Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators

Abstract

To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10(-323)). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10(-13)). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications.

Figure 1

GWA study of serum MCP-1. (A) Signal-intensity plot showing the age- and sex-adjusted association of SNPs across all autosomes with MCP-1 in the GWA analysis. The coordinate provides the number of the chromosome. Lines indicate P value thresholds for P = 5 * 10⁻⁸. (B) Regional plots of associations with lowest P value for the DARC gene. Based on HapMapCEU, NCBI Build 36. The color coding represents r² with lead SNP (rs12075), white indicates r² < 0.01; gray, 0.01 ≤ r² < 0.1; yellow, 0.1 ≤ r² < 0.2; orange, 0.2 ≤ r² < 0.5. Signal intensity for multivariable-adjusted serum concentrations and results for plasma are presented in supplemental Figure 2.

Figure 2

Linkage analysis of serum MCP-1. Age- and sex-adjusted multipoint linkage of MCP-1 on chromosome 1 (154.8 Mb) with an LOD score of 14.3; genome-wide P = 8.0 * 10⁻¹³. The DARC gene is located between 157 441 134 and 157 442 914 base pairs (bp) on chromosome 1.

Figure 3

Molecular weight estimates of MCP-1 in serum and plasma. Results of size exclusion chromatography for MCP-1 (dashed lines) in (A) EDTA plasma, (B) serum, and (C) heparin plasma. Arrows indicate elution of molecular weight standards, in kDa; solid line indicates absorbance at 280 nm. (A) In EDTA plasma MCP-1 predominantly elutes from the gel filtration column at a molecular weight of approximately 13 500 kDa. The slight shoulder suggests possible dimerization. (B) MCP-1 in serum elutes predominantly as a single peak similar to EDTA plasma. (C) MCP-1 in heparin plasma elutes at a large molecular weight range, suggesting binding of the MCP-1 to the various molecular weight glycosaminoglycans present in unfractionated heparin.

Figure 4

Darc expression on erythrocytes. (A) A representative field showing Darc expression on erythrocytes visualized by confocal microscopy (“Darc expression”). (B) Flow cytometric histograms of red cell Darc expression from representative persons with A/A (blue) and G/G (red) genotypes. The corresponding isotype controls are shown as red and blue lines. The table indicates the average values for the percentage of erythrocytes expressing Darc and the mean fluorescent intensity from persons with G/G, A/G, and A/A genotypes, as described in “Darc expression.”