RESUMEN
CCL18 and CXCL12 are homeostatic chemokines with high constitutive concentrations in serum. Elevated levels of CCL18 have been described in various diseases including childhood acute lymphocytic leukemia (ALL) but its functions remain poorly characterized. Its receptor has not been identified, but functional cellular responses like lymphocyte chemotaxis have been described. CXCL12 is a pivotal chemokine for hematopoiesis and B cell homing processes. We demonstrate that CCL18 interferes with CXCL12-mediated pre-B ALL cell activation. CXCL12-induced calcium mobilization, chemotaxis, pseudo-emperipolesis and cellular proliferation could be significantly reduced by CCL18 in pre-B ALL cell lines. The results could be observed in primary cells from patients suffering from pre-B ALL, but not in cells from patients suffering from common ALL. Direct effects of CCL18 on the receptor for CXCL12, CXCR4, could be excluded. Moreover, we found that CCL18 modulations of CXCL12-induced responses are mediated through the chemokine-like receptor GPR30. CCL18 bound to GPR30 expressing cells, and antibodies against GPR30 abolished this binding as well as CCL18-mediated functional effects. We also observed that, CCL18 interferes with the activation of GPR30 by previously identified ligands (17ß-estradiol and chemical agonists). We therefore suggest that CCL18 is an important modulator of CXCR4-dependent responses in pre-B ALL cells via interactions with GPR30.
Asunto(s)
Quimiocinas CC/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Células Precursoras de Linfocitos B/inmunología , Receptores CXCR4/metabolismo , Transducción de Señal , Animales , Apoptosis , Células COS , Señalización del Calcio , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito , Chlorocebus aethiops , Estradiol/metabolismo , Antagonistas de Estrógenos/farmacología , Humanos , Ligandos , Activación de Linfocitos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/patología , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to G(i). In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.
Asunto(s)
Toxinas Botulínicas , Quimiocinas CXC , Endotelio Vascular/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/farmacología , Proteínas de Unión al GTP Monoméricas/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , ADP Ribosa Transferasas , Actinas/metabolismo , Anticuerpos/farmacología , Células Cultivadas , Quimiocina CXCL1 , Factores Quimiotácticos/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Pulmón/irrigación sanguínea , Pulmón/citología , Microcirculación/citología , Microcirculación/efectos de los fármacos , Microcirculación/metabolismo , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Toxina del Pertussis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8B/antagonistas & inhibidores , Transducción de Señal/fisiología , Fibras de Estrés/metabolismo , Factores de Virulencia de Bordetella/farmacología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-alpha (MGSA/GROalpha), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROalpha was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.
Asunto(s)
Comunicación Autocrina/inmunología , Quimiocinas CXC , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/metabolismo , Monocitos/enzimología , Monocitos/inmunología , Anticuerpos Monoclonales , Quimiocina CXCL1 , Factores Quimiotácticos/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Citometría de Flujo , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Humanos , Interleucina-8/genética , Interleucina-8/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/química , Neumonía/inmunología , Neumonía/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/inmunología , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Interleucina-8A/análisis , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8B/análisis , Receptores de Interleucina-8B/inmunología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
A human pancreatic cancer cell line, Capan-1, secretes the chemokines interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha). Capan-1 cells also express the chemokine receptor 2 (CXCR2), which is a Gialpha-protein coupled receptor. Growth of Capan-1 cells was inhibited when anti-IL-8 or anti-GROalpha monoclonal antibody was added into the culture medium. Pertussis toxin, which blocks Gialpha also demonstrated a growth-inhibitory effect on Capan-1 cells. These results indicated that IL-8 and GROalpha act on Capan-1 cells as growth factors in an autocrine manner through CXCR2.
Asunto(s)
División Celular , Quimiocinas CXC , Factores Quimiotácticos/fisiología , Inhibidores de Crecimiento/fisiología , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/fisiología , Transcripción Genética , Anticuerpos Monoclonales/farmacología , División Celular/efectos de los fármacos , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Inhibidores de Crecimiento/genética , Sustancias de Crecimiento/genética , Humanos , Interleucina-8/genética , Neoplasias Pancreáticas , Toxina del Pertussis , Receptores de Interleucina-8B/genética , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The CXC-chemokines Groalpha and interleukin-8 (IL-8) are well characterized growth factors for melanoma cells. Here the constitutive expression of Groalpha, IL-8 and their receptors (CXCR1 and CXCR2) as well as their functional involvement in the proliferation response were analyzed in normal keratinocytes and epidermoid carcinoma cell lines A431 and KB. Flow cytometric measurements, ELISA and semi-quantitative RT-PCR revealed low constitutive protein secretion and mRNA expression of both CXC-chemokines as well as CXCR1 and 2 in normal keratinocytes, whereas significant higher levels of CXC-chemokines and CXCR2 were deteced in epidermoid carcinoma cells. Proliferation of epidermoid carcinoma cells could be induced by CXC-chemokines and constitutive proliferation could be inhibited by neutralizing antibodies against CXC-chemokines and CXCR2. These studies indicate that constitutive Groalpha, IL-8 and CXCR2 protein expression enable an autocrine growth mechanism in epidermoid carcinoma cells.
Asunto(s)
Antígenos CD/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Factores Quimiotácticos/biosíntesis , Sustancias de Crecimiento/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/biosíntesis , Queratinocitos/metabolismo , Receptores de Quimiocina/biosíntesis , Receptores de Interleucina/biosíntesis , Neoplasias Cutáneas/metabolismo , Comunicación Autocrina , Carcinoma de Células Escamosas/patología , División Celular , Quimiocina CXCL1 , Quimiocinas CXC/biosíntesis , Humanos , Queratinocitos/patología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Interleucina-8A , Receptores de Interleucina-8B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
The chemokine receptor CXCR2 is the closest homologue to Kaposi's sarcoma herpesvirus-G protein-coupled receptor (KSHV-GPCR), which is known to be constitutively activated and able to cause oncogenic transformation. Among G protein-coupled receptors, a DRY sequence in the second intracellular loop is highly conserved. However, the KSHV-GPCR shows a VRY sequence instead. In this study, we exchanged Asp138 of the DRY sequence in the CXCR2 with a Val (D138V), the corresponding amino acid in KSHV-GPCR, or with a Gln (D138Q), and investigated the functional consequences of these mutations. In focus formation and soft agar growth assays in NIH 3T3 cells, the D138V mutant exhibited transforming potential similar to the KSHV-GPCR. Surprisingly, the CXCR2 wild type itself showed transforming activity, although not as potently, due to continuous autocrine stimulation, whereas the D138Q mutant formed no foci. In agreement with these results were high levels of inositol phosphate accumulation in the D138V mutant and the KSHV-GPCR, indicating constitutive activity. These data emphasize the importance of the DRY sequence for G protein-coupled signaling of the CXCR2. Either constitutive activation or persistent autocrine stimulation of the CXCR2 causes transformation similar to KSHV-GPCR-transfected cells, probably activating the same signal transduction cascade that can abrogate normal growth control mechanisms.
Asunto(s)
Transformación Celular Neoplásica/genética , Quimiocinas CXC , Herpesvirus Humano 8/genética , Mutación Puntual , Receptores de Quimiocina/genética , Sarcoma de Kaposi/genética , Transducción de Señal/genética , Células 3T3 , Actinas/metabolismo , Agar , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , División Celular/genética , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Quimiocinas CXC/genética , Inhibición de Contacto/genética , Humanos , Fosfatos de Inositol/metabolismo , Ratones , Datos de Secuencia Molecular , Ratas , Sarcoma de Kaposi/inmunología , Sarcoma de Kaposi/metabolismo , Transducción de Señal/inmunología , Células Tumorales CultivadasRESUMEN
As a model of the meconium aspiration syndrome (MAS) of human infants, adult rabbits and newborn rhesus monkeys received intratracheal instillation of human meconium to induce pulmonary injury. Injured rabbits were ventilated with 100% O2 and divided into four treatment groups, receiving: 1) bronchoalveolar lavages (BAL) with dilute KL4-Surfactant; 2) lavages with equal volumes of sterile saline; 3) a single intratracheal bolus of KL4-Surfactant, 100 mg/kg; and 4) no treatment. The untreated rabbits developed atelectasis, a fall in pressure-volume levels and in partial pressure of O2 in arterial blood (PaO2) from approximately 500 to < 100 mm Hg, and severe pulmonary inflammation between 3 and 5 h after instillation of meconium. Rabbits treated by BAL with dilute KL4-Surfactant showed rapid and sustained recovery of PaO2 to approximately 300 mm Hg within minutes, a return toward normal pressure-volume levels, and diminished inflammation. Rabbits receiving BAL with saline failed to show recovery, and rabbits treated with a bolus of surfactant intratracheally exhibited a transient response by 1-2 h after treatment, but then returned to the initial atelectatic state. Newborn rhesus monkeys, after receiving human meconium intratracheally before the first breath, developed severe loss of pulmonary function. Treatment of these monkeys 1-5 h after birth with BAL with dilute KL4-Surfactant produced clearing of chest radiographs and a rapid improvement in pulmonary function with ratios of partial pressure of O2 in arterial blood to the fraction of O2 in the inspired air rising into the normal range where they remained through the 20-h period of study. The studies indicate that pulmonary function in two models of severe meconium injury respond rapidly to BAL with dilute KL4-Surfactant.
Asunto(s)
Lavado Broncoalveolar , Modelos Animales de Enfermedad , Síndrome de Aspiración de Meconio/terapia , Péptidos/uso terapéutico , Surfactantes Pulmonares/uso terapéutico , Animales , Animales Recién Nacidos , Humanos , Recién Nacido , Instilación de Medicamentos , Péptidos y Proteínas de Señalización Intercelular , Macaca mulatta , Neumonía/prevención & control , Intercambio Gaseoso Pulmonar , Conejos , TráqueaRESUMEN
The work presented here demonstrates that human complement factor H is an adhesion ligand for human neutrophils but not for eosinophils. The adherence of polymorphonuclear leukocytes (PMNs) to plastic wells coated with factor H depended on divalent metal ions and was augmented by C5a and TNF-alpha. PMN adhesion to factor H in the presence or absence of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity purification using factor H Sepharose followed by immunoprecipitation using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as a factor H binding receptor. The presence of surface bound factor H enhanced neutrophil activation resulting in a two- to fivefold increase in the generation of hydrogen peroxide by PMNs stimulated by C5a or TNF-alpha. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells adhered to immobilized heparin or chondroitin A. In addition, augmented adhesion of PMNs was measured when factor H, but not HSA or C9, was absorbed to wells that were first coated with heparin or chondroitin A. The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs to CD11b and CD18. These studies demonstrate that factor H is an adhesion molecule for human neutrophils and suggest that the interaction of factor H with glycosaminoglycans may facilitate the tethering of this protein in tissues allowing factor H to serve as a neutrophil adhesion ligand in vivo.
Asunto(s)
Antígenos CD18/metabolismo , Factor H de Complemento/metabolismo , Antígeno de Macrófago-1/metabolismo , Neutrófilos/inmunología , Anticuerpos Monoclonales/farmacología , Proteínas Sanguíneas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Complemento C5a/farmacología , Factor H de Complemento/farmacología , Glicosaminoglicanos/inmunología , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacología , Humanos , Técnicas In Vitro , Interleucina-8/farmacología , Lactoferrina/metabolismo , Ligandos , Antígeno de Macrófago-1/aislamiento & purificación , Neutrófilos/citología , Neutrófilos/metabolismo , Oxidantes/metabolismo , Unión Proteica , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The CXCR2 is phosphorylated at the C-terminal intracytoplasmic portion within 15 sec following the addition of IL-8 or MGSA. Cells transfected with a truncated form of the receptor missing the last 12 amino acids (T3) showed normal binding affinity, but were no longer phosphorylated; individual alanine replacement indicated that Ser346 and 348 were the primary sites of phosphorylation. In studies of the importance of phosphorylation in CXCR2 desensitization, cells expressing wild type CXCR2 lost GTP gamma S binding above basal rate after the first exposure to IL-8, while cells with the T3 mutant retained 60% of their capacity to induce GTP gamma S exchange upon a second exposure to IL-8. In contrast, receptor internalization was not affected by the loss of phosphorylation of the T3 mutant. Further receptor truncation led to decreasing binding affinities for IL-8 and MGSA and a decreased rate of GTP gamma S exchange following addition of excess ligand which suggests involvement of this region in G-protein coupling.
Asunto(s)
Interleucina-8/metabolismo , Receptores de Quimiocina/fisiología , Receptores de Interleucina/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Leucemia Basofílica Aguda , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Ratas , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8B , Eliminación de Secuencia , Serina/genética , Serina/fisiología , Transducción de Señal/genética , Radioisótopos de Azufre/metabolismo , Células Tumorales CultivadasAsunto(s)
Inflamación/fisiopatología , Interleucina-8/fisiología , Neutrófilos/fisiología , Receptores de Interleucina/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Heparitina Sulfato/metabolismo , Humanos , Técnicas In Vitro , Interleucina-8/química , Ligandos , Datos de Secuencia Molecular , Receptores de Interleucina/químicaRESUMEN
The chemokines interleukin-8 (IL-8) and GRO alpha bind in neutrophils to the interleukin-8 receptor alpha and beta (IL-8R alpha and beta) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R alpha and beta to pertussis toxin-insensitive G alpha 16-proteins as well as to pertussis toxin-sensitive G alpha i2- or G alpha i3-proteins. To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing G alpha 16-, G alpha i2- and G alpha i3-proteins were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R alpha and beta was confirmed by binding studies with radiolabeled ligands. IL-8R alpha bound IL-8 with high affinity (Kd approximately 1 nM) and GRO alpha with low affinity (Kd approximately 1 microM), whereas IL-8R beta bound both IL-8 and GRO alpha with high affinity (Kd approximately 1nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of G alpha i2- or G alpha i3-proteins, but not G alpha 16-proteins in this response.
Asunto(s)
Actinas/metabolismo , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Células HL-60/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/farmacología , Adenosina Difosfato Ribosa/química , Antígenos CD/genética , Antígenos CD/metabolismo , Secuencia de Bases , Unión Competitiva , Quimiocina CXCL1 , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiocinas/farmacología , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , ADN Complementario/química , ADN Complementario/metabolismo , Activación Enzimática/efectos de los fármacos , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Células HL-60/citología , Células HL-60/metabolismo , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Toxina del Pertussis , Reacción en Cadena de la Polimerasa , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8A , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/toxicidadRESUMEN
The chemokine GRO alpha is an autocrine growth factor for melanoma cells. Although GRO alpha has been identified as a high affinity ligand for the IL-8 receptor beta (IL-8R beta) in recombinant systems, the receptor mediating its action in melanoma cells has been a matter of debate. Here, we show by reverse transcription and PCR expression of IL-8R beta, mRNA transcripts in different melanoma cell lines and in normal human melanocytes. To characterize the role of the IL-8R beta in melanoma cells, antiserum was raised in rabbits against a fusion protein containing the NH2-terminal portion of the receptor. Its specificity was shown by flow cytometry with IL-8R beta-transfected HL60 cells. A specific epitope could be mapped with IL-8R beta mutants to the peptide sequence between ASP-4 and ASP-14 of this receptor. Binding studies with [125I]GRO alpha in IL-8R beta transfectants indicated ligand antagonistic properties of this Ab. Expression of IL-8R beta protein at the cell surface of various melanoma cell lines could be shown by flow cytometry with F(ab')2 fragments of the IL-8R beta antiserum. Moreover, anti-IL-8R beta Ab partially blocked specific binding of [125I]GRO alpha in various melanoma cell lines. Addition of F(ab')2 fragments of the IL-8R beta antiserum or of neutralizing anti-GRO alpha mAb to different melanoma cell lines identified this GRO alpha-IL-8R beta interaction as a major component required for serum-independent melanoma cell growth.
Asunto(s)
Antígenos CD/fisiología , Quimiocinas CXC , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Melanoma/inmunología , Receptores de Interleucina/fisiología , Antígenos CD/inmunología , Secuencia de Bases , Unión Competitiva/inmunología , División Celular/efectos de los fármacos , División Celular/fisiología , Quimiocina CXCL1 , Factores Quimiotácticos/fisiología , Sustancias de Crecimiento/inmunología , Humanos , Melanoma/genética , Melanoma/patología , Datos de Secuencia Molecular , Receptores de Interleucina/inmunología , Receptores de Interleucina-8A , Células Tumorales CultivadasRESUMEN
Interleukin-8 (IL-8) and GRO alpha are leukocyte-attracting peptides of the chemokine family. To study the priming potential of these chemokines, we measured superoxide anion production and up-regulation of N-formyl peptide receptors in human neutrophils. IL-8 and GRO alpha themselves did not stimulate production of significant amounts of superoxide anions but potentiated N-formyl peptide-induced superoxide anion production in a concentration-dependent manner. Binding measurements by flow cytometry at 37 degrees C with fluorescein-labeled N-formyl peptide revealed enhanced total N-formyl peptide binding after pretreatment of neutrophils with IL-8 and GRO alpha. Binding measurements performed at 4 degrees C indicated that the chemokines stimulated the up-regulation of N-formyl peptide receptors at the cell surface but did not alter their affinity for the ligand. This study indicates that IL-8 and GRO alpha, in addition to their known chemotactic activity, prime neutrophils for superoxide anion production, presumably by up-regulating the number of receptors for strong superoxide-anion-triggering stimuli.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/farmacología , Neutrófilos/metabolismo , Receptores Inmunológicos/fisiología , Receptores de Péptidos/fisiología , Superóxidos/metabolismo , Quimiocina CXCL1 , Humanos , Neutrófilos/efectos de los fármacos , Receptores de Formil Péptido , Regulación hacia Arriba/fisiologíaRESUMEN
Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His13 and Thr15 with Tyr13 and Lys15 of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca2+ mobilization. As a corollary, replacement of the Tyr13 and Lys15 of the human IL-8 with His13 and Thr15 of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr13 and Lys15 sequence was found to be in the NH2-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.
Asunto(s)
Quimiocinas CXC , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/química , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Calcio/metabolismo , Quimiocina CXCL1 , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Interleucina-8/metabolismo , Células L , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Conejos , Receptores de Interleucina-8A , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
The neutrophil chemotaxins, complement fragment C5a (C5a) and GRO alpha, induced the mobilization of Ca2+ from intracellular stores and the polymerization of actin in human neutrophils as assayed by flow cytometric measurements. [Ca2+]i-transients developed as an "all-or-none" response. Individual neutrophils required different threshold concentrations of added ligand to induce [Ca2+]i-transients which were then always maximal. In contrast, chemotaxin-induced formation of actin filaments in single neutrophils occurred in a dose-dependent manner. Pertussis toxin blocked chemotaxin-induced actin polymerization and [Ca2+]i-transients indicating that both cell responses shared initial activation steps such as ligand binding and activation of guanine nucleotide-binding proteins (G-proteins).
Asunto(s)
Actinas/biosíntesis , Calcio/metabolismo , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Complemento C5a/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Neutrófilos/metabolismo , Quimiocina CXCL1 , Complemento C5a/antagonistas & inhibidores , Citometría de Flujo , Proteínas de Unión al GTP/metabolismo , Humanos , Técnicas In Vitro , Cinética , Neutrófilos/efectos de los fármacos , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The cytokine melanoma growth-stimulating activity (MGSA) is a growth factor for melanoma cells and a chemotaxin for neutrophils. Known purification procedures of MGSA from human sources or expression systems give a low yield and require multiple chromatography steps. Here, a fast and high-yield method for the purification of recombinant MGSA is described. Approximately 500 micrograms MGSA were recovered from the bacterial lysate of a 10 liter culture within a day. For this purpose, total mRNA of Hs294T melanoma cells was isolated and cDNA of MGSA was obtained by reverse transcription and polymerase chain reaction. The cDNA of MGSA was subcloned into the expression vector pGEX-2T, generating a fusion with the Schistosoma japonicum glutathione S-transferase gene. The fusion protein was expressed in E. coli DH5a and purified from the bacterial lysate using glutathione-sepharose beads. MGSA was cleaved from the complex of fusion protein and glutathione-sepharose beads with thrombin and purified to homogeneity by anion-exchange high-performance liquid chromatography with a Mono-S-column. The bioactivity of the recombinant MGSA was assessed by chemotactic migration and triggered [Ca2+]i-transients in human neutrophils. In addition, [125I]MGSA bound specifically to undifferentiated human leukemia cells HL-60 transfected with the cDNA of the interleukin-8 (IL-8) receptor beta with similar properties as [125I]IL-8. Thus, this described method might be a powerful tool to generate large amounts of cytokines in a short time.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Sustancias de Crecimiento/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular , Secuencia de Aminoácidos , Secuencia de Bases , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Clonación Molecular , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Amplificación de Genes , Sustancias de Crecimiento/genética , Humanos , Leucemia Mieloide/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Signal transduction of interleukin-8 (IL-8) was analyzed in neutrophils, and compared with the well known neutrophil activator N-formyl peptide. Stimulation of human neutrophils with IL-8 induced a rapid polymerization of actin as detected by 7-nitrobenz-2-oxa-1,3-diazol-(NBD)-phallacidin staining of f-actin and reduction of monitored right-angle light scatter. Actin polymerization peaked within 10 seconds after the addition of IL-8 and was short-lived as compared to N-formyl peptide-induced stimulation. Analysis of phospholipids by thin-layer chromatography and analysis of deacylation products of lipid extracts by high-pressure liquid chromatography (HPLC) showed that IL-8 triggered a rapid rise of [32P]phosphatidyl-inositol(3,4,5)trisphosphate (PtdInsP3) followed by a slower increase of [32P]phosphatidylinositol(3,4)bisphosphate (PtdIns-3,4-P2) along with a rapid decrease of [32P]phosphatidylinositol(4,5)bisphosphate (PtdIns-4,5-P2). Changes in polyphosphoinositide metabolism were more moderate and transient than those obtained by N-formyl peptide. Moreover, [32P]phosphatidic acid (PA) production stimulated by IL-8 was minimal and transient as compared to the response activated by N-formyl peptide. Both IL-8 and N-formyl peptide induced Ca++ mobilization from intracellular stores, but IL-8 in contrast to N-formyl peptide failed to trigger the secondary influx of Ca++ from the extracellular medium. In summary, IL-8 and N-formyl peptide stimulated similar and distinct patterns of intracellular activation steps. This study indicates that IL-8 is a potent activator of intracellular events presumably required for chemotaxis, but a relatively weak activator for events associated with superoxide anion generation and proinflammatory activity.
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Interleucina-8/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Fosfolípidos/metabolismo , Humanos , Luz , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/sangre , Radioisótopos de Fósforo/sangre , Polímeros , Proteínas Recombinantes/farmacología , Dispersión de RadiaciónRESUMEN
To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Quimiocina CXCL1 , Humanos , Interleucina-8/química , Datos de Secuencia Molecular , Mutación , Receptores de Interleucina-8A , Relación Estructura-ActividadRESUMEN
Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastoma cells for therapy in combination with reactive oxygen intermediates. In the present study the effects of 6-hydroxydopamine (6-OHDA) and H2O2 on metabolic parameters critical for cell survival were investigated in cells with low and high ferritin content in the presence and absence of ascorbate. Human neuroblastoma SK-N-SH cells were pretreated with 100 microM FeSO4 and 10 microM desferrioxamine, respectively, for 24 h yielding cells with different ferritin contents. The effects of 6-OHDA and H2O2 (25 microM-250 microM) in the absence and presence of 1 mM ascorbic acid on DNA strand break formation, activation of poly(ADP-ribose) polymerase, and finally decrease in NAD+ and ATP concentration were investigated. All these parameters were influenced by 6-OHDA and H2O2 in a concentration-dependent manner in a similar way. The effects were most pronounced in ferritin-rich cells and in the presence of ascorbic acid. Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid caused strand breaks that were prevented in the presence of mannitol or desferrithiocine. H2O2-mediated strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by others a model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application of a high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.
Asunto(s)
Ácido Ascórbico/farmacología , Daño del ADN , ADN/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Neuroblastoma/metabolismo , Oxidopamina/farmacología , Adenosina Trifosfato/análisis , Ácido Ascórbico/farmacocinética , Humanos , Hierro/análisis , NAD/análisis , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Poli(ADP-Ribosa) Polimerasas/análisis , Células Tumorales CultivadasRESUMEN
HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.