Cloning of rat TARC cDNA and analysis of tissue-specific mRNA expression

Thymus and activation-regulated chemokine (TARC) is one that selectively controls the migration of type 2-helper T lymphocytes into inflammatory lesions. TARC is a CC chemokine, and plays an essential role in recruiting CC chemokine receptor 4-positive Th2 cells to allergic lesions. We cloned TARC cDNA from rat thymus using RT-PCR. The rat TARC clone contained a full-length open reading frame encoding 93 amino acids that showed 83% and 66% homology with mouse and human homologs, respectively. The expression of TARC mRNA was mainly in the lymphoid organs, for example, the thymus, spleen, and lymph node. The recombinant TARC was expressed in Escherichia coli and purified in an active form. In addition, the purified rat TARC with S-tagged specifically binds to human CCR4 in CD4.CCR4-transfected HOS cells by Cell-binding assay using flow-cytometry. The TARC cDNA clones obtained in this study will be valuable for future studies on allergic diseases in rats.     

Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.     

Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.     

C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4(+) thymocytes and on a major fraction of blood nonintestinal (alpha(4)beta(7)(-)) memory CD4 lymphocytes, including almost all skin memory CD4(+) cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4(+) memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4(+) CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4(+) memory CD4 lymphocytes (including both CLA(+) and CLA(-) fractions) readily express the Th1 cytokine IFN-gamma after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.     

Interleukin-4 induction of the CC chemokine TARC (CCL17) in murine macrophages is mediated by multiple STAT6 sites in the TARC gene promoter

Background  

Macrophages (Mθ) play a central role in the innate immune response and in the pathology of chronic inflammatory diseases. Macrophages treated with Th2-type cytokines such as Interleukin-4 (IL-4) and Interleukin-13 (IL-13) exhibit an altered phenotype and such alternatively activated macrophages are important in the pathology of diseases characterised by allergic inflammation including asthma and atopic dermatitis. The CC chemokine Thymus and Activation-Regulated Chemokine (TARC/CCL17) and its murine homologue (mTARC/ABCD-2) bind to the chemokine receptor CCR4, and direct T-cell and macrophage recruitment into areas of allergic inflammation. Delineating the molecular mechanisms responsible for the IL-4 induction of TARC expression will be important for a better understanding of the role of Th2 cytokines in allergic disease.     

Thymus and activation-regulated chemokine (TARC/CCL17) produced by mouse epidermal Langerhans cells is upregulated by TNF-alpha and IL-4 and downregulated by IFN-gamma

Thymus and activation-regulated chemokine (TARC/CCL17) is a Th2-type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Langerhans cells (LC) are immature dendritic cells (DC) in the epidermis of the skin and play vital roles in immune response. In this study, we investigated TARC expression by murine freshly isolated LC and 48 h cultured (mature) LC, and the regulation of TARC production in cultured LC by various cytokines. Murine LC was prepared using a panning method from BALB/c mice. RT-PCR was performed using fresh and cultured LC to evaluate TARC mRNA levels. ELISA was carried out using supernatant of cultured LC to calculate secreted TARC protein levels. TARC mRNA was strongly upregulated during maturation of murine LC. TARC production by murine LC was upregulated by TNF-alpha and IL-4 and downregulated by IFN-gamma, dose-dependently. Th1 and Th2 cytokines reciprocally regulate the production of Th2-type chemokine TARC by murine LC. Th2 cytokine microenvironments in skin may increase TARC production by mature LC, providing attraction of Th2 cells in skin. This may be an amplification circuit in Th2-dominant inflammatory skin disease like atopic dermatitis.     

Inducible expression of a Th2-type CC chemokine thymus- and activation-regulated chemokine by human bronchial epithelial cells

CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-alpha and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-gamma. The enhancement by IFN-gamma was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-alpha, IL-4, and IFN-gamma. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.     

Two C-terminal peptides of human CKLF1 interact with the chemokine receptor CCR4

Human chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. A previous study demonstrated that CKLF1 is a functional ligand for human CC chemokine receptor 4 (CCR4). In this study, N-terminal amino acid sequencing of secreted CKLF1 protein showed that it contains at least two peptides, C27 and C19. To examine whether C27 or C19 play a role via CCR4, C27 and C19 were chemically synthesized and analyzed by chemotaxis, calcium mobilization, and receptor internalization assays in CCR4-tranfected HEK293 cells or Hut78 cells. The chemotaxis assay showed that C27 could induce chemotaxis to CCR4-transfected HEK293 cells or Hut78 cells while C19 had weaker chemotactic activity, especially in Hut78 cells. C27- or C19-induced chemotaxis was abolished by pertussis toxin, suggesting the involvement of a Gi/o pathway. C27- or C19-induced chemotaxis was also inhibited by an antagonist of CCR4 that show good binding potency, excellent chemotaxis inhibitory activity and selectivity toward CCR4, suggesting that their chemotactic activity specifically involved CCR4. The chemotactic response of CCR4-tranfected HEK293 cells to C27 or C19 was markedly inhibited by preincubation with TARC/CCL17. TARC/CCL17 effectively desensitized the calcium mobilization induced by C27 or C19. Similarly, both of C27 or C19 also desensitized the calcium mobilization and chemotaxis of CCR4-tranfected HEK293 cells in response to TARC/CCL17, suggesting that they might interact with a common receptor. Both C27- and C19-induced clear internalization of CCR4-EGFP. These results confirm that the secreted peptides of CKLF1, C27 and C19, have functional activation via CCR4.     

Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity

Thymic stromal lymphopoietin (TSLP) is said to increase expression of chemokines attracting Th2 T cells. We hypothesized that asthma is characterized by elevated bronchial mucosal expression of TSLP and Th2-attracting, but not Th1-attracting, chemokines as compared with controls, with selective accumulation of cells bearing receptors for these chemokines. We used in situ hybridization and immunohistochemistry to examine the expression and cellular provenance of TSLP, Th2-attracting (thymus and activation-regulated chemokine (TARC)/CCL17, macrophage-derived chemokine (MDC)/CCL22, I-309/CCL1) and Th1-attracting (IFN-gamma-inducible protein 10 (IP-10)/CXCL10, IFN-inducible T cell alpha-chemoattractant (I-TAC)/CXCL11) chemokines and expression of their receptors CCR4, CCR8, and CXCR3 in bronchial biopsies from 20 asthmatics and 15 normal controls. The numbers of cells within the bronchial epithelium and submucosa expressing mRNA for TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in asthmatics as compared with controls (p 相似文献   

Differential regulation of CC chemokine receptors by 9-cis retinoic acid in the human mast cell line, HMC-1

Mast cells are well known as effector cells in a variety of inflammatory diseases, including asthma as well as other allergic disorders. The precise role of 9-cis retinoic acid (9CRA) in mast cells is not understood despite the accepted fact that 9CRA regulates inflammatory responses and neutrophil differentiation. In this study, we investigated the effects of 9CRA on the expression of CC chemokine receptors in the human mast cell line, HMC-1. 9CRA selectively inhibits the CCR2 mRNA level and increases the CCR3 mRNA level in both a time and dose dependent manner. Other CC chemokine receptors, including CCR1, CCR4 and CCR5 are not altered by treatment with 9CRA. Both TNF-alpha and LPS, known pro-inflammatory molecules, have no effect on mRNA levels of CC chemokine receptors. For surface expression, 9CRA decreased the CCR2 level but had no effect on the CCR3 level. 9CRA inhibited the chemotactic activity in response to the CCR2-dependent chemokine, MCP-1/CCL2 but not in response to CCR3-specific chemokine, eotaxin/CCL11. 9CRA decreased spontaneous homotype clustering. Therefore, our results demonstrate that 9CRA differentially decreases both CCR2 expression and chemotactic ability of HMC-1 cells, and may regulate the inflammatory effects of mast cells.     

The identification, characterization, and distribution of guinea pig CCR4 and epitope mapping of a blocking antibody.

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.     

Variations in the human Th2-specific chemokine TARC gene

Th2-specific chemokine thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL)17 is highly implicated in the pathogenesis of Th-2-dominated allergic diseases such as bronchial asthma (BA) and atopic dermatitis (AD). We performed polymorphism screening of the coding and promoter regions of the TARC gene. We found two rare variations in the coding region of exon 3 (2134C>T and 2037G>A) and a single nucleotide polymorphism (SNP) in the 5'-flanking region (-431C>T). Individuals carrying the 431T allele showed significantly increased serum levels of TARC compared with those not carrying the 431T allele, suggesting that this SNP has functional significance. However, when the genotypes at the SNP site were determined for 158 healthy individuals, 105 patients with BA and 148 patients with AD, we observed no significant association of the SNP with susceptibility to BA or AD.     

IL-4, but not IL-13, modulates TARC (thymus and activation-regulated chemokine)/CCL17 and IP-10 (interferon-induced protein of 10kDA)/CXCL10 release by TNF-alpha and IFN-gamma in HaCaT cell line

It is known that both interleukin-4 (IL-4) and IL-13 are produced by Th2-type cells and share similar biological functions with each other. However, recently accumulated evidences have revealed that IL-4 may be involved in the Th1-type response. Both thymus and activation-regulated chemokine (TARC/CCL17), a ligand for CC chemokine receptor 4 that is mainly expressed on Th2-type cells, and interferon-induced protein of 10kDa (IP-10/CXCL10), a ligand for CXC chemokine receptor 3 that is mainly expressed on Th1-type cells, are produced by keratinocytes after the stimulation with the primary cytokines such as tumor necrotic factor-alpha (TNF-alpha) and/or interferon-gamma (IFN-gamma). In this study, we investigated the regulation of TARC or IP-10 production from HaCaT cells, an immortalized human keratinocyte cell line, after stimulation with TNF-alpha, IFN-gamma, IL-4 and/or IL-13. Without stimulation, HaCaT cells did not produce TARC. When both TNF-alpha and IFN-gamma were added, they increased synergistically (P<0.003). In addition, when HaCaT cells were stimulated with IL-4, but not IL-13, in combination with TNF-alpha and IFN-gamma, the supernatant TARC levels significantly decreased compared to those with both TNF-alpha and IFN-gamma (P<0.009). This inhibition was completely abolished with the addition of neutralizing anti-IL-4 antibody. The supernatant IP-10 levels also increased synergistically by stimulation with TNF-alpha and IFN-gamma for 24h (P<0.001). When IL-4, but not IL-13, was added to the medium and the cells were co-cultured with these cytokines, the IP-10 levels significantly increased compared to those with both TNF-alpha and IFN-gamma (P<0.04). Furthermore, the effects of IL-4 on TARC and IP-10 production in these cells were detected in a dose-dependent manner. These data strongly suggest that IL-4 may act not only as a mediator of Th1-type response but also as a down-regulator of Th2-type response in terms of the regulation of chemokine production by HaCaT cells.     

Eotaxin potentiates antigen-dependent basophil IL-4 production.

Basophils are a major source of IL-4, which is a critical factor in the generation of allergic inflammation. Eotaxin induces chemotaxis mediated through the CC chemokine receptor 3 (CCR3) present on basophils as well as eosinophils and Th2 cells, thereby promoting cell recruitment. To determine whether eotaxin has other proinflammatory activity, we examined the effect of eotaxin on basophil IL-4 expression by flow cytometry. Eotaxin alone had no effect on basophil IL-4 production, but further increased allergen-stimulated IL-4 expression. Eotaxin also enhanced IL-4 release from purified basophils 2- to 4-fold, as determined by ELISA (p < 0.01). Addition of eotaxin to cultures resulted in a 40-fold left shift in the dose response to Ag. This effect was obtained with physiologic concentrations of eotaxin (10 ng/ml), was abrogated by an Ab to the CCR3 receptor, and was noted with other chemokine ligands of CCR3. Additionally, eotaxin augmented IL-3 priming of basophil IL-4 production in a synergistic manner (p < 0.01). In contrast, no priming was observed with either IL-5 or GM-CSF. These results establish a novel function for eotaxin and other chemokine ligands of CCR3: the potentiation of Ag-mediated IL-4 production in basophils, and suggest a potential nonchemotactic role for CC chemokines in the pathogenesis and amplification of inflammation.     

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.     

Cutting edge: C-C chemokine receptor 6 is essential for arrest of a subset of memory T cells on activated dermal microvascular endothelial cells under physiologic flow conditions in vitro

Memory T cells (mTC) express multiple chemokine receptors (including CCR4 and CCR6) that may potentially be involved in their arrest on inflamed endothelia. Herein, we specifically addressed whether CCR6 is required for mTC to arrest on TNF-alpha-activated human dermal microvascular endothelial cells (HDMEC) in vitro under shear stress conditions. Recombinant liver and activation-regulated chemokine (LARC)/CCL20 (a CCR6 ligand) induced firm arrest of cutaneous lymphocyte Ag(+) mTC in a flow chamber system using purified substrates. Strikingly, desensitization of CCR6 with LARC, but not thymus and activation-regulated chemokine/CCL17 or secondary lymphoid tissue chemokine/CCL21, caused a 50-75% decrease (p < 0. 001) in arrest of mTC on HDMEC, which was indistinguishable from the reduction observed when total mTC were treated with pertussis toxin (p > 0.5). CCR6-depleted mTC also had a markedly reduced ability to arrest on HDMEC. Our results suggest that LARC production by activated endothelial cells and CCR6 expression by mTC may be critical components in the pertussis toxin-sensitive arrest of mTC on activated HDMEC.     

Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells

Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis.     

Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK

Thymus-expressed chemokine (TECK) has been reported to chemoattract dendritic cells, thymocytes, and activated macrophages. Here, we show that TECK is a specific agonist for a human orphan receptor called GPR-9-6. We have determined the cDNA sequence of human GPR-9-6 and cloned the corresponding murine cDNA. Human and murine GPR-9-6 expression is very high in the thymus and low in lymph nodes and spleen. RT-PCR analysis of murine GPR-9-6 expression on murine FACS-sorted thymocyte subpopulations showed that this gene is expressed in both immature and mature T cells. Additions of human or murine TECK to HEK 293/human GPR-9-6 and HEK 293/murine GPR-9-6 transfectants provoked intracytoplasmic calcium mobilization. Human TECK also induced the in vitro migration of HEK 293/human GPR-9-6 cells. These results confirm that GPR-9-6 is a specific receptor for TECK. According to the established nomenclature system, we propose to rename GPR-9-6 as CC chemokine receptor 9 (CCR9).