Figure 1.
TNF-α, CCL2 and CXCL8 production in human PBMCs exposed to IL-6.
Human PBMCs were exposed to IL-6 (10 ng/ml) in combination with sIL-6R (125 ng/ml) (IL-6/sIL-6R) for 18 hours. TNF-α, CCL2 and CXCL8 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R stimulated compared with not treated (NT) cells.
Figure 2.
Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to TLR ligands.
Human PBMCs were pre-exposed to IL-6/sIL-6R for 1 hour. Cells were then stimulated with poly(I-C) (20 µg/ml), CpG (5 µg/ml), PAM (200 ng/ml), MDP (10 µg/ml), LPS (10 ng/ml), S100A8 (5 µg/ml) for 18 hours. IL-1β (A) and CXCL8 (B) levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.
Figure 3.
Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to IL-1β.
Human PBMCs were pre-exposed to IL-6/sIL-6R for 1 hour. Cells were then stimulated with IL-1β (1 ng/ml) for 18 hours. CXCL8 (A), CCL2 (B), and IL-6 (C) levels were measured by ELISA. In (C), the levels of exogenous IL-6 were subtracted after the ELISA measurement. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.
Figure 4.
Increased p65 NF-κB activation in response to inflammatory stimuli in human PBMCs pre-exposed to IL-6/IL-6R.
Western blots showing the expression of phospho–p65 NF-kB (Ser536) in total lysates from human PBMCs pre-exposed to IL-6 (10 ng/ml) in combination with sIL-6R (125 ng/ml) for 1 hour and stimulated with LPS, poly(I-C), MDP, CpG, IL-1β, PAM (used at the same concentrations as in Figure 2) for 90 minutes. Expression of α-tubulin was used as a loading control. Results of densitometric analysis are shown above each blot. Data are representative of 3 independent experiments.
Figure 5.
Exposure to IL-6 enhances cytokine and chemokine production in SFMC.
SFMC were left to adhere on plastic for 3 hours in DMEM supplemented with 10% fetal calf serum (FCS). SFMC were pre-exposed to IL-6/sIL-6R in combination with IgG1 or with TCZ (50 µg/ml) for 1 hour. Cells were then left untreated (A) or stimulated with poly(I-C) and MDP (B), IL-1β (1ng/ml) (C). CXCL8, CCL2 and IL-1β levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.
Figure 6.
Exposure to IL-6 enhances cytokine and chemokine production in SFMC.
SFMC were left to adhere on plastic for 3 hours in DMEM supplemented with 10% fetal calf serum (FCS). SFMC were pre-exposed to IL-6/sIL-6R in combination with IgG1 or with TCZ for 1 hour. Cells were then stimulated for 18 hours with LPS (10 ng/ml) (A), and S100A8 (5 µg/ml) (B). IL-1β, CXCL8 and CCL2 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R -stimulated compared with NT cells.
Figure 7.
Exposure to sIL-6R enhances cytokine and chemokine production in RA synoviocytes.
(A) Cells were treated with control IgG1, with sIL-6R in combination with IgG1 (sIL-6/IgG1) or with TCZ (sIL-6R/TCZ) for 18 hours. IL-6, CXCL8 and CCL2 levels were measured by ELISA. (B–C) RA synoviocytes were treated with control IgG1, with sIL-6/IgG1 or with sIL-6R/TCZ for 1 hour. Cells were then left untreated or stimulated with IL-1β (1 ng/ml) (B) or LPS (10 ng/ml) (C). CXCL8 and CCL2 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R -stimulated compared with NT cells.
Figure 8.
Western blots showing the expression of phospho–STAT3 (Tyr705), phospho–NF-kB (Ser536) in total lysates of RA synoviocytes pretreated as in Figure 7B for 1 hour and then stimulated with IL-1β (1 ng/ml) or LPS (10 ng/ml) for 90 minutes.
Expression of α-tubulin was used as a loading control. Results of densitometric analysis are shown above each blot. Data are representative of 3 independent experiments.
Figure 9.
IL-6 amplifies the inflammatory response through a direct effect on stromal and innate immunity cells.