Scavenging roles of chemokine receptors: chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues

Abstract

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1(-/-) mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2(-/-) mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3(-/-) mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Figure 1

Comparative analysis of circulating CX3CL1 and CCL2 levels. Serum samples from Cx3cr1^−/−, Ccr2^−/−, and Cx3cr1^−/−Ccr2^−/− double-knockout mice were assayed by ELISA for the presence of CX3CL1 and CCL2, and results are shown in the left y-axis (open symbols) and right y-axis (filled symbols), respectively. Bars show mean value with 95% confidence interval (CI) of chemokine in pg/mg of protein. Each point represents an individual mouse.

Figure 2

Reconstitution with wild-type bone marrow was sufficient to clear excess ligand. Serum levels of CX3CL1 (A) and CXCL1 (B) were measured by ELISA before and 6 weeks after bone marrow transfer. The results reveal that reconstitution with wild-type cells significantly decreased serum levels of ligands to levels comparable with wild-type mice. Similarly, measurement of chemokines in CNS tissue of mice shows that CX3CL1 (C) and CXCL1 (D) were reduced 6 weeks after reconstitution with wild-type bone marrow. Clearance of CX3CL1 by CX3CR1 was evaluated in an in vitro brain slice preparation (E). Bars show mean value with 95% CI of CX3CL1 in pg/mL. Each point represents a value from individual wells in 2 independent experiments.

Figure 3

Impaired CCL3 binding in CCR2-deficient PBMCs. Peripheral blood mononuclear cells were assayed for CCL3 binding as a measurement of CCR1/CCR5 function by flow cytometry. When comparing wild-type (A) and Ccr2^−/− (B) mice, a decreased percentage of CD11b⁺ cells shows CCL3 binding in cells lacking CCR2; a specificity control using a CCL3-blocking antibody is shown (C). Decreased CCL3 binding was not observed in other chemokine receptor–deficient lines with intact CCR2 expression, such as Cxcr3^−/− mice (D). Graphed results (E) revealed a 50% reduction in CCL3 binding in cells lacking CCR2. Bars show mean value with SD. Each point in the bar represents an individual mouse.

Figure 4

Decreased CCL3 binding and CCL3-induced biologic responses in CCR2-deficient resident peritoneal cells. Resident peritoneal cells were assayed for CCL3 binding as described and the macrophage population was gated (A; solid oval). Plots of an unstained sample (B), wild-type cells (C), and Ccr2^−/− cells (D) are shown. Similar to the previous results, a higher number of CCL3-bound cells was found in wild-type cells (C) compared with Ccr2^−/− resident peritoneal cells (D,E). Ccr2^−/− cells also exhibited a defective migratory response to CCL3 (F) but not to CXCL12 (not shown). Bars show mean value with SD in panels E and F. Each point in panel E represents an individual mouse. Each point in panel F represents the mean of 2 independent experiments.