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Biomedical subjects

I F Charo

Publications and source records attributed to I F Charo.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. Rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation.

Fractalkine is a novel multidomain protein expressed on the surface of activated endothelial cells. Cells expressing the chemokine receptor CX3CR1 adhere to fractalkine with high affinity, but it is not known if adherence requires G-protein activation and signal transduction. To investigate the cell adhesion properties of fractalkine, we created mutated forms of CX3CR1 that have little or no ability to transduce intracellular signals. Cells expressing signaling-incompetent forms of CX3CR1 bound rapidly and with high affinity to immobilized fractalkine in both static and flow assays. Video microscopy revealed that CX3CR1-expressing cells bound more rapidly to fractalkine than to VCAM-1 (60 versus 190 ms). Unlike VCAM-1, fractalkine did not mediate cell rolling, and after capture on fractalkine, cells did not dislodge. Finally, soluble fractalkine induced intracellular calcium fluxes and chemotaxis, but it did not activate integrins. Taken together these data provide strong evidence that CX3CR1, a seven-transmembrane domain receptor, mediates robust cell adhesion to fractalkine in the absence of G-protein activation and suggest a novel role for this receptor as an adhesion molecule.

Animals↗
PubMed1999

Enhanced myeloid progenitor cell cycling and apoptosis in mice lacking the chemokine receptor, CCR2.

Chemokines regulate hematopoiesis in part by influencing the proliferative status of myeloid progenitor cells (MPC). Human MCP-1/murine JE, a myelosuppressive chemokine, specifically binds C-C chemokine receptor 2 (CCR2). Transgenic mice containing a targeted disruption in CCR2 that prevents expression of CCR2 mRNA and protein and have MPC that are insensitive to inhibition by MCP-1 and JE in vitro were assessed for potential abnormalities in growth of bone marrow (BM) and spleen MPC. MPC in both unseparated and c-kit+lin- populations of BM from CCR2-deficient (-/-) mice were in a greatly increased proliferation state compared with CCR2 littermate control (+/+) mice, an effect not apparent with progenitors from spleens of CCR2 (-/-) mice. Increased cycling status of CCR2 (-/-) BM MPC did not result in increased numbers of nucleated cells or MPC in BM or spleens of CCR2 (-/-) mice. Possible reasons for this apparent discrepancy were highlighted by flow cytometric analysis of c-kit+lin- BM cells and colony formation by MPC subjected to delayed addition of growth factors. The c-kit+lin- population of BM cells from CCR2 (-/-) mice had a significantly higher percentage of apoptotic cells than those from CCR2 (+/+) BM. However, elevated apoptosis was not associated with decreased numbers of c-kit+lin- cells. The increased percentage of apoptotic c-kit+lin- cells was due to elevated apoptosis within the c-kitdimlin-, but not the c-kitbrightlin-, subpopulations of cells. Consistent with enhanced apoptosis of phenotypically defined cells, MPC from CCR2 (-/-) BM and purified c-kit+lin- cells demonstrated decreased cell survival in vitro upon delayed addition of growth factors. The data suggest that signals received by CCR2 limit proliferation of progenitor cells in the BM, but also enhance survival of these cells.

Animals↗
PubMed1999

MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B.

The earliest recognizable atherosclerotic lesions are fatty streaks composed of lipid-laden macrophages (foam cells). Circulating monocytes are the precursors of these foam cells, but the molecular mechanisms that govern macrophage trafficking through the vessel wall are poorly understood. Monocyte chemoattractant protein-1 (MCP-1), a member of the chemokine (chemotactic cytokine) family, is a potent monocyte agonist that is upregulated by oxidized lipids. Recent studies in hypercholesterolemic mice lacking apo E or the low-density lipoprotein receptor have suggested a role for MCP-1 in monocyte recruitment to early atherosclerotic lesions. To determine if MCP-1 is critically involved in atherogenesis in the setting of elevated physiological plasma cholesterol levels, we deleted the MCP-1 gene in transgenic mice expressing human apo B. Here we report that the absence of MCP-1 provides dramatic protection from macrophage recruitment and atherosclerotic lesion formation in apo B transgenic mice, without altering lipoprotein metabolism. Taken together with the results of earlier studies, these data provide compelling evidence that MCP-1 plays a critical role in the initiation of atherosclerosis.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Identification of C-C chemokine receptor 1 (CCR1) as the monocyte hemofiltrate C-C chemokine (HCC)-1 receptor.

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1alpha. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1alpha activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1alpha for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1alpha). Similarly, HCC-1 was less potent than MIP-1alpha in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was approximately 100-fold lower than that of MIP-1alpha (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.

Cell Line↗
PubMed1997

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by betagamma dimers released by activation of Galphai-coupled receptors.

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Galphaq (the m3 muscarinic receptor), Galphai (the kappa-opioid receptor), and Galphas (the beta-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Galphai, but not to Galphaq or Galphas, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Galpha transducin, which binds to and inactivates free Gbetagamma dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gbetagamma dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Galphai and the release of Gbetagamma dimers, and that Galphai-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.

B-Lymphocytes↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Dissociation of chemotaxis from agonist-induced receptor internalization in a lymphocyte cell line transfected with CCR2B. Evidence that directed migration does not require rapid modulation of signaling at the receptor level.

To investigate the role of the carboxyl-terminal region (52 amino acids) of the monocyte chemoattractant protein 1 receptor (CCR2B) in chemotaxis, we created a series of mutants and expressed them in a murine pre-B lymphocyte cell line. Truncation of the cytoplasmic carboxyl tail to 20 amino acids had little or no effect on chemotaxis or signal transduction, but further truncation resulted in marked functional defects. Upon incubation with monocyte chemoattractant protein 1, CCR2B underwent rapid and extensive internalization, and this was impaired progressively as the carboxyl tail was truncated from 52 to 8 amino acids. Mutation of all of the serine and threonine residues in the carboxyl tail to alanine also resulted in markedly impaired receptor internalization but did not affect signaling or chemotaxis. We conclude that the membrane-proximal portion of the cytoplasmic carboxyl tail of CCR2B is critically involved in chemotaxis and signal transduction, but neither phosphorylation of carboxyl serines or threonines nor internalization of the receptor is required for robust chemotaxis.

Adenylyl Cyclases↗
PubMed1997

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.

Adenylyl Cyclase Inhibitors↗
PubMed1997

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity.

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

Amino Acid Sequence↗
PubMed1997

Organization and differential expression of the human monocyte chemoattractant protein 1 receptor gene. Evidence for the role of the carboxyl-terminal tail in receptor trafficking.

Two forms of the monocyte chemoattractant protein-1 receptors (the type A monocyte chemoattractant protein 1 (MCP-1) receptor CCR-2A and the type B MCP-1 receptor (CCR-2B) have been recently cloned and found to differ only in their terminal carboxyl tails. Here, we report that the two isoforms are alternatively spliced variants of a single MCP-1 receptor gene. Sequencing of the gene revealed that the 47-amino acid carboxyl tail of CCR2B was located in the same exon as the seven transmembrane domains of the receptor, and the 61-amino acid tail of CCR2A was in a downstream exon. Examination of freshly isolated human monocytes by reverse transcriptase-polymerase chain reaction revealed that CCR2B was the predominant isoform and that message levels of both CCR2A and CCR2B decreased as the monocytes differentiated into macrophages. In stably transfected cell lines, CCR2B trafficked well to the cell surface, but CCR2A was found predominantly in the cytoplasm. Equilibrium binding studies revealed that those CCR2A receptors that successfully trafficked to the cell surface bound MCP-1 with high affinity (Kd = 310 pM), similar to CCR2B. In signaling studies, both CCR2A and CCR2B mediated agonist-dependent calcium mobilization, as well as inhibition of adenylyl cyclase. Creation of chimeras between CCR2A and the human thrombin receptor revealed that the cytoplasmic retention of CCR2A was due to its terminal carboxyl tail. Progressive truncation of the carboxyl tail indicated that a cytoplasmic retention signal(s) was located between residues 316 and 349. These data indicate that the alternatively spliced form of the human MCP-1 receptor (CCR2A) binds MCP-1 with high affinity and is a functional receptor and that expression at the cell surface is controlled by amino acid sequences located in the terminal carboxyl tail.

Amino Acid Sequence↗
PubMed1997

Murine monocyte chemoattractant protein (MCP)-5: a novel CC chemokine that is a structural and functional homologue of human MCP-1.

The chemokines are a large family of cytokines that control the recruitment of leukocytes in immune and inflammatory responses. We describe the isolation of a novel murine CC chemokine that, based on its biological and structural features, we have named monocyte chemoattractant protein (MCP)-5. MCP-5 mapped to the CC chemokine cluster on mouse chromosome 11 and was most closely related to human MCP-1 in structure (66% amino acid identity). Purified recombinant MCP-5 protein was a potent chemoattractant for peripheral blood monocytes, was only weakly active on eosinophils at high doses, and was inactive on neutrophils. MCP-5 induced a calcium flux in peripheral blood mononuclear cells, but not in purified murine eosinophils or neutrophils. Consistent with these results, MCP-5 induced a calcium flux in human embryonic kidney (HEK)-293 cells transfected with human and murine CCR2, a CC chemokine receptor expressed on monocytes. MCP-5 did not induce a calcium flux in HEK-293 cells transfected with CCR1, CCR3, or CCR5. Constitutive expression of MCP-5 mRNA was detected predominantly in lymph nodes, and its expression was markedly induced in macrophages activated in vitro and in vivo. Moreover, MCP-5 expression was up-regulated in the lungs of mice following aerosolized antigen challenge of sensitized mice, and during the host response to infection with Nippostrongylus brasiliensis. These data indicate that MCP-5 is a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens.

Amino Acid Sequence↗
PubMed1997

Molecular approaches to identifying ligand binding and signaling domains of C-C chemokine receptors.

The construction of chimeric receptors provides a useful starting point for the identification of ligand-binding and G-protein-coupling sites on chemokine receptors. Correlation of the binding and signaling properties of a set of complementary receptor chimeras is a powerful approach for probing structure-function relationships. Further molecular resolution can subsequently be achieved by site-directed mutagenesis and/or alanine scanning.

Amino Acid Sequence↗
PubMed1997

Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop.

The chemokine receptor CCR5 acts as an essential cofactor for cell entry by macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains, whereas CXCR4 acts as an essential cofactor for T-cell-line-adapted strains. We demonstrated that the specific amino acids in the V3 loop of the HIV-1 envelope protein that determine cellular tropism also regulate chemokine coreceptor preference for cell entry by the virus. Further, a strong correlation was found between HIV-1 strains classified as syncytium inducing in standard assays and those using CXCR4 as a coreceptor. These data support the hypothesis that progressive adaptation to additional coreceptors is a key molecular basis for HIV-1 phenotypic evolution in vivo.

Amino Acid Sequence↗
PubMed1996

Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor. Critical role of carboxyl-tail serines/threonines in receptor function.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of chemotactic cytokines and signals via activation of a G protein-coupled seven-transmembrane domain receptor to mediate chemotaxis. Monocyte activation is limited by desensitization and internalization of the MCP-1R, but these mechanisms are not well understood. In this study, we show that the type B MCP-1R (MCP-1RB/CCR2B) is rapidly phosphorylated and internalized in response to nanomolar concentrations of MCP-1. Co-expression of CCR2B in Xenopus oocytes with beta-adrenergic receptor kinase 2 (beta ark2), but not beta ark1 or rhodopsin kinase, specifically blocked receptor activation by MCP-1. Mutation of serine (Ser) and threonine (Thr) residues in the terminal carboxyl-tail of the receptor, which are potential targets of beta ark-mediated phosphorylation, prevented inhibition of receptor activation by beta ark2 in microinjected oocytes. Finally, a construct in which multiple Ser and Thr residues in the carboxyl-tail were changed to alanine significantly prolonged the agonist-dependent intracellular calcium flux and inhibited receptor internalization in transfected human embryonic kidney (HEK)-293 cells. These studies demonstrate that phosphorylation of Ser and Thr residues in the carboxyl-tail of CCR2B mediates receptor desensitization and internalization and may serve to limit the chemotactic response of leukocytes to MCP-1 and related chemokines.

Amino Acid Sequence↗
PubMed1996

Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines.

The human beta-chemokine receptor CCR5 is an important cofactor for entry of human immunodeficiency virus-type 1 (HIV-1). The murine form of CCR5, despite its 82 percent identity to the human form, was not functional as an HIV-1 coreceptor. HIV-1 entry function could be reconstituted by fusion of various individual elements derived from the extracellular region of human CCR5 onto murine CCR5. Analysis of chimeras containing elements from human CCR5 and human CCR2B suggested that a complex structure rather than single contact sites is responsible for facilitation of viral entry. Further, certain chimeras lacking the domains necessary to signal in response to their natural chemokine ligands retained vigorous HIV-1 coreceptor activity.

Animals↗