Background IL-6 signals via a receptor consisting of gp130 (CD130) and either membrane-bound IL-6 receptor α (CD126) present on leukocytes and hepatocytes or soluble (sIL-6Rα) on all other cells, the latter process being called trans-signalling.
Objectives To investigate the IL-6/IL-6 receptor system in SLE and compare it to healthy individuals (HC) and RA patients.
Methods We compared lymphocytes of 33 SLE, 16 RF and ACPA positive (RA+) and 12 seronegative (RA-) RA patients, and 50 HC. PBMC were stained with PE-labelled or control antibodies for CD126 and CD130. For determining Stat3 phosphorylation, PBMC were stimulated with IL-6 (250 ng/ml) for 10 min and 15 min, fixed with formaldehyde, permeabilized with methanol, and stained with PE-labelled anti-pStat3 or control antibodies. For in vitro experiments, HC PBMC were incubated for 24 hours in complete RPMI with or without the addition of IL-6, IL-10, TNF, interferon-α (IFNα) or combinations of these cytokines. CD126 staining was performed as above. Cells were analyzed on a Becton Dickinson FACSCalibur fluorocytometer, gating for lymphocytes. Increase in pStat3 mean fluorescence intensity (mfi) was used as an estimate of IL-6 signalling. Serum levels of IL-6 and soluble IL-6 receptor (sIL-6R) were measured by ELISA.
Results As compared to IL-6 serum levels of HC (0.5 (0.5-12.8) median (range) pg/ml), serum IL-6 was increased in SLE (3.7 (0.97-69.3) pg/ml, p<0.0001), RA+ (7.9 (2.4-158.5) pg/ml, p<0.0001) and RA- (5.1 (1.2-17.5) pg/ml, p<0.0001); RA+ IL-6 levels were significantly higher than those of SLE patients (p=0.02). The percentage of CD130+ lymphocytes, as compared to HC (61 (17-76) %), was significantly decreased in SLE (48% (15-85), p=0.003), but not RA+ (55 (41-73) %, p=0.1) or RA- patients (60 (49-77), p=0.7). The percentage of CD126+ lymphocytes, again compared to HC (61±9 (mean ± SD) %), was decreased in SLE (47±16%, p<0.0001) and RA+ (54±9%, p=0.007), but not RA- (61±13%, p=0.9) patients. Percentages of CD126+ and CD130+ lymphocytes were highly correlated with each other (r=0.82, (p<0.0001) for SLE; r=0.76 (p=0.0006) for RA+; r=0.9 (p<0.0001) for RA-). In contrast to the membrane receptor, sIL-6R serum levels (38.7 (18.1-66.9) ng/ml in HC) were increased in SLE (42.3 (26.9-109.6) ng/ml, p=0.03), RA+ (55.4±18.2 ng/ml, p=0.004), but also RA- (50.2±14 ng/ml, p=0.006). The decrease in surface receptor was functional in that IL-6, which induced a pStat3 mfi increase of 16.11±7.6 in HC lymphocytes, led to a significantly lesser increase in pSTAT3 in RA+ lymphocytes of 7.01±12.7 (p=0.02), and a trend (p=0.07) towards a diminished increase in SLE lymphocytes of 11.3±12.17. In vitro experiments showed that combinations of IL-6 with either TNF or interferon-α led to a pronounced reduction in the percentage of lymphocytes carrying CD126.
Conclusions IL-6 is increased in SLE, albeit less than in RA+. Cytokine combinations released in response to immune complexes induced a decrease in IL-6 receptors in vitro, which was similar to the in vivo situation. The concomitant increase in sIL-6R in SLE and RA+ sera points to receptor shedding. Reduction in surface receptors appeared to reduce IL-6 induced Stat3 phosphorylation. Thus, shedding may decrease leukocyte (and possibly hepatocyte) effects of IL-6, while increasing effects on other cells that rely on trans-signalling.
Disclosure of Interest M. Skwarek: None declared, J. Fantana: None declared, B. Heschel: None declared, M. Aringer Consultant for: Roche, Speakers bureau: Roche