RESUMO
BACKGROUND: KL-6, and surfactant protein A (SP-A) and surfactant protein D (SP-D) derived from alveolar type II cells and/or bronchiolar epithelial cells have been reported to be useful markers for interstitial lung diseases. OBJECTIVE: The aim of this study was to measure the levels of these molecules in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis to investigate their relationship with other markers of inflammatory activity. METHODS: We measured KL-6, SP-A and SP-D levels in BALF from patients with pulmonary sarcoidosis using an ELISA. RESULTS: KL-6 and SP-D, but not SP-A levels were significantly increased in pulmonary sarcoidosis compared with controls. KL-6, SP-A and SP-D levels were significantly correlated with each other. KL-6 and SP-D levels were relatively and significantly correlated with the percentage of lymphocytes in BALF. KL-6, SP-D, but not SP-A levels were significantly correlated with the concentration of albumin in BALF. There was no significant correlation between KL-6, SP-A, or SP-D levels and chest X-ray findings, angiotensin-converting enzyme levels, or CD4/CD8 ratio in BALF. CONCLUSIONS: We conclude that KL-6 and SP-D levels in BALF were increased in pulmonary sarcoidosis. Since these markers are specifically derived from epithelial cells, it is considered that KL-6 and SP-D levels are reflecting damage or release of these markers from epithelial cells due to the inflammatory response.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Glicoproteínas/metabolismo , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Sarcoidose Pulmonar/metabolismo , Adulto , Idoso , Antígenos , Antígenos de Neoplasias , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mucina-1 , Mucinas , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Sarcoidose Pulmonar/epidemiologia , Estatística como AssuntoRESUMO
We previously demonstrated essential roles of the Fas-Fas ligand (FasL) pathway in bleomycin-induced pneumopathy in mice. T lymphocytes and natural killer cells express FasL on activation and use it as a cytotoxic effector molecule. Because interleukin (IL)-12 is known to play a critical role in cell-mediated immunity, we investigated whether anti-IL-12 antibody treatment suppresses the development of this model. The anti-IL-12 antibody treatment decreased the number of apoptotic cells and the degree of inflammation and fibrosis in lung tissue. The results of RT-PCR showed that IL-12p40, IL-12 receptor (R) beta2, interferon-gamma, tumor necrosis factor-alpha and FasL mRNAs were upregulated after bleomycin instillation. The upregulation of FasL, IL-12Rbeta2, and tumor necrosis factor-alpha mRNA expression in lung tissue was suppressed by anti-IL-12 antibody treatment. The results of enzyme-linked immunosorbent assay showed that the levels of IL-12p40, but not of IL-12p70, were increased in lung tissue after bleomycin instillation. Although the increase in IL-12Rbeta2 mRNA levels suggests that the T helper type 1 cell response may participate in lung injury, the increase in IL-12p40 supports T helper type 2 cell predominance in the fibrotic process of this model. The administration of anti-IL-12 antibody could be a novel therapy against lung injury and pulmonary fibrosis.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Interleucina-12/antagonistas & inibidores , Fibrose Pulmonar/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Bleomicina , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Citocinas/análise , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Proteína Ligante Fas , Hidroxiprolina/metabolismo , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Interleucina-12/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , RNA Mensageiro/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
The caspase cascade is an executioner of apoptosis, mediated by Fas. Fas-associating protein with death domain (FADD) interacts with Fas and initiates apoptosis through activating caspase-8. It has previously been demonstrated that the Fas-Fas ligand pathway may be involved in the pathophysiology of idiopathic pulmonary fibrosis (IPF). The aim of this study was to investigate Fas-signalling molecules in epithelial cells in IPF. The immunohistochemistry for FADD and caspase-1 and -3 and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick endlabelling (TUNEL) methods were performed in lung tissues from 10 patients with IPF obtained by thoracoscopic biopsy and in seven normal lung parenchyma specimens. The induction of caspases expression and activation by Fas-ligation on lung epithelial cell line A549 was also investigated. The immunoreactivity grade for FADD and caspase-1 and -3, and positive signals for TUNEL were significantly increased in epithelial cells of IPF compared with controls. Fas-ligation induced upregulation of caspase-1 and -3 expression in the nucleus and cytoplasm in A549 cells. Procaspase-1, -3, and -8 were activated in apoptotic cells, but not in viable cells. Although direct measurement of the caspase activity in lung epithelial cells of idiopathic pulmonary fibrosis could not be made, these results suggest that the Fas-signalling pathway is upregulated in lung epithelial cells of idiopathic pulmonary fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/análise , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Idoso , Western Blotting , Caspase 1/análise , Caspase 3 , Caspases/análise , Linhagem Celular , Ativação Enzimática , Células Epiteliais/metabolismo , Proteína de Domínio de Morte Associada a Fas , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/patologia , Regulação para CimaRESUMO
Caspases have been implicated in the effector process of apoptosis in several systems including the Fas-Fas ligand pathway. We previously demonstrated that excessive apoptosis of lung epithelial cells and the Fas-Fas ligand pathway were essential in the pathogenesis of bleomycin-induced pneumopathy in mice. Therefore, the purpose of this study was to investigate whether a caspase inhibitor could prevent the development of this model. The expression of caspase-1 and caspase-3 was upregulated on lung epithelial cells, alveolar macrophages, and infiltrating inflammatory cells in this model. We demonstrated that a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, decreased the caspase-1- and caspase-3-like activity, the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues in this model. We conclude that caspase inhibitors could be a new therapeutic approach against lung injury and pulmonary fibrosis.
Assuntos
Clorometilcetonas de Aminoácidos/administração & dosagem , Bleomicina/toxicidade , Inibidores de Caspase , Fibrose Pulmonar/prevenção & controle , Administração por Inalação , Animais , Western Blotting , Caspase 1/metabolismo , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Inibidores de Cisteína Proteinase/administração & dosagem , Fragmentação do DNA/efeitos dos fármacos , Hidroxiprolina/análise , Hidroxiprolina/metabolismo , Marcação In Situ das Extremidades Cortadas , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos ICR , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologiaRESUMO
LPS (lipopolysaccharide) is one of the major factors that induce acute lung injury. Recently, it was reported that LPS induced disseminated endothelial apoptosis, preceding nonendothelial tissue damage. Caspases play important roles in apoptosis, including tumor necrosis factor-alpha-induced apoptosis, in several systems. We therefore investigated whether the injection of a caspase inhibitor prevents LPS-induced apoptosis and acute lung injury in mice. LPS (30 mg/kg) was administered intravenously to Institute for Cancer Research mice. Electron microscopic findings demonstrated characteristic features of apoptosis in endothelial cells and alveolar epithelial cells. The caspase-3 activity and the number of terminal dUTP nick-end labeling-positive cells in lung tissues were significantly increased after LPS administration. Benzyloxycarbonil-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), which is a broad-spectrum caspase inhibitor, was injected before and after the administration of LPS. The injection of Z-VAD.fmk suppressed the caspase-3 activity in lung tissues, and significantly decreased the number of terminal dUTP nick-end labeling-positive cells. Furthermore, the survival rate of mice was prolonged significantly by the injection of Z-VAD.fmk. These results indicate that apoptosis may play an important role in acute lung injury, and thus that inhibition of caspase activity may constitute a new therapeutic approach for treatment of this disease.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Lipopolissacarídeos/farmacologia , Pneumopatias/prevenção & controle , Pulmão/efeitos dos fármacos , Doença Aguda , Animais , Caspase 1/metabolismo , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Interleucina-1/sangue , Pulmão/patologia , Pulmão/ultraestrutura , Pneumopatias/induzido quimicamente , Pneumopatias/enzimologia , Camundongos , Camundongos Endogâmicos ICR , Análise de SobrevidaRESUMO
Interstitial lung diseases are thought to be associated with the infiltration of activated T-lymphocytes. To induce an effective immune response, antigen-presenting cells have to not only present antigenic peptide with major histocompatibility complex (MHC) molecules to T-lymphocytes but also express B7 molecules. Therefore, the expression of B7-1, B7-2 and class II MHC molecules was investigated in lung tissues from patients with idiopathic pulmonary fibrosis (IPF) and bronchiolitis obliterans-organizing pneumonia (BOOP), and in normal lung parenchyma as a control, using immunohistochemical localization. B7-1 and B7-2 were aberrantly expressed in bronchiolar and alveolar epithelial cells, and class II MHC molecules were also aberrantly expressed in bronchiolar epithelial cells in IPF. B7-1 was aberrantly expressed in bronchiolar epithelial cells in BOOP. There was no significant difference in the expression of these proteins in alveolar macrophages between IPF and control subjects. However, B7-2 and class II MHC molecule expression in alveolar macrophages was decreased in BOOP compared with that in control subjects. Expression of CD28 and CTLA4, receptors for B7 molecules, was detected in infiltrating lymphocytes in lung tissues in IPF and BOOP. It was concluded that bronchiolar and alveolar epithelial cells may actively participate in the pathophysiology of idiopathic pulmonary fibrosis through the aberrant expression of B7 and class II major histocompatibility complex molecules. The dysregulation of these molecules in epithelial cells may lead to the activation of autoreactive T-lymphocytes, which might contribute to the pathogenesis of fibrosing lung diseases.
Assuntos
Antígenos CD/análise , Antígeno B7-1/análise , Bronquiolite Obliterante/patologia , Pneumonia em Organização Criptogênica/patologia , Antígenos de Histocompatibilidade Classe II/análise , Glicoproteínas de Membrana/análise , Fibrose Pulmonar/patologia , Adulto , Idoso , Antígeno B7-2 , Biópsia , Brônquios/imunologia , Brônquios/patologia , Bronquiolite Obliterante/imunologia , Pneumonia em Organização Criptogênica/imunologia , Feminino , Humanos , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Up-regulation of Fas and Fas ligand and excessive apoptosis of bronchiolar and alveolar epithelial cells were identified in bleomycin-induced pulmonary fibrosis in mice. This study hypothesized that apoptosis-regulatory genes other than Fas-Fas ligand, such as p53, p21 (Waf1/Cip1), bcl-2, bcl-x, and bax, may also participate in epithelial cell apoptosis in this model. The expression of these genes was assessed by reverse transcription polymerase chain reaction (RT-PCR), RT in situ PCR, or immunohistochemistry. The expression of p53 and p21 mRNA was concurrently up-regulated in the alveolar epithelial cells at 1 h to 7 days after intratracheal instillation of bleomycin. The expression of bcl-2 mRNA was weakly up-regulated at 1 h to 14 days, while the expression level of bcl-2 protein was not changed. The expression of bcl-x(L) and bax mRNA was strongly up-regulated at 1 h to 7 days. The expression of bcl-x protein was up-regulated in lymphocytes and macrophages, whereas bax protein was up-regulated in both epithelial and inflammatory cells. It is concluded that epithelial cell apoptosis in this model may also be induced by the up-regulation of p53 and bax and by the imbalance between apoptosis-inducible and -inhibitory genes, in addition to the up-regulation of the Fas-Fas ligand pathway.
Assuntos
Apoptose/genética , Células Epiteliais/fisiologia , Fibrose Pulmonar/genética , Animais , Brônquios/patologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Células Epiteliais/patologia , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos ICR , Proteínas Proto-Oncogênicas/metabolismo , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
Sarcoidosis is a chronic inflammatory disease of unknown aetiology characterized by the formation of non-necrotizing granulomas. The course of disease is usually self-limiting with the spontaneous resolution of granuloma. In the immune system, Fas antigen (Fas) and Fas ligand (FasL) are involved in the down regulation of immune reactions by inducing apoptosis. Therefore, it was hypothesized that the Fas/FasL pathway and apoptosis may be associated with the course of granulomatous inflammation in sarcoidosis. Terminal deoxynucleotidyl transferase-mediated biotin nick end-labelling (TUNEL) was performed to assess deoxyribonucleic acid strand breakages as a characteristic of apoptosis. Immunohistochemistry was also performed to detect Fas and FasL protein, and reverse transcriptase polymerase chain reaction (RT-PCR) and RT in situ PCR to detect FasL messenger ribonucleic acid (mRNA). Positive signals for TUNEL were detected in epithelioid histiocytes and lymphocytes within granulomas and in bronchoalveolar lavage (BAL) lymphocytes from patients with sarcoidosis. Positive signals for Fas were also detected in these cells. FasL mRNA was expressed in BAL lymphocytes from 15 of 20 patients with sarcoidosis, but from only one of 10 patients with normal lung parenchyma. FasL protein was expressed in lymphocytes surrounding and within the granuloma. There was a significant correlation between the result of TUNEL and clinical course in patients with sarcoidosis. Apoptosis in epithelioid histiocytes and inflammatory cells seems to participate in the course of granulomatous inflammation. Further studies are needed to determine the role of Fas, FasL and other regulatory factors in apoptosis in the granulomatous inflammation in pulmonary sarcoidosis.
Assuntos
Apoptose , Pulmão/patologia , Sarcoidose Pulmonar/patologia , Adulto , Idoso , Líquido da Lavagem Broncoalveolar/citologia , Proteína Ligante Fas , Feminino , Granuloma/metabolismo , Granuloma/patologia , Histiócitos/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Pulmão/metabolismo , Linfócitos/metabolismo , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Sarcoidose Pulmonar/metabolismo , Receptor fas/análiseRESUMO
Epithelial cell injury is the common manifestation of lung injury. Contributing to such injury of epithelial cells is apoptosis. Although apoptosis is part of the normal process of epithelial renewal, in excess it is pathologic. We previously demonstrated the excessive apoptosis of lung epithelial cells and the upregulation of Fas and Fas ligand (FasL) in fibrosing lung diseases. We also showed that inhalation of anti-Fas antibody induced lung injury and fibrosis in mice. Interleukin (IL)-8 is one of the most important cytokines in the pathophysiology of acute lung injury and pulmonary fibrosis. In this study we investigated whether Fas ligation induces IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Bronchiolar epithelial cells underwent apoptosis and also secreted IL-8 in response to tumor necrosis factor (TNF)-alpha or Fas ligation. New gene expression and protein synthesis were not necessary for Fas ligation- and TNF-alpha- mediated apoptosis, but were necessary for IL-8 secretion. We further found that Fas ligation induced activation of nuclear factor-kappa B. We conclude that the Fas/FasL pathway not only mediates apoptosis but also plays a proinflammatory role, and that stimulation of the Fas/FasL pathway in bronchiolar epithelial cells leads to IL-8 production, which may amplify the inflammatory cascade in lung injury and pulmonary fibrosis.
Assuntos
Apoptose , Brônquios/metabolismo , Interleucina-8/metabolismo , Glicoproteínas de Membrana/farmacologia , Receptor fas/farmacologia , Anticorpos Monoclonais/farmacologia , Brônquios/citologia , Núcleo Celular/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Dactinomicina/farmacologia , Relação Dose-Resposta Imunológica , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteína Ligante Fas , Citometria de Fluxo , Humanos , Interferon gama/farmacologia , Interleucina-8/farmacologia , Microscopia Eletrônica , NF-kappa B/metabolismo , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Alveolar macrophages (AM) of sarcoidosis have an enhanced capacity to mediate antigen-induced T lymphocyte proliferation. To induce an effective immune response, antigen-presenting cells have to not only present antigenic peptide with MHC molecules to T lymphocytes, but also express B7 costimulating molecules. OBJECTIVE: The purpose of this study was to investigate the expression of B7 and MHC molecules in lung tissues from patients with sarcoidosis. METHODS: We performed immunohistochemistry for B7-1, B7-2 and MHC class II antigens using transbronchial lung biopsy specimens obtained from patients with sarcoidosis and normal lung parenchyma obtained by lobectomy for solitary pulmonary nodule as controls. RESULTS: B7-1, B7-2 and MHC class II antigen were expressed in epithelioid cells in granulomas in 14 (93.3%), 2 (13.3%) and 9 (60.0%) of 15 patients with sarcoidosis, respectively. These were also expressed in AM in 14 (93. 3%), 5 (33.3%) and 12 (80.0%) of 15 patients with sarcoidosis, respectively. The positivity of B7-1 was significantly higher than that of B7-2 in both epithelioid cells and AM in sarcoidosis (p < 0. 01). Positive signals for B7-1, B7-2 and MHC class II antigen were also found in AM in 9 (90%), 8 (80%) and 8 (80%) of 15 of controls, respectively. However, the intensity of positive signals for B7-1, but not B7-2 or MHC class II antigen in AM was significantly increased in sarcoidosis compared to controls (p < 0.05). CONCLUSIONS: These results suggested that epithelioid cells in granulomas and AM from patients with sarcoidosis had the capability to act as accessory cells and that the accessory function of these cells was shifted to B7-1 rather than B7-2 in sarcoidosis.
Assuntos
Antígeno B7-1/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos Alveolares/imunologia , Sarcoidose Pulmonar/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-IdadeRESUMO
The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.
Assuntos
Apoptose , Glicoproteínas de Membrana/fisiologia , Fibrose Pulmonar/prevenção & controle , Receptor fas/fisiologia , Animais , Bleomicina/toxicidade , Proteína Ligante Fas , Humanos , Hidroxiprolina/análise , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Marcação In Situ das Extremidades Cortadas , Células Matadoras Naturais/imunologia , Pulmão/química , Pulmão/patologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Camundongos Endogâmicos MRL lpr , Camundongos Mutantes , Fagocitose , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/genéticaRESUMO
BACKGROUND: We have previously reported that the inhalation of anti-Fas antibody induced pulmonary fibrosis in mice. To induce an effective immune response, antigen-presenting cells have to not only present antigenic peptide with MHC molecules to T lymphocytes, but also express B7 costimulating molecules. The purpose of this study is to investigate whether B7 family costimulating molecules and interleukin-12 (IL-12), which primarily promote cellular immunity, are associated with anti-Fas antibody-induced pulmonary fibrosis. METHODS: We examined the expression of B7-1, B7-2, and IL-12 using the revese transcription-polymerase chain reaction (RT-PCR), RT-in situ PCR, and immunohistochemistry. RESULTS: We observed the upregulation of B7-1, B7-2, and IL-12 p40 mRNA after anti-Fas antibody inhalation. B7-2 and IL-12 p40 mRNA appeared to be expressed in mononuclear cells, while B7-1 mRNA and protein were expressed in bronchiolar epithelial cells as well as macrophages. CONCLUSION: These findings indicate that the T-cell-mediated immune response in this model involved the upregulation of B7-1, B7-2, and IL-12, and that the aberrant expression of B7-1 in bronchiolar epithelial cells may induce autoreactive T cell proliferation against themselves.
Assuntos
Antígenos CD/genética , Antígeno B7-1/genética , Interleucina-12/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Fibrose Pulmonar/imunologia , Administração por Inalação , Animais , Anticorpos/administração & dosagem , Anticorpos/efeitos adversos , Apoptose/fisiologia , Antígeno B7-2 , Brônquios/citologia , Células Epiteliais/química , Células Epiteliais/citologia , Proteína Ligante Fas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos ICR , Fibrose Pulmonar/genética , Fibrose Pulmonar/fisiopatologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Therapeutic efficacy and the treatment days for cure of imipenem/cilastatin sodium (IPM/CS) in treatment of pulmonary infections were prospectively determined in comparison with those of beta-lactams other than carbapenems mainly ceftazidime (CAZ) or sulbactam/cefoperazone (SBT/CPZ). The overall response rate was 84.9% (62/73) in the IPM/CS group and 74.7% (56/75) in the beta-lactam group, the difference not being significant. In the subjects having underlying respiratory diseases, the response rate was 91.1% (41/45) and 73.9% (34/46) in the IPM/CS and beta-lactam groups, respectively. In patients with infections secondary to chronic respiratory disease, the rate was 91.2% (31/34) in the former group and 66.7% (24/36) in the latter group, respectively. The differences were significant for both stratified analyses. The treatment days for cure judged by the attending physician were 12.9 +/- 0.6 days in the IPM/CS group, and 14.5 +/- 0.7 days in the beta-lactam group. The difference was not, however, significant. In patients with mild to moderate infections, the treatment days for cure was 12.0 +/- 0.6 days (n = 64) in the IPM/CS group and 14.3 +/- 0.7 days (n = 70) in the beta-lactam group. In patients with underlying respiratory disease, the treatment days for cure were 11.8 +/- 0.7 days (n = 45) and 14.7 +/- 0.9 days (n = 46) in the IPM/CS and beta-lactam groups, respectively. In patients with infections secondary to chronic respiratory disease, the days were 11.1 +/- 0.7 days (n = 34) and 14.7 +/- 1.1 days (n = 36), respectively. Thus, IPM/CS therapy significantly reduced the number of treatment days until cure. There was, however, no significant difference between the two therapy groups in treatment of the patients with severe infections, those without underlying respiratory disease, or those with pneumonia and/or lung abscess. The treatment days for cure were also assessed by the members of review committee taking into consideration of body temperature, leukocyte count, and C-reactive protein. As the result, it was 6.9 +/- 0.5 days in the IPM/ CS and 10.3 +/- 0.7 days in the beta-lactam groups; respectively, and the difference was significant. Time (days) until cure was also compared between the two groups using survival time analysis, confirming a more rapid response in the IPM/CS group. Although IPM/CS therapy was associated with a shorter response time as assessed by both the attending physicians and the review committee, there were considerable differences between the results of these judgements. Thus, the duration of treatment with injectable antibiotics requires reevaluation in the future. No significant differences were observed between the groups with respect to parameters indicating side effects and laboratory abnormalities. There were no severe symptoms or laboratory findings, and symptoms and changes in laboratory values, if any resolved during the course of therapy or after the withdrawal of treatment. In conclusion, IPM/CS seems to be very useful as first-line therapy for respiratory tract infections and for shortening the duration of treatment.
Assuntos
Antibacterianos/uso terapêutico , Quimioterapia Combinada/uso terapêutico , Infecções Respiratórias/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Cilastatina/efeitos adversos , Cilastatina/uso terapêutico , Combinação Imipenem e Cilastatina , Combinação de Medicamentos , Avaliação de Medicamentos , Quimioterapia Combinada/efeitos adversos , Feminino , Humanos , Imipenem/efeitos adversos , Imipenem/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Pulmonary fibrosis begins with alveolitis, which progresses to destruction of lung tissue and excess collagen deposition. This process could be the result of DNA damage and a form of apoptosis. Therefore, we hypothesized that Fas ligand (FasL), which induces apoptosis in cells expressing Fas antigen (Fas), is associated with pulmonary fibrosis. We examined frozen lung tissues from seven patients with idiopathic pulmonary fibrosis (IPF), and bronchoalveolar lavage fluid (BALF) cells from 19 patients with IPF and from 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP). We used five frozen lungs with normal lung parenchyma and BALF cells from 10 patients with solitary pulmonary nodule as controls. Reverse transcription-polymerase chain reaction (RT-PCR) showed that FasL messenger RNA (mRNA) was expressed in BALF cells from all patients with IPF and from 15 of 16 patients with CVD-IP. FasL mRNA was not detected in BALF cells except in one of 10 controls. RT in situ PCR detected FasL mRNA in inflammatory cells in BALF from patients with IPF. Immunohistochemistry detected FasL protein in infiltrating lymphocytes and granulocytes in all of seven frozen lung tissues of IPF, but in none of five control lung tissues. Additionally, the expression of Fas appeared to be upregulated in bronchiolar and alveolar epithelial cells in IPF compared with normal lung parenchyma by immunohistochemistry. We conclude that Fas and FasL were upregulated in fibrosing lung diseases and may associate with DNA damage or apoptosis of bronchiolar and alveolar epithelial cells in this disorder.
Assuntos
Glicoproteínas de Membrana/metabolismo , Fibrose Pulmonar/metabolismo , Receptor fas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Líquido da Lavagem Broncoalveolar , Antígenos CD4/análise , Antígenos CD8/análise , Colágeno/metabolismo , Criopreservação , Proteína Ligante Fas , Feminino , Humanos , Imuno-Histoquímica , Antígenos CD15/análise , Doenças Pulmonares Intersticiais/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor fas/genéticaRESUMO
BACKGROUND: Studies have shown the importance of apoptosis in vascular injury in vitro. We postulated that apoptosis of the endothelium contributes to vascular injury in vivo and may be involved in acute lung injury. METHODS: To test this hypothesis, we investigated the incidence of endothelial cell apoptosis in acute lung injury induced in mice by the administration of lipopolysaccharide (LPS). Male ICR mice were administered LPS (20 mg/kg body weight) intravenously and sacrificed at specified times thereafter. RESULTS: Histologic findings were consistent with acute lung injury which increased with time from 3 to 48 h after injection. Electrophoretic analysis of DNA that was extracted from lung tissue and 3'-end-labeled with digoxigenin demonstrated a fragmentation of DNA starting at 6 h. In situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) demonstrated DNA strand breaks in the endothelial cells. TUNEL also revealed DNA strand breaks in bronchial and alveolar epithelial cells as well as inflammatory cells in the interstitium. These TUNEL-positive cells appeared 6 h after injection. Electron-microscopic examination of the endothelium strongly suggested the morphological characteristics of apoptosis. CONCLUSION: Apoptosis was induced by LPS administration in endothelial cells in vivo. A role for such apoptosis is suggested in acute lung injury.
Assuntos
Pulmão/patologia , Síndrome do Desconforto Respiratório/patologia , Animais , Apoptose/fisiologia , Fragmentação do DNA , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Marcação In Situ das Extremidades Cortadas , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Síndrome do Desconforto Respiratório/induzido quimicamenteRESUMO
Fas is expressed in various cells and transduces the cell death signal. p21 is a mediator of p53-dependent G1 arrest associated with deoxyribonucleic acid (DNA) damage. The upregulation of p53 and p21 associated with DNA damage in idiopathic pulmonary fibrosis has been described previously. In this study, p53, p21, and Fas expression and DNA damage were examined in interstitial pneumonia associated with collagen vascular diseases (CVD-IP). DNA damage was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL) and p53, p21 and Fas proteins were detected by immunohistochemistry in 13 cases of CVD-IP, 13 of sarcoidosis, seven of hypersensitivity pneumonitis (HP) and eight control patients with normal lung parenchyma. TUNEL-positive signals were found in bronchiolar or alveolar epithelial cells in 11 of 13 (85%) specimens of CVD-IP, but not in sarcoidosis, HP or controls, except for a case of chronic HP with pulmonary fibrosis. p53, p21 and Fas were detected in bronchiolar or alveolar epithelial cells in nine (69%), 10 (77%) and 12 (92%) of 13 specimens of CVD-IP, respectively, but not in sarcoidosis, HP or controls, except for a case of chronic HP. These results suggest that the upregulation of p53, p21 and Fas in bronchiolar and alveolar epithelial cells associated with deoxyribonucleic acid damage may participate in the process of pulmonary fibrosis in interstitial pneumonia associated with collagen vascular diseases and chronic hypersensitivity pneumonitis.
Assuntos
Alveolite Alérgica Extrínseca/patologia , Proteínas de Ligação ao GTP/análise , Fibrose Pulmonar/patologia , Sarcoidose Pulmonar/patologia , Proteína Supressora de Tumor p53/análise , Receptor fas/análise , Adulto , Idoso , Alveolite Alérgica Extrínseca/genética , Biópsia por Agulha , Técnicas de Cultura , Dano ao DNA , Feminino , Humanos , Imuno-Histoquímica , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/patologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/genética , Valores de Referência , Sarcoidose Pulmonar/genética , Sensibilidade e Especificidade , Transdução de Sinais , Regulação para Cima , Proteína rhoA de Ligação ao GTPRESUMO
BACKGROUND: The bleomycin-induced pneumopathy involves a T cell-mediated immune response. T cell activation requires both antigen/MHC recognition and costimulatory signals. The CD28 receptor on T cells with its ligand B7 represents one of the most important examples of this costimulation. Interleukin 12 (IL-12) has a strong synergistic effect with the B7-1/CD28 interaction on inducing proliferation and cytokine production in T cells. METHODS: In this study, we investigated the expression of B7-1, B7-2, and IL-12 in bleomycin-induced pneumopathy in mice using reverse transcription polymerase chain reaction (RT-PCR), RT in situ PCR, and immunohistochemistry. RESULTS: We observed concurrent upregulation of B7-1, B7-2, and IL-12p40 mRNA in the lung tissues at 1 h to 7 days after bleomycin instillation into the trachea. B7-1 mRNA and protein were found in bronchiolar epithelial cells as well as macrophages, B7-2 and IL-12p40 mRNA appeared to be expressed in mononuclear cells. CONCLUSIONS: These findings indicate that T cell-mediated immune response in this model involves the upregulation of B7-1, B7-2, and IL-12p40 mRNA, and also demonstrate the aberrant expression of B7-1 in bronchiolar epithelial cells.
Assuntos
Antibacterianos/imunologia , Antibacterianos/toxicidade , Antígenos CD/imunologia , Antígeno B7-1/imunologia , Bleomicina/imunologia , Bleomicina/toxicidade , Interleucina-12/imunologia , Pneumopatias/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Interleucina-12/biossíntese , Pneumopatias/induzido quimicamente , Pneumopatias/metabolismo , Glicoproteínas de Membrana/biossíntese , CamundongosRESUMO
Deoxyribonucleic acid (DNA) strand breaks as a characteristic of apoptosis, and Fas antigen (Fas)/Fas ligand (FasL) expression may participate in acute immune complex alveolitis in mice. Male Institute for Cancer Research (ICR) mice were injected intravenously with immunoglobulin G (IgG) antibodies against ovalbumin and inhaled an aerosolized oval albumin (OA) solution. They were killed at 4, 6, 12, 24, 48 h and 7 days after aerosolization. We assessed DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL). The expression of Fas and FasL messenger ribonucleic acid (mRNA) in lung tissues was assessed by reverse transcriptase (RT) polymerase chain reaction, and by in situ hybridization (ISH) to localize Fas mRNA, and RT in situ polymerase chain reaction to localize FasL mRNA. The fragmentation of DNA extracted from lung tissue was found 6-24 h after OA inhalation. TUNEL detected positive signals in bronchial and alveolar epithelial, endothelial and inflammatory cells in the lung tissue. These positive signals had disappeared 7 days after OA inhalation. TUNEL also detected positive signals in apoptotic neutrophils in bronchoalveolar lavage fluid at 6-12 h. Fas mRNA was expressed in the alveolar epithelial and inflammatory cells, while the expression of FasL mRNA appeared to be upregulated in infiltrating inflammatory cells at 6-24 h. These results suggest that apoptosis may be associated with the resolution of inflammation and with tissue repair and also suggest the involvement of the Fas antigen/Fas ligand pathway in acute immune complex alveolitis in mice.
Assuntos
Alveolite Alérgica Extrínseca/fisiopatologia , Apoptose , Doenças do Complexo Imune/fisiopatologia , RNA Mensageiro/análise , Receptor fas/análise , Doença Aguda , Alveolite Alérgica Extrínseca/patologia , Animais , Sequência de Bases , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Modelos Animais de Doenças , Doenças do Complexo Imune/patologia , Ligantes , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Fas antigen is a cell surface protein that mediates apoptosis, and it is expressed in various cells and tissues. Fas ligand binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells. Malfunction of the Fas-Fas ligand system causes lymphoproliferative disorders and autoimmune diseases, whereas its exacerbation may cause tissue destruction. We hypothesize that excessive apoptosis mediated by Fas-Fas ligand interaction may damage alveolar epithelial cells and result in pulmonary fibrosis. Mice were allowed to inhale repeatedly an aerosolized anti-Fas antibody for 14 days. The nuclei of bronchial and alveolar epithelial cells were positively stained by in situ DNA nick end labeling. Electron microscopy demonstrated apoptotic changes in bronchial and alveolar epithelial cells. Histologic findings and hydroxyproline content showed the development of pulmonary fibrosis, which was dependent on the dose of anti-Fas antibody. The repeated inhalation of control antibody (isotype-matched control hamster IgG) did not induce apoptosis of epithelial cells or pulmonary fibrosis. The expression of TGF-beta mRNA was upregulated from day 7 to day 28 in lung tissues of anti-Fas antibody-treated mice but not in those of control mice. In this report, we present the evidence that repeated inhalation of anti-Fas antibody mimicking Fas-Fas ligand crosslinking induces excessive apoptosis and inflammation, which results in pulmonary fibrosis in mice.
Assuntos
Apoptose/fisiologia , Fibrose Pulmonar/fisiopatologia , Receptor fas/genética , Receptor fas/imunologia , Animais , Anticorpos/farmacologia , Biotina , Líquido da Lavagem Broncoalveolar/citologia , Reagentes de Ligações Cruzadas/metabolismo , Fragmentação do DNA , Nucleotídeos de Desoxiuracil , Células Epiteliais , Epitélio/imunologia , Epitélio/ultraestrutura , Expressão Gênica/imunologia , Hidroxiprolina/análise , Ligantes , Linfotoxina-alfa/genética , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Alvéolos Pulmonares/química , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Coloração e Rotulagem , Fator de Necrose Tumoral alfa/genética , Receptor fas/metabolismoRESUMO
The history of patients with idiopathic pulmonary fibrosis (IPF) shows that the disease may be preceded by a viral-like illness. Although viruses have not been demonstrated, it is possible that viruses were not detected in culture because they do not replicate during latency. We investigated the presence of adenovirus in IPF and interstitial pneumonia associated with collagen vascular disease (CVD-IP), using the nested polymerase chain reaction (PCR) and in situ hybridization (ISH) for the E1A region of the adenovirus genome. Studies were performed on lung tissues obtained by transbronchial lung biopsy from 19 patients with IPF, 10 patients with CVD-IP and, for comparison, from 20 patients with sarcoidosis. The E1A DNA was present in 3 out of 19 (16%) cases of IPF, in 5 of 10 (50%) cases of CVD-IP, and in 2 of 20 (10%) cases of sarcoidosis. The incidence of E1A DNA in CVD-IP was significantly higher than that in sarcoidosis (p<0.05). In patients with IPF and CVD-IP, E1A DNA was more prevalent in patients treated with corticosteroids (6 out of 9 cases; 67%) than in those without it (2 out of 20 cases; 10%) (p<0.01). ISH studies showed that 1 out of 8 cases of IPF and CVD-IP, in which E1A DNA was detected by PCR, was positive for E1A DNA. We conclude that adenovirus E1A is unlikely to be aetiologically involved in the pathogenesis of idiopathic pulmonary fibrosis or interstitial pneumonia associated with collagen vascular disease. However, a latent adenovirus infection may be reactivated or may newly infect the host following corticosteroid administration.