Ex vivo effects of proNGF and NGF on JIA mononuclear cells.

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Ex vivo effects of proNGF and NGF on JIA mononuclear cells. Ex vivo effects of proNGF and NGF on JIA mononuclear cells. (A) No changes in cell viability were observed when mononuclear cells from synovial fluid (SFMC) of patients with JIA were treated for 24 hours with proNGF or mNGF, with or without LPS stimulation. Apoptosis was assessed by Annexin V flow cytometry analysis. The results represent one of three independent experiments performed in duplicate. (B) While proNGF addition alone did not induce the production of interleukin (IL)-6 in unstimulated SFMC, in LPS-activated cells proNGF induced IL-6 release in a dose-dependent manner with a maximal effect at 200 ng/mL (n=4). (C) proNGF stimulation significantly increased IL-6, IL-1β, IL-8 and tumor necrosis factor-α (TNF-α) mRNA expression levels in LPS-stimulated SFMC (n=18) of patients with JIA after 3 hours of incubation, but did not modify IL-10 mRNA expression in either CTRL or JIA mononuclear cells. Mature NGF treatment did not significantly modify proinflammatory cytokine levels or IL-10 mRNA expression in JIA mononuclear cells, while it increased IL-10 mRNA levels in CTRL PBMC. Results are expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene GAPDH. (D) proNGF treatment significantly increased IL-6 release after 18 hours (measured by ELISA) in LPS-stimulated SFMC from patients with JIA (n=16) compared with PBMC of healthy CTRL (n=6). Mature NGF treatment did not affect IL-6 protein levels. *p<0.05. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood. Gaetana Minnone et al. RMD Open 2017;3:e000441 Copyright © BMJ Publishing Group & EULAR. All rights reserved.