RESUMEN
BACKGROUND: Neuropeptides, such as substance P (SP), have long been seen as mediators of widespread continuous airway inflammation, a process known as neurogenic inflammation. However, this has been difficult to demonstrate clinically, suggesting an alternative role for these signaling molecules. OBJECTIVES: We sought to examine the role of SP in nasal infection by assessing the release of SP in response to viral stimulation and characterizing the effects of SP on innate immunity, with the latter reflected in changes in local Toll-like receptor (TLR) expression. METHODS: The distribution of SP and TLRs in the nasal mucosa and local airway neurons was assessed with immunohistochemistry. The TLR7 agonists R-837 and R-848 were used to mimic a viral insult in the upper airways represented by primary human nasal epithelial cells (HNECs) and murine nasal epithelial cells (MNECs) and isolated murine trigeminal ganglial neurons. SP release from HNECs, MNECs, and trigeminal ganglial neurons was quantified with EIA. The effects of SP on TLR expression on HNECs were determined by using flow cytometry and confocal microscopy. RESULTS: SP was released from the sensory neurons, MNECs, and HNECs within 15 minutes of local TLR7 stimulation. Subsequently, stimulation with SP induced upregulation of TLR expression in HNECs within 30 minutes through induction of TLR movement within HNECs. Upregulation of TLR expression was not evident when cells were treated with the neurokinin 1 receptor antagonist aprepitant before SP stimulation. CONCLUSIONS: This highlights a novel role for sensory neuropeptides as acute and local mediators of pathogen-driven inflammation, rapidly priming innate immune defenses in the airway.
Asunto(s)
Células Epiteliales/inmunología , Inmunidad Innata , Mucosa Nasal/inmunología , Neuronas Aferentes/inmunología , Sustancia P/inmunología , Animales , Células Epiteliales/citología , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Mucosa Nasal/citología , Mucosa Nasal/inervación , Neuronas Aferentes/citología , Receptor Toll-Like 7/inmunologíaRESUMEN
The concept of functional neutrophil subsets is new and their clinical significance in malignancies is unknown. Our study investigated the role of CD16dim CD62Lhigh , CD16high CD62Lhigh and CD16high CD62Ldim neutrophil subsets in head and neck squamous cell carcinoma (HNSCC) patients. These neutrophil subsets may play different roles in immune-related activity in cancer, based on their profile, activation state and migration ability within a tumor site, which may be important in predicting cancer prognoses. Tumor biopsies and blood were obtained from newly diagnosed untreated HNSCC patients and healthy controls. Neutrophil subsets and their phenotype were characterized using flow cytometry. Isolated granulocytes were assessed for anti-tumor immune functions. Compared to controls HNSCC patients exhibited increased CD16high CD62Ldim neutrophils in blood; this subset displayed a distinct phenotypes with high expression of CD11b and CD18. This subset was prone to migrate into the tumor facilitated by tumor-derived IL-8. Furthermore, IL-8 was also found to activate neutrophils and thereby promoting subset transition. Various assays demonstrated that activated CD16high CD62Ldim neutrophils inhibited migration, proliferation and induced apoptosis of FaDu cancer cells. Neutrophil elastase detected in activated CD16high CD62Ldim neutrophils and tumor biopsies suggested that CD16high CD62Ldim neutrophils impart anti-tumoral activity via neutrophil extracellular traps. Furthermore, increased fraction of CD16high CD62Ldim neutrophils was shown to correlate with an increased survival rate. Our study demonstrates the clinical relevance of the CD16high CD62Ldim neutrophil subset, providing evidence for its increased migration capacity, its anti-tumor activity including increased NET formation and finally its correlation with increased survival in HNSCC patients.
Asunto(s)
Carcinoma de Células Escamosas/patología , Movimiento Celular/fisiología , Neoplasias de Cabeza y Cuello/patología , Selectina L/metabolismo , Neutrófilos/patología , Receptores de IgG/metabolismo , Anciano , Anciano de 80 o más Años , Apoptosis/fisiología , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular/fisiología , Femenino , Proteínas Ligadas a GPI/metabolismo , Granulocitos/metabolismo , Granulocitos/patología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Tasa de SupervivenciaRESUMEN
CONCLUSION: The capability of Nod1 to recognize bacteria along with its altered expression and ability to cause an immunological response in head and neck cancer suggest a novel pathway for bacteria to interfere with ongoing cancer inflammation. OBJECTIVE: Nucleotide oligomerization domain (Nod)-like receptors (NLRs) comprise a recently discovered family of pattern-recognition receptors. In addition to their protective function against infections, accumulating evidence suggests a role for these receptors in various diseases, including cancer. The present study was designed to explore the presence of NLRs in head and neck squamous cell carcinoma, and to determine if these cells have the ability to respond immunologically to ligand stimulation. METHODS: The pharyngeal squamous cell carcinoma cell lines Detroit-562 and FaDu were used as a model for head and neck cancer, and compared to healthy primary human nasal epithelial cells. Analyses were performed using immunohistochemistry, real-time RT-PCR, Luminex Multiplex Immunoassay, ELISA, and flow cytometry. RESULTS: The expression profile of NLRs in head and neck cancer cells differed from that seen in healthy epithelial cells. Further, Nod1 stimulation induced an immunological response in tumor cells that differed from the response in normal epithelial cells, especially regarding the expression of ß-defensin 2, granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), intercellular adhesion molecule-1 (ICAM-1), and cell survival.
Asunto(s)
Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Proteína Adaptadora de Señalización NOD1/genética , ARN Neoplásico/genética , Apoptosis , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Proteína Adaptadora de Señalización NOD1/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , Células Tumorales CultivadasRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69(+), CD71(+) and CD98(+) T cell subsets and NK cells, and a reduced expression of L-selectin in CD14(high)CD16(+) monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14(high) CD16(+) monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98(+) Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis.
Asunto(s)
Carcinoma de Células Escamosas/sangre , Neoplasias de Cabeza y Cuello/sangre , Leucocitos/inmunología , Análisis de Supervivencia , Antígenos CD/inmunología , Femenino , Citometría de Flujo , Humanos , MasculinoRESUMEN
BACKGROUND: S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR). METHODS: Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC). RESULTS: Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC. CONCLUSIONS: The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.
Asunto(s)
Citocinas/metabolismo , Rinitis Alérgica Estacional/metabolismo , Proteínas S100/metabolismo , Células Th2/metabolismo , Adulto , Alérgenos , Línea Celular , Desensibilización Inmunológica , Femenino , Histamina/farmacología , Humanos , Interleucina-4/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos , Masculino , Persona de Mediana Edad , Líquido del Lavado Nasal/química , Mucosa Nasal/metabolismo , Pruebas de Provocación Nasal , Neutrófilos/efectos de los fármacos , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100/análisis , Proteínas S100/farmacología , Adulto JovenRESUMEN
BACKGROUND: Retinoic acid-inducible gene 1-like receptors (RLRs) are a novel family of pattern recognition receptors that include retinoic acid-inducible gene 1 (RIG-1), melanoma differentiation-associated gene 5 (MDA-5), and laboratory of genomics and physiology 2 (LGP-2). The knowledge of RLRs and their function in the human airway is limited. This study explores the role of RLRs in the upper respiratory tract. METHODS: Tonsils, adenoids, nasal polyps, and biopsy specimens from the nasal mucosa were examined for the occurrence of the RIG-1, MDA-5, and LGP-2 using real-time reverse-transcription polymerase chain reaction and immunohistochemistry. The nasopharyngeal epithelial cell line FaDu was cultured with the RIG-1/MDA-5 ligand poly(I:C)/LyoVec (Invivogen, San Diego, CA) and analyzed for cytokine release using ELISA. RESULTS: RIG-1, MDA-5, and LGP-2 mRNA were found in all tissues tested. The airway epithelium appeared to be their most prominent location. The RIG-1 and MDA-5 mRNA levels were higher in nasal polyps than in normal nasal mucosa, a state that seemed to be reversed by local steroid treatment. Culture of FaDu with poly(I:C)/LyoVec resulted in IL-6 and IL-8 release. No alteration in RLR expression in tonsils was seen on infection. CONCLUSION: This study shows the presence and functional activity of RLRs in the human upper airways. It also suggests a role for RLRs in nasal polyposis.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasales/metabolismo , ARN Helicasas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Tonsila Faríngea/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Línea Celular , Niño , Preescolar , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Lactante , Helicasa Inducida por Interferón IFIH1 , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/inmunología , Mucosa Nasal/patología , Pólipos Nasales/genética , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Tonsila Palatina/patología , Poli I-C/farmacología , ARN Helicasas/genética , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/genética , Adulto JovenAsunto(s)
Proteínas Portadoras/biosíntesis , Expresión Génica , Membrana Mucosa/patología , Proteína Adaptadora de Señalización NOD2/biosíntesis , Otitis Media/patología , Receptores Toll-Like/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Enfermedad Crónica , Humanos , Lactante , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR , Adulto JovenRESUMEN
Nitric oxide (NO) is a key mediator in the local immune response of human airways. Inducible NO-synthases (iNOS), and endothelial NO-synthases (eNOS) are two enzymes known to regulate its production. The role of NO in middle ear disease is not fully known. Previous studies suggest that NO might have a dual role, both promoting and suppressing middle ear inflammation. The aim of the present study was to compare the eNOS and iNOS expression in adenoids obtained from children with otitis media with effusion (OME) with the expression seen in adenoids derived from children without middle ear disease. In addition, the expression of IL-1ß and TNF-α were analyzed, because of their role in the iNOS-induction pathway. The iNOS and eNOS expression were analyzed with real-time PCR in 8 OME and 11 control adenoids. The corresponding proteins were demonstrated by immunohistochemical staining of adenoid tissue. A Luminex(®) assay was performed to analyze IL-1ß and TNF-α in nasopharyngeal secretion in 10 OME and 8 controls, and immunohistochemistry was performed on adenoid tissue and imprints from the adenoid surface. Children with OME exhibited lower levels of iNOS than controls without middle ear disease. No such difference was seen for eNOS. The corresponding proteins were found mainly in conjunction with surface epithelium. No significant changes were seen among the cytokines tested. The present results indicate that local induction of iNOS in adenoids might be of importance for preventing development of OME.
Asunto(s)
Tonsila Faríngea/patología , Hipertrofia/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Otitis Media con Derrame/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Tonsila Faríngea/inmunología , Tonsila Faríngea/metabolismo , Adolescente , Niño , Preescolar , Regulación hacia Abajo/inmunología , Femenino , Humanos , Hipertrofia/complicaciones , Hipertrofia/diagnóstico , Hipertrofia/genética , Inflamación , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Otitis Media con Derrame/complicaciones , Otitis Media con Derrame/diagnóstico , Otitis Media con Derrame/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND/AIM: Viral respiratory infections are increasingly implicated in allergic exacerbations. Virus-induced activation of eosinophils through Toll-like receptors (TLRs) could be involved. The present study was designed to examine TLR3 expression in eosinophils from bone marrow (BM) and peripheral blood (PB) during symptomatic allergic rhinitis, and to evaluate the functional responsiveness of TLR3 in purified eosinophils. METHODS: BM and PB samples were obtained from healthy volunteers and patients with seasonal allergic rhinitis outside and during the pollen season. Eosinophils were analyzed for TLR3 expression by flow cytometry. Polyinosinic:polycytidylic acid [poly(I:C)], an agonist for TLR3, was used to assess its functional role in purified eosinophils and the intracellular signaling pathways involved. RESULTS: TLR3 expression was demonstrated in BM and PB eosinophils. It was higher in BM-derived than in circulating cells and it was downregulated in both compartments during symptomatic allergic rhinitis. TLR3 expression was also downregulated in the presence of interleukin (IL)-4 and IL- 5. Stimulation with poly(I:C) increased the percentage of CD11b+ cells and enhanced the secretion of IL-8, effects mediated via the p38 mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways. Moreover, pretreatment with IL-5 augmented the poly(I:C)-induced IL-8 release. CONCLUSIONS: Eosinophils activated via TLR3 might be more able to home and recruit leukocytes to sites of inflammation. The decreased TLR3 expression during symptomatic allergic rhinitis and in the presence of Th2 cytokines indicates a role in allergic airway inflammation. Thus, eosinophils might function as a link between viral infections and exacerbations of allergic disease.
Asunto(s)
Eosinófilos/metabolismo , Rinitis Alérgica Estacional/metabolismo , Receptor Toll-Like 3/metabolismo , Virosis/inmunología , Adulto , Recuento de Células Sanguíneas , Células de la Médula Ósea/citología , Antígeno CD11b/metabolismo , Recuento de Células , Inhibidores de Cisteína Proteinasa/farmacología , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Femenino , Expresión Génica/genética , Humanos , Imidazoles/farmacología , Interleucina-4/farmacología , Interleucina-5/farmacología , Interleucina-8/metabolismo , Leupeptinas/farmacología , Masculino , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Neutrófilos/metabolismo , Fosforilación/efectos de los fármacos , Poli I-C/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Receptor Toll-Like 3/genética , Adulto Joven , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
CONCLUSION: Toll-like receptor 7 (TLR7) is present in the adenoids in young children and might play a role in the immunological response behind the development of otitis media with effusion (OME). OBJECTIVES: To investigate the expression of the TLRs TLR4 and TLR7 in adenoids from children with OME and to compare the results with data obtained from healthy controls. SUBJECTS AND METHODS: This was a controlled, prospective study. Eleven young children with long-standing OME and 10 controls with healthy middle ears were recruited consecutively when scheduled for adenoidectomy. mRNA was quantified using real-time polymerase chain reaction (PCR) and the localization of the corresponding proteins was assessed by immunohistochemistry. RESULTS: mRNA for TLR4 and TLR7 could be obtained from all samples tested along with their corresponding proteins. The mRNA levels for TLR7 were increased among the children with a history of OME. No such increase was found for TLR4.
Asunto(s)
Tonsila Faríngea/patología , Infecciones por Haemophilus/genética , Haemophilus influenzae , Otitis Media con Derrame/genética , Infecciones Neumocócicas/genética , Receptor Toll-Like 7/genética , Adenoidectomía , Niño , Preescolar , Femenino , Expresión Génica/genética , Infecciones por Haemophilus/patología , Infecciones por Haemophilus/cirugía , Humanos , Técnicas para Inmunoenzimas , Masculino , Otitis Media con Derrame/patología , Otitis Media con Derrame/cirugía , Infecciones Neumocócicas/patología , Infecciones Neumocócicas/cirugía , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/genéticaRESUMEN
Toll-like receptors (TLRs) are increasingly implicated in the pathogenesis of cancer. The present study describes TLR expression and function in healthy and malignant airway epithelial cells. The squamous cell carcinoma cell line Detroit-562 was compared with the healthy bronchial epithelial cell line NL-20 and primary human nasal epithelial cells (HNECs). TLR2, TLR3 and TLR5 were present in primary head and neck squamous cell carcinomas (HNSCCs). Consistent with this, Detroit-562 expressed TLR2, TLR3 and TLR5, whereas NL-20 expressed mainly TLR3 and HNECs expressed TLR2-5. In Detroit-562, Pam(3)CSK(4), poly(I:C) and flagellin, ligands for TLR2, TLR3 and TLR5, respectively, induced an up-regulation of intercellular adhesion molecule 1 (ICAM-1), an increase in interleukin (IL)-6 and IL-8 secretion and a decrease in cell viability. Additionally, poly(I:C) affected IL-1beta production and the migratory behaviour of Detroit-562. NL-20 responded with a slight increase in IL-8 secretion upon poly(I:C) stimulation. Poly(I:C) induced a small increase in IL-1beta, IL-6 and IL-8 production in HNECs, while Pam(3)CSK(4) increased viability. The TLR signalling was transcription-dependent, but the pathways involved differed among TLRs as well as cells. In Detroit-562, TLR2 and TLR5 activation was mediated via c-jun N-terminal kinase (JNK)-, p38-, phosphatidylinositol 3-kinase (PI3K)- and nuclear factor (NF)-kappaB-related pathways, while TLR3 was dependent on NF-kappaB. In NL-20, TLR3 signalled via p38, and in HNECs, NF-kappaB, JNK and extracellular signal-regulated kinase (ERK) appeared to be involved. We found that TLR agonists induced a robust response in HNSCCs, characterized by generation of inflammation and cell death. A similar response was not seen in normal epithelial cells. Thus, the TLR system should be considered an important target in future antitumour immunotherapy.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 5/agonistas , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Flagelina/farmacología , Neoplasias de Cabeza y Cuello/patología , Humanos , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Inductores de Interferón/farmacología , Interleucina-6/agonistas , Interleucina-6/metabolismo , Interleucina-8/agonistas , Interleucina-8/inmunología , Interleucina-8/metabolismo , Lipopéptidos/farmacología , Poli I-C/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 5/metabolismoRESUMEN
Cytokines like interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), released during the inflammatory process, play important roles in the development of airway hyperresponsiveness. The effects of these cytokines are mediated by cell surface receptors, specific for each cytokine. The expression of cytokine receptors is a dynamic process, where receptors can be up- or down-regulated in response to changes in the environment. One such environmental factor is the presence of cytokines per se. The present study was designed to evaluate the effects of IL-1beta on the expression of its corresponding receptor IL-1 RI, as well as on the closely related TNFalpha receptors TNF RI and TNF RII in airways using a mouse organ culture assay and intranasal inoculation model. Immunohistochemical staining was used to quantify expressional differences between fresh and cultured tracheal segments. In the fresh, uncultured, segments, IL-1 RI and TNF RI were seen in the epithelial layer and TNF RI in the smooth muscle layer. After 4 days of culture, the expression of TNF RI decreased in the epithelial layer, whereas the corresponding expression of IL-1 RI and TNF RI in the smooth muscle remained unchanged. When culture was performed in the presence of IL-1beta, the expression of IL-1 RI and TNF RI in the epithelial cells and TNF RI in the smooth muscle cells increased. TNF RII was not detected in either fresh or cultured trachea, but after treatment with IL-1beta an expression was found in both the epithelial layer and in the smooth muscle cells. The IL-1beta-induced increased expression, on TNF RI and TNF RII in the smooth muscle ex vivo and in the lung parenchyma after intranasal challenge in vivo, was verified at the mRNA level using real-time RT PCR. To summarize, presence of IL-1beta increases the expression of IL-1 R1 and TNF RI and induces expression of TNF RII in the airway wall. It is not inconceivable that these alterations of the IL-1 and TNF receptors may have important functional implications for the development of hyperresponsiveness in inflammatory airway diseases like asthma.
Asunto(s)
Interleucina-1beta/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Administración Intranasal , Animales , Epitelio/metabolismo , Inmunohistoquímica , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/metabolismo , Técnicas de Cultivo de Órganos , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/metabolismo , Regulación hacia ArribaRESUMEN
The sensory innervation of intracranial vessels originates in the trigeminal ganglion with calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase activating peptide (PACAP) as frequent neuronal messengers. The present study was designed to study the expression of these neuropeptides (a) in primary culture of adult rat trigeminal ganglion neuronal cells and (b) in organ culture of sections of the trigeminal ganglion. In cell culture, axons grow in the peripheral direction for up to 48 h. Immunocytochemistry revealed that the cell bodies showed increased expression of CGRP at 24 h and SP at 24-48 h (p<0.05), whereas cell culture did not increase the expression of PACAP at 24 h (p>0.05), but at 48 h (p<0.05). A significant elevation of CGRP mRNA was seen at 12 h, i.e. before the increased CGRP immunoreaction was observed. In organ culture of sections of trigeminal ganglia, the number of CGRP immunoreactive (-ir) cells and the mRNA expression were significantly increased at 24 and 48 h of incubation as compared to control (p<0.05), while the number of SP-ir cells was not altered (p>0.05). In conclusion, neurons of rat trigeminal ganglia alter their expression of neuropeptides during cell and organ culture differently, but it is mainly the CGRP system that is up-regulated. We have compared two methods for future studies of underlying molecular mechanisms responsible for regulation of neuropeptide expression in the trigeminal system.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Neuronas/metabolismo , Ganglio del Trigémino/citología , Regulación hacia Arriba/fisiología , Animales , Axones/fisiología , Péptido Relacionado con Gen de Calcitonina/genética , Recuento de Células/métodos , Técnicas de Cultivo de Célula , Masculino , Neuroglía/metabolismo , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia P/metabolismo , Factores de TiempoRESUMEN
CONCLUSION: The rich supply of nerve fibres containing neurotransmitters, particularly those containing SP and CGRP, is suggested to be a prerequisite for the recognition of chemical irritants as part of a chemical sense. OBJECTIVE: The present study was designed to examine the distribution of different neurotransmitter candidates in the vomeronasal organ (VNO) of rats. MATERIALS AND METHODS: The distribution of neurotransmitter candidates was studied in the vomeronasal organ of the rat using immunocytochemistry. RESULTS: The neuronal marker protein gene product 9.5 revealed a very rich supply of nerve fibres within and beneath the sensory epithelium, around blood vessels and glands. A moderate supply of nerve fibres containing tyrosine hydroxylase and neuropeptide Y was mostly seen close to blood vessels. Numerous nerve fibres containing nitric oxide synthase and vasoactive intestinal peptide were seen around blood vessels and in the subepithelial layer, with occasional fibres within the epithelium. Only few fibres located in the subepithelial layer contained pituitary adenylate cyclase activating peptide. Nerve fibres containing substance P and in particular calcitonin gene-related peptide were abundant in and beneath the epithelium and scattered in the submucosal layers around blood vessels.
Asunto(s)
Fibras Nerviosas/metabolismo , Órgano Vomeronasal/metabolismo , Animales , Biomarcadores , Péptido Relacionado con Gen de Calcitonina/metabolismo , Femenino , Inmunohistoquímica , Neuropéptido Y/metabolismo , Óxido Nítrico Sintasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia P/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Órgano Vomeronasal/inervaciónRESUMEN
CONCLUSION: Lipopolysaccharide (LPS) challenge of the human nose has the capacity to reduce the amount of natural anti-inflammatory proteins, such as uteroglobin. OBJECTIVES: Nasal challenge with LPS, an activator of innate immunity, has been shown to increase the amount of pro-inflammatory mediators in nasal lavage fluid. Uteroglobin is a newly described anti-inflammatory mediator that is secreted in the nose. This study examined the effect of nasal LPS application on the level of uteroglobin in nasal lavage fluid as well as on the expression of uteroglobin in nasal mucosa. MATERIALS AND METHODS: Thirty-eight volunteers were challenged nasally with either 50 microg LPS or vehicle; 6 h later, nasal lavage fluid was collected and a nasal biopsy was obtained. Levels of uteroglobin, albumin and the pro-inflammatory mediators interleukin (IL)-6 and IL-8 were analysed in the lavage fluids using enzyme-linked immunosorbent assays (ELISAs). Biopsies were used for either quantification of uteroglobin mRNA by real-time PCR or for localization of the corresponding protein with immunohistochemistry. RESULTS: The uteroglobin level decreased in nasal lavage fluid following LPS challenge, whereas the levels of IL-6 and albumin increased. Uteroglobin was mainly seen in the respiratory epithelium and its mRNA expression decreased as a consequence of the LPS challenge.
Asunto(s)
Lipopolisacáridos/inmunología , Mucosa Nasal/inmunología , Uteroglobina/metabolismo , Adulto , Biopsia , Regulación hacia Abajo/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunidad Innata/inmunología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Líquido del Lavado Nasal/inmunología , ARN Mensajero/genética , Albúmina Sérica/metabolismo , Uteroglobina/genéticaRESUMEN
BACKGROUND: Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis. METHODS: The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively. RESULTS: TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis. CONCLUSION: The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.
Asunto(s)
Médula Ósea/metabolismo , Leucocitos/metabolismo , Mucosa Nasal/metabolismo , Rinitis Alérgica Estacional/metabolismo , Receptor Toll-Like 9/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVES: Endothelin is a potent peptide mediator that is synthesized by a number of cells. Previous studies have revealed the occurrence of endothelin in nerve cell bodies of some peripheral ganglia. Endothelin mediates its effects via two distinct receptor subtypes ETA and ETB. The present study was designed to investigate the presence of these two receptors in the human trigeminal ganglion. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show the presence of mRNA encoding ETA and ETB receptors in the human trigeminal ganglion. To localize the protein immunocytochemistry with antibodies against the endothelin receptors was used. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed mRNA for both receptor subtypes in the human trigeminal ganglion. Immunocytochemistry revealed numerous cell bodies containing the ETA and the ETB receptor proteins. CONCLUSIONS: The expression of ETA and ETB receptors in the human trigeminal ganglion suggests a role for endothelin in autonomic and sensory neural transmission.
Asunto(s)
Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Ganglio del Trigémino/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
CONCLUSIONS: Topical steroids did not affect expression of growth-related oncogene-alpha (GRO-alpha) in nasal polyps. The results of this study suggest roles for steroid-resistant gene expression in the pathogenesis of nasal polyps and point to the need for additional pharmacological strategies. OBJECTIVE: Infiltration of inflammatory cells is believed to play a role in the development of nasal polyps. GRO-alpha is a chemokine that recruits and activates neutrophils and also possesses growth stimulatory and angiogenetic properties. An increased presence of GRO-alpha has been demonstrated in nasal polyps compared with normal nasal tissue. In this study we evaluate the presence and expression levels of GRO-alpha in nasal polyps before and after glucocorticoid treatment. MATERIAL AND METHODS: Nasal polyps were surgically removed in patients before and 6 weeks after treatment with topically applied fluticasone. GRO-alpha gene expression and the presence of GRO-alpha peptide were detected in polyp tissue by means of in situ hybridization, quantitative real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: Strong GRO-alpha gene expression and the presence of GRO-alpha peptide were seen in both the epithelium and stromal inflammatory cells of nasal polyps. No differences in gene expression levels in tissue homogenates were found when untreated polyp tissue was compared with polyps treated for 6 weeks with topically applied steroids.
Asunto(s)
Androstadienos/farmacología , Antiinflamatorios/farmacología , Quimiocinas CXC/biosíntesis , Expresión Génica/genética , Pólipos Nasales/metabolismo , Oncogenes/fisiología , Administración Tópica , Adulto , Androstadienos/administración & dosificación , Androstadienos/metabolismo , Antiinflamatorios/administración & dosificación , Antiinflamatorios/metabolismo , Quimiocina CXCL1 , Quimiocinas/biosíntesis , Quimiocinas/genética , Quimiocinas CXC/genética , Femenino , Fluticasona , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Pólipos Nasales/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) alpha, betadelta and gamma are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested. METHODS: Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification. RESULTS: mRNA expression of PPARalpha, PPARbetadelta, PPARgamma was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARalpha and PPARgamma than normal nasal mucosa and these levels were, for PPARgamma, further reduced following steroid treatment. PPARgamma immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARgamma immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment. CONCLUSION: The down-regulation of PPARgamma, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARgamma might be of importance in long standing inflammations.