RESUMO
Human airway trypsin-like protease (HAT) has been isolated from mucoid sputum of patients with chronic airway diseases. In order to clarify the cellular source of this novel protease in the human airway, we examined the localization of immunoreactive HAT in bronchial tissues obtained at surgery and fixed in 4% paraformaldehyde using an extremely sensitive immunohistochemical technique called a catalyzed signal amplification method and a monoclonal antibody against recombinant HAT. HAT immunoreactivity was demonstrated in cytoplasm of ciliated cells of bronchial epithelium and/or at the basal part of cilia. No positive reaction was found in submucosal glands or mast cells. The heterogeneous distribution of HAT immunoreactivity within the bronchial epithelium indicates that its expression might be changeable and that it might be closely related to the physiological status of the airway epithelium. Non-specific but intense reaction caused by endogenous avidin-binding activity (EABA) was selectively detected in submucosal glands, but was effectively blocked by successive treatments with avidin and biotin. These results indicate that HAT may be synthesized in the ciliated cells and that it may play some physiological roles within the epithelial layer and on the airway surface. It is necessary to keep in mind that some cells show strong EABA, especially when a highly sensitive immunohistochemical technique is applied.
Assuntos
Brônquios/enzimologia , Serina Endopeptidases/metabolismo , Anticorpos Monoclonais , Avidina , Biotina , Western Blotting , Brônquios/citologia , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica/métodos , Peroxidase , Proteínas Recombinantes/imunologia , Serina Endopeptidases/imunologiaRESUMO
To clarify the pathophysiological role of cathepsins in rheumatoid arthritis (RA), we investigated whether cathepsin B or cathepsin L was increased in synovial fluid (SF) of RA joints, and whether the cathepsin isolated from SF of RA patients activated pro-urokinase or not. Thus, we estimated the content of cathepsins in SF of RA patients by measuring their activities by fluorospectrometry, using Z-Phe-Arg-MCA as the substrate. Cathepsin activity was approximately 4-fold higher in the SF of RA patients than in those of patients with osteoarthritis. Cathepsin B and cathepsin L were separated by cation-exchange column chromatography. As a result, a large peak corresponding to cathepsin B and a very small peak corresponding to cathepsin L were detected. Biochemical sequential fractionation of the cathepsin purified from the SF showed that the large peak was mainly composed of cathepsin B. This purified enzyme induced conversion of pro-urokinase to urokinase, and the Km for pro-urokinase was approximately 8.27 microM. These findings indicated that an imbalance between cathepsin B and its inhibitors occurred due to increased concentrations of active cathepsin B in RA articular lesions, and that cathepsin B might be related to the degradation of cartilage in RA by activating the fibrinolytic cascade.
Assuntos
Artrite Reumatoide/enzimologia , Catepsina B/metabolismo , Catepsinas/metabolismo , Endopeptidases , Precursores Enzimáticos/metabolismo , Líquido Sinovial/enzimologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Catepsina B/antagonistas & inibidores , Catepsina B/isolamento & purificação , Catepsina B/farmacologia , Catepsina L , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Ativação Enzimática , Feminino , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/enzimologia , Proteínas Recombinantes/metabolismoRESUMO
The effect of biochemical components on the viscoelasticity of nasal mucus from 24 patients with chronic sinusitis (CS) was investigated by multiple stepwise regression analysis. The dynamic viscosity (eta') and the elastic modulus (G') of nasal mucus were determined with an oscillating sphere magnetic rheometer at oscillatory frequencies of 1 and 10 Hz. The eta' and G' values of mucus determined at 1 Hz were 1.6 +/- 1.5 Pa/s and 31.8 +/- 31.0 Pa, respectively, and these values were much higher than optimal viscoelasticity for mucociliary transport. The concentrations of fucose, N-acetyl neuraminic acid, albumin, IgG, secretory-IgA, and lysozyme were measured in the same mucus samples. The multiple regression analysis showed that the concentration of fucose, a marker of mucous glycoproteins, was the most important determinant of eta' and G'. The analysis also revealed that the level of IgG was the next important determinant. The coefficients of multiple determination for fucose and IgG were 0.732 and 0.733 when the response variables were eta' and G', respectively. The results indicate that locally produced mucous glycoproteins may largely contribute to the high viscoelasticity of nasal mucus in CS.
Assuntos
Glicoproteínas/fisiologia , Muco/fisiologia , Sinusite/fisiopatologia , Adolescente , Adulto , Idoso , Criança , Doença Crônica , Feminino , Fucose/análise , Fucose/fisiologia , Glicoproteínas/análise , Humanos , Imunoglobulina G/análise , Imunoglobulina G/fisiologia , Masculino , Pessoa de Meia-Idade , Muco/química , Análise de Regressão , Reologia , ViscosidadeRESUMO
We examined the relationships among the activity of daily living (ADL), oral cavity care, and the number of oral cavity microorganisms in 40 patients with cerebrovascular diseases (CVD). The CVD patients were classified into 4 groups, I, II, III and IV based on their ADL and the method used for oral cavity care. The ADL was highest in group I and lowest in group III. Only the patients of only group III could not eat by themselves and were receiving naso-esophageal feeding. Oral cavity care was performed by the patients themselves in groups I and IV, but was performed by caregivers in groups II and III. The group IV patients had no teeth, but could eat by themselves using full dentures. The numbers of microorganisms in the pharyngeal swabs from the 4 groups were measured and expressed as colony-forming units (cfu). The numbers of both Staphylococci spp. and Candida spp. were significantly higher in group III than in the other groups. Moreover, Pseudomonas aeruginosa was isolated only from patients of group III (in about 66%). The oral cavity care by caregivers was almost the same in groups II and III, but the numbers of oral cavity microorganisms were significantly higher in group III than in group II. These results indicated that microorganisms grow more easily in the oral cavities of CVD patients with low ADL compared with CVD patients with higher ADL, and that eating is thought to be important for the prevention of an increase of microorganisms in the oral cavity.
Assuntos
Atividades Cotidianas , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Candida/isolamento & purificação , Candidíase/microbiologia , Transtornos Cerebrovasculares/microbiologia , Saúde Bucal , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Bacterianas/etiologia , Candidíase/etiologia , Transtornos Cerebrovasculares/complicações , Transtornos Cerebrovasculares/psicologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Boca/microbiologiaRESUMO
OBJECTIVE: To clarify which proteases are specifically activated in the lesions of rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The activity levels of the serine proteases of the coagulation and fibrinolytic systems, and of elastase and collagenase as controls, in synovial fluid from 27 RA patients and 28 OA patients were measured using fluorogenic synthetic substrates which had methylcoumarylamide (MCA) at their COOH-termini. The thrombin-antithrombin III complex (TAT) content was also measured by ELISA. RESULTS: Among the proteases, thrombin-like activity was the highest in both RA and OA. The profiles of protease activity were similar in RA and OA, but their activities were in general significantly higher in RA than in OA (p < 0.01). The levels of both thrombin-like activity and TAT were about 7.5-fold higher in RA than in OA, while the levels of CRP and fibrinogen were only about 2-fold higher. Biochemical characterization of the thrombin-like activity in the synovial fluid of RA patients showed that this activity was due to thrombin. Thrombin-like activity positively correlated with the TAT concentration in RA (r = 0.750, p < 0.0001), but not in OA. CONCLUSION: Activation of the coagulation system was more marked in RA than in OA, strongly suggesting that in RA there is an imbalance between thrombin and its inhibitors, and that thrombin is more closely linked to the pathogenesis of RA than to that of OA. Our results also show that analysis of the synovial fluid may be useful to estimate the activation of the coagulation system in RA, but not that of the fibrinolytic system.
Assuntos
Artrite Reumatoide/enzimologia , Osteoartrite/enzimologia , Serina Endopeptidases/metabolismo , Líquido Sinovial/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/patologia , Colagenases/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator Xa/metabolismo , Feminino , Fibrinolisina/metabolismo , Hirudinas/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Elastase Pancreática/metabolismo , Proteínas Recombinantes/farmacologia , Líquido Sinovial/efeitos dos fármacos , Trombina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The kinetics of apoptotic fragmentation of the chromosomal DNA was determined in the influenza virus-infected MDCK, HeLa and KB cells, respectively. Comparison of these kinetics with the kinetics of virus multiplication revealed that the multiplication of influenza virus was observed only when apoptosis was induced after the production of progeny virus in the infected cells. The extent of apoptotic response was reversely correlated with the permissiveness of the cells.
Assuntos
Apoptose , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Replicação Viral , Animais , Linhagem Celular , Fragmentação do DNA , Cães , Humanos , Cinética , VirulênciaRESUMO
Previously we isolated a new trypsin-like enzyme designated human airway trypsin-like protease (HAT) from human sputum. In this study, we examined in vitro whether HAT was related to the prevention of fibrin deposition in the airway lumen by cleaving fibrinogen. In mucoid sputum samples from patients with chronic airway diseases, the concentration of fibrinogen, as measured by ELISA, was in the range of 2-20 micrograms/ml, and trypsin-like activity, as measured by spectrofluorometry was in the range of 10-50 milliunits (mU)/ml. We showed by gel filtration that the trypsin-like activity of mucoid sputum was mainly due to HAT. We examined the effects of HAT on human fibrinogen at pH 7.4 and 8.6. Fibrinogen was used at concentrations of 4-2,000 micrograms/ml and HAT purified from sputum at concentrations of 0.6-10 mU/ml. As shown by SDS-polyacrylamide gel electrophoresis, HAT cleaved fibrinogen, especially its alpha-chain, regardless of the concentration of fibrinogen. Pretreatment of fibrinogen with HAT resulted in a decrease or complete loss of its thrombin-induced clotting capacity, depending on the duration of pretreatment with HAT and the concentration of HAT. From these results we postulated that HAT may participate in the anticoagulation process within the airway, especially at the level of the mucous membrane, by cleaving fibrinogen transported from the blood stream.
Assuntos
Fibrinogênio/metabolismo , Serina Endopeptidases/metabolismo , Escarro/enzimologia , Albuminas/análise , Asma/enzimologia , Cromatografia em Gel , Fibrinopeptídeo A/análise , Humanos , Pneumopatias Obstrutivas/enzimologia , Elastase Pancreática/análise , Serina Endopeptidases/isolamento & purificação , Trombina/análiseRESUMO
Previously we isolated a trypsin-like enzyme designated human airway trypsin-like protease from the sputum of patients with chronic airway diseases. This paper describes the cDNA cloning, characterization of the primary protein structure deduced from the cDNA, and gene expression of this enzyme in various human tissues. We obtained an entire 1517-base pair sequence of cDNA with an open reading frame encoding a polypeptide with 418-amino acid residues. The polypeptide consisted of a 232-residue catalytic region and a 186-residue noncatalytic region with a hydrophobic putative transmembrane domain near the NH2 terminus. The polypeptide was suggested to be a type II integral membrane protein in which the COOH-terminal catalytic region is extracellular. Therefore, this protein is thought to be synthesized as a membrane-bound precursor and to mature to a soluble and active protease by limited proteolysis. It showed 29-38% identity in the sequence of the catalytic region with human hepsin, enteropeptidase, acrosin, and mast cell tryptase. The noncatalytic region had little similarity to other known proteins. In Northern blot analysis a transcript of 1.9 kilobases was detectable most prominently in the trachea among 17 human tissues examined.
Assuntos
Sistema Respiratório/enzimologia , Doenças Respiratórias/enzimologia , Serina Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Fibrinogênio/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solubilidade , EscarroRESUMO
A novel trypsin-like protease was purified to homogeneity from the sputum of patients with chronic airway diseases, by sequential chromatographic procedures. The enzyme migrated on SDS-polyacrylamide gel electrophoresis to a position corresponding to a molecular weight of 28 kDa under both reducing and non-reducing conditions, and showed an apparent molecular weight of 27 kDa by gel filtration, indicating that it exists as a monomer. It had an NH2-terminal sequence of Ile-Leu-Gly-Gly-Thr-Glu-Ala-Glu-Glu-Gly-Ser-Trp-Pro-Trp-Gln-Val-Ser-Leu- Arg-Leu, which differed from that of any known protease. Studies with model peptide substrates showed that the enzyme preferentially cleaves the COOH-terminal side of arginine residues at the P1 position of certain peptides, cleaving Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide most efficiently and having an optimum pH of 8.6 with this substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate, leupeptin, antipain, aprotinin, and soybean trypsin inhibitor, but hardly inhibited by secretory leukocyte protease inhibitor at 10 microM. An immunohistochemical study indicated that the enzyme is located in the cells of the submucosal serous glands of the bronchi and trachea. These results suggest that the enzyme is secreted from submucosal serous glands onto the mucous membrane in patients with chronic airway diseases.
Assuntos
Brônquios/enzimologia , Broncopatias/enzimologia , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Traqueia/enzimologia , Sequência de Aminoácidos , Cumarínicos/metabolismo , Fibrinogênio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos/metabolismo , Análise de Sequência , Serina Endopeptidases/análise , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologia , Membrana Serosa/enzimologia , Escarro/enzimologia , Especificidade por Substrato , Tripsina/químicaRESUMO
Bone change induced by knee immobilization was assessed on dissected femurs and tibias to clarify the influences upon the mechanical properties and their demands. Fifty-eight Wistar-Imamichi male rats (11-12 weeks old, body weight 350-450 g) were subjected to knee joint immobilization (150 degrees flexed position) on one side while the opposite side served as a control. Animals were killed in seven groups at time intervals of 1, 2, 3, 4, 5, 7, and 10 weeks. The hind leg was extirpated and prepared for (1) biomechanical analysis by the indentation method at the articular surfaces of the femoral condyle and head and at the subchondral bone of the proximal tibia, and for (2) dual-energy X-ray absorptiometry of the distal metaphysis of the femur. The biomechanical parameters measured induced dynamic stiffness and phase lag derived from forced oscillation (preload 3 N, cyclic load 2 N and 11 Hz, 35 Hz), and bone mineral density was analyzed. These were compared between the immobilized side and control side, and among the seven time groups. The biomechanical results showed an early change of osteocartilaginous properties at the femoral condyle, a late response at the tibial subchondral bone, and no change at the femoral head. The measurement of bone mineral density revealed that a very sensitive reaction started within 1-2 weeks. This study provides objective data demonstrating that disuse or lack of mechanical stress greatly affects the remodeling activity for homeostasis of joints, and dramatically impairs normal bone mineral density next to the immobilized joint in young animals.
Assuntos
Densidade Óssea/fisiologia , Imobilização , Articulação do Joelho/fisiologia , Absorciometria de Fóton , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Homeostase , Masculino , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
We previously reported finding a novel trypsin-like enzyme in sputum samples from patients with chronic airway diseases, and named it human airway tryptase (HAT). We also obtained data suggesting that HAT is secreted from submucosal serous glands onto the mucous membrane of the airway, and that fibrinogen is cleaved by HAT. We studied whether HAT can act as an anticoagulant in the airway by breaking down fibrinogen. The concentration of fibrinogen in sputum samples was measured by enzyme-linked immunosorbent assay. The concentration was higher in mucoid sputum from patients with bronchial asthma (6.3 +/- 5.5) than in mucoid sputum from patients with chronic bronchitis (1.9 +/- 1.1), and it was higher in purulent sputum from patients with bronchiectasis (18.8 +/- 8.8) than in mucoid sputum from patients with asthma. The trypsin-like activity in sputum (milliunit/ml) was 23.4 +/- 18.0 in mucoid sputum from patients with chronic bronchitis, 46.9 +/- 43.9 in mucoid sputum from patients with asthma, and 14.9 +/- 8.23 in purulent sputum from patients with bronchiectasis. The effect of HAT on human fibrinogen at concentrations from 4 to 2000 micrograms/ml was examined at pH 7.4 and 8.6, with purified HAT at concentrations from 0.5 to 10 milliunit/ml. SDS-polyacrylamide gel electrophoresis showed that HAT can cleave fibrinogen. especially the alpha-chain, at low concentrations (4 to 16 micrograms/ml) and at a high concentration (2000 micrograms/ml) of fibrinogen. Exposure of fibrinogen to HAT resulted in the loss of thrombin-induced clotting capacity; the strength of that effect depended on the duration of exposure to HAT and on the concentration of HAT. From these results we postulate that HAT acts at an anticoagulant in the airways, especially on the mucous membrane, by cleaving fibrinogen transported from blood.
Assuntos
Fibrinogênio/metabolismo , Sistema Respiratório/enzimologia , Serina Endopeptidases/metabolismo , Asma/enzimologia , Bronquiectasia/enzimologia , Bronquite/enzimologia , Fibrinólise , Humanos , Escarro/enzimologiaRESUMO
OBJECTIVE: Influence of low-level (810 nm, Ga-Al-As semiconductor) laser on bone and cartilage during joint immobilization was examined with rats' knee model. MATERIALS AND METHODS: The hind limbs of 42 young Wistar rats were operated on in order to immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/cm2, 5.8 W/cm2, and sham treatment. After 6 times of treatment for another 2 weeks both hind legs were prepared for 1) indentation of the articular surface of the knee (stiffness and loss tangent), and for 2) dual energy X-ray absorptiometry (bone mineral density) of the focused regions. RESULTS AND CONCLUSIONS: The indentation test revealed preservation of articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy. Soft laser treatment has a possibility for prevention of biomechanical changes by immobilization.
Assuntos
Remodelação Óssea/efeitos da radiação , Cartilagem Articular/fisiopatologia , Imobilização/efeitos adversos , Articulações/fisiopatologia , Lasers , Animais , Fenômenos Biomecânicos , Densidade Óssea/efeitos da radiação , Cartilagem Articular/efeitos da radiação , Modelos Animais de Doenças , Articulações/efeitos da radiação , Terapia a Laser , Masculino , Dosagem Radioterapêutica , Ratos , Ratos WistarRESUMO
We measured contents of tracheobronchial and alveolar components contained in the newborn airway aspirates (NAA) obtained from 54 healthy newborns, with bronchoalveolar lavage fluid (BALF) and bronchial lavage fluid (BLF) from healthy adults, and with mucoid sputum from adults with chronic bronchitis as controls. Fucose (a parameter of tracheobronchial mucus mucoprotein) and tryptase activity were used as tracheobronchial components, and pulmonary surfactant apoprotein A (SP-A) as an alveolar component. The ratio of the content of each component to that of total protein (TP) was compared among NAA, BLF, BALF, and the mucoid sputum. The SP-A/TP ratio in the NAA was similar to that in BLF but 1/24 of that in BALF. The fucose/TP ratio in NAA was about 1/2 of that in BLF and about 6 times higher than that in BALF. The tryptase/TP ratio in NAA was about twice that in BLF and about 80 times that in BALF. The ratios of the above components to TP in the mucoid sputum were closer to those in NAA and BLF than those in BALF. These results suggest that NAA contain both tracheobronchial and alveolar components, and their contents of the formers are larger than those of the latters, and that NAA may be useful for diagnosing pathologic conditions in the airway of newborns, and for comparing the biological defence mechanism between newborns and adults.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Recém-Nascido/metabolismo , Adulto , Bronquite/metabolismo , Quimases , Feminino , Fucose/análise , Humanos , Concentração de Íons de Hidrogênio , Masculino , Proteínas/análise , Proteolipídeos/análise , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/análise , Serina Endopeptidases/análise , Escarro/química , TriptasesRESUMO
We found a novel trypsin-like enzyme (tryptase) in sputum from patients with chronic airway diseases, and named this enzyme human airway tryptase (HAT). To clarify its physiological significance in the airway, we compared biochemical properties of purified HAT with those of purified lung mast cell tryptase (MCT). Studies with model peptide substrates showed that both the HAT and MCT preferentially cleaved the COOH-terminal side of arginine residues of certain peptides, but substrate specificities to nine synthetic model substrates of HAT differed from those of MCT. Effects of protease inhibitors on the two enzymes were examined at a concentration of 10 microM. Both the HAT and MCT were strongly inhibited by the trypsin inhibitors leupeptin, antipain, and aprotinin. An alpha-1-protease inhibitor inhibited HAT by 50%, but it did not inhibit MCT. In contrast, a secretory leukocyte protease inhibitor strongly inhibited MCT, but not HAT. Mucoid sputum from patients with chronic bronchitis contained much more HAT than MCT. These differences in biochemical properties between HAT and MCT indicate that they play different physiological roles in the airways.
Assuntos
Pulmão/citologia , Mastócitos/enzimologia , Serina Endopeptidases/metabolismo , Escarro/enzimologia , Asma/enzimologia , Bronquite/enzimologia , Quimases , Humanos , Serina Endopeptidases/análise , TriptasesRESUMO
We compared lung fibroblast growth-stimulating activity (FGA) of several serine proteases including thrombin in vitro, and examined the mechanism of FGA. FGA was measured by incorporation of 3H-thymidine into lung fibroblasts (IMR-90). The activities of the enzymes were measured by spectrofluorometric method with synthetic peptides specific for each enzyme, and these enzymes were added to the assay system for FGA at concentrations of 7 x 10(0)-7 x 10(5) unit/ml. Human thrombin, bovine trypsin and bovine alpha-chymotrypsin showed clear FGA, but that of alpha-chymotrypsin was lower than those of thrombin and trypsin. On the other hand, porcine pancreatic elastase and human neutrophil elastase did not show any FGA, and had a cytotoxic effect on fibroblasts. A specific low molecular-weight thrombin inhibitor, argatroban (MW. 562), inhibited not only the enzyme (peptidolytic) activity of thrombin, but also its FGA at the same concentration. These results suggest that serine proteases can be classified into at least two groups, showing FGA and cytotoxic activity, respectively, and that the FGA of the former group is mediated by their catalytic (enzymatic) action.
Assuntos
Antígenos de Neoplasias , Biomarcadores Tumorais , Substâncias de Crescimento/farmacologia , Serina Endopeptidases/farmacologia , Animais , Catálise , Bovinos , Células Cultivadas , Endopeptidases , Fibroblastos/efeitos dos fármacos , Gelatinases , Humanos , Proteínas de Membrana , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/etiologia , Serina Endopeptidases/fisiologia , SuínosRESUMO
In order to clarify the mechanisms of pulmonary fibrosis, we produced bleomycin (BLM)-induced pulmonary fibrosis in rats and examined the ability of alveolar macrophages (AM) to produce interleukin-1 (IL-1). BLM (0.9 mg) was administered once via the trachea to male Wistar rats weighing about 200 g. Bronchoalveolar lavage was performed at 1, 3, 6, 9 and 12 days after administration. AM were incubated for 24 hours, then extracellular IL-1 in the supernatants and cell-associated IL-1 of AM were measured by proliferation assay of mouse thymocytes. Cell-associated IL-1 activity was measured after fixation by paraformaldehyde (PFA). Extracellular IL-1 was detected in the culture media of AM at only day 1 after administration. On the other hand, cell-associated IL-1 was detected in AM fixed by PFA on days 1, 3, 6 and 9 after administration. AM from BLM-induced pulmonary fibrosis in rats were fixed by PFA and then were treated with anti-IL-1 alpha antibody or anti-IL-1 beta antibody. Cell-associated IL-1 activity was neutralized by treatment with anti-IL-1 alpha antibody and was not neutralized by treatment with anti-IL-1 beta antibody. Following this, the effect of cell-associated IL-1 on pulmonary fibroblasts was examined. This was estimated by the proliferation of pulmonary fibroblasts using incorporation of 3H-thymidine. When pulmonary fibroblasts were incubated with AM fixed by PFA from BLM-induced pulmonary fibrosis in rats, proliferation of pulmonary fibroblasts was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bleomicina/efeitos adversos , Interleucina-1/biossíntese , Macrófagos Alveolares/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Interleucina-1/fisiologia , Masculino , Fibrose Pulmonar/induzido quimicamente , Ratos , Ratos WistarRESUMO
To investigate the mechanism of eosinophilia in patients with eosinophilic pleural effusions, we measured the activities of eosinophil colony-stimulating factor (Eo-CSF) and stimulating factor for eosinophil survival in the eosinophilic pleural fluids of six patients (two with tuberculous pleuritis, two with drug allergy, and one each with chronic eosinophilic pneumonia and pleuritis associated with rheumatoid arthritis). The number of eosinophil colonies formed by the pleural fluid of patients with eosinophilic pleural effusions significantly exceeded that of control patients with noneosinophilic pleural effusions (7.5 +/- 1.9 colonies/10(5) bone marrow cells, n = 6, versus 0.3 +/- 0.1 colonies/10(5) bone marrow cells, n = 6, P < 0.01). Similarly, eosinophil survival evaluated on day 4 of culture with pleural fluid of patients with eosinophilic pleural effusions significantly exceeded that of patients with noneosinophilic pleural effusions (83.9 +/- 9.8% versus 46.1 +/- 11.2%, P < 0.001). Both activities were inhibited mainly by anti-IL-5 antibody and partially by anti-GM-CSF antibody and anti-IL-3 antibody. Mononuclear cells obtained from eosinophilic pleural fluid released the activities of Eo-CSF and stimulating factor for eosinophil survival in vitro. These findings suggest that GM-CSF, IL-5, and IL-3 are important to eosinophil accumulation in pleural cavity as stimulators of proliferation and survival of eosinophils.
Assuntos
Eosinófilos/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Linfocinas/metabolismo , Derrame Pleural/patologia , Adulto , Idoso , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Humanos , Pessoa de Meia-Idade , Monócitos , Derrame Pleural/metabolismoRESUMO
Changes in the content and molecular state of secretory leukocyte protease inhibitor (SLPI) in the airways in chronic airway diseases were studied. SLPI in bronchoalveolar and bronchial lavage fluids (BALF and BLF) from normal subjects and patients with diffuse panbronchiolitis (DPB), and in mucoid sputa from patients with emphysema and in purulent sputa from DPB patients were examined by ELISA and Western blotting. Results showed that in the BALF and BLF of normal subjects, the SLPI/alpha 1-protease inhibitor (alpha 1-PI) ratios (M/M) are about 0.6 and 6, respectively and the neutrophil elastase inhibitory capacity of BLF is mainly due to SLPI. In the BALF and BLF of DPB patients, both the elastase activity and alpha 1-PI level are increased, but the SLPI level is decreased. In purulent sputa, the elastase activity was found to be 430-fold that in mucoid sputa and the alpha 1-PI level to be 3.5-fold that in mucoid sputa, but the SLPI level was slightly lower in the mucoid sputa. Analysis by Western blotting showed that in BLF from normal subjects and mucoid sputa, SLPI is present in an intact form and in complexes with other substances, whereas in BLF and purulent sputa from DPB patients, SLPI is present in a degraded form and in complexes with other substances, but not in the intact form. These results indicate that SLPI is the main protease inhibitor in the airways of both normal subjects and patients with hypersecretory respiratory diseases without infection, but that its level is insufficient to overcome the increased protease burden in the airways of DPB patients.
Assuntos
Proteínas , Doenças Respiratórias/fisiopatologia , Inibidores de Serina Proteinase/metabolismo , Adulto , Idoso , Brônquios/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Elastase Pancreática/metabolismo , Proteínas Secretadas Inibidoras de Proteinases , Inibidor Secretado de Peptidases Leucocitárias , alfa 1-Antitripsina/metabolismoRESUMO
Fibroblasts are important for maintenance of the structural frame network for most tissues, and they also play an important role in the inflammatory process via production of various mediators. In this study, we demonstrated that pulmonary fibroblasts may participate in pulmonary inflammation by production of neutrophil chemotactic factor (NCF). Pulmonary fibroblasts were stimulated with various cytokines (IGF-1, PDGF, IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF, IFNr). Fibroblasts stimulated with either TNF, IL-1 alpha or IL-beta but not IGF, PDGF, IL-2 or IL-6 demonstrated a kinetic and dose-dependent increase in NCF activity. The NCF activity of crude supernatant was heat-stable and was not changed by anti-C5 antibody treatment or ether extraction. Characterization of the NCF activity by gel-filtration using high pressure liquid chromatography showed two active fractions, one with MW greater than 100 kD and the other with MW less than 10 kD. NCF activity in the small molecular weight fraction was demonstrated by inhibition of chemotaxis by addition of anti-IL-8 antibody. These data suggest that cytokine-treated fibroblast-derived NCF may be important in the pathogenesis and expression of a variety of pulmonary disease processes associated with neutrophil accumulation and activation.
Assuntos
Citocinas/farmacologia , Fibroblastos/efeitos dos fármacos , Interleucina-8/biossíntese , Pulmão/citologia , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Estimulação QuímicaRESUMO
The in vitro production of granulocyte/macrophage colony-stimulating factor (GM-CSF) by mononuclear cells (MNC) from the peripheral blood of patients with bronchial asthma (BA) was examined by enzyme-linked immunosorbent assay (ELISA). GM-CSF concentrations in the media of MNC from patients with BA cultured with interleukin-2 (IL-2) was 80.2 +/- 52.0 pg/ml (mean +/- SE, n = 12), and that in cultures without a stimulant was 12.1 +/- 11.3 pg/ml. The GM-CSF concentrations in the media of MNC from patients with other diseases (n = 13) and from healthy volunteers (n = 6) cultured with or without IL-2 were less than 7.5 pg/ml (the minimum detectable value). The culture media of MNC from patients with BA demonstrated activities for stimulating the proliferation and survival of eosinophils, and these activities were partially inhibited by anti-GM-CSF antibodies. GM-CSF production by MNC of patients with BA treated with glucocorticoids was lower than that of MNC from untreated patients with BA, and it was inhibited by coculture with glucocorticoids in vitro. These results suggest that GM-CSF production by MNC is increased in patients with BA, is modulated by glucocorticoids, and may play an important role in the pathogenesis of BA.