ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints. A novel pathogenic mechanism in chronic arthritis

To identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammatio

NGF and Its Receptors in the Regulation of Inflammatory Response

There is growing interest in the complex relationship between the nervous and immune systems and how its alteration can affect homeostasis and result in the development of inflammatory diseases. A key mediator in cross-talk between the two systems is nerve growth factor (NGF), which can influence both neuronal cell function and immune cell activity. The up-regulation of NGF described in inflamed tissues of many diseases can regulate innervation and neuronal activity of peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters, but can also directly influence innate and adaptive immune responses. Expression of the NGF receptors tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) is dynamically regulated in immune cells, suggesting a varying requirement for NGF depending on their state of differentiation and functional activity. NGF has a variety of effects that can be either pro-inflammatory or anti-inflammatory. This apparent contradiction can be explained by considering NGF as part of an endogenous mechanism that, while activating immune responses, also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression, such as that recently demonstrated in immune cells of arthritis patients, might prevent the activation by NGF of regulatory feed-back mechanisms, thus contributing to the development and maintenance of chronic inflammation

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

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

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

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 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

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