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. 2003 Apr;111(8):1241-50.
doi: 10.1172/JCI16790.

Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans

David H McDermott  1 , Alan M Fong, Qiong Yang, Joan M Sechler, L Adrienne Cupples, Maya N Merrell, Peter W F Wilson, Ralph B D'Agostino, Christopher J O'Donnell, Dhavalkumar D Patel, Philip M Murphy
Affiliations

Chemokine receptor mutant CX3CR1-M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans

David H McDermott et al. J Clin Invest. 2003 Apr.

Abstract

The chemokine receptor CX3CR1 is a proinflammatory leukocyte receptor specific for the chemokine fractalkine (FKN or CX3CL1). In two retrospective studies, CX3CR1 has been implicated in the pathogenesis of atherosclerotic cardiovascular disease (CVD) based on statistical association of a common receptor variant named CX3CR1-M280 with lower prevalence of atherosclerosis, coronary endothelial dysfunction, and acute coronary syndromes. However, the general significance of CX3CR1-M280 and its putative mechanism of action have not previously been defined. Here we show that FKN-dependent cell-cell adhesion under conditions of physiologic shear is severely reduced in cells expressing CX3CR1-M280. This was associated with marked reduction in the kinetics of FKN binding as well as reduced FKN-induced chemotaxis of primary leukocytes from donors homozygous for CX3CR1-M280. We also show that CX3CR1-M280 is independently associated with a lower risk of CVD (adjusted odds ratio, 0.60, P = 0.008) in the Offspring Cohort of the Framingham Heart Study, a long-term prospective study of the risks and natural history of this disease. These data provide mechanism-based and consistent epidemiologic evidence that CX3CR1 may be involved in the pathogenesis of CVD in humans, possibly by supporting leukocyte entry into the coronary artery wall. Moreover, they suggest that CX3CR1-M280 is a genetic risk factor for CVD.

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Figures

Figure 1
Figure 1
CX3CR1-M280 binds FKN with delayed kinetics. (a) Receptor expression on transfected HEK 293 cell lines. The name of the transfected receptor is at the top of each FACS plot. Thick lines: FITC-conjugated CX3CR1-specific mAb 2A9. Thin lines: isotype antibody controls. Data are representative of three independent experiments. (b) Equilibrium competition binding of 125I-FKN to recombinant CX3CR1 variants. Cell lines analyzed in a were incubated with 0.25 nM 125I-FKN in the presence of azide and the indicated amount of unlabeled FKN for 2 hours at 37°C. Inset: Scatchard analysis of the binding data. (c) Kinetic binding of 125I-FKN to recombinant CX3CR1 variants. Cell lines analyzed in a were incubated with 0.25 nM 125I-FKN at 37°C in the presence of azide for the indicated time period. Data in a and b are presented as mean ± SEM and are representative of three independent experiments. (d) Kinetic binding of 125I-FKN to recombinant CX3CR1 variants. Cell lines analyzed in c were incubated with 125I-FKN for 48 minutes at 37°C, at which time a 1,000-fold molar excess of unlabeled FKN was added. Serial samples of cells were then removed at the indicated timepoints after addition of the unlabeled ligand, and radioactivity was counted. Data are presented as mean ± SEM and are representative of three independent experiments.
Figure 2
Figure 2
CX3CR1-M280 has defective adhesion activity. (a) Receptor expression in transfected K562 cell lines. The name of the transfected receptor is at the top of each FACS plot. Thick lines: CX3CR1-specific mAb 2A9. Thin lines: isotype antibody control. Data are representative of three independent experiments. (b) Flow-based adhesion assay. K562 cells were perfused over monolayers of untransfected (shown as 926) and FKN-transfected (shown as 926-FKN) human endothelial cells at room temperature (RT) or 37°C and allowed to capture at a shear stress of 0.25 dynes/cm2, then washed at progressively higher shear stresses up to 10 dynes/cm2. The numbers of cells remaining firmly bound to 926 cell monolayers at 10 dynes/cm2 are shown. Data represent the mean ± SD of results from three separate experiments at 37°C and four separate experiments at room temperature.
Figure 3
Figure 3
Calcium mobilization by CX3CR1-M280 is defective. Data represent the mean ± SEM of one representative experiment (a) and three independent experiments (b). The cell lines used were the same as those analyzed in Figure 1. (a) Kinetics. Shown are real-time fura-2 AM fluorescence traces of untransfected HEK 293 cells or HEK 293 cells expressing the recombinant receptor indicated by the key at upper right in response to 1 μM soluble FKN added at 19 seconds. (b) Concentration dependence. Shown is the maximal fura-2 AM fluorescence change of HEK 293 cells stably transfected with the indicated construct in response to the indicated concentration of FKN. P values at the top of each set of bars compare results for CX3CR1-M280 and CX3CR1-WT by two-tailed Student t test.
Figure 4
Figure 4
Leukocytes from CX3CR1-M280 homozygotes have defective calcium flux and chemotactic responses to FKN. PBMCs from donors homozygous for CX3CR1-WT (i.e., genotype 1 in Table 2) and CX3CR1-M280 (i.e., genotype 3 in Table 2) were used. (a) Calcium flux: kinetics. Shown are real-time fura-2 AM fluorescence traces in response to the addition of 1 μM soluble FKN (arrow). Data represent the mean ± SEM of triplicates, and are representative of four independent experiments. (b) Calcium flux: FKN concentration dependence. Shown is the maximal fura-2AM fluorescence change of PBMCs in response to the indicated concentration of FKN. Data are summarized as the mean ± SEM of results from four independent experiments, two for each CX3CR1-M280 homozygote, in which each condition was tested in triplicate. P values at the top of each set of bars compare results for CX3CR1-M280 and CX3CR1-WT by two-tailed t test. (c) Chemotaxis. PBMCs from the indicated donors were added to the top of Transwell chemotaxis chambers. Specific chemotaxis of CD56+ NK cells (>90% mAb 2A9–positive for all five donors) was determined in response to the indicated concentration of FKN or 10 nM SDF-1 (CXCL12). Data are summarized as the mean ± SEM of results from four independent experiments, two for each CX3CR1-M280 homozygote, in which each condition was tested in triplicate. P values at the top of each set of bars compare results for CX3CR1-M280 and CX3CR1-WT by two-tailed Student t test.
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