AN ACTIVE PRONGF-P75NTR AXIS PLAYS A PRO-INFLAMMATORY ROLE IN CHRONIC ARTHRITIS

Introduction: Inflammation has been associated with a marked increase in the production of nerve growth factor (NGF) in tissues of patients with a variety of chronic inflammatory diseases, including rheumatoid arthritis and juvenile idiopathic arthritis (JIA). However, the effects of NGF, its immature form proNGF, and their receptors, TrkA and p75NTR, in regulating cells and mediators during inflammatory responses and their contribution to the pathogenesis of arthritis are not yet defined. Objectives: To evaluate the role of proNGF, mature NGF and their receptors in the modulation of inflammatory response in chronic arthritis patients. Methods: Seventy-five JIA patients, according to the International League of Associations for Rheumatology classification criteria, were divided in persistent oligoarticular, extended oligoarticular or polyarticular subtypes. The expression of the NGF receptors, p75NTR and TrkA, in peripheral blood (PBMC) and synovial fluid (SFMC) mononuclear cells from JIA patients and healthy children was analyzed using real time-PCR and Western Blot. In ex vivo experiments JIA SFMC were treated with mature NGF or proNGF and the effects on intracellular signaling pathways and cytokine production were assessed by Western Blot, real time-PCR and ELISA Results: p75NTR is the main NGF receptor expressed in JIA PBMC and SFMC while TrkA is the prevalent NGF receptor in healthy donor mononuclear cells. The increased expression of p75NTR and the concomitant decrease in TrkA, at both the mRNA and protein levels, resulted in a highly abnormal ratio between p75NTR and TrkA in JIA cells. The highest expression of, not only p75NTR, but also sortilin, the p75NTR co-receptor, was found in JIA SFMC. When patients were divided according to JIA subtypes the highest p75NTR expression levels were found in SFMC of patients with polyarticular JIA. The lowest p75NTR expression levels characterized SFMC from persistent oligoarticular patients while patients with extended oligoarticular JIA showed intermediate p75NTR levels. A significant correlation was found in JIA patients between the p75NTR mRNA expression levels in SFMC and the number of active joints at sampling and the levels of C-reactive protein. In the synovial fluid of JIA patients the concentration of proNGF, the specific ligand of p75NTR, far exceeded that of mNGF. In ex vivo experiments, LPS-stimulated SFMC of JIA patients were treated with proNGF. We found that proNGF specifically enhanced phosphorylation of p38, JNK and IkB, all involved in inflammatory cytokine production in immune cells. The addition of NGF did not induce the activation of these pathways and proNGF, but not mNGF, induced in SFMC a higher production of pro-inflammatory cytokine. This production was blocked using p75NTR inhibitors. Conclusion: Our results show that high levels of p75NTR mRNA expression in the SFMC of JIA patients are associated with the most severe subtype and with disease activity. We also found that proNGF is the predominant form of NGF in the synovial fluid of JIA patients. Blocking of p75NTR in ex vivo experiments with JIA SFMC showed that proNGF acts through p75NTR. Altogether these results suggest that an active proNGF-p75NTR axis is involved in sustaining inflammatory production and may provide the rationale for specifically targeting p75NTR in chronic arthritides. Disclosure of