Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice Noah Saederup1., Astrid E. Cardona2.¤, Kelsey Croft1, Makiko Mizutani2, Anne C. Cotleur2, Chia-Lin Tsou 1 , Richard M. Ransohoff2*, Israel F. Charo1,3* 1 Gladstone Institute of Cardiovascular Disease, San Francisco, California, United States of America, 2 Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States of America, 3 Cardiovascular Research Institute, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America 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 Ly6Chi/CCR2hi monocytes. Surprisingly, neutrophils, not Ly6Clo monocytes, largely replaced Ly6Chi 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. Citation: Saederup N, Cardona AE, Croft K, Mizutani M, Cotleur AC, et al. (2010) Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice. PLoS ONE 5(10): e13693. doi:10.1371/journal.pone.0013693 Editor: Derya Unutmaz, New York University, United States of America Received June 28, 2010; Accepted September 11, 2010; Published October 27, 2010 Copyright: 2010 Saederup et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sources of funding are: National Institutes of Health (HL52773 and HL63894 to IFC, NIH R01NS32151 to RMR), the Multiple Sclerosis Society (TA3121-A1/T to AEC), and the National MS Society (RG3980 to RMR). Competing Interests: The authors have declared that no competing interests exist. * E-mail: icharo@gladstone.ucsf.edu (IFC); ransohr@ccf.org (RMR) . These authors contributed equally to this work. ¤ Current address: Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, Texas, United States of America Introduction Monocytes, the blood-borne precursors of tissue macrophages and dendritic cells (DCs) [1–5], are critical mediators of innate and adaptive immune functions [6,7]. Monocytes can be divided into at least two subsets based on expression of the surface marker Ly6C in mice [2,8] and three subsets based on CD14/CD16 expression in humans [9–11]. The Ly6Chi [12,13] and CD14+/CD162 subsets [14] express chemokine receptor 2 (CCR2) and low levels of CX3CR1 [2] and appear to be functionally equivalent across species. Conversely, the Ly6Clo and CD14/CD16+ subsets lack CCR2 but express high levels of CX3CR1 [2,3,14,15]. Ly6Chi cells enter tissues in response to injury or infection and traffic back to draining lymph nodes [2,3]. Within inflamed tissues, Ly6Chi cells differentiate into macrophages or conventional DCs [1,16]. In contrast, Ly6Clo cells patrol the vascular endothelial lumen, respond early to inflammatory insults, and are hypothesized to contribute to wound healing in the later stages of inflammation [1,16]. CCR2 is thought to mediate the trafficking of Ly6Chi cells [2,12,13,16,17], while CX3CR1 may contribute to Ly6Clo cell migration [16], although other chemokine receptors such as CCR5 also may be important [18]. The roles of particular monocyte subsets likely vary in different disease models and different organs [19–22]. Further complicating the picture, resident macrophages and DCs are difficult to distinguish from macrophages and DCs derived from infiltrating monocytes. The study of monocyte subsets has been greatly aided by the use of CX3CR1-GFP knock-in reporter mice [2,3,23], but the lack of reagents to visualize CCR2-expressing cells, particularly in tissue sections, has restricted tracking monocyte subsets in vivo. To investigate monocyte trafficking in vivo, we generated red fluorescent protein (RFP)-CCR2 knock-in mice, crossed them with CX3CR1-GFP mice, and analyzed them in models of sterile peritonitis and multiple sclerosis using flow cytometry and evaluation of tissue sections. The results provide direct evidence that the presence of CCR2-directed Ly6Chi monocytes dictates the distribution and composition of leukocyte infiltrates in both the brain and spinal cord of mice with experimental autoimmune PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13693