Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells.

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas.

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.

Efficacy of Chemokine Receptor Inhibition in Treating IL-36α-Induced Psoriasiform Inflammation.

Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare, but severe, psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of preactivated IL-36α. We found the immune cells infiltrating IL-36α-injected mouse skin to be of dramatically different composition than those infiltrating imiquimod-treated skin. The IL-36α-induced leukocyte population comprised nearly equal numbers of CD4+ αß T cells, neutrophils, and inflammatory dendritic cells, whereas the imiquimod-induced population comprised γδ T cells and neutrophils. Ligands for chemokine receptors CCR6 and CXCR2 are increased in both GPP and IL-36α-treated skin, which led us to test an optimized small-molecule antagonist (CCX624) targeting CCR6 and CXCR2 in the IL-36α model. CCX624 significantly reduced the T cell, neutrophil, and inflammatory dendritic cell infiltrates and was more effective than saturating levels of an anti-IL-17RA mAb at reducing inflammatory symptoms. These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.

Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

IL-17-Secreting γδ T Cells Are Completely Dependent upon CCR6 for Homing to Inflamed Skin.

mAbs that neutralize IL-17 or its receptor have proven efficacious in treating moderate-to-severe psoriasis, confirming IL-17 as an important driver of this disease. In mice, a rare population of T cells, γδT17 cells, appears to be a dominant source of IL-17 in experimental psoriasis. These cells traffic between lymph nodes and the skin, and are identified by their coexpression of the TCR variable regions γ4 and δ4. These cells are homologous to the Vγ9Vδ2 T cell population identified in human psoriatic plaques. In this study we report that a potent and specific small molecule antagonist of the CCR6 chemokine receptor, CCX2553, was efficacious in reducing multiple aspects of psoriasis in two different murine models of the disease. Administration of CCX2553 ameliorated skin inflammation in both the IL-23-induced ear swelling model and the topical imiquimod model, and significantly reduced the number of γδT17 cells in inflamed skin. γδT17 cells were greatly reduced in imiquimod-treated skin of CCR6-/- mice, but adoptively transferred wild-type (CCR6+/+) γδT17 cells homed normally to the skin of imiquimod-treated CCR6-/- mice. Our data suggest that γδT17 cells are completely dependent on CCR6 for homing to psoriasiform skin. Thus, CCR6 may constitute a novel target for a mechanistically distinct therapeutic approach to treating psoriasis.

Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection.

West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory monocytes are a critical component of the cellular infiltrate found in the CNS during WNV encephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes monocyte migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8(+) T cells into the CNS compared with WT or Ccl2(-/-) mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl7(-/-) mice and observed a significant increase in monocytes and neutrophils, but not CD8(+) T cells, within the CNS, as well as an enhancement in survival compared with Ccl7(-/-) mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS.

Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation.

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.

Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells.

Under most physiological circumstances, monocytes are excluded from parenchymal CNS tissues. When widespread monocyte entry occurs, their numbers decrease shortly after engraftment in the presence of microglia. However, some disease processes lead to focal and selective loss, or dysfunction, of microglia, and microglial senescence typifies the aged brain. In this regard, the long-term engraftment of monocytes in the microglia-depleted brain remains unknown. Here, we report a model in which a niche for myeloid cells was created through microglia depletion. We show that microglia-depleted brain regions of CD11b-HSVTK transgenic mice are repopulated with new Iba-1-positive cells within 2 wk. The engrafted cells expressed high levels of CD45 and CCR2 and appeared in a wave-like pattern frequently associated with blood vessels, suggesting the engrafted cells were peripheral monocytes. Although two times more numerous and morphologically distinct from resident microglia up to 27 wk after initial engraftment, the overall distribution of the engrafted cells was remarkably similar to that of microglia. Two-photon in vivo imaging revealed that the engrafted myeloid cells extended their processes toward an ATP source and displayed intracellular calcium transients. Moreover, the engrafted cells migrated toward areas of kainic acid-induced neuronal death. These data provide evidence that circulating monocytes have the potential to occupy the adult CNS myeloid niche normally inhabited by microglia and identify a strong homeostatic drive to maintain the myeloid component in the mature brain.

Alkylsulfone-containing trisubstituted cyclohexanes as potent and bioavailable chemokine receptor 2 (CCR2) antagonists.

We describe novel alkylsulfones as potent CCR2 antagonists with reduced hERG channel activity and improved pharmacokinetics over our previously described antagonists. Several of these new alkylsulfones have a profile that includes functional antagonism of CCR2, in vitro microsomal stability, and oral bioavailability. With this improved profile, we demonstrate that two of these antagonists, 2 and 12, are orally efficacious in an animal model of inflammatory recruitment.

The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.

Microglial cells are difficult to track during development because of the lack of specific reagents for myeloid subpopulations. To further understand how myeloid lineages differentiate during development to create microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1(+/GFP)CCR2(+/RFP) knockin fluorescent protein reporter mice. The principal findings include: 1) CX3CR1(+) cells localized to the aorta-gonad-mesonephros region, and visualized at embryonic day (E)9.0 in the yolk sac and neuroectoderm; 2) at E10.5, CX3CR1 single-positive microglial cells were visualized penetrating the neuroepithelium; and 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 a 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.

Dual effect of chemokine CCL7/MCP-3 in the development of renal tubulointerstitial fibrosis.

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis - TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0-8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0-3 days) decrease of inflammatory cell infiltration followed (3-8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10-14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.

Macrophage polarization and insulin resistance: PPARgamma in control.

Macrophages orchestrate an inflammatory response that contributes to glucose intolerance in diet-induced obesity and plaque instability in atherosclerosis. Within this heterogeneous group of cells are proinflammatory (M1) and anti-inflammatory (M2) macrophages. Recent work has identified the nuclear hormone receptor PPARgamma as a critical signaling molecule in determining macrophage phenotype in vitro and in adipose tissue. In the current issue of Cell Metabolism, Bouhlel et al. (2007) extend this paradigm to the vessel wall by showing that both M1 and M2 macrophages are present in atherosclerotic lesions and that activation of PPARgamma polarizes circulating blood monocytes to become M2 macrophages.

Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications.

The following manuscript was published as a Fast Forward article on February 29, 2012: Sullivan TJ, Dairaghi DJ, Krasinski A, Miao Z, Wang Y, Zhao BN, Baumgart T, Berahovich R, Ertl LS, Pennell A, Seitz L, Miao S, Ungashe S, Wei Z, Johnson D, Boring L, Tsou C-L, Charo IF, Bekker P, Schall TJ, and Jaen JC, Characterization of CCX140-B, an orally bioavailable antagonist of the CCR2 chemokine receptor, for the treatment of type 2 diabetes and associated complications. J Pharmacol Exp Ther jpet.111.190918; doi:10.1124/jpet.111.190918 It was later found that the chemical identity of a compound cited in the article, CCX140-B, was not sufficiently disclosed. The authors are unable, at this time, to provide the chemical identity of CCX140-B in accordance with the editorial policies of The Journal of Pharmacology and Experimental Therapeutics. As a result, the authors have voluntarily withdrawn this manuscript from publication. We apologize for any inconvenience this may cause JPET's readers.

Resolving postoperative neuroinflammation and cognitive decline.

OBJECTIVE: Cognitive decline accompanies acute illness and surgery, especially in the elderly. Surgery engages the innate immune system that launches a systemic inflammatory response that, if unchecked, can cause multiple organ dysfunction. We sought to understand the mechanisms whereby the brain is targeted by the inflammatory response and how this can be resolved. METHODS: C57BL/6J, Ccr2(RFP/+)Cx3cr1(GFP/+), Ikk(F/F) mice and LysM-Cre/Ikk(F/F) mice underwent stabilized tibial fracture operation under analgesia and general anesthesia. Separate cohorts of mice were tested for systemic and hippocampal inflammation, integrity of the blood-brain barrier (BBB), and cognition. The putative resolving effects of the cholinergic pathway on these postoperative responses were also studied. RESULTS: Peripheral surgery disrupts the BBB via release of tumor necrosis factor-alpha (TNFα), which facilitates the migration of macrophages into the hippocampus. Macrophage-specific deletion of Ikappa B kinase (IKK)ß, a central coordinator of TNFα signaling through activation of nuclear factor (NF) κB, prevents BBB disruption and macrophage infiltration in the hippocampus following surgery. Activation of the α7 subtype of nicotinic acetylcholine receptors, an endogenous inflammation-resolving pathway, prevents TNFα-induced NF-κB activation, macrophage migration into the hippocampus, and cognitive decline following surgery. INTERPRETATION: These data reveal the mechanisms for bidirectional communication between the brain and immune system following aseptic trauma. Pivotal molecular mechanisms can be targeted to prevent and/or resolve postoperative neuroinflammation and cognitive decline.

Macrophages recruited via CCR2 produce insulin-like growth factor-1 to repair acute skeletal muscle injury.

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.

Zmynd15 encodes a histone deacetylase-dependent transcriptional repressor essential for spermiogenesis and male fertility.

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes.

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1.

The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.

Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.

Monocyte recruitment to sites of inflammation is regulated by members of the chemokine family of chemotactic cytokines. However, the mechanisms that govern the migration of monocytes from bone marrow to blood and from blood to inflamed tissues are not well understood. Here we report that CC chemokine receptor 2 (CCR2) is highly expressed on a subpopulation of blood monocytes whose numbers are markedly decreased in CCR2(-/-) mice. In bone marrow, however, CCR2(-/-) mice had an increased number of monocytes, suggesting that CCR2 is critical for monocyte egress. Intravenous infusion of ex vivo-labeled WT or CCR2(-/-) bone marrow into WT recipient mice demonstrated that CCR2 is necessary for efficient monocyte recruitment from the blood to inflamed tissue. Analysis of mice lacking monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-5, or MCP-2 plus MCP-5 revealed that MCP-3 and MCP-1 are the CCR2 agonists most critical for the maintenance of normal blood monocyte counts. These findings provide evidence that CCR2 and MCP-3/MCP-1 are critical for monocyte mobilization and suggest new roles for monocyte chemoattractants in leukocyte homeostasis.