Fig 1.
CCL18 is a poorly potent recruiter of 4DE4-CCR8 cells and the parental 4DE4 line in chemotaxis assays.
(A) Cell surface levels of CCR8 expressed stably in the 4DE4 cell line maintained in 0.1 mg/mL G418. The green line denotes detection with an anti-CCR8 mAb and the solid histogram reflects isotype control staining. (B) Comparative chemotaxis responses of the same 4DE4-CCR8 line to CCL1 (blue) and CCL18 (red) and the parental 4DE4 line (maintained without G418) to CCL18 (black). (C) Inhibition of chemotactic responses to 1 nM CCL1 and 2 μM CCL18 from 4DE4-CCR8 cells (blue) and the parental 4DE4 line (black), respectively. Striped bars denote pre-incubation of cells for 30 min at 37°C with 500 nM MC148. Data represent mean chemotactic indices ± S.E.M. from 3 independent experiments. * denotes p<0.05 as examined by a paired t-test.
Fig 2.
CCL18 is a poorly potent recruiter of various mouse pre-B cells expressing CCR8.
(A, B) Chemotactic responses to CCL1 (blue) and CCL18 (red) from two independently rederived clones of the 4DE4-CCR8 cell line maintained in 0.1 mg/mL G418. (C, D) Chemotactic responses to CCL1 (blue) and CCL18 (red) from L1.2 cells stably (C) or transiently (D) expressing CCR8. Data represent mean chemotactic indices ± S.E.M. from 3 (panels A -B) or 4 (panels C-D) independent experiments.
Fig 3.
CCL18 fails to induce significant endocytosis of CCR8 in transfectants.
(A) Representative cell surface levels of CCR8 on the 4DE4-CCR8 stable cell line which were untreated (green line) or incubated with 100 nM CCL1 (magenta line) for 30 min at 37°C, then assayed by flow cytometry. Isotype staining of untreated cells is shown as a comparator (filled histogram). (B) Levels of CCR8 expression on the 4DE4-CCR8 cells following incubation with CCL1 (100 nM), CCL18 (1 μM), or CCL17 (100 nM) for 30 minutes at 37°C. Data represent mean % basal CCR8 expression levels ± S.E.M. from 6 independent experiments. **** represents p<0.0001 and NS represents no significant difference compared with untreated cells, as examined by one-way ANOVA and Bonferroni’s multiple comparisons test. (C) CCR8 expression detected by 433H (anti-CCR8) or isotype control on 4DE4-CCR8 cells following incubation with either buffer for 30 min at 4°C or 100 nM CCL1, for 30 min at 4°C or 37°C. Data show median fluorescence levels ± S.E.M. from 6 independent experiments. *** represents p<0.001 and ** represents p<0.01 compared with buffer treated cells stained with 433H, as examined by one-way ANOVA and Bonferroni’s multiple comparisons test.
Fig 4.
CCL18 can antagonize chemotaxis of 4DE4-CCR3 but not 4DE4-CCR8 transfectants.
(A) Representative cell surface levels of CCR3 (green line) on the 4DE4-CCR3 stable cell line. Isotype control staining is shown as a shaded histogram. (B, C) Inhibition of chemotactic responses of 4DE4-CCR3 (B) and 4DE4-CCR8 (C) transfectants to a fixed 1 nM concentration of CCL11 or CCL1, respectively, by increasing concentrations of CCL18. Data shown are mean % of migrating levels ± S.E.M. from 5 independent experiments. **** represents p<0.0001 when compared with responses to buffer. (D) Binding of increasing concentrations of AF-CCL18 to 4DE4-CCR3 (green), 4DE4-CCR8 (red) and parental 4DE4 (black) cells. Data represent mean fluorescent indices ± S.E.M. from 5 independent experiments. ** represents p<0.01 when compared with buffer treatment, as examined by one-way ANOVA and Bonferroni’s multiple comparisons test.
Fig 5.
CCR8 mediates signal transduction in response to CCL1 but not CCL18.
Concentration-response curves for chemokine-induced (A) G protein activation, (B) cAMP inhibition and (C) β-arrestin2 recruitment in Flp-In CHO cells stably expressing CCR8. The presented data are mean ± S.E.M. of at least three independent experiments, each performed in triplicate.
Fig 6.
Four CCL18 variants fail to activate or inhibit G protein activation mediated by CCR8.
(A) Sequences of CCL18 variants. (B) C4 reversed-phase HPLC traces for purified CCL18 variants. (C) Mass spectrometry data for the purified CCL18 variants. Expected masses assume two disulfide bonds. Observed masses were obtained from the 5+ charged peaks in electrospray ionization spectra. (D) G protein activation data for CCL18 variants (red) and CCL1 (positive control, blue), each at 100 nM. (E) The CCL18 variants (red), each at 300 nM, fail to inhibit G protein activation stimulated by 100 nM CCL1. The data in panels D and E were obtained using Flp-In CHO cells stably expressing CCR8. The presented data are mean ± S.E.M. of at least three independent experiments, each performed in triplicate.
Fig 7.
mCCR8 mediates signal transduction in response to mCCL1 but not mCCL8.
Concentration-response curves for chemokine-induced (A) G protein activation, (B) cAMP inhibition and (C) β-arrestin2 recruitment in Flp-In CHO cells stably expressing mCCR8. The presented data are mean ± S.E.M. of at least three independent experiments, each performed in triplicate.