Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice
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{"title"=>"Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice", "type"=>"journal", "authors"=>[{"first_name"=>"Noah", "last_name"=>"Saederup", "scopus_author_id"=>"6506570927"}, {"first_name"=>"Astrid E.", "last_name"=>"Cardona", "scopus_author_id"=>"8357901400"}, {"first_name"=>"Kelsey", "last_name"=>"Croft", "scopus_author_id"=>"36070373000"}, {"first_name"=>"Makiko", "last_name"=>"Mizutani", "scopus_author_id"=>"24473505600"}, {"first_name"=>"Anne C.", "last_name"=>"Cotleur", "scopus_author_id"=>"6507181476"}, {"first_name"=>"Chia Lin", "last_name"=>"Tsou", "scopus_author_id"=>"7006319405"}, {"first_name"=>"Richard M.", "last_name"=>"Ransohoff", "scopus_author_id"=>"7004271760"}, {"first_name"=>"Israel F.", "last_name"=>"Charo", "scopus_author_id"=>"7006052028"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"doi"=>"10.1371/journal.pone.0013693", "sgr"=>"78149438403", "issn"=>"19326203", "pui"=>"359931325", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "pmid"=>"21060874", "scopus"=>"2-s2.0-78149438403"}, "id"=>"3ed91a0c-62fc-3f7b-b547-97da6f4930b6", "abstract"=>"BACKGROUND: Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents. METHODOLOGY/PRINCIPAL FINDINGS: We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C(hi)/CCR2(hi) monocytes. Surprisingly, neutrophils, not Ly6C(lo) monocytes, largely replaced Ly6C(hi) cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia. CONCLUSION/SIGNIFICANCE: These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations", "link"=>"http://www.mendeley.com/research/selective-chemokine-receptor-usage-central-nervous-system-myeloid-cells-ccr2red-fluorescent-protein", "reader_count"=>193, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>10, "Researcher"=>54, "Student > Doctoral Student"=>13, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>7, "Other"=>5, "Student > Master"=>12, "Student > Bachelor"=>15, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>8}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>10, "Researcher"=>54, "Student > Doctoral Student"=>13, "Student > Ph. D. Student"=>66, "Student > Postgraduate"=>7, "Other"=>5, "Student > Master"=>12, "Student > Bachelor"=>15, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>8}, "reader_count_by_subject_area"=>{"Unspecified"=>9, "Agricultural and Biological Sciences"=>96, "Arts and Humanities"=>1, "Philosophy"=>1, "Chemistry"=>1, "Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>11, "Materials Science"=>1, "Medicine and Dentistry"=>32, "Neuroscience"=>21, "Pharmacology, Toxicology and Pharmaceutical Science"=>3, "Psychology"=>1, "Social Sciences"=>1, "Immunology and Microbiology"=>13}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>32}, "Social Sciences"=>{"Social Sciences"=>1}, "Psychology"=>{"Psychology"=>1}, "Unspecified"=>{"Unspecified"=>9}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>3}, "Arts and Humanities"=>{"Arts and Humanities"=>1}, "Engineering"=>{"Engineering"=>2}, "Chemistry"=>{"Chemistry"=>1}, "Neuroscience"=>{"Neuroscience"=>21}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>13}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>96}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>11}, "Philosophy"=>{"Philosophy"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "United States"=>5, "Italy"=>1, "United Kingdom"=>2, "Mexico"=>1, "Portugal"=>1, "Germany"=>1}, "group_count"=>5}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/822311"], "description"=>"<p>(A) CD115<sup>+</sup> monocytes were sorted by flow cytometry into populations that did or did not express cell-surface CCR2. Total RNA was isolated, pooled from three mice per genotype, and analyzed for CCR2 mRNA and RFP mRNA by quantitative RT-PCR. Probe specificity was confirmed with WT and <i>Ccr2<sup>−/−</sup></i> cells. Expression levels were normalized to beta-actin, and the results are expressed relative to the concentration of CCR2 mRNA in monocytes expressing cell-surface CCR2. (B) NK and T cells were sorted by flow cytometry into RFP<sup>+</sup> and RFP<sup>–</sup> fractions. Expression of CCR2 and RFP mRNA was characterized as in (A) and normalized to the amount of CCR2 RNA in the RFP<sup>+</sup> T-cell fraction from <i>Ccr2<sup>+/RFP</sup></i> mice. (C to G) WT, <i>Ccr2<sup>RFP/+</sup></i>, and <i>Ccr2<sup>RFP/RFP</sup></i> mice were bred onto the <i>Apoe<sup>−/−</sup></i> background and fed a high-fat diet for 8 weeks. Peripheral blood monocytes were stained for CD115, CCR2, and Ly6C and analyzed by flow cytometry. (C) Gating on monocytes. (D) Ly6C and RFP expression of monocytes. Dashed line indicates the cutoff for a positive RFP signal. (E) Mean RFP fluorescence intensity in Ly6C<sup>hi</sup> and Ly6C<sup>lo</sup> subsets. Bars indicate SEM. n = 4 mice/group. P<0.008. (F) CCR2 and RFP expression of Ly6C<sup>hi</sup> monocytes. (G) CCR2 and RFP expression in Ly6C<sup>lo</sup> monocytes. CCR2-positive and CCR2-negative gates were based on CCR2-deficient <i>Ccr2<sup>RFP/RFP</sup></i> mice. (H to K) Flow cytometric analysis of Ly6C, RFP, GFP and cell-surface CCR2 expression on monocytes from <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> mice. CD115+ monocytes were gated (H) by Ly6C/GFP expression and analyzed for RFP (I) and CCR2 (J) expression. <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i> monocytes (K) were used as a negative control for CCR2 surface-staining. Results are representative of four mice in two similar experiments.</p>", "links"=>[], "tags"=>["ccr2", "rfp", "sorted"], "article_id"=>492672, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g002", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_CCR2_and_RFP_in_sorted_cell_populations_/492672", "title"=>"Expression of CCR2 and RFP in sorted cell populations.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:44:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/822816"], "description"=>"<p>Confocal images of the periventricular lesions were obtained after staining with the 7/4 antibody. Images represent the same lesion (A) Merged images of red/CCR2, green/CX3CR1, and blue/7/4. (B) Red/CCR2 and green/CX3CR1. (C) Blue/7/4 and green/CX3CR1. (D) Red/CCR2 and blue/7/4. The majority of CCR2<sup>+</sup> cells are 7/4<sup>+</sup> and CX3CR1<sup>lo</sup> or negative, suggesting that they are classical infiltrating monocytes (see individual cell “1”). Ly6C<sup>lo</sup> monocytes (CX3CR1<sup>hi</sup> and CCR2) are also present (cell “2”), as well as T cells (“3”) and activated microglial cells (“4”).</p>", "links"=>[], "tags"=>["monocyte", "subsets", "lesions", "eae", "mice"], "article_id"=>493170, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g006", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_monocyte_subsets_in_brain_lesions_of_EAE_Ccr2_RFP_Cx3cr1_GFP_mice_at_peak_EAE_/493170", "title"=>"Analysis of monocyte subsets in brain lesions of EAE <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> mice at peak EAE.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:52:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/822452"], "description"=>"<p>WT, <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i>, <i>Ccr2<sup>RFP/+</sup>Cxc3r1<sup>GFP/GFP</sup></i>, Ccr2<i><sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i>, and <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/GFP</sup></i> mice were injected intraperitoneally with thioglycollate, and inflammatory exudate cells were harvested 24 h later. Cells were stained with F4/80. (A) RFP/GFP profiles of F4/80<sup>+</sup> peritoneal cells from naive mice (top panels) and 24 hr after injection (bottom panels). (B) Percentage of exudate cells staining positive for F4/80. Values are mean ± SEM. n = 4 mice/group. Results are representative of two similar experiments.</p>", "links"=>[], "tags"=>["peritoneal", "exudate", "cells", "ccr2-rfp", "cx3cr1-gfp", "mice", "sterile", "peritonitis"], "article_id"=>492805, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g003", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_peritoneal_exudate_cells_from_Ccr2_RFP_Cx3cr1_GFP_mice_in_a_sterile_peritonitis_model_/492805", "title"=>"Analysis of peritoneal exudate cells from Ccr2-RFP Cx3cr1-GFP mice in a sterile peritonitis model.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:46:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/822581"], "description"=>"<p>(A) <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> and <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i> mice were immunized with MOG peptide 33–55 and scored daily for neurological signs. (B) EAE symptom onset and peak disease. (C) Total number of brain mononuclear cells and CD45<sup>hi</sup> infiltrating cells at peak disease. (D) CD45<sup>hi</sup> infiltrating cells were analyzed to quantify total neutrophils (SSC<sup>hi</sup>, CD115<sup>−</sup>, Ly6C<sup>lo</sup>), monocytes (CD115<sup>+</sup>), and monocyte subsets (CD115<sup>+</sup>/Ly6C<sup>hi/lo</sup>), (E) CX3CR1 GFP and CCR2-RFP fluorescence intensities in monocyte subsets and microglia. Data points represent individual mice. Values are mean ± SEM. Results are representative of two similar experiments.</p>", "links"=>[], "tags"=>["mice", "delayed", "onset", "eae", "monocyte", "recruitment"], "article_id"=>492937, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCR2_deficient_mice_exhibit_delayed_onset_EAE_and_decreased_monocyte_recruitment_to_the_CNS_/492937", "title"=>"CCR2-deficient mice exhibit delayed onset EAE and decreased monocyte recruitment to the CNS.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:48:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/822678"], "description"=>"<p>(A–C) Low-magnification epifluorescence images of naive <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> (A), diseased <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> (B), and <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i> (C) brains show the extent of inflammation and anatomical location of the lesions. Higher magnification views of the boxed areas are shown in panels D–F. Confocal image of healthy perivascular (D) tissue shows GFP<sup>+</sup> microglia (asterisks), small round RFP<sup>+</sup> cells (arrowhead), and double-positive cells (circles). (E) Parenchymal lesion in a <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i>mouse and (F) a perivascular lesion from a <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i> mouse at peak EAE disease. Dashed line in (F) indicates the approximate perivascular boundary. Within EAE lesions, note RFP/GFP double-positive cells (circles), GFP<sup>+</sup> activated microglia (E, F; asterisks), large elongated RFP<sup>+</sup> cells (E, F; arrows), and small round RFP<sup>+</sup> cells (E, F; arrowheads). Small RFP<sup>+</sup> cells were CD3<sup>+</sup> (not shown). The particularly elongated RFP<sup>+</sup> cells in the <i>Ccr2<sup>RFP/+</sup>Cx3cr1<sup>GFP/+</sup></i> lesion (E) were much less abundant in the <i>Ccr2<sup>RFP/RFP</sup>Cx3cr1<sup>GFP/+</sup></i> lesion (F).</p>", "links"=>[], "tags"=>["cx3cr1", "ccr2", "lesions", "mice"], "article_id"=>493034, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g005", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_CX3CR1_and_CCR2_in_brain_lesions_of_mice_with_EAE_/493034", "title"=>"Expression of CX3CR1 and CCR2 in brain lesions of mice with EAE.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:50:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/409702", "https://ndownloader.figshare.com/files/409741", "https://ndownloader.figshare.com/files/409777", "https://ndownloader.figshare.com/files/409803"], "description"=>"<div><h3>Background</h3><p>Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track <em>in vivo</em>, partly due to lack of CCR2 reagents.</p><h3>Methodology/Principal Findings</h3><p>We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C<sup>hi</sup>/CCR2<sup>hi</sup> monocytes. Surprisingly, neutrophils, not Ly6C<sup>lo</sup> monocytes, largely replaced Ly6C<sup>hi</sup> cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.</p><h3>Conclusion/Significance</h3><p>These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.</p></div>", "links"=>[], "tags"=>["selective", "chemokine", "receptor", "usage", "myeloid", "cells", "ccr2-red", "fluorescent", "knock-in", "mice"], "article_id"=>140925, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0013693.s001", "https://dx.doi.org/10.1371/journal.pone.0013693.s002", "https://dx.doi.org/10.1371/journal.pone.0013693.s003", "https://dx.doi.org/10.1371/journal.pone.0013693.s004"], "stats"=>{"downloads"=>21, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Selective_Chemokine_Receptor_Usage_by_Central_Nervous_System_Myeloid_Cells_in_CCR2_Red_Fluorescent_Protein_Knock_In_Mice/140925", "title"=>"Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-10-27 00:15:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/822127"], "description"=>"<p>(A) Recombinant targeting strategy. Hatched box represents region of CCR2 deleted by recombination. Triangles represent <i>loxP</i> sites. (B) Southern blot analysis of WT, <i>Ccr2<sup>RFP/+</sup></i> heterozygous, and <i>Ccr2<sup>RFP/RFP</sup></i> homozygous genomic DNA. DNA was digested with <i>Pst</i>I, and hybridized with the indicated probe. (C) Flow cytometry analysis of peripheral blood leukocytes (PBL) from WT, <i>Ccr2<sup>RFP/+</sup></i>, and <i>Ccr2<sup>RFP/RFP</sup></i> mice. Cells were stained with anti-CCR2 antibody MC21. The results are representative of four similar experiments.</p>", "links"=>[], "tags"=>["characterization"], "article_id"=>492495, "categories"=>["Neuroscience", "Hematology", "Immunology"], "users"=>["Noah Saederup", "Astrid E. Cardona", "Kelsey Croft", "Makiko Mizutani", "Anne C. Cotleur", "Chia-Lin Tsou", "Richard M. Ransohoff", "Israel F. Charo"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0013693.g001", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_and_characterization_of_CCR2_RFP_mice_/492495", "title"=>"Generation and characterization of CCR2<sup>RFP</sup> mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-27 00:41:35"}

PMC Usage Stats | Further Information

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