Figure 1.
Duffy antigen mediates CXCL1 binding and endocytosis in DIH cells.
MIH cells and DIH cells were incubated with 125I-CXCL1/GRO-α at the designated time points then were washed with binding buffer or acid wash buffer, as described in Materials and Methods. (A) Internalized 125I-CXCL1/GRO-α counts in MIH and DIH cells. * p<0.01 MIH cells vs DIH cells, paired t test. (B) Percentage of 125I-CXCL1/GRO-α internalization in MIH and DIH cells. * p<0.001 MIH cells vs DIH cells, paired t test. Experiments were conducted in triplicates and the mean ± SEM from two independent experiments is shown. (C) DIH cells were labeled with anti-Duffy 2C3 Fy6 antibody or isotype mouse IgG1 antibody and surface expression of Duffy examined by flow cytometry. (D) DIH cells do not show detectable surface CXCR2 expression following immune-labeling with anti-human CXCR2 or isotype mouse IgG1 antibody. (E) Peripheral leukocytes were isolated from a healthy volunteer and immune-labeled with anti-human CXCR2 or isotype IgG1 control antibody. Histogram shows gated population of granulocytes expressing CXCR2. Flow cytometric examination of surface DARC and CXCR2 expression on DIH cells was performed 2 independent times. CXCR2 expression on human peripheral granulocytes was performed once.
Figure 2.
Cholesterol is required for CXCL1 endocytosis in DIH cells.
(A) DIH cells were pre-treated with 2-hydroxypropyl-β-Cyclodextrin prior to incubation with 125I -CXCL1/GRO-α for various time points, as described in the Materials and Methods. 2-hydroxypropyl-β-cyclodextrin showed inhibition of chemokine internalization that could be reversed with cholesterol repletion. * p<0.01, 10 mM 2-hydroxypropyl-β-Cyclodextrin treatment vs control, two way ANOVA. ** p<0.05 10 mM 2-hydroxypropyl-β-Cyclodextrin treatment vs repletion with cholesterol, two way ANOVA. (B) DIH cells were pre-treated with 2-hydroxypropyl-β-Cyclodextrin, prior to the addition of 125I -CXCL1/GRO-α with or without unlabeled excess CXCL1 for 120 minutes, as described in the Materials and Methods. Data presented are the combined results from at least three independent experiments.
Figure 3.
Duffy antigen co-localizes with caveolin-1 but not with clathrin.
The DIH cells were stimulated with or without CCL2 100 ng/ml for 120 minutes. Figure A shows a typical single plane confocal optical section from the lamellipodial region of a cell, with Panel B focusing at a specific area on the cell outlined by the rectangle in A. Panel B is then separated into specific color signals to accentuate the localization status of each protein. Cells are stained for Duffy (Panel C, blue), caveolin (Panel D, green) and clathrin (Panel E, red). Some Duffy positive foci colocalize with the caveolin signal (arrows) while no Duffy signal is seen colocalized with the red clathrin signal(arrowheads). Figure (F-H), by immuno-EM, caveolin-1 was labeled with 10 nm gold particle- (▾), Duffy antigen was labeled with 5 nm gold particle- (↓), colocalization between caveolin and Duffy is observed in flask-like membrane structures- (Δ).
Figure 4.
Duffy mediated chemokine endocytosis in DIH cells is caveolin-1, clathrin, flotillin-1 independent.
DIH cells were transfected with SiRNA targeted against caveolin-1 (A&B), flotillin-1(C&D), clathrin (E, F &G), and control scramble sequence RNA (ScRNA). At 72 hour post-transfection, endocytosis experiments were performed and cells lysates were analysed by SDS-PAGES and immunoblotted with caveolin-1, clathrin, flotillin-1 and β-actin antisera. (E) DIH cells transfected with SiCLAC or control SiRNA and were incubated with 125I-transferrin for various time points then were washed with binding buffer or acid wash buffer, as described in the Materials and Methods. The mean ± SEM from three independent experiments is shown.
Figure 5.
Majority of Duffy antigen is present on the surface of endothelial cells within vesicles and plasma membrane extensions.
Duffy antigen was detected on membrane surfaces by pre-embedding TEM in DIH cells as described in Materials and Methods. All panels are imaged from different cells. Duffy antigen (5 nm gold, arrows) was seen located associated with plasma membrane extensions and invaginations. These structures are large (>100 nm) and have no consistent morphology, suggesting they are macropinosomes.
Figure 6.
PDGF enhances chemokine internalization in DIH cells and co-localization with early endosomal antigen.
(A) DIH cells were stimulated with or without PDGF for 10 min, then DARC (red) and EEA (green) or LAMP (green) co-localization were detected using confocal imaging. Blue, actin. (B) DIH cells were pretreated with PDGF 50 ng/ml for 10 min prior to endocytosis experiments. By 120 minutes, PDGF increased DARC-mediated CXCL1 internalization. * p<0.05 PDGF treatment vs control, paired t test. Cells incubated in the absence of PDGF served as a control for internalization. Data presented are the combined results of at least three independent experiments. (C) DIH cells were pretreated with or without PDGF 50 ng/ml for 10 min prior to surface staining for DARC as described in the Materials and Methods.
Figure 7.
CXCL1 endocytosis is CtBP1 and PAK1 independent.
DIH cells were transfected with SiRNA targeted against CtBP1 (A) or PAK1 (B) and control scramble sequence RNA (ScRNA). At 72 hour post-transfection, endocytosis experiment was performed and cells lysates were analysed by SDS-PAGES and immunoblotted with CtBP1, PAK1 and β-actin antisera. The mean ± SEM from three independent experiments is shown.
Figure 8.
Silencing of Dynamin II expression partially inhibited chemokine endocytosis.
(A) DIH cells were pretreated with dynasore prior to incubation with 125I-CXCL1/GRO-α for the specified time points, as described in Materials and Methods. Dynasore inhibited CXCL1 internalization in DIH cells. * p<0.05 dynasore treatment vs control, paired t test. (B) DIH cells were transfected with SiRNA targeted against dynamin II and control SiRNA (ScRNA). At 72 hour post-transfection, endocytosis experiments were performed and cells lysates were analysed by SDS-PAGES and immunoblotted with dynamin II and β-actin antisera. The mean ± SEM from three independent experiments is shown.
Figure 9.
Addition of amiloride, inhibition of actin polymerization, or PKC inhibition decreased CXCL1 internalization in DIH cells.
DIH cells were pre-treated with various inhibitors prior to incubation with 125I-CXCL1/GRO-α for the specified time points, as described in Materials and Methods. (A) Amiloride significantly inhibited DARC-mediated CXCL1 internalization in DIH cells. * p<0.05 amiloride treatment vs control, paired t test. (B) Actin polymerization inhibitor cytochalasin D decreased CXCL1 internalization in DIH cells. * p<0.05 cytochalasin D treatment vs control, paired t test. (C) PKC inhibitor Gö6976 also showed inhibition of CXCL1 internalization. * p<0.05 Gö6976 treatment vs control, paired t test. Shown is the mean ± SEM from three experiments.