Figure 1.
Real-time chemotaxis of Bio-Gel elicited macrophages to murine CCL5.
(A) (A) Representative trace from the RTCA-DP software of 4×105 Bio-Gel elicited macrophages from C57BL/6J male mice in the top chamber with murine CCL5 (5 nM) (red) or no chemokine (green) in the bottom chamber. A control of no macrophages and no chemokine was also run (blue). By 70 mins max CI had been reached, the plate was removed and membranes were cut and fixed with 2.5% gluteraldehyde and processed for scanning electron microscopy (SEM). Representative images are shown of macrophages adhered to the underside of filters in response to 5 nM murine CCL5 (C & D) no chemokine (E & F) or control where no cells or chemokine were added (G & H). Pores in the membrane are clearly visible in panel H. The number of cells adhered to individual gold discs on the underside of the membrane (seen as light grey regions in the SEM images) were quantitated (n = 1 experiment with technical replicates of each condition analysed) (B).
Table 1.
Primer sequences used in RT-PCR.
Table 2.
Chemokine receptor mRNA expression in Bio-Gel and thioglycollate elicited macrophages.
Figure 2.
Real-time chemotaxis of different murine macrophage populations to murine CCL3.
Panels A, D, G & J are representative FACS plots of the four different murine macrophage populations tested in the RTCA-DP system chemotaxis assay. (A) Injection with 2% Bio-Gel (polyacrylamide beads) typically yields 60–70% monocyte/macrophages which are collected after 4 days following the initial injection (assessed by staining with F4/80, 7/4 & CD11b antibodies). (B) 4×105 Bio-Gel elicited peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (C) Migration was measured for each curve of each concentration using slope (set between 15–60 mins) analysis. (D) Injection with 4% Thioglycollate generates 70% F4/80+ 7/4− macrophages which were harvested 4 days post injection. (E) 4×105 Thioglycollate-elicited peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h (G). (F) Migration was measured for each curve of each concentration using slope analysis. (G) Lavage of the peritoneal cavity in non-elicited conditions yields ∼30% F4/80hi resident peritoneal macrophages. (H) 4×105 Resident peritoneal macrophages were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (I) Migration was measured for each curve of each concentration using slope analysis. (J) Bone marrow cells cultured over 7 days in the presence of L929 conditioned media +10% FBS generated a 92% pure population of macrophages which expressed both CD11b and F4/80. (K) 0.5×105 BMDM were allowed to migrate toward indicated concentrations of murine CCL3 for 3 h. (L) Migration was measured for each curve of each concentration using slope analysis. Data are shown as migration index (i.e. fold change over media alone) for slope analysis. Data are the mean ± SEM of 3–7 independent experiments, each concentration with 2–3 technical replicates per experiment. Statistical analysis was performed by one-way ANOVA and Dunnett's multiple comparison post-test. *, p<0.05, **, p<0.01, ***, p<0.01 relative to media alone.
Figure 3.
Bio-Gel and thiogclycollate elicited macrophage chemotaxis to CCL2 and CCL5.
Thioglycollate and Bio-Gel elicited macrophages (4×105 total) were allowed to migrate toward indicated concentrations of murine CCL2 (A & E) and CCL5 (C & G) for 4 h. Migration was measured for each curve of each concentration using slope analysis (B & F- D & H). Data are shown as migration index (i.e. fold change over media alone) for each parameter of analysis. Data are expressed as mean ± SEM of 3–5 independent experiments, each concentration with two technical replicates per experiment. Statistical analysis performed by one-way ANOVA and Dunnett's multiple comparison post-test. *, p<0.05, **, p<0.01, ***, p<0.01 relative to media alone.
Figure 4.
Demonstration of chemotaxis and its inhibition by Pertussis toxin (PTX) and cytochalasin D.
To test that the assay was detecting true macrophage chemotaxis, rather than chemokinesis or fugetaxis, the following three conditions were tested; CCL5 added to the bottom well alone (test chemotaxis), equal concentrations (5 nM) of murine CCL5 added to both top and bottom wells (test chemokinesis) or CCL5 added to the top well alone (test fugetaxis) (A) Single trace of a single experiment with four technical replicates shows Bio-Gel elicited macrophages migrate towards murine CCL5 (green), but display no chemokinesis (red) or fugetaxis (blue). Control (pink) had chemotaxis buffer alone in the bottom well. Bio-Gel elicited macrophages were pre-incubated for 90 mins at 37°C with PTX (200 ng/ml). (B) A representative trace shows that pre-treatment of Bio-gel elicited macrophages with PTX significantly inhibits chemotaxis toward both murine CCL2 and CCL5. Data are shown as a percentage of 10 nM CCL2 or CCL5 induced migration for (C) Slope (D) Max-min CI and (E) AUC. Data are the mean ± SEM of three independent experiments with 3–4 technical replicates per experiment. Vehicle alone was omitted in these sets of experiments in order to have all five conditions on a single CIM-16 plate. (F) Bio-Gel elicited macrophages were pre-incubated for 90 mins with either 200 nM cytochalasin D, an inhibitor of F-actin polymerisation. A representative trace is shown (F). Data are shown as a percentage of 5 nM CCL2 induced migration for (G) Slope (H) Max-min CI and (I) AUC. Data are the mean ± SEM of four independent experiments with 3–4 technical replicates per experiment. Statistical analysis was performed by one-way ANOVA and Dunnett's multiple comparison post-test. *, p<0.05, **, p<0.01, ***, p<0.01 relative to CCL2 or CCL5 alone.
Figure 5.
Bio-Gel elicited macrophage chemotaxis to non CC chemokine chemoattractants.
Bio-Gel elicited macrophages from C57BL/6J (4×105 total) were allowed to migrate toward indicated concentrations of murine C5a, chemerin and LTB4 for 4 h. Migration was measured for each curve of each concentration using slope (A, D, G), max-min (B, E, H) and AUC (C, F, I) analysis. Data are shown as migration index (i.e. fold change over media alone) for each parameter of analysis. Data are expressed as mean ± SEM of 3–5 independent experiments, each concentration with two technical replicates per experiment. Statistical analysis performed by one-way ANOVA and Dunnett's multiple comparison post-test. *, p<0.05, **, p<0.01, ***, p<0.01 relative to media alone. (J) A schematic representation of the plate designed used to test whether the chemotactic effects of chemerin are ChemR23 dependent. Representative traces of Bio-Gel elicited macrophages from ChemR23-/- and WT controls in response to (K) chemerin and (L) CCL5 are shown. Migration was measured for each curve using slope (M) and Max-min CI (N). Data are the mean ± SEM of three independent experiments with 2–4 technical replicates per experiment. Statistical analysis was performed by two-way ANOVA and Bonferroni multiple comparison post-test. *, p<0.05, **, p<0.01, relative to WT.
Figure 6.
Analysis of chemotaxis responses in human monocytes reveals distinct donor to donor variation.
Panel (A) shows a representative FACS plot of human monocytes following isolation by CD14 positive selection (Inset panel red = CD14+; blue = isotype control Ab). Panels B, C, and D are individual traces from three independent donors measuring monocyte (4×105 total) chemotaxis toward 5 nM human CCL2. (E) The inhibitory action of an anti-CCR2 drug (INCB-3284 dimesylate) was also tested with human monocytes. Human monocytes were pre-incubated with INCB-3284 (1 µM) for 90 mins at 37°C and allowed to migrate toward 5 nM CCL2 for 3 h. Data are shown as a percentage of 5 nM human CCL2 induced migration for (F) Slope (G) Max-min CI and (H) (AUC). Data are the mean ± SEM of three independent experiments with four technical replicates per experiment. Statistical analysis was performed by one-way ANOVA and Dunnett's multiple comparison post-test. *,p<0.05 relative to CCL2 alone.