The present study is a descriptive comprehensive quantitative expression analysis of mRNA transcripts encoding the four known human neurotrophins and their four corresponding receptors in the synovial compartment of arthritis patients.
The presence of neurotrophic factors in the inflamed joint has been described earlier [2
]. Focusing on the NGF/trkA-p75 axis in our own and other work, we could previously demonstrate high trkA and p75 NGF receptor expression at the protein level in the inflamed ST in peripheral SpA synovitis. This expression was correlated with inflammatory disease activity and was downregulated by TNF-blocking treatment indicating that their expression is not constitutive but actively modulated in inflammation [7
]. However, the high-affinity receptors trkB and trkC as well as the ligands NGF, NT-3, and NT-4 were expressed in the minority of patients or not detectable by immunohistochemistry.
In order to investigate the NGF/trkA-p75 axis as well as all other neurotrophic ligands and receptors at the transcript level, we used quantitative real-time RT-PCR to determine their expression in a larger panel of SpA and RA patients with active peripheral synovitis of the knee. Our data confirm the high expression of both the trkA and p75 NGF receptors at the transcript level in the synovial compartment of SpA and RA patients. Of note, we now provide evidence that the NGF ligand is also expressed in the SF and tissue biopsy samples of peripheral synovitis indicating that this system is active in chronic inflammatory arthritis. However, we need to state, that the high NGF transcript expression is in contrast to our previous ELISA data in SF samples [7
]. This discrepancy between mRNA and protein expression has been reported earlier in studies on brain tissue [19
]. Reasons for this phenomenon might involve post-transcriptional modifications of NGF [1
]. We also can not exclude technical reasons such as the NGF antibody used for the previous quantitative immunoassay.
The cellular source of NGF in humans has been investigated in several studies. Under unstimulated conditions, NGF is produced mainly by CD4+ T and B lymphocytes [1
]. Under inflammatory conditions such as allergy and arthritis, NGF can be produced, stored, and released by eosinophils, mast cells, lymphocytes, and synovial fibroblasts, as well as monocytes and macrophages [22
]. Using intracellular FACS analysis, we could demonstrate the presence of the NGF protein in the two inflammatory arthritis groups on both CD3+ and CD14+ cells, that is, T lymphocytes and monocytes/macrophages, which are known to be involved in the major pathways of both SpA and RA. However, ST-derived FLS do not seem to produce NGF as measured by ELISA. This finding might indicate the mere pro-inflammatory potential of NGF as opposed to factors released by fibroblasts, which are predominantly involved in structural damage. On the other hand, we can not definitely rule out the production of NGF by FLS. One explanation would be, that FLS loose their ability to produce NGF when cultured over three passages in vitro
. Another explanation would be that NGF is produced but not secreted, at least not in large amounts. Nevertheless, FLS are most likely one of the targets of NGF.
To date, the functional role of neurotrophins in inflammatory joint disorders is unclear. A pathogenic role for the NGF/trkA-p75 axis and other neurotrophins has been postulated for airway inflammation [23
], atopic dermatitis [24
], psoriasis [25
], inflammatory bowel disease [26
], and arthritis [2
]. In inflammatory syndromes, NGF has been attributed to upregulating TNF-alpha, promoting the differentiation of B cells to plasma cells, enhancing chemotaxis and production of superoxide by neutrophils [22
]. NGF is also involved in humoral immune responses by acting as an autocrine survival factor maintaining the viability of memory B-cells and macrophages [27
]. NGF and its receptors have also tissue remodeling capacities exerting a strong fibrotic stimulus on skin and lung fibroblasts [28
]. Upon binding to trkA, NGF induces its auto-phosphorylation and subsequently the activation of both phospholipase PLCγ and protein kinase C, which in turn activates the mitogen-activated protein kinase pathway involving the c-jun N-terminal, the p38, and the extracellular-regulated protein kinases (ERK1/2) all of which have been identified in arthritis as well. Interestingly, the wnt proteins, which have been described as regulating neurotrophin expression [29
], have recently also been found to play a pathogenic role in spondyloarthritis [30
]. In addition, NGF has been identified as a proangiogenic factor, another significant pathogenic pathway in chronic inflammatory arthritis such as SpA and RA [31