Resistance to Experimental Autoimmune Encephalomyelitis in Mice Lacking the Cc Chemokine Receptor (Ccr2)

Monocyte recruitment to the central nervous system (CNS) is a necessary step in the development of pathologic inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Monocyte chemoattractant protein (MCP)-1, a potent agonist for directed monocyte migration, has been implicated in the pathogenesis of EAE. Here we report that deficiency in CC chemokine receptor (CCR)2, the receptor for MCP-1, confers resistance to EAE induced with a peptide derived from myelin oligodendrocyte glycoprotein peptide 35–55 (MOGp35–55). CCR2−/− mice immunized with MOGp35–55 failed to develop mononuclear cell inflammatory infiltrates in the CNS and failed to increase CNS levels of the chemokines RANTES (regulated on activation, normal T cell expressed and secreted), MCP-1, and interferon (IFN)-inducible protein 10 (IP-10) as well the chemokine receptors CCR1, CCR2, and CCR5. Additionally, T cells from CCR2−/− immunized mice showed decreased antigen-induced proliferation and production of IFN-γ compared with wild-type immunized controls, suggesting that CCR2 enhances the T helper cell type 1 immune response in EAE. These data indicate that CCR2 plays a necessary and nonredundant role in the pathogenesis of EAE

Interrelations of platelet aggregation and secretion.

A B S T R A C T The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of y-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of y-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are -consistent with a simple model of platelet stimulusresponse coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described

Selective Chemokine Receptor Usage by Central Nervous System Myeloid Cells in CCR2-Red Fluorescent Protein Knock-In Mice

Background: Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents. Methodology/Principal Findings: We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C hi /CCR2 hi monocytes. Surprisingly, neutrophils, not Ly6C lo monocytes, largely replaced Ly6C hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia. Conclusion/Significance: These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations

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)-1α. 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-1α 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-1α for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1α). Similarly, HCC-1 was less potent than MIP-1α 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 ∼100-fold lower than that of MIP-1α (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Leishmania major Infection

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7−/− C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7−/− mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control

Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE−/− mice

AngII (angiotensin II) induces atherosclerosis and AAAs (abdominal aortic aneurysms) through multiple proposed mechanisms, including chemotaxis. Therefore, we determined the effects of whole-body deficiency of the chemokine receptor CCR2 (CC chemokine receptor 2) on these diseases. To meet this objective, apoE (apolipoprotein E)−/− mice that were either CCR2+/+ or CCR2−/−, were infused with either saline or AngII (1000 ng·kg−1 of body weight·min−1) for 28 days via mini-osmotic pumps. Deficiency of CCR2 markedly attenuated both atherosclerosis and AAAs, unrelated to systolic blood pressure or plasma cholesterol concentrations. During the course of the present study, we also observed that AngII infusion led to large dilatations that were restricted to the ascending aortic region of apoE−/− mice. The aortic media in most of the dilated area was thickened. In regions of medial thickening, distinct elastin layers were discernable. There was an expansion of the distance between elastin layers in a gradient from the intimal to the adventitial aspect of the media. This pathology differed in a circumscribed area of the anterior region of ascending aortas in which elastin breaks were focal and almost transmural. All regions of the ascending aorta of AngII-infused mice had diffuse medial macrophage accumulation. Deficiency of CCR2 greatly attenuated the AngII-induced lumen dilatation in the ascending aorta. This new model of ascending aortic aneurysms has pathology that differs markedly from AngII-induced atherosclerosis or AAAs, but all vascular pathologies were attenuated by CCR2 deficiency

Role of CCR8 and Other Chemokine Pathways in the Migration of Monocyte-derived Dendritic Cells to Lymph Nodes

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs

Progress Toward a Human CD4/CCR5 Transgenic Rat Model for De Novo Infection by Human Immunodeficiency Virus Type 1

The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection