, but promoted the activation of latent TGF-β1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the differentiation of lung myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.

The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-β1 (TGF-β1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-β1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1-expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of HO by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor. HO-1⁻/⁻ mice did not develop organized granuloma in their lungs, had higher lung colony forming unit of M. avium when infected with intratracheal M. avium, and had loose collections of inflammatory cells in the lung parenchyma. Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1⁺/⁺ mice. In HO-1⁻/⁻ mice, mycobacteria were also found in the liver and spleen and showed increased mortality. Peripheral blood monocytes isolated from GFP⁺ mice and given intravenously to HO-1⁺/⁺ mice localized into tight granulomas, while in HO-1⁻/⁻ mice they remained diffusely scattered in areas of parenchymal inflammation. Higher MCP-1 levels were found in bronchoalveolar lavage fluid of M. avium infected HO-1(-/-) mice and CCR2 expression was higher in HO-1⁻/⁻ alveolar macrophages when compared with HO-1⁺/⁺ mice. CCR2 expression localized to granuloma in HO-1⁺/⁺ mice but not in the HO-1⁻/⁻ mice. These findings strongly suggest that HO-1 plays a protective role in the control of M. avium infection.

Idiopathic pulmonary fibrosis (IPF) is characterised by myofibroblast proliferation leading to architectural destruction. Neither the origin nor the continued proliferation of myofibroblasts is well understood. Explanted human IPF lungs were stained by immunohistochemistry for calretinin, a marker of pleural mesothelial cells (PMCs). Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) lungs acted as controls. The number of PMCs per 100 nucleated cells and per photomicrograph was estimated along with the Ashcroft score of fibrosis. Mouse PMCs expressing green fluorescent protein (GFP) or labelled with nanoparticles were injected into the pleural space of mice given intranasal transforming growth factor (TGF)-β1. Mouse lungs were lavaged and examined for the presence of GFP, smooth muscle α-actin (α-SMA) and calretinin. Calretinin-positive PMCs were found throughout IPF lungs, but not in COPD or CF lungs. The number of PMCs correlated with the Ashcroft score. In mice, nanoparticle-laden PMCs were recoverable by bronchoalveolar lavage, depending on the TGF-β1 dose. Fluorescent staining showed α-SMA expression in GFP-expressing PMCs, with co-localisation of GFP and α-SMA. PMCs can traffic through the lung and show myofibroblast phenotypic markers. PMCs are present in IPF lungs, and their number correlates with IPF severity. Since IPF presumably begins subpleurally, PMCs could play a pathogenetic role via mesothelial-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is the most common and lethal of the idiopathic interstitial pneumonias. There are currently no effective pharmacological therapies approved for the treatment of IPF. Despite the focus on targeting fibrogenic pathways, recent clinical trials have been largely disappointing. Progress is being made in elucidating key cellular processes and molecular pathways critical to IPF pathogenesis, and this should facilitate the development of more effective therapeutics for this recalcitrant disease. Emerging pathobiological concepts include the role of aging and cellular senescence, oxidative stress, endoplasmic reticulum stress, cellular plasticity, microRNAs and mechanotransduction. Therapeutic approaches that target molecular pathways to modulate aberrant cellular phenotypes and promote tissue homeostasis in the lung must be developed. Heterogeneity in biological and clinical phenotypes of IPF warrants a personalized medicine approach to diagnosis and treatment of this lung disorder.

Receptor EphA2 over-expression is associated with the aggressive nature of growth in malignant mesothelioma (MM) and silencing EphA2 with interference RNA suppressed MM proliferation. The mechanisms associated with targeting the EphA2 gene in MM were not clear. We sought to determine whether silencing EphA2 induces apoptosis in MM cells by either extrinsic or intrinsic pathways. The receptor EphA2 signaling pathway may provide attractive therapeutic strategy for MM. Apoptosis was determined by Cell Death ELISA in MM Cells transfected with siRNA-EphA2 and control siRNA. The gene expression profile of apoptosis pathways were analyzed by GEArray. Selected genes were further studied by quantitative PCR, Western analysis, and immunofluorescence. Caspases activities were measured by fluorescence spectrometer. Silencing EphA2 expression induced apoptosis in MMC. Apoptosis was characterized by FADD expression, activated caspase-8, caspase-3 and induction of Bax, Bak, and Bid as revealed by GEArray and protein fractionation assays. The expression of FADD, Bid, caspase-8, cytochrome-c and apaf-1 were significantly higher in the cytosolic fractions of EphA2-siRNA transfected cells. Furthermore, blocking the expression of caspase-8 by an inhibitor blunted FADD expression, indicating that caspase-8 is implicated in EphA2-siRNA induced apoptosis in MMC. Our data indicates that targeting the EphA2 gene by siRNA induced both extrinsic and intrinsic apoptotic pathways in MM Cells. Receptor EphA2 inhibition may be an effective approach for inhibiting MM growth and a promising direction for MM therapy.

Rationale: Nontuberculous mycobacterial (NTM) infection is a growing problem in the United States and remains underrecognized in the developing world. The management of NTM infections is further complicated by several factors, including the need to use high systemic doses of toxic agents, the length of therapy, and the development of drug resistance., Objectives: We have evaluated the use of monocyte-derived dendritic cells (DCs) as a delivery vehicle for a luminescent derivative of amikacin prepared by conjugation to fluorescein isothiocyanate (FITC) (amikacin-FITC) into granulomas formed in the tissues of mice infected with Mycobacterium avium., Methods: Amikacin-FITC was prepared and quantitative fluorescence was used to track the intracellular uptake of this modified antibiotic. The antibiotic activity of amikacin-FITC was also determined to be comparable to unmodified amikacin against M. avium. Amikacin-FITC-loaded DCs were first primed with M. avium, and then the cells were injected into the tail vein of infected mice. After 24 hours, the mice were sacrificed and the tissues were analyzed under fluorescence microscope., Measurements and Main Results: We found that we were able to deliver amikacin into granulomas in a mouse model of disseminated mycobacterial infection. No increase in levels of monocyte chemoattractant protein-1 and its CCR2 as markers of inflammation were found when DCs were treated with amikacin-FITC., Conclusions: DC-based drug delivery may be an adjunct and useful method of delivering high local concentrations of antibiotics into mycobacterial granulomas.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis noted in the pulmonary parenchyma. Pleural mesothelial cells (PMC) are metabolically dynamic cells that cover the lung and chest wall as a monolayer and are in intimate proximity to the underlying lung parenchyma. The precise role of PMC in the pathogenesis of pulmonary parenchymal fibrosis remains to be identified. Transforming growth factor (TGF)-beta1, a cytokine known for its capacity to induce proliferative and transformative changes in lung cells, is found in significantly higher quantities in the lungs of patients with IPF. High levels of TGF-beta1 in the subpleural milieu may play a key role in the transition of normal PMC to myofibroblasts. Here we demonstrate that PMC activated by TGF-beta1 undergo epithelial-mesenchymal transition (EMT) and respond with haptotactic migration to a gradient of TGF-beta1 and that the transition of PMC to myofibroblasts is dependent on smad-2 signaling. The EMT of PMC was marked by upregulation of alpha-smooth muscle actin (alpha-SMA), fibroblast specific protein-1 (FSP-1), and collagen type I expression. Cytokeratin-8 and E-cadherin expression decreased whereas vimentin remained unchanged over time in transforming PMC. Knockdown of smad-2 gene by silencing small interfering RNA significantly suppressed the transition of PMC to myofibroblasts and significantly inhibited the PMC haptotaxis. We conclude that PMC undergo EMT when exposed to TGF-beta1, involving smad-2 signaling, and PMC may be a possible source of myofibroblasts in IPF.

Background: Gastrointestinal manifestations are frequently encountered in cystic fibrosis patients. Gastroparesis evidenced by a variety of diagnostic methods has been described in patients with cystic fibrosis, predominantly in children and in individuals with advanced lung disease. The presence of gastroparesis in adult patients with different degrees of lung involvement and its response to the acute and chronic administration of macrolides have not been reported., Methods: Using the University of Florida Cystic Fibrosis database we identified symptomatic patients who had gastroparesis confirmed by a prolonged half-time during gastric emptying scintigraphy., Results: Of 86 cystic fibrosis patients, periodically followed in our institution, we found five who had classical symptoms and prolonged gastric emptying half-time. Age 25.2+/-8 years, 80% females, BMI 22+/-9 kg/m(2), HbA1c 5.8+/-0.6 g/dl, FEV1 53.2+/-15% of predicted. Gastric emptying half-time was 191.4+/-91.4 min (range 100-300 min) and decreased to 12.2+/-6 min (range 5-20 min) after IV administration of erythromycin (p=0.043). Patients were followed up for 3+/-2.1 years. All patients but one, who was taking opiods, had good clinical response to PO macrolides., Conclusions: Gastroparesis occurs in patients with cystic fibrosis, even in patients with relatively preserved lung function and in those without cystic-fibrosis related diabetes. Macrolides may be an effective therapy in cystic fibrosis patients with gastroparesis when administered acutely or chronically.

The innate immune response is mediated in part by pattern recognition receptors including Toll-like receptors (TLRs). The pleural mesothelial cells (PMCs) that line the pleural surface are in direct contact with pleural fluid and accordingly carry the risk of exposure to infiltrating microorganisms or their components in an event of a complicated parapneumonic effusion. Here we show that murine primary PMCs constitutively express TLR-1 through TLR-9 and, upon activation with peptidoglycan (PGN), mouse PMC produce antimicrobial peptide beta-defensin-2 (mBD-2). Treatment of PMCs with staphylococcal PGN, a gram-positive bacterial cell wall component and a TLR-2 agonist, resulted in a significant increase in TLR-2 and mBD-2 expression. Silencing of TLR-2 expression by small interfering RNA led to the downregulation of PGN-induced mBD-2 expression, thereby establishing causal relationship between the activation of TLR-2 receptor and mBD-2 production. PMCs exposed to PGN showed increased p38 MAPK activity. In addition, PGN-induced mBD-2 expression was attenuated by SB203580, a p38 MAPK inhibitor, underlining the importance of p38 MAPK in mBD-2 induction. Inhibition of erk1/erk2 or phosphatidylinositol 3-kinase did not block PGN-induced mBD-2 expression in PMC. PGN-activated PMC-derived mBD-2 significantly killed Staphylococcus aureus, and mBD-2-neutralizing antibodies blunted this antimicrobial activity. Taken together, these data indicate that PMCs may contribute to host innate immune defense upon exposure to gram-positive bacteria or their products within the pleural space by upregulating TLR-2 and mBD-2 expression.

Primary graft dysfunction and rejection are common complications in lung transplant recipients. Increased expression of thioredoxin-1 (Trx), a 12-kDa redox-regulatory protein, has been reported in multiple lung pathophysiological conditions involving oxidative and inflammatory mediated injury including graft rejection in canine and rat models of lung transplantation. Our objective was to determine whether increased Trx expression is associated with progression of rejection pathophysiology in human lung transplant recipients. Bronchoalveolar lavage (BAL) fluid and transbronchial biopsy samples were collected as a routine part of post-transplant clinical care from 18 lung transplant patients from our adult lung transplant programme. Lung transplant recipient profile included age/sex, ethnic background, days on ventilator, total ischaemic time, and cytomegalovirus (CMV) status. Based on histopathological grading criteria, patients were divided into two groups, rejecting (A1/A2 or B1) and non-rejecting (A0/B0). Rejecting and non-rejecting group total BAL cell counts and differential cell counts for neutrophils, macrophages, lymphocytes and eosinophils as well as total BAL cell Trx levels were analysed. Total BAL cell counts were significantly (p <0.05) elevated in graft rejecting versus non-rejecting patients. Differential BAL macrophage counts were comparable in rejection and non-rejection groups, whereas there were significant increases in neutrophils and lymphocytes but not eosinophils in patients with rejection versus non-rejection pathology (p <0.05). Total ischaemic time and days on ventilator in rejection and non-rejection groups were comparable. However, Trx levels were significantly elevated in BAL cells from graft-rejecting patients compared with non-rejecting patients (p <0.05). These data suggest that surveillance monitoring of BAL Trx levels after lung transplantation can serve as a biomarker to assess severity of graft rejection.

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 (c) 2008 S. Karger AG, Basel)

The objective of this study was to understand the possible mechanisms of activation of receptor EphA2 by its ligand ephrinA1 in malignant mesothelioma cell (MMC) growth. Activation of receptor EphA2 by its ligand ephrinA1 triggered the phosphorylation of EphA2. Ligand activation of EphA2 also induced phosphorylation of ERK1/2 and significantly decreased MMC proliferation. Ligand activated and ephrinA1 vector (pcDNA/EFNA1) transfected MMC demonstrated decreased clonal growth in 3-D matrigels when compared to resting MMC. These studies suggest that EphA2 activation by its ligand ephrinA1 transmits intracellular signals from cell membrane to nucleus via ERK1/2 signaling cascade and inhibits MM growth.

In bacterial empyema the pleural mesothelium is constantly exposed to microorganisms. Staphylococcus aureus (S. aureus) is one of the most frequent pathogens associated with empyema. In an earlier study we demonstrated that S. aureus induced barrier dysfunction in pleural mesothelial cell monolayers. In the present study we report that S. aureus activates the early response genes c-fos and c-jun and activator protein-1 (AP-1), and induces proapoptosis genes Bad and Bak in primary mouse pleural mesothelial cells (PMCs). Our data indicate that in PMCs S. aureus induces apoptosis in a time- and multiplicity of infection (MOI)-dependent manner. Staphylococcus aureus induced Bcl (2), Bcl-X (L), c-fos, c-jun, and AP-1 expression in PMCs during the initial phase of infection. In S. aureus-infected PMCs, Bad and Bak gene expression was increased and correlated with DNA fragmentation and cytochrome-c release. Bcl (2) and Bcl-X (L) gene expression was significantly lower in S. aureus-infected PMCs than in uninfected PMCs 12 h postinfection. We conclude that at the initial stage of infection S. aureus modulates expression of early response genes c-fos and c-jun, and in the late phase of infection S. aureus induces expression of proapoptotic genes Bak and Bad in PMCs. Silencing AP-1 significantly inhibited S. aureus-induced Bak and Bad expression in PMCs. The upregulation of early response genes during the early phase of infection may contribute to the activation of proapoptotic genes Bak and Bad and release of cytochrome-c, caspase-3 thereby resulting in apoptosis in PMCs.

Bronchial airway epithelial cells (BAEpC) are among the first cells to encounter M. tuberculosis following airborne infection. However, the response of BAEpC to M. tuberculosis infection has been little studied. This study investigates the response of a human BAEpC cell line (BEAS-2B) to infection with Mycobacterium bovis Bacille Calmette Guerin (BCG). Cultured human BEAS-2B cells were experimentally infected with BCG. Uninfected BEAS-2B cultures were included as controls. Following infection, BEAS-2B cells were evaluated by various methods at various time points up to 3 days. Cell proliferation was evaluated by cellular bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Distribution of cells along the cell cycle was evaluated by FACS analysis of cellular DNA. Apoptotic cells were identified by cell death ELISA and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method. Eighty-four apoptosis-relevant genes were screened by PCR gene microarray. Translation of Fas, Fas ligand (Fas-L), and Fas-associated death domain (FADD) were evaluated quantitatively by real-time PCR. Expression of Fas and FADD proteins was evaluated by immunofluorescence and Western blot. Activity of caspase-3 and caspase-8 was evaluated by colorimetric assay of their enzymatic activity. BCG infection of BEAS-2B cells inhibits proliferation, induces cell cycle arrest at the G(0)/G(1) phase, causes apoptosis, modulates transcription of multiple apoptosis-relevant genes, promotes translation of Fas, Fas-L, and FADD, upregulates expression of Fas and FADD proteins, and increases activity of caspase-3 and caspase-8. Infection with BCG does not cause any significant change in the secretion of TGF-beta. The roles of Fas and FADD as mediators of BCG-induced apoptosis in BEAS-2B cells were tested by partial blockade of Fas and FADD expression with silencing RNA. Partial blockade of Fas or FADD expression results in a decreased apoptotic response to BCG infection. In conclusion, BCG induces cell cycle arrest and apoptosis in BEAS-2B cells. BCG induced apoptosis of BEAS-2B cells via the Fas death receptor pathway.

Talc remains the most effective sclerosing agent for pleurodesis. However, its mechanism of action in resolving pleural malignant disease remains unclear. The present study evaluated the angiogenic balance in the pleural space in patients with malignant pleural effusions (MPE) following talc insufflation. Patient pleural fluid samples were collected both before and after talc insufflation. The ability of pleural mesothelial cells (PMC) and malignant mesothelioma cells (MMC) to produce endostatin in vitro was compared. The biological effects of pleural fluids and conditioned media from talc-activated PMC on endothelial cells were evaluated by performing proliferation, invasion, tube formation and apoptosis assays. Pleural fluids from patients with MPE who received thoracoscopic talc insufflation contained significantly higher levels of endostatin (median 16.75 ng.mL(-1)) compared with pre-talc instillation (1.06 ng.mL(-1)). Talc-activated PMC released significantly greater amounts of endostatin (mean+/-SEM 1052.39+/-38.66 pg.mL(-1)) when compared with a MMC line (134.73+/-8.72 pg.mL(-1)). In conclusion, talc alters the angiogenic balance in the pleural space from a biologically active and angiogenic environment to an angiostatic milieu. Functional improvement following talc poudrage in patients with malignant pleural effusions may, in part, reflect these alterations in the pleural space.

Mechanical strain of lung tissue is an important stimulus for the production of growth factors that are critical for lung growth and development. However, excessive mechanical strain, as may occur during mechanical ventilation, may produce an increase in growth factors that may contribute to lung injury. We hypothesized that mechanical strain of primary bronchial airway epithelial cells (BAEpCs) induced the production of placental growth factor (PlGF), a member of the VEGF family. BAEpCs were cultured on a deformable silicoelastic membrane and exposed to different magnitudes of stretch. Stretch induced PlGF and nitric oxide (NO) production that increased with increasing magnitude of stretch. Stretch also induced PlGF and inducible NO synthase (iNOS) gene expression. The stretch-induced PlGF production and NO synthesis were attenuated by PD98059, a specific mitogen-activated protein kinase kinase-1 and -2 inhibitor. Inhibition of NO generation by l-NAME or l-NMMA or scavenging NO by carboxy-PTIO prevented stretch-mediated erk1/2 activation. In addition, in unstretched BAEpCs, exogenous NO enhanced erk1/erk2 activation. Our data suggest that mechanical stretch of BAEpCs induces iNOS expression and induces PlGF release in an erk1/2 activation-dependent manner.

Background: The over-expression of the ephrin-A1 ligand receptor EphA2 is associated with the growth and metastatic potential of tumors. Although EphA2 is expressed in a variety of tumors, its expression and function in malignant mesothelioma (MM) remain unknown. The authors hypothesized that expression of the receptor EphA2 in MM cells (MMCs) plays a key role in the growth and haptotactic migration of MM. They also hypothesized that silencing EphA2 expression by using small-interfering RNA (siRNA) inhibits the proliferation and haptotaxis of MMCs and induces apoptosis in MMCs., Methods: The expression of EphA2 in MMCs and in normal pleural mesothelial cells (PMCs) was studied by using real-time quantitative polymerase chain reaction analysis and Western blot analysis. The growth of MMCs was determined with the WST-1 cell-proliferation assay. The haptotactic migration of MMCs and PMCs was determined with a Boyden chamber assay. Expression of caspases was determined with calorimetric assays., Results: The results demonstrated that silencing the receptor EphA2 by siRNA significantly reduced the proliferation and haptotactic migration of MMCs compared with controls. Over-expression of EphA2 with plasmid pcDNA/EphA2 enhanced the proliferation and haptotaxis of MMCs significantly. Knocking down EphA2 expression initiated caspase-9-mediated apoptosis in MMCs., Conclusions: The current results suggested that constitutive expression of EphA2 may contribute to the aggressive behavior and cellular survival of MMCs. EphA2 may be an effective therapeutic target in patients with mesothelioma. Silencing the receptor EphA2 gene is a novel approach for the containment of growth and migration of tumor in patients with malignant mesothelioma.

Pleural fibrosis can result from a variety of inflammatory processes. The response of the pleural mesothelial cell to injury and the ability to maintain its integrity are crucial in determining whether normal healing or pleural fibrosis occurs. The pleural mesothelial cell, various cytokines, and disordered fibrin turnover are involved in the pathogenesis of pleural fibrosis. The roles of these mediators in producing pleural fibrosis are examined. This article reviews the most common clinical conditions associated with the development of pleural fibrosis. Fibrothorax and trapped lung are two unique and uncommon consequences of pleural fibrosis. The management of pleural fibrosis, including fibrothorax and trapped lung, is discussed.

Active, nonanesthetized, tracheotomized rabbits were subjected to continuous positive airway pressure (CPAP) for 4 days to determine the effects of chronic mechanical strain on lung and airway function. Rabbits were maintained for 4 days at a CPAP of 6 cmH(2)O (high CPAP), at a CPAP of 0 cmH(2)O (low CPAP), or without tracheostomy (no CPAP). After treatment with CPAP, changes in respiratory resistance in response to increasing concentrations of inhaled ACh were measured during mechanical ventilation to evaluate respiratory system responsiveness in vivo. Intraparenchymal bronchial segments were isolated from the lungs of all animals to evaluate airway smooth muscle responsiveness and bronchial compliance in vitro. Rabbits maintained for 4 days at high CPAP demonstrated significantly lower responsiveness to ACh compared with rabbits that were maintained at low CPAP or with no CPAP. Airways isolated from the lungs of animals subjected to the chronic application of high CPAP were also less responsive to ACh in vitro than the airways isolated from animals subjected to low CPAP or no CPAP. The persistence of the decreased responsiveness in the excised airway tissues suggests that the decreased respiratory system responsiveness observed in vivo results primarily from direct effects on the airways. The results demonstrate that the application of prolonged mechanical strain in vivo can reduce airway reactivity.

Brush cells, also termed tuft, caveolated, multivesicular, and fibrillovesicular cells, are part of the epithelial layer in the gastrointestinal and respiratory tracts. The cells are characterized by the presence of a tuft of blunt, squat microvilli (approximately 120-140/cell) on the cell surface. The microvilli contain filaments that stretch into the underlying cytoplasm. They have a distinctive pear shape with a wide base and a narrow microvillous apex. The function of the pulmonary brush cell is obscure. For this reason, a working group convened on August 23, 2004, in Bethesda, Maryland, to review the physiologic role of the brush (microvillous) cell in normal airways and alveoli and in respiratory diseases involving the alveolar region (e.g., emphysema and fibrosis) and airway disease characterized by either excessive or insufficient amounts of airway fluid (e.g., cystic fibrosis, chronic bronchitis, and exercise-induced asthma). The group formulated several suggestions for future investigation. For example, it would be useful to have a panel of specific markers for the brush cell and in this way separate these cells for culture and more direct examination of their function (e.g., microarray analysis and proteomics). Using quantitative analysis, it was suggested to examine the number and location of the cells in disease models. Understanding the function of these cells in alveoli and airways may provide clues to the pathogenesis of several disease states (e.g., cystic fibrosis and fibrosis) as well as a key for new therapeutic modalities.

RSV infection is characterized by airway edema. Stabilization of hypoxia inducible factor-1alpha (HIF-1alpha) is important in both inflammation and edema formation. In this study we evaluated whether RSV induced release of nitric oxide (NO) by bronchial airway epithelial cells leading to the stabilization of HIF-1alpha and subsequent transcription of VEGF(165). Primary human bronchial epithelial cells (HBEpC) were used; cell supernatants were analyzed. Western blot analysis was used for the detection of HIF-1alpha. Bronchial airway epithelial monolayer permeability was assessed using electric cell-substrate impedance sensing (ECIS) in real time. There was increased stabilization of HIF-1alpha in RSV infected cells. Addition of an NO inhibitor blocked RSV mediated HIF-1alpha expression. Antagonism of NO also inhibited VEGF production and HBEpC monolayer permeability. Our results demonstrate that in HBEpC, RSV induced NO causes stabilization of HIF-1alpha in vitro.

Background: Respiratory syncytial virus (RSV)-induced diseases are mediated through active cytokines released during infection. We hypothesized that RSV infection causes bronchial epithelial monolayer permeability in vitro via induction of vascular endothelial growth factor (VEGF)., Methods: Human bronchial epithelial cells were infected with RSV. In some cultures, VEGF antibody was included to block VEGF response; in other cultures, palivizumab was added to block RSV infection. Permeability was assessed in real-time using electric cell-substrate impedance sensing. VEGF release was assessed using enzyme-linked immunosorbent assay. Gap formation was assessed using live cell imaging., Results: RSV-infected cells demonstrated a decrease in the resistance of the monolayer indicating an increase in permeability; this increase was blocked with VEGF-specific antibody, and palivizumab. Intercellular gap formation developed in RSV-infected epithelial monolayers., Conclusion: RSV increases permeability of the bronchial airway epithelial monolayer via VEGF induction.

Asthma is characterized by infiltration and shedding of the bronchial epithelium. The Th2 cytokines IL-4 and IL-13 are involved in the cellular recruitment and infiltration seen in asthma. The effects of IL-4 and IL-13 on cell-matrix interactions and epithelial shedding are unknown. We hypothesize that bronchial airway epithelial cells (BAEC) express paxillin, a structural focal adhesion protein, and downregulation of paxillin by Th2 cytokines lead to BAEC hyperpermeability. We showed by confocal microscopy the presence of paxillin in BAEC. We demonstrated by Western blot analysis that IL-4 and IL-13 stimulation results in downregulation of paxillin production. IL-4 and IL-13 stimulation decreased epithelial cell-matrix attachment as measured by electrical cell-substrate impedance sensing system (ECIS). Our results suggest that Th2 cytokines IL-4 and IL-13 downregulate paxillin production by BAEC, thereby disrupting the cell-matrix interactions. This may help explain the epithelial shedding and epithelial membrane hyperpermeability that occurs in asthma.

The pleura responds to the presence of infecting organisms with a vigorous inflammatory response associated with an exudation of white blood cells and proteins. The development and outcome of pleural infections is a function of a balance between the virulence of the invading microorganism and the immune reaction involving professional immune cells as well as the pleural mesothelial cells. Most commonly, pleural infection occurs after invasion through the lung parenchyma and a breach in the viscera pleura resulting in the formation of a parapneumonic process. Upon infection, the microorganisms are recognized by the pleural mesothelial cell, which remains the first line of defence. Pleural responses to infection include those of innate immunity as well as adaptive or acquired immunity. Innate and acquired immune responses are closely linked. In this review, we discuss the different virulence factors that allow microorganisms to infect the pleura and the role of the pleural mesothelial cells in bridging the innate and acquired immune responses.

Study Objectives: Sialomucin complex (SMC) is a heterodimeric glycoprotein, and is found on the surfaces of the mesothelia of the pleura, pericardium, and peritoneum. Sialomucins play a significant role in adhesion as well as in defense. In this study, we hypothesized that pleural mesothelial cells (PMCs) express SMC and thus prevent the adherence of ovarian cancer cells (HTB-77) to the pleura., Methods: PMCs were plated, and the adherence of HTB-77 cells was observed using a cytofluor. The PMC monolayer was pretreated with sialidase, and HTB-77 adherence was observed using cytofluor. In another set of HTB-77 cells, adherence was observed when the PMC monolayer was pretreated with supernatants of HTB-77 cells. Last, supernatants of HTB-77 cells were assayed for sialidase activity., Results: The removal of SMC by sialidase greatly increased the adherence of HTB-77 cells to the PMC monolayer, which was statistically significant (p < 0.05). Similar results were obtained when the PMC monolayer was pretreated with the supernatants of HTB-77 cells. Supernatants of HTB-77 cells showed the presence of sialidase., Conclusions: The presence of SMC on the PMC acts as a defense layer, and its removal by sialidase increases the susceptibility of the PMC layer to the adherence of malignant cells and to increased metastasis. HTB-77 cells also express sialidase, which by its action on the monolayer aids in the adherence of tumor cells to the pleural surface.

The pleural membrane consisting of pleural mesothelial cells and its underlying connective tissue layers play a critical role in immunological responses in both local and systemic diseases. The pleura, because of its intimate proximity to the lung, is positioned to respond to inflammatory changes in the lung parenchyma. Importantly, several systemic diseases have a predilection for expression on the pleural surface. Immunological responses in the pleura include the development of pleural permeability and pleural effusion formation as well as the development of pleural fibrosis and scarring. Under either circumstance, the normal functioning of the pleura is impaired and has multiple consequences leading to increased morbidity and even mortality for the patient. During infections in the pleural space, the pleural mesothelium responds by actively recruiting inflammatory phagocytic cells and allowing the movement of proteins from the vascular compartment into the pleural space. The release of chemokines by the pleural mesothelium allows for directed migration of phagocytic cells from the basilar surface of the pleura towards the apical surface. In malignant disease, the pleura may be the site of primary tumours such as mesothelioma and also the site for malignant metastatic deposits. Certain cancers such as cancers of the breast, ovary, lung, and stomach have a predilection for the pleural mesothelium. The process whereby malignant cells attach to the pleural mesothelium and develop autocrine mechanisms for survival in the pleural space are elucidated in this review. The pleura functions not only as a mechanical barrier, but also as an immunologically and metabolically responsive membrane that is involved in maintaining a dynamic homeostasis in the pleural space.

In bacterial empyema, the recruited polymorphonuclear leukocytes (PMN) represent important phagocytic cells involved in antibacterial defense. In this study we demonstrate that pleural fluids (PF) obtained from patients with empyema (EMP) contains significantly higher levels of granulocyte colony stimulating factor (GM-CSF), and PMN incubated in empyema (EMP) pleural fluid (PF) showed significantly less apoptosis than congestive heart failure (CHF) PF. Staphylococcus aureus-stimulated PMC released significantly (P < 0.001) higher levels of GM-CSF than resting PMC. Staphylococcus aureus-stimulated PMC (SPMC)-CM significantly (P < 0.001) inhibited PMN apoptosis. In SPMC-CM-incubated PMN the antiapoptotic gene Bcl-xL mRNA and protein expression was up-regulated; Bak mRNA and protein expression was down-regulated compared to control PMN. The active caspases activity significantly decreased. When SPMC-CM and EMP PF were immunodepleted with GM-CSF antibody, PMN apoptosis was significantly higher. The delay in apoptosis of PMN is in part attributable to the release of cytokine GM-CSF by activated PMC. These findings suggest that S. aureus-activated PMC extend PMN life span by modulating Bcl-xL and Bak gene expression and active caspases activity during acute inflammation and empyema.

Patients with pulmonary tuberculosis develop pleural effusions with a high protein content. Pleural mesothelial adherens junctions promote mesothelial cell-cell adhesion and contribute to pleural integrity. In the present study we have investigated the effect of mycobacterium (BCG) on mesothelial cell adherens junction proteins and pleural permeability. BCG enhanced pleural mesothelial cell (PMC) release of vascular endothelial growth factor (VEGF), and decreased electrical resistance across the PMC monolayer. Neutralizing antibodies to VEGF significantly restored the drop in PMC electrical resistance caused by BCG. BCG infection down regulated beta-catenin (adherens junction protein) expression and caused increased permeability across confluent mesothelial monolayer. Our results suggest that in TB pleurisy, mycobacteria cause VEGF release from mesothelial cells and leads to protein exudation by altering mesothelial adherens junction proteins.

Tumors such as ovarian, lung, and breast have been found to have a predilection for the pleura. Pleural mesothelial cells (PMCs) play an active role in pleural inflammation via release of cytokines. However, mechanisms whereby PMCs defend themselves against invading malignant cells are unknown. In the present study, we hypothesized that PMCs release the antiangiogenic factor endostatin and inhibit malignant cell invasion. We evaluated the endostatin levels in malignant (MAL) and congestive heart failure (CHF) pleural fluids (PF). Endostatin expression by PMC was also demonstrated by Western analysis and confocal microscopy. Our results demonstrate that CHF PF contained significantly higher levels of endostatin when compared with MAL PF. PMCs alone released a significantly greater amount of endostatin when compared with ovarian cancer cells (OCCs). When the PMC were cocultured with OCCs without contact, there was an increase in the endostatin production. However, when the PMCs were cocultured in direct contact with OCCs the endostatin levels significantly decreased. Endostatin production was upregulated in the presence of tumor cells but not when OCCs were adherent to underlying PMC monolayer. Immunofluorescent staining of PMCs for endostatin correlated with endostatin release. These findings suggest that PMCs play a key role in the antiangiogenesis process by producing endostatin in the pleural space, and thus preventing tumor spread and metastasis in the pleura.

The repair of an injured bronchial epithelial cell (BEC) monolayer requires proliferation and migration of BECs into the injured area. We hypothesized that BEC monolayer injury results in monocyte chemoattractant protein-1 (MCP-1) production, which initiates the repair process. BECs (BEAS-2B from ATCC) were utilized in this study. MCP-1 interacts with CCR2B receptor (CCR2B), resulting in cell proliferation, haptotaxis, and healing of the monolayer. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to verify the presence of CCR2B. CCR2B was not merely present but also inducible by interleukin-2 (IL-2) and lipopolysaccharide (LPS). We demonstrated by immunohistochemistry that BECs express MCP-1 after injury and that receptor expression can be regulated by exposure to IL-2 and LPS. Haptotactic migration of cells was enhanced in the presence of MCP-1 and reduced in the presence of CCR2B antibody. This enhanced or depressed ability of the BECs to perform haptotactic migration was shown to be statistically significant (P < 0.05) when compared to controls. Finally, BECs proliferate in response to MCP-1 as proven by electric cell-substrate impedance sensing (ECIS) technology. MCP-1-specific antibodies were shown to neutralize the MCP-1-mediated BEC proliferation. This cascade of events following injury to the bronchial epithelium may provide insight into the mechanism of the repair process.

Current knowledge implicates pleural mesothelial cells as mainly responsible for inflammatory responses in the pleural space. However, a vast body of recent evidence underscores the important role of fibroblasts in the process of inflammation in several types of tissues. We hypothesize that HPFBs (human pleural fibroblasts) play an important role in pleural responses and also when activated by bacterial endotoxin LPS (lipopolysaccharide), IL-1 beta (interleukin-1 beta), or TNF-alpha (tumor necrosis factor-alpha) release of C-C and C-X-C chemokines-specifically, MCP-1 and IL-8. Our results show that pleural fluid-isolated human fibroblasts release IL-8 and MCP-1 upon stimulation with IL-1 beta, TNF-alpha, and LPS in both a concentration- and time-dependent manner. RT-PCR (reverse-transcriptase-polymerase chain reaction) studies have also confirmed IL-8- and MCP-1-specific mRNA expression in activated pleural fibroblasts. On the time-dependent response curve, IL-8 was found in maximum concentrations at 144 hr, whereas MCP-1 continued to increase even after 196 hr following stimulation. IL-1 beta induced the maximum release of IL-8 (800-fold) and MCP-1 (164-fold), as compared to the controls. TNF-alpha induced a 95-fold increase in IL-8 and an 84-fold increase in MCP-1 levels, as compared to the controls. Collectively, our results show that human pleural fibroblasts contribute to the inflammatory cascade in the pleural space.

Malignant pleural effusion (MPE) carries a grave prognosis with median survival after diagnosis being 5 months. The major causes of MPE are lung, breast, ovary,and gastric cancer. It is still unclear how cancer cells penetrate the pleural mesothelial monolayer and reach the pleural space. In this study we examined the effect of ovarian epithelial cancer cells on a confluent pleural mesothelial cell (PMC) monolayer. We demonstrate that ovarian cancer cells adhere to the mesothelial monolayer in a time-dependent manner and induce PMC barrier dysfunction as evidenced by a drop in electrical resistance on electrical cell substrate impedance-sensing system (ECIS) and increased protein permeability. Barrier dysfunction is attenuated by addition of vascular endothelial growth factor (VEGF) antibody. Significant release of VEOF was noted when ovarian cancer cells were cocultured with PMC. Electron microscopy demonstrated gap formation in PMC monolayer only at the site of cancer cell attachment with surrounding areas remaining confluent.

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that is secreted in significant quantities by pleural mesothelial cells (PMC) and malignant mesotheioma cells (MMC). The functional significance of HA deposition in the pleural space has not been fully elucidated. In this study, we hypothesized that low molecular weight but not high molecular weight hyaluronan induces proliferation and migration of MMC, and that the hyaluronan receptor (CD44S) expressed on the mesothelioma cell surface is involved in this process. We evaluated the effect of low molecular weight hyaluronan (LMWHA) and high molecular weight hyaluronan (HMWHA) on four MMC lines (CRL-2081, CRL-5915, CRL-5830, CRL-5820) proliferation and haptotactic migration. We also studied the expression of HA receptor CD44S on MMC by Northern hybridization and flow cytometry. The binding of LMWHA and HMWHA t o MMC surface was determined by FACS analysis using FITC-conjugated hyaluronan. Our results indicate that the MMC line that expressed the highest amount of CD44 receptor showed increased proliferation and haptotactic migration of MMC when stimulated with LMWHA but not HMWHA. Monoclonal antibody against CD44 inhibited proliferation by about 12-40% and migration by 10-35% in the MMC lines that were studied, and thus in part inhibited LMWHA-induced proliferation and migration in MMC. LMWHA binding to MM cell surface was significantly higher than HMWHA. This directly correlated with their CD44 receptor expression. Neutralization of CD44 receptor significantly reduced the LMMHA binding to MMC. These results provide evidence that the interaction between the adhesive protein receptor CD44 and extracellular matrix component (HA) transmits regulatory signals for mediating the locomotion and proliferation of MMC, and thus plays an important role in localized extension of tumor growth.

Pneumonia remains one of the most common infectious causes of mortality. Patients with pneumonia develop parapneumonic effusions with a high neutrophil count as well as high protein concentrations. We hypothesized that pulmonary parenchymal bacterial infection causes a permeability change in the pleural mesothelium by inducing the production of vascular endothelial growth factor (VEGF). Complicated parapneumonic pleural effusions (empyema) have a 19-fold higher VEGF level than pleural fluids secondary to congestive heart failure and a 4-fold higher level than pleural fluids secondary to uncomplicated parapneumonic effusions. We also analyzed the influence of live Staphylococcus aureus on mesothelial barrier function using a model of confluent mesothelial monolayers. There was a significant drop in electrical resistance across S. aureus-infected pleural mesothelial cell (PMC) monolayers. Recombinant VEGF also decreases PMC electrical resistance. Neutralizing antibodies to VEGF significantly inhibited the drop in PMC electrical resistance caused by S. aureus. S. aureus infection also caused a significant increase in protein leak across confluent mesothelial monolayers. Our results suggest that bacterial pathogens induce VEGF release in mesothelial cells and alter mesothelial permeability, leading to protein exudation in empyema.

Migration of polymorphonuclear neutrophils (PMNL) from the vascular compartment into the pleural space occurs rapidly during the development of parapneumonic effusions. This study investigated the polarized secretion of interleukin (IL)-8 in activated pleural mesothelial cells (PMC) and the migration of PMNL across resting, activated PMC monolayers. Results show that PMC produce IL-8 in a polar manner. When PMC were stimulated with Staphylococcus aureus or IL-1beta at the basal or at the apical surface, significantly (P< .05) more IL-8 was released toward the apical surface. This polarized production of IL-8 was confirmed by in situ hybridization. PMNL migration was higher from the basilar to apical than from the apical to basilar surface of PMC. Neutralizing antibodies against IL-8 and intercellular adhesion molecule (ICAM)-1 significantly (P< .001) blocked PMNL migration across activated monolayers. Thus, during pleural inflammation, PMC regulate the influx of PMNL into the pleural space by polar production of IL-8 and expression of ICAM-1.

Bacterial empyema is a frequent complication of pneumonia in patients with acquired immunodeficiency syndrome (AIDS). A model of Staphylococcus aureus empyema was developed that closely resembles bacterial empyema in patients infected with human immunodeficiency virus (HIV). Results show a compartmentalized chemokine response in bacterial empyema. The chemokine levels were higher in the pleural compartment than in the peripheral circulation. Polymorphonuclear leukocyte counts, murine GRO-alpha (KC), and macrophage inflammatory protein-2 levels were significantly (P<.001) lower in CD4+ knockout (CD4 KO) mice pleural fluid than in CD4+ wild-type (CD4 WT) mice. The CD4 KO mice had poorer bacterial clearance than CD4 WT mice. During S. aureus infection, interleukin-10 levels increased in the CD4 KO mice, whereas interferon-gamma levels were increased in CD4 WT mice. CD4+ T cell depletion results in a decreased pleural chemokine response, decreased neutrophil influx into pleural space, and impaired bacterial clearance in empyema.

Pleural injury results in the death of mesothelial cells and denudation of the mesothelial basement membrane. Repair of the mesothelium without fibrosis requires proliferation and migration of mesothelial cells into the injured area. We hypothesized that monocyte chemoattractant protein-1 (MCP-1) induces proliferative and haptotactic responses in pleural mesothelial cells (PMCs) and that the MCP-1 binding receptor CCR2 mediates the pleural repair process. We demonstrate that PMCs exhibited MCP-1-specific immunostaining on injury. MCP-1 induced proliferative and haptotactic responses in PMCs. PMCs express CCR2 in a time-dependent manner. Fluorescence-activated cell sorting analysis demonstrated that interleukin (IL)-2 upregulated CCR2 protein expression in PMCs, whereas lipopolysaccharide (LPS) downregulated the response at the initial period compared with that in resting PMCs. However, the inhibitory potential of LPS was lost after 12 h and showed a similar response at 24 and 48 h. Haptotactic migration was upregulated in PMCs that were cultured in the presence of IL-2. The increased haptotactic capacity of mesothelial cells in the presence of IL-2 correlated with increased CCR2 mRNA expression. PMCs cultured in the presence of LPS showed decreased haptotactic activity to MCP-1. Blocking the CCR2 with neutralizing antibodies decreased the haptotactic response of PMCs to MCP-1. These results suggest that the haptotactic migration of mesothelial cells in response to MCP-1 are mediated through CCR2, which may play a crucial role in reepithelialization of the denuded basement membrane at the site of pleural injury and may thus contribute to the regeneration of the mesothelium during the process of pleural repair.

Pleurodesis with talc is an accepted method for the treatment of symptomatic pleural effusions secondary to mesotheliomas. Patients with mesothelioma who have talc-induced pleurodesis have a lower morbidity than do those who do not have pleurodesis. The mechanisms whereby talc mediated these effects were considered to be secondary to a decrease or absence of a pleural effusion. The possibility that talc may directly affect malignant cells was not considered. The present study was designed to evaluate if talc directly effects cell death of malignant mesothelioma cells (MMC) or normal pleural mesothelial cells (PMC). Three confluent MMC and PMC were exposed to talc for 24, 48, and 72 h. In parallel experiments, glass beads similar in size to talc were included as control. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and DNA electrophoresis. Our results demonstrated that talc at a therapeutically achievable concentration (6 microg/cm(2)) induces significant apoptosis in MMC. Talc-induced maximum apoptosis in MMC (39.50 +/- 2.55%, 31.87 +/- 4.69%, and 15.10 +/- 3.93% in CRL-2081, CRL-5820, and CRL-5915, respectively) at 48 h, which was significantly (p < 0.05) greater than that in control cells. Electrophoresis of DNA isolated from talc-exposed MMC demonstrated the typical ladder pattern of internucleosomal DNA cleavage. Talc did not induce apoptosis in PMC, and glass beads did not cause significant apoptosis in either MMC or PMC. The present study has demonstrated that talc induces apoptosis in MMC without affecting normal mesothelial cells of the pleura.

The pleural mesothelium is a dynamic cellular membrane with multiple key functions. It plays a pivotal role in pleural inflammation through its release of several cytokines and the expression of cell-surface molecules. The expression of intercellular adhesion molecule (ICAM)-1 in the pleural mesothelium of patients with active pleural tuberculosis and the role of ICAM-1 in monocyte transmigration across pleural mesothelium during tuberculous inflammation was investigated. Results indicate pleural mesothelial cells (PMCs) express ICAM-1 in tuberculous pleuritis. When PMCs were stimulated with bacille Calmette-Guérin (BCG) in vitro, they expressed ICAM-1 in a time-dependent manner. Monocyte transmigration was higher across PMC monolayers that had been stimulated with BCG. Blocking ICAM-1 on BCG-activated PMC monolayers inhibited monocyte transmigration against chemotactic gradient generated by macrophage inflammatory protein 1-alpha or monocyte chemotactic protein-1. These results indicate that ICAM-1 expression in PMCs facilitates monocyte transmigration during tuberculous pleural inflammation.

The recruitment of leukocytes to an area of injury or inflammation site is one of the most fundamental host defenses. Pulmonary tuberculosis is characterized by granulomatous inflammation with an extensive infiltration of mononuclear cells. In tuberculous pleurisy pleural mesothelial cells are exposed to mycobacteria in the pleural space. In this study we demonstrate that mouse pleural mesothelial cells (PMCs), when stimulated with BCG or IFN-gamma, produced MIP-1alpha and MCP-1 in vitro. IFN-gamma enhanced the BCG-mediated MIP-1alpha and MCP-1 expression in a concentration-dependent manner. The RT-PCR studies also confirmed that both BCG and IFN-gamma induce chemokine expression. IL-4 inhibited the BCG-mediated MIP-1alpha and MCP-1 expression in a concentration-dependent manner. The lower concentrations of IL-4 were ineffective; however, at higher concentrations, the inhibitory effect of IL-4 persisted for 24 h and decreased thereafter. BCG stimulation resulted in an increase of IFN-gamma and IL-4 receptors on PMCs. Our results demonstrate that Th1 and Th2 cytokines may regulate the C-C chemokine expression in PMCs and thus play a biologically important role in mononuclear cell recruitment to the pleural space.

The pleura responds to the presence of infecting organisms with a vigorous inflammatory response associated with an exudation of white blood cells and proteins. Changes in pleural permeability lead to formation of an exudative pleural effusion. The pleural mesothelial cell is the primary cell lining the pleural space and, when activated by the presence of organisms, initiates the inflammatory response by releasing a battery of chemokines and cytokines. Mesothelial cells are actively phagocytic and also release oxidants and proteases. The acute inflammatory process may resolve with appropriate antibiotic therapy and drainage leaving minimal fibrosis. However, under certain circumstances vigorous pleural fibrosis with scarring and loss of delineation of pleural surfaces can occur. Recognition of the stage of development of the empyema is an important clinical judgement that can affect outcome. The pathogenesis of infections of the pleural space and the role played by the various cell types is delineated in this article.