RESUMEN
Scratching triggers skin flares in atopic dermatitis. We demonstrate that scratching of human skin and tape stripping of mouse skin cause neutrophil influx. In mice, this influx was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1(-/-) mice and required expression of BLT1 on both T cells and non-T cells. Cotransfer of wild-type (WT) neutrophils, but not neutrophils deficient in BLT1 or the LTB4-synthesizing enzyme LTA4H, restored the ability of WT CD4(+) effector T cells to transfer allergic skin inflammation to Ltb4r1(-/-) recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.
Asunto(s)
Dermatitis/inmunología , Leucotrieno B4/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Animales , Biopsia , Dermatitis/patología , Modelos Animales de Enfermedad , Humanos , Leucotrieno B4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/inmunologíaRESUMEN
Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.
Asunto(s)
Proliferación Celular/fisiología , Colitis/metabolismo , Mucosa Intestinal/metabolismo , Receptores de Leucotrieno B4/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colitis/inducido químicamente , Colitis/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Indometacina/toxicidad , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Antagonistas de Leucotrieno/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Leucotrieno B4/antagonistas & inhibidores , Receptores de Leucotrieno B4/deficiencia , Tetrazoles/farmacologíaRESUMEN
In an experimental asthma model, the activation of TLR4 by bacterial LPS occasionally exacerbates allergic inflammation through the production of Th2 cytokines, and mast cells have been suggested to play a central role in this response. However, the detailed mechanism underlying how LPS/TLR4 stimulates the production of Th2 cytokines, especially IL-13, remains unclear in mast cells. In the current study, we observed that the expression levels of leukotriene B4 receptor-2 (BLT2) and the synthesis of its ligands were highly upregulated in LPS-stimulated bone marrow-derived mast cells and that BLT2 blockade with small interfering RNA or a pharmacological inhibitor completely abolished IL-13 production, suggesting a mediatory role of the BLT2 ligand-BLT2 axis in LPS/TLR4 signaling to IL-13 synthesis in mast cells. Moreover, we demonstrated that MyD88 lies upstream of the BLT2 ligand-BLT2 axis and that this MyD88-BLT2 cascade leads to the generation of reactive oxygen species through NADPH oxidase 1 and the subsequent activation of NF-κB, thereby mediating IL-13 synthesis. Interestingly, we observed that costimulation of LPS/TLR4 and IgE/FcεRI caused greatly enhanced IL-13 synthesis in mast cells, and blockading BLT2 abolished these effects. Similarly, in vivo, the IL-13 level was markedly enhanced by LPS administration in an OVA-induced asthma model, and injecting a BLT2 antagonist beforehand clearly attenuated this increase. Together, our findings suggest that a BLT2-linked cascade plays a pivotal role in LPS/TLR4 signaling for IL-13 synthesis in mast cells, thereby potentially exacerbating allergic response. Our findings may provide insight into the mechanisms underlying how bacterial infection worsens allergic inflammation under certain conditions.
Asunto(s)
Hipersensibilidad/inmunología , Interleucina-13/biosíntesis , Lipopolisacáridos/inmunología , Mastocitos/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Leucotrieno B4/metabolismo , Receptor Toll-Like 4/inmunología , Animales , Asma/inmunología , Asma/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , Especies Reactivas de Oxígeno/metabolismo , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
Leukotriene B4 (LTB4) and its functional receptor BLT1 are closely involved in tissue inflammation by primarily mediating leukocyte recruitment and activation. Elevated LTB4 was reported in patients with lung fibrosis; however, the role of the LTB4/BLT1 axis in lung fibrosis remains unknown. In this study, we demonstrated that BLT1-/- mice exhibited significantly attenuated bleomycin (BLM)-induced lung fibrosis. Interestingly, BLT1 blockade with its specific antagonist U75302 in the acute injury phase (days 0-10 after BLM treatment) significantly attenuated lung fibrosis, which was accompanied by significant decreases in early infiltrating neutrophils and later infiltrating CD4+ T cells and the production of TGF-ß, IL-13, and IL-17A. In contrast, BLT1 blockade in the fibrotic phase (days 10-21 after BLM treatment) had no effect on lung fibrosis and TGF-ß production, although it significantly decreased CD4+ T cell infiltration. Furthermore, depletion of neutrophils or CD4+ T cells had no effect on BLM-induced lung fibrosis, suggesting the independence of profibrotic activity of the LTB4/BLT1 axis on BLT1-dependent lung recruitment of these two leukocytes. Finally, although BLT1 blockade had no effect on the recruitment and phenotype of macrophages in BLM-induced lung fibrosis, the LTB4/BLT1 axis could promote TGF-ß production by macrophages stimulated with BLM or supernatants from BLM-exposed airway epithelial cells in an autocrine manner, which further induced collagen secretion by lung fibroblasts. Collectively, our study demonstrates that the LTB4/BLT1 axis plays a critical role in acute injury phase to promote BLM-induced lung fibrosis, and it suggests that early interruption of the LTB4/BLT1 axis in some inflammatory diseases could prevent the later development of tissue fibrosis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Neutrófilos/inmunología , Fibrosis Pulmonar/inmunología , Receptores de Leucotrieno B4/metabolismo , Animales , Bleomicina , Linfocitos T CD4-Positivos/fisiología , Alcoholes Grasos/administración & dosificación , Glicoles/administración & dosificación , Inflamación , Interleucina-13/biosíntesis , Interleucina-13/inmunología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Pulmón/inmunología , Pulmón/patología , Ratones , Infiltración Neutrófila , Neutrófilos/fisiología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/prevención & control , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Immunotherapies have shown considerable efficacy for the treatment of various cancers, but a multitude of patients remain unresponsive for various reasons, including poor homing of T cells into tumors. In this study, we investigated the roles of the leukotriene B4 receptor, BLT1, and CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, under endogenous as well as vaccine-induced antitumor immune response in a syngeneic murine model of B16 melanoma. Significant accelerations in tumor growth and reduced survival were observed in both BLT1(-/-) and CXCR3(-/-) mice as compared with wild-type (WT) mice. Analysis of tumor-infiltrating leukocytes revealed significant reduction of CD8(+) T cells in the tumors of BLT1(-/-) and CXCR3(-/-) mice as compared with WT tumors, despite their similar frequencies in the periphery. Adoptive transfer of WT but not BLT1(-/-) or CXCR3(-/-) CTLs significantly reduced tumor growth in Rag2(-/-) mice, a function attributed to reduced infiltration of knockout CTLs into tumors. Cotransfer experiments suggested that WT CTLs do not facilitate the infiltration of knockout CTLs to tumors. Anti-programmed cell death-1 (PD-1) treatment reduced the tumor growth rate in WT mice but not in BLT1(-/-), CXCR3(-/-), or BLT1(-/-)CXCR3(-/-) mice. The loss of efficacy correlated with failure of the knockout CTLs to infiltrate into tumors upon anti-PD-1 treatment, suggesting an obligate requirement for both BLT1 and CXCR3 in mediating anti-PD-1 based antitumor immune response. These results demonstrate a critical role for both BLT1 and CXCR3 in CTL migration to tumors and thus may be targeted to enhance efficacy of CTL-based immunotherapies.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Movimiento Celular/inmunología , Regulación de la Expresión Génica , Melanoma Experimental/inmunología , Receptores CXCR3/metabolismo , Receptores de Leucotrieno B4/metabolismo , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/inmunología , Quimiotaxis de Leucocito , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genéticaRESUMEN
Cisplatin is an effective chemotherapeutic agent and widely used in treatment of various solid organ malignancies, including head and neck, ovarian, and testicular cancers. However, the induction of acute kidney injury (AKI) is one of its main side effects. Leukotriene B4 receptor 1 (BLT1) mediates the majority of physiological effects of leukotriene B4 (LTB4), a potent lipid chemoattractant generated at inflammation sites, but the role of the LTB4-BLT1 axis in cisplatin-induced AKI remains unknown. Here we found upregulated LTB4 synthesis and BLT1 expression in the kidney after cisplatin administration. Cisplatin was found to directly upregulate gene expression of leukotriene A4 hydrolase and stimulate LTB4 production in renal tubular epithelial cells. Reduced kidney structural/functional damage, inflammation, and apoptosis were observed in BLT1-/- mice, as well as in wild-type mice treated with the LTA4H inhibitor SC-57461A and the BLT1 antagonist U-75302. Neutrophils were likely the target of this pathway, as BLT1 absence induced a significant decrease in infiltrating neutrophils in the kidney. Adoptive transfer of neutrophils from wild-type mice restored kidney injury in BLT1-/- mice following cisplatin challenge. Thus, the LTB4-BLT1 axis contributes to cisplatin-induced AKI by mediating kidney recruitment of neutrophils, which induce inflammation and apoptosis in the kidney. Hence, the LTB4-BLT1 axis could be a potential therapeutic target in cisplatin-induced AKI.
Asunto(s)
Lesión Renal Aguda/metabolismo , Quimiotaxis de Leucocito , Cisplatino , Riñón/metabolismo , Leucotrieno B4/metabolismo , Infiltración Neutrófila , Neutrófilos/metabolismo , Receptores de Leucotrieno B4/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Lesión Renal Aguda/prevención & control , Traslado Adoptivo , Animales , Apoptosis , Línea Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Riñón/efectos de los fármacos , Riñón/patología , Antagonistas de Leucotrieno/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Neutrófilos/trasplante , Fenotipo , Receptores de Leucotrieno B4/antagonistas & inhibidores , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Transducción de Señal , Factores de TiempoRESUMEN
BACKGROUND: Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation, and its biological effects are mediated primarily through the high affinity LTB4 receptor BLT1. Although numerous studies have reported that LTB4-BLT1 signaling is involved in inflammatory diseases, the role of BLT1 signaling in pain remains undefined. To clarify the role of LTB4-BLT1 signaling in acute inflammatory pain induced by tissue injury, we performed pain behavioral analysis and assessment of local inflammation induced by peripheral formalin injections in BLT1 knockout mice. We examined the phosphorylation of cAMP response element-binding protein (CREB) in the spinal cord both in wild-type and BLT1 knockout mice because phosphorylation of CREB in spinal cord neurons is important for nociceptive sensitization following peripheral injury. We also examined the effect of a BLT1 antagonist on formalin-induced pain responses in mice. RESULTS: BLT1 knockout mice exhibited markedly attenuated nociceptive responses induced by intraplantar formalin injections. Edema formation and neutrophil infiltration in the paw were significantly decreased in BLT1 knockout mice compared with wild-type mice. Phosphorylation of CREB in the spinal cord after the intraplantar formalin injection was decreased in BLT1 knockout mice. In addition, mice pretreated with a BLT1 antagonist showed reduced nociception and attenuated CREB phosphorylation in the spinal cord after the formalin injection. CONCLUSIONS: Our data suggest that LTB4-BLT1 axis contributes not only to the peripheral inflammation but also to the neuronal activation in the spinal cord induced by intraplantar formalin injections. Thus, LTB4-BLT1 signaling is a potential target for therapeutic intervention of acute and persistent pain induced by tissue injury.
Asunto(s)
Formaldehído/toxicidad , Dolor/metabolismo , Receptores de Leucotrieno B4/metabolismo , Médula Espinal/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inflamación/metabolismo , Ratones , Ratones Noqueados , Receptores de Leucotrieno B4/deficiencia , Transducción de Señal/efectos de los fármacosRESUMEN
BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF-induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4(+) T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF-sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4(+) T subsets and increasing numbers of Th17 and memory CD44(hi)CD4(+) T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4(+) T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF-induced antitumor memory CD4(+) T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Leucemia Experimental/inmunología , Receptores de Leucotrieno B4/inmunología , Transducción de Señal/inmunología , Inmunidad Adaptativa , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/patología , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Inmunidad Innata , Memoria Inmunológica , Leucemia Experimental/genética , Leucemia Experimental/patología , Leucotrieno B4/inmunología , Leucotrieno B4/metabolismo , Activación de Linfocitos , Masculino , Ratones , Ratones Noqueados , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Transducción de Señal/genética , Transducción GenéticaRESUMEN
Leukotriene B4 (LTB4) has been implicated in the pathogenesis of allergic diseases. BLT2, a low-affinity LTB4 receptor, is activated by LTB4 and 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). Although the high-affinity LTB4 receptor BLT1 has been shown to exert proinflammatory roles, the role of BLT2 in allergic inflammation has not been clarified. To study the function of BLT2 in development of asthma, we used mice model of ovalbumin (OVA)-induced allergic airway disease. The 12-HHT levels were elevated in bronchoalveolar lavage (BAL) fluids of OVA-sensitized/challenged wild-type mice. BLT2-deficient mice exhibited enhanced eosinophilia in BAL fluids after OVA exposure. Interleukin (IL)-13 levels in BAL fluids and IL-13-producing CD4(+) T cells in the lungs were elevated in BLT2-deficient mice compared to wild-type mice, whereas the levels of IL-4, IL-5, and interferon (IFN)-γ in BAL fluids and serum OVA-specific IgE were comparable. Transfection of BLT2-specific small interfering RNA enhanced IL-13 production in CD4(+) T cells in vitro. Expression of BLT2 mRNA in CD4(+) T cells was significantly reduced in patients with asthma compared to healthy control subjects. These findings indicate that BLT2 has a protective role in allergic airway inflammation and that diminished BLT2 expression in CD4(+) T cells may contribute to the pathophysiology of asthma.
Asunto(s)
Asma/inmunología , Eosinofilia/inmunología , Pulmón/inmunología , Receptores de Leucotrieno B4/inmunología , Adulto , Anciano , Animales , Asma/genética , Asma/metabolismo , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células CHO , Calcio/inmunología , Calcio/metabolismo , Cricetinae , Cricetulus , Citocinas/inmunología , Citocinas/metabolismo , Eosinofilia/genética , Eosinofilia/metabolismo , Ácidos Grasos Insaturados/inmunología , Ácidos Grasos Insaturados/metabolismo , Humanos , Leucotrieno B4/inmunología , Leucotrieno B4/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Interferencia de ARN , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B(4) (LTB(4)). The influence of G protein-coupled receptor ligands such as LTB(4) on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB(4) signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB(4) production and signaling through its high-affinity G protein-coupled receptor leukotriene B(4) receptor 1 (BLT1) direct dectin-1-dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wild-type counterparts. LTB(4) increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB(4)-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB(4) effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF(-/-) macrophages. Our results identify LTB(4)-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Lectinas Tipo C/biosíntesis , Leucotrieno B4/fisiología , Macrófagos Alveolares/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Animales , Candida albicans/inmunología , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leucotrieno B4/biosíntesis , Leucotrieno B4/deficiencia , Macrófagos Alveolares/microbiología , Macrófagos Peritoneales/microbiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/fisiología , Transactivadores/biosíntesis , Transcripción Genética/inmunologíaRESUMEN
Leukotrienes (i.e., products of the 5-lipoxygenase pathway) are thought to be contributors to lung pathologies. Moreover, eosinophils have been linked with pulmonary leukotriene activities both as potential sources of these mediators and as responding effector cells. The objective of the present study was to define the role(s) of leukotrienes in the lung pathologies accompanying eosinophil-associated chronic respiratory inflammation. A transgenic mouse model of chronic T helper (Th) 2-driven inflammation expressing IL-5 from T cells and human eotaxin-2 locally in the lung (I5/hE2) was used to define potential in vivo relationships among eosinophils, leukotrienes, and chronic Th2-polarized pulmonary inflammation. Airway levels of cys-leukotrienes and leukotriene B4 (LTB4) are both significantly elevated in I5/hE2 mice. The eosinophil-mediated airway hyperresponsiveness (AHR) characteristic of these mice was abolished in the absence of leukotrienes (i.e., 5-lipoxygenase-deficient I5/hE2). More importantly, the loss of leukotrienes led to an unexpectedly significant decrease in collagen deposition (i.e., pulmonary fibrosis) that accompanied elevated levels of IL-4/-13 and TGF-ß in the lungs of I5/hE2 mice. Further studies using mice deficient for the LTB4 receptor (BLT-1(-/-)/I5/hE2) and I5/hE2 animals administered a cys-leukotriene receptor antagonist (montelukast) demonstrated that the AHR and the enhanced pulmonary fibrosis characteristic of the I5/hE2 model were uniquely cys-leukotriene-mediated events. These data demonstrate that, similar to allergen challenge models of wild-type mice, cys-leukotrienes underlie AHR in this transgenic model of severe pulmonary Th2 inflammation. These data also suggest that an underappreciated link exists among eosinophils, cys-leukotriene-mediated events, and fibrotic remodeling associated with elevated levels of IL-4/-13 and TGF-ß.
Asunto(s)
Eosinófilos/inmunología , Leucotrienos/inmunología , Neumonía/etiología , Fibrosis Pulmonar/etiología , Animales , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/inmunología , Hiperreactividad Bronquial/inmunología , Quimiocina CCL24/genética , Quimiocina CCL24/inmunología , Modelos Animales de Enfermedad , Eosinófilos/patología , Humanos , Interleucina-5/inmunología , Leucotrieno B4/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neumonía/inmunología , Neumonía/patología , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/inmunología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Lipid mediators derived from arachidonic acid through the cyclooxygenase and lipoxygenase pathways are known to be important mediators of inflammation. Studies in mouse models demonstrated an important role for the high-affinity leukotriene B(4) receptor BLT1 in arthritis, atherosclerosis, and asthma. BLT2, a low-affinity leukotriene B(4) receptor, was also shown to be a high-affinity receptor for cyclooxygenase-1 derived 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. However, its biochemical activities and physiological roles remain unknown. In this study, we developed mice deficient in BLT2 by targeted disruption. The BLT2(-/-) mice developed normally, and analysis of immune cells showed that disruption of BLT2 did not alter BLT1 expression or function. Mast cells from the C57BL/6 mice but not from the BLT2(-/-) mice showed intracellular calcium mobilization in response to 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. In an autoantibody-induced inflammatory arthritis model, the BLT2(-/-) mice showed reduced incidence and severity of disease, including protection from bone and cartilage loss. Reciprocal bone marrow transplant experiments identified that loss of BLT2 expression on a bone marrow-derived cell lineage offers protection against severe disease. Thus, BLT2, a unique receptor for 5-lipoxygenase- and cyclooxygenase-1-derived lipid mediators, represents a novel target for therapies directed at treating inflammation associated with arthritis.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/patología , Mediadores de Inflamación/fisiología , Leucotrieno B4/metabolismo , Receptores de Leucotrieno B4/fisiología , Animales , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Articulación de la Rodilla/inmunología , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Leucotrieno B4/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Receptores de Leucotrieno B4/biosíntesis , Receptores de Leucotrieno B4/deficienciaRESUMEN
Although leukotriene B(4) (LTB(4)) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB(4) receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB(4) receptor BLT2, and produced LTB(4). LTB(4) changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB(4) increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.
Asunto(s)
Resorción Ósea/etiología , Receptores de Leucotrieno B4/fisiología , Animales , Calcio/metabolismo , Comunicación Celular , Forma de la Célula/efectos de los fármacos , Leucotrieno B4/farmacología , Ratones , Ratones Noqueados , Osteoclastos/metabolismo , Receptores de Leucotrieno B4/deficiencia , Transducción de SeñalRESUMEN
Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.
Asunto(s)
Bronquiolitis Obliterante/etiología , Linfocitos T CD8-positivos/fisiología , Rechazo de Injerto/etiología , Trasplante de Pulmón/efectos adversos , Receptores de Leucotrieno B4/fisiología , Animales , Secuencia de Bases , Bronquiolitis Obliterante/inmunología , Bronquiolitis Obliterante/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Colágeno/metabolismo , Modelos Animales de Enfermedad , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Trasplante de Pulmón/inmunología , Trasplante de Pulmón/patología , Trasplante de Pulmón/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Tráquea/trasplanteAsunto(s)
Aminoquinolinas , Leucotrienos/metabolismo , Psoriasis/metabolismo , Piel/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/deficiencia , Araquidonato 5-Lipooxigenasa/genética , Modelos Animales de Enfermedad , Imiquimod , Leucotrienos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Psoriasis/inducido químicamente , Psoriasis/genética , Psoriasis/inmunología , Psoriasis/patología , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Transducción de Señal , Piel/inmunología , Piel/patologíaRESUMEN
BACKGROUND: Leukotriene B4 (LTB4) is a potent inflammatory lipid mediator that binds to LTB4 receptor 1 (BLT1). Ligation of BLT1 by LTB4 plays an important role in the recruitment of effector memory CD8+ T cells into the airways of sensitized and challenged mice. OBJECTIVES: The effects of the corticosteroid dexamethasone (DEX) on BLT1-expressing effector memory CD8+ T cells and effector memory CD8+ T cell-mediated airway hyperresponsiveness (AHR) and allergic inflammation were determined. METHODS: Effector memory CD8+ T cells were generated from ovalbumin(257-264)-primed mononuclear cells from OT-1 mice in the presence of IL-2. In some cultures DEX was added. The effects of DEX on BLT1 expression, LTB4-induced Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, chemotaxis, and effector memory CD8+ T cell-mediated AHR were examined. RESULTS: DEX-treated effector memory CD8+ T cells showed significant increases in surface expression of BLT1, LTB4-induced intracellular Ca2+ influx, phosphorylation of extracellular signal-regulated kinase 1/2, and chemotaxis. Upregulation of BLT1 by DEX was accompanied by increased IL-2 receptor expression. Adoptive transfer of DEX-treated effector memory CD8+ T cells into ovalbumin-sensitized and ovalbumin-challenged CD8-/- mice resulted in significant increases in AHR, allergic inflammation, goblet cell metaplasia, and numbers of both CD8+ and CD4+ T cells in the bronchoalveolar lavage fluid and lungs. CONCLUSIONS: Corticosteroids upregulate BLT1 on effector memory CD8+ T cells and related signaling pathways and potentiate allergic airway inflammation and AHR induced by these cells.
Asunto(s)
Alérgenos/fisiología , Hiperreactividad Bronquial/inmunología , Linfocitos T CD8-positivos/inmunología , Dexametasona/farmacología , Mediadores de Inflamación/fisiología , Ovalbúmina/inmunología , Receptores de Leucotrieno B4/biosíntesis , Regulación hacia Arriba/inmunología , Adyuvantes Inmunológicos/fisiología , Animales , Hiperreactividad Bronquial/patología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Proteínas del Huevo/fisiología , Memoria Inmunológica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/fisiología , Fragmentos de Péptidos , Fosforilación/efectos de los fármacos , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Receptores de Leucotrieno B4/fisiologíaRESUMEN
Leukotrienes (LTs), metabolites of arachidonic acid through 5-lipoxygenase (5-LOX), have been known to play a role in leukocyte recruitment. However, the contribution of LTB4 to liver microcirculatory dysfunction during endotoxemia remains unknown. LTB4 receptor (BLT1) has been identified as a high-affinity receptor specific for LTB4. The present study was conducted to examine the roles of LTB4 and BLT1 in hepatic microcirculatory dysfunction elicited by LPS in mice. The number of leukocytes adhering to the endothelial cells of the hepatic microvessels and perfused sinusoids was determined 4 h after the administration of LPS (0.3 mg/kg, i.v.) to male C57Bl6 mice by in vivo microscopy. A 5-LOX synthase inhibitor, AA-861 (10 or 100 mg/kg, s.c.), was administered 30 min before LPS injection. BLT1 knockout mice were used to investigate whether LPS-induced hepatic microcirculatory dysfunction is mediated by BLT1 signaling. The expression of 5-LOX, intercellular adhesion molecule (ICAM) 1, and TNF-[alpha] in the liver was measured by real-time reverse-transcriptase-polymerase chain reaction. The administration of LPS caused significant accumulation of leukocyte adhesion to the hepatic microvessels and reduced sinusoidal perfusion when compared with saline-treated mice. The hepatic microcirculatory dysfunction elicited by LPS was minimized in mice pretreated with AA-861 or in BLT1 knockout mice. This was associated with the suppression of hepatic expression of 5-LOX, ICAM-1, and TNF-[alpha]. These findings suggest that the LTB4/BLT1 pathway mediates hepatic microcirculatory dysfunction by enhanced expression of ICAM-1 and TNF-[alpha] in a murine model of endotoxemia.
Asunto(s)
Leucotrieno B4/fisiología , Microcirculación/efectos de los fármacos , Receptores de Leucotrieno B4/fisiología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Molécula 1 de Adhesión Intercelular/biosíntesis , Lipopolisacáridos , Inhibidores de la Lipooxigenasa , Hígado/irrigación sanguínea , Hígado/fisiopatología , Masculino , Ratones , Ratones Noqueados , Receptores de Leucotrieno B4/deficienciaRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce inflammation by suppressing cyclooxygenases (COXs). NSAID eye drops are frequently prescribed after ocular surgery to reduce inflammation and pain, but this treatment has clinically significant side effects, including corneal ulcer and perforation. The molecular mechanisms underlying these side effects remain unknown. Recently, the COX product 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) was identified as an endogenous ligand for leukotriene B4 receptor 2 (BLT2), which is important in maintenance of epithelial homeostasis. We hypothesized that NSAID-dependent corneal damage is caused by reduced production of 12-HHT. Diclofenac eye drops decreased the abundance of downstream products of COX and delayed corneal wound healing in BALB/c mice. Expression of BLT2 was observed in murine ocular tissues including cornea, and in human corneal epithelial cell line and human primary corneal epithelial cells. In BLT2-knockout mice, corneal wound healing was delayed, but the diclofenac-dependent delay in corneal wound healing disappeared. 12-HHT accelerated wound closure both in BLT2-transfected corneal cell line and human primary corneal epithelial cells. Thus, our results reveal that NSAIDs delay corneal wound healing by inhibiting 12-HHT production, and suggest that stimulation of the 12-HHT/BLT2 axis represents a novel therapeutic approach to corneal wound healing.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Lesiones de la Cornea/metabolismo , Ácidos Grasos Insaturados/metabolismo , Receptores de Leucotrieno B4/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Animales , Lesiones de la Cornea/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Leucotrieno B4/deficiencia , Receptores de Leucotrieno B4/genética , Cicatrización de Heridas/genéticaRESUMEN
BACKGROUND: It is known that 5-lipoxygenase and its product, leukotriene B4 (LTB4), are highly expressed in several human pathologies, including atherosclerotic plaque. LTB(4) signals primarily through its high-affinity G protein-coupled receptor BLT1, which is expressed on specific leukocyte subsets. BLT1 receptor expression and function on other atheroma-associated cell types is unknown. METHODS AND RESULTS: To directly assess the role of the LTB4-BLT1 pathway in atherogenesis, we bred BLT1(-/-) mice into the atherosclerosis-susceptible apoE(-/-) strain. Compound-deficient apoE(-/-)/Blt1(-/-) mice fed a Western-type diet had a marked reduction in plaque formation compared with apoE(-/-) controls. Immunohistochemical analysis of atherosclerotic lesions in compound-deficient mice revealed a striking decrease in smooth muscle cells (SMCs) and significant decreases in macrophages and T cells. We report here novel evidence of the expression and function of BLT1 on vascular SMCs. LTB4 triggered SMC chemotaxis, which was pertussis toxin sensitive in Blt1(+/+) SMCs and absent in Blt1(-/-) cells, suggesting that BLT1 was the dominant receptor mediating effector functions through a G protein-coupled signaling pathway. Furthermore, BLT1 colocalized with SMCs in human atherosclerotic lesions. CONCLUSIONS: These new findings extend the role of inducible BLT1 to nonleukocyte populations and suggest an important target for intervention to modulate the response to vascular injury.
Asunto(s)
Aterosclerosis/prevención & control , Leucotrieno B4/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Receptores de Leucotrieno B4/fisiología , Receptores Purinérgicos P2/fisiología , Animales , Apolipoproteínas E/fisiología , Aterosclerosis/etiología , Movimiento Celular , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Receptores CCR2 , Receptores de Quimiocina/fisiología , Receptores de Leucotrieno B4/deficiencia , Receptores Purinérgicos P2/deficiencia , Transducción de SeñalRESUMEN
OBJECTIVE: Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice. METHODS AND RESULTS: Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. CONCLUSIONS: The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.