Monocyte Chemoattractant Protein-1 Receptor Research Articles

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.

Monocyte chemoattractant protein-1 (MCP-1), secreted by bone marrow endothelial cells, induces chemoattraction of 5T multiple myeloma cells.

Homing of multiple myeloma (MM) cells to the bone marrow (BM) requires transendothelial migration. In the present work we tested whether monocyte chemoattractant protein-1 (MCP-1) and CCR2, the high affinity receptor for MCP-1, are involved in this process. Murine 5T2 and 5T33MM cell lines were selected as source of MM cells and STR4, 10 and 12 of BM endothelial cells (BMEC). RT-PCR demonstrated transcripts for MCP-1 in BMEC and ELISA the presence of MCP-1 protein in culture medium. RNase protection assay showed mRNA expression for CCR2, and FACS analysis the presence of CCR2 protein on the MM cells. EC conditioned medium induced chemoattraction of MM cells, a phenomenon inhibited by anti-MCP-1 antibodies. In conclusion, MM cells express CCR2 and are attracted by MCP-1 secreted by BMEC. We suggest that local MCP-1 production by BMEC is one of the mechanisms involved in homing of myeloma cells to the BM.

CCR2A and CCR2B, the two isoforms of the monocyte chemoattractant protein-1 receptor are up-regulated and expressed by different cell subsets in idiopathic inflammatory myopathies.

The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyosititis (PM) and inclusion body myositis (IBM), are a group of autoimmune diseases characterized by chronic lymphocytic and macrophagic infiltration in muscle. The mechanism for recruitment of these cells probably involves chemokines. We have previously reported that monocyte chemoattractant protein-1 (MCP-1), a beta chemokine, seems to play a major role in mononuclear cell recruitment especially in DM. Here we have investigated the distribution of the main MCP-1 receptors CCR2A and CCR2B in IIM by polymerase chain reaction (PCR), immunohistochemistry and in situ hybridization. We have shown by reverse transcription-PCR that both CCR2A and CCR2B were expressed at low level in normal muscle and that CCR2A was up-regulated in IIM (P=0.02) and was higher in PM and IBM than in DM (P=0.04). By immunohistochemistry and in situ hybridization we have observed that CCR2 isoforms were expressed by different cell subsets in both normal and IIM muscle. CCR2A was expressed in vessel walls and by some mononuclear cells, especially in cells involved in partial invasion in PM and IBM. CCR2B expression was observed in all satellite cells, in the muscular domain of neuromuscular junctions and in some regenerative fibers of IIM, but not in inflammatory exudates. In conclusion, the present study highlights the major role played by MCP-1 and its counter-receptor CCR2 in the pathophysiology of IIM, and shows that the CCR2 receptors are cell specific. The variation of the total amount of CCR2A and its local distribution according to the type of IIM might be a new path towards the understanding of the constitution of mononuclear infiltrates in IIM.

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: possible involvement of oxygen free radicals

Homocysteinaemia is an independent risk factor for atherosclerosis. The development of atherosclerosis involves monocyte chemoattractant protein 1 (MCP-1)-mediated monocyte recruitment to the lesion site. The action of MCP-1 is mostly via its interaction with MCP-1 receptor (CCR2), which is the major receptor for MCP-1 on the surface of monocytes. The objective of the present study was to investigate the effect of homocysteine on CCR2 expression in human THP-1 monocytes. Cells were incubated with various concentrations of homocysteine for 6, 12, 24 and 48h. The expression of CCR2 mRNA was determined by nuclease protection assay and the CCR2 protein was measured by Western immunoblotting analysis. The binding of MCP-1 to CCR2 as a functional receptor on the monocyte surface was determined by flow cytometry. Homocysteine (0.05–0.2mM) significantly enhanced the expression of CCR2 mRNA (129–209% of the control) and CCR2 protein (up to 183% of control) in these cells after 24h of incubation. Stimulation of CCR2 expression was associated with a parallel increase in the binding activity of CCR2 (129–191% of control) as well as an enhanced chemotactic response of homocysteine-treated monocytes. Further investigation revealed that the levels of superoxide were significantly elevated in cells incubated with homocysteine for 12–48h. The addition of superoxide dismutase, a scavenger of superoxide, to the culture medium abolished the stimulatory effect of homocysteine on CCR2 expression as well as the binding activity of the receptor. The stimulatory effect of homocysteine on the expression of CCR2 mRNA and the levels of CCR2 protein was also observed in human peripheral blood monocytes. In conclusion, the present study has clearly demonstrated that homocysteine stimulates CCR2 expression in monocytes, leading to an enhanced binding activity and chemotatic response. Homocysteine-induced superoxide formation might serve as one of the underlying mechanisms for this effect.

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: possible involvement of oxygen free radicals.

Homocysteinaemia is an independent risk factor for atherosclerosis. The development of atherosclerosis involves monocyte chemoattractant protein 1 (MCP-1)-mediated monocyte recruitment to the lesion site. The action of MCP-1 is mostly via its interaction with MCP-1 receptor (CCR2), which is the major receptor for MCP-1 on the surface of monocytes. The objective of the present study was to investigate the effect of homocysteine on CCR2 expression in human THP-1 monocytes. Cells were incubated with various concentrations of homocysteine for 6, 12, 24 and 48 h. The expression of CCR2 mRNA was determined by nuclease protection assay and the CCR2 protein was measured by Western immunoblotting analysis. The binding of MCP-1 to CCR2 as a functional receptor on the monocyte surface was determined by flow cytometry. Homocysteine (0.05-0.2 mM) significantly enhanced the expression of CCR2 mRNA (129-209% of the control) and CCR2 protein (up to 183% of control) in these cells after 24 h of incubation. Stimulation of CCR2 expression was associated with a parallel increase in the binding activity of CCR2 (129-191% of control) as well as an enhanced chemotactic response of homocysteine-treated monocytes. Further investigation revealed that the levels of superoxide were significantly elevated in cells incubated with homocysteine for 12-48 h. The addition of superoxide dismutase, a scavenger of superoxide, to the culture medium abolished the stimulatory effect of homocysteine on CCR2 expression as well as the binding activity of the receptor. The stimulatory effect of homocysteine on the expression of CCR2 mRNA and the levels of CCR2 protein was also observed in human peripheral blood monocytes. In conclusion, the present study has clearly demonstrated that homocysteine stimulates CCR2 expression in monocytes, leading to an enhanced binding activity and chemotatic response. Homocysteine-induced superoxide formation might serve as one of the underlying mechanisms for this effect.

An anti-inflammatory drug, propagermanium, may target GPI-anchored proteins associated with an MCP-1 receptor, CCR2.

Monocyte chemoattractant protein-1 (MCP-1) promotes the migration and activation of monocytes and plays a pivotal role in the development of chronic inflammation. Propagermanium (3-oxygermylpropionic acid polymer) has been used as a therapeutic agent against chronic hepatitis B in Japan. We report here that propagermanium specifically inhibits in vitro chemotactic migration of monocytes by MCP-1. Propagermanium did not inhibit binding of MCP-1 to a human monocytic cell line, THP-1 cells, or affect intracellular Ca(2+) mobilization or the cAMP concentration in MCP-1-treated THP-1 cells. The effect of propagermanium seems to require glycosylphosphatidylinositol (GPI)-anchored proteins, as cleavage of GPI anchors by phosphatidylinositol-phospholipase C (PI-PLC) eliminated the inhibitory activity of propagermanium. Anti-GPI-anchored protein antibodies, such as anti-CD55 and anti-CD59, reduced staining of C-C chemokine receptor 2 (CCR2) with an anti-CCR2 antibody against the N-terminus of CCR2 in a flow cytometric analysis, and these antibodies also selectively inhibited MCP-1-induced migration of THP-1 cells. Furthermore, under fluorescence microscopy, GPI-anchored proteins colocalized with CCR2 on THP-1 cells. These results suggest that propagermanium may target GPI-anchored proteins that are closely associated with CCR2 to selectively inhibit the MCP-1-induced chemotaxis, thus providing a mechanistic basis for the anti-inflammatory effects of the drug.

Peptide mimics of monocyte chemoattractant protein-1 (MCP-1) with an antagonistic activity.

In this study, we attempted to analyze the peptide motifs recognized by 24822.111 and F9, monoclonal antibodies (mAbs) that inhibit the chemotactic activity of monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines. We isolated phage clones from a phage display library and identified six peptide motifs. One of these clones, C27, was strongly and specifically recognized by 24822.111 mAb, while another, G25, was similarly recognized by F9 mAb. Both the C27 motif and the G25 motif contain two cysteines in their sequences and have little homology to the primary amino acid sequence of MCP-1. These clones, however, bound to THP-1 cells, and the binding was competitively inhibited by MCP-1. The clones strongly inhibited the MCP-1-induced chemotaxis of human monocytes. The synthetic and intramolecularly disulfide-linked peptides of C27 and G25 (sC27 and sG25) also inhibited the chemotaxis induced by MCP-1, while their derivatives with serine in place of cysteine did not, suggesting the importance of the loop structure for the inhibition. These results suggest that sC27 and sG25 may mimic the MCP-1-binding domain to the MCP-1 receptor.

A mechanism for the impaired IFN-gamma production in C-C chemokine receptor 2 (CCR2) knockout mice: role of CCR2 in linking the innate and adaptive immune responses.

We have recently shown that mice with a targeted disruption of CCR2, the receptor for monocyte chemoattractant protein-1, have markedly impaired recruitment of macrophages to sites of inflammation. An unexpected finding in the CCR2(-/-) mice was a dramatic decrease in the production of IFN-gamma after challenge with purified protein derivative of Mycobacterium bovis. In this study, we have investigated the mechanism of this cytokine production defect. In vitro, direct activation of splenocytes with CD3/CD28 Abs failed to reveal any differences in IFN-gamma production between CCR2(+/+) and CCR2(-/-) mice. However, after immunization, the number of Ag-specific, IFN-gamma-producing cells in the draining lymph nodes was decreased by 70% in the CCR2(-/-) mice, suggesting an in vivo trafficking defect. Direct measurement of cell trafficking with fluorescently labeled CFA revealed a marked decrease in the number of monocytes/macrophages migrating to the site of immunization and to the draining lymph nodes in the CCR2(-/-) mice. The data suggest that impaired trafficking of APCs in the CCR2(-/-) mice contributes to the defect in IFN-gamma production. These data support the idea that CCR2-positive monocytes/macrophages are critical in linking the innate and adaptive immune responses.

CC chemokine receptor 2 is required for macrophage infiltration and vascular hypertrophy in angiotensin II-induced hypertension.

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 approximately 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.

Enhanced pulmonary allergic responses to Aspergillus in CCR2-/- mice.

Allergic responses to Aspergillus species exacerbate asthma and cystic fibrosis. The natural defense against live Aspergillus fumigatus spores or conidia depends on the recruitment and activation of mononuclear and polymorphonuclear leukocytes, events that are dependent on chemotactic cytokines. In this study, we explored the relative contribution of the monocyte chemoattractant protein-1 receptor, CCR2, in the pulmonary response to A. fumigatus conidia. Following sensitization to soluble A. fumigatus Ags, mice lacking CCR2 due to targeted deletion were markedly more susceptible to the injurious effects of an intrapulmonary challenge with live conidia compared with mice that expressed CCR2 or CCR2+/+. CCR2-/- mice exhibited a major defect in the recruitment of polymorphonuclear cells, but these mice also had significantly more eosinophils and lymphocytes in bronchoalveolar lavage samples. CCR2-/- mice also had significant increases in serum levels of total IgE and whole lung levels of IL-5, IL-13, eotaxin, and RANTES compared with CCR2+/+ mice. Airway inflammation, hyper-responsiveness to spasmogens, and subepithelial fibrosis were significantly enhanced in CCR2-/- mice compared with CCR2+/+ mice after the conidia challenge. Thus, these findings demonstrate that CCR2 plays an important role in the immune response against A. fumigatus, thereby limiting the allergic airway inflammatory and remodeling responses to this fungus.

Increased severity of glomerulonephritis in C-C chemokine receptor 2 knockout mice

Increased severity of glomerulonephritis in C-C chemokine receptor 2 knockout mice. The C-C chemokine receptor 2 (CCR2) is expressed on monocytes and facilitates monocyte migration. CCR2 is a prominent receptor for monocyte chemoattractant protein-1 (MCP-1). This chemokine recruits monocytes to sites of inflammation. It has been suggested that CCR2 and its ligand, MCP-1, play a role in the pathogenesis of glomerulonephritis. The goal of this study was to determine the contribution of CCR2 in a murine model of accelerated nephrotoxic nephritis. We measured the extent of development of renal disease in CCR2 wild-type and knockout mice after the administration of antiglomerular basement membrane antibody. Eight groups of animals were treated (N = 10 per group). Four days after IgG immunization, CCR2 wild-type and knockout mice received control serum or nephrotoxic serum. The urinary protein/creatinine ratio was measured on days 1 and 3; plasma and kidneys were collected on days 4 and 7. Kidneys were evaluated by light microscopy, immunohistochemistry, and immunofluorescence. The genotype of mice was confirmed by tissue analysis. Protective effects of CCR2 knockout on the urinary protein/creatinine ratio were observed on day 1, as values for this parameter were significantly lower (35 +/- 3.6) than in nephritic wild-type mice (50 +/- 6.8). There was a marked increase in proteinuria in nephritic wild-type mice on day 1 compared with vehicle-treated, wild-type animals (5 +/- 1.0). On day 3, the ameliorative effects of CCR2 knockout were not observed; the increase in the urinary protein/creatinine ratio was similar in nephritic CCR2 wild-type (92 +/- 11.2) and knockout mice (102 +/- 9. 2). Plasma markers of disease were evaluated on days 4 and 7. At these time points, there were no beneficial effects of CCR2 receptor knockout on plasma levels of urea nitrogen, creatinine, albumin, or cholesterol. On day 7, blood urea nitrogen (248 +/- 19.9 mg/dL) and plasma cholesterol were higher in nephritic CCR2 knockout mice than in wild-type mice (142 +/- 41.7 mg/dL) that received nephrotoxic serum. Histopathologic injury was more severe in nephritic CCR2 knockout mice than nephritic wild-type mice on day 4 (3.1 +/- 0.3 vs. 2.0 +/- 0.3) and day 7 (3.6 +/- 0.2 vs. 2.9 +/- 0.3). By immunohistochemical analysis at day 4, there were significantly fewer mac-2-positive cells, representative of macrophages in the glomeruli of nephritic CCR2 knockout (2.1 +/- 0.6) mice than nephritic wild-type (3.9 +/- 0.5) animals. By indirect immunofluorescence, there was a moderate, diffuse linear IgG deposition of equivalent severity present in glomeruli of both wild-type and CCR2 knockout nephritic mice. These results suggest that our strategy was successful in reducing macrophage infiltration, but this model of glomerulonephritis is not solely dependent on the presence of CCR2 for progression of disease. After a transient ameliorative effect on proteinuria, CCR2 knockout led to more severe injury in nephritic mice. This raises the intriguing possibility that a CCR2 gene product ameliorates glomerulonephritis in this murine model. Although effects that occur in chemokine knockout mice are not equivalent to those expected with prolonged use of a chemokine antagonist, this study may nevertheless have implications for consideration of long-term use of chemokine antagonists in renal disease.

Experimental Gliosarcoma Induces Chemokine Receptor Expression in Rat Brain

Macrophage/microglial infiltration is a characteristic feature of brain tumors. The functional role(s) of these cells is complex and could include both trophic and suppressive effects on tumors. Information has recently emerged about the molecular signals that regulate the accumulation and function of monocytes in pathological disorders. Recent data indicate that the chemokine, monocyte chemoattractant protein-1 (MCP-1), a potent monocyte activating and chemotactic factor, is a primary regulator of the macrophage response in brain tumors. We hypothesized that if MCP-1 regulates macrophage/microglial infiltration, then expression of the specific MCP-1 receptor, CCR2, will be induced in peritumoral tissue and/or within brain tumors. Identification of a specific receptor that is preferentially expressed in brain tumors could be important both in terms of tumor biology and as a potential therapeutic target. We used an established experimental gliosarcoma model, induced by intracranial transplantation of cultured 9L cells into adult rat brain, to test this hypothesis. RT-PCR analysis showed high levels of both MCP-1 and CCR2 mRNA and Western blot analysis demonstrated increased CCR2 protein in tumor extracts. Immunocytochemistry showed CCR2 immunoreactive microglia in peritumoral tissue and, unexpectedly, that intrinsic tumor cells, rather than monocytes, were the predominant source of CCR2. These results demonstrate that CCR2 expression is markedly upregulated in this brain tumor model.

Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice

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.

Cloning and Functional Characterization of the 5′-Flanking Region of the Human Monocyte Chemoattractant Protein-1 Receptor (CCR2) Gene: ESSENTIAL ROLE OF 5′-UNTRANSLATED REGION IN TISSUE-SPECIFIC EXPRESSION

The human monocyte chemoattractant protein-1 receptor designated hCCR2 is an essential co-receptor in cell entry by the human immunodeficiency virus as well as a receptor for monocyte chemoattractant protein-1, a member of the family of C-C chemokines that mediate monocyte chemotaxis. To elucidate the molecular mechanisms underlying the transcriptional regulation of hCCR2, we cloned and sequenced the hCCR2 gene; it was approximately 8 kilobase pairs in length and consisted of three exons divided by two introns. In the 5'-flanking region, there were the typical mammalian promoter consensus elements, a CAAT box and a TATA box, resulting in a single transcription initiation site. In addition, we found clustered tissue-specific cis-regulatory elements such as GATA consensus sequences, Oct-1 binding sequences, and CAAT/enhancer-binding protein binding sequences. Luciferase assays with various promoter deletions and gel mobility shift assays indicated that three cis-regulatory elements located within the region from -89 to +118 are required for basal activity in THP-1 cells. One element is an octamer sequence 36-base pair upstream from the TATA box; it binds mainly to Oct-1 and is capable of increasing transcriptional activity. The other two elements, which are tandem recognition sites of the CAAT/enhancer-binding protein family, are located in the 5'-untranslated region and account for the transcriptional activation as well as the tissue specificity of hCCR2.

Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis.

Chemokines are proinflammatory cytokines that function in leukocyte chemoattraction and activation and have recently been shown to block the HIV-1 infection of target cells through interactions with chemokine receptors. In addition to their function in viral disease, chemokines have been implicated in the pathogenesis of atherosclerosis. Expression of the CC chemokine monocyte chemoattractant protein-1 (MCP-1) is upregulated in human atherosclerotic plaques, in arteries of primates on a hypercholesterolaemic diet; and in vascular endothelial and smooth muscle cells exposed to minimally modified lipids. To determine whether MCP-1 is causally related to the development of atherosclerosis, we generated mice that lack CCR2, the receptor for MCP-1 (ref. 7), and crossed them with apolipoprotein (apo) E-null mice which develop severe atherosclerosis. Here we show that the selective absence of CCR2 decreases lesion formation markedly in apoE-/- mice but has no effect on plasma lipid or lipoprotein concentrations. These data reveal a role for MCP-1 in the development of early atherosclerotic lesions and suggest that upregulation of this chemokine by minimally oxidized lipids is an important link between hyperlipidaemia and fatty streak formation.

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis

Monocyte chemoattractant protein-1 (MCP-1) is a member of the β chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13–35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1–10), second loop (amino acids 37–51), and carboxy-terminus (amino acids 56–71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.

Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice.

Monocyte chemoattractant protein-1 (MCP-1) is a potent agonist for mononuclear leukocytes and has been implicated in the pathogenesis of atherosclerosis and granulomatous lung disease. To determine the role of MCP-1 and related family members in vivo, we used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of C-C chemokine receptor 2 (CCR2), the receptor for MCP-1. CCR2-/- mice were born at the expected Mendelian ratios and developed normally. In response to thioglycollate, the recruitment of peritoneal macrophages decreased selectively. In in vitro chemotaxis assays, CCR2-/- leukocytes failed to migrate in response to MCP-1. Granulomatous lung disease was induced in presensitized mice by embolization with beads coupled to purified protein derivative (PPD) of Mycobacterium bovis. As compared with wild-type littermates, CCR2-/- mice had a decrease in granuloma size accompanied by a dramatic decrease in the level of interferon gamma in the draining lymph nodes. Production of interferon gamma was also decreased in PPD-sensitized splenocytes from CCR2-/- mice and in naive splenocytes activated by concanavalin A. We conclude that CCR2-/- mice have significant defects in both delayed-type hypersensitivity responses and production of Th1-type cytokines. These data suggest an important and unexpected role for CCR2 activation in modulating the immune response, as well as in recruiting monocytes/macrophages to sites of inflammation.

Tissue Factor Is Induced by Monocyte Chemoattractant Protein-1 in Human Aortic Smooth Muscle and THP-1 Cells

Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine thought to play a major role in recruiting monocytes to the atherosclerotic plaque. Tissue factor (TF), the initiator of coagulation, is found in the atherosclerotic plaque, macrophages, and human aortic smooth muscle cells (SMC). The exposure of TF during plaque rupture likely induces acute thrombosis, leading to myocardial infarction and stroke. This report demonstrates that MCP-1 induces the accumulation of TF mRNA and protein in SMC and in THP-1 myelomonocytic leukemia cells. MCP-1 also induces TF activity on the surface of human SMC. The induction of TF by MCP-1 in SMC is inhibited by pertussis toxin, suggesting that the SMC MCP-1 receptor is coupled to a Gi-protein. Chelation of intracellular calcium and inhibition of protein kinase C block the induction of TF by MCP-1, suggesting that in SMC it is mediated by activation of phospholipase C. SMC bind MCP-1 with a Kd similar to that previously reported for macrophages. However, mRNA encoding the macrophage MCP-1 receptors, CCR2A and B, is not present in SMC, indicating that they possess a distinct MCP-1 receptor. These data suggest that in addition to being a chemoattractant, MCP-1 may have a procoagulant function and raise the possibility of an autocrine pathway in which MCP-1, secreted by SMC and macrophages, induces TF activity in these same cells.

The Amino-terminal Domain of CCR2 Is Both Necessary and Sufficient for High Affinity Binding of Monocyte Chemoattractant Protein 1: RECEPTOR ACTIVATION BY A PSEUDO-TETHERED LIGAND

High affinity binding of monocyte chemoattractant protein 1 (MCP-1) requires the presence of the amino-terminal domain of CCR2, the MCP-1 receptor. Here we report that the 35 amino-terminal residues of CCR2, expressed as a membrane-bound fusion protein, bound MCP-1 with an affinity similar to that of the intact, wild-type receptor. Furthermore, the amino-terminal fusion protein enhanced, in trans, agonist-dependent activation of a CCR2 variant that was engineered to lack the high affinity binding sites for MCP-1. Mutation of highly conserved cysteines in the amino-terminal domain and third extracellular loop of CCR2, but not in the fusion protein, resulted in a dramatic loss of MCP-1 binding, suggesting the existence of a critical intramolecular disulfide bond that positions the amino-terminal protein for ligand interaction. These data indicate that the amino-terminal region of CCR2 is both necessary and sufficient for the high affinity binding of MCP-1 and provide the first direct evidence for activation of a chemokine receptor by a pseudo-tethered ligand. In this model, high affinity binding by the relatively short amino-terminal domain of CCR2 serves to tether MCP-1 and enhance low affinity interactions with distal regions of the receptor.