Table 1.
Clinical Data and Pleural Fluid characteristics*.
Definition of abbreviations: TBP, tuberculous pleuritis; CHF, congestive heart failure; LDH, lactate dehydrogenase; ADA, adenosine deaminase; RPT, residual pleural thickening.
Fig 1.
Effusion levels of TNF-α, MMPs and their relationship with effusion radiological scores in TBP patients.
Levels of (A), TNF-α, (B), MMP-1 and (C), MMP-9 in pleural effusion of patients with TBP (n = 18) or CHF (n = 18), and the correlation between effusion levels of (D), TNF-α, (E), MMP-1 or (F), MMP-9 and effusion shadowing on chest radiograph in patients with TBP. TNF, tumor necrosis factor; MMP, matrix metalloproteinase; TBP, tuberculous pleuritis; CHF, congestive heart failure.
Fig 2.
Relationship between effusion levels of TNF-α and MMPs and residual pleural thickening in TBP patients.
Correlation between pleural fluid levels of (A), TNF-α, (B), MMP-1 or (C), MMP-9 and pleural thickening on chest radiograph in patients with TBP (n = 18), and pleural fluid levels of (D), TNF-α, (E), MMP-1 and (F), MMP-9 between TBP patients with RPT ≥10 mm (n = 6) and those with RPT <10 mm (n = 12). TNF, tumor necrosis factor; MMP, matrix metalloproteinase; TBP, tuberculous pleuritis; RPT, residual pleural thickening.
Fig 3.
Expression and release of TNF-α in MTBRa-Stimulated Human PMCs.
Levels of TNF-α in the culture supernatants of MeT-5A cells stimulated by (A), MTBRa (1 ng/ml) for the indicated times or (B), MTBRa with various concentrations (0.1–10 ng/ml) for 6 h. TNF-α protein expression in MeT-5A cells treated with (C), MTBRa (1 ng/ml) for the indicated times or (D), MTBRa (0.1–10 ng/ml) for 6 h. (E), TNF-α mRNA expression in MeT-5A cells treated with MTBRa (0.1–10 ng/ml) for 4 h. (F), TNF-α protein expression in primary-cultured human PMCs stimulated with MTBRa (0.1–10 ng/ml) for 6 h. Supernatant TNF-α level was analyzed by ELISA and cell lysate TNF-α protein level was assessed by Western blot. TNF-α mRNA concentrations were analyzed by semiquantitative reverse transcriptase PCR and normalized with GAPDH mRNA. Data are shown as mean ± SD of three to five independent experiments. #p<0.05 and ###p<0.001 compared with the control or resting group. PMC, pleural mesothelial cell; MTBRa, heat-killed M. tuberculosis H37Ra; TNF, tumor necrosis factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig 4.
Signal pathways mediating TNF-α expression in MTBRa-stimulated human PMCs.
(A), MeT-5A cells were pretreated with vehicle, SP600125 (SP), SB20358 (SB), LY294002 (LY), Parthenolide (Par), PD98059 (PD), respectively, then stimulated with MTBRa (1 ng/ml) for 24 h. TNF-α protein expression was assessed by Western blot. (B), MeT-5A cells were pretreated with PD98059 (PD) (10 μM), followed by stimulation with MTBRa (1 ng/ml) for 4h. TNF-α mRNA concentrations were analyzed by RT-PCR. (C), MeT-5A cells were pretreated with PD98059 (PD) (5 and 10 μM) following stimulation with MTBRa (1 ng/ml) for 6 h, supernatant TNF-α level was analyzed by ELISA. (D), MeT-5A cells were treated with MTBRa (1 ng/ml) for the indicated times. ERK expression was analyzed by Western blotting with antibodies specific for phosphorylated or total proteins. ##p <0.01, ###p<0.001 compared with the resting group; **p<0.001, ***p<0.001 compared with the vehicle (DMSO) group. PMC, pleural mesothelial cell; TLR, toll-like receptor; MTBRa, heat-killed M. tuberculosis H37Ra; TNF, tumor necrosis factor; ERK, extracellular-signal-regulated kinase.
Fig 5.
TLR/ERK Signaling Activating TNF-α Expression in MTBRa-Stimulated Human PMCs.
(A), Flow cytometric analyses of expression of TLR2 or TLR4 (green) on cell surface of MeT-5A cells treated with MTBRa (1 ng/ml) for 3 h or left untreated (control). Isotype control IgG (red). Quantitative analysis of TLR2 or TLR4 expression was expressed as percentage of the total cells. A representative of three experiments is depicted. ###p<0.001 compared with the control group. MeT-5A cells were transfected with scrambled siRNA, TLR2 siRNA (25 nM) or TLR4 siRNA (25 nM). After 30 min, 3 and 6 h of stimulation with MTBRa, the cellular extracts were prepared and the protein amounts of (B), TLR2 and TLR4, (C) ERK, and (D), TNF-α were determined by Western blotting. The data represent four independent experiments. ##p <0.01, ###p<0.001 compared with the control (scrambled RNA) group; **p<0.01, ***p<0.001 compared with scrambled RNA-transfected MTBRa-treated group. PMC, pleural mesothelial cell; TLR, toll-like receptor; MTBRa, heat-killed M. tuberculosis H37Ra; ERK, extracellular-signal-regulated kinase; TNF, tumor necrosis factor.
Fig 6.
Relationship between pleural fluid levels of TNF-α and MMP-1/-9 in TBP.
Correlation between TNF-α and (A), MMP-1 and (B), MMP-9 in TBP (n = 18). Effects of Heat-killed M. tuberculosis H37Ra or TNF-α on the expression of MMP-1 and MMP-9 in human pleural mesothelial cells. (C) and (D), MeT-5A cells were pretreated with vehicle (control), control IgG (12 μg/ml) or anti-TNF-α Ab (12 μg/ml) for 30 min, then stimulated with MTBRa for 24 hours. (E) and (F), MeT-5A cells were stimulated with TNF-α (5–20 ng/ml) for 24 h. The level of MMP-1 and MMP-9 was assessed by Western blot. Data are shown as mean ± SD of three to four independent experiments. ###p<0.001 compared with the resting group; ***p<0.001 compared with the MTBRa-treated group (control). TNF, tumor necrosis factor; MMP, matrix metalloproteinase; TBP, tuberculous pleuritis; MTBRa, heat-killed M. tuberculosis H37Ra.
Fig 7.
Schematic diagram of the activated signalings in human pleural mesothelial cells upon Mycobacterium tuberculosis infection.
MTBRa induces TNF-α expression in human pleural mesothelial cells through activation of TLR2/ERK signaling pathway, and increases MMP-1 and MMP-9 production (see text for further explanation). MTBRa, heat-killed M. tuberculosis H37Ra; TLR, toll-like receptor; ERK, extracellular-signal-regulated kinase; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; Ⓟ, phosphorylate