THU0112 Effects of inflammatory activity and therapeutic interleukin-6 receptor (IL-6R) blockade on IL-6R alpha expression and STAT3 phosphorylation in rheumatoid arthritis (RA)
Authors: M. Skwarek, B. Heschel, M. Winzer, M. Aringer
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> IL-6 is one of the main proinflammatory cytokines involved in RA. IL-6 drives CRP and may downmodulate its own receptor. IL-6 signalling centrally involves STAT3 tyrosine phosphorylation. <h3>Objectives</h3> To analyse the effects of systemic inflammation and the influence of tocilizumab on IL-6Rα (CD126) expression and STAT3 phosphorylation in RA patients. <h3>Methods</h3> Peripheral venous blood was taken from 37 RA patients, 10 of whom subsequently underwent tocilizumab therapy, and from 26 healthy individuals (HC). Clinical disease activity measured by CDAI and serum CRP were determined in clinical routine. Peripheral blood mononuclear cells (PBMC) were prepared immediately after blood drawing. Anti-CD126 staining was performed with PE-labelled or control antibodies. For the determination of phosphorylated (p) STAT3, cells were analyzed unstimulated or stimulated with IL-6 (0.25μg/ml) for 10 and 15 minutes, fixed with formaldehyde (2%), permeabilized with methanol (80%), and stained with PE-labelled anti-pSTAT3 or control antibodies. Cells were analyzed on a Becton Dickinson FACSCalibur fluorocytometer, gating for both lymphocytes and monocytes. Mean fluorescence intensity (mfi) was used as a measure for pSTAT3 contents. <h3>Results</h3> At baseline, the percentage of CD126+ lymphocytes was decreased in RA vs. HC (mean±SD 46±18% vs. 55±10%, p=0.02), while the percentage of CD126+ RA monocytes was increased (median (range) 90% (54-98%) vs. 82% (13-95%) HC, p=0.005). Nevertheless, both showed negative correlations with CRP (lymphocytes: Pearson r=-0.72, p=0.002; monocytes: Spearman r=-0.32, p=0.006), while neither correlated with CDAI. Baseline pSTAT3 was increased in both RA lymphocytes (mfi 16.6 (8.8-60.5) vs. 13.8 (8.8-27.8), p=0.02) and monocytes (mfi 39.6 (15.5-148.8) vs. 41.3 (22.6-65.7), p=0.91), and again correlated with CRP (r=0.36, p=0.003 and r=0.27, p=0.025, respectively), but not CDAI. In contrast, the maximum increase in STAT3 was less pronounced in lymphocytes (delta mfi 5.6±5.5 vs. 16.1±7.6, p&lt;0.0001) and monocytes (delta mfi 21.1 (-4.9-52.2) vs. 42.4 (5.6-105.2), p=0.002). Following tocilizumab, CD126+ monocytes increased (to 97% (81-100%), p&lt;0.0001), CD126+ lymphocytes stayed constant (57±18%). Under tocilizumab, the pSTAT3 increase upon IL-6 stimulation was almost completely abolished in lymphocytes (delta mfi 3.5±3.4, p&lt;0.0001) and severely reduced in monocytes (delta mfi 7.66 (-7.1-33.9), p&lt;0.0001). Basal pSTAT3 was reduced to normal in both (lymphocytes: mfi 14.26 (8.8-36.3), monocytes: 33.4 (24.9-62.7)). <h3>Conclusions</h3> Chronic influence of IL-6 induces differential effects on lymphocyte and monocyte CD126 expression, while increasing basal, and decreasing stimulated, pSTAT3 in both cells. These changes expectedly correlate well with CRP, but were not significantly correlated with clinical disease activity. As expected, therapeutic IL-6R blockade with tocilizumab blocks both IL-6 induced STAT3 phosphorylation and IL-6 dependent chronic effects on basal pSTAT3, while the increased monocyte CD126 expression in RA may stem from a different mechanism. <h3>Disclosure of Interest</h3> M. Skwarek: None Declared, B. Heschel: None Declared, M. Winzer: None Declared, M. Aringer Consultant for: Advisory boards Roche/Chugai (Tocilizumab)