Søren Ribel-Madsen

This study aimed at determining if synovial cell cultures from rheumatoid arthritis (RA), osteoarthritis (OA), and healthy controls (HC) differ and are suitable disease models in pharmacological studies, and tested their response to some anti-inflammatory drugs. Synovial cells were isolated from synovial membrane or joint fluid. Cells were cultivated and exposed to no or TNF-α stimulation without, or in the presence of, betamethasone, ibuprofen, or a standardized ginger extract. Concentrations of a panel of cytokines, growth factors, and chemokines were mapped for each culture and condition. Our cells secreted an increased amount of the cytokines IL-1β, IL-6, and IL-8 in response to TNF-α stimulation in all conditions. OA cells showed a higher IL-6 and IL-8 and a lower IL-1β production, when not stimulated, than RA and HC cells, which were similar. TNF-α stimulation caused similar IL-1β, IL-6, and IL-8 release in all groups. Ibuprofen showed no effect on cytokine production, while ginger extract was similar to betamethasone. Ginger extract was as effective an anti-inflammatory agent as betamethasone in this in vitro model. Cultured fibroblast-like synoviocytes from OA and RA subjects promise to be a useful pharmacological disease model, but further studies, to support results from such a model are needed.

Our aim was to clarify if anti‐tumour necrosis factor (TNF) drugs have effect on expression of three splice forms of FoxP3 mRNA in blood CD4+ T cells from rheumatoid arthritis (RA) patients compared with healthy controls. Forty‐five rheumatoid arthritis patients treated with anti‐TNF therapy were investigated in a 12‐week prospective cohort study. FoxP3 isoforms, CD25 and CTLA‐4 mRNA in blood CD4+ T cells were measured with quantitative real‐time PCR. Patients benefitting from the treatment, based on changes in DAS28 scores, revealed a significant decrease in expression of full‐length FoxP3 following 12 weeks treatment with TNF receptor 2 fusion protein (Etanercept), but not following treatment with anti‐TNF antibodies (Adalimumab or Infliximab). A partial normalization of the CTLA‐4/FoxP3fl ratio and a correlation between clinical improvement and change in FoxP3 mRNA expression were also seen in Etanercept responders. These changes were not observed in responsive patients treated with the antibody therapies. Our data suggest that TNF decoy receptor and anti‐TNF antibodies differ in their effect on FoxP3 expression in responsive patients. As Etanercept binds both TNF‐α and Lymphotoxin‐α (LT‐α), whereas the antibodies only target TNF‐α, LT‐α may regulate FoxP3 expression in a subset of RA patients. Our findings support the view that anti‐TNF treatment is mainly symptomatic.

Ryder LR, Bartels EM, Woetmann A, Madsen HO, Ødum N, Bliddal H, Danneskiold‐Samsøe B, Ribel‐Madsen S, Ryder LP. FoxP3 mRNA splice forms in synovial CD4+ T cells in rheumatoid arthritis and psoriatic arthritis. APMIS 2012; 120: 387–96. Our aim was to elucidate the relative amount of the different splice forms of FoxP3 mRNA in CD4+ T cells in peripheral blood (PB) compared to synovial fluid (SF) in RA and PsA patients. FoxP3 mRNA was measured using a quantitative real‐time PCR method. CD4+ T cells were isolated from 17 paired samples of PB and SF from RA and PsA patients, and PB from 10 controls. FoxP3fl and FoxP3Δ2 mRNA was significantly increased (6.7 and 2.1‐fold, respectively) in PB CD4+ T cells from RA patients compared to controls. FoxP3fl and Δ2 mRNA in SF CD4+ T cells was increased compared to controls in sero‐negative RA and PsA, but not in sero‐positive RA patients, who had a high FoxP3 expression in both PB and SF. The FoxP3Δ2Δ7 mRNA was barely detectable in patient samples, and not at all in healthy individuals. We provide evidence of an increased expression of FoxP3 splice forms in synovial CD4+ T cells from RA patients. A skewed, high expression profile of FoxP3, but not CTLA‐4, in sero‐negative RA and PsA, indicates that synovial CD4+ T cells may represent unique subsets of T cells which have been induced locally or selectively recruited to the joint.

Use of cytokines as biomarkers for disease is getting more widespread. Cytokines are conveniently determined by immunoassay, but interference from present antibodies is known to cause problems. In rheumatoid arthritis (RA), interference of rheumatoid factor (RF) may be problematic. RF covers a group of autoantibodies from immunoglobulin subclasses and is present in 65–80% of RA patients. Partly removal of RF is possible by precipitation. This study aims at determining the effects of presence of RF in blood and synovial fluid on cytokine measurements in samples from RA patients and finding possible solutions for recognized problems. IL-1β, IL-4, IL-6, and IL-8 were determined with multiplex immunoassays (MIA) in samples from RA patients prior to and after polyethylene glycol (PEG 6000) precipitation. Presence of RF does interfere with MIA. PEG 6000 precipitation abolishes this RF interference. We recommend PEG precipitation for all immunoassay measurements of plasma samples from RA patients.