Abstract
BackgroundThe Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.Methodology/Principal FindingsWe investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated 125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. 125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression. 125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.Conclusions/SignificanceThese results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.
Highlights
The malarial parasite receptor and minor blood group antigen, Duffy, is a chemokine binding protein expressed on erythrocytes and the surface of post-capillary venular endothelial cells [1,2,3]
Conclusions/Significance: These results show Duffy antigen receptor for chemokines (DARC)-mediated chemokine endocytosis occurs through a macropinocytosislike process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization
We previously reported that the Duffy-expressing immortalized human umbilical vein endothelial cell (HUVEC) (DIH) shows saturable binding of 125I-CXCL1/GRO-a with equilibrium dissociation constant (Kd) of,5 nM and binds multiple chemokines with a binding profile consistent with what is reported in vivo [3,14,15,16,17,18]
Summary
The malarial parasite receptor and minor blood group antigen, Duffy, is a chemokine binding protein expressed on erythrocytes and the surface of post-capillary venular endothelial cells [1,2,3]. The findings of enhanced expression of DARC on post capillary venular endothelium and capillaries of the lungs during inflammatory states further support DARC’s role in inflammation [10]. We have shown that DARC facilitates the movement of radiolabeled 125I -CXCL1/GRO-a across an endothelial monolayer and augments neutrophil recruitment to inflammatory sites [5]. Consistent with this finding, others have shown that Duffy antigen mediates chemokine endocytosis [8]. The mechanism of chemokine endocytosis by DARC remains unclear
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