RESUMEN
INTRODUCTION: SPL84 is an inhaled antisense oligonucleotide (ASO) in development for the treatment of cystic fibrosis (CF) patients carrying the 3849 + 10kb C->T (3849) mutation. To support the initiation of the first clinical study, a full battery of safety and toxicology studies were performed. RESEARCH DESIGN AND METHODS: SPL84 was administered by inhalation to mice and monkeys to determine the no observed adverse effect level (NOAEL) and establish sufficient safety margins for the starting clinical dose. RESULTS: There were no preclinical safety findings with SPL84; no related clinical signs, nor any effect on body weight, food consumption, or clinical pathology. The microscopic changes in the lungs were regarded as non-adverse and reflected a normal clearance process for inhaled compounds. Systemic exposure in both species was low. The NOAEL for mice and monkeys was the highest administered dose in both species, resulting in safety margins ~ 40X the proposed starting clinical dose. CONCLUSION: These successful results supported the initiation of a phase 1/2 clinical study of SPL84 (ongoing), assessing the safety, tolerability, and pharmacokinetics of a single ascending dose in healthy subjects to be followed by assessment of safety, tolerability, pharmacokinetics, and preliminary efficacy of multiple ascending doses in CF patients carrying the 3849 mutation.
Asunto(s)
Fibrosis Quística , Oligonucleótidos Antisentido , Animales , Humanos , Ratones , Administración por Inhalación , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Pulmón , Mutación , Oligonucleótidos Antisentido/efectos adversos , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como AsuntoRESUMEN
Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led to increased numbers of ASO regulatory approvals. In this study, we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease cystic fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when targeting the cell nucleus. The distribution of SPL84 throughout the lungs, penetration into the epithelial cells and nucleus, and structural stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of our inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL84 through hyperconcentrated mucus was also demonstrated. Our results, supported by a promising preclinical pharmacological effect of full restoration of cystic fibrosis transmembrane conductance regulator channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients. In addition, successfully tackling the lung distribution of SPL84 offers immense opportunities for further development of SpliSense's inhaled ASO-based drugs for unmet needs in pulmonary diseases.
Asunto(s)
Fibrosis Quística , Humanos , Ratones , Animales , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Oligonucleótidos Antisentido/farmacología , Preparaciones Farmacéuticas , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , PulmónRESUMEN
Daily injections of growth hormone (GH) as replacement therapy in GH-deficient (GHD) patients may cause poor compliance and inconvenience. C-terminal peptide-modified human GH (MOD-4023) has been developed for once-weekly administration in GHD adults and children. In the present study, the pharmacokinetics (PK) and pharmacodynamics (PD) of a single subcutaneous dose of MOD-4023 were evaluated in healthy Caucasian and Japanese adults, using a phase 1 double-blind, vehicle-controlled, randomized study design. The study was conducted in 42 healthy Japanese (n = 21) and Caucasian (n = 21) men receiving either MOD-4023 at a dose of 2.5, 7.5, or 15 mg or vehicle. In the 2.5- and 7.5-mg cohorts, no differences in mean MOD-4023 serum concentration were found between Japanese and Caucasian subjects. A comparison of PK parameters in the 15-mg group suggests a slower absorption rate of MOD-4023 in Japanese subjects. PD analysis showed no apparent differences in IGF-1 and IGFBP-3 plasma concentrations between the Japanese and Caucasian subjects and indicated that a dose of 15 mg achieved the maximal effect in both ethnic groups. MOD-4023 demonstrated a favorable safety profile and local tolerance following single-dose subcutaneous administration. This study provides additional support for the development of MOD-4023 as a long-acting human growth hormone formulation for once-weekly administration.
Asunto(s)
Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/farmacocinética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Adulto , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Composición de Medicamentos , Femenino , Voluntarios Sanos , Hormona de Crecimiento Humana/farmacología , Humanos , Inyecciones Subcutáneas , Japón , Masculino , Cooperación del Paciente , Población Blanca/estadística & datos numéricos , Adulto JovenRESUMEN
BACKGROUND/AIMS: MOD-4023 is a long-acting human growth hormone (hGH) in clinical trials for the treatment of growth hormone deficiency (GHD). A key goal is maintenance of serum concentrations of insulin-like growth factor (IGF) 1 within normal range throughout GH dosing. The study aimed to develop a pharmacokinetic model for MOD-4023 and a pharmacodynamic model for the effect of MOD-4023 on IGF-1 to allow estimation of peak and mean IGF-1 and to identify the optimal IGF-1 sampling day. METHODS: MOD-4023 (0.25, 0.48, or 0.66 mg/kg) was administered weekly for 12 months to 41 GH-naive GHD children (age 3-11 years). The control group (n = 11, age 4-9 years) received daily recombinant human growth hormone (r-hGH; 34 µg/kg). Sparse samples (4/subject) were obtained to determine serum concentrations of MOD-4023 or r-hGH and IGF-1. RESULTS: A 2-compartment pharmacokinetic model with first-order absorption fit MOD-4023 data well; a 1-compartment model was appropriate for r-hGH. For both, weight-normalized systemic parameters were preferred over allometric scaling. For MOD-4023, an indirect model fit IGF-1 SDS data well; baseline IGF-1 increased over time. At steady state, samples obtained 4 days following dose administration predicted mean IGF-1 SDS during the dosing interval well. CONCLUSION: The IGF-1 profile is consistent with the weekly dosing interval. Sampling 4 days following dose administration allows estimation of mean IGF-1 SDS during the dosing interval in GHD patients.
Asunto(s)
Hormona de Crecimiento Humana , Factor I del Crecimiento Similar a la Insulina/metabolismo , Modelos Biológicos , Niño , Preescolar , Femenino , Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/farmacocinética , Humanos , MasculinoRESUMEN
Context: Daily injections are required for growth hormone (GH) replacement therapy, which may cause low compliance as a result of inconvenience and distress in patients. Objective: C-terminal peptide-modified human GH (MOD-4023) is developed for once-a-week dosing regimen in GH-deficient (GHD) adults and children. The present trial was a safety and dose-finding study for weekly MOD-4023 in GHD children. Design: A multicenter, open-label, randomized, controlled phase 2 study in children with GHD, evaluating the safety, tolerability, pharmacokinetics/pharmacodynamics, and efficacy of three different weekly MOD-4023 doses, compared with daily recombinant human GH (r-hGH). Setting: The trial was conducted in 14 endocrinology centers in Europe. Patients: Fifty-three prepubertal children with GHD completed 12 months of treatment with either MOD-4023 (N = 42) or r-hGH (N = 11). Interventions: C-terminal peptide-modified hGH (MOD-4023) was administered weekly at a dose of either 0.25, 0.48, or 0.66 mg/kg/wk and compared with daily hGH at a dose of 0.24 mg/kg/wk. Results: MOD-4023 showed an estimated half-life approximately fivefold to 10-fold longer when compared with daily r-hGH. Insulin-like growth factor (IGF)-I and IGF-binding peptide 3 showed a dose-dependent increase during MOD-4023 treatment. IGF-I standard deviation score for MOD-4023 did not exceed +2. All MOD-4023 cohorts demonstrated adequate catch-up growth. The 0.66 mg/kg/wk dose demonstrated efficacy closest to daily r-hGH. No serious adverse events were observed during MOD-4023 treatment, and its tolerability was consistent with known properties of r-hGH. Conclusions: This study confirms the long-acting properties of MOD-4023 and shows a promising safety and tolerability profile. This provides support for initiation of a phase 3 study in GHD children using a single weekly injection of MOD-4023.
Asunto(s)
Enanismo Hipofisario/tratamiento farmacológico , Hormona de Crecimiento Humana/administración & dosificación , Niño , Preescolar , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Enanismo Hipofisario/metabolismo , Europa (Continente) , Femenino , Terapia de Reemplazo de Hormonas , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Proteínas RecombinantesRESUMEN
OBJECTIVE: Growth hormone (GH) replacement therapy currently requires daily injections, which may cause distress and low compliance. C-terminal peptide (CTP)-modified growth hormone (MOD-4023) is being developed as a once-weekly dosing regimen in patients with GH deficiency (GHD). This study's objective is to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of MOD-4023 administered once-weekly in GHD adults. DESIGN: 54 adults with GHD currently treated with daily GH were normalized and randomized into 4 weekly dosing cohorts of MOD-4023 at 18.5%, 37%, 55.5% or 123.4% of individual cumulative weekly molar hGH dose. The study included 2 stages: Stage A assessed the effectiveness and PK/PD profiles of the 4 dosing regimens of MOD-4023. Stage B was an extension period of once-weekly MOD-4023 administration (61.7% molar hGH content) to collect further safety data and confirm the results from Stage A. RESULTS: Dose-dependent response was observed for both PK and PD data of weekly MOD-4023 treatment. Insulin-like growth factor I (IGF-I) SDS levels were maintained within normal range. The 18.5% cohort was discontinued due to low efficacy. MOD-4023 was well tolerated and exhibited favorable safety profile in all dose cohorts. The reported adverse events were consistent with known GH-related side effects. CONCLUSIONS: Once-weekly MOD-4023 administration in GHD adults was found to be clinically effective while maintaining a favorable safety profile and may obviate the need for daily injections. Weekly GH injections may improve compliance and overall outcome. The promising results achieved in this Phase 2 study led to a pivotal Phase 3 trial, which is currently ongoing.
Asunto(s)
Terapia de Reemplazo de Hormonas/métodos , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/uso terapéutico , Hipopituitarismo/tratamiento farmacológico , Adulto , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023's binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults.
Asunto(s)
Hormona de Crecimiento Humana/farmacocinética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fragmentos de Péptidos/química , Receptores de Somatotropina/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Western Blotting , Proliferación Celular , Femenino , Semivida , Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/química , Humanos , Técnicas In Vitro , Macaca mulatta , Masculino , Ratones , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.
Asunto(s)
Movimiento Celular/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Inflamación/inmunología , Linfocitos T/inmunología , Tetraspanina 24/fisiología , Animales , Estudios de Casos y Controles , Movimiento Celular/fisiología , Estudios de Seguimiento , Proteínas de Homeodominio/fisiología , Humanos , Antígenos Comunes de Leucocito/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Receptores CCR2/fisiologíaRESUMEN
AIM: To investigate the expression and function of CD74 in normal murine colon epithelial cells (CEC) and colon carcinoma cells. METHODS: Expression of CD74 mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and fluorescence-activated cell sorter (FACS). The effect of migration inhibitory factor (MIF) on the survival of normal CEC from C57BL/6, NOD/SCID, and CD74 deficient mice both in vitro and in vivo, and on the CT26 carcinoma cell line was analyzed by (quantitative) qRT-PCR, RT-PCR, Western blotting and FACS. RESULTS: CD74 was found to be expressed on normal CEC. Stimulation of CD74 by MIF induced a signaling cascade leading to up-regulation of Bcl-2 expression, resulting in a significant increased survival of CEC. CD74 was also expressed on the CT26 colon carcinoma cell line and its stimulation by MIF resulted in enhanced cell survival, up-regulation of Akt phosphorylation and Bcl-2 expression. CONCLUSION: CD74 is expressed on CEC and colon carcinoma cells and serves as a survival receptor in these cells. These results may have implications on colorectal cancer research.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Colon/citología , Colon/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colon/efectos de los fármacos , Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Cartilla de ADN/genética , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Técnicas In Vitro , Factores Inhibidores de la Migración de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Immature B cells migrate to the spleen where they differentiate into mature cells. This final maturation step is crucial to enable B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that Id2 acts as a negative regulator of the differentiation of immature B cells occurring in the spleen. Id2 expression has been found to depend on Myc-Max-Mad transcriptional complexes in mammary epithelial cells. Nearly all studies to date have shown that Mad proteins inhibit proliferation, presumably by antagonizing the function of Myc proteins. In the current study, we followed the Mad family members during peripheral B cell differentiation. We show that Mad3 actively regulates B cell differentiation. Our results demonstrate that high expression levels of Mad3 in immature B cells induce Id2 expression, which inhibits transcription of genes essential for B cell differentiation. During their differentiation to mature cells, B cells reduce their Mad3 expression, enabling the maturation process to occur.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular/fisiología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Represoras/metabolismo , Bazo/citología , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Linfocitos B/citología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genéticaRESUMEN
Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1- and LFA-1-mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.
Asunto(s)
Adhesión Celular , Quimiocina CCL21/fisiología , Quimiocina CCL2/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Artritis/prevención & control , Asma/prevención & control , Diferenciación Celular/efectos de los fármacos , Humanos , Inmunidad , Ganglios Linfáticos , Linfocitos/citología , Ratones , Receptores CCR7/metabolismo , BazoRESUMEN
To complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or to sites of inflammation, enabling their targeting to the spleen for final maturation. This inhibition is mediated by IFN-gamma, which is transcribed and secreted at low levels by these immature B cells; IFN-gamma expression is extinguished following B-cell maturation. Stimulation of the MHC class I receptor, Ly49D, triggers a signaling cascade that increases transcription of both IL-12 (p40) and IL-18; these, in turn, induce the secretion of IFN-gamma. In the present study, we demonstrate that Ly49D-dependent secretion of IL-12 and IL-18 induces IL-15 expression by immature B cells, and that these 3 factors together regulate IFN-gamma production that inhibits their ability to home to the lymph nodes or to sites of inflammation. Thus, IL-15 controls immature B-cell homing, resulting in shaping the B-cell repertoire to enable an efficient immune response.
Asunto(s)
Antígenos Ly/inmunología , Subunidad p40 de la Interleucina-12/inmunología , Interleucina-15/inmunología , Interleucina-18/inmunología , Lectinas Tipo C/inmunología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Animales , Antígenos Ly/metabolismo , Comunicación Autocrina/inmunología , Movimiento Celular/inmunología , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Regulación hacia Abajo/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Subunidad p40 de la Interleucina-12/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-18/metabolismo , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Subfamilia A de Receptores Similares a Lectina de Células NK , Células Precursoras de Linfocitos B/metabolismo , Receptores Similares a Lectina de Células NK , Transducción de Señal/inmunología , Bazo/citología , Transcripción Genética/inmunologíaRESUMEN
In order to complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to LN or to sites of inflammation, enabling their targeting to the spleen. This inhibition is mediated by IFN-gamma, which is transcribed and secreted at low levels by these immature B cells; its expression is subsequently down-regulated following B cell maturation. The activating and inhibitory MHC class I receptors, Ly49D and Ly49G2, regulate IFN-gamma secretion in B cells, preventing their migration to antigen-enriched sites and their premature encounter with an antigen, while enabling their entry into the LN when mature. In the present study, we elucidate the pathways by which the Ly49 receptors regulate IFN-gamma levels. We show that Ly49D stimulation triggers a signaling cascade that increases transcription of both IL-12B and IL-18; these, in turn, can interact with their specific receptors, which are expressed at elevated levels on immature B cells. Ligation of the IL-12B and IL-18 receptors induces the secretion of IFN-gamma, thereby regulating their cytoskeleton rearrangement and migration.
Asunto(s)
Antígenos Ly/inmunología , Linfocitos B/inmunología , Quimiotaxis de Leucocito/inmunología , Interleucina-12/inmunología , Interleucina-18/inmunología , Lectinas Tipo C/inmunología , Animales , Antígenos Ly/metabolismo , Linfocitos B/citología , Western Blotting , Diferenciación Celular/inmunología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Lectinas Tipo C/metabolismo , Ratones , Ratones Endogámicos C57BL , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NK , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Transcripción Genética/inmunologíaRESUMEN
To complete their maturation and to participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or sites of inflammation, enabling their targeting to the spleen to allow their final maturation. This inhibition is mediated by IFN-gamma, which is transcribed and secreted at low levels by these immature B cells and is down-regulated at the mature stage. The activating MHC class I receptor, Ly49D, which is expressed at high levels on immature B cells, stimulates this IFN-gamma secretion. In this study we show that B cells coexpress the inhibitory MHC class I receptor, Ly49G2. In addition, we demonstrate a tight regulation in the expression of the Ly49 family members on B cells that depends on their cell surface levels. High levels of Ly49G2 have a dominant inhibitory effect on Ly49D expressed at low levels on immature bone marrow and mature B cells, resulting in inhibition of IFN-gamma secretion. However, low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, on the immigrating immature B cells enable the secretion of specific low levels of IFN-gamma. This expression pattern insures the inhibitory control of peripheral immature B cell to prevent premature encounter with an Ag while enabling entry to the lymph nodes during the mature stage.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Receptores Inmunológicos/fisiología , Animales , Linfocitos B/citología , Células Cultivadas , Citoesqueleto/metabolismo , Regulación hacia Abajo/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Interferón gamma/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/biosíntesis , Regulación hacia Arriba/inmunologíaRESUMEN
Immature B cells are targeted to specific areas in the spleen, where a fraction of these cells receive signals that induce them to mature and participate in the immune response. In this study, we show that the C-C chemokine receptor 2 (CCR2) is transcribed in immature B cells, while its message is dramatically down-regulated at the mature stage. CCR2-deficient cells exhibit up-regulation of chemokine-induced actin polymerization, migration, and homing to the lymph nodes of immature B cells. In addition, we demonstrate that control of homing by CCR2 is mediated by its ligand, CCL2/JE, which is secreted by B cells and down-regulates the stromal derived factor-1 (SDF-1) signaling cascade. Thus, this study describes an additional, previously uncharacterized, role for CCR2 and its ligand as negative regulators of the homing of immature B cells.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Quimiotaxis de Leucocito , Citoesqueleto/metabolismo , Receptores de Quimiocina/genética , Actinas/metabolismo , Animales , Linfocitos B/fisiología , Quimiocina CCL2/fisiología , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Regulación de la Expresión Génica , Ganglios Linfáticos/citología , Ratones , Ratones Noqueados , Receptores CCR2 , Receptores de Quimiocina/fisiología , Transcripción GenéticaRESUMEN
Low levels of IFN-gamma secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN-gamma secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN-gamma transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN-gamma gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN-gamma and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.