Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.     

Essential involvement of CX3CR1-mediated signals in the bactericidal host defense during septic peritonitis

Cecal ligation and puncture (CLP) caused septic peritonitis in wild-type (WT) mice, with approximately 33% mortality within 7 days after the procedure. Concomitantly, the protein level of intraperitoneal CX3CL1/fractalkine was increased, with infiltration by CX3CR1-expressing macrophages into the peritoneum. CLP induced 75% mortality in CX3CR1-deficient (CX3CR1(-/-)) mice, which, however, exhibited a similar degree of intraperitoneal leukocyte infiltration as WT mice. Despite this, CX3CR1(-/-) mice exhibited impairment in intraperitoneal bacterial clearance, together with a reduction in the expression of intraperitoneal inducible NO synthase (iNOS) and bactericidal proinflammatory cytokines, including IL-1beta, TNF-alpha, IFN-gamma, and IL-12, compared with WT mice. Bactericidal ability of peritoneal phagocytes such as neutrophils and macrophages was consistently attenuated in CX3CR1(-/-) mice compared with WT mice. Moreover, when WT macrophages were stimulated in vitro with CX3CL1, their bactericidal activity was augmented in a dose-dependent manner, with enhanced iNOS gene expression and subsequent NO generation. Furthermore, CX3CL1 enhanced the gene expression of IL-1beta, TNF-alpha, IFN-gamma, and IL-12 by WT macrophages with NF-kappaB activation. Thus, CX3CL1-CX3CR1 interaction is crucial for optimal host defense against bacterial infection by activating bacterial killing functions of phagocytes, and by augmenting iNOS-mediated NO generation and bactericidal proinflammatory cytokine production mainly through the NF-kappaB signal pathway, with few effects on macrophage infiltration.     

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.     

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.     

The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

Air pollution is known to exacerbate chronic inflammatory conditions of the lungs including pulmonary hypertension, cardiovascular diseases and autoimmune diseases. Directly pathogenic antibodies bind pro-inflammatory cell receptors and cause or exacerbate inflammation. In contrast, anti-inflammatory antibody isotypes (e.g. mouse immunoglobulin G1, IgG1) bind inhibitory cell receptors and can inhibit inflammation. Our previous studies showed that co-exposure to antigen and urban ambient particulate matter (PM_2.5) induced severe pulmonary arterial thickening and increased right ventricular systolic pressures in mice via T-cell produced cytokines, Interleukin (IL)-13 and IL-17A. The aim of the current study was to understand how B cell and antibody responses integrate into this T cell cytokine network for the pulmonary hypertension phenotype. Special focus was on antigen-specific IgG1 that is the predominant antibody in the experimental response to antigen and urban ambient PM_2.5. Wild type and B cell-deficient mice were primed with antigen and then challenged with antigen and urban particulate matter and injected with antibodies as appropriate. Our data surprisingly showed that B cells were necessary for the development of increased right ventricular pressures and molecular changes in the right heart in response to sensitization and intranasal challenge with antigen and PM_2.5. Further, our studies showed that both, the increase in right ventricular systolic pressure and right ventricular molecular changes were restored by reconstituting the B cell KO mice with antigen specific IgG1. In addition, our studies identified a critical, non-redundant role of B cells for the IL-17A-directed inflammation in response to exposure with antigen and PM_2.5, which was not corrected with antigen-specific IgG1. In contrast, IL-13-directed inflammatory markers, as well as severe pulmonary arterial remodeling induced by challenge with antigen and PM_2.5 were similar in B cell-deficient and wild type mice. Our studies have identified B cells and antigen specific IgG1 as potential therapeutic targets for pulmonary hypertension associated with immune dysfunction and environmental exposures.     

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells

Chemokines play an important role in the recruitment of leukocytes and have recently been shown to also attract regulatory T cells. Using blocking mAbs, we analyzed the role of the chemokine receptor CCR2 during initiation and progression of collagen-induced arthritis in mice. Blockade of CCR2 from days 0 to 15 markedly improved clinical signs of arthritis and histological scores measuring leukocyte infiltration, synovial hyperplasia, and bone and cartilage erosion. CCR2 blockade during disease initiation significantly reduced plasma titers of collagen Abs in vivo. In vitro CCR2 blockade also interfered with collagen-specific activation and proliferation of T cells. Surprisingly, CCR2 blockade from days 21 to 36 markedly aggravated clinical and histological signs of arthritis and increased the humoral immune response against collagen. We show that CCR2 is expressed on regulatory T cells. Purified CCR2+ T cells are fully anergic toward polyclonal and collagen-specific activation and potently suppress activation of other T and B cells. The subpopulation of CCR2+ CD25+ regulatory T cells increases approximately 5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice

Background

Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.

Methodology/Principal Findings

Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr−/− and abr−/− macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.

Conclusions

Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.     

Hydrogen sulfide inhibits the development of atherosclerosis with suppressing CX3CR1 and CX3CL1 expression

Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H(2)S affects atherosclerosis remain unclear. Therefore, the present study aimed to investigate the potential role of H(2)S in atherosclerosis and the underlying mechanism with respect to chemokines (CCL2, CCL5 and CX3CL1) and chemokine receptors (CCR2, CCR5, and CX3CR1) in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS (50 μM, 100 μM, 200 μM), an H(2)S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H(2)S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H(2)S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE(-/-) mice were fed a high-fat diet and then randomly given NaHS (1 mg/kg, i.p., daily) or DL-propargylglycine (PAG, 10 mg/kg, i.p., daily). NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H(2)S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H(2)S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H(2)S hampers the progression of atherosclerosis in fat-fed apoE(-/-) mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.     

T cell antiviral effector function is not dependent on CXCL10 following murine coronavirus infection

The chemokine CXCL10 is expressed within the CNS in response to intracerebral infection with mouse hepatitis virus (MHV). Blocking CXCL10 signaling results in increased mortality accompanied by reduced T cell infiltration and increased viral titers within the brain suggesting that CXCL10 functions in host defense by attracting T cells into the CNS. The present study was undertaken to extend our understanding of the functional role of CXCL10 in response to MHV infection given that CXCL10 signaling has been implicated in coordinating both effector T cell generation and trafficking. We show that MHV infection of CXCL10(+/+) or CXCL10(-/-) mice results in comparable levels of T cell activation and similar numbers of virus-specific CD4+ and CD8+ T cells. Subsequent analysis revealed no differences in T cell proliferation, IFN-gamma secretion by virus-specific T cells, or CD8+ T cell cytolytic activity. Analysis of chemokine receptor expression on CD4+ and CD8+ T cells obtained from MHV-immunized CXCL10(+/+) and CXCL10(-/-) mice revealed comparable levels of CXCR3 and CCR5, which are capable of responding to ligands CXCL10 and CCL5, respectively. Adoptive transfer of splenocytes acquired from MHV-immunized CXCL10(-/-) mice into MHV-infected RAG1(-/-) mice resulted in T cell infiltration into the CNS, reduced viral burden, and demyelination comparable to RAG1(-/-) recipients of immune CXCL10(+/+) splenocytes. Collectively, these data imply that CXCL10 functions primarily as a T cell chemoattractant and does not significantly influence T cell effector response following MHV infection.     

Atherogenic lipids induce adhesion of human coronary artery smooth muscle cells to macrophages by up-regulating chemokine CX3CL1 on smooth muscle cells in a TNFalpha-NFkappaB-dependent manner

Recent genetic evidence has implicated the adhesive chemokine CX3CL1 and its leukocyte receptor CX3CR1 in atherosclerosis. We previously proposed a mechanism involving foam cell anchorage to vascular smooth muscle cells because: 1) CX3CL1 and CX3CR1 are expressed by both cell types in mouse and human atherosclerotic lesions; 2) foam cells are reduced in lesions in cx3cr1(-/-)apoE(-/-) mice; and 3) proatherogenic lipids (oxidized low density lipoprotein [oxLDL] and oxidized linoleic acid derivatives) induce adhesion of primary human macrophages to primary human coronary artery smooth muscle cells (CASMCs) in vitro in a macrophage CX3CR1-dependent manner. Here we analyze this concept further by testing whether atherogenic lipids regulate expression and function of CX3CL1 and CX3CR1 on CASMCs. We found that both oxLDL and oxidized linoleic acid derivatives indirectly up-regulated CASMC CX3CL1 at both the protein and mRNA levels through an autocrine feedback loop involving tumor necrosis factor alpha production and NF-kappaB signaling. Oxidized lipids also up-regulated CASMC CX3CR1 but through a different mechanism. Oxidized lipid stimulation also increased adhesion of macrophages to CASMCs when CASMCs were stimulated prior to assay, and a synergistic pro-adhesive effect was observed when both cell types were prestimulated. Selective inhibition with a CX3CL1-specific blocking antibody indicated that adhesion was strongly CASMC CX3CL1-dependent. These findings support the hypothesis that CX3CR1 and CX3CL1 mediate heterotypic anchorage of foam cells to CASMCs in the context of atherosclerosis and suggest that this chemokine/chemokine receptor pair may be considered as a pro-inflammatory target for therapeutic intervention in atherosclerotic cardiovascular disease.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

Chemokines regulate small leucine-rich proteoglycans in the extracellular matrix of the pressure-overloaded right ventricle

Chemokines have been suggested to play a role during development of left ventricular failure, but little is known about their role during right ventricular (RV) remodeling and dysfunction. We have previously shown that the chemokine (C-X-C motif) ligand 13 (CXCL13) regulates small leucine-rich proteoglycans (SLRPs). We hypothesized that chemokines are upregulated in the pressure-overloaded RV, and that they regulate SLRPs. Mice with RV pressure overload following pulmonary banding (PB) had a significant increase in RV weight and an increase in liver weight after 1 wk. Microarray analysis (Affymetrix) of RV tissue from mice with PB revealed that CXCL10, CXCL6, chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-C motif) ligand 5 (CCL5), CXCL16, and CCL2 were the most upregulated chemokines. Stimulation of cardiac fibroblasts with these same chemokines showed that CXCL16 increased the expression of the four SLRPs: decorin, lumican, biglycan, and fibromodulin. CCL5 increased the same SLRPs, except decorin, whereas CX3CL1 increased the expression of decorin and lumican. CXCL16, CX3CL1, and CCL5 were also shown to increase the levels of glycosylated decorin and lumican in the medium after stimulation of fibroblasts. In the pressure-overloaded RV tissue, Western blotting revealed an increase in the total protein level of lumican and a glycosylated form of decorin with a higher molecular weight compared with control mice. Both mice with PB and patients with pulmonary stenosis had significantly increased circulating levels of CXCL16 compared with healthy controls measured by enzyme immunoassay. In conclusion, we have found that chemokines are upregulated in the pressure-overloaded RV and that CXCL16, CX3CL1, and CCL5 regulate expression and posttranslational modifications of SLRPs in cardiac fibroblasts. In the pressure-overloaded RV, protein levels of lumican were increased, and a glycosylated form of decorin with a high molecular weight appeared.     

Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation

Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.     

Effective Elicitation of Human Effector CD8(+) T Cells in HLA-B*51:01 Transgenic Humanized Mice after Infection with HIV-1

Humanized mice are expected to be useful as small animal models for in vivo studies on the pathogenesis of infectious diseases. However, it is well known that human CD8(+) T cells cannot differentiate into effector cells in immunodeficient mice transplanted with only human CD34(+) hematopoietic stem cells (HSCs), because human T cells are not educated by HLA in the mouse thymus. We here established HLA-B*51:01 transgenic humanized mice by transplanting human CD34(+) HSCs into HLA-B*51:01 transgenic NOD/SCID/Jak3(-/-) mice (hNOK/B51Tg mice) and investigated whether human effector CD8(+) T cells would be elicited in the mice or in those infected with HIV-1 NL4-3. There were no differences in the frequency of late effector memory and effector subsets (CD27(low)CD28(-)CD45RA(+/-)CCR7(-) and CD27(-)CD28(-)CD45RA(+/-)CCR7(-), respectively) among human CD8(+) T cells and in that of human CD8(+) T cells expressing CX3CR1 and/or CXCR1 between hNOK/B51Tg and hNOK mice. In contrast, the frequency of late effector memory and effector CD8(+) T cell subsets and of those expressing CX3CR1 and/or CXCR1 was significantly higher in HIV-1-infected hNOK/B51Tg mice than in uninfected ones, whereas there was no difference in that of these subsets between HIV-1-infected and uninfected hNOK mice. These results suggest that hNOK/B51Tg mice had CD8(+) T cells that were capable of differentiating into effector T cells after viral antigen stimulation and had a greater ability to elicit effector CD8(+) T cells than hNOK ones.     

A non-glycosaminoglycan-binding variant of CC chemokine ligand 7 (monocyte chemoattractant protein-3) antagonizes chemokine-mediated inflammation

A non-glycosaminoglycan (GAG)-binding variant of the pleiotropic chemokine CCL7 was generated by mutating to alanine the basic (B) amino acids within an identified (44)BXBXXB(49) GAG-binding motif. Unlike wild-type (wt) CCL7, the mutant sequence had no affinity for heparin. However, the mutant retained a normal affinity for CCR1, CCR2b, and CCR3, and produced a normal calcium flux in mononuclear leukocytes. Both the wt and mutant proteins elicited an equal leukocyte chemotactic response within a solute diffusion gradient but, unlike the wt protein, the mutant failed to stimulate cell migration across a model endothelium. The number of leukocytes recruited to murine air pouches by the mutant sequence was lower than that recruited by wt CCL7. Furthermore, the presence of a mixture of a mutant and wt CCL7 within the air pouch elicited no significant cell accumulation. Cell recruitment also failed using a receptor-sharing mixture of mutant CCL7 and wt CCL5 or a nonreceptor sharing mixture of mutant CCL7 and wt CXCL12. The potential of the mutant sequence to modulate inflammation was confirmed by demonstration of its ability to inhibit the chemotactic response generated in vitro by synovial fluid from patients with active rheumatoid arthritis. A further series of experiments suggested that the non-GAG-binding mutant protein could potentially induce receptor desensitization before, and at a site remote from, any physiological recognition of GAG-bound chemokines. These data demonstrate that GAG binding is required for chemokine-driven inflammation in vivo and also suggest that a non-GAG-binding chemokine receptor agonist can inhibit the normal vectorial leukocyte migration mediated by chemokines.