Exposure to IL-6 enhances cytokine and chemokine production in SFMC.

<p>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 (<b>A</b>) or stimulated with poly(I-C) and MDP (<b>B</b>), IL-1β (1ng/ml) (<b>C</b>). 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.</p

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

<div><p>The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.</p></div

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.

<p>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.</p

Exposure to IL-6 enhances cytokine and chemokine production in SFMC.

<p>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) (<b>A</b>), and S100A8 (5 µg/ml) (<b>B</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.</p

Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to TLR ligands.

<p>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 (<b>A</b>) and CXCL8 (<b>B</b>) levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R-stimulated compared with NT cells.</p

Exposure to sIL-6R enhances cytokine and chemokine production in RA synoviocytes.

<p>(<b>A</b>) 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>B–C</b>) 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>B</b>) or LPS (10 ng/ml) (<b>C</b>). CXCL8 and CCL2 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R -stimulated compared with NT cells.</p

Exposure to IL-6 affects the production of inflammatory cytokines and chemokines in human PBMCs in response to IL-1β.

<p>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 (<b>A</b>), CCL2 (<b>B</b>), and IL-6 (<b>C</b>) levels were measured by ELISA. In (<b>C</b>), 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.</p

Increased p65 NF-κB activation in response to inflammatory stimuli in human PBMCs pre-exposed to IL-6/IL-6R.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107886#pone-0107886-g002" target="_blank">Figure 2</a>) 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.</p

IL-6 amplifies the inflammatory response through a direct effect on stromal and innate immunity cells.

<p>IL-6 amplifies the inflammatory response through a direct effect on stromal and innate immunity cells.</p

TNF-α, CCL2 and CXCL8 production in human PBMCs exposed to IL-6.

<p>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.</p