Defective expression of the monocyte chemotactic protein-1 receptor CCR2 in macrophages associated with human ovarian carcinoma

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-alpha restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.     

Differential expression of the isoforms for the monocyte chemoattractant protein-1 receptor, CCR2, in monocytes.

Two isoforms of human CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), have been identified but their relative expression in monocytes and contribution to inflammatory responses mediated by MCP-1 remain uncertain. All available information on CCR2 expression is based on mRNA data because isoform-specific antibodies were not available until now. To analyze the relative expression of each isoform, we made two antibodies that specifically recognized CCR2A and CCR2B. Examination of receptor protein with these isoform-specific antibodies showed that the total expression of CCR2B in monocytes was about 10-fold higher than that of CCR2A with an equal distribution between the cell surface and intracellular pools. A detailed analysis using purified plasma membranes demonstrated that about 90% of all CCR2 on the cell surface were composed of CCR2B. The relatively abundant expression of CCR2B on the cell surface suggests a principal role of this isoform as a mediator of monocyte responses to MCP-1 in inflammation.     

EXPRESSION OF MONOCYTE CHEMOTACTIC PROTEIN-3 IN HUMAN MONOCYTES EXPOSED TO THE MYCOBACTERIAL CELL WALL COMPONENT LIPOARABINOMANNAN

Monocyte chemotactic protein-3 (MCP-3) is a C–C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovisBCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.     

CCR5 Expression Correlates with Susceptibility of Maturing Monocytes to Human Immunodeficiency Virus Type 1 Infection

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1_BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.     

High-content analysis of CCR2 antagonists on human primary monocytes

The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.     

Pharmacological characterization of INCB3344, a small molecule antagonist of human CCR2

The chemokine receptor 2 (CCR2) directs migration of monocytes and has been proposed to be a drug target for chronic inflammatory diseases. INCB3344 was first published as a small molecule nanomolar inhibitor of rodent CCR2. Here, we show that INCB3344 can also bind human CCR2 (hCCR2) with high affinity, having a dissociation constant (K_d) of approximately 5 nM. The binding of the compound to the receptor is rapid and reversible. INCB3344 potently inhibits hCCR2 binding of monocyte chemoattractant protein-1 (MCP-1) and MCP-1-induced signaling and function in hCCR2-expressing cells, including ERK phosphorylation and chemotaxis, and is competitive against MCP-1 in vitro. INCB3344 also blocks MCP-1 binding to monocytes in human whole blood, with potency consistent with in vitro studies. The whole blood binding assay described here can be used for monitoring pharmacodynamic activity of CCR2 antagonists in both preclinical models and in the clinic.     

CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines

Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.     

Thioredoxin specifically cross-desensitizes monocytes to MCP-1

Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca(2+) induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca(2+) responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca(2+) response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.     

The role of the chemokines MCP-1, GRO-alpha, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.     

Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.     

Irbesartan attenuates ischemic brain damage by inhibition of MCP-1/CCR2 signaling pathway beyond AT₁ receptor blockade

Angiotensin II type 1 (AT(1)) receptor blockers (ARBs) are known to prevent the onset of stroke and to attenuate neural damage. Additional beneficial effects of ARBs, independent of AT(1) receptor blockade, have been highlighted. Irbesartan is reported to act as an antagonist of the monocyte chemoattractant protein-1 (MCP-1) receptor, C-C chemokine receptor 2 (CCR2), due to its molecular structure. We examined the possible synergistic effects of co-administration of irbesartan with propagermanium, a CCR2 antagonist, on ischemic brain damage. Administration of propagermanium decreased ischemic brain area after middle cerebral artery occlusion (MCAO). To study the possible synergistic effects of propagermanium with ARBs, we employed non-effective lower doses of irbesartan and losartan. Administration of irbesartan with propagermanium decreased the ischemic brain area more markedly compared with propagermanium alone, but co-administration of losartan did not. MCP-1 mRNA level was significantly increased on the ipsilateral side after MCAO, and administration of irbesartan with propagermanium decreased the MCP-1 level, whereas co-administration of losartan did not. Similar results were obtained for MCP-1 protein level. CCR2 mRNA expression was significantly elevated on the ipsilateral side; however, no significant difference was observed between each group. mRNA levels of other inflammatory cytokines such as TNF-α and IL-1β also significantly increased on the ipsilateral side, but the expression levels were not changed by each drug treatment. Taking these findings together, irbesartan exerts more beneficial effects on ischemic brain damage with an MCP-1 receptor blocker, at least due to its inhibitory effects on MCP-1/CCR2 signaling beyond AT(1) receptor blockade.     

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.     

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.     

Combinatorial model of chemokine involvement in glomerular monocyte recruitment: role of CXC chemokine receptor 2 in infiltration during nephrotoxic nephritis

A sequential model involving chemokines has been proposed for leukocyte extravasation into areas of inflammation; however, site-specific aspects remain to be elucidated. Hence, we studied the role of chemokines produced by mesangial (MC) or glomerular endothelial cells (GEC) and their receptors in glomerular recruitment of monocytes. Stimulation of MC with TNF-alpha up-regulated mRNA and protein of CC and CXC chemokines but not constitutive expression of the CX(3)C chemokine fractalkine. While growth-related activity (GRO)-alpha was immobilized to MC proteoglycans, monocyte chemotactic protein (MCP)-1 was secreted into the soluble phase. Firm adhesion and sequestration of monocytes on activated MC was supported by the GRO-alpha receptor CXCR2 and to a lesser extent by CX(3)CR, whereas the MCP-1 receptor CCR2 contributed to their transendothelial chemotaxis toward activated MC. In contrast, fractalkine mRNA and protein was induced by TNF-alpha in transformed rat GEC, and both CXCR2 and CX(3)CR mediated monocyte arrest on GEC in shear flow. The relevance of these mechanisms was confirmed in a rat nephrotoxic nephritis model where acute glomerular macrophage recruitment was profoundly inhibited by blocking CXCR2 or CCR2. In conclusion, our results epitomize a combinatorial model in which chemokines play specialized roles in driving glomerular monocyte recruitment and emphasize an important role for CXCR2 in macrophage infiltration during early phases of nephrotoxic nephritis.     

HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes.

AIDS dementia is characterized by neuronal loss in association with synaptic damage. A central predictor for clinical onset of these symptoms is the infiltration of monocytes and macrophages into CNS parenchyma. Chronic HIV-1 infection of monocytes also allows these cells to serve as reservoirs for persistent viral infection. Using a coculture of endothelial cells and astrocytes that models several aspects of the human blood-brain barrier, we examined the mechanism whereby the HIV-derived factor Tat may facilitate monocyte transmigration. We demonstrate that treatment of cocultures on the astrocyte side with HIV-1 Tat induced significant monocyte chemoattractant protein (MCP)-1 protein. Astrocytes, but not endothelial cells, were the source of this MCP-1 expression. Supernatants from Tat-treated cocultures induced significant monocyte transmigration, which was detected by 2.5 h after the addition of PBMC. Pretreatment of the supernatants from Tat-stimulated cocultures with an Ab to MCP-1 completely blocked monocyte transmigration. Flow cytometric analysis of Tat-stimulated PBMC demonstrated that Tat up-regulated expression of the chemokine receptor, CCR5, on monocytes in a time-dependent manner. Taken together, our data indicate that HIV-1 Tat may facilitate the recruitment of monocytes into the CNS by inducing MCP-1 expression in astrocytes. These recruited monocytes may contribute to the pathogenesis of HIV-1-associated AIDS encephalitis and dementia.     

瑞舒伐他汀对颈动脉粥样硬化患者单个核细胞CCR2表达的影响及机制

目的：探讨瑞舒伐他汀对颈动脉粥样硬化患者单个核细胞CC类趋化因子受体2（CCR2）表达的影响及上游机制。方法：选择颈动脉粥样硬化患者20例。予瑞舒伐他汀5~20 mg/d治疗。在基线、3月时采集血标本,测定单核细胞趋化蛋白-1（MCP-1）及血脂水平;分离单个核细胞,流式细胞学检测单个核细胞上的CCR2表达;荧光定量逆转录PCR法检测CCR2、过氧化物酶体增生物激活受体（PPAR）β mRNA的表达;Western印迹法检测PPAR β蛋白的表达。结果：瑞舒伐他汀治疗3个月后,患者低密度脂蛋白胆固醇（LDL-C）水平明显降低（P<0.01）,MCP-1及单个核细胞CCR2表达显著下降（P<0.05）,PPAR β mRNA及蛋白表达均较前增加（P<0.05）。结论：瑞舒伐他汀可能通过PPAR β途径降低MCP-1,抑制单个核细胞CCR2的表达。