Abstract
Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high‐quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa‐Fluor‐labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa‐Fluor‐labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment.
Highlights
In vivo leukocyte migration is regulated, in the main, by proteins belonging to the chemokine family of chemotactic cytokines [1, 2]
ACKR2 is predominantly expressed on lymphatic endothelial cells [31, 32] and placental trophoblasts [23, 33, 34] with expression being detected on subsets of splenic B cells [35]
Further reasons for selecting CCL22 for this analysis include the fact that this ligand does not display the broad receptor binding profiles of other ACKR2 ligands and expression of CCR4 is more limited than the receptors that bind the other ACKR2 ligands [14]
Summary
In vivo leukocyte migration is regulated, in the main, by proteins belonging to the chemokine family of chemotactic cytokines [1, 2]. We have had a particular interest in ACKR2, which is the prototypic member of the ACKR family [19] This receptor binds, internalizes, and degrades all inflammatory chemokines belonging to the CC-chemokine subfamily and plays an essential role in the resolution of the inflammatory response [20,21,22,23,24]. This has implications for tumorigenesis [25,26,27,28] as well as for branching morphogenesis in a number of developmental contexts [29, 30]. The ACKRs are central regulators of chemokine activity in vivo
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