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.
Subject(s)
Endothelial Cells/immunology , Lung/immunology , Receptors, Chemokine/immunology , Stromal Cells/immunology , Animals , Carbocyanines/chemistry , Endothelial Cells/cytology , Epithelial Cell Adhesion Molecule/genetics , Epithelial Cell Adhesion Molecule/immunology , Flow Cytometry , Fluorescent Dyes/chemistry , Gene Expression , Lung/blood supply , Lung/cytology , Mice , Mice, Knockout , Receptors, Chemokine/genetics , Staining and Labeling/methods , Stromal Cells/cytologyABSTRACT
Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2-/-embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as 'intermediate' cells. CC chemokine receptor 2 (CCR2)-/-embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2-/-embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences.
Subject(s)
Chemokines/metabolism , Mammals/metabolism , Placenta/metabolism , Animals , Cell Movement , Decidua/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Macrophages/metabolism , Mice, Inbred C57BL , Monocytes/metabolism , Pregnancy , Receptors, Chemokine/deficiency , Receptors, Chemokine/metabolism , Skin/embryology , Skin/metabolism , Transcription, Genetic , Yolk Sac/metabolismABSTRACT
Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
Subject(s)
Eosinophils/immunology , Inflammation/immunology , Monocytes/immunology , Receptors, CCR/immunology , Animals , Chemokines/immunology , Chemokines/metabolism , Eosinophils/metabolism , Gene Expression Profiling/methods , Inflammation/genetics , Inflammation/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Receptors, CCR/genetics , Receptors, CCR/metabolism , Receptors, CCR1/immunology , Receptors, CCR1/metabolism , Receptors, CCR2/immunology , Receptors, CCR2/metabolism , Receptors, CCR3/immunology , Receptors, CCR3/metabolism , Receptors, CCR5/immunology , Receptors, CCR5/metabolismABSTRACT
Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2-/- mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.
Subject(s)
Killer Cells, Natural/immunology , Neoplasms, Experimental/immunology , Receptors, Chemokine/metabolism , Animals , Carcinoma, Lewis Lung , Cell Movement , Chemokine CCL2/metabolism , Cytotoxicity, Immunologic , Lectins, C-Type , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Metastasis , Receptors, CCR2/metabolism , Receptors, Chemokine/genetics , Receptors, Immunologic/metabolismABSTRACT
Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2-/- mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes.
