Israel F. Charo

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.

CCR2 modulates inflammatory and metabolic effects of high-fat feeding

The C-C motif chemokine receptor-2 (CCR2) regulates monocyte and macrophage recruitment and is necessary for macrophage-dependent inflammatory responses and the development of atherosclerosis. Although adipose tissue expression and circulating concentrations of CCL2 (also known as MCP1), a high-affinity ligand for CCR2, are elevated in obesity, the role of CCR2 in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity, has not been studied. To determine what role CCR2 plays in the development of metabolic phenotypes, we studied the effects of Ccr2 genotype on the development of obesity and its associated phenotypes. Genetic deficiency in Ccr2 reduced food intake and attenuated the development of obesity in mice fed a high-fat diet. In obese mice matched for adiposity, Ccr2 deficiency reduced macrophage content and the inflammatory profile of adipose tissue, increased adiponectin expression, ameliorated hepatic steatosis, and improved systemic glucose homeostasis and insulin sensitivity. In mice with established obesity, short-term treatment with a pharmacological antagonist of CCR2 lowered macrophage content of adipose tissue and improved insulin sensitivity without significantly altering body mass or improving hepatic steatosis. These data suggest that CCR2 influences the development of obesity and associated adipose tissue inflammation and systemic insulin resistance and plays a role in the maintenance of adipose tissue macrophages and insulin resistance once obesity and its metabolic consequences are established.

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.

Langerhans cells renew in the skin throughout life under steady-state conditions

Langerhans cells (LCs) are bone marrow (BM)–derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.

Chemokines in the Pathogenesis of Vascular Disease

Our increasing appreciation of the importance of inflammation in vascular disease has focused attention on the molecules that direct the migration of leukocytes from the blood stream to the vessel wall. In this review, we summarize roles of the chemokines, a family of small secreted proteins that selectively recruit monocytes, neutrophils, and lymphocytes to sites of vascular injury, inflammation, and developing atherosclerosis. Chemokines induce chemotaxis through the activation of G-protein-coupled receptors, and the receptors that a given leukocyte expresses determines the chemokines to which it will respond. Monocyte chemoattractant protein 1 (MCP-1), acting through its receptor CCR2, appears to play an early and important role in the recruitment of monocytes to atherosclerotic lesions and in the formation of intimal hyperplasia after arterial injury. Acute thrombosis is an often fatal complication of atherosclerotic plaque rupture, and recent evidence suggests that MCP-1 contributes to thrombin generation and thrombus formation by generating tissue factor. Because of their critical roles in monocyte recruitment in vascular and nonvascular diseases, MCP-1 and CCR2 have become important therapeutic targets, and efforts are underway to develop potent and specific antagonists of these and related chemokines.

Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites

Monocyte recruitment to sites of inflammation is regulated by members of the chemokine family of chemotactic cytokines. However, the mechanisms that govern the migration of monocytes from bone marrow to blood and from blood to inflamed tissues are not well understood. Here we report that CC chemokine receptor 2 (CCR2) is highly expressed on a subpopulation of blood monocytes whose numbers are markedly decreased in CCR2(-/-) mice. In bone marrow, however, CCR2(-/-) mice had an increased number of monocytes, suggesting that CCR2 is critical for monocyte egress. Intravenous infusion of ex vivo-labeled WT or CCR2(-/-) bone marrow into WT recipient mice demonstrated that CCR2 is necessary for efficient monocyte recruitment from the blood to inflamed tissue. Analysis of mice lacking monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-5, or MCP-2 plus MCP-5 revealed that MCP-3 and MCP-1 are the CCR2 agonists most critical for the maintenance of normal blood monocyte counts. These findings provide evidence that CCR2 and MCP-3/MCP-1 are critical for monocyte mobilization and suggest new roles for monocyte chemoattractants in leukocyte homeostasis.

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.

Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha

Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1β, and MIP-1α with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P. (1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1β.

Differential roles of microglia and monocytes in the inflamed central nervous system

Phagocytic monocyte-derived macrophages associate with the nodes of Ranvier and initiate demyelination while microglia clear debris and display a suppressed metabolic gene signature in EAE.

Multiple Extracellular Elements of CCR5 and HIV-1 Entry: Dissociation from Response to Chemokines

The human β-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.