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Abstract
Objective To analyze the IL-6 signaling pathway in SLE to better understand why CRP levels are disproportionally low for the increased IL-6 levels in active SLE, but increase in bacterial infection in the same patients.
Methods PBMC and sera of 41 SLE patients and 71 healthy individuals (HC) were investigated. IL-6 and soluble IL-6 receptor (sIL-6R) were measured by ELISA. CD126 and phosphorylated Stat3 (pStat3) were stained with phycoerythrin (PE)-labelled antibodies. PBMC were incubated short term with IL-6 or for 24 hours with or without IL-6, IL-10, TNF, interferon-α (IFNα), or combinations of these. HEK293T cells transfected with wildtype IL-6R or a shedding resistant IL-6R mutant were incubated with or without IL-6, IFNα, or their combination. Immunoprecipitation (IP) and immunoblotting was used to detect sIL-6R in supernatants. Flow cytometry was used for the other analyses.
Results IL-6 was increased in SLE (median 3.64 vs 0.89 pg/ml in HC, p<0.0001). ECLAM correlated with IL-6 (Spearman r=0.40, p<0.01), but not CRP (r=0.29). CD126+ lymphocytes were decreased (median 46% SLE vs. 61% for HC, Mann Whitney p<0.0001), and IL-6-induced Stat3 phosphorylation was reduced (Δmfi 14.2 vs. 18.8, p=0.0044). In a mirror image of CD126, sIL-6R was increased in SLE (median 42.2 ng/mL vs. 38.6 ng/mL in HC, p=0.02). Stimulation of healthy PBMC with IL-6 plus IFNα led to a 39±13% reduction (p<0.0001) in CD126+ cells (figure 1A) and to an increase in sIL-6R (p=0.0055) (figure 1B), mimicking the in vivo situation. IL-6-induced increases in supernatant sIL-6R were found for HEK293T transfected with IL-6R, but not those transfected with shedding-resistant IL-6R.
Conclusions The combination of type I interferon and IL-6, both of which are well known to be increased in SLE, leads to shedding of membrane bound CD126 to sIL-6R. This moves IL-6 effects from the membrane CD126 and gp130 receptors of the liver, which produces CRP, to other cells that bind the IL-6/sIL-6R complex with their gp130. This effectively increases peripheral IL-6 inflammatory effects while limiting CRP levels. As such, the data offer a molecular explanation for the (relatively) low CRP levels in SLE, which have been found associated with the type I interferon signature.
Acknowledgements This project was in large parts funded by Deutsche Forschungsgemeinschaft (DFG) grant AR-757/1–1 to Martin Aringer.
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