RESUMO
BACKGROUND: Allergic asthma is a Th2-type chronic inflammatory disease of the lung. It is characterized by infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2 cytokines like interleukin (IL)-4, IL-5 and chemokines like eotaxin are increased in the asthmatic response. The processing and presentation of exogenous antigens is important in the sensitization to an allergen. Cathepsin E (Ctse) is an intracellular aspartic endoprotease which is expressed in immune cells like dendritic cells (DCs). It was found to play an essential role in the processing and presentation of ovalbumin (OVA). The aim of the present study was to investigate the inhibition of Ctse in two different experimental models of allergic airway inflammation. METHODS: Ctse wild-type (Ctse(+/+)) and Ctse-deficient (Ctse(-/-)) bone marrow-derived DCs (BMDCs) were pulsed with OVA/OVA peptide and cocultured with OVA transgenic T II (OT II) cells whose proliferation was subsequently analyzed. Two different in vivo asthma models with Ctse(+/+) and Ctse(-/-) mice were performed: an acute OVA-induced and a subchronic Phleum pratense-induced airway inflammation. RESULTS: Proliferation of OT II cells was decreased when cocultured with BMDCs of Ctse(-/-) mice as compared to cells cocultured with BMDCs of Ctse(+/+) mice. In vivo, Ctse deficiency led to reduced lymphocyte influx after allergen sensitization and challenge in both investigated airway inflammation models, compared to their control groups. CONCLUSION: Ctse deficiency leads to a reduced antigen presentation in vitro. This is followed by a distinct effect on lymphocyte influx in states of allergic airway inflammation in vivo.
Assuntos
Asma/imunologia , Catepsina E/deficiência , Células Dendríticas/imunologia , Pulmão/imunologia , Pneumonia/imunologia , Linfócitos T/imunologia , Doença Aguda , Alérgenos/imunologia , Animais , Asma/complicações , Asma/enzimologia , Asma/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Catepsina E/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Modelos Animais de Doenças , Pulmão/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Knockout , Ovalbumina/imunologia , Ovalbumina/farmacologia , Peptídeos/imunologia , Peptídeos/farmacologia , Phleum/imunologia , Pneumonia/complicações , Pneumonia/enzimologia , Pneumonia/patologia , Baço/imunologia , Baço/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologiaRESUMO
AIMS: Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases. MAIN METHODS: A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured. KEY FINDINGS: We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV. SIGNIFICANCE: Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.
Assuntos
Antígenos CD13/antagonistas & inibidores , Citocinas/biossíntese , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Inibidores de Proteases/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Dióxido de Silício/toxicidade , Animais , Linhagem Celular , Inibidores da Dipeptidil Peptidase IV/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Interleucina-6/biossíntese , Interleucina-8/farmacologia , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/etiologia , Fator de Crescimento Transformador beta/biossínteseRESUMO
PURPOSE: Lung fibrosis can be caused by radiation therapy during cancer treatment and therefore can be the limiting factor of the treatment. The factors that cause the actual fibrosis and the interaction between different cell types were investigated. MATERIALS AND METHODS: Epithelial lung cells and fibroblasts were irradiated and different cytokines were measured in the supernatant. Also effects of radiation on the matrix production of fibroblasts were investigated. RESULTS: Irradiation of isolated lung fibroblasts did not cause increased extracellular matrix production; however, the co-culturing of fibroblasts and irradiated lung epithelial cells or the treatment of fibroblasts with supernatants of irradiated epithelial cells did result in an increase. We were able to show that increased interleukin-8 (IL-8) levels led to increased matrix production. CONCLUSIONS: IL-8 is not only a proinflammatory cytokine but it also stimulates collagen synthesis and matrix production and therefore could be a possible drug target in preventing radiation damage during cancer therapy.
Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/efeitos da radiação , Interleucina-8/fisiologia , Linhagem Celular Tumoral , Colágeno/biossíntese , Citocinas/análise , Citocinas/fisiologia , Matriz Extracelular/efeitos da radiação , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Pulmão/citologiaRESUMO
The expression of the trefoil factor family (TFF) genes (TFF1, TFF2, and TFF3) was systematically analyzed in 18 different organs from male or female mice using RT-PCR analysis. The expression patterns showed some gender-specific differences, e.g., TFF3 transcripts in the urinary bladder and liver. Furthermore, the murine expression profile differed from that in human, e.g., in the respiratory tract and uterine cervix. As a hallmark, an aberrant TFF1-related transcript was detected specifically in the kidney and liver of several mouse strains. Molecular characterization of this rare 1.8kb long transcript from the kidney clearly revealed that its 3' region originated from the antisense strand of the TFF1 locus containing particularly large parts of the antisense strands of introns 1 and 2. Homology searches using various databases revealed that this antisense TFF1-related transcript is subject of intense alternative splicing and no protein product encoded by this antisense TFF1-related transcript could be identified. Although the function of this transcript is not known currently, we can speculate that this antisense TFF1-related transcript might have a gene silencing effect particularly on TFF1 expression in the murine kidney and liver.
Assuntos
Rim/metabolismo , Fígado/metabolismo , Mucinas/genética , Proteínas Musculares/genética , Peptídeos/genética , Sequência de Aminoácidos , Animais , Feminino , Inativação Gênica/fisiologia , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual/fisiologia , Fator Trefoil-1 , Fator Trefoil-2 , Fator Trefoil-3RESUMO
Trefoil factor family (TFF) peptides, besides their prominent expression in mucous epithelia, are also synthesized in the central nervous system. Previously TFF1 expression was observed in mouse brain astrocytes, while oxytocinergic neurons of the hypothalamo-pituitary axis are recognized sites of TFF3 synthesis. Here, the expression of TFF1, TFF2, and TFF3 was systematically studied using reverse transcription-polymerase chain reaction (RT-PCR) analysis of dissected adult mouse brain regions including the pituitary. Additionally, the developmental profile of TFF expression in murine cerebral cortex and cerebellum was monitored. Overall, the expression patterns of the three TFF genes differed. The TFF1 and TFF2 profiles shared some similarities, whereas the TFF3 expression pattern was completely different. TFF1 was nearly uniformly, but weakly expressed in all brain regions tested. The TFF1 and TFF2 expression patterns differed characteristically in the pituitary where abundant TFF2 transcription was detected in the anterior and not the posterior lobe and the expression level in males was higher than in females. In contrast, TFF3 expression was limited to the hippocampus, the temporal cortex, and the cerebellum, the latter being surprisingly the major site of expression. Here, TFF3 mRNA appeared to be restricted mainly to neurons and not glial cells. Cerebellar TFF3 expression is clearly developmentally regulated (maximum at P15), indicating a role for TFF3 during postnatal cerebellar development.
Assuntos
Encéfalo/metabolismo , Mucinas/metabolismo , Proteínas Musculares/metabolismo , Peptídeos/metabolismo , Hipófise/metabolismo , Animais , Córtex Cerebelar/metabolismo , Cerebelo/metabolismo , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Mucinas/genética , Proteínas Musculares/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Peptídeos/genética , RNA Mensageiro/genética , Fator Trefoil-1 , Fator Trefoil-2 , Fator Trefoil-3RESUMO
BACKGROUND: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed. METHODOLOGY/PRINCIPAL FINDINGS: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/-) mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh(-/-) mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+) and Ctsh(-/-) mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/-) mice. CONCLUSIONS/SIGNIFICANCE: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.
Assuntos
Catepsina H/genética , Marcação de Genes , Surfactantes Pulmonares/metabolismo , Animais , Catepsina H/deficiência , Catepsina H/metabolismo , Regulação da Expressão Gênica , Humanos , Pulmão/enzimologia , Pulmão/patologia , Camundongos , Fenótipo , Proteínas Associadas a Surfactantes Pulmonares/metabolismoRESUMO
Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory products of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known for their motogenic and anti-apoptotic effects, and they modulate the inflammatory response of bronchial epithelial cells. Here, an established mouse model of asthma was investigated (i.e., exposure to Aspergillus fumigatus [AF] antigens). RT-PCR analysis of lung tissue showed elevated levels particularly of TFF1 transcripts in AF-sensitized/challenged animals. In contrast, transcripts encoding Clara cell secretory protein (CCSP/CC10) were strongly diminished in these animals. For comparison, the expression of the goblet cell secretory granule marker mCLCA3/Gob-5, the mucins Muc1-Muc6 and Muc19, and the secretoglobins ScgB3A1 and ScgB3A2, as well as the mammalian ependymin-related gene MERP2, were monitored. Immunohistochemistry localized TFF1 mainly in cells with a mixed phenotype (e.g., TFF1-positive cells stain with the lectin wheat germ agglutinin (WGA), which recognizes mucins characteristic of goblet cells). In addition, these cells express CCSP/CC10, a Clara cell marker. When compared with mucins or CCSP/CC10, TFF1 was stored in a different population of secretory granules localized at the more basolateral portion of these cells. Thus, the results presented indicate for the first time that allergen exposure leads to the trans-differentiation of Clara cells toward a TFF1-expressing mucous phenotype.
Assuntos
Asma/patologia , Diferenciação Celular , Regulação da Expressão Gênica , Pulmão/citologia , Peptídeos/genética , Animais , Aspergillus fumigatus , Asma/induzido quimicamente , Asma/genética , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunofluorescência , Imunoglobulina E/sangue , Inflamação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mucinas/metabolismo , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator Trefoil-1 , Uteroglobina/metabolismoRESUMO
Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Transdução de Sinais , Linhagem Celular , Movimento Celular , Quimiotaxia , Dimerização , Relação Dose-Resposta a Droga , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Humanos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mucinas/fisiologia , Proteínas Musculares/fisiologia , NF-kappa B/metabolismo , Peptídeos/química , Peptídeos/fisiologia , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/química , Mucosa Respiratória/citologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Frações Subcelulares/metabolismo , Fatores de Tempo , Fator Trefoil-2 , Fator Trefoil-3 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelia and are aberrantly secreted during chronic inflammatory diseases. They are known to enhance the migration of intestinal, corneal, and bronchial epithelial cells. Using the human bronchial epithelial cell line BEAS-2B as a model, it is shown here for the first time that TFF-peptides are capable of modulating the inflammatory response in vitro by regulating tumor necrosis factor-alpha-induced secretion of interleukin (IL)-6 and IL-8. In contrast, TFF2 itself does not change IL-6 and IL-8 secretion but triggers sustained activation of the extracellular signal-regulated kinases (ERK1/2) as well as phosphorylation of c-Jun N-terminal kinase (JNK). A complex differential regulation of tumor necrosis factor-alpha-induced IL-6 and IL-8 secretion by TFF2 is observed that involves signaling via protein kinase C and ERK1/2. Furthermore, the motogenic effect of TFF2 on BEAS-2B cells is analyzed using a modified Boyden chamber assay. This migratory effect is shown to be dependent not only on protein kinase C and ERK1/2 but also on the activation of the Src family of tyrosine kinases. Taken together, the data presented indicate an important physiological role of TFF-peptides during inflammatory conditions of mucous epithelia.