Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis

Abstract

Introduction: We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment. Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF).

Methods: mRNA expression levels of four ligands (NGF, brain derived growth factor (BDNF), neurotrophin (NT)-3, NT-4) and their four corresponding receptors (tyrosine kinase (trk) A, trkB, trkC, NGFRp75) were determined in the synovial fluid (SF) cells of 9 patients with rheumatoid arthritis (RA) and 16 with spondyloarthritis (SpA) and compared with 7 osteoarthritis (OA) patients. NGF was also determined in synovial tissue (ST) biopsies of 10 RA and 10 SpA patients. The production of NGF by monocytes and lymphocytes was assessed by flow cytometry of SF cells, synovial tissue derived fibroblast-like synoviocytes (FLS) were assessed by ELISA on culture supernatant.

Results: SF cell analysis revealed a clear BDNF and NGF mRNA expression, with significantly higher NGF expression in RA and SpA patients than in the OA group. NGF expression was higher in ST samples of RA as compared to SpA. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ T lymphocytes and CD14+ cells, i.e. monocytes/macrophages, whereas cultured FLS did not produce NGF in vitro.

Conclusions: Neurotrophins and especially NGF are expressed in the synovial fluid and tissue of patients with peripheral synovitis. The presence of neurotrophins as well as their receptors, in particular the NGF/trkA-p75 axis in peripheral synovitis warrants further functional investigation of their active involvement in chronic inflammatory arthritis.

Figure 1

Scatter plots showing mRNA expression of the neurotrophic ligands. The scatter plots a to d depict the expression levels of the four neurotrophic ligands. (a) nerve growth factor (NGF). (b) Brain-derived growth factor (BDNF). (c) Neurotrophin (NT)-3. (d) NT-4). Bold horizontal lines represent the median. The highest levels were found for BDNF and NGF. Significantly higher expression was revealed for NGF in both spondyloarthritis (SpA) and rheumatoid arthritis (RA) as compared with osteoarthritis (OA; P = 0.001 for both comparisons).

Figure 2

Scatter plots showing mRNA expression of neurotrophin receptors. The scatter plots a to d depict the expression levels of the four neurotrophin receptors. (a) Tyrosine kinase (trk)A. (b) p75. (c) trkB. (d) trkC. Bold horizontal lines represent the median. The highest levels were found for trkA and p75, revealing significantly higher expression levels in spondyloarthritis (SpA; P = 0.0003, P = 0.003 respectively) and rheumatoid arthritis (RA; P = 0.004 and P = 0.001, respectively) vs osteoarthritis (OA).

Figure 3

mRNA expression of NGF in synovial tissue samples. mRNA expression of nerve growth factor (NGF) as the prototype of neurotrophins was measured in the synovium of both 10 spondyloarthritis (SpA) and 10 rheumatoid arthritis (RA) patients; the expression levels were compared with each other (relative expression) showing a twice as high and thus significantly higher NGF expression in RA as compared with SpA (P = 0.02).

Figure 4

NGF staining by flow cytometry. PBMC of (a) healthy controls (HC) and (b) synovial fluid mononuclear cells (SFMC) from spondyloarthritis (SpA), and (c) rheumatoid arthritis (RA) patients were first stained with surface markers (CD3 and CD14) and permeabilized in order to enable intracellular detection of nerve growth factor (NGF). The cells were analysed by flow cytometry after setting lymphocyte (left column) and monocyte (right column) gates according to forward scatter vs side scatter properties of the cells. Dot plots of one representative individual of each group are shown.