Recombinant human TGF-β1 and human IL-1β were used as the main cytokines. Monoclonal antibodies against E-cadherin (R&D systems, Minneapolis, MN, USA), pancytokeratin (Sigma Aldrich, St. Louis, MO, USA), vimentin (Sigma Aldrich, St. Louis, MO, USA), and rhodamine-phalloidin (Molecular probes, Inc., California, USA) were used for immunofluorescence studies.

In order to obtain normal pleural mesothelial cells, we collected the irrigation fluid of five primary spontaneous pneumothorax patients during bullectomy operations with video-assisted thoracoscopic surgery. To obtain the effluents of tuberculous pleury, thoracentesis from thirteen patients were done. If the pleural fluid adensine deaminase (ADA) level is above 40 U per L and the pleural fluid has a lymphocyte-to-neutrophil ratio greater than 0.75, a diagnosis of tuberculous pleurisy was made. Then, the cells were isolated by centrifugation and cultured until grown to confluence for 7-10 days. Finally, seven of the thirteen effluents reached confluences. The morphologic features of cells in confluent cultures were compared and remained stable during the two cell passages. All cells were cultured in Earle's M199 medium, 20 percent fetal-calf serum, 50 U of penicillin per milliliter, 50 µg of streptomycin per milliliter, and 2 percent Biogro-2 (containing insulin, transferrin, ethanolamine, and putrescine) (Biological Industries). For the first 24 to 48 hours of the culture with irrigation fluid of primary spontaneous pneumothorax patients, media were supplemented with 100 µg/mL cis-OH-proline (Sigma, St. Louis, MO, USA) to selectively eliminate fibroblasts from cultures. Mesothelial cells from spontaneous pneumothorax were treated with TGF-β1 at 0.5 ng/mL and/or IL-1β at 2 ng/mL for 72 hours. The concentration level of IL-1β used in this study was determined based on previous studies showing IL-1β induced EMT.10 Cells cultured from tuberculous pleurisy were stained after cell growth to confluence without cytokine supplement. For immunofluorescence studies, the cells were seeded on cover slips without Biogro-2. The procedures were performed in accordance with institutional guidelines, and were approved by an institutional review board. Informed consent was obtained from all study participants.

Total RNA was isolated using Trizol (Invitrogen, California, USA). The complementary DNA was obtained from 500 ng-1 µg of total RNA with the use of a random primer according to the manufacturer's instructions, the Superscript™ III First-Strand Synthesis system, and Platinum PCR SuperMix (Invitrogen, California, USA). Amplification of snail, E-cadherin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed according to the procedures described in a previous study.10

Immunofluorescence staining was used to detect E-cadherin, cytokeratin, vimentin, and F-actin in the cells from primary spontaneous pneumothorax and tuberculous pleurisy. Cells from effluents of tuberculous pleurisy and the Immunofluorescence studies were performed as described previously.10 The fluorescent images were obtained by confocal laser scanning microscope (Zeiss LSM 510, Oberkochen, Germany).

Cultured mesothelial cells from primary spontaneous pneumothorax had an almost round shape under the phase-contrast microscopy. The cells were stained strongly with epithelial markers, E-cadherin and cytokeratin. Mesenchymal markers vimentin and F-actin stained only some cortical areas of the cells. Our data ascertained that mesothelial cells from primary spontaneous pneumothorax were characterized by epithelial phenotype. After TGF-β1 treatment with 0.5 ng/mL, the round cell morphology gradually changed to elongated fibroblast-like cells under phase-contrast microscopy. Immunofluorescence microscopy demonstrated the loss of E-cadherin, cytokeratin with the replacement by vimentin, and the stress fiber reorganization by F-actin (Fig. 1). Our data suggested that TGF-β1 might induce the mesothelial cells to undergo EMT in vitro.

To determine whether IL-1β alone or in combination with TGF-β1 could induce EMT or have additive effects in EMT, cultured mesothelial cells from primary spontaneous pneumothorax were treated with IL-1β alone and in combination with 0.5 ng/mL concentration of TGF-β1. The treatment with IL-1β alone induced the cells to undergo morphologic changes to the elongated fibroblast-like cells under phase-contrast light microscopy (Fig. 1). The immunofluorescence stain after treatment with IL-1β alone also demonstrated the loss of E-cadherin, cytokeratin and replacement by vimentin and F-actin, although cytokeratin in some cells was still positive (Fig. 1). The treatment with TGF-β1 also induced morphologic changes to fibroblast-like cells, but the treatment IL-1β in combination with TGF-β1 did not reveal additional morphologic changes (Fig. 1).

The transcription factor snail is known as an inducer of EMT process. To determine whether the snail expression was associated with the phenotype changes during EMT, RT-PCR analysis was used. The expression of mRNA encoding E-cadherin was down-regulated with 0.5 ng/mL TGF-β1 treatments in cultured cells of primary spontaneous pneumothorax, and more down-regulation was detected with TGF-β1 treatment in combination with IL-1β (Fig. 2). While mRNA encoding snail was scarcely found without TGF-β1 treatment, the expression of mRNA encoding snail was revealed after 0.5 ng/mL TGF-β1 treatment. The combination of TGF-β1 with IL-1β tended to have an additional effect on snail expression in a patient with spontaneous pneumothorax (Fig. 2).

The cultured cells from tuberculous pleurisy revealed an expression of snail mRNA in all cases (Fig. 4), whereas the expression of E-cadherin mRNA was detected in only two cases (Fig. 4) (column 1 and 2), showing both characteristics of epithelial and mesenchymal phenotype in immunofluorescence stains (Fig. 3A and B). Our data suggest that EMT in mesothelial cells might be associated with the snail expression.

The mean age of seven patients with tuberculous pleurisy was 42.1 ± 15.6. No one had experienced any medical illness. On the chest radiographs or CT images, four patients had pulmonary tuberculosis with tuberculous pleurisy. Three of them showed remnant pleural thickening on their chest radiographs after six month treatment.

Cultured cells from the effluents of tuberculous pleurisy revealed two phenotypes. The first phenotype, which was found in five cases (Fig. 3C, D and E), demonstrated fibroblast-like cell characteristics that had elongated cell morphology, mesenchymal characteristics positive for vimentin, and stress fiber reorganization by F-actin. The second phenotype, which was found in two cases (Fig. 3A and B), had both characteristics of epithelial and mesenchymal cells, relatively round cell morphology, positive result for E-cadherin and cytokeratin, as well as stress fiber reorganization by F-actin under immunofluorescence study. In the second phenotype group, results of RT-PCR also revealed both expression of mRNA encoding E-cadherin and snail (Fig. 4) (column 1 and 2), whereas the first phenotype group (Fig. 4) (column 3-7) only revealed snail expression. Clinically, three patients with the first phenotype showed pleural thickening on their chest radiographs at the end of the treatment, but all patients with the second phenotype did not show remnant pleural thickening.

The results of immunofluorescence and RT-PCR studies indicated that some cases of tuberculous pleurisy have both characteristics of epithelial and mesenchymal cell. Therefore, our data suggest that EMT process might be involved in tuberculous pleurisy.