RESUMEN
BACKGROUND: Eosinophils and T lymphocytes represent constant features in the airways of subjects with exacerbated chronic bronchitis. Eotaxin is the most potent and selective eosinophil chemoattractant which can also attracts lymphocytes. The aim of the study was to evaluate the expression of eotaxin and its receptor, CCR3, in bronchial airways during exacerbation of chronic bronchitis. METHODS: By immunohistochemistry we studied eotaxin and CCR3 expression in the lamina propria of 14 subjects with acute exacerbation of chronic bronchitis. 20 asthmatics, and 8 healthy subjects. We determined the cell types expressing the CCR3 receptor by colocalization experiments. We finally studied the relationship between eotaxin and CCR3 and eosinophils and T lymphocytes. RESULTS: The number of eotaxin+ and CCR3+ cells was significantly higher in exacerbated chronic bronchitis (P<0.003 and P<0.002) and asthma (P<0.002 and P<0.0001) when compared to healthy subjects. CCR3 was mainly expressed by eosinophils and to a lesser extent by CD4+ and CD8+ lymphocytes. In exacerbated chronic bronchitis the number of CCR3+ cells was strongly correlated to the number of eosinophils (P<0.0002. r=0.85) and to the number of CD4+ lymphocytes (P<0.05, r=0.57). CONCLUSION: Our study suggests that eotaxin and CCR3 are up-regulated and could be involved in the eosinophil and CD4+ lymphocyte recruitment into the airways which occur during acute exacerbations of chronic bronchitis.
Asunto(s)
Bronquitis Crónica/inmunología , Bronquitis Crónica/fisiopatología , Quimiocinas CC/metabolismo , Receptores de Quimiocina/metabolismo , Regulación hacia Arriba , Adulto , Anciano , Asma/inmunología , Asma/fisiopatología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Quimiocina CCL11 , Eosinófilos/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Receptores CCR3RESUMEN
BACKGROUND & AIMS: Thymus-expressed chemokine (TECK) or CCL25) is selectively expressed in the small bowel (SB), where lamina propria lymphocytes (LPL) and intraepithelial leukocyte expressing the cognate chemokine receptor CCR9 predominate. We characterize the role of TECK and CCR9-expresing lymphocytes in small intestinal Crohn's disease. METHODS: CCR9 expression on lymphocytes from lamina propria, mesenteric lymph node, and peripheral blood was analyzed by flow cytometry and by Northern blotting for LPL. TECK expression was analyzed in inflamed SB and colon by reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: The fraction of CCR9(+) T cells in inflamed SB was significantly lower than in uninvolved SB mucosa. In contrast, in peripheral blood lymphocytes, CCR9(+) lymphocytes were markedly elevated in patients with small bowel Crohn's or celiac disease, but not in patients with purely colonic Crohn's. Also, TECK expression is altered in inflamed small bowel, being intensely expressed in a patchy distribution in crypt epithelial cells in proximity to lymphocytic infiltrates. TECK is not expressed in either normal or inflamed colon. CONCLUSIONS: In SB immune-mediated diseases, there is repartitioning of CCR9(+) lymphocytes between SB and blood and an altered pattern of TECK expression in SB Crohn's. The TECK/CCR9 ligand/receptor pair may play an important role in the pathogenesis of SB Crohn's disease.
Asunto(s)
Quimiocinas CC/análisis , Colon/patología , Enfermedad de Crohn/patología , Intestino Delgado/patología , Receptores de Quimiocina/análisis , Linfocitos T/química , Enfermedad de Crohn/inmunología , Diagnóstico Diferencial , Expresión Génica/inmunología , Humanos , Mucosa Intestinal/patología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , ARN Mensajero/análisis , Receptores CCR , Receptores de Quimiocina/genética , Linfocitos T/inmunologíaRESUMEN
CC chemokine receptors are expressed on hematopoietic cells, and these may impart selective homing of monocyte, leukocyte, and lymphocyte subsets to sites of inflammation. CC chemokine receptor 3 is the major receptor on eosinophils and is also expressed on other inflammatory cells suggesting its important role for allergic diseases such as atopic dermatitis and bronchial asthma. Eotaxin, eotaxin-2 and eotaxin-3 have been identified as ligands that only activate CC chemokine receptor 3. CC chemokine receptor 3 is also activated by other promiscuous ligands, however, such as RANTES and monocyte chemotactic protein 4. To date, CC chemokine receptor 3 has not been reported to be expressed on nonhematopoietic cells. In this study, we investigated whether keratinocytes possess autocrine and paracrine mechanisms for CC chemokine secretion and receptor expression as reported for the expression of interleukin 8 and its receptors. Reverse transcriptase polymerase chain reaction analysis demonstrated that CC chemokine receptor 3 mRNA is expressed constitutively in cultured keratinocytes. The signal quantities of the CC chemokine receptor 3 amplicons showed lower intensities for keratinocytes than for eosinophils. In situ hybridization techniques exhibited that basal cell layers of the epidermis were stained homogeneously for CC chemokine receptor 3 mRNA with a decreasing signal to the upper epidermis showing that differentiating and proliferating keratinocytes did express mRNA specific for CC chemokine receptor 3. Immunohistochemical studies confirmed low expression of CC chemokine receptor 3 protein on epidermal keratinocytes compared to the high level observed on infiltrating eosinophils. Furthermore, stimulation of cultured keratinocytes with eotaxin resulted in an increased [3H]thymidine incorporation indicating a role of CC chemokine receptor 3 in epidermal proliferation and differentiation. These data demonstrate that CC chemokine receptor 3 is expressed not only on hematopoietic cells but also on keratinocytes as nonhematopoietic cells with ectodermal origin. Therefore, the identification of CC chemokine receptor 3 on epidermal keratinocytes may indicate a role for CC chemokine receptor 3 and its ligands in skin physiology and pathophysiology.
Asunto(s)
Queratinocitos/metabolismo , Receptores de Quimiocina/metabolismo , Células Cultivadas , Células Epidérmicas , Epidermis/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Hibridación in Situ , ARN Mensajero/metabolismo , Receptores CCR3 , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The lymphatic vessels (lymphatics) play an important role in channeling fluid and leukocytes from the tissues to the secondary lymphoid organs. In addition to driving leukocyte egress from blood, chemokines have been suggested to contribute to leukocyte recirculation via the lymphatics. Previously, we have demonstrated that binding sites for several pro-inflammatory beta-chemokines are found on the endothelial cells (ECs) of lymphatics in human dermis. Here, using the MIP-1alpha isoform MIP-1alphaP, we have extended these studies to further support the contention that the in situ chemokine binding to afferent lymphatics exhibits specificity akin to that observed in vitro with the promiscuous beta-chemokine receptor D6. We have generated monoclonal antibodies to human D6 and showed D6 immunoreactivity on the ECs lining afferent lymphatics, confirmed as such by staining serial skin sections with antibodies against podoplanin, a known lymphatic EC marker. In parallel, in situ hybridization on skin with antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic ECs. D6-immunoreactive lymphatics were also abundant in mucosa and submucosa of small and large intestine and appendix, but not observed in several other organs tested. In lymph nodes, D6 immunoreactivity was present on the afferent lymphatics and also in subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also D6-positive. Peripheral blood cells and the ECs of blood vessels and high endothelial venules were consistently nonreactive with anti-D6 antibodies. Additionally, we have demonstrated that D6 immunoreactivity is detectable in some malignant vascular tumors suggesting they may be derived from, or phenotypically similar to, lymphatic ECs. This is the first demonstration of chemokine receptor expression by lymphatic ECs, and suggests that D6 may influence the chemokine-driven recirculation of leukocytes through the lymphatics and modify the putative chemokine effects on the development and growth of vascular tumors.
Asunto(s)
Endotelio Linfático/metabolismo , Neoplasias de Tejido Vascular/metabolismo , Receptores de Quimiocina/biosíntesis , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Quimiocina CCL3 , Quimiocina CCL4 , Dermis/inmunología , Femenino , Humanos , Intestinos/inmunología , Tejido Linfoide/inmunología , Proteínas Inflamatorias de Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Neoplasias de Tejido Vascular/clasificación , Neoplasias de Tejido Vascular/patología , Receptores CCR10 , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Piel/inmunología , Piel/metabolismo , Transcripción Genética , Receptor de Quimiocina D6RESUMEN
Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-alpha stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-gamma potentiated the TNF-alpha-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca(2+) flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, (125)I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.
Asunto(s)
Bronquios/inmunología , Bronquios/metabolismo , Quimiocinas CC/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Receptores de Quimiocina/biosíntesis , Unión Competitiva/inmunología , Bronquios/citología , Bronquios/patología , Calcio/metabolismo , Señalización del Calcio/inmunología , Línea Celular , Quimiocina CCL11 , Quimiocinas CC/farmacología , Citocinas/agonistas , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Citocinas/farmacología , Células Epiteliales/patología , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Líquido Intracelular/metabolismo , ARN Mensajero/biosíntesis , Receptores CCR3 , Receptores de Quimiocina/agonistas , Receptores de Quimiocina/genéticaRESUMEN
Among the inflammatory cells infiltrating the lungs of asthmatic patients, eosinophils and Th2 cells are thought to play a central role in the pathogenesis of this disease. Several studies have implicated that chemokines are prime candidates for being responsible for the selective recruitment of the leukocyte subsets found in atopic diseases. Regulated upon activation, normal T-cell-expressed and secreted (RANTES), monocyte chemoattractant protein-3 (MCP-3), MCP-4 and the eotaxins, for example, have been shown in vitro to potently induce eosinophil chemotaxis as well as initiate several other pro-inflammatory activities such as integrin activation, lipid mediator biosynthesis and degranulation. Ligand binding and chemotaxis experiments with these chemokines demonstrated that a G-protein coupled-receptor (GPCR) cloned from eosinophils, termed CCR3, was responsible for producing a chemokine selectivity profile identical to that of eosinophils. In addition, blocking CCR3 on eosinophils, with a monoclonal antibody, completely abolished eosinophil responses to these chemokines. Together these studies strongly suggest a central role for this receptor in eosinophil trafficking. CCR3 has also been found on in vitro derived Th2 cells and on T-cells co-localising with eosinophils in diseased tissue, thus revealing a possible pathogenic mechanism for T-cell recruitment into the airways. Therefore, blockade of CCR3 represents a highly attractive and innovative strategy for asthma therapy.
Asunto(s)
Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Quimiocinas CC , Factores Quimiotácticos Eosinófilos , Citocinas , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Antiasmáticos/uso terapéutico , Anticuerpos Monoclonales , Quimiocina CCL11 , Humanos , Inflamación , Ligandos , Receptores CCR3RESUMEN
Chemokines play an important role in the migration of leukocytes at sites of inflammation, and some constitutively expressed chemokines may direct lymphocyte trafficking within lymphoid organs and peripheral tissues. Thymus-expressed chemokine (TECK or Ckbeta-15/CCL25), which signals through the chemokine receptor CCR9, is constitutively expressed in the thymus and small intestine but not colon, and chemoattracts a small fraction of PBLs that coexpress the integrin alpha(4)beta(7). Here we show that TECK is expressed in the human small bowel but not colon by endothelial cells and a subset of cells in intestinal crypts and lamina propria. CCR9 is expressed in the majority of freshly isolated small bowel lamina propria mononuclear cells (LPMC) and at significantly higher levels compared with colonic LPMC or PBL. TECK was selectively chemotactic for small bowel but not colonic LPMC in vitro. The TECK-induced chemotaxis was sensitive to pertussis toxin and partially inhibited by Abs to CCR9. TECK attracts predominantly the T cell fraction of small bowel LPMC, whereas sorted CD3(+)CCR9(+) and CD3(+)CCR9(-) lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Collectively, our data suggest that the selective expression of TECK in the small bowel underlie the homing of CCR9(+) intestinal memory T cells to the small bowel rather than to the colon. This regional specialization implies a segregation of small intestinal from colonic immune responses.
Asunto(s)
Quimiocinas CC/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Receptores de Quimiocina/fisiología , Timo/inmunología , Timo/metabolismo , Movimiento Celular/inmunología , Quimiocinas CC/biosíntesis , Quimiotaxis de Leucocito/inmunología , Colon/citología , Colon/inmunología , Colon/metabolismo , Citocinas/biosíntesis , Humanos , Inmunidad Mucosa , Inmunofenotipificación , Mucosa Intestinal/citología , Intestino Delgado/citología , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Leucocitos Mononucleares/inmunología , Receptores CCR , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/sangre , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Timo/citologíaRESUMEN
TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.
Asunto(s)
Quimiocinas CC/farmacología , Quimiotaxis/inmunología , Mucosa Intestinal/inmunología , Receptores de Quimiocina/inmunología , Linfocitos T/metabolismo , Timo/inmunología , Anticuerpos Monoclonales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Calcio , Línea Celular , Quimiocinas CC/genética , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Humanos , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/inmunología , ARN Mensajero/inmunología , Receptores CCR , Receptores de Quimiocina/genética , Receptores Mensajeros de Linfocitos/inmunología , TransfecciónRESUMEN
To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1alpha induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37 degrees C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1alpha in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1alpha signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.
Asunto(s)
Quimiocinas CC , Quimiocinas/farmacología , Eosinófilos/efectos de los fármacos , Receptores de Quimiocina/fisiología , Calcio/metabolismo , Quimiocina CCL11 , Quimiocina CCL3 , Quimiocina CCL4 , Citocinas/farmacología , Eosinófilos/fisiología , Citometría de Flujo , Humanos , Proteínas Inflamatorias de Macrófagos/farmacología , Proteínas Quimioatrayentes de Monocitos/farmacología , Monocitos/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Receptores CCR1 , Receptores CCR3 , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Certain C-C chemokines, signaling via the eotaxin receptor C-C chemokine receptor-3 (CCR3), are thought to be central mediators of eosinophil accumulation in allergic inflammation. To investigate the role of CCR3 in vivo, we cloned the guinea pig eotaxin receptor (guinea pig CCR3) from a genomic DNA library. We isolated a single-exon open reading frame coding for a 358-amino acid chemokine receptor protein with 67 and 69% homology to human and murine CCR3, respectively. When expressed in stable transfectants, this receptor bound 125I-labeled guinea pig eotaxin, 125I-labeled human monocyte chemotactic protein-3, and 125I-labeled human RANTES. In chemotaxis assays, guinea pig CCR3 transfectants responded only to guinea pig eotaxin, with a maximal effect at 100 nM. mAbs were raised that bound selectively to both guinea pig CCR3 transfectants and guinea pig eosinophils. One of these mAbs, 2A8, blocked both ligand binding to transfectants and their chemotaxis in response to eotaxin. The Ab also inhibited chemotaxis and the elevation of cytosolic calcium in guinea pig eosinophils in response to eotaxin. F(ab')2 fragments of 2A8 were prepared that retained the ability to inhibit eosinophil calcium responses to eotaxin. Pretreatment of (111)In-labeled eosinophils in vitro with F(ab')2 2A8 selectively inhibited their accumulation in response to eotaxin in vivo. These data demonstrate that functional blockade of eosinophil chemokine receptors can be achieved in vivo and provide further support for the development of novel anti-inflammatory drugs targeting eosinophil recruitment through chemokine receptor antagonism.
Asunto(s)
Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Quimiocinas CC , Citocinas/metabolismo , Eosinófilos/metabolismo , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/farmacología , Quimiocina CCL11 , Factores Quimiotácticos Eosinófilos/metabolismo , Quimiotaxis de Leucocito/inmunología , Clonación Molecular , Eosinófilos/trasplante , Cobayas , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/farmacología , Radioisótopos de Indio/metabolismo , Inyecciones Intravenosas , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica/inmunología , Receptores CCR3 , Receptores de Quimiocina/química , Receptores de Quimiocina/inmunología , Transducción de Señal/inmunología , TransfecciónRESUMEN
CCR1 and CCR3 are seven-transmembrane domain G protein-coupled receptors specific for members of the CC chemokine subgroup of leukocyte chemoattractants. Both have been implicated in the inflammatory response, and CCR3, through its expression on eosinophils, basophils, and Th2 lymphocytes, may be especially important in allergic inflammation. CCR1 and CCR3 are 54% identical in amino acid sequence and share some ligands but not others. In particular, macrophage inflammatory protein 1alpha (MIP-1alpha) is a ligand for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1. To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1alpha and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1alpha nor eotaxin were effective agonists at either chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites determine chemokine selectivity, but others are needed for receptor activation. Agents that bind to the N-terminal segments of CCR1 and CCR3 may be useful in blocking receptor function.
Asunto(s)
Citocinas/metabolismo , Proteínas Inflamatorias de Macrófagos/metabolismo , Receptores de Quimiocina/química , Animales , Linfocitos B/fisiología , Sitios de Unión/fisiología , Quimiocina CCL11 , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/química , Proteínas de Unión al GTP/metabolismo , Ratones , Unión Proteica/fisiología , Receptores CCR1 , Receptores CCR3 , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Chemokines are widely hypothesized to stimulate firm adhesion of leukocytes on endothelium in shear flow. Thus far, this has been demonstrated experimentally for exogenously added chemoattractants, but not for those released by endothelium. We found that human umbilical cord endothelial cells (HUVEC) stimulated with TNF-alpha and IFN-gamma secreted eosinophil chemoattractants into the culture supernatant. This material induced transendothelial chemotaxis, stimulated eosinophil binding to purified intercellular adhesion molecule 1, and augmented binding to purified vascular cell adhesion molecule 1 in a 3-min static assay. Chemotaxis and stimulation of adhesion were abrogated completely by the pretreatment of eosinophils with an mAb to the C-C chemokine receptor 3 (CCR3). Eosinophils accumulated efficiently on HUVEC stimulated with TNF-alpha and IFN-gamma in shear flow at 1.5 dyn/cm2. CCR3 mAb slightly but significantly reduced eosinophil arrest and accumulation, by preventing development of firm adhesion by some of the tethered eosinophils, so that they detached within 30 s after the initial tethering. In the presence of mAb to the alpha4 integrin subunit, the effect of CCR3 mAb was more prominent, and approximately half of eosinophil arrest and accumulation was abolished. Inhibition by CCR3 mAb in the presence of beta2 integrin mAb was similar to that in control eosinophils. This is the first evidence that endothelial cell-derived chemokines can activate firm adhesion through alpha4 and beta2 integrins even in the presence of shear flow.
Asunto(s)
Endotelio Vascular/fisiología , Eosinófilos/fisiología , Receptores de Quimiocina/fisiología , Antígenos CD/metabolismo , Antígenos CD18/metabolismo , Adhesión Celular/fisiología , Quimiotaxis de Leucocito/fisiología , Endotelio Vascular/efectos de los fármacos , Humanos , Inflamación , Integrina alfa4 , Integrinas/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/farmacología , Factor de Activación Plaquetaria/antagonistas & inhibidores , Receptores CCR3 , Reología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used (111)In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of (111)In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-alpha4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when (111)In-eosinophils were pretreated in vitro with anti-alpha4 integrin or anti-beta2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-alpha or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24-28 h) but not the early phase (0-4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on alpha4 integrin/vascular cell adhesion molecule-1 and beta2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.
Asunto(s)
Antígenos CD/fisiología , Antígenos CD18/fisiología , Quimiocinas CC , Citocinas/farmacología , Eosinófilos/inmunología , Integrinas/fisiología , Interleucina-4/farmacología , Piel/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD18/inmunología , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CCL11 , Factores Quimiotácticos Eosinófilos/farmacología , Citocinas/administración & dosificación , Citocinas/biosíntesis , Edema/etiología , Edema/inmunología , Edema/patología , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Humanos , Radioisótopos de Indio , Inyecciones Intradérmicas , Inyecciones Intravenosas , Integrina alfa4 , Integrinas/inmunología , Líquido Intracelular/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Piel/patología , Factores de TiempoRESUMEN
To date almost 40 human chemokines have been described, modulating a complex and overlapping set of biological activities important for basal leukocyte trafficking, as well as the extravasation and tissue infiltration of leukocytes in response to inciting agents. Chemokines appear to be key mediators in a number of inflammatory pathologies as a result of their chemotactic effects on almost every leukocyte type, including neutrophils, eosinophils, basophils, monocytes, dendritic cells, NK cells, B-cells and T-cells, together with their ability to stimulate effector functions, such as granule release and superoxide radical production from these cells. With the recent discovery that some chemokine receptors function as HIV-1 co-receptors and with accumulating evidence that chemokines may also regulate certain aspects of haematopoiesis, angiogenesis and apoptosis, the chemokines and their receptors have emerged as a new set of drug discovery targets for the pharmaceutical and biotechnology industry.
RESUMEN
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.
Asunto(s)
Basófilos/fisiología , Quimiocinas CC , Quimiocinas/farmacología , Citocinas/farmacología , Proteínas Quimioatrayentes de Monocitos/farmacología , Receptores de Quimiocina , Receptores de Citocinas/fisiología , Quimiocina CCL11 , Quimiocina CCL5/farmacología , Quimiotaxis , Liberación de Histamina , Humanos , Receptores CCR3 , Receptores de Citocinas/análisisRESUMEN
Monocyte chemotactic protein-4 (MCP-4) is a newly identified C-C chemokine with potent eosinophil chemoattractant properties. We describe studies of its biological activity in vitro to induce chemotaxis of peripheral blood eosinophils and to induce histamine release from IL-3-primed peripheral blood basophils. MCP-4 and eotaxin caused a similar rise in eosinophil intracytoplasmic Ca2+ and complete cross-desensitization. MCP-4 also abolished the eosinophil Ca2+ response to MCP-3 and partially desensitized the response to macrophage inflammatory protein-1alpha. MCP-4 activated cell migration via either CCR2b or CCR3 in mouse lymphoma cells transfected with these chemokine receptors. MCP-4 inhibited binding of 125I-eotaxin to eosinophils and CCR3-transfected cells and inhibited 125I-MCP-1 binding to CCR2b-transfectants. MCP-4 mRNA was found in cells collected in bronchoalveolar lavage of asthmatic and nonasthmatic subjects and was prominently expressed in human lung and heart. MCP-4 mRNA was expressed in several human bronchial epithelial cell lines after cytokine stimulation. Pretreatment of BEAS-2B epithelial cells with the glucocorticoid budesonide inhibited MCP-4 mRNA expression. These features make MCP-4 a candidate for playing a role in eosinophil recruitment during allergic respiratory diseases.
Asunto(s)
Quimiocinas CC , Eosinófilos/metabolismo , Eosinófilos/fisiología , Proteínas Quimioatrayentes de Monocitos/metabolismo , Proteínas Quimioatrayentes de Monocitos/fisiología , Secuencia de Aminoácidos , Animales , Basófilos/inmunología , Basófilos/metabolismo , Northern Blotting , Líquido del Lavado Bronquioalveolar/citología , Budesonida , Calcio/metabolismo , Movimiento Celular , Células Cultivadas/metabolismo , Quimiocina CCL11 , Quimiocina CCL4 , Quimiocina CCL5/genética , Quimiocina CCL5/farmacología , Quimiocina CCL5/fisiología , Quimiocina CCL7 , Quimiotaxis , Citocinas/genética , Citocinas/farmacología , Citocinas/fisiología , ADN Complementario/análisis , Eosinófilos/inmunología , Células Epiteliales , Liberación de Histamina , Humanos , Interleucina-3/farmacología , Proteínas Inflamatorias de Macrófagos/farmacología , Ratones , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/genética , Proteínas Quimioatrayentes de Monocitos/farmacología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Reacción en Cadena de la Polimerasa , Pregnenodionas/farmacología , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales Cultivadas/metabolismoRESUMEN
We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Productos del Gen env/inmunología , Receptores de Citocinas/inmunología , Receptores del VIH/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Secuencia de Bases , ADN Viral , Productos del Gen env/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Receptores CCR5 , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificaciónRESUMEN
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.