BackgroundMonocyte 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 FindingsWe 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/SignificanceThese 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.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2+ inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2+ monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.

CX3CR1 expression is associated with the commitment of CSF-1R+ myeloid precursors to the macrophage/dendritic cell (DC) lineage. However, the relationship of the CSF-1R+ CX3CR1+ macrophage/DC precursor (MDP) with other DC precursors and the role of CX3CR1 in macrophage and DC development remain unclear. We show that MDPs give rise to conventional DCs (cDCs), plasmacytoid DCs (PDCs), and monocytes, including Gr1+ inflammatory monocytes that differentiate into TipDCs during infection. CX3CR1 deficiency selectively impairs the recruitment of blood Gr1+ monocytes in the spleen after transfer and during acute Listeria monocytogenes infection but does not affect the development of monocytes, cDCs, and PDCs.

Microglial cells are difficult to track during development due to the lack of specific reagents for myeloid sub-populations. To further understand how myeloid lineages differentiate during development to give rise to microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1+/GFPCCR2+/RFP knock-in fluorescent protein reporter mice. The principal findings include: 1) CX3CR1+ cells localized to the AGM region, and visualized at E9.0 in the yolk sac and neuroectoderm, 2) At E10.5 CX3CR1 single positive microglial cells were visualized penetrating the neuroepithelium, 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as source of microglial precursors. We provide a novel model to monitor chemokine receptor expression changes in microglia and myeloid cells early (E8.0-E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues.

CC chemokine receptor 2 (CCR2) is essential to acute skeletal muscle injury repair. We studied the subpopulation of inflammatory cells recruited via CCR2 signaling and their cellular functions with respect to muscle regeneration. Mobilization of monocytes/macrophages (MOs/MPs), but not lymphocytes or neutrophils, was impaired from bone marrow to blood and from blood to injured muscle in Ccr2(-/-) mice. While the Ly-6C(+) but not the Ly-6C(-) subset of MOs/MPs was significantly reduced in blood, both subsets were drastically reduced in injured muscle of Ccr2(-/-) mice. Expression of insulin-like growth factor-1 (IGF-I) was markedly up-regulated in injured muscle of wild-type but not Ccr2(-/-) mice. IGF-I was strongly expressed by macrophages within injured muscle, more prominently by the Ly-6C(-) subset. A single injection of IGF-I, but not PBS, into injured muscle to replace IGF-I remarkably improved muscle regeneration in Ccr2(-/-) mice. CCR2 was not detected in myogenic cells or capillary endothelial cells in injured muscle to suggest its direct involvement in muscle regeneration or angiogenesis. We conclude that CCR2 is essential to acute skeletal muscle injury repair primarily by recruiting Ly-6C(+) MOs/MPs. Within injured muscle, these cells conduct phagocytosis, contribute to accumulation of intramuscular Ly-6C(-) macrophages, and produce a high level of IGF-I to promote muscle regeneration.

Cytomegalovirus is a widespread opportunistic pathogen affecting immunocompromised individuals in whom neutrophils may mediate virus dissemination and contribute to progression of disease. Recent sequence analysis suggests that genes absent or altered in attenuated strains may influence pathogenesis. We have found two genes, UL146 and UL147, whose products have sequence similarity to ␣ (CXC) chemokines. UL146 encodes a protein, designated vCXC-1, that is a 117-aa glycoprotein secreted into the culture medium as a late gene product, where its presence correlates with the ability to attract human neutrophils. Recombinant vCXC-1 is a fully functional chemokine, inducing calcium mobilization, chemotaxis, and degranulation of neutrophils. High-affinity vCXC-1 binding is shown to be mediated via CXCR2, but not CXCR1. vCXC-1 exhibits a potency approaching that of human IL-8. As the first example of a virus-encoded ␣ chemokine, vCXC-1 may ensure the active recruitment of neutrophils during cytomegalovirus infection, thereby providing for efficient dissemination during acute infection and accounting for the prominence of this leukocyte subset in cytomegalovirus disease.

Background-Monocyte-derived foam cells are the hallmark of early atherosclerosis, and recent evidence indicates that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Genetic deletions of monocyte chemoattractant protein-1 (MCP-1, CCL2), fractalkine (CX3CL1), or their cognate receptors, CCR2 and CX3CR1, markedly reduce atherosclerotic lesion size in murine models of atherosclerosis. The aim of this study was to determine whether these 2 chemokines act independently or redundantly in promoting atherogenesis. Methods and Results-We crossed CX3CL1Ϫ/Ϫ ApoE Ϫ/Ϫ and CCR2triple knockouts and performed a 4-arm atherosclerosis study. Here, we report that deletion of CX3CL1 in CCR2 A therosclerosis is a multigenic disease that begins as fatty streaks in early life and progresses to complex plaques that are vulnerable to rupture. Circulating blood monocytes are the precursors of the lipid-laden macrophages that are characteristic of early lesions, 1 and it is now widely appreciated that macrophages contribute to the inflammation in both early and advanced lesions. 2 Intense investigation of the molecular basis for recruitment of monocytes to atherosclerotic lesions has revealed critical roles for chemokines. Clinical Perspective p 1648Chemokines are chemotactic cytokines that are synthesized and secreted by vascular wall endothelial and smooth muscle cells in response to cytokines, lipopolysaccharide, or oxidized lipids. 3,4 Monocytes undergo chemotaxis in response to several chemokines, including monocyte chemotactic protein-1 (CCL2) and fractalkine (CX3CL1), but the mechanisms by which chemokines recruit and capture circulating monocytes are not well understood. Among the circulating monocytes are "inflammatory" monocytes that are thought to move rapidly from the blood to sites of inflammation, such as atherosclerotic lesions. 5 Recent studies have found that these monocytes are uniformly CCR2 ϩ 6,7 and CX3CR1 lo . 8 In contrast, CX3CR1 is most highly expressed on CCR2 Ϫ monocytes that are destined to become resident macrophages. 8 These monocytes also traffic to atherosclerotic lesions, albeit somewhat less efficiently than the inflammatory monocytes. 9 Nonetheless, deletion of either CCR2 10 or CX3CR1 11,12 markedly reduces macrophage recruitment to early atherosclerotic lesions, which results in significant atheroprotection.In the present study, we sought to determine whether CCR2 and CX3CR1 act independently or in concert to promote atherosclerosis. The close proximity of CCR2 and CX3CR1 on chromosome 9 makes the creation of mice genetically deficient in both receptors technically difficult. We therefore took advantage of the fact that CX3CL1 is the only known ligand for CX3CR1 and crossed CX3CL1 Ϫ/Ϫ mice with CCR2 Ϫ/Ϫ mice to investigate the relative contributions of CCL2/CCR2 and CX3CL1/CX3CR1 in atherosclerotic lesion development. We performed a 4-arm atheroscle- background. Here, we report that the combined genetic deletion of CCR2 and CX3CL1 resulted in dramatic ...

Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (␤) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1͞MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1͞MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.