IL-23 of 10.2 pg/ml induced 3209 250 c.p.m. low levels of endogenous IL-23 are sufficient to support cytokine production and/or that this relevant Th17 cells were not present. Conclusions. These results suggest that although IL-23 may have pathogenic activity in a proportion of patients with late-stage RA, it is not abundantly produced in this inflammatory tissue, nor does it have a dominant role in all patient tissues analysed. = 7) were stained for IL-23R and -12R1 and cell surface markers, then analysed by circulation cytometry as explained in Materials and methods section (C). The CD4, -45RA and -45RO populations were analysed from your lymphocyte gate as determined by forward and side scatter, whereas the CD14 populace was determined from your macrophage gate. Per cent of positive cells in each quadrant is usually indicated. Data shown are representative plots from one (Donor 4) of seven patients. GM-CSF-differentiated macrophages produced 400C600 pg/ml IL-23 in lysates, but only 50 pg/ml was secreted into the supernatants (data not shown). The secreted IL-23 protein increased (up to 300 pg/ml) upon toll-like receptor (TLR) ligation with LPS, and/or R848 with 600 pg/ml in the lysates. This suggests that 10-fold more IL-23 protein is found in cell lysates compared with secreted IL-23 (again, suggesting that this IL-23 is surface bound), and in comparison with other macrophage-derived cytokines (e.g. TNF-), picogram but not nanogram amounts of IL-23 are produced. IL-17A message has been detected in some, but not all, synovial membrane biopsies [26, 36]. In our synovial cultures, IL-17A message was expressed at very low levels [0.03 (0C1.1)] and was undetectable in 5 of 18 samples GLPG0974 (Table 1). Lack of IL-17A mRNA was also reflected in low levels of IL-17A protein in culture supernatants as determined by ELISA (detection limit 10 pg/ml). Protein was detectable in GLPG0974 6 of 16 samples [10 (0C100) pg/ml] (Fig. 1A). Low levels of IL-17A were also present in synovial lysates from five of seven samples examined [15.2 (3.3C23.1) pg/ml]. Somewhat surprisingly, in two cultures where protein was detected in supernatants and lysates, IL-17A mRNA was undetectable, suggesting that IL-17A transcripts, like some other T-cell cytokines, could be targeted for quick Reln degradation. IL-27 is usually involved in suppressing Th17 development [37], and low levels of IL-27 message (Table 2) were detected in all donors tested [2.1 (0.8C11.8)]. Table 2. Cytokine and cytokine receptor mRNA expression in freshly isolated mononuclear cells from rheumatoid synovium = 2087.3 (6.8C1627)IL-23R, = 160.6 (0C3.6)IL-12p40, = 130.1 (0C2.0)IL-12p35, = 174.7 (0.1C24)IL-12R1, = 179.6 (0.8C35.2)IL-12R2, = 173.8 (0C135.4)IL-17A, = 180.03 (0C1.1)IL-17RA, = 17153 (19.1C349)IL-17RC, = 14106 (10.3C509)IL-27, = 72.1 (0.8C11.8) Open in a separate windows Cytokine mRNA expression of IL-23p19 and -23 receptor, IL-12p35, common chain p40, IL-12 receptor (2) and common receptor chain (1), IL-27 and -17A together with IL-17 receptor models A and C were assessed by Taqman PCR as described in Materials and methods section in freshly isolated synovial membrane mononuclear cells from patients with RA. Results are expressed relative to ubiquitin. IL-23 receptor message was more variable with low levels of IL-23R [0.58 (0C3.6)] and moderate levels of IL-12R1 [9.6 (0.79C35.2)]. High expression of the IL-17A receptors was observed [152.6 (19.1C348.4) for IL-17RA and 105.9 (10.3C508.8) for IL-17RC] GLPG0974 in synovial cells. No correlations were found between percentage of CD3, -14 or GLPG0974 -45 cells detected by FACS and any of the cytokine subunits or receptor subunits detected by mRNA (Spearman test). The presence and relative large quantity of IL-23, and to a lesser extent IL-23R, in RA synovial tissue was confirmed by staining of tissue sections. Abundant IL-23p19 was observed in cells in both the lining.