In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-γ and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.
The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.
T lymphocytes; chemokines; allergy
CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4−/−) mice by gene targeting. CCR4−/− mice developed normally. Splenocytes and thymocytes isolated from the CCR4−/− mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1α. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4−/− mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4+/+ mice. After high dose LPS treatment, serum levels of tumor necrosis factor α, interleukin 1β, and MIP-1α were reduced in CCR4−/− mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4−/− mice by flow cytometry also revealed a significant decrease in the F4/80+ cell population. This may reflect a defect in the ability of the CCR4−/− macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.
CC chemokine receptor 4; lipopolysaccharide; endotoxic shock; F4/80 antigen; T helper type 2 cells
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
rodent; T cells; cytokines; chemokines; lung
Recruitment of antigen-specific Th2 cells into the lung is critical for the development of allergic airway inflammation. Although CCR4 and CCR8 are preferentially expressed on Th2 cells and CCR4, CCR8 and CXCR3 ligands are increased in asthma, the specific relative contribution of these receptors to antigen-specific Th2 cell trafficking into the allergic lung is not known.
To determine the relative contribution of CCR4, CCR8 and CXCR3 to antigen-specific Th2 cell trafficking in a murine model of allergic pulmonary inflammation.
We used adoptive transfer experiments to compare the trafficking of wild type antigen-specific Th2 cells with antigen-specific Th2 cells deficient in CCR4, CCR8 or CXCR3.
CCR4-deficient antigen-specific Th2 cells failed to traffic efficiently into the lung and the airways. In contrast, CCR8-deficient antigen-specific Th2 cells accumulated in these sites. Trafficking of CXCR3-deficient antigen-specific Th2 cells and CCR4-deficient and CCR8-deficient antigen-specific Th1 cells were comparable to their wild type counterparts. Approximately 60% of IL-4 producing antigen-specific T cells expressed CCR4. Disruption of CCR4-mediated antigen-specific Th2 cell trafficking decreased the levels of Th2-type cytokines in the airways and reduced airway eosinophila and mucus production.
Our study demonstrates that CCR4 is required for the efficient entry of antigen-specific Th2 cells into the lung and the airways in murine model of allergic pulmonary inflammation.
Inhibition of CCR4-mediated Th2 cell trafficking may contribute to asthma therapy.
CCR4 is required for efficient entry of Th2 cells into the allergic lung. Disruption of CCR4-mediated antigen-specific Th2 cell trafficking decreases airway Th2-type cytokines, eosinophilia and mucus production,
CCR4; CCR8; CXCR3; asthma; chemokine; T cell trafficking
Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell–rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3–/– and CCR3+/+ eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86-dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4+ T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4+ T cells.
The underlying inflammation present in chronic airway diseases is orchestrated by increased expression of CC chemokines that selectively recruit leukocyte populations into the pulmonary system. Human CCL26 signals through CC chemokine receptor 3 (CCR3), is dramatically upregulated in challenged asthmatics, and stimulates recruitment of eosinophils (EOSs) and other leukocytes. CCL26 participates in regulation of its receptor CCR3 and modulates expression of a variety of chemokines in alveolar type II cells. Utilizing the A549 alveolar type II epithelial cell culture model, we carried out studies to test the hypothesis that CCL26-siRNA treatment of these cells would ameliorate Th2-driven release of the eotaxins and other CCR3 ligands that would, in turn, decrease recruitment and activation of EOSs. Results demonstrate that CCL26-siRNA treatments decreased interleukin-4-induced CCL26 and CCL24 expression by >70%. CCL26-directed small-interfering RNA (siRNA) treatments significantly decreased release of CCL5 (RANTES), CCL15 (MIP-1δ), CCL8 (MCP-2), and CCL13 (MCP-4). In bioactivity assays it was shown that EOS migration and activation were reduced up to 80% and 90%, respectively, when exposed to supernatants of CCL26-siRNA-treated cells. These results provide evidence that CCL26 may be an appropriate target for development of new therapeutic agents designed to alleviate the underlying inflammation associated with chronic diseases of the airways.
The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.
Expression of chemokine receptors on T helper 2 cells and eosinophils has been postulated to be the mechanism by which these cells are selectively recruited to the lung during allergic inflammatory reactions. Mouse models have provided evidence to show that blocking the ligands for these receptors is successful in abrogating the pathophysiological effects of allergen challenge. However, recent studies describing the effect of genetic deletions of these chemokine receptors have not confirmed the results obtained with ligand knockouts or neutralising antibodies. Coupled with the realisation that, because of a lack of species cross-reactivity, it is not possible to test small molecule antagonists against human receptors in the original in vivo animal models, the future of chemokine receptor therapeutics is in question. However, recent advances have been made regarding the therapeutic potential of blocking the chemokine receptors CCR3, CCR4 and CCR8 in allergic airway disease.
Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are upregulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bi-specific antagonist of CCR1 and CCR3 named UCB35625, which was proposed to interact with the transmembrane residues Y41, Y113 and E287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/Eotaxin suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Y113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1:CCR3 chimaeric receptor lacking the CCR3 amino-terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Y41, Y113 and E287 which resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.
The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.
Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.
Chemokine CCL24 is the second member of eotaxins, a group of eosinophils’ selectively chemoattractants. Via binding to its only receptor CCR3, CCL24 mainly mediates atopic disorders, parasitic infections and systemic diseases. It is well-known that CCR3 is expressed at the maternal-fetal interface; nevertheless whether CCL24 is located there and which role CCL24/CCR3 axis played is unclear. In this article, we assessed the expression of CCL24 and CCR3 in decidual stromal cells (DSCs) and trophoblasts, investigated the effects of DSCs-trophoblasts contact and pregnancy-associated hormones on the expression of CCR3 by DSCs, and last examined the role of trophoblasts-derived CCL24 on the proliferation, cell numbers and apoptosis of DSCs in vitro. We found that trophoblasts secrete chemokine CCL24, whereas DSCs express receptor CCR3. DSCs and trophoblasts co-culture had an raised level of CCL24 in culture supernatants, and the expression of CCR3 on DSCs was also obviously improved. Estrogen, progesterone and hCG up-regulated the expression of CCR3 on DSCs at appropriate concentration. CCL24 increased the proliferation and apoptosis of DSCs, whereas on the whole it promoted the number of DSCs. Thus, we conclude that by secreting CCL24 trophoblasts could promote the growth of DSCs; pregnancy associated environments such as DSCs-trophoblasts contact and hormones increased local CCL24/CCR3, which means a beneficial factor for the process of decidualization in human early pregnancy.
CCL24; CCR3; DSCs; maternal-fetal interface; trophoblasts
Chemokine receptors have been suggested to be preferentially expressed on CD4+ T cells with CCR3 and CCR8 linked to the T helper (Th) 2 subset and CCR5 and CXCR3 to the Th1 subset, however this remains controversial.
Our aim was to compare the CCR3, CCR5, CCR8 and CXCR3 expression in memory Th cells from allergic, asymptomatically sensitized and healthy individuals.
Peripheral blood mononuclear cells from 8 pollen allergic rhinitis patients, 10 asymptomatically sensitized and 10 healthy individuals were stimulated for 7 days with allergen or tetanus toxoid. CCR3, CCR5, CCR8, CXCR3, CD4 and CD45RO were detected by flow cytometry.
No differences in chemokine receptor expression were observed between the three groups on day 0, and seven days of unstimulated culture did not change the expression. Both antigenic stimuli increased the chemokine receptor expression, tetanus toxoid being the most potent. No differences in percentage chemokine receptor positive memory Th cells were observed between the three groups on day 7. Only a change in MFI for CCR5 was significantly different between the three groups after allergen stimulation of the Th cells.
We conclude that even though allergen and antigen induced increased chemokine receptor expression, no differences in profiles were identified in memory Th cells from patient groups with different atopic status.
CC Chemokine Receptor 3 (CCR3), the major chemokine receptor expressed on eosinophils, binds promiscuously to several ligands including eotaxins 1, 2, and 3. Even though the only cells that consistently accumulate following eotaxin administration in vivo are myeloid cells (primarily eosinophils), other cell types have recently been shown to express CCR3. It is therefore important to elucidate the molecular mechanisms regulating receptor expression.
In order to define regions responsible for CCR3 transcription, a DNAse hypersensitive site was identified in the vicinity of exon 1. Coupled with our previous data implicating exon 1 in CCR3 transcription, we hypothesized that transcription factors bind to exon-1. Electrophoretic mobility shift analysis revealed that nuclear proteins in eosinophilic cells bound to exon 1. Furthermore, antibody interference and mutation studies demonstrated GATA-1 binding to exon 1. In order to test the 1.6-kb CCR3 promoter element (that includes exon 1) for in vivo function, this region was used to generate transgenic mice that expressed a reporter protein. Strong transgene expression was achieved, with the pattern of expression suggesting a broad acting promoter.
The transcription factor GATA-1 binds to CCR3 exon 1. The 1.6-kb CCR3 promoter element, that includes exon 1, is a strong promoter in vivo.
Chemokine receptor expression is exquisitely regulated on T cell subsets during the course of their migration to inflammatory sites. In the present study we demonstrate that CCR4 expression marks a pathogenic population of autoimmune T cells. CCR4 was found exclusively on memory CD4+ T cells during the progression of disease in NOD mice. Cells expressing the CCR4 ligand TARC (thymus- and activation-regulated chemokine) were detected within infiltrated islets from prediabetic mice. Interestingly, neutralization of macrophage-derived chemokine (MDC) with Ab caused a significant reduction of CCR4-positive T cells within the pancreatic infiltrates and inhibited the development of insulitis and diabetes. Furthermore, enhanced recruitment of CCR4-bearing cells in NOD mice resulting from transgenic expression of MDC resulted in acceleration of clinical disease. Cumulatively, the results demonstrate that CCR4-bearing T cells participate in the development of such tissue-driven autoimmune reactions.
Allergic asthmatic responses in the airway are associated with airway hyperreactivity, eosinophil accumulation in the lung, and cytokine production by allergen-specific, T helper cell type 2 (Th2) lymphocytes. Here, we show that in a cockroach antigen (CA) model of allergic pulmonary inflammation, the chemokine macrophage inflammatory protein (MIP)-3α is expressed in the lung within hours of allergen challenge. To determine the biologic relevance of this expression, mice lacking CCR6, the only known receptor for MIP-3α, were studied for their response to CA. CCR6-deficient mice were immunized to the same extent as their wild-type counterparts, as judged by cytokine production in antigen-challenged lymphocytes. However, compared with CA-challenged wild-type mice, challenged CCR6-deficient mice had reduced airway resistance, fewer eosinophils around the airway, lower levels of interleukin 5 in the lung, and reduced serum levels of immunoglobulin E. Together, these data demonstrate that MIP-3α and CCR6 function in allergic pulmonary responses and suggest that these molecules might represent novel therapeutic targets for treatment of asthma.
CCR6; MIP-3α; chemokine; asthma; lung
In some autistic children, there is an imbalance of T helper (Th)1/Th2 lymphocytes toward Th2, which may be responsible for the induction of the production of autoantibodies in these children. Th2 lymphocytes express CCR4 receptors. CCR4 ligands include macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC). They direct trafficking and recruitment of Th2 cells. We are the first to measure serum levels of CCR4 ligands in relation to the degree of the severity of autism.
Serum concentrations of MDC and TARC were measured, by quantitative sandwich enzyme immunoassay technique, in 56 autistic children and 32 healthy matched children.
Autistic children had significantly higher serum levels of MDC and TARC than healthy controls (P <0.001 and P <0.001, respectively). Children with severe autism had significantly higher serum levels of MDC and TARC than patients with mild to moderate autism (P <0.001 and P = 0.01, respectively). In addition, there were significant positive correlations between CARS and serum levels of both MDC (P <0.001) and TARC (P <0.001) in children with autism. There were significant positive correlations between serum levels of MDC and TARC in autistic children (P <0.001).
Serum levels of CCR4 ligands were elevated in autistic children and they were significantly correlated to the degree of the severity of autism. However, further research is warranted to determine the pathogenic role of CCR4 ligands in autism and to shed light on the therapeutic role of CCR4-ligand antagonism in autistic children.
Autism; Autoimmunity; Childhood Autism Rating Scale; CCR4 ligands; Macrophage-derived chemokine; Thymus and activation-regulated chemokine
Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (TH2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of TH2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5–enriched TH2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of TH2 cell homing that drives IL-5–mediated chronic allergic inflammation.
This report shows that interleukin (IL) 17–producing T helper type 17 (Th17) cells predominantly express CC chemokine receptor (CCR) 6 in an animal model of rheumatoid arthritis (RA). Th17 cells induced in vivo in normal mice via homeostatic proliferation similarly express CCR6, whereas those inducible in vitro by transforming growth factor β and IL-6 additionally need IL-1 and neutralization of interferon (IFN) γ and IL-4 for CCR6 expression. Forced expression of RORγt, a key transcription factor for Th17 cell differentiation, induces not only IL-17 but also CCR6 in naive T cells. Furthermore, Th17 cells produce CCL20, the known ligand for CCR6. Synoviocytes from arthritic joints of mice and humans also produce a large amount of CCL20, with a significant correlation (P = 0.014) between the amounts of IL-17 and CCL20 in RA joints. The CCL20 production by synoviocytes is augmented in vitro by IL-1β, IL-17, or tumor necrosis factor α, and is suppressed by IFN-γ or IL-4. Administration of blocking anti-CCR6 monoclonal antibody substantially inhibits mouse arthritis. Thus, the joint cytokine milieu formed by T cells and synovial cells controls the production of CCL20 and, consequently, the recruitment of CCR6+ arthritogenic Th17 cells to the inflamed joints. These results indicate that CCR6 expression contributes to Th17 cell function in autoimmune disease, especially in autoimmune arthritis such as RA.
Trafficking and inflammation in airway diseases are, in part, modulated by members of the CC chemokine family, eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), which transduce signals through their CCR3 receptor. In this context, we hypothesized that transfecting alveolar type II epithelial cells with CCR3-targeted siRNA or antisense (AS-ODN) sequences will downregulate cellular synthesis and release of the primary CCR3 ligands CCL26 and CCL24 and will modulate other CCR3 ligands. The human A549 alveolar type II epithelium-like cell culture model was used for transfection and subsequent effects on CCR3 agonists. siRNAs were particularly effective. PCR showed a 60-80% decrease in mRNA and immunoblots showed up to 75-84% reduction of CCR3 in siRNA treated cells. CCR3-siRNA treatments reduced IL-4 stimulated CCL26 release and constitutive CCL24 release by 65% and 80%, respectively. Release of four additional CCR3 agonists RANTES, MCP-2, MCP-3 and MCP-4 was also significantly reduced by CCR3-siRNA treatments of the alveolar type II cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced when eosinophils were treated with supernatants of A549 cells pretreated with CCR3-targeted siRNAs or AS-ODNs. Collectively, the data suggest that post-transcriptional regulation of CCR3 receptors may be a potential therapeutic approach for interrupting proinflammatory signaling.
alveolar type II cells; CCR3 receptor; eotaxins; siRNA; antisense oligonucleotides
Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.
chemokine receptors; chemokines; T helper type 2 cells; allergy; granulomas
Chemokines bind and signal through G-protein coupled seven transmembrane receptors. Various chemokine receptors are expressed on leukocytes, and these may impart selective homing of leukocyte subsets to sites of inflammation. Human eosinophils express the eotaxin receptor, CCR3, but respond to a variety of CC chemokines apart from eotaxin, including RANTES, monocyte chemotactic protein (MCP)-2, MCP-3, and MCP-4. Here we describe a mAb, 7B11, that is selective for CCR3 and has the properties of a true receptor antagonist. 7B11 blocked binding of various radiolabeled chemokines to either CCR3 transfectants, or eosinophils. Pretreatment of eosinophils with this mAb blocked chemotaxis and calcium flux induced by all CCR3 ligands. In all individuals examined, including allergic and eosinophilic donors, > 95% of the response of eosinophils to eotaxin, RANTES, MCP-2, MCP-3, and MCP-4 was shown to be mediated through CCR3. The IL-8 receptors, particularly CXCR2, were induced on IL-5 primed eosinophils, however these eosinophils responded to CC chemokines in the same manner as unprimed eosinophils. These results demonstrate the importance of CCR3 for eosinophil responses, and the feasibility of completely antagonizing this receptor.
To determine whether the ocular surface inflammation in uveitis mimics or counteracts intraocular inflammatory pathways by directly comparing T-helper (Th) lymphocytes Th1 and Th2 markers in conjunctival and ciliary body expression in endotoxin-induced uveitis (EIU). This study used the following inflammatory markers: chemokine receptor, CC chemokine receptor 4 (CCR4), and its ligand, macrophage-derived chemokine (MDC), to evaluate Th2 participation; chemokine receptor, CCR5, to evaluate the Th1 system; and its ligand, regulated on activation normal T cell expressed and secreted (RANTES), to evaluate both Th1 and Th2 systems.
Immunohistochemistry and real-time polymerase chain reaction (RT–PCR) were used to compare protein and RNA expression of CCR4, MDC, CCR5, and RANTES in the conjunctiva and ciliary body in EIU 6 h and 24 h after the lipopolysaccharide (LPS) injection and in control (without injection) Lewis rats.
Immunohistochemistry with CCR5, RANTES, and MDC showed an increase in fluorescent staining in the conjunctiva and ciliary body in the rats with uveitis compared to the control rats. For CCR4, immunostaining was comparable in the conjunctiva and ciliary body and did not show any clear differences between control rats and rats with EIU. For RANTES, MDC, and CCR5, RT-PCR showed a significantly higher RNA expression in conjunctiva and in ciliary body at 6 h compared to 24 h and controls. For CCR4, RT-PCR did not illustrate any significant differences in conjunctiva and in ciliary body between all groups of animals.
Protein and RNA expressions of RANTES, MDC, and CCR5 were higher in EIU rats than in control rats in the conjunctiva and ciliary body whereas the CCR4 level was not modified in the conjunctiva and ciliary body of EIU rats when compared to controls. Th1 activation seemed to predominate in this model with high levels of CCR5 expression and no increased expression of CCR4, but Th2 participation with MDC was noted. The expression of RANTES, MDC, CCR4 and CCR5 in EIU was quite similar between the conjunctiva and the ciliary body, so conjunctival inflammation might reproduce the intraocular inflammation, probably generated by local extension and diffusion in this model. If the ocular surface mimics intraocular inflammatory pathways, the conjunctiva may provide a new and easier access for uveitis studies.
CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-γ and IL-4 producers. Studies of CCR4 knockout (CCR4−/−) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-γ production during secondary but not primary challenge. Adoptive transfer indicated CCR4−/−CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4+/+) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4+/+CD4+ T cells and CCR4−/− dendritic cells revealed intact IL-2 but impaired IFN-γ production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.