Background and objectives Low serum vitamin D associates with chronic inflammatory diseases such as rheumatoid arthritis (RA), thus vitamin-D supplementation has been suggested for their treatment. T-cells have a pathogenic role in RA. We have shown that active vitamin-D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), has potent anti-inflammatory effects on T-cells. However the efficacy of vitamin-D in an inflammatory setting is unclear. This study was designed to assess the effect of 1,25(OH)₂D₃ upon inflammatory responses by CD4⁺T-cells from the blood and synovial fluid (SF) of RA patients.

Methods Ethical approval was granted by Solihull Research Ethics Committee. Patients who fulfilled 1987 ACR criteria for RA and age-matched healthy controls were recruited with informed consent. CD4⁺T-cell responses to 100nM 1,25(OH)₂D₃ were assessed by flow cytometry after stimulation. Mononuclear cells from blood and SF were stimulated with antiCD3 and magnetically isolated naïve (CD45-RA) and memory (CD45-RO) CD4⁺T cells were stimulated with monocytes+antiCD3. Regulators of 1,25(OH)₂D₃signalling were measured in purified T-cells by qPCR before and after stimulation with antiCD3/CD28 beads. Significance was tested by Wilcoxon analysis.

Results 1,25(OH)₂D_{3 ­}significantly reduced IL-17 and IFNy production by T-cells from the blood of RA patients and healthy controls. By contrast, SF T-cells from RA patients_, showed reduced suppression of IL-17 by 1,25(OH)₂D₃ and no inhibition of IFNγ. 98(±1.8)% of SF T-cells were memory compared to 49(±14)% for blood. Unlike SF T-cells, 1,25(OH)₂D₃ suppressed IFNγ production by memory T-cells from blood. However across several targets: IL-17, IFNγ, IL-21, CTLA-4 and FoxP3, naive cells showed greater sensitivity than memory cells to 1,25(OH)₂D₃. Interestingly, the vitamin D receptor (VDR) was elevated in SF memory T-cells compared to equivalent cells from blood, suggesting that lack of VDR does not explain the diminished responsiveness of SF T-cells. Furthermore, stimulation, increased VDR, RXR and the VDR co-enhancer/co-repressor ratio to similar levels in T-cells from both sites and suppressed their expression of the 1,25(OH)₂D₃-inactivating enzyme, CYP24A1 equally.

Conclusion Stimulation primes CD4⁺T-cells from RA patient blood and SF to respond to 1,25(OH)₂D₃. However, SF T-cells are less sensitive to 1,25(OH)₂D₃. This is only partially explained by their shift to memory status. Such insensitivity of joint T-cells to 1,25(OH)₂D₃ has important implications regarding the use of vitamin D to treat RA.