Monocyte/macrophage chemokine receptor CCR2 mediates diabetic renal injury

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN). C-C chemokine receptor (CCR)2 regulates monocyte/macrophage migration into injured tissues. However, the direct role of CCR2-mediated monocyte/macrophage recruitment in diabetic kidney disease remains unclear. We report that pharmacological blockade or genetic deficiency of CCR2 confers kidney protection in Ins2(Akita) and streptozotocin (STZ)-induced diabetic kidney disease. Blocking CCR2 using the selective CCR2 antagonist RS504393 for 12 wk in Ins2(Akita) mice significantly attenuated albuminuria, the increase in blood urea nitrogen and plasma creatinine, histological changes, and glomerular macrophage recruitment compared with vehicle. Furthermore, mice lacking CCR2 (CCR2(-/-)) mimicked CCR2 blockade by reducing albuminuria and displaying less fibronectin mRNA expression and inflammatory cytokine production compared with CCR2(+/+) mice, despite comparable blood glucose levels. Bone marrow-derived monocytes from CCR2(+/+) or CCR2(-/-) mice adoptively transferred into CCR2(-/-) mice reversed the renal tissue-protective effect in diabetic CCR2(-/-) mice as evaluated by increased urinary albumin excretion and kidney macrophage recruitment, indicating that CCR2 is not required for monocyte migration from the circulation into diabetic kidneys. These findings provide evidence that CCR2 is necessary for monocyte/macrophage-induced diabetic renal injury and suggest that blocking CCR2 could be a novel therapeutic approach in the treatment of DN.     

Additive roles for MCP-1 and MCP-3 in CCR2-mediated recruitment of inflammatory monocytes during Listeria monocytogenes infection

Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6C(high) inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2(-/-) mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1(-/-) mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3(-/-) mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3(-/-) mice have significantly fewer Ly6C(high) monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3(-/-) mice, like MCP-1(-/-) mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.     

Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules

Influenza A virus pneumonia is characterized by severe lung injury and high mortality. Early infection elicits a strong recruitment of monocytes from the peripheral blood across the endo-/epithelial barrier into the alveolar air space. However, it is currently unclear which of the infected resident lung cell populations, alveolar epithelial cells or alveolar macrophages, elicit monocyte recruitment during influenza A virus infection. In the current study, we investigated whether influenza A virus infection of primary alveolar epithelial cells and resident alveolar macrophages would elicit a basal-to-apical monocyte transepithelial migration in vitro. We found that infection of alveolar epithelial cells with the mouse-adapted influenza A virus strain PR/8 strongly induced the release of monocyte chemoattractants CCL2 and CCL5 followed by a strong monocyte transepithelial migration, and this monocytic response was strictly dependent on monocyte CCR2 but not CCR5 chemokine receptor expression. Analysis of the adhesion molecule pathways demonstrated a role of ICAM-1, VCAM-1, integrin-associated protein (CD47), and junctional adhesion molecule-c on the epithelial cell surface interacting with monocyte beta(1) and beta(2) integrins and integrin-associated protein in the monocyte transmigration process. Importantly, addition of influenza A virus-infected alveolar macrophages further enhanced monocyte transmigration across virus-infected epithelium in a TNF-alpha-dependent manner. Collectively, the data show an active role for virus-infected alveolar epithelium in the regulation of CCL2/CCR2-dependent monocyte transepithelial migration during influenza infection that is essentially dependent on both classical beta(1) and beta(2) integrins but also junctional adhesion molecule pathways.     

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.     

Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2(+) alveolar macrophage population, and CCR2-deficient mice transfused with CCR2(+) mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.     

Distinct differentiation potential of blood monocyte subsets in the lung

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1(high)CX(3)CR1(int) and Gr1(low)CX(3)CR1(high), respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1(low)CX(3)CR1(high) monocytes, but not Gr1(high)CX(3)CR1(int) cells, had the potential to differentiate into lung macrophages. However, Gr1(high)CX(3)CR1(int) monocytes could acquire this potential upon conversion into Gr1(low)CX(3)CR1(high) cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.     

Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses

Monocytes can have important effects on the polarization and expansion of lymphocytes and may contribute to shaping primary and memory T-cell responses in humans and mice. However, their precise contribution in terms of cellular subsets and the molecular mechanisms involved remains to be determined. Mouse monocytes originate from a bone marrow progenitor, the macrophage and DC precursor (MDP), which also gives rise to conventional dendritic cells through a separate differentiation pathway. Mouse monocytes may be grouped in different functional subsets. The CD115(+) Gr1(+) 'inflammatory' monocyte subset can give rise not only to immunostimulatory 'TipDCs' in infected mice but also to immunosuppressive 'myeloid-derived suppressor cells' in tumor-bearing mice. CD115(+) Gr1(+) monocytes can also contribute to the renewal of several resident subsets of macrophages and DCs, such as microglia and Langerhans cells, in inflammatory conditions. The CD115(+) Gr1(-) 'resident' monocyte subset patrols blood vessels in the steady state and extravasates during infection with Listeria monocytogenes or in the healing myocardium. CD115(+) Gr1(-) monocytes are responsible for an early and transient inflammatory burst during Lm infection, which may play a role in the recruitment of other effector cells and subsequently differentiate toward 'M2'-like macrophages that may be involved in wound healing. More research will no doubt confirm the existence of more functional subsets, the developmental relationship between mouse subsets as well as the correspondence between mouse subsets and human subsets of monocytes. We will discuss here the potential roles of monocytes in the immune response, the existence of functional subsets and their relationship with other myeloid cells, including dendritic cells.     

A MyD88-dependent IFNγR-CCR2 signaling circuit is required for mobilization of monocytes and host defense against systemic bacterial challenge

Monocytes are mobilized to sites of infection via interaction between the chemokine MCP-1 and its receptor, CCR2, at which point they differentiate into macrophages that mediate potent antimicrobial effects. In this study, we investigated the mechanisms by which monocytes are mobilized in response to systemic challenge with the intracellular bacterium Francisella tularensis. We found that mice deficient in MyD88, interferon-γ (IFNγ)R or CCR2 all had defects in the expansion of splenic monocyte populations upon F. tularensis challenge, and in control of F. tularensis infection. Interestingly, MyD88-deficient mice were defective in production of IFNγ, and IFNγR-deficient mice exhibited defective production of MCP-1, the ligand for CCR2. Transplantation of IFNγR-deficient bone marrow (BM) into wild-type mice further suggested that mobilization of monocytes in response to F. tularensis challenge required IFNγR expression on BM-derived cells. These studies define a critical host defense circuit wherein MyD88-dependent IFNγ production signals via IFNγR expressed on BM-derived cells, resulting in MCP-1 production and activation of CCR2-dependent mobilization of monocytes in the innate immune response to systemic F. tularensis challenge.     

CCR2-positive monocytes recruited to inflamed lungs downregulate local CCL2 chemokine levels

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.     

Increased expression levels of monocyte CCR2 and monocyte chemoattractant protein-1 in patients with diabetes mellitus

Increased monocyte recruitment into subendothelial space in atherosclerotic lesions is one of the hallmarks of diabetic angiopathy. The aim of this study was to determine the state of peripheral blood monocytes in diabetes associated with atherosclerosis. Diabetic patients treated with/without an oral hypoglycemic agent and/or insulin for at least 1 year were recruited (n=106). We also included 24 non-diabetic control subjects. We measured serum levels of monocyte chemoattractant protein (MCP)-1, fasting plasma glucose (FPG), HbA1c, total cholesterol, triglyceride, body mass index (BMI), high sensitivity CRP (hs-CRP) and evaluated CCR2, CD36, CD68 expression on the surface of monocytes. Serum MCP-1 levels were significantly (p<0.05) higher in diabetic patients than in normal subjects. In diabetic patients, serum MCP-1 levels correlated significantly with FPG, HbA1c, triglyceride, BMI, and hs-CRP. The expression levels of CCR2, CD36, and CD68 on monocytes were significantly increased in diabetic patients and were more upregulated by MCP-1 stimulation. Our data suggest that elevated serum MCP-1 levels and increased monocyte CCR2, CD36, CD68 expression correlate with poor blood glucose control and potentially contribute to increased recruitment of monocytes to the vessel wall in diabetes mellitus.     

Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.     

The pathological effects of CCR2+ inflammatory monocytes are amplified by an IFNAR1-triggered chemokine feedback loop in highly pathogenic influenza infection

Background

Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection.

Results

We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/β receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2−/− mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice.

Conclusions

Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.     

Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells

Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2(-/-), or CCR2(-/-) mice were transplanted with allogeneic marrow and spleen cells. WT and (-/-) recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4(+) and CD8(+) T cells in lungs post-BMT did not differ between WT and (-/-) recipients. However, the T cell dependency of the host CD11b(+) major histocompatibility complex class II(+) cell influx was lost in CCR2(-/-) recipients. In CCR2(-/-) mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of (-/-) mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2(-/-) mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Alpha-aminothiazole-gamma-aminobutanoic amides as potent, small molecule CCR2 receptor antagonists

A series of racemic and homochiral alpha-aminothiazole-gamma-aminobutyroamides that display high affinities for human and murine CCR2 and functional antagonism by inhibition of monocyte recruitment are described. A representative example is (2S)-2-[2-(acetylamino)-1,3-thiazol-4-yl]-N-[3-methyl-5-(trifluoromethyl)benzyl]-4-(4-phenylpiperidin-1-yl)butanamide, which shows 5 nM affinity for human monocytes and CHO cells expressing the human CCR2b receptor. It also inhibited MCP-1 initiated chemotaxis of human monocytes with an IC50 of 0.69 nM.     

Lipoapoptosis induced by saturated free fatty acids stimulates monocyte migration: a novel role for Pannexin1 in liver cells

Recruitment of monocytes in the liver is a key pathogenic feature of hepatic inflammation in nonalcoholic steatohepatitis (NASH), but the mechanisms involved are poorly understood. Here, we studied migration of human monocytes in response to supernatants obtained from liver cells after inducing lipoapoptosis with saturated free fatty acids (FFA). Lipoapoptotic supernatants stimulated monocyte migration with the magnitude similar to a monocyte chemoattractant protein, CCL2 (MCP-1). Inhibition of c-Jun NH₂-terminal kinase (JNK) in liver cells with SP600125 blocked migration of monocytes in a dose-dependent manner, indicating that JNK stimulates release of chemoattractants in lipoapoptosis. Notably, treatment of supernatants with Apyrase to remove ATP potently inhibited migration of THP-1 monocytes and partially blocked migration of primary human monocytes. Inhibition of the CCL2 receptor (CCR2) on THP-1 monocytes with RS102895, a specific CCR2 inhibitor, did not block migration induced by lipoapoptotic supernatants. Consistent with these findings, lipoapoptosis stimulated pathophysiological extracellular ATP (eATP) release that increased supernatant eATP concentration from 5 to ~60 nM. Importantly, inhibition of Panx1 expression in liver cells with short hairpin RNA (shRNA) decreased supernatant eATP concentration and inhibited monocyte migration, indicating that monocyte migration is mediated in part by Panx1-dependent eATP release. Moreover, JNK inhibition decreased supernatant eATP concentration and inhibited Pannexin1 activation, as determined by YoPro-1 uptake in liver cells in a dose-dependent manner. These results suggest that JNK regulates activation of Panx1 channels, and provide evidence that Pannexin1-dependent pathophysiological eATP release in lipoapoptosis is capable of stimulating migration of human monocytes, and may participate in the recruitment of monocytes in chronic liver injury induced by saturated FFA.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9456-5) contains supplementary material, which is available to authorized users.     

Ly-6G+CCR2- myeloid cells rather than Ly-6ChighCCR2+ monocytes are required for the control of bacterial infection in the central nervous system

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-6C(high)CCR2(+) but not Ly-6C(low)CCR2(-) monocytes were recruited in high numbers to the brain as early as 12 h after bacterial challenge. Surprisingly, CD11b(+)Ly-6C(high)CCR2(+) inflammatory monocytes modulated local CXCL2 and IL-1beta 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-6C(high)CCR2(+) 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.     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

CCR2 Defines a Distinct Population of NK Cells and Mediates Their Migration during Influenza Virus Infection in Mice

Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.     

Bone marrow subsets differentiate into endothelial cells and pericytes contributing to Ewing's tumor vessels

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.