Mature and immature NGF forms in patients with JIA and RA

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Mature and immature NGF forms in patients with JIA and RA Mature and immature NGF forms in patients with JIA and RA. (A,B) Only proNGF forms of different molecular weights but not mature NGF were detected by western blot in synovial fluids of 12 patients with JIA (40 μg total protein). (B) Blocking the anti-NGF antibody by adding mature NGF results in the disappearance of the specific proNGF bands observed in synovial fluids of three patients with JIA, as well as of the band of the 25 kDa commercial proNGF that was added as positive control. (C) In JIA and RA synovial fluids, proNGF and mature NGF (mNGF) concentrations were assessed using a newly developed ELISA showing that proNGF is 4.8-fold higher in JIA (n=27) and 16.8-fold higher in patients with RA (n=5) than mNGF. (D) Real-time PCR shows a very low expression of NGF mRNA in mononuclear cells obtained from peripheral blood of healthy donors (CTRL PBMC) or in mononuclear cells from peripheral blood (JIA PBMC) and from synovial fluids of patients with JIA (JIA SFMC). Mature and immature NGF forms in patients with JIA and RA. (A,B) Only proNGF forms of different molecular weights but not mature NGF were detected by western blot in synovial fluids of 12 patients with JIA (40 μg total protein). (B) Blocking the anti-NGF antibody by adding mature NGF results in the disappearance of the specific proNGF bands observed in synovial fluids of three patients with JIA, as well as of the band of the 25 kDa commercial proNGF that was added as positive control. (C) In JIA and RA synovial fluids, proNGF and mature NGF (mNGF) concentrations were assessed using a newly developed ELISA showing that proNGF is 4.8-fold higher in JIA (n=27) and 16.8-fold higher in patients with RA (n=5) than mNGF. (D) Real-time PCR shows a very low expression of NGF mRNA in mononuclear cells obtained from peripheral blood of healthy donors (CTRL PBMC) or in mononuclear cells from peripheral blood (JIA PBMC) and from synovial fluids of patients with JIA (JIA SFMC). High NGF mRNA expression levels characterised synovial tissues (RA synovia; n=5). Fibroblast-like synoviocytes (FLS; n=3) of patients with RA express more NGF mRNA than FLS from osteoarthritis patients (OA FLS) (n=4) and control fibroblasts (CTRL FB; n=4). Results were expressed as arbitrary units (AU) and obtained after normalisation with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (E) Both unstimulated (US) and LPS-stimulated JIA SFMC do not release proNGF or mNGF in the supernatants. Commercial proNGF and mNGF were added as positive controls. (F) Western blot shows the stability of exogenous added mNGF and proNGF. After its addition to culture media, proNGF is still detected after 3 hours and undergoes a minor maturation into mNGF in the first 2 hours of incubation (left side of F). mNGF is not degraded in the first 3 hours after its addition (right side of F). Commercial proNGF and mNGF were included as positive controls. (G) Neither proNGF nor mNGF were released in culture media of US or LPS-stimulated JIA SFMC after 18 hours of incubation. Exogenous proNGF or mNGF were detected only at time of supplementation (in agreement with F) but were no longer detectable after 18 hours of incubation. Commercial proNGF and mNGF were included as positive controls. (H) ELISA instead shows that conditioned media of RA FLS (n=5) have higher concentrations of proNGF than control fibroblasts (CTRL FB; n=5) and that the concentration of proNGF is higher than mNGF after 18 hours of incubation. *p<0.05, **p<0.01, ***p<0.001. JIA, juvenile idiopathic arthritis; LPS, Lipopolysaccharide; NGF, nerve growth factor; PBMC, mononuclear cells isolated from peripheral blood; RA rheumatoid arthritis; SFMC, mononuclear cells isolated from synovial fluids. Gaetana Minnone et al. RMD Open 2017;3:e000441 Copyright © BMJ Publishing Group & EULAR. All rights reserved.