RESUMEN
OBJECTIVE: To identify interleukin (IL)-7Rα expression in the labial salivary gland (LSG) of patients with primary Sjögren's syndrome (pSS) and non-Sjögren's syndrome sicca (nSS-sicca) and to study its correlation with glandular inflammation and IL-7 expression. METHODS: The presence of infiltrating immune cells and IL-7Rα cells in inflamed LSG of patients with pSS (n=12) and nSS-sicca controls (n=7) was studied by immunohistochemistry and fluorescence activated cell sorting analysis upon tissue digestion (n=15 and n=13, respectively). Additionally, the correlations of IL-7Rα cells with hallmark disease parameters of pSS, major infiltrating inflammatory cells and IL-7 were assessed. RESULTS: In the LSG of patients with pSS increased numbers of IL-7Rα cells were found as compared with nSS-sicca patients. IL7Rα cells strongly correlated with the lymphocytic focus score, IL-7 expression, the decrease in percentage of IgA plasma cells and numbers of CD3 T cells, CD20 B cells, and CD1a and CD208 myeloid dendritic cells. Analysis of isolated cells from the LSG demonstrated strongly increased percentages of IL-7Rα CD3 T cells in pSS as compared with nSS, showing abundant IL-7Rα expression on both CD4 and CD8 T cells. Other CD45 leucocytes and CD45- tissue cells scarcely expressed IL-7Rα. Percentages of IL-7Rα T cells also significantly correlated with glandular inflammation. CONCLUSIONS: This study shows the presence of increased IL-7Rα T cells in the LSG of patients with pSS and their association with the severity of sialadenitis, disease parameters and IL-7 expression. Considering the immunostimulatory ability of IL-7Rα T cells and IL-7, this suggests that IL-7(R)-dependent T cell-driven immune activation plays an important role in inflammation in pSS.
Asunto(s)
Interleucina-7/inmunología , Receptores de Interleucina-7/inmunología , Glándulas Salivales/inmunología , Sialadenitis/inmunología , Síndrome de Sjögren/inmunología , Linfocitos T/inmunología , Adulto , Biomarcadores/metabolismo , Biopsia , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Femenino , Humanos , Interleucina-7/metabolismo , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Receptores de Interleucina-7/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Sialadenitis/metabolismo , Sialadenitis/patología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
OBJECTIVES: In the salivary glands of patients with primary Sjögren Syndrome (pSjS) an accumulation of dendritic cells (DCs) is seen, which is thought to play a role in stimulating local inflammation. Aberrancies in subsets of monocytes, generally considered the blood precursors for DCs, may play a role in this accumulation of DCs. This study is aimed at determining the level of mature CD14lowCD16+ monocytes in pSjS and their contribution to the accumulation of DCs in pSjS. METHODS: Levels of mature and immature monocytes in patients with pSjS (n = 19) and controls (n = 15) were analysed by flow cytometry. The reverse transmigration system was used for generation of DCs generated from monocyte subsets. The phenotype of DCs in pSjS salivary glands was analysed using immunohistochemistry. In vivo tracking of monocyte subsets was performed in a mouse model. RESULTS: Increased levels of mature CD14lowCD16+ monocytes were found in patients with pSjS (mean (SD) 14.5 (5.5)% vs 11.4 (3.4)%). These cells showed normal expression of chemokine receptor and adhesion molecules. Mature monocytes partly developed into DC-lysosome-associated membrane glycoprotein (LAMP)+ (19.6 (7.5)%) and CD83+ (16 (9)%) DCs, markers also expressed by DCs in pSjS salivary glands. Monocyte tracking in the non-obese diabetic (NOD) mouse showed that the homologue population of mature mouse monocytes migrated to the salivary glands, and preferentially developed into CD11c+ DCs in vivo. CONCLUSIONS: Mature monocytes are increased in pSjS and patient and mouse data support a model where this mature monocyte subset migrates to the salivary glands and develops into DCs.
Asunto(s)
Células Dendríticas/inmunología , Monocitos/inmunología , Receptores de IgG/sangre , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Animales , Antígenos CD/sangre , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/sangre , Proteínas Ligadas a GPI , Humanos , Inmunoglobulinas/sangre , Inmunofenotipificación , Proteínas de Membrana de los Lisosomas/sangre , Glicoproteínas de Membrana/sangre , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Antígeno CD83RESUMEN
In the early development of type 1 diabetes macrophages and dendritic cells accumulate around the islets of Langerhans at sites of fibronectin expression. It is thought that these macrophages and dendritic cells are derived from blood monocytes. Previously, we showed an increased serum level of MRP8/14 in type 1 diabetes patients that induced healthy monocytes to adhere more strongly to fibronectin (FN). Here we show that MRP8/14 is expressed and produced at a higher level by type 1 diabetes monocytes, particularly after adhesion to FN, creating a positive feedback mechanism for a high fibronectin-adhesive capacity. Also adhesion to endothelial cells was increased in type 1 diabetes monocytes. Despite this increased adhesion the transendothelial migration of monocytes of type 1 diabetes patients was decreased towards the proinflammatory chemokines CCL2 and CCL3. Because non-obese diabetic (NOD) mouse monocytes show a similar defective proinflammatory migration, we argue that an impaired monocyte migration towards proinflammatory chemokines might be a hallmark of autoimmune diabetes. This hampered monocyte response to proinflammatory chemokines questions whether the early macrophage and dendritic cell accumulation in the diabetic pancreas originates from an inflammatory-driven influx of monocytes. We also show that the migration of type 1 diabetes monocytes towards the lymphoid tissue-related CCL19 was increased and correlated with an increased CCR7 surface expression on the monocytes. Because NOD mice show a high expression of these lymphoid tissue-related chemokines in the early pancreas it is more likely that the early macrophage and dendritic cell accumulation in the diabetic pancreas is related to an aberrant high expression of lymphoid tissue-related chemokines in the pancreas.
Asunto(s)
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Citocinas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Monocitos/inmunología , Páncreas/inmunología , Adulto , Estudios de Casos y Controles , Adhesión Celular , Quimiotaxis de Leucocito , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Inmunofenotipificación , MasculinoRESUMEN
BACKGROUND: Corticosteroids are routinely used in patients with pulmonary fibrosis. The timing for initiation of treatment is likely to be crucial for corticosteroids to exert an antifibrotic effect. Experimental studies in animals have examined the effect of corticosteroid treatment starting before or at the time of lung injury. However, this is not representative of the human condition as treatment only begins after disease has been established. We examined the effect of a short course corticosteroid treatment starting 3 days after bleomycin induced lung injury on the development of pulmonary fibrosis. METHODS: Bleomycin (1.5 mg/kg) was instilled intratracheally into rats to induce pulmonary fibrosis. The effect of a 3-day course of dexamethasone (0.5 mg/kg) initiated 3 days after bleomycin induced lung injury on cell proliferation and collagen deposition was examined by analysing bronchoalveolar lavage (BAL) fluid and lung tissue. RESULTS: Treating bleomycin exposed animals after injury with dexamethasone for 3 days inhibited lung collagen deposition compared with animals exposed to bleomycin without dexamethasone treatment (15.2 (2.2) mg collagen/lung v 22.5 (2.1) mg/lung; p<0.05). Dexamethasone treatment reduced pulmonary parenchymal cell proliferation in bleomycin exposed rats but did not influence BAL fluid mitogenic activity for lung fibroblasts or alter the BAL fluid levels of the fibrogenic mediators transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin. CONCLUSIONS: A 3 day course of dexamethasone treatment initiated 3 days after bleomycin induced lung injury reduces lung cell proliferation and collagen deposition by mechanisms other than through reduction of transforming growth factor-beta(1), platelet derived growth factor-AB, and thrombin levels in BAL fluid. We propose that an early short course treatment with dexamethasone may be useful in inhibiting pulmonary fibrosis.
Asunto(s)
Dexametasona/administración & dosificación , Glucocorticoides/administración & dosificación , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos , Bleomicina , Peso Corporal , Líquido del Lavado Bronquioalveolar/citología , Colágeno/química , Fibroblastos , Masculino , Fibrosis Pulmonar/inducido químicamente , Ratas , Ratas Endogámicas LewRESUMEN
Pulmonary fibrosis results from excessive fibroblast proliferation and increased collagen deposition and occurs in chronic lung disease of prematurity (CLD). Platelet-derived growth factor (PDGF)-BB is mitogenic for fibroblasts and levels are increased in fibrotic lung disorders. Systemic dexamethasone (DEX) treatment improves pulmonary function and reduces inflammation in infants with or at risk of CLD. However, the effect of DEX treatment on fibroblast activity, PDGF-BB and collagen synthesis in the lungs of CLD patients is uncertain. Bronchoalveolar lavage (BAL) fluids, obtained from 15 infants at risk of CLD before and after DEX treatment, were analysed for fibroblast mitogenicity, PDGF-BB, N-terminal propeptide of collagen type III (PIIINP) and interleukin (IL)-1beta levels and inflammatory cell numbers. After DEX treatment, the mitogenic activity of BAL fluid for fibroblasts was not reduced but increased. The change in mitogenicity correlated with a change in BAL fluid PDGF-BB levels. Furthermore, BAL fluid-induced fibroblast proliferation was blocked using an inhibitor of the PDGF receptor. DEX treatment did not influence PIIINP levels, but reduced IL-1beta levels and inflammatory cell numbers in BAL fluid. This study suggests that dexamethasone treatment does not reduce fibroblast proliferation despite apparent downregulation of inflammation. The present findings do not support the use of dexamethasone for prevention of the fibrotic response in infants at risk of chronic lung disease of prematurity.
Asunto(s)
Antiinflamatorios/uso terapéutico , Líquido del Lavado Bronquioalveolar/inmunología , Dexametasona/uso terapéutico , Fibrosis Pulmonar/prevención & control , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Líquido del Lavado Bronquioalveolar/citología , Fibroblastos/fisiología , Humanos , Recién Nacido , Recien Nacido Prematuro , Fibrosis Pulmonar/etiología , Síndrome de Dificultad Respiratoria del Recién Nacido/complicacionesRESUMEN
Bronchopulmonary dysplasia (BPD) can evolve in prematurely born infants who require mechanical ventilation because of hyaline membrane disease (HMD). The development of BPD can be divided in an acute, a regenerative, a transitional, and a chronic phase. During these different phases, extensive remodeling of the lung parenchyma with re-epithelialization of the alveoli and formation of fibrosis occurs. Matrix metalloproteinase-1 (MMP-1) is an enzyme that is involved in re-epithelialization processes, and dysregulation of MMP-1 activity contributes to fibrosis. Localization of MMP-1 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were investigated in lung tissue obtained from infants who died during different phases of BPD development. In all studied cases (n = 50) type-II pneumocytes were found to be immunoreactive for MMP-1, TIMP-1, and TIMP-2. During the acute and regenerative phase of BPD, type-II pneumocytes re-epithelialize the injured alveoli. This may suggest that MMP-1 and its inhibitors, expressed by type-II pneumocytes, play a role in the re-epithelialization process after acute lung injury. Although MMP-1 staining intensity remained constant in type-II pneumocytes during BPD development, TIMP-1 increased during the chronic fibrotic phase. This relative elevation of TIMP-1 compared with MMP-1 is indicative for reduced collagenolytic activity by type-II pneumocytes in chronic BPD and may contribute to fibrosis. Fibrotic foci in chronic BPD contained fibroblasts immunoreactive for MMP-1 and TIMP-1 and -2. This may indicate that decreased collagen turnover by fibroblasts contributes to fibrosis in BPD development.
Asunto(s)
Displasia Broncopulmonar/fisiopatología , Pulmón/patología , Metaloproteinasa 1 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Autopsia , Displasia Broncopulmonar/enzimología , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , Recién Nacido , Recien Nacido Prematuro , Pulmón/enzimología , MasculinoRESUMEN
OBJECTIVES: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues. METHODS: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice. RESULTS: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein. DISCUSSION: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Glándula Submandibular/crecimiento & desarrollo , Glándula Submandibular/patología , Envejecimiento/inmunología , Alelos , Animales , Animales Congénicos , Animales Recién Nacidos , Apoptosis , Enfermedades Autoinmunes/enzimología , División Celular , Mapeo Cromosómico , Células Epiteliales/patología , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos NOD , Morfogénesis , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/fisiopatología , Glándula Submandibular/enzimología , Glándula Submandibular/inmunologíaAsunto(s)
Asma/enzimología , Bronquios/enzimología , Antígenos CD13/fisiología , Dipeptidil Peptidasa 4/fisiología , Neprilisina/fisiología , Inflamación Neurogénica/enzimología , Neuropéptidos/metabolismo , Animales , Antiasmáticos/farmacología , Antiinflamatorios/farmacología , Asma/patología , Líquido del Lavado Bronquioalveolar , Células Dendríticas/enzimología , Eosinófilos/enzimología , Glucocorticoides/farmacología , Antagonistas de Hormonas/farmacología , Humanos , Mifepristona/farmacología , Inflamación Neurogénica/patología , Fagocitos/enzimología , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/fisiología , Fumar/efectos adversos , SolubilidadRESUMEN
Sjögren's syndrome is an autoimmune disease that primarily affects the salivary and lacrimal glands. In these glands, focal lymphocytic infiltrates develop. Little is known about the initiation of this autoimmune disease. Antigen-presenting cells (APC) such as dendritic cells (DC) can play a role in the initiation of autoimmunity. To date, no data on the presence of DC in Sjögren's syndrome are available. Several mouse strains, the nonobese diabetic (NOD) and the MRL/Ipr mouse, can be used as models for Sjögren's syndrome. We compared the development of sialoadenitis in the submandibular glands (SMG) of NOD and MRL/Ipr mice with particular focus on the presence of APC. DC, macrophages, T cells, and B cells in the SMG were studied by means of immunohistochemistry, after which positively stained cells were quantified. NOD-severe combined immunodeficiency (SCID) mice were used to study the presence of APC in the SMG in the absence of lymphocytes. Before lymphocytic infiltration, increased numbers of DC were detected in the SMG of NOD mice compared with those numbers in control mice and MRL/Ipr mice, which suggests that DC play a role in the initiation of sialoadenitis in NOD mice. In the SMG of NOD mice, lymphocytic infiltrates organized in time. In MRL/Ipr mice, however, lymphocytic infiltrates were already organized at the time of appearance. This organization was lost over time. In conclusion, two types of sialoadenitis are described in two mouse models for Sjögren's syndrome. Differences exist with regard to early events that may lead to the development of sialoadenitis and to the composition and organization of inflammatory infiltrates. It is possible that different types of sialoadenitis also exist in humans and that the pathogenetic process in both the early and late phases of the autoimmune reaction differs among patients.
Asunto(s)
Células Dendríticas/inmunología , Síndrome de Sjögren/inmunología , Animales , Presentación de Antígeno , Autoinmunidad/genética , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos NOD , Síndrome de Sjögren/etiología , Síndrome de Sjögren/genética , Especificidad de la Especie , Glándula Submandibular/inmunología , Glándula Submandibular/patologíaRESUMEN
Sjögren's syndrome is an autoimmune disease in which lymphocytic infiltrates develop in the salivary and lacrimal glands. We have shown that dendritic cells (DC) infiltrate the submandibular gland of the nonobese diabetic (NOD) mouse, a mouse model for Sjögren's syndrome, before lymphocytic infiltration, suggesting that these antigen-presenting cells (APC) may play a role in the initiation of Sjögren's syndrome. In later stages, DC and macrophages also form an important part of the infiltrate of the NOD sialoadenitis. To find out if DC and macrophages form part of the infiltrate in Sjögren's syndrome as well, and to determine whether they may be useful in the histopathological diagnosis of Sjögren's syndrome, we studied their presence in minor salivary glands (MSG) of patients with Sjögren's syndrome and patients with focal lymphocytic sialoadenitis (FLS), but without clinical or serological criteria of Sjögren's syndrome. Immunohistochemistry was applied, followed by semiquantitative analysis. DC and macrophages were present in all MSG; however, there were clear differences in marker expression between Sjögren's syndrome and FLS, on the one hand, and control tissue, on the other hand. CD1a+ DC and RFD9+ macrophages were mainly observed in MSG in which a focal lymphocytic infiltrate was present. In fact, the diffuse presence of single CD1a+ DC and RFD9+ macrophages correlated closely with the presence of a focal lymphocytic infiltrate in the MSG. This indicates that these cells could be of help during the evaluation of a MSG. Because the detection of APC is technically less cumbersome than a focal score, this parameter may perhaps replace the focal score in the histopathological diagnosis of Sjögren's syndrome. This study therefore prompts further investigation focusing on the presence of CD1a+ and RFD9+ cells in the MSG of a large cohort of patients.
Asunto(s)
Células Presentadoras de Antígenos/patología , Glándulas Salivales/patología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Adulto , Anciano , Anticuerpos Monoclonales , Células Presentadoras de Antígenos/inmunología , Antígenos CD/análisis , Antígenos CD1/análisis , Biopsia , Femenino , Humanos , Inmunoglobulinas/análisis , Labio/inmunología , Labio/patología , Linfocitos/inmunología , Linfocitos/patología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Glicoproteínas de Membrana/análisis , Persona de Mediana Edad , Glándulas Salivales/inmunología , Antígeno CD83RESUMEN
BACKGROUND: Neuropeptides may be involved in the pathogenesis of asthma by evoking neurogenic inflammation. Since the effects of neuropeptides are limited by peptidases, reduced activity of peptidases may contribute to the inflammatory process. OBJECTIVE: We hypothesized that soluble peptidase activities are decreased in asthmatics and that inhaled glucocorticoids exert part of their anti-inflammatory action by increasing soluble peptidase activities. METHODS: Serum and bronchoalveolar lavage (BAL) fluid was obtained from non-smoking and smoking volunteers and from allergic asthmatics both before and after treatment for 12 weeks with placebo or inhaled fluticasone propionate. Activities of neutral endopeptidase (NEP), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV) were determined using colourometric assays. RESULTS: Reduced DPP IV activity in serum and reduced NEP activity in BAL fluid were found in healthy smokers compared with non-smokers. In contrast, no differences in peptidase activities in serum or BAL fluid were observed between allergic asthmatics and healthy non-smokers. Fluticasone propionate treatment did not affect peptidase activities in the asthmatic patients. CONCLUSIONS: We conclude that reduced peptidase activities in serum or BAL fluid can be found in healthy smokers, but not in allergic asthmatics, and that inhaled glucocorticoids do not affect peptidase activities in BAL fluid or serum of asthmatics. Our results do not support the hypothesized dysfunction of peptidases in the asthmatic airways.
Asunto(s)
Asma/enzimología , Líquido del Lavado Bronquioalveolar/química , Antígenos CD13/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Hipersensibilidad/enzimología , Neprilisina/metabolismo , Administración por Inhalación , Administración Tópica , Adolescente , Adulto , Androstadienos/administración & dosificación , Androstadienos/uso terapéutico , Antialérgicos/administración & dosificación , Antialérgicos/uso terapéutico , Antiasmáticos/administración & dosificación , Antiasmáticos/uso terapéutico , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Asma/tratamiento farmacológico , Antígenos CD13/análisis , Antígenos CD13/sangre , Dipeptidil Peptidasa 4/análisis , Dipeptidil Peptidasa 4/sangre , Femenino , Fluticasona , Glucocorticoides , Humanos , Hipersensibilidad/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Neprilisina/análisis , Neprilisina/sangre , Valores de Referencia , FumarRESUMEN
Asthma is considered a Th2-like disease, characterized by locally increased levels of interleukin (IL) 4. The bronchial epithelium plays an important role in the initiation and perpetuation of inflammatory reactions within the airways. However, little is known about the presence of IL-4 receptors on human bronchial epithelial cells, or the effects of IL-4 on these cells. In this report, definitive evidence of IL-4 receptor expression on human bronchial epithelial cells using several methods is presented. IL-4 receptor expression on human bronchial epithelial cells in vivo was demonstrated using in situ hybridization and immunohistochemistry. No difference in IL-4 receptor protein expression was observed between bronchial biopsies of healthy subjects compared to allergic asthmatics. Cultured human bronchial epithelial cells also expressed IL-4 receptor mRNA and protein (as determined by RT-PCR analysis and flow cytometry, respectively). IL-4 receptor protein expression by bronchial epithelial cells could be increased by stimulation with PMA+calcium ionophore, whereas IL-1beta and IL-6 decreased IL-4 receptor expression. A cyclic AMP analogue and IL-4 had no effect. Finally, it is shown that the IL-4 receptor is functionally active as IL-4 stimulates the release of IL-8, monocyte chemoattractant protein 1, and particularly IL-1 receptor antagonist by human bronchial epithelial cells. It is concluded that human bronchial epithelial cells express IL-4 receptors both in vivo and in vitro. Stimulation of human bronchial epithelial cells by IL-4 may result in the release of both pro- and anti-inflammatory mediators known to be upregulated in asthmatic airways.
Asunto(s)
Asma/metabolismo , Bronquios/metabolismo , Regulación de la Expresión Génica , Receptores de Interleucina-4/metabolismo , Bucladesina/farmacología , Calcimicina/farmacología , Células Cultivadas , Quimiocina CCL2/metabolismo , Células Epiteliales/metabolismo , Humanos , Hibridación in Situ , Molécula 1 de Adhesión Intercelular/metabolismo , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-4/farmacología , Interleucina-8/metabolismo , Interleucinas/farmacología , ARN Mensajero/metabolismo , Receptores de Interleucina-1/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Previously, we found that inflammatory mediators modulated the number and binding affinity of glucocorticoid receptors (GR) in human bronchial epithelial cell lines. In this study we investigated whether smoking and chronic obstructive pulmonary disease (COPD), both characterized by airway inflammation with increased levels of inflammatory mediators, affect GR characteristics in cultured human bronchial epithelial cells (HBEC). A statistically significant difference was found between the dissociation constant (Kd) values in HBEC from smoking (Kd = 0.98+/-0.08 nM; n = 6) and nonsmoking controls (Kd = 0.76+/-0.10 nM, P = 0.03; n = 5), but no significant difference was found between the mean number of binding sites. Our results are the first indication that cultured HBEC from smokers possess GR with a lower binding affinity. This may result from the inflammation found in the airways from smokers. Furthermore, these results provide further evidence that the bronchial epithelium may be an actual target for inhaled glucocorticoid therapy.
Asunto(s)
Bronquios/química , Enfermedades Pulmonares Obstructivas/metabolismo , Receptores de Glucocorticoides/análisis , Fumar/metabolismo , Anciano , Sitios de Unión , Línea Celular , Dexametasona/metabolismo , Células Epiteliales/química , Humanos , Persona de Mediana EdadRESUMEN
BACKGROUND: Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and chronic inflammation of the airways. Since peptides are able to produce many of the pathophysiological features which are characteristic of asthma, peptide-mediated inflammation is thought to play a role in this disease. The effects of peptides are modulated by peptidases, which are able to degrade peptides, mostly resulting in their inactivation. OBJECTIVES: In this study, we investigated the distribution of two peptidases, aminopeptidase N and dipeptidyl peptidase IV, in the human bronchus and determined whether their expression was altered in allergic asthmatics. METHODS: We first determined the distribution of aminopeptidase N and dipeptidyl peptidase IV in the human bronchus using immuno- and enzymehistochemistry and compared this with the distribution of neutral endopeptidase. Secondly, the expression of aminopeptidase N and dipeptidyl peptidase IV was determined in bronchial biopsies of healthy subjects (n = 8) and allergic asthmatics (n = 12). RESULTS: Aminopeptidase N was localized in connective tissue, blood vessels, gland ducts, perichondrium, nerves and leucocytes (mainly mononuclear phagocytes, dendritic cells, and eosinophils). Dipeptidyl peptidase IV was localized in serosal glands, blood vessels, and T cells. Immunohistochemistry and enzymehistochemistry gave similar results. Comparison of the expression of aminopeptidase N and dipeptidyl peptidase IV in bronchial biopsies of healthy controls and atopic asthmatics revealed no significant differences in the lamina propria. In contrast, in the bronchial epithelium of atopic asthmatics an increased number of aminopeptidase N-positive cells could be found. Double-staining identified these cells as L25+ dendritic cells and eosinophils. CONCLUSIONS: We conclude that expression of aminopeptidase N and dipeptidyl peptidase IV is restricted to specific sites within the human bronchus. Furthermore, in the bronchial epithelium of allergic asthmatics an increased number of aminopeptidase N-expressing dendritic cells and eosinophils can be found.
Asunto(s)
Asma/enzimología , Bronquios/enzimología , Antígenos CD13/biosíntesis , Dipeptidil Peptidasa 4/biosíntesis , Adulto , Asma/patología , Biopsia , Bronquios/química , Bronquios/patología , Antígenos CD13/análisis , Dipeptidil Peptidasa 4/análisis , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neprilisina/análisis , Neprilisina/metabolismo , Distribución TisularRESUMEN
Peptidases play an important role in the regulation of peptide-mediated effects. Modulation of peptidase activity may therefore be a major mechanism to control peptide actions. Our aim was to analyse the effects of cytokines and glucocorticoids on peptidases expressed by human bronchial epithelial cells, which have been shown to be an important site for peptidase activity. The effects of cytokines [interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), IL-4, interferon gamma (INF-gamma), and epidermal growth factor (EGF)] and/or dexamethasone (DEX) on both expression and activity of neutral endopeptidase (NEP) and aminopeptidase N (APN) by BEAS 2B cells were determined using flow cytometry and activity assays, respectively. IL-1 beta, and to a lesser extent, TNF-alpha and IL-4 increased NEP activity and expression, whereas IFN-gamma decreased NEP. The effect of IL-1 beta was mediated, at least in part, via a cAMP-dependent pathway which did not involve prostaglandin E2 synthesis. APN was increased after 24-h stimulation with IFN-gamma, whereas other stimuli had no effect. DEX strongly increased NEP and APN expression and activity, both in the absence and in the presence of cytokines. We conclude that cytokines and glucocorticoids are able to modulate the activity of NEP and APN on BEAS 2B cells. Our results suggest a role for the human bronchial epithelium in the control of inflammation and indicate that one beneficial effect of glucocorticoids on asthma may be upregulation of peptidases expressed by bronchial epithelial cells.
Asunto(s)
Antígenos CD13/metabolismo , Citocinas/farmacología , Dexametasona/farmacología , Células Epiteliales/enzimología , Glucocorticoides/farmacología , Neprilisina/metabolismo , Bronquios/citología , Recuento de Células/efectos de los fármacos , Línea Celular Transformada , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/efectos de los fármacos , Humanos , Sistemas de Mensajero SecundarioRESUMEN
Cytogenetic deletions of the short arm of chromosome 9, 9p, have been detected in cell lines of malignant mesothelioma as well as in tumor material. Many tumor types carry deletions of chromosome 9 or more specifically of 9p21. The tumor-suppressor genes, CDKN2A and CDKN2B, each of which encodes a structurally and functionally similar cyclin-dependent kinase inhibitor, were mapped to the commonly deleted region. The tumor-suppressive effect of these genes, or of CDKN2A alone, requires functional retinoblastoma protein, pRb. Malignant mesothelioma expresses pRb, which, together with the cytogenetic data, suggests the involvement of CDKN2A and/or CDKN2B in its tumorigenesis. We present data on the deletion status of chromosome 9 in malignant mesothelioma cell lines and tumor tissue. A deletion map of the 9p21.3-p23 region was constructed for 12 cell lines. Homozygous deletions of chromosomal regions containing CDKN2A were detected in all cell lines. The smallest region of overlap for deletion is approximately 24 kb, and does not include CDKN2B. The frequency of deletion of the centromeric region of chromosome 9 was compared with that of chromosomes 1, 6, and 10 by genomic in situ hybridization. Deletion of the centromere of chromosome 9 is the predominant event at a frequency of 73 +/- 3%. Our data show that deletions of a critical region of chromosome 9, including the CDKN2A but not the CDKN2B locus, are common among malignant mesothelioma. Such deletions may be involved in tumorigenesis of mesothelium.
Asunto(s)
Cromosomas Humanos Par 9 , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Eliminación de Gen , Mesotelioma/genética , Aberraciones Cromosómicas/genética , Deleción Cromosómica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Hibridación in Situ , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
Previous studies have demonstrated mitogenic effects of several mediators on mesothelial cells in vitro, but their effects in vivo have not been investigated. The aim of this study was to examine the effects of various cytokines on normal mesothelial cell proliferation in vitro and in vivo and correlate the findings in both assay systems. In vitro proliferation was assessed using a technique based on the uptake and subsequent release of methylene blue. Autoradiographic methods were applied in a murine model to assess mitogenic activity of these factors on mesothelium in vivo. In vitro data demonstrated a dose-dependent increase in human mesothelial cell proliferation by all mediators examined: at optimal concentrations, proliferation was enhanced between 26.53 +/- 3.77% standard deviation (SD), p < 0.001 for fibroblast growth factor-2 (FGF-2) and 114.58 +/- 6.97%, p < 0.001 for platelet-derived growth factor-AB (PDGF-AB) above control medium. In vivo, DNA synthesis in mesothelial cells was stimulated by FGF-2 (29.52 +/- 5.85% labeled cells, compared with 7.04 +/- 4.36% for control medium; p < 0.001), tumor necrosis factor-alpha (TNF-alpha; 13.14 +/- 4.55% compared with 7.23 +/- 2.85; p < 0.005) and PDGF-BB (11.53 +/- 4.74% compared with 4.67 +/- 3.48%; p < 0.005). Transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) had no effect on DNA synthesis in mesothelial cells in vivo. It is concluded that FGF2, TNF-alpha, and PDGF stimulate mesothelial cell proliferation in vitro and in vivo, whereas TGF-beta1 and EGF only had a mitogenic effect in vitro at the concentrations examined. The mitogenic potency of the different PDGF isoforms in vitro was consistent with PDGF-alpha and beta receptor expression.
Asunto(s)
División Celular/fisiología , Citocinas/fisiología , Testículo/citología , Animales , División Celular/efectos de los fármacos , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Epitelio/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor. Coexpression of the PDGF beta-receptor and its ligand may lead to an autocrine growth stimulating loop in the malignant cell type. In nuclear run off experiments, PDGF B-chain mRNA was detectable in MM cells only, indicating an increased level of transcription in this cell type. The proximal promoter of the PDGF B-chain gene contains DNaseI hypersensitive (DH) sites and mediates reporter gene activation in both normal and malignant cells. Nuclear proteins, extracted from both cell types, interact with DNA sequences within the proximal promoter around bp-64 to -61 relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) indicate that these factors are more abundantly present in the malignant than in the normal cell type. A DH site around -9.9 kb was found in both cell types. When tested in CAT assays, this region exerted a stimulatory effect on transcription in malignant cells. The elevated level of transcription of the PDGF B-chain gene in malignant cells may well be the result of interaction of regulatory sites in the proximal promoter and an enhancing element located at -9.9 kb from the transcription start site.
Asunto(s)
Mesotelioma/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción Genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Bronchial epithelium plays a major role in the regulation of inflammatory reactions in the airways. It is thought to be a possible target for glucocorticoid therapy. Glucocorticoid responsiveness requires the presence of specific glucocorticoid receptors (GR). Until now, little was known about the presence of such receptors in the human bronchial epithelium. In this study we demonstrated the expression of GR messenger ribonucleic acid (mRNA) in two simian virus (SV)-40/adenovirus-transformed human bronchial epithelial cell lines, BEAS S6 and BEAS 2B. In a whole cell dexamethasone binding assay, BEAS S6 and BEAS 2B cells were found to possess (mean +/- SEM) 28.9 +/- 4.4 x 10(3) and 32.1 +/- 5.7 x 10(3) binding sites per cell, respectively, with dissociation constant (Kd) values of 8.2 +/- 1.5 and 8.6 +/- 2.4 nM, respectively. Using electrophoretic mobility shift assays we demonstrated the binding of nuclear translocated GR to specific sites on deoxyribonucleic acid (DNA), named glucocorticoid responsive elements (GRE). Lipopolysaccharide (LPS) and interleukin-1 beta (IL-1 beta) significantly increased the number of GR per cell (median = 312% and 171% of control, respectively; p < 0.05), but significantly reduced the ligand affinity of these receptors, i.e. increased the Kd (median = 410% and 145% of control, respectively; p < 0.05) in BEAS 2B cells. These results indicate that the bronchial epithelium may be an actual target for glucocorticoid therapy. Inflammatory mediators, such as IL-1 beta and LPS, modulate the number and ligand affinity of these GR. Therefore, the response of bronchial epithelium to glucocorticoid therapy may be modulated by airway diseases associated with inflammation.