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Abstract
In active SLE, CRP levels are disproportionally low for increased IL-6 levels. While a molecular explanation is lacking, these low CRP levels have been found associated with increased type I interferon signature in SLE. IL-6 signalling requires gp130 and IL-6 receptor- α (IL-6R). Leukocytes and hepatocytes carry surface IL-6R (CD126). Other cells express only gp130 and require soluble IL-6R receptor (sIL-6R) for IL-6 signalling. This sIL-6R derives from membrane-bound CD126 shedded by metalloproteinases. We therefore comprehensively analyze IL-6 and its signal transduction via signal transducer and activator of transcription (Stat) 3 in patients with SLE and in healthy individuals (HC).
We analyzed peripheral blood mononuclear cells (PBMC) and sera of 41 SLE patients and 71 HC. IL-6 and soluble IL-6 receptor (sIL-6R) were measures by ELISA. CD126 was stained with phycoerythrin (PE)-labelled antibodies. PBMC were stimulated with recombinant human IL-6, fixed, permeabilized and stained with PE-labelled antibodies to phosphorylated Stat3 (pStat3) or control antibodies. Healthy PBMC were incubated for 24 hours with or without the addition of IL-6, IL-10, tumor necrosis factor (TNF), interferon-α (IFNα), or combinations of these cytokines. Flow cytometry was performed on a Becton Dickinson FACSCalibur fluorocytometer, determining percentages of CD126+ lymphocytes or the increase in pStat3 mean fluorescence intensity (Δmfi). Immunoprecipitation (IP) and immunoblotting was used to detect sIL-6R in supernatants.
IL-6 was increased in SLE (median 3.64 vs 0.89 pg/ml in HC, p<0.0001). Significant correlations with SLE disease activity by ECLAM were found for serum IL-6 (Spearman r=0.40, p<0.01), but not CRP (r=0.29). CD126+ lymphocytes were decreased in SLE (median 46% vs. 61% for HC, Mann Whitney p<0.0001), in line with reduced IL-6 induced phosphorylation of Stat3 in SLE (median Δmfi 14.2 vs. 18.8 in HC, p=0.0044). In a mirror image of CD126, sIL-6R serum levels were increased in SLE (median 42.2 ng/mL vs. 38.6 ng/mL in HC, p=0.02). Stimulation of healthy PBMC with the combination of IL-6 and IFNα led to a reduction in CD126+ cells by 39±13% (p<0.0001)(A) and to an increase in sIL-6R (normalized for IgG) (p=0.0055)(B) detected by IP and Western blotting, mimicking the in vivo situation (figure 1).
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 liver, which produces CRP, to other cells, effectively increasing IL-6 inflammatory effects while limiting CRP levels.
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