Figure 1.
rs2228145 is the major determinant of circulating sIL-6R levels at the IL6R locus.
(A) X2 statistics for the association (additive allelic effects model of inheritance; see Methods) of 45 SNPs genotyped using the Illumina ImmunoChip with sIL-6R concentrations are plotted against the physical position of the SNPs on chromosome 1 (hg.19). Recombination rates and linkage disequilibrium (r2) values are based on CEU HapMap. Inset depicts exonic structure of the membrane-bound IL-6R (fl-IL6R) and the differentially spliced soluble isoform (ds-IL6R). (B) Results from a regression model containing all three independent IL6R SNPs. Mean differences in sIL-6R concentration (%) compared to the common homozygote reference groups are plotted for the 3 SNPs independently associated with sIL-6R levels. Box size is proportional to the number of individuals in each group and error bars represent the standard error of the mean. SE = standard error of the mean difference. R2 = variance explained by the individual SNP.
Figure 2.
rs2228145 regulates the expression of the ds-IL6R but not the fl-IL6R isoform.
(A) Expression of fl-IL6R and (B) ds-IL6R relative to the housekeeping gene B2M was measured by quantitative real-time PCR of RNA purified from PBMCs of 88 healthy volunteers from the Cambridge BioResource. Individual expression levels and their mean (±SEM) are plotted according to rs2228145 genotype. Differences in the mean expression levels relative to the common homozygotes group (Asp/Asp) are indicated above the black horizontal lines. P-values represent test for an association of rs2228145 with the expression levels of fl-IL6R or ds-IL6R, using an additive allelic effects model. AU, arbitrary units.
Figure 3.
The 358Ala allele is associated with decreased levels of membrane-bound IL-6R.
Surface expression of IL-6R was quantified by flow cytometry in cryopreserved PBMCs from 128 volunteers from the Cambridge BioResource. Donors were sampled according to rs2228145 genotype. IL-6R surface expression was measured in four distinct immune cell subsets: CD4+ naïve and memory T cells, CD4+ regulatory T cells (Treg) and monocytes. Scatter plots depict the individual normalized IL-6R fluorescence intensity values measured as molecules of equivalent fluorochrome (MEF; see Methods for details). Error bars represent the standard error of the mean as shown by the middle horizontal line. The horizontal grey dotted reference line represents the average background fluorescence signal of the isotype control group. Differences in the mean expression levels, relative to the common homozygote group (Asp/Asp) are indicated above the horizontal black lines. P-values represent test for an association of rs2228145 with surface IL-6R levels, using an additive allelic effects model (see Methods for details).
Figure 4.
The 358Ala allele is associated with reduced IL-6 signaling potential.
(A) Frequency of pSTAT3 and (B) pSTAT1 positive cells following stimulation of PBMCs with 0, 0.1, 1 or 10 ng/ml of IL-6. Intracellular levels of pSTAT3 and pSTAT1 were measured by flow cytometry in three distinct immune cell subsets: CD4+ naïve T cells, CD4+ memory T cells and monocytes in 14 Asp/Asp and 14 Ala/Ala volunteers from the Cambridge BioResource. Median and interquartile range of the distribution of the frequency of pSTAT3 and pSTAT1 positive events in the two genotype groups for each dose of IL-6 stimulation are plotted. P-values represent tests for differences between rs2228145 genotype groups in pSTAT activation compared to control across doses. (see Methods and Figure S6 for details).