RESUMO
BACKGROUND: The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. OBJECTIVE: The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. METHOD: We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. RESULTS: Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. CONCLUSIONS AND CLINICAL RELEVANCE: There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.
Assuntos
Regulação para Baixo/imunologia , Receptores CCR4/antagonistas & inibidores , Hipersensibilidade Respiratória/tratamento farmacológico , Células Th2/imunologia , Transferência Adotiva , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Caliciformes/imunologia , Células Caliciformes/patologia , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores CCR6/antagonistas & inibidores , Receptores CCR6/genética , Receptores CCR6/imunologia , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/patologiaRESUMO
The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.
Assuntos
Memória Imunológica , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Virais/imunologia , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Glicoproteínas/imunologia , Proteínas de Fluorescência Verde , Técnicas In Vitro , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Proteínas Luminescentes/genética , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Proteínas Virais/imunologiaRESUMO
Cytokine LD78 is a human counterpart of the mouse macrophage inflammatory protein 1 alpha/hematopoietic stem cell inhibitor. Promoters of the LD78 alpha and LD78 beta genes showed similar inducible activities in two leukemic cell lines, K562 and Jurkat, but the induction mechanisms differed between the two cell lines. Further characterization of the LD78 alpha promoter indicated that multiple positive and negative regulatory elements are present, some of which are differentially required for induction and repression of the promoter activity in different cells. One of the negative regulatory elements, ICK-1, functioned in both cell lines in the absence and presence of stimulation and was shown to be a recognition site for positive and negative transcriptional factors. This ICK-1 element contained a direct repeat, and similar repeats were also found in the negative regulatory elements of hematopoietic growth factor interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoters. Nuclear extracts from K562 and Jurkat cells formed several protein-DNA complexes with the LD78 alpha ICK-1 element, one of which was also observed with the IL-3 and GM-CSF ICK-1 elements. Results from in vivo and in vitro analyses suggested that the protein forming this complex functions as a negative factor. The binding affinity of this protein, ICK-1A, to the LD78 alpha ICK-1 element was low and was significantly affected by the incubation temperature and the salt concentration in the binding buffer. ICK-1B, another protein bound specifically by the LD78 alpha ICK-1 element, was shown to be a positive factor important for induction of the promoter. These results suggested that ICK-1A plays an important role in balanced expression of LD78, IL-3, and GM-CSF during hematopoietic cell growth and differentiation.
Assuntos
Citocinas/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-3/genética , Monocinas/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Sondas de DNA , Humanos , Leucemia Eritroblástica Aguda , Leucemia de Células T , Luciferases/genética , Luciferases/metabolismo , Proteínas Inflamatórias de Macrófagos , Metilação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas/efeitos dos fármacos , Mapeamento por Restrição , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
We have determined the entire sequence of human cDNA encoding a novel CC chemokine NCC-4 by 5' and 3' RACE methods. Two types of transcripts, 579 bp and 1503 bp long, respectively, are generated through alternative polyadenylation sites. Both species contain an open reading frame encoding 120 amino acids with 19-38% identity to other human CC chemokines. The short and long transcripts are expressed highly selectively in the liver at nearly equivalent levels. There seems to be one copy of the gene per haploid genome. We now designate NCC-4 as LEC from liver-expressed chemokine.
Assuntos
Quimiocinas CC/genética , DNA Complementar/isolamento & purificação , Fígado/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Quimiocinas CC/isolamento & purificação , Clonagem Molecular , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we have identified a novel CC chemokine whose gene exists next to the IL-11 receptor alpha (IL-11Ralpha) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL-11Ralpha has been assigned. We term this novel chemokine IL-11Ralpha-locus chemokine (ILC). ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place. Thus, ILC is likely to be the original chemokine of MC148R.
Assuntos
Quimiocinas CC/genética , Cromossomos Humanos Par 9/genética , Interleucina-11/metabolismo , Vírus do Molusco Contagioso/genética , Receptores de Interleucina/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Quimiocina CCL27 , Quimiocinas CC/biossíntese , Quimiocinas CC/isolamento & purificação , Clonagem Molecular , DNA Complementar/isolamento & purificação , Humanos , Subunidade alfa de Receptor de Interleucina-11 , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Receptores de Interleucina-11 , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Alinhamento de SequênciaRESUMO
Macrophage inflammatory protein (MIP)-1 alpha, also known as LD78, is a member of a family of chemokines which recruit leukocytes to sites of inflammation. We have shown by Northern blot analyses that MIP-1 alpha mRNA is expressed in several human tissues, including brain. To explore MIP-1 alpha distribution in brain tissue, we immunohistochemically examined brain tissues from 13 neuropsychiatric patients. Glial cells in the white matter of brain tissues from four patients with schizophrenia and one with manic depressive illness were MIP-1 alpha positive. Glial cells in the cortex from these patients were negative, except in one patient with schizophrenia in whom neurones as well as glial cells in the cortex stained positively for MIP-1 alpha. In situ hybridization showed that MIP-1 alpha mRNA was expressed in both neurones as well as glial cells in this patient. These results suggest a heterogeneous distribution of MIP-1 alpha in human brain.
Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Transtornos Psicóticos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Quimiocina CCL4 , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Valor Preditivo dos TestesRESUMO
LD78 is a member of a new cytokine superfamily, consisting of at least twelve small proteins, which are involved in inflammation and cell growth. Depending on their primary structure, these cytokines can be divided into two families, one of which contains LD78 and is known as the CC family. The protein and gene structures and the physiological functions of LD78 in comparison with the other CC family members are summarized here. Furthermore, the generation mechanism of the three LD78 genes and their expression are discussed.
Assuntos
Citocinas/fisiologia , Regulação da Expressão Gênica , Monocinas/fisiologia , Proteínas de Neoplasias/fisiologia , Sequência de Aminoácidos , Animais , Quimiocina CCL4 , Citocinas/genética , Humanos , Proteínas Inflamatórias de Macrófagos , Dados de Sequência Molecular , Monocinas/genética , Proteínas de Neoplasias/genéticaAssuntos
Sistema de Condução Cardíaco/fisiopatologia , Adulto , Idoso , Arritmias Cardíacas/fisiopatologia , Flutter Atrial/fisiopatologia , Função Atrial , Complexos Cardíacos Prematuros/fisiopatologia , Eletrocardiografia , Feminino , Bloqueio Cardíaco/fisiopatologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Primary ATL cells frequently express CCR4 at high levels. Since HTLV-1 Tax does not induce CCR4 expression, transcription factor(s) constitutively active in ATL may be responsible for its strong expression. We identified an activator protein-1 (AP-1) site in the CCR4 promoter as the major positive regulatory element in ATL cells. Among the AP-1 family members, Fra-2, JunB and JunD are highly expressed in fresh primary ATL cells. Consistently, the Fra-2/JunB and Fra-2/JunD heterodimers strongly activated the CCR4 promoter in Jurkat cells. Furthermore, Fra-2 small interfering RNA (siRNA) or JunD siRNA, but not JunB siRNA, effectively reduced CCR4 expression and cell growth in ATL cells. Conversely, Fra-2 or JunD overexpression promoted cell growth in Jurkat cells. We identified 49 genes, including c-Myb, BCL-6 and MDM2, which were downregulated by Fra-2 siRNA in ATL cells. c-Myb, BCL-6 and MDM2 were also downregulated by JunD siRNA. As Fra-2, these proto-oncogenes were highly expressed in primary ATL cells but not in normal CD4+ T cells. Collectively, aberrantly expressed Fra-2 in association with JunD may play a major role in CCR4 expression and oncogenesis in ATL.
Assuntos
Proliferação de Células , Antígeno 2 Relacionado a Fos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Receptores CCR4/genética , Sítios de Ligação , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Antígeno 2 Relacionado a Fos/antagonistas & inibidores , Antígeno 2 Relacionado a Fos/metabolismo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/fisiologia , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno/farmacologiaRESUMO
Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1beta or tumor necrosis factor (TNF)-alpha strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1alpha or TNF-alpha. Transient transfection assays using murine LARC promoter-reporter constructs identified a region essential for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-kappaB site located between -96 and -87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta- or TNF-alpha-induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-kappaB bound to this site in 293T cells upon treatment with IL-1beta and TNF-alpha, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1beta. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-kappaB site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB.
Assuntos
Quimiocinas CC/biossíntese , Quimiocinas CC/metabolismo , Mucosa Intestinal/imunologia , Proteínas Inflamatórias de Macrófagos/biossíntese , NF-kappa B/metabolismo , Receptores de Quimiocinas , Animais , Sequência de Bases , Linhagem Celular , Quimiocina CCL20 , Quimiocinas CC/genética , Humanos , Interleucina-1/farmacologia , Camundongos , Dados de Sequência Molecular , Subunidade p50 de NF-kappa B , Receptores CCR6 , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.
Assuntos
Quimiocinas CC , Quimiocinas/farmacologia , Linfócitos/fisiologia , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Linfócitos B/fisiologia , Ligação Competitiva , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL20 , Quimiocinas/metabolismo , Quimiotaxia , Clonagem Molecular , Células-Tronco Hematopoéticas , Humanos , Rim , Cinética , Fígado/metabolismo , RNA Mensageiro/biossíntese , Receptores CCR6 , Receptores de Citocinas/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Liver-and activation-regulated chemokine (LARC)/macrophage inflammatory protein (MIP)-3alpha/CCL20 is a CC chemokine which is constitutively expressed by follicle-associated epithelial cells in the mucosa, and attracts cells expressing CCR6 such as immature dendritic cells and alpha(4)beta(7)(high) intestine-seeking memory T cells. Here, we examine LARC/CCL20 expression in the skin. LARC/CCL20 mRNA and protein were induced in primary human keratinocytes upon stimulation with proinflammatory cytokines such as IL-1alpha and tumor necrosis factor (TNF)-alpha. In mice, intradermal injection of IL-1alpha and TNF-alpha rapidly induced a local accumulation of transcripts for LARC/CCL20 and its receptor CCR6 with a lag of several hours in the latter. In humans, immunostaining of LARC/CCL20 was weak if any in normal skin tissues but strongly augmented in lesional skin tissues with atopic dermatitis. Furthermore, massive infiltration of cells with markers such as CD1a, CD3 or HLA-DR was present in atopic skin lesions. Many infiltrating cells were also found to be CCR6(+) by a newly generated monoclonal anti-CCR6. However, Langerhans cells residing within the epidermis were hardly stained by anti-CCR6 in normal and atopic skin tissues. Furthermore, plasma levels of LARC/CCL20 were found to be elevated in patients with atopic dermatitis. Collectively, our results suggest that epidermal keratinocytes produce LARC/CCL20 upon stimulation with proinflammatory cytokines such as IL-1alpha and TNF-alpha, and attract CCR6-expressing immature dendritic cells and memory/effector T cells into the dermis of inflamed skin such as atopic dermatitis. LARC/CCL20 may not, however, play a major role in homeostatic migration of Langerhans cells into the skin.
Assuntos
Quimiocinas CC/biossíntese , Dermatite Atópica/imunologia , Queratinócitos/metabolismo , Proteínas Inflamatórias de Macrófagos/biossíntese , Adulto , Animais , Linhagem Celular , Células Cultivadas , Quimiocina CCL20 , Quimiocinas CC/sangue , Quimiocinas CC/genética , Quimiotaxia de Leucócito/imunologia , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Humanos , Injeções Intradérmicas , Interleucina-1/administração & dosagem , Queratinócitos/imunologia , Proteínas Inflamatórias de Macrófagos/sangue , Proteínas Inflamatórias de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , Receptores CCR6 , Receptores de Quimiocinas/metabolismo , Pele/imunologia , Pele/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Partial overlapping cDNA sequences likely to encode a novel human CC chemokine were identified from the GenBank Expressed Sequence Tag data base. Using these sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By Northern blot, this chemokine was mainly expressed in liver among various tissues and strongly induced in several human cell lines by phorbol myristate acetate. We thus designated this chemokine as LARC from Liver and Activation-Regulated Chemokine. We mapped the LARC gene close to the chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and radiation hybrid mappings and isolated two yeast artificial chromosome clones containing the LARC gene from this region. To prepare LARC, we subcloned the cDNA into a baculovirus vector and expressed it in insect cells. The secreted protein started at Ala-27 and was significantly chemotactic for lymphocytes. At a concentration of 1 microg/ml, it also showed a weak chemotactic activity for granulocytes. Unlike other CC chemokines, however, LARC was not chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged with secreted alkaline phosphatase-(His)6 bound specifically to lymphocytes, the binding being competed only by LARC and not by other CC or CXC chemokines. Scatchard analysis revealed a single class of receptors for LARC on lymphocytes with a Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new group of CC chemokines localized on chromosome 2.
Assuntos
Quimiocinas CC , Quimiocinas/genética , Cromossomos Humanos Par 2 , Fígado/química , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas , Sequência de Aminoácidos , Baculoviridae , Sequência de Bases , Quimiocina CCL20 , Quimiocinas/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/química , Humanos , Dados de Sequência Molecular , Receptores CCR6 , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21-33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC from secondary lymphoid-tissue chemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nM) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligand chemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.
Assuntos
Quimiocinas CC/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 9 , Linfócitos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL21 , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/biossínteseRESUMO
By searching the expressed sequence tag (EST) data base, we identified partial cDNA sequences encoding a novel human CC chemokine. We determined the complete cDNA sequence that encodes a highly basic polypeptide of a total 98 amino acids with 20 to 30% identity to other human CC chemokines. We termed this novel chemokine from EBI1-Ligand Chemokine as ELC (see below). The ELC mRNA was most strongly expressed in the thymus and lymph nodes. Recombinant ELC protein was expressed as a fusion protein with the Flag tag (ELC-Flag). For receptor-binding assays, recombinant ELC protein fused with the secreted form of alkaline phosphatase (SEAP) was used. By stably expressing five CC chemokine receptors (CCR1 to 5) and five orphan receptors, ELC-SEAP was found to bind specifically to an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3, eotaxin, MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed and secreted), thymus and activation-regulated chemokine (TARC), or liver and activation-regulated chemokine (LARC), competed with ELC-SEAP for EBI1. ELC-Flag-induced transient calcium mobilization and chemotactic responses in EBI1-transfected cells. ELC-Flag also induced chemotaxis in HUT78 cells expressing endogenous EBI1 at high levels. By somatic hybrid and radiation hybrid analyses, the gene for ELC (SCYA19) was mapped to chromosome 9p13 instead of chromosome 17q11.2 where the genes for CC chemokines are clustered. Taken together, ELC is a highly specific ligand for EBI1, which is known to be expressed in activated B and T lymphocytes and strongly up-regulated in B cells infected with Epstein-Barr virus and T cells infected with herpesvirus 6 or 7. ELC and EBI1 may thus play roles in migration and homing of normal lymphocytes, as well as in pathophysiology of lymphocytes infected with these herpesviruses. We propose EBI1 to be designated as CCR7.
Assuntos
Quimiocinas CC , Quimiocinas/genética , Cromossomos Humanos Par 9 , Receptores de Superfície Celular/metabolismo , Receptores de Quimiocinas , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Quimiocina CCL19 , Quimiocinas/metabolismo , Quimiotaxia , Bandeamento Cromossômico , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , Bases de Dados Factuais , Biblioteca Gênica , Humanos , Ligantes , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Receptores CCR7 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Leukocyte trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. Fractalkine, a novel transmembrane molecule with a CX3C-motif chemokine domain atop a mucin stalk, induces both adhesion and migration of leukocytes. Here we identify a seven-transmembrane high-affinity receptor for fractalkine and show that it mediates both the adhesive and migratory functions of fractalkine. The receptor, now termed CX3CR1, requires pertussis toxin-sensitive G protein signaling to induce migration but not to support adhesion, which also occurs without other adhesion molecules but requires the architecture of a chemokine domain atop the mucin stalk. Natural killer cells predominantly express CX3CR1 and respond to fractalkine in both migration and adhesion. Thus, fractalkine and CX3CR1 represent new types of leukocyte trafficking regulators, performing both adhesive and chemotactic functions.
Assuntos
Adesão Celular/imunologia , Movimento Celular/imunologia , Quimiocinas CX3C , Quimiocinas/metabolismo , Leucócitos/citologia , Proteínas de Membrana/metabolismo , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Antígenos CD/análise , Receptor 1 de Quimiocina CX3C , Membrana Celular/imunologia , Células Cultivadas , Quimiocina CX3CL1 , Endotélio Vascular/imunologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Leucemia Eritroblástica Aguda , Subpopulações de Linfócitos , RNA Mensageiro/análise , Receptores de Quimiocinas/metabolismo , Receptores de Citocinas/genética , Receptores de HIV/genética , Transdução de Sinais/imunologia , Células Tumorais Cultivadas , Veias UmbilicaisRESUMO
Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3--4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses.
Assuntos
Quimiocinas CC/metabolismo , Hepatócitos/metabolismo , Receptores CCR5/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Sinalização do Cálcio/imunologia , Linhagem Celular , Quimiocinas CC/biossíntese , Quimiocinas CC/sangue , Quimiocinas CC/fisiologia , Quimiotaxia/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Células de Kupffer , Ligantes , Fígado/metabolismo , Camundongos , Ligação Proteica/imunologia , RNA Mensageiro/biossíntese , Receptores CCR1 , Receptores CCR2 , Receptores de Quimiocinas/fisiologia , Células Tumorais CultivadasRESUMO
By searching the expressed sequence tag (EST) database, we identified partial cDNA sequences encoding a polypeptide with significant sequence identity to the human CC chemokine macrophage-inflammatory protein-1 alpha (MIP-1 alpha)/LD78 alpha. We determined the complete cDNA sequence that contained a reading frame of 89 amino acids with 61% identity to human MIP-1 alpha/LD78 alpha. The mRNA was expressed constitutively at high levels in human lung and at low levels in some lymphoid tissues. Furthermore, the mRNA was strongly induced in several human cell lines, including monocytic U937 cells, by PMA. From these results, we designated this novel CC chemokine as PARC from pulmonary and activation-regulated chemokine. In situ hybridization analyses showed that alveolar macrophages, follicular dendritic cells in the germinal centers of regional lymph nodes, and peripheral blood monocytes stimulated with LPS express PARC mRNA. Using the human CC chemokine yeast artificial chromosome contig that we constructed recently, we mapped the PARC gene (SCYA18) within one of the two subregions of the CC chemokine gene cluster at chromosome 17q11.2. To investigate its biologic activity, the PARC protein was expressed in insect cells. PARC was chemotactic for both activated (CD3+) T cells and nonactivated (CD14-) lymphocytes, but not for monocytes or granulocytes. Binding analysis using PARC fused with alkaline phosphatase-(His)6 showed the presence of a single class of receptors for PARC on lymphocytes with a Kd of 1.9 nM and 590 sites/cell. Thus, PARC is a novel CC chemokine with a close phylogenic relationship with MIP-1 alpha/LD78 alpha, but with a highly selective activity on lymphocytes.