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. 2013 Sep 23;210(10):1889-98.
doi: 10.1084/jem.20130240. Epub 2013 Sep 2.

Identification of human CCR8 as a CCL18 receptor

Sabina A Islam  1 , Morris F Ling, John Leung, Wayne G Shreffler, Andrew D Luster
Affiliations

Identification of human CCR8 as a CCL18 receptor

Sabina A Islam et al. J Exp Med. .

Abstract

The CC chemokine ligand 18 (CCL18) is one of the most highly expressed chemokines in human chronic inflammatory diseases. An appreciation of the role of CCL18 in these diseases has been hampered by the lack of an identified chemokine receptor. We report that the human chemokine receptor CCR8 is a CCL18 receptor. CCL18 induced chemotaxis and calcium flux of human CCR8-transfected cells. CCL18 bound with high affinity to CCR8 and induced its internalization. Human CCL1, the known endogenous CCR8 ligand, and CCL18 competed for binding to CCR8-transfected cells. Further, CCL1 and CCL18 induced heterologous cross-desensitization of CCR8-transfected cells and human Th2 cells. CCL18 induced chemotaxis and calcium flux of human activated highly polarized Th2 cells through CCR8. Wild-type but not Ccr8-deficient activated mouse Th2 cells migrated in response to CCL18. CCL18 and CCR8 were coexpressed in esophageal biopsy tissue from individuals with active eosinophilic esophagitis (EoE) and were present at markedly higher levels compared with esophageal tissue isolated from EoE patients whose disease was in remission or in normal controls. Identifying CCR8 as a chemokine receptor for CCL18 will help clarify the biological role of this highly expressed chemokine in human disease.

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Figures

Figure 1.
Figure 1.
CCR8 is sufficient for CCL18-induced migration. (a and b) Dose–response chemotaxis of hCCR8-transfected and untransfected 4DE4 cells to CCL18 (a) and hCCL1 (b). (c) Chemotaxis of PTX treated hCCR8-transfected cells to CCL18 (*, P < 0.05 by unpaired two-tailed t test in a–c). (d) Chemotaxis of hCCR8-transfected 4DE4 cells in a checkerboard-type transwell chemotaxis assay with varying concentrations of CCL18 in the bottom and top chamber. (e) Chemotaxis of hCCR8-transfected 4DE4 cells to varying concentrations of CCL1 mixed with varying concentrations of CCL18 in the bottom chamber (P = NS for CCL18 by two-way ANOVA). Data in all panels are representative of at least three independent experiments (mean ± SEM).
Figure 2.
Figure 2.
CCL18 induces calcium flux in CCR8-transfected cells. (a) Dose–response calcium flux of hCCR8-transfected cells to CCL18. (b) Calcium flux of untransfected 4DE4 cells to CCL18 and CXCL12. (c and d) Heterologous cross-desensitization of hCCR8-transfected cells to CCL18 and CCL1 (c), and to CCL18 and vMIP-I (d). a–d are representative of three to eight independent experiments.
Figure 3.
Figure 3.
CCL18 induces internalization of CCR8 and inhibits 125I-CCL18 and 125I-hCCL1 binding to CCR8. (a) Representative FACS plots of CCR8 surface expression on hCCR8-transfected and untransfected cells. (b) Dose-dependent internalization of surface CCR8 in hCCR8-transfected cells stimulated with CCL18 and hCCL1 but not CCL17 (*, P < 0.05; **, P = 0.06, for CCL18 vs. CCL17 and CCL1 vs. CCL17 by unpaired two-tailed t test). (c) Kinetics of internalized versus surface CCR8 expression in hCCR8-transfected cells stimulated with CCL18 (10−8 M) or hCCL1 (10−8 M) for 1, 5, 10, or 20 min at 37°C (*, P < 0.05 for internalized or surface CCR8 treated with CCL18 or CCL1 at 37°C compared with 4°C control by unpaired two-tailed Student’s t test). Data are expressed as mean ± SEM, representative of at least five experiments in a–c. (d) Inhibition of 0.2 nM 125I-CCL18 binding to hCCR8-transfected cells with increasing concentrations of unlabeled CCL18 and hCCL1. Representative of two (cold hCCL1) and five (cold CCL18) experiments (P = 0.01 for CCL18 versus CCL3 and P ≤ 0.0001 for CCL1 versus CCL3 by two-way ANOVA). (e) Inhibition of 0.1 nM 125I-hCCL1 binding to hCCR8-transfected cells with unlabeled CCL18 and hCCL1. Representatives of three (cold hCCL1) and five (cold CCL18) experiments are shown; data are shown as mean ± SEM in d and e.
Figure 4.
Figure 4.
CCL18 induces calcium flux and CCR8-dependent migration of activated highly polarized Th2 cells. (a) Representative histograms of CCR8 expression on human Th2 cells that have undergone one (Th2 R1), two (Th2 R2), or three (Th2 R3) rounds of Th2 polarization. (Left) CCR8 mAb staining and isotype control are depicted with colored and gray lines, respectively. (Right) Quantitation of CCR8 surface expression on human Th2 cells. (*, P < 0.05 for CCR8 on Th2 R1 vs. Th2 R3 by unpaired two-tailed Student’s t test). (b) Representative cytokine profiles of Th2 cells. (c) Dose–response chemotaxis of activated human Th2 cells to CCL18 (left) or hCCL1 (right; *, P < 0.05 for Th2 R1 vs. Th2 R3 by unpaired two-tailed Student’s t test). (d) Comparison of chemotaxis of 100 ng/ml PTX–treated activated human Th2 R3 cells and untreated controls. (e) Activated human Th2 R3 cells were treated with 1 µg/ml CCR8 mAb and compared with untreated activated Th2 R3 cells in chemotaxis assay to CCL18. a–e are representative of at least three independent experiments (mean ± SEM). (f) Dose-dependent calcium flux response of human Th2 R3 cells to CCL18 stimulation. (g) Heterologous cross-desensitization of the calcium flux response of human Th2 R3 cells to CCL18 and hCCL1 stimulation. f and g are representative of three to five separate experiments. (h) Representative histograms of surface expression of the LFA-1 activation epitope m24 on activated human Th2 R3 cells treated with CCL18, CXCL12, PMA, or not treated (left). Quantitation of m24 expression on activated Th2 R3 cells after CCL18 or CXCL12 stimulation (right). (i) Chemotaxis of activated wild-type and Ccr8-deficient mouse Th2 cells to CCL18. (j) Chemotaxis of mCcr8-transfected and untransfected Baf/3 cells to CCL18 stimulation. h–j are representative of at least three independent experiments (mean ± SEM; *, P < 0.05 by unpaired two-tailed Student’s t test for d, e, and h–j).
Figure 5.
Figure 5.
CCL18 expression in AAM and in human EoE. (a) Induction of CCL18 and CCL17 RNA in human AAM by IL-4 at 24 h. (b) Comparison of 24- and 72-h CCL18 and CCL17 induction in human AAM after IL-4, IL-10, and TNF treatment. (c) 24-h induction of Ccl8 and Ccl17 in mouse AAM by IL-4. (d) Comparison of 24 and 72 h Ccl8 and Ccl17 induction in mouse AAM after treatment with IL-4, IL-10, and TNF. a–d are representative of at least three independent experiments (mean ± SEM; *, P < 0.05 by unpaired two-tailed Student’s t test for a–d). (e) Expression of CCL18 and CCR8 and other Th2-associated chemokines (top) and receptors (bottom) in esophageal biopsy tissue recovered from individuals with active EoE (A), individuals with EoE in remission (R), and normal controls (N). Data are presented in box-and-whiskers plots with bars indicating fifth and 95th percentiles (*, P < 0.05 by Mann-Whitney test). (f) Relationship of CCR8 mRNA expression to the expression of CCL1 (blue square), CCL18 (green circle), and CCL26 (red diamond) in the human esophageal samples from all groups studied (r represents Spearman correlation).
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