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Mononuclear phagocytes have been attributed a crucial role in the host defense toward influenza virus (IV), but their contribution to influenza-induced lung failure is incompletely understood. We demonstrate for the first time that lung-recruited “exudate” macrophages significantly contribute to alveolar epithelial cell (AEC) apoptosis by the release of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a murine model of influenza-induced pneumonia. Using CC-chemokine receptor 2–deficient (CCR2−/−) mice characterized by defective inflammatory macrophage recruitment, and blocking anti-CCR2 antibodies, we show that exudate macrophage accumulation in the lungs of influenza-infected mice is associated with pronounced AEC apoptosis and increased lung leakage and mortality. Among several proapoptotic mediators analyzed, TRAIL messenger RNA was found to be markedly up-regulated in alveolar exudate macrophages as compared with peripheral blood monocytes. Moreover, among the different alveolar-recruited leukocyte subsets, TRAIL protein was predominantly expressed on macrophages. Finally, abrogation of TRAIL signaling in exudate macrophages resulted in significantly reduced AEC apoptosis, attenuated lung leakage, and increased survival upon IV infection. Collectively, these findings demonstrate a key role for exudate macrophages in the induction of alveolar leakage and mortality in IV pneumonia. Epithelial cell apoptosis induced by TRAIL-expressing macrophages is identified as a major underlying mechanism.

Myeloid cell recruitment is a characteristic feature of bacterial meningitis. However, the cellular mechanisms important for the control of Streptococcus pneumoniae infection remain largely undefined. Previous pharmacological or genetic studies broadly depleted many myeloid cell types within the meninges, which did not allow defining the function of specific myeloid subsets. Herein we show that besides CD11b+Ly-6G+CCR2− granulocytes, also CD11b+Ly-6ChighCCR2+ but not Ly-6ClowCCR2− monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b+Ly-6ChighCCR2+ inflammatory monocytes modulated local CXCL2 and IL-1β production within the meninges but did not provide protection against bacterial infection. Consistent with these results, CCR2 deficiency strongly impaired monocyte recruitment to the infected brains but was redundant for disease pathogenesis. In contrast, specific depletion of polymorphonuclear granulocytes caused elevated local bacterial titer within the brains, led to an aggravated clinical course, and enhanced mortality. These findings demonstrate that Ly-6ChighCCR2+ inflammatory monocytes play a redundant role for the host defense during bacterial meningitis and that predominantly CD11b+Ly-6G+CCR2− myeloid cells are involved in the restriction of the extracellular bacteria.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPCmolecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Members of the chemokine system, play a central role in inflammatory processes that underlie the pathogenesis of atherosclerosis and possibly, aortic valve sclerosis. Here we show that genetic inactivation of CC chemokine receptor 5 (CCR5) in the atherosclerosis-prone Apoe-/- mice (Apoe-/- Ccr5-/-) fed a normal chow or a high-fat diet (HFD) are protected against advanced atherosclerosis as well as age-associated aortic valve thickening (AAAVT)--a murine correlate of aortic valve sclerosis. Notably, human sclerotic valves contained CCR5+ cells. We confirm that Apoe-/- Ccr5-/- mice does not influence early-atherosclerotic stage. Adoptive transfer studies showed that the atheroprotective effect of CCR5 inactivation resided in the bone marrow compartment, but was not dependent on T-cells. The CCR5-null state was associated with phenotypes postulated to be atheroprotective such as reduced macrophage accumulation in the plaque, and lower circulating levels of IL-6 and MCP-5. The lack of CCR5 expression in Apoe-/- mice was also associated with higher numbers of endothelial progenitor cells (EPCs)--another postulated athero-protective factor. Compared with controls, carriers of a polymorphism in the Ccr5 gene that leads to the lack of CCR5 in the cell surface had an increased mean percentage of EPCs, but this difference did not reach statistical significance. Collectively, these findings underscore a critical role of CCR5 in age-associated cardiovascular diseases, and highlight that the effects of the chemokine system can be temporally constrained to distinct stages of these disease processes.

Increased numbers of pulmonary dendritic cells (DCs) are recruited to the lungs during allergic airway inflammation and contribute to the maintenance of the inflammatory immune response. The chemokine receptors that directly control DC accumulation into the lungs are largely unknown. To explore this issue, we generated mixed bone marrow chimeric mice containing both wild-type and knockout cells for a given chemokine receptor. After induction of allergic airway inflammation, we specifically tracked and compared chemokine receptor knockout vs wild-type DC populations through various lung compartments. Using this approach, we show that CCR2, but not CCR5 or CCR6, directly controls the accumulation of DCs into allergic lungs. Furthermore, the size of inflammatory monocyte populations in peripheral blood was strikingly CCR2 dependent, suggesting that CCR2 primarily mediates the release of monocytic DC precursors into the bloodstream.

Following genital herpes simplex virus type 2 (HSV-2) exposure, NK cells and T cells are mobilized to sites of infection to control viral replication and spread. The present investigation sought to determine the role of the chemokine receptor CCR5 in this process. Mice deficient in CCR5 (CCR5−/−) displayed a significant reduction in cumulative survival following infection in comparison to wild-type, HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5−/−mice yielded significantly more virus and expressed higher levels of tumor necrosis factor alpha, CXCL1, CCL2, CCL3, and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. Whereas there was no difference in absolute number of leukocytes (CD45high), CD4 T cells, or CD8 T cells residing in the draining lymph nodes, spleen, spinal cord, or brain stem comparing HSV-2-infected wild-type to CCR5−/−mice prior to or after infection, there were significantly more NK cells (NK1.1+CD3−) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5−/−mice. In addition, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord, and brain stem tissue of wild-type but not CCR5−/−mice. Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation, and spread associated with a specific reduction in NK cell expansion, infiltration, and activity in the nervous system.

Chemokines recruit inflammatory cells to sites of injury, but the role of the CC chemokine receptor 2 (CCR2) during regenerative processes following ischemia is poorly understood. We studied injury, inflammation, perfusion, capillary formation, monocyte chemotactic protein-1 (MCP-1) levels, muscle regeneration, fat accumulation, and transcription factor activation in hindlimb muscles of CCR2−/− and wild-type (WT) mice following femoral artery excision (FAE). In both groups, muscle injury and restoration of vascular perfusion were similar. Nevertheless, edema and neutrophil accumulation were significantly elevated in CCR2−/− compared with WT mice at day 1 post-FAE and fewer macrophages were present at day 3. MCP-1 levels in post-ischemic calf muscle of CCR2−/− animals were significantly elevated over baseline through 14 days post-FAE and were higher than WT mice at days 1, 7, and 14. In addition, CCR2−/− mice exhibited impaired muscle regeneration, decreased muscle fiber size, and increased intermuscular adipocytes with similar capillaries/mm2postinjury. Finally, the transcription factors, MyoD and signal transducers of and activators of transcription-3 (STAT3), were significantly increased above baseline but did not differ significantly between groups at any time point post-FAE. These findings suggest that increases in MCP-1, and possibly, MyoD and STAT3, may modulate molecular signaling in CCR2−/− mice during inflammatory and regenerative events. Furthermore, alterations in neutrophil and macrophage recruitment in CCR2−/− mice may critically alter the normal progression of downstream regenerative events in injured skeletal muscle and may direct myogenic precursor cells in the regenerating milieu toward an adipogenic phenotype.

To clarify the role of the monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) signalling pathway in hyperoxia-induced acute lung injury, CCR2-deficient (CCR2-/-) and wild-type (CCR2+/+) mice were exposed to 85% O(2) for up to 6 days. At day 3, body weight significantly decreased and total protein concentration in bronchoalveolar lavage fluid (BALF) was higher in CCR2-/- mice compared with CCR2+/+ mice. Cumulative survivals were significantly lower in CCR2-/- mice than in CCR2+/+ mice. However, the two groups showed no significant differences in both histological changes and number of macrophages in BALF. Real-time reverse transcriptase-polymerase chain reaction revealed increased mRNA levels of MCP-1, interleukin-1beta thioredoxin-1, and inducible nitric oxide synthase (iNOS) in lung tissues in CCR2-/- mice compared with CCR2+/+ mice. Increased iNOS mRNA levels in alveolar macrophages exposed to 85% O(2) for 48 h in vivo or in vitro were significantly higher in CCR2-/- mice than in CCR2+/+ mice. These results suggest that the MCP-1/CCR2 signalling pathway is protective against hyperoxia-induced tissue injury by suppressing induction of iNOS and consequent production of reactive oxygen species by activated alveolar macrophages.

The host factors that influence autoimmune arthritides such as rheumatoid arthritis have not been fully elucidated. We previously found that genetic inactivation of CC chemokine receptor 2 (CCR2) in the arthritis-prone DBA/1j mouse strain significantly increases the susceptibility of this strain to autoimmune arthritis induced by immunization with collagen type II (CII) and complete Freund's adjuvant (CFA). Here, we show that following intradermal infection with Mycobacterium avium, a similar arthritis phenotype was detected in Ccr2-null mice in the DBA/1j, but not in the BALB/c background. The failure to develop arthritis in Ccr2-null BALB/c mice occurred in the face of high bacterial burdens and low interferon gamma (IFNgamma) production. By contrast, Ccr2-null DBA/1j mice had low bacterial burdens, produced normal amounts of IFNgamma, and had high titers of autoantibodies against CII. Thus, the Ccr2-null state in an arthritic-prone genetic background leads to increased arthritis susceptibility following infectious (M. avium) and noninfectious (CII/CFA) challenges. Because CCR2 serves as a negative regulator of murine arthritis, caution might need to be exercised while testing CCR2 blockers in human arthritis or other diseases. These findings also indicate that Ccr2-null DBA/1j mice might serve as a valuable model system to uncover the immunological determinants of arthritis and to test novel antiarthritic agents.

Background Monocyte chemoattractant protein-1 (MCP-1) and its major receptor, CC chemokine receptor 2 (CCR2), have been shown to contribute to left ventricular remodeling after myocardial infarction. However, it is unknown whether CCR2 deficiency protects the myocardium after myocardial ischemia-reperfusion. The purpose of the present study was to investigate the effects of CCR2 deficiency on myocardial ischemia-reperfusion injury in mice. Methods and Results Experiments were performed in CCR2-/- and wild-type mice subjected to 45 min of ischemia followed by reperfusion. Macrophage infiltration in ischemic lesions was markedly reduced in CCR2-/- mice compared with wild-type mice (p

Microarray analysis of human alcoholic brain and cultured cells exposed to ethanol showed significant changes in expression of genes related to immune or inflammatory responses, including chemokines and chemokine receptors. To test the hypothesis that chemokines exhibit previously undiscovered pleiotropic effects important for the behavioral actions of ethanol, we studied mutant mice with deletion of the Ccr2, Ccr5, Ccl2 or Ccl3 genes. Deletion of Ccr2, Ccl2 (females) or Ccl3 in mice resulted in lower preference for alcohol and consumption of lower amounts of alcohol in a two-bottle choice test as compared with wild-type mice. Ethanol treatment (2.5 g/kg, i.p.) induced stronger conditioned taste aversion in Ccr2, Ccl2 or Ccl3 null mutant mice than in controls. Ccr2 and Ccr5 null mutant mice did not differ from wild-type mice in ethanol-induced loss of righting reflex (LORR), but mice lacking Ccl2 or Ccl3 showed longer LORR than wild-type mice. There were no differences between mutant strains and wild-type mice in severity of ethanol-induced withdrawal. Genetic mapping of chromosome 11 for the Ccl2 and Ccl3 genes (46.5 and 47.6 cM, respectively) revealed that an alcohol-induced LORR QTL region was contained within the introgressed region derived from 129/SvJ, which may cause some behavioral phenotypes observed in the null mice. On the contrary, known QTLs on Chr 9 are outside of 129/SvJ region in Ccr2 and Ccr5 (71.9 and 72.0 cM, respectively) null mutant mice. These data show that disruption of the chemokine network interferes with motivational effects of alcohol.

Homing of mast cell progenitors (MCps) to the mouse small intestine involves the interaction of α4β7 integrin with mucosal addressin cellular adhesion molecule-1 (MAdCAM-1). We now demonstrate the dependence of this process on CXC chemokine receptor 2 (CXCR2) and vascular cell adhesion molecule-1 (VCAM-1) using null strains and mice sublethally irradiated and bone marrow (BM) reconstituted (SIBR) with wild-type or null BM or with wild-type BM followed by administration of blocking antibody. The intestinal MCp concentration in CXCR2-/- mice was reduced by 67%, but was unaltered in CC chemokine receptor 2-/- (CCR2-/-), CCR3-/-, or CCR5-/- mice. SIBR mice given CXCR2-/- BM had an intestinal MCp concentration that was 76% less than that in BALB/c BM reconstituted mice. Antibody blockade of VCAM-1 or of CXCR2 in SIBR mice reduced intestinal MCp reconstitution, and mice lacking endothelial VCAM-1 also had a marked reduction relative to wild-type mice. Finally, the half-life of intestinal MCps in wild-type mice was less than one week on the basis of a more than 50% reduction by administration of anti-α4β7 integrin or anti-CXCR2. Thus, the establishment and maintenance of MCps in the small intestine is a dynamic process that requires expression of the α4β7 integrin and the α-chemokine receptor CXCR2.

IFN-α/β-mediated functions promote production of MIP-1α (or CCL3) by mediating the recruitment of MIP-1α-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-γ delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-α/β and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1α during infection and was dependent on IFN-α/β effects for induction. Resident F4/80+ liver leukocytes were identified as primary IFN-α/β responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1α and IFN-γ in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.

The CC chemokine ligand-2 (CCL2) and its receptor CCR2 are essential for monocyte trafficking under inflammatory conditions. However, the mechanisms that determine the intensity and duration of alveolar monocyte accumulation in response to CCL2 gradients in inflamed lungs have not been resolved. To determine the potential role of CCR2-expressing monocytes in regulating alveolar CCL2 levels, we compared leukocyte recruitment kinetics and alveolar CCL2 levels in wild-type and CCR2-deficient mice in response to intratracheal LPS challenge. In wild-type mice, LPS elicited a dose- and time-dependent alveolar monocyte accumulation accompanied by low CCL2 levels in bronchoalveolar lavage fluid (BALF). In contrast, LPS-treated CCR2-deficient mice lacked alveolar monocyte accumulation, which was accompanied by relatively high CCL2 levels in BALF. Similarly, wild-type mice that were treated systemically with the blocking anti-CCR2 antibody MC21 completely lacked LPS-induced alveolar monocyte trafficking that was associated with high CCL2 levels in BALF. Intratracheal application of anti-CCR2 antibody MC21 to locally block CCR2 on both resident and recruited cells did not affect LPS-induced alveolar monocyte trafficking but led to significantly increased BALF CCL2 levels. Reciprocally bone marrow-transplanted, LPS-treated wild-type and CCR2-deficient mice showed a strict inverse relationship between alveolar monocyte recruitment and BALF CCL2 levels. In addition, freshly isolated human and mouse monocytes were capable of integrating CCL2 in vitro. LPS-induced alveolar monocyte accumulation is accompanied by monocytic CCR2-dependent consumption of CCL2 levels in the lung. This feedback loop may limit the intensity of monocyte recruitment to inflamed lungs and play a role in the maintenance of homeostasis.

Circulating murine monocytes comprise two largely exclusive subpopulations that are responsible for seeding normal tissues (Gr-1−/CCR2−/CX3CR1high) or responding to sites of inflammation (Gr-1+/CCR2+/CX3CR1lo). Gr-1+ monocytes are recruited to the site of infection during the early stages of immune response to the intracellular pathogen Toxoplasma gondii. A murine model of toxoplasmosis was thus used to examine the importance of Gr-1+ monocytes in the control of disseminated parasitic infection in vivo. The recruitment of Gr-1+ monocytes was intimately associated with the ability to suppress early parasite replication at the site of inoculation. Infection of CCR2−/− and MCP-1−/− mice with typically nonlethal, low doses of T. gondii resulted in the abrogated recruitment of Gr-1+ monocytes. The failure to recruit Gr-1+ monocytes resulted in greatly enhanced mortality despite the induction of normal Th1 cell responses leading to high levels of IL-12, TNF-α, and IFN-γ. The profound susceptibility of CCR2−/− mice establishes Gr-1+ monocytes as necessary effector cells in the resistance to acute toxoplasmosis and suggests that the CCR2-dependent recruitment of Gr-1+ monocytes may be an important general mechanism for resistance to intracellular pathogens.

Monocyte chemoattractant protein-1 (also known as CC chemokine ligand 2 [CCL2]) and its receptor CC chemokine receptor 2 (CCR2) play a central role in the inflammatory response and neointimal formation after vascular injury. In the context of hyperlipidemia, this appears to involve neointimal monocyte infiltration. Hence, we investigated the function of the CCL2/CCR2 axis in early monocyte recruitment to injured arteries. Wire-induced injury of the carotid artery in apoE −/− mice caused a rapid increase of JE/CCL2 protein in the vessel wall peaking at 24 hours after injury, whereas serum JE/CCL2 was increased solely at 6 hours and blood cell-associated levels were unaltered, as demonstrated by enzyme-linked immunosorbent assay. Immunohistochemistry revealed intense staining for JE/CCL2 in smooth muscle cells (SMCs) and in association with platelets adherent to the denuded vessel wall 24 hours after injury. In vitro, exogenous or SMC-derived JE/CCL2 binds to the platelet surface and triggers monocyte arrest on adherent platelets but not on SMCs in flow assays. Accordingly, monocyte arrest in ex vivo perfused apoE −/− carotid arteries isolated 24 hours after injury was profoundly inhibited by pretreatment with a JE/CCL2 antibody. In CCR2 −/− /apoE −/− mice, neointimal plaque area was reduced by 47% compared with CCR2 +/+ /apoE −/− mice. Moreover, CCR2 deletion markedly decreased neointimal macrophage content while expanding SMC content. Vascular JE/CCL2 expressed by SMCs and immobilized by adherent platelets after endothelial denudation is crucial for mediating early monocyte recruitment to injured arteries in hyperlipidemic mice. This mechanism may explain reduced neointimal macrophage infiltration and lesion formation in CCR2-deficient apoE −/− mice.

We have recently described a new subfamily of Fox genes, Foxp1/2/4, which are transcriptional repressors and are thought to regulate important aspects of development in several tissues, including the lung, brain, thymus and heart. Here, we show that Foxp1 is expressed in the myocardium as well as the endocardium of the developing heart. To further explore the role of Foxp1 in cardiac development, we inactivated Foxp1 through gene targeting in embryonic stem cells. Foxp1 mutant embryos have severe defects in cardiac morphogenesis, including outflow tract septation and cushion defects, a thin ventricular myocardial compact zone caused by defects in myocyte maturation and proliferation, and lack of proper ventricular septation. These defects lead to embryonic death at E14.5 and are similar to those observed in other mouse models of congenital heart disease, including Sox4 and Nfatc1 null embryos. Interestingly, expression of Sox4 in the outflow tract and cushions of Foxp1 null embryos is significantly reduced, while remodeling of the cushions is disrupted, as demonstrated by reduced apoptosis and persistent Nfatc1 expression in the cushion mesenchyme. Our results reveal a crucial role for Foxp1in three aspects of cardiac development: (1) outflow tract development and septation, (2) tissue remodeling events required for cardiac cushion development, and (3) myocardial maturation and proliferation.

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication after allogeneic stem cell transplantation (allo-SCT) that responds poorly to standard immunosuppressive therapy. The pathophysiology of IPS involves the secretion of inflammatory cytokines including IFN-γ and TNF-α along with the recruitment of donor T cells to the lung. CXCR3 is a chemokine receptor that is expressed on activated Th1/Tc1 T cell subsets and the expression of its ligands CXCL9 (monokine induced by IFN-γ (Mig)) and CXCL10 (IFN-γ-inducible protein 10 (IP-10)) can be induced in a variety of cell types by IFN-γ alone or in combination with TNF-α. We used a lethally irradiated murine SCT model (B6 → bm1) to evaluate the role of CXCR3 receptor:ligand interactions in the development of IPS. We found that Mig and IP-10 protein levels were significantly elevated in the bronchoalveolar lavage fluid of allo-SCT recipients compared with syngeneic controls and correlated with the infiltration of IFN-γ-secreting CXCR3+ donor T cells into the lung. The in vivo neutralization of either Mig or IP-10 significantly reduced the severity of IPS compared with control-treated animals, and an additive effect was observed when both ligands were blocked simultaneously. Complementary experiments using CXCR3−/− mice as SCT donors also resulted in a significant decrease in IPS. These data demonstrate that interactions involving CXCR3 and its primary ligands Mig and IP-10 significantly contribute to donor T cell recruitment to the lung after allo-SCT. Therefore, approaches focusing on the abrogation of these interactions may prove successful in preventing or treating lung injury that occurs in this setting.

Chemokines regulate the recruitment of leukocytes to sites of inflammation and may therefore play an important role in lymphocyte trafficking between draining lymph nodes and pancreatic islet tissue allografts. The intragraft expression of alpha- and beta-chemokine mRNA during the rejection of BALB/c proislet (fetal precursor islet tissue) allografts in CBA/H mice was assessed quantitatively and semiquantitatively by RT-PCR analyses. Allograft rejection was associated with the strongly enhanced (from day 4) and prolonged expression (up to day 10) of the alpha-chemokine IP-10 and enhanced intragraft mRNA expression of the beta-chemokines MCP-1, MIP-lalpha, MIP-1beta, RANTES, and eotaxin. Peak transcript expression was identified at day 4 (IP-10, MCP-1), day 5 (eotaxin), day 6 (MIP-1alpha, MIP-1beta), and day 14 (RANTES). To examine the role of beta-chemokine receptors in allograft rejection, additional allografts to CCR2-/- , CCR5-/-, and wild-type CCR+/+ mice were analyzed by histology, immunohistochemistry, and morphometry. In CCR5-/- mice, the intragraft recruitment of T cells and macrophages was slower and allograft destruction was delayed; in CCR2-/- mice, the initial entry of macrophages was retarded but graft survival was not prolonged. These findings suggest that IP-10 regulates the initial influx of T cells into proislet allografts, MCP-1/CCR2 signaling controls initial macrophage entry, and the MIP-1alpha, MIP-1beta, and RANTES/CCR5 pathway contributes to the rejection response by subsequently amplifying the recruitment of T cell subpopulations required for graft destruction.

Fibrosis is a hallmark of progressive organ diseases. Monocyte chemoattractant protein (MCP)-1, also termed as macrophage chemotactic and activating factor (MCAF/CCL2) and its receptor, CCR2 are presumed to contribute to progressive fibrosis. However, the therapeutic efficacy of MCP-1/CCR2 blockade in progressive fibrosis remains to be investigated. We hypothesized that blockade of CCR2 may lead to the improvement of fibrosis. To achieve this goal, we investigated renal interstitial fibrosis induced by a unilateral ureteral obstruction in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393, CCR2 inhibitors. Cell infiltrations, most of which were F4/80-positive, were reduced in CCR2 knockout mice. In addition, dual staining revealed that CCR2-positive cells were mainly F4/80-positive macrophages. Importantly, CCR2 blockade reduced renal interstitial fibrosis relative to wild-type mice. Concomitantly, renal transcripts and protein of MCP-1, transforming growth factor-beta, and type I collagen were decreased in CCR2-null mice. Further, this CCR2-dependent loop for renal fibrosis was confirmed by treatment with CCR2 antagonists in a unilateral ureteral obstruction model. These findings suggest that the therapeutic strategy of blocking CCR2 may prove beneficial for progressive fibrosis via the decrease in infiltration and activation of macrophages in the diseased kidneys.

The purpose of this study was to determine whether certain chemokines, which are highly expressed in injured skeletal muscle, are involved in the repair and functional recovery of the muscle after traumatic injury. In wild-type control mice, mRNA transcripts of macrophage inflammatory protein (MIP)-1α, MIP-1β, and monocyte chemoattractant protein (MCP)-1 as well as their major receptors, CCR5 and CCR2, increased after freeze injury and gradually returned to control (uninjured) levels by 14 days. Muscle function and histological characteristics were monitored in injured mice that were genetically deficient for the CCR5 receptor (a major receptor for MIP-1α and MIP-1β) and also rendered MCP-1 deficient with neutralizing antibodies. To dissect the role of these chemokines, additional studies were conducted in CCR5- and CCR2-deficient mice. CCR5-/- mice injected with MCP-1 antiserum for the first 3 days after injury exhibited a twofold greater maximal isometric tetanic torque deficit at 14 days after injury than did controls (i.e., 33% vs. 17%; P = 0.002). The impaired functional recovery was accompanied with an increased fat infiltration within the regenerating muscle without a significant difference in the influx of inflammatory cells, including macrophages. Strength recovery was also impaired in mice deficient for the receptor of MCP-1, CCR2, but not in CCR5-/- mice that were not injected with MCP-1 antiserum. The data suggest that MCP-1/CCR2 plays a role in the regeneration and recovery of function after traumatic muscle injury.

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. Induced macrophage migration in vivo is driven by the binding of monocyte chemoattractant protein-1 (MCP-1, or CCL2 in the new nomenclature) to the CCR2-chemokine receptor on macrophages. To determine whether the CCL2-CCR2 signaling pathway is involved in the accumulation of macrophages in growing collateral vessels, we used mice that are deficient in CCR2 in a model of experimental arterial occlusion and collateral vessel growth. In an in vitro CCL2-driven chemotaxis assay, mononuclear cells isolated from wild-type BALB/c mice exhibited CCL2 concentration–dependent migration, whereas this migration was abolished in cells from CCR2−/−mice on a BALB/c genetic background. In vivo, blood flow recovery as measured by laser Doppler (LDI) and MRI (MRI) was impaired in CCR2−/−mice on either the BALB/c or C57BL/6 genetic backgrounds. Three weeks after femoral artery ligation, LDI perfusion ratio of operated versus nonoperated distal hindlimb in BALB/c wild-type mice increased to 0.45±0.06 and in CCR2−/−animals only to 0.21±0.03 (PPP−/−mice. Morphometric analysis showed a lesser increase in collateral vessel diameters in CCR2−/−mice. Importantly, the number of invaded monocytes/macrophages in the perivascular space of collateral arteries of CCR2−/−animals was dramatically reduced in comparison to wild-type mice. In conclusion, our results demonstrate that the CCR2 signaling pathway is essential for efficient collateral artery growth.

Objective Chemokines have been postulated to play a role in the pathogenesis of graft-vs-host disease (GVHD) after allogeneic hematopoietic transplantation. Recent reports have indicated that the absence of donor expression of CCR5 on T cells ameliorates GVHD in models using no conditioning of the recipient. We therefore assessed the role of CCR5 on donor cells in models where intensive conditioning of the recipient occurs, thus more appropriately mirroring the clinical experience. Methods Lethally irradiated mice received allogeneic bone marrow transplants. Recipients were given full MHC-mismatched donor bone marrow and splenocytes from CCR5 knockout (KO) mice vs wild-type (WT) control donors. Results Recipients of CCR5 KO donor cells succumbed to acute GVHD at an accelerated rate compared to mice receiving WT cells. Donor CD8 + T cells expanded to a significantly greater extent in recipients of CCR5 KO vs WT control cells. T cells recovered from recipients of CCR5 KO cells produced more IFN-γ and TNF-α and proliferated to a T-cell mitogen at a significantly greater level then T cells from recipients of WT cells, indicating that CCR5 plays a role in downregulating donor alloreactive CD8 + T-cell expansion. Histological assessment of the mice indicated pathological lesions in the kidneys and a greater degree of liver pathological changes in mice that received CCR5 KO donor grafts. Conclusions These results indicate that the role of CCR5 in allogeneic bone marrow transplants and GVHD is more complex than initially thought. In a murine transplant model with intensive conditioning, the overall effect of absent CCR5 expression on donor cells results in greater GVHD and donor T-cell expansion.

Chemokine receptor 5 (CCR5) binds macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, and members of the monocyte chemotactic protein family and is also a receptor for human immunodeficiency virus (HIV). CCR5 ligands can suppress HIV-1 entry into cells. In humans, homozygous mutations of theccr5gene confer resistance to HIV-1 infection. The role of CCR5 in defense against microbial infection is unclear. In this study we examined the innate and adaptive immune responses of CCR5-deficient mice to the intracellular bacterial pathogenListeria monocytogenes.We found that migration of monocytic cells, formation ofL. monocytogenes-containing lesions, and bacterial clearance occurred normally in the spleens and livers of CCR5-deficient animals. Activation of macrophages and dendritic cells during the first 3 days postinfection was normal in the absence of CCR5, as demonstrated by intact expression of inducible nitric oxide synthase (iNOS) and production of the cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-12. Priming ofL. monocytogenes-specific CD8 T cells also occured independently of CCR5 expression. Previously immunized, CCR5-deficient animals mounted normal secondary CD8 T-cell responses and cleared bacteria from infected organs similarly to wild-type controls, suggesting that CCR5 is dispensable for migration and activation of memory CD8 T cells. Our data indicate that CCR5-mediated chemotaxis is not required for defense against infection withL. monocytogenes.

Microbial infections induce chemokine and cytokine cascades that coordinate innate immune defenses. Infection with the intracellular bacterial pathogen Listeria monocytogenes induces CCR2-dependent monocyte recruitment and activation, an essential response for host survival. Herein we show that invasive L. monocytogenes, but not killed or noninvasive bacteria, induce secretion of MCP-1, the requisite chemokine for monocyte recruitment. Induction of MCP-1, but not TNF or IL-12, following L. monocytogenes infection is MyD88 independent. Consistent with these results, MyD88 deficiency does not impair monocyte recruitment to L. monocytogenes infected spleens, but prevents monocyte activation. Our results indicate that distinct microbial signals activate innate immune responses in an ordered, step-wise fashion, providing a mechanism to specify and modulate antimicrobial effector functions.

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.

Examined were CCR2-deficient mice to clarify the contribution of macrophages via monocyte chemoattractant protein 1 (MCP-1 or CCL2)/CCR2 signaling to the pathogenesis of renal ischemia-reperfusion injury. Also evaluated was the therapeutic effects via the inhibition of MCP-1/CCR2 signaling with propagermanium (3-oxygermylpropionic acid polymer) and RS-504393. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells and marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury was observed. Ischemia-reperfusion induced the expression of MCP-1 mRNA and protein in injured kidneys, followed by CCR2-positive macrophages in interstitium in wild-type mice. The expression of MCP-1 was decreased in CCR2-deficient mice compared with wild-type mice. The number of interstitial infiltrated macrophages was markedly smaller in the CCR2-deficient mice after ischemia-reperfusion. CCR2-deficient mice decreased the number of interstitial inducible nitric oxide synthase-positive cells after ischemia-reperfusion. The area of tubular necrosis in CCR2-deficient mice was significantly lower than that of wild-type mice after ischemia-reperfusion. In addition, CCR2-deficient mice diminished KC, macrophage inflammatory protein 2, epithelial cell-derived neutrophil-activating peptide 78, and neutrophil-activating peptide 2 expression compared with wild-type mice accompanied with the reduction of interstitial granulocyte infiltration. Similarly, propagermanium and RS-504393 reduced the number of interstitial infiltrated cells and tubular necrosis up to 96 h after ischemia-reperfusion injury. These results revealed that MCP-1 via CCR2 signaling plays a key role in the pathogenesis of renal ischemia-reperfusion injury through infiltration and activation of macrophages, and it offers a therapeutic target for ischemia-reperfusion.

Innate responses in the CNS are critical to first line defense against infection and injury. Leukocytes migrate to inflammatory sites in response to chemokines. We studied leukocyte migration and glial chemokine expression within the denervated hippocampus in response to axonal injury caused by entorhinodentate lesions. A population of Mac1/CD11b+ CD45highmacrophages (distinct from CD45lowmicroglia) was specifically detected within the lesion-reactive hippocampus by 12 hr after injury. Significant infiltration by CD3+ T cells did not occur in the denervated hippocampus until 24 hr after axotomy. A broad spectrum of chemokines [RANTES/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, interferon γ inducible protein (IP)-10/CXCL10, macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4, and MIP-2/CXCL2] was induced at this time. RANTES/CCL5 was not significantly elevated until 24 hr after axotomy, whereas MCP-1/CCL2 was significantly induced before leukocyte infiltration occurred. Neither T cells nor macrophages infiltrated the denervated hippocampus of CCR2-deficient mice, arguing for a critical role for the CCR2 ligand MCP-1/CCL2 in leukocyte migration. Both T cells and macrophages infiltrated CCR5-deficient hippocampi, showing that CCR5 ligands (including RANTES/CCL5) are not critical to this response.In situhybridization combined with immunohistochemistry for ionized binding calcium adapter molecule (iba)1 or glial fibrillary acidic protein (GFAP) identified iba1+ microglia and GFAP+ astrocytes as major sources of MCP-1/CCL2 within the lesion-reactive hippocampus. We conclude that leukocyte responses to CNS axonal injury are directed via innate glial production of chemokines.

Solomon MF, Kuziel WA, Mann DA, Simeonovic CJ. The role of chemokines and their receptors in the rejection of pig islet tissue xenografts. Xenotransplantation 2003; 10: 164–177. © Blackwell Munksgaard, 2003 The mechanism by which inflammatory cells are recruited to pig islet tissue (proislet) xenografts was investigated by examining the intragraft mRNA expression of murine α- and β-chemokines in CBA/H mice from days 3 to 10 post-transplant. Xenograft rejection was associated with early intragraft transcript expression for monocyte chemotactic protein-1 (MCP-1) (3 to 5 days), IP-10 (3 to 4 days) and macrophage inflammatory protein-1α (MIP-1α) (3 to 5 days) and subsequent expression of eotaxin (days 4 to 10), MIP-1β (days 4 and 5) and regulated on activation, normal T cell expressed and secreted (RANTES) (days 4 to 6) mRNA. This pattern was consistent with the early recruitment of macrophages (MCP-1, MIP-1α), the influx of CD4 T cells (MCP-1, MIP-1α, MIP-1β, IP-10 and RANTES) and the characteristic infiltrate of eosinophils (eotaxin and RANTES) associated with islet xenograft rejection. Inhibition of β-chemokine signaling in CCR2−/− mice (which lack the major co-receptor for MCP-1) resulted in retarded macrophage and CD4 T cell recruitment, enhanced eosinophil influx and a minor delay in rejection, compared with wildtype mice; there was little effect on leukocyte infiltration in xenografts harvested from CCR5−/− mice (lacking the co-receptor for MIP-1α, MIP-1β and RANTES). The impeded migration of leukocytes into xenografts in CCR2−/− hosts was associated with delayed intragraft expression of MCP-1 and RANTES mRNA; absence of MCP-1/CCR2-mediated signaling led to enhanced intragraft expression of MCP-1, MIP-1α and MIP-1β mRNA. These findings suggest that MCP-1 plays an important role in regulating macrophage and CD4 T cell infiltration to xenograft sites via the CCR2 signaling pathway. Additional treatment of xenografted CCR2−/− transplant recipients with anti-interleukin-(IL)-4 and anti-IL-5 mAbs further delayed xenograft rejection demonstrating the potential for combined antirejection strategies in facilitating pig islet xenotransplantation.

CC chemokine receptor 2 (CCR2) −/− mice are protected from experimental pulmonary fibrosis, a disease increasingly recognized as being mediated by dysfunctional interactions between epithelial cells and fibroblasts. We have sought to investigate the interactions between alveolar epithelial cells (AECs) and fibroblasts in these fibrosis-resistant (CCR2 −/−) and fibrosis-sensitive (CCR2 +/+) mice. AECs from CCR2 −/− mice suppress fibroblast proliferation more than AECs from CCR2 +/+ mice (77 vs. 43%). Exogenous administration of the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) to the fibroblast-AEC cocultures reverses the suppression mediated by CCR2 +/+ AECs but has no effect with CCR2 −/− AECs. MCP-1 regulates AEC function but not fibroblast function. AEC inhibition of fibroblast proliferation was mediated by a soluble, aspirin-sensitive factor. Accordingly, AECs from CCR2 −/− mice produce greater quantities of PGE2than do AECs from CCR2 +/+ mice, and MCP-1 inhibits AEC-derived PGE2synthesis. Diminished PGE2production by AECs results in enhanced fibroproliferation. Thus an important profibrotic mechanism of MCP-1/CCR2 interactions is to limit PGE2production in AECs after injury, thus promoting fibrogenesis.

Chemokines are low molecular weight cytokines which act as chemoattractants for infiltrating cells bearing appropriate receptors (CCR) to sites of inflammation. It has been proposed that CCR2 on monocytes is responsible for their recruitment into the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, and two previous reports have described resistance of CCR2−/− mice to EAE. The present study examined three different mouse strains with CCR2 deletions for susceptibility to EAE. Animals were studied up to 4 months post-sensitization and were examined by neuropathology, RNase protection assay, in situ hybridization, and in vitro assays. All three strains were found to be susceptible to EAE: C57BL/6 × J129 and Balb c strains, 100%; and C57BL/6, 67%. Unusual in CNS lesions of CCR2−/− mice was an overabundance of neutrophils versus monocytes in wild-type animals. An attempt of the immune system to develop compensatory mechanisms for the lack of CCR2 was evidenced by a corresponding increase in mRNA for other chemokines and CCR. Inasmuch as neutrophils replaced monocytes and led to demyelination, our findings support the concept that promiscuity of chemokines and CCR was able to surmount the deletion of CCR2, still resulting in full expression of this autoimmune disease.

The CC chemokine ligand 2 (CCL2) (JE, monocyte chemotactic protein-1 [MCP-1]) and its CC chemokine receptor 2 (CCR2) are critical regulators of monocyte/macrophage trafficking. Recently, we demonstrated that application of exogenous CCL2 in the lungs of mice induced monocyte accumulation in the airspace, whereas combined bronchoalveolar instillation of CCL2 and Escherichia coli endotoxin provoked both enhanced monocyte accumulation and extensive neutrophil influx associated with loss of pulmonary endothelial/epithelial barrier function. In this study, we investigated the role of the CCL2 receptor CCR2 in alveolar leukocyte traffic. In CCR2 knockout mice or wild-type mice treated with the anti-CCR2-blocking monoclonal antibody MC21, monocyte accumulation in response to alveolar CCL2 or CCL2 plus endotoxin was inhibited by more than 90%. Unexpectedly, alveolar neutrophil accumulation in the CCL2/lipopolysaccharide (LPS) model was also drastically reduced by both approaches of CCR2 function interference. When wild-type mice treated with anti-Gr-1 monoclonal antibody to deplete neutrophils selectively or treated with antileukinate, a CXC receptor inhibitor, were challenged with alveolar CCL2 plus LPS, alveolar monocyte accumulation was markedly decreased. Wild-type mice treated with MC21 to block CCR2 function or with anti-Gr-1 to deplete neutrophils did not exhibit the vascular leakage that typically accompanies inflammation triggered by CCL2 and LPS in wild-type mice. These findings confirm a central role for CCR2 in the process of alveolar monocyte recruitment in response to CCL2 alone and combined CCL2 plus LPS and reveal a previously unobserved interdependence between monocyte and neutrophil trafficking that has important implications for the concomitant increase in vascular permeability.

Cultured mouse astrocytes respond to the CC chemokine RANTES by production of chemokine and cytokine transcripts. Stimulation of astrocytes with 1 nM RANTES or 3–10 nM of the structurally related chemokines (eotaxin, macrophage inflammatory protein-1α and -β [MIP-1α, MIP-1β]) induced transcripts for KC, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), MIP-1α, MIP-2, and RANTES in a chemokine and cell-specific fashion. Synthesis of chemokine (KC and MCP-1) and cytokine (TNF-α) proteins was also demonstrated. RANTES-mediated chemokine synthesis was specifically inhibited by pertussis toxin, indicating that G-protein-coupled chemokine receptors participated in astrocyte signaling. Astrocytes expressed CCR1 and CCR5 (the redundant RANTES receptors). Astrocytes derived from mice with targeted mutations of either CCR1 or CCR5 respond after RANTES stimulation, suggesting multiple chemokine receptors may separately mediate RANTES responsiveness in astrocytes. Preliminary data suggest activation of the MAP kinase pathway is also critical for RANTES-mediated signaling in astrocytes. Treatment with RANTES specifically modulated astrocyte receptors upregulating intercellular adhesion molecule 1 (ICAM-1) and downregulating CX3CR1 expression. Thus, after chemokine treatment, astrocytes release proinflammatory mediators and reprogram their surface molecules. The combined effects of RANTES may serve to amplify inflammatory responses within the central nervous system. GLIA 39:19–30, 2002. © 2002 Wiley-Liss, Inc.

Many transcription factors regulate specific temporal-spatial events during cardiac differentiation; however, the mechanisms that regulate such events are largely unknown. Using a modified subtractive hybridization method to identify specific genes that influence early cardiac development, we found that Bop is expressed specifically in cardiac and skeletal muscle precursors before differentiation of these lineages. Bop encodes a protein containing MYND and SET domains, which have been shown to regulate transcription by mediating distinct chromatin modifications. We show that m-Bop is a histone deacetylase-dependent transcriptional repressor. Targeted deletion of Bop in mice disrupted maturation of ventricular cardiomyocytes and interfered with formation of the right ventricle. Normal expression of Hand2, a transcription factor essential for right ventricular development, in cardiomyocyte precursors is dependent upon m-Bop. These results indicate that m-Bop is essential for cardiomyocyte differentiation and cardiac morphogenesis.

Uterine NK (uNK) cells are abundant in human and murine uteri during decidualization. It is unclear whether precursors of uNK (pre-uNK) cells self-renew or are recruited from other sites. To assess self-renewal of pre-uNK cells, uterine segments from NK cell-competent mice were grafted orthotopically into NK/uNK cell-deficient or wild-type mice. Only in wild-type recipients did decidualized grafts contain uNK cells, indicating that pre-uNK cells do not self-renew in uterus. To identify pre-uNK cell sources, thymus, bone marrow, lymph node, or spleen cells were grafted from virgin or pregnant NK cell-competent donors into mated NK/uNK cell-deficient recipients. Cells from secondary lymphoid tissues of pregnant donors gave high level uNK cell reconstitution, which was independent of chemokine receptors CCR2 or CCR5. Pregnancy-induced changes to lymphocyte-endothelial cell interactions were documented using adhesion of human lymphocytes to frozen mouse tissue sections under shear. A dynamic increase was observed in L-selectin- and α4 integrin-dependent adhesion of CD56bright NK cells to decidualizing uterus and in human PBL adhesion to lymph node endothelium. These data support a model that attributes the dramatic increases in human and murine uNK cells during decidualization to precursor cell recruitment.

In the present study, we evaluated the role of CCR2 in a model of viral-induced neurologic disease. An orchestrated expression of chemokines, including the CCR2 ligands monocyte chemoattractant protein-1/CCL2 and monocyte chemoattractant protein-3/CCL7, occurs within the CNS following infection with mouse hepatitis virus (MHV). Infection of mice lacking CCR2 (CCR2−/−) with MHV resulted in increased mortality and enhanced viral recovery from the brain that correlated with reduced (p ≤ 0.04) T cell and macrophage/microglial (determined by F4/80 Ag expression, p ≤ 0.004) infiltration into the CNS. Moreover, MHV-infected CCR2−/− mice displayed a significant decrease in Th1-associated factors IFN-γ (p ≤ 0.001) and RANTES/CCL5 (p ≤ 0.002) within the CNS as compared with CCR2+/+ mice. Further, peripheral CD4+ and CD8+ T cells from immunized CCR2−/− mice displayed a marked reduction in IFN-γ production in response to viral Ag and did not migrate into the CNS of MHV-infected recombination-activating gene (RAG)1−/− mice following adoptive transfer. In addition, macrophage/microglial infiltration into the CNS of RAG1−/− mice receiving CCR2−/− splenocytes was reduced (p ≤ 0.05), which correlated with a reduction in the severity of demyelination (p ≤ 0.001) as compared with RAG1−/− mice receiving splenocytes from CCR2+/+ mice. Collectively, these results indicate an important role for CCR2 in host defense and disease by regulating leukocyte activation and trafficking.

Several chemoattractants mediate Natural Killer (NK) cell migration. The CC-chemokines, monocyte inflammatory protein (MIP)-1 alpha, regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage chemotactic protein (MCP)-1 are the most potent. Peripheral NK cells express the CC-chemokine receptor CCR2, for MCP-1 and CCR5, for MIP-1 alpha and RANTES. These chemokines are detected in the uterus during the estrous cycle and become elevated during pregnancy. To assess the roles of CCR2, CCR5 and MIP-1 alpha in NK cell migration to the uterus and localization within implantation sites, histological analysis was conducted on implantation sites from mice genetically-ablated for CCR2, CCR5, MIP-1 alpha or CCR2 and MIP-1 alpha. Uterine NK (uNK) cell densities in both the decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp) of all mutant strains matched wildtype controls. Ratios of vascular: non-vascular uNK cell position were identical in mutants and controls. In the decidua basalis, 25-35% and in the MLAp, 15-20% of uNK cells were perivascular. Intravascular uNK cells were observed in the decidua basalis but not in the MLAp and were more numerous at gestation day 10 than 12. Two measures of uNK cell activation, cell diameter and cytoplasmic granule number, were similar in the mutants and controls. Thus, migration, distribution and activation of NK cells within the pregnant uterus are independent of CCR2, CCR5 and MIP-1 alpha.

Abstract —Recent studies have identified the presence of macrophages in the arterial wall of hypertensive animals and suggested that as is the case in atherosclerosis, macrophage products may be important mediators of the adaptive response of the arterial wall. In support of this, we have previously shown that the expression of monocyte chemoattractant protein-1 is upregulated in the arteries of hypertensive animals. We hypothesized that macrophage recruitment is a critical step in the pathogenesis of hypertension. To obtain insights into this potential mechanism, we made use of mice deficient in the CC chemokine receptor 2 (CCR2), the receptor for monocyte chemoattractant protein-1. Hypertension was induced with the subcutaneous administration of angiotensin II (0.75 mg · kg −1 · d −1 ) for 7 days. Using in situ hybridization with a probe for c- fms to identify macrophages, we found that hypertension-induced macrophage infiltration of the arterial wall was virtually eliminated in CCR2-deficient mice. In addition, vascular hypertrophy was reduced by ≈65% compared with wild-type animals. These data demonstrate that CCR2 is essential for the recruitment of macrophages into the arterial wall in the setting of hypertension. Furthermore, the decreased hypertrophic response suggests that vascular hypertrophy occurs in part as a consequence of macrophage infiltration. In angiotensin II–induced hypertension, CCR2-mediated responses are critical to the process of macrophage recruitment and vascular hypertrophy and may represent one mechanism by which at least some forms of hypertension may lead to the development of atherosclerosis.

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1α, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1α led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8α1 T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection–induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell–specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine–biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major–resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major–susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. The objective of this study was to determine the role of CCR2 in leukocyte recruitment and development of T1-type cell-mediated immunity during pulmonary C. neoformans infection. Intratracheal inoculation of C. neoformans into CCR2 knockout (CCR2−/−) mice produced a prolonged pulmonary infection (5000-fold CFU at 6 wk compared with CCR2+/+ mice) and significant dissemination to the spleen and brain (160- and 800-fold greater). In addition, CCR2 deficiency resulted in significantly reduced recruitment of macrophages (weeks 1–3) and CD8+ T cells (weeks 1–2) into the lungs. The immune response in CCR2−/− mice was characterized by chronic pulmonary eosinophilia, crystal deposition in the lungs, pulmonary leukocyte production of IL-4 and IL-5 but not IFN-γ, lack of anticryptococcal delayed-type hypersensitivity, and high levels of serum IgE. These results demonstrate that expression of CCR2 is required for the development of a T1-type response to C. neoformans infection and lack of CCR2 results in a switch to a T2-type response. Thus, CCR2 plays a critical role in promoting the development of T1- over T2-type immune responses in the lung following cryptococcus infection.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1α (MIP-1α), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1α, or CCR2 had reduced IFN-γ responses following ligation of the TCR. Reduced IFN-γ responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5−/− and CCR2−/− mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-γ responses. However, this reduced IFN-γ response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-γ response was evident in CCR5−/− and MIP-1α−/− mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1α, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1α, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

After intratracheal inoculation of the AIDS-associated pathogen Cryptococcus neoformans, 12-wk survival was >90% for CCR5+/+ mice but

The accumulation of circulating monocytes in the arterial wall is an early event in atherosclerotic plaque formation. Monocyte chemoattractant protein-1 (MCP-1) has been implicated as the primary source of monocyte chemoattractant functioning in these early stages of atherogenesis. To determine whether the receptor for MCP-1, CC chemokine receptor CCR2, plays a role in atherogenesis, CCR2-deficient animals were crossed with mice lacking apo E, a well characterized model of atherosclerosis. While lipid levels were unchanged, the double knockout mice exhibited a 3-fold reduction in mean aortic lesion area compared to apo E-deficient control mice. Furthermore, the lesions in the double mutants were less advanced, consisting primarily of foam cell deposits and fatty streaks located on or directly adjacent to the aortic valve attachment sites. These studies directly demonstrate that the MCP-1 receptor, CCR2, plays an important role in atherogenesis.

Purpose. To determine if genetic ablation of the chemokine receptor CCR2 (involved in leukocyte and endothelial chemotaxis) inhibits the development of corneal neovascularization. Methods. Wild-type C57BL/6J mice, as well as species-specific counterparts with targeted homozygous disruption of the CCR2, underwent chemical and mechanical denudation of corneal and limbal epithelium. Corneas were harvested 2 weeks after injury. Neovascularization was quantified by CD31 immunostaining. Results. The mean percentages of neovascularized corneal area in control mice and CCR2-deficient mice 2 weeks after denudation were 58.3% and 38.8% (P = 0.047), respectively. Conclusions. Development of corneal neovascularization is inhibited in CCR2-deficient mice.

Cocaine-induced sensitization induces long-term neuroplastic changes in the striatum. Among these, extracellular signal-regulated kinase (ERK) is a fundamental component in striatal gene and epigenetic regulation and plays an important role in reward processes. As previous studies suggested that the chemokine CCL2 enhanced striatal dopamine release and as its cognate CCR2 receptor was located in brain structures implicated in cocaine reward, we tested the hypothesis that CCR2/CCL2 could be involved in cocaine-induced behavioral response. We used CCR2 knockout mice (CCR2(-/-)) and studied two crucial steps in cocaine sensitization: locomotor activity in sensitized mice and ERK activation in the striatum. We show that locomotor sensitization is significantly reduced in CCR2(-/-) mice as well as the dopamine transporter regulation and the cocaine-induced p-ERK striatal activation. Taken together, our results suggest that CCR2 receptor is involved in cocaine sensitization.

Macrophage inflammatory protein-1α (MIP-1α) is a member of the chemokine family of proinflammatory mediators. In addition to its inflammatory roles, MIP-1α has been shown to be active as an inhibitor of primitive hemopoietic cell proliferation. Indeed, a dysfunction in this inhibitory process has been postulated to contribute to leukemogenesis. Research has been aimed at characterizing the receptor involved in cellular inhibition by MIP-1α. This study demonstrates that of all the β-chemokines tested, only MIP-1α is capable of inhibiting primitive hemopoietic cell proliferation. Because no MIP-1α–specific receptors have been identified, this suggests that inhibition is mediated by an uncharacterized receptor. Further evidence for the involvement of a novel receptor in this process is the equivalent potencies of MIP-1αS and MIP-1αP variants of human MIP-1α and the fact that primitive cells from bone marrow derived from individual MIP-1α receptor null mice display a full response to MIP-1α inhibition.

Background: Acute lung injury (ALI) is a frequent complication of gram-negative pneumonia and is characterized by high alveolar levels of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and alveolar barrier dysfunction. However, the contribution of IL-1β to epithelial injury[for full text, please go to the a.m. URL], 10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010)

Short-term bulk cultures and some long-term clones and lines of murine T cell receptor (TcR) gamma/delta-bearing epidermal T cells (dEC) were found to express an Fc gamma receptor II (Fc gamma RII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the Fc gamma RII expressed on dEC is encoded solely by the Fc gamma R alpha gene. While all the various cultured dEC cell populations analyzed exhibit lectin-dependent cellular cytotoxicity, only those which expressed Fc gamma R alpha were also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of Fc gamma R alpha on mouse natural killer cells support an essential role for Fc gamma R alpha in ADCC and extend an analogy with surface CD16 (Fc gamma RIII) expression and ADCC in human natural killer cells and peripheral TcR gamma/delta T cells.

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2(-/-) BMCs (ob/ob + CCR2(-/-) BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2(-/-) BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.