RESUMEN
The chemokine signaling system, which coordinates the basal and emergency trafficking of leukocytes, presumably coevolved with the hematopoietic system. To study its phylogenetic origins, we used the open reading frame (ORF) of the human chemokine receptor CXCR4 as a genomic probe, since in mammals it is the most highly conserved chemokine receptor known. CXCR4 cross-hybridized to genomic DNA from mouse and chicken, but not zebrafish, Drosophila, or Caenorhabditis elegans. Accordingly, we cloned the corresponding chicken cDNA. The ORF is 359 codons long versus 352 for human CXCR4, and encodes a protein 82% identical to human CXCR4. In a calcium flux assay of receptor function, CHO-K1 cells stably transfected with the chicken cDNA responded specifically to human SDF-1, the specific ligand for CXCR4, but not to a panel of other chemokines tested at 100 nM. SDF-1 activated the cells in a dose-dependent manner (EC50 approximately 5 nM), whereas parental CHO-K1 cells did not respond. The CHO-K1 cell transfectants also bound 125I-SDF-1 specifically. Leukocytes from chicken peripheral blood expressed chCXCR4 mRNA and responded to human SDF-1 in a calcium flux assay with an EC50 similar to that for chCXCR4-transfected CHO cells, suggesting that this response is mediated by native chCXCR4. Analysis of chicken genomic DNA with the chicken cDNA as probe revealed a pattern consistent with a single copy gene, and the absence of any closely related genes. mRNA was detected in brain, bursa, liver, small and large intestine, embryonal fibroblasts, and blood leukocytes, but not in stomach or pancreas. These results, which identify the first functional non-viral, non-mammalian chemokine receptor, suggest that the origins of a functional chemokine system extend at least to birds and suggest that, as in mammals, CXCR4 functions in many avian tissues.
Asunto(s)
Pollos/genética , Pollos/inmunología , ARN Mensajero/metabolismo , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Secuencia de Aminoácidos , Animales , Células CHO , Quimiocina CXCL12 , Quimiocinas CXC/agonistas , Clonación Molecular , Secuencia Conservada , Cricetinae , Dosificación de Gen , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Filogenia , Receptores CXCR4/metabolismo , Receptores CXCR4/fisiología , Receptores del VIH/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
T-20, a synthetic peptide corresponding to the heptad repeat sequence of HIV-1 gp41, blocks HIV-1 entry by targeting gp41, and is currently in clinical trials as an anti-retroviral agent. We recently reported that in vitro T-20 also functions as a phagocyte chemoattractant and a chemotactic agonist at the phagocyte N-formylpeptide receptor (FPR). Here we show that T-20 is also a potent chemotactic agonist in vitro at a related human phagocyte receptor FPRL1R. To test the relative importance of FPR and FPRL1R in primary cells, we identified the corresponding mouse T-20 receptors, mFPR and FPR2, which are both expressed in neutrophils, and compared T-20 action on neutrophils from wild type and mFPR knockout mice. Surprisingly, although T-20 activates mFPR and FPR2 in transfected cells with equal potency and efficacy in both calcium flux and chemotaxis assays, neutrophils from mFPR knockout mice did not respond to T-20. These results provide genetic evidence that FPR is the major phagocyte T-20 receptor in vivo and point to the potential feasibility of studying T-20 effects on immunity in a mouse model. This may help define the cause of local inflammation after T-20 injection that has recently been reported in Phase I clinical trials.
Asunto(s)
Fármacos Anti-VIH/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Proteína gp41 de Envoltorio del VIH/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Animales , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/química , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quimiotaxis de Leucocito/inmunología , Relación Dosis-Respuesta a Droga , Enfuvirtida , Proteína gp41 de Envoltorio del VIH/efectos adversos , Proteína gp41 de Envoltorio del VIH/química , Humanos , Inflamación/inducido químicamente , Ratones , Ratones Noqueados , Familia de Multigenes/genética , N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/inmunología , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/química , Receptores de Formil Péptido , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Receptores de Péptidos/agonistas , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , TransfecciónRESUMEN
The N-formylpeptide receptor (FPR) is a G protein-coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC(50)s, approximately 5 microM for calcium flux and chemotaxis, were approximately 100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.