Figure 1.
Inhibition of TNF-α secretion by different human cell types in presence of Ro 32-7315, TQ and Celastrol.
Cells (5×105/ml) were incubated for 4 hours (for human monocytes and THP-1 cells) or 24 hours (for PBMC) with 1or 5 µg/ml LPS respectively, in the presence of ranging compounds concentrations or 0.1% (v/v) DMSO as a control. The TNF-α levels were then assessed in culture supernatant by ELISA technique. Dose-response curves were fitted on GraphPad PRISM (v.4.0, GraphPad Software, Inc. La Jolla, CA,USA) sigmoidal dose-response curve-fit model (n = 3 independent experiments); s-TNF-α correspond to the secreted form of TNF-α. Statistical difference between IC50 of Ro 32-7315, TQ and Celastrol are indicated in Table 1.
Table 1.
IC50 (µM) values of tested compounds for TNF-α release by human monocytes, THP-1 cells and PBMC (n = 3 independent experiments) are estimated using GraphPad PRISM® 5.0 d (GraphPad Software, Inc., San Diego CA).
Figure 2.
THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment.
Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p<0.05).
Figure 3.
PBMC surface accumulation of the membrane-bound TNF-α after Ro 32-7315, Celastrol and TQ treatment.
Cells were stimulated for 24 hours with 5 µg/ml of LPS in the presence of 10 µM of Ro 32-7315, TQ and Celastrol. 0.1% (v/v) DMSO was used as a control (silver histogram). The non-permeabilized cells were then stained with FITC-anti-m-TNF-α MAb. Membrane TNF-α expression was evaluated by flow cytometry. The representative histograms are shown. The fluorescence intensity (AU) is plotted against the number of cells (y-axis). For all 3 compounds, membrane–bound TNF-α expression increased after THP-1 cells were treated (p<0.05, n = 3).
Figure 4.
Immunofluorescence of membrane-bound TNF-α on the surface of THP-1 cells and human monocytes after Ro 32-7315 treatment.
Cells were stimulated for 4 hours with LPS (1 µg/ml) in the presence of Ro 32-7315. Then, cells were stained by FITC-anti-m-TNF-α MAb. Image acquisition was made on a confocal microscope. The numerical recording of the images and analysis were carried out in Image J (Wayne Rasband, National Institute of Mental Health, Bethesda, Maryland, USA.).
Figure 5.
TACE expression on surface of THP-1 Cells and PBMCs.
Non-activated or LPS stimulated cells (4 hours by 1 µg/ml of LPS for THP-1 cells or 24 hours by 5 µg/ml of LPS for PBMC) were stained with Phycoerythrin labelled anti-TACE MAb. Cell surface TACE expression was then evaluated by flow cytometric analysis. The ordinate relates to the relevant cell number, while fluorescence intensity can bee seen on the abscissa, representing cell surface TACE level. Silver histogram show unstained cells. Green and red lines present stained cells, non-activated and activated, respectively. Difference is statistically significant (p = 0.049 for both THP-1 and PBMC, n = 3).
Figure 6.
Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by human monocytes as compared to the THP-1 cell line (inset).
The enzymatic activity was determined for viable intact cells using MOCA and ABZ substrates (n = 3). Thus, 0.5×106/ml of viable cells were incubated with 5 µM of the substrate in Ca2+ and Mg2+ free HBSS in the presence of compounds (10 µM). Fluorescence intensity was monitored for 600 s, and the rates of the peptide hydrolysis were then calculated from the linear section of the fluorescence curve. The inhibitory potency of the compounds was calculated by divining the reaction rates of substrate hydrolysis obtained for different compounds concentrations (V(substrate)[inhibitor]) by substrate hydrolysis reaction rate with DMSO addition (V(substrate)[0.1% DMSO]). Inhibitory potency (%) = 100%−(V(substrate)[inhibitor]/V(substrate)[0.1% DMSO])×100%.
Figure 7.
Effects of Ro 32-7315, Celastrol and TQ on MOCA and ABZ substrates hydrolysis by THP-1 cells.
The viable intact THP-1 cells (0.5×106 cells/ml) were incubated with 5 µM of the MOCA or ABZ substrate in Ca2+ and Mg2+ free HBSS in the presence of various concentrations of Ro 32-7315, Celastrol or TQ. The final DMSO concentration in the reaction mixture was 0.1%. The fluorescence intensity was monitored for 600 s and the rates of the substrates hydrolysis were calculated from the linear section of the fluorescence curve. To estimate the accurate inhibitory potency of the tested compounds, the reaction rates of MOCA hydrolysis obtained for different compounds concentrations (V(MOCA)[inhibitor]) were divided by MOCA hydrolysis reaction rate in the presence of 0.1%DMSO (V(MOCA)[0.1% DMSO]). Inhibitory potency (%) = 100%−(V(MOCA)[inhibitor]/V(MOCA)[0.1% DMSO])×100%.
Figure 8.
Effects of Ro 32-7315, Celastrol and TQ on rh TACE activity.
100 ng/ml of rh TACE were incubated with 5 µM of MOCA substrate in 25 mM Tris, pH 9.0, 2.5 µM ZnCl2, 0.005% Brij 35, in the presence of different compounds concentrations. The compounds were injected directly into the reaction solution every 600 s and the fluorescence intensity was monitored at λ excitation/λ emission: 325/400 nm, at room temperature. To estimate the accurate inhibitory potency of the tested compounds, the reaction rates of MOCA hydrolysis obtained for different compounds concentrations (V(MOCA)[inhibitor]) were divided by MOCA hydrolysis reaction rate with DMSO addition (V(MOCA)[DMSO]). Inhibitory potency (%) = 100%−(V(MOCA)[inhibitor]/V(MOCA)[DMSO])×100%.