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Rationale: Imatinib is an inhibitor of platelet-derived growth factor receptors. We have reported that treatment with imatinib inhibited bleomycin-induced pulmonary fibrosis in mice. However, late treatment with imatinib had no effect., Objectives: To clarify why imatinib had no antifibrotic effect when its administration was delayed, we focused on alpha(1)-acid glycoprotein (AGP), because it was reported to bind imatinib and mediate drug resistance., Methods: The concentration of AGP in serum of mice and patients with idiopathic pulmonary fibrosis was measured by radial immunodiffusion testing. The effects of AGP in vitro were evaluated by assaying the growth of lung fibroblasts. We examined the combined effects of erythromycin (EM) or clarithromycin (CAM) on bleomycin-induced pulmonary fibrosis in mice., Measurements and Main Results: Addition of AGP abrogated imatinib-mediated inhibition of the growth of fibroblasts. However, treatment with EM or CAM restored the growth-inhibitory effects of imatinib. The elevated level of AGP was detected in serum and lung homogenates in bleomycin-exposed mice and reached a plateau on Day 14. Imatinib alone did not ameliorate pulmonary fibrosis when treatment was started on Day 15, whereas coadministration of imatinib and EM or CAM significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts in vivo. Serum levels of AGP were higher in patients with idiopathic pulmonary fibrosis than in healthy subjects., Conclusions: AGP is an important regulatory factor modulating the ability of imatinib to prevent pulmonary fibrosis in mice, and combined therapy with imatinib and EM or CAM might be useful for treatment of pulmonary fibrosis.

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.

To clarify the clinical significance of autoantibodies to interleukin-1 alpha (IL-1 alpha autoantibodies) in rapidly progressive idiopathic pulmonary fibrosis (IPF), we measured the level of IL-1 alpha autoantibodies in serum of 11 patients on the first hospital day, when patients were admitted due to severe symptoms, and on the 21st hospital day. IL-1 alpha autoantibodies in serum were measured using radioimmunoassay, and the limitation of this assay for IL-1 alpha autoantibodies was 5 ng/ml. These antibodies were detected in 5 of 11 patients on the first hospital day. On the 21st hospital day, these antibodies were detected in all patients, and its level was increased compared with that on the first hospital day. IL-1 alpha autoantibodies that appeared in patients corresponded to that of IgG. The half life of exogenous autoantibodies was investigated following administration of autoantibody rich plasma obtained from healthy blood donors to 6 control patients (CP) and 6 progressive IPF patients. These autoantibody levels in their serum were less than 5 ng/ml before administration. Serum was obtained at the indicated time after administration of IL-1 alpha autoantibodies and the level of these autoantibodies in serum was measured, then the half life was calculated. Half life of exogenous IL-1 alpha autoantibodies in progressive IPF patients was significantly shorter than that in CP (71.3 +/- 31.8 hr vs 352.0 +/- 98.3 hr, p < 0.01). These findings suggested that IL-1 alpha autoantibodies were generated in response to the inflammatory process of rapidly progressive IPF and may act as a regulatory factor for IL-1 alpha.

In order to clarify the mechanism of pulmonary fibrosis, we examined the functional changes of lung fibroblasts in bleomycin (BLM)-induced pulmonary fibrosis. Lung fibroblastic cells were obtained from rat lungs after an intratracheal treatment of BLM or saline. The spontaneous proliferation of BLM-treated rat fibroblasts (BRF), which was estimated by 3H-TdR incorporation and direct cell counting, was significantly more rapid than that of normal saline-treated rat fibroblasts (NRF). Next, we investigated prostaglandin (PG) E2 synthesis by BRF and NRF, with or without stimulation by interleukin (IL)-1 alpha, and found that PGE2 production by BRF was significantly less than that by NRF. There was no significant difference in cyclooxygenase (COX) activity and COX-2 mRNA level between BRF and NRF, indicating that the change in PGE2 production was independent of COX, a rate-limiting enzyme for the production of PGE2. These results suggest that the proliferation of fibroblasts is down-regulated by PGE2 released from themselves in normal lungs in an autocrine fashion, thus the decreased PGE2 production observed in lung fibroblasts from rats with BLM-induced pulmonary fibrosis may result in the excessive fibroblast proliferation in this disorder. Overall, these findings throw some light on the mechanism of development of BLM-induced pulmonary fibrosis.

This study was designed to clarify the contribution of IL-8 as a specific neutrophil chemotactic factor in the human respiratory tract in various pulmonary diseases. The neutrophil chemotactic activity (NCA), neutrophil counts and IL-8 concentration in the bronchoalveolar lavage fluid (BALF) obtained from normal volunteers (NV), control patients (CP), patients with diffuse panbronchiolitis (DPB) and patients with idiopathic pulmonary fibrosis (IPF) were examined. Neutrophil counts, NCA and IL-8 concentration in BALF obtained from patients with DPB or IPF was significantly higher than that from NV or CP. The IL-8 concentration correlated with neutrophil count and also correlated with NCA in BALF from patients with IPF, whereas there was no correlation between these factors in BALF from DPB. These results suggest that the contribution of IL-8 to neutrophil accumulation of the lower respiratory tract is different between IPF and DPB.

Thrombin is a multifunctional enzyme generated at sites of vascular injury, and is known to be increased in the lungs in some types of fibrotic lung disease. In this study, to determine whether thrombin is associated with fibroblast growth and pulmonary fibrosis in these disorders, we examined whether a growth factor for fibroblasts (platelet-derived growth factor, PDGF) was released by thrombin-stimulated alveolar macrophages (AM). The culture supernatants of rat AM stimulated with 1 or 10 U/ml of thrombin showed a significant increase in fibroblast growth-stimulating activity (FGA). Pretreatment of the AM supernatant with anti-PDGF-AA antibody significantly decreased the FGA, but pretreatment with anti-PDGF-BB antibody did not. The supernatants of AM stimulated with thrombin also increased the growth of fibroblasts from the lungs of rats with bleomycin-induced lung injury. These results indicate that thrombin stimulates AM to release PDGF-AA, which is responsible, at least in part, for fibroblast growth and the development of pulmonary fibrosis in some types of fibrotic lung disease.

The activities of cell-associated IL-1 (IL-1 alpha) and extracellular IL-1 (IL-1 beta) in alveolar macrophages (AM) from rats with bleomycin-induced pulmonary fibrolosis were measured to determine their role in fibroblast growth. AM were obtained on Days 1, 3, 6, 9 and 12 after a intratracheal injection of bleomycin, and the IL-1 activity and fibroblast growth-stimulating activity in fixed AM and AM supernatants were measured. Higher cell-associated IL-1 activity was detected in AM from bleomycin-treated rats than in those of control on Day 1 through 9. But extracellular IL-1 activity in the supernatant of AM from bleomycin-treated rats significantly higher only on Day 1. Expression of IL-1 alpha mRNA in AM from bleomycin-treated rats was significantly higher than that in AM of control, but there was no significant difference in the mRNA levels of IL-1 beta in AM of these two groups. Fixed AM from bleomycin-treated rats caused growth-inhibition of fibroblasts in a density-dependent manner. The inhibitory activity was decreased by pretreatment of fixed AM with anti IL-1 alpha antibody, but not anti IL-1 beta antibody. These results suggest that cell-associated IL-1 (IL-1 alpha) is produced continuously in AM from rats with bleomycin-induced pulmonary fibrosis and may be important in regulation of this disorder by inhibiting fibroblast growth.

To clarify the mechanism of pulmonary fibrosis, the growth rate of fibroblasts obtained from bleomycin (BLM)-treated rats was compared with that of fibroblasts obtained from control rats. Proliferation of fibroblasts obtained from rats at 4 days after BLM instillation (BRF) was significantly more rapid than that of fibroblasts obtained from rats at 4 days after saline instillation (CRF), as assessed by cell counting and 3H-TdR incorporation. CRF and BRF were separated by the method of discontinuous Percoll gradients. Three fibroblast fractions of specific gravity (S.G.), S.G. < 1.036, 1.036 < or = S.G. < 1.050, 1.050 < or = S.G. < 1.062, were obtained. Percentages of the densest fibroblast fraction were 46.2 +/- 5.2 in BRF and 6.4 +/- 2.8 in CRF. The densest fibroblasts in BRF proliferated more rapidly than the least dense fibroblasts. Rat rIL-1 alpha suppressed the proliferation of CRF and BRF, dose dependently. These results suggest that an increase in the densest fibroblasts in the lung might correlate with BLM-induced pulmonary fibrosis and that IL-1 alpha suppresses the proliferation of pulmonary fibroblasts.

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)

This study was designed to clarify the contributions of specific neutrophil chemotactic factors (NCF) in neutrophil accumulation in the human respiratory tract associated with various diseases. The activity and characteristics of the NCF in the bronchoalveolar lavage (BAL) fluid and culture media of alveolar macrophages obtained from normal volunteers, control patients, patients with chronic airway diseases (CAD) and patients with idiopathic pulmonary fibrosis (IPF) were examined. The BAL fluid from normal volunteers contained NCF comparable with the chemotactic factors interleukin-8 (IL-8) and leukotriene B4 (LTB4). Analysis of the biochemical characteristics of NCF released from alveolar macrophages suggests that they are derived from alveolar macrophages. The NCF activities in BAL fluids from patients with CAD and IPF were higher than those in BAL fluids from normal volunteers and control patients. Biochemical analysis demonstrated that several kinds of NCF, including those derived from the complement component C5 and alveolar macrophages, were present in the BAL fluid from patients with CAD and respiratory infections. The especially marked increase of C5-derived NCF indicate their importance in neutrophil accumulation in the respiratory tract of patients with CAD. Alveolar macrophages released different types of NCF after different lengths of culture periods (4 h and 24 h). Alveolar macrophages from patients with IPF released larger amounts of NCF than alveolar macrophages from normal volunteers, indicating the importance of alveolar-macrophage-derived NCF as well as C5-derived NCF in neutrophil accumulation in the respiratory tract of patients with IPF. These results suggest that various types of NCF increase in response to different disease states of the respiratory tract and serve to regulate the accumulation of neutrophils.

To clarify the role of thrombin in fibroblast growth and the development of pulmonary fibrosis in bleomycin-induced interstitial lung disease, we examined the relationship of thrombin activity to fibroblast growth-stimulating activity (FGA) in bronchoalveolar lavage (BAL) fluid from bleomycin-treated rats. Male Wistar rats were given a single intratracheal injection of bleomycin, BAL was performed 2, 6, and 15 days later, and the BAL fluid was assayed for thrombin activity and FGA. Higher FGA than the control value was detected in the BAL fluid from rats on day 6 after bleomycin administration. In bleomycin-treated rats, thrombin activity in the BAL fluid was significantly elevated on day 2 and maximal on day 6. The FGA of the BAL fluid from bleomycin-treated rats on day 6 was significantly decreased by its treatment with various thrombin inhibitors, such as alpha 1-protease inhibitor, antithrombin III, hirudin, and MD-805. In our assay, purified rat thrombin also showed FGA in vitro, and its FGA was inhibited by the same concentrations of these thrombin inhibitors as those inhibiting the activity in the BAL fluid. On ammonium sulfate fractionation, most of the thrombin activity was recovered in the fraction of 35 to 50% saturation in which most of the FGA was detected. These results suggest that the FGA of the BAL fluid from bleomycin-treated rats was at least partly due to thrombin is responsible, at least in part, for fibroblast growth and pulmonary fibrosis in bleomycin-induced interstitial lung disease.

In order to clarify the role of thrombin on the development of pulmonary fibrosis in diffuse interstitial lung diseases, we examined the relationship between fibroblast growth-stimulating activity (FGA) and thrombin activity in bronchoalveolar lavage fluid (BALF) from rats with bleomycin-induced interstitial lung disorders. Male Wistar strain rats (body weight about 200 g) were given a single intratracheal injection of 0.9 mg bleomycin, and bronchoalveolar lavage was performed on days 2, 6 and 15. The BALF was centrifuged at 250 X g for 10 min to remove cells, and then the supernatant was recentrifugation at 27,000 X g for 40 min to remove pulmonary surfactants. The supernatant (10 ml) was dialyzed overnight against distilled water, frozen at -70 degrees C, freeze-dried, and resuspended in 2 ml of Dulbecco's modified Eagle medium (concentrated 5-fold). The 5-fold concentrated BALF was added to rat lung fibroblast culture media, and assayed for cytotoxic activity and FGA. Thrombin activity in 250 X g supernatant was measured by using fluorescence assay with the synthetic peptide substrate, Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. Histological examination showed a prominent increase in fibroblast number in the pulmonary interstitium on day 6, and transformation of fibroblasts into mature forms, fibrocytes, on day 15. On day 2 after bleomycin administration, no FGA was seen but cytotoxic activity was detected in the BALF. On day 6, the cytotoxic activity was not found, whereas FGA showed a significantly higher level than the control value. On day 15, the FGA decreased to the control value.(ABSTRACT TRUNCATED AT 250 WORDS)

The cell profiles of bronchial and bronchoalveolar lavage fluids (BLF and BALF) of patients with idiopathic pulmonary fibrosis (IPF) were compared with those of normal volunteers (NV) and age-matched control patients (CP), to characterize the cell profiles of the bronchoalveolar region in normals and patients with IPF. In BALF of nonsmokers from both control groups (NV and CP), alveolar macrophages (AM) were predominant and the percentage of neutrophil leukocytes and that of eosinophil leukocytes below 1% of the total cells. The percentage of neutrophils and that of bronchial epithelial cells were higher in BLF than in BALF of both control groups. Of the immune and inflammatory cells in BLF, the mean percentage of neutrophils was 12% in NV group and 42% in CP group. The percentage of neutrophils and that of eosinophils in BALF were higher in IPF group than in CP group, but the percentage of neutrophils in BLF of IPF group was comparable to that of CP group. In the IPF group, the percentage of neutrophils in BALF was lower than that in BLF. These results indicated that even in healthy subjects, a considerable number of neutrophils are present in the bronchial region and that the cell profile of the lavage fluid of the bronchoalveolar tree changes depending on the method of lavage. Presumably the higher percentage of neutrophils in BALF of patients with IPF is partly due to derangements of the alveolar structure, because the amount of saline infused into this region is limited.