RESUMO
Respiratory Syncytial Virus (RSV) infection is an important cause of severe infant bronchiolitis, partly due to lower airway inflammation orchestrated by virus-induced chemokine secretion. Chemokine receptors may therefore be therapeutic targets. We investigated RSV-induced chemokine receptor (CCR) 1, 2 and 5 surface expressions in a cellular model and in infants. RSV infection increased human monocytic CCR1, 2 and 5 expression, as assessed by FACS, via replication-dependent mechanisms. CCR1 and CCR5 levels peaked at 36 h and CCR2 levels at 48 h. Monocytes from infants with RSV-bronchiolitis significantly increased CCR1 expression after ex vivo RSV infection compared to controls. Expression of CCR5 also increased, and correlated with CCR1 expression (r=0.78, p<0.0001). CCR1 upregulation correlated with disease severity markers. Monocyte CCR1 receptors were functionally active as stimulation resulted in calcium influx. CCR1/5 blocking strategies may be useful in decreasing cellular inflammation in RSV infection.
Assuntos
Monócitos/metabolismo , Receptores CCR1/biossíntese , Receptores CCR2/biossíntese , Receptores CCR5/biossíntese , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Lactente , Masculino , Regulação para CimaRESUMO
Respiratory syncytial virus (RSV) infection can cause extensive airway inflammation, which is orchestrated by chemokines and their receptors. RSV-infected epithelial cells secrete many cytokines and chemokines, but little is known about regulation of chemokine receptors on target cells. We investigated the effects of conditioned media (CM) from RSV-infected epithelial cells on monocyte CCR1, CCR2, and CCR5 expression. RSV-CM but not control-CM stimulated a biphasic increase in cell-surface CCR1, and levels peaked at 36 h and 96 h poststimulation. Similar CCR1 up-regulation occurred on monocyte-derived macrophages. Cytochlasin D and colchicine blocked both peaks of expression, demonstrating requirement of a functional cytoskeleton. Intracellular staining revealed little internal sequestration of CCR1 protein, and CCR1 up-regulation was inhibited by actinomycin D and cycloheximide, indicating that both waves of RSV-CM-induced surface CCR1 expression were dependent on de novo transcription and protein synthesis. Cytokine-neutralizing experiments showed that the effects of RSV-CM were decreased by blocking TNF-alpha (percent inhibition=51+/-2.3% at 36 h peak and 42+/-7.7% at 96 h peak) and to a lesser extent, IL-1 (percent inhibition=32+/-7.2% at 36 h and 23+/-2.9% at 96 h). In summary, RSV-CM causes a biphasic up-regulation of surface CCR1 on monocytes, which is dependent on an intact cytoskeleton, requires new gene transcription and protein synthesis, and is mediated in part by the proinflammatory cytokines TNF-alpha and IL-1.
Assuntos
Células Epiteliais/metabolismo , Monócitos/metabolismo , Receptores de Quimiocinas/biossíntese , Mucosa Respiratória/metabolismo , Vírus Sinciciais Respiratórios/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Cicloeximida/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Dactinomicina/farmacologia , Células Epiteliais/virologia , Humanos , Interleucina-1/fisiologia , Macrófagos/imunologia , Macrófagos/virologia , Receptores CCR1 , Receptores CCR2 , Receptores CCR3 , Receptores de Quimiocinas/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Regulação para CimaRESUMO
We report here the genomic organization and phylogenic relationships of CD109, a member of the the alpha2-macroglobulin/complement (AMCOM) gene family. CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors. We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence. The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence. Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members. CD109 also shares motifs with certain other AMCOM members including: (1) a thioester 'GCGEQ" motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus. Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C. elegans (ZK337.1) and E. coli homologs, we propose CD109 represents a novel and independent branch of the alpha2-macroglobulin/complement gene family (AMCOM) and may be its oldest member.
Assuntos
Antígenos CD/genética , Proteínas do Sistema Complemento/genética , alfa-Macroglobulinas/genética , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Células CHO , Linhagem Celular Tumoral , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , DNA Complementar/química , DNA Complementar/genética , Evolução Molecular , Éxons , Proteínas Ligadas por GPI , Genes/genética , Humanos , Íntrons , Dados de Sequência Molecular , Família Multigênica/genética , Proteínas de Neoplasias , Fosfatidilinositol Diacilglicerol-Liase/metabolismo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.