Joyce Hardwick

Induction of MCP-1 expression in airway epithelial cells: Role of CCR2 receptor in airway epithelial injury

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.

Polar Production of Interleukin-8 by Mesothelial Cells Promotes the Transmesothelial Migration of Neutrophils: Role of Intercellular Adhesion Molecule–1

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.

Mycobacteria Induces Pleural Mesothelial Permeability by Down-Regulating β-Catenin Expression

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 β-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.

Pleural Mesothelial Cell (PMC) Defense Mechanisms Against Malignancy

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.

Inflammatory Cytokines Mediate C–C (Monocyte Chemotactic Protein 1) and C–X–C (Interleukin 8) Chemokine Expression in Human Pleural Fibroblasts

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 β (interleukin-1 β), or TNF-α (tumor necrosis factor-α) 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 β, TNF-α, 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 β induced the maximum release of IL-8 (800-fold) and MCP-1 (164-fold), as compared to the controls. TNF-α 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.

Pleural mesothelial cells modulate polymorphonuclear leukocyte apoptosis in empyema

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.

Defensive role of pleural mesothelial cell sialomucins in tumor metastasis.

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.

An aggressive form of transthyretin amyloidosis

In 1990, Ueno et al. identified a new transthyretin (TTR) mutation in a 45-year-old Japanese man with peripheral neuropathy of six-years duration [1]. This mutation, Ser50Arg, was the result of a single nucleotide change of T!G in the third position of the codon for Ser50 (AGT!AGG). Recently, we have had occasion to identify a number of patients with systemic amyloidosis (ATTR) associated with a Ser50Arg mutation, however, all of these patients share a different nucleotide exchange with AGT50!AGA.