Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells.
BACKGROUND
The 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.


RESULTS
We 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 (125)I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. (125)I-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.(125)I-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
These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Yani
Y
Zhao
Nilam S
NS
Mangalmurti
Zeyu
Z
Xiong
Bharat
B
Prakash
Fengli
F
Guo
Donna B
DB
Stolz
Janet S
JS
Lee
D000584:Amiloride / Q000494:pharmacology; D018925:Chemokines / Q000378:metabolism*; D004375:Duffy Blood-Group System / Q000276:immunology*; D004705:Endocytosis; D004727:Endothelium / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D046529:Microscopy, Electron, Transmission; D010873:Pinocytosis; D011493:Protein Kinase C / Q000378:metabolism; D011946:Receptors, Antigen / Q000276:immunology* / Q000378:metabolism
D018925:Chemokines; D004375:Duffy Blood-Group System; D011946:Receptors, Antigen; D000584:Amiloride; D011493:Protein Kinase C