The Role of c-Jun N-terminal Kinase in the Radiation-Induced Lung Fibrosis / 결핵

BACKGROUND: The undrlying pathogenesis of radiation-induced lung fibrosis (RTLF) has not been very well defined. However, the role of TGF-β in the generation of RTLE has been a major focus because there is an increase in the expression of both the TGE-β stimulated lung fibrosis includes the activation of many mediators such as Smad and c-Jun N-terminal kinase (JNK) through TAK1. It is we hypothesized that JNK activation may play a pivotal role in RTLF pathogensis through increased transcription of the fibrogenic cytokines. The present study evaluates JNK activity in alveolar macrophages after irradiation and the relationship between JNK activity and the amount of collagen in the lung tissues. METHODS: C57BL/6 mice(20-25 gr. males) received cholorotetracycline(2g/L) in their drinking water 1 week prior to irradiation and continuously there after. The mice were irradiated once with 1400 cGy of 60COγ-ray over the whole chest. The cellular composition of the whole lung bronchoalveolar lavage fluids(BALF), elastin expression in the lung tissues, the level of hydroxyproline in lung tissues, and an in vitro JNK assay was measured before irradiation and one, four, and eight weeks after irradiation (RT). RESULTS: The volumes of BALF retrieved from instilled 4ml of saline with 2% heparin were 3.7-3.8ml for each group. The cell numbers were similar before(4.1×10(4)±0.5±10(4)/ml) and 1 week(3.1×10(4)±0.5±10(4)/ml) after RT. At four and eight weeks after RT, the cell number reached to 14.0×10(4)±1.5±10(4)/ml and 10.0×10(4)±1.3±10(4)/ml, respectively. There we no changes in the lymphocytes and neutrophils population obseved in the BALF after RT. The H-E stain of the lung tissues did not show any structural and fibrotic change in the lung tissues at 4 and 8 weeks after RT. In addition, the amount of elastin and collagen were not different on Verhoeff staining of the lung tissues before RT to eight weeks after RT. The hydroxyproine content was measured with the left lung dissected from the left main bronchus. The lung were homogenized and hydrolyzed with 6 N HCI for 12 hours at 110℃ then measured as previously described. The content of hydroxyproline, standardized with a lung protein concentration, reached a peak 4 weeks after RT. and thereafter showed a plateau. An In vitro JNK assay using c-Jun(1-79)-GST sepharose beads were performed with the alveolar macrophages obtained from the BAL. JNK activity was not detected prior to RT, However, the JNk activity increased from one week after RT and reached a peak four weeks after RT. CONCLUSION: JNK may be involved in the pathogensis because the JNK activity showed similar pattern observed with the hydroxyproine content. However, it is necessary to clarify that the JNK increases the transcription of fibrogenic cytokines through the transcription factor.

The Activity of c-Jun N-terminal Kinase (JNKb) in Patients with UIP / 결핵

BACKGROUND: TNF-alpha is related to the generation of lung fibrosis in patients with UIP. The precise mechanism leading to lung fibrosis by TNF-alpha is unknown. However, the activation of a transcription factor like AP-1(down stream of c-jun N-terminal kinase, JNK) by TNF-alpha may be related to the induction of fibrogenic cytokines like PDGF or IGF-I. Furthermore, JNK was reported to be activated in the radiation-in-duced lung fibrosis model. This study examined JNK activity in patients with UIP. METHODS: The expression of phosphorous JNK(p-JNK), macrophage/moncoyte specific markers, CD68, and cytokeratin was evaluated by immunohistochemical (IHC) staining of lung tissues from patients with UIP and lung cancer. An in vitro kinase assay was performed with alveolar macrophages obtained by a bronchollung cancer. An in vitro kinase assay was performed with alvolar macrophages obrtained by a bronchol avleolar lavage from patients with UIP and healthy persons as the control. RESULTS: The IHC stain showed that p-JNK is expressed in the almost all of the alveolar macrophages and smooth muscle cells in patients with UIP. In case of the normal areas of the lung from patients with lung cancer, the alveolar macrophages showed little p-JNK expression. Interestingly, increased JNK activity was not found in the in vitro kinase assay of the alveolar macrophages obtained from both patients with UIP and healthy persons as the control. Furthermore, 10 ng/ml of TNF-alpha failed to increase the JNK activity of the alveolar macrophages in both patients with UIP and healthy people. CONCLUSION: The JNK was activated constitutionally in patients with UIP. However, the role of JNK in the pathogenesis of lung fibrosis needs to be clarified.

Lipopolysaccharide-induced Synthesis of IL-1beta, IL-6, TNF-alpha and TGF-beta by Peripheral Blood Mononuclear Cells / 결핵

BACKGROUND: Endotoxin (LPS lipopolysaccharide), a potent activator of immune system, can induce acute and chronic inflammation through the production of cytokincs by a variety of cells, such as monocytes, endothelial cells, lymphocytes, eosinophils, neutrophils and fibroblasts. LPS stimulate the mononucelar cells by two different pathway, the CD14 dependent and independent way, of which the former has been well documented, but not the latter. LPS binds to the LPS-binding protein (LBP), in serum, to make the LPS-LBP complex which interacts with 0D14 molecules on the mononuclear cell surface in peripheral blood or is transported to the Ussues. In case of high concentration of LPS, LPS can stimulate directly the macrophages without LBP. We investigated to detect, the generation of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-alpha, and fibrogenic cytokine, TGF-beta, by peripheral blood mononuclear cells (PBMC) after LB'S stimulation under serum-free conditions, which lacks LBPs. METHODS: PBMC were obtained by centrifugation on Ficoll Hypaque solution of peripheral venous bloods from healthy normal subjects, then stimulated in the presence of LPS (0.1 microgram/mL to 100 microgram/mL). The activities of IL-1, IL-6, TNF, and TGF-betawere measured by bioassaies using cytokines - dependent proliferating or inhibiting cell lines. The cellular sources producing the cytokines was investigated by immunohistochemical stains and in situ hybridization. RESULTS: PBMC started to produce IL-6, TNF-alpha and TGF-beta in 1 hr, 4 hrs and 8hrs, respectively, after LPS stimulation The production of IL-6, TNF-alpha and TGF-beta continuously increased 96 His after stimulation of LPS. The amount of production was 19.8 ng/ml of IL-6 by 105 PEMC, 4.1 ng/mL of TNF by 106 PBMC and 344 pg/mL of TGF-betaby 2 x 106 PBMC. The immunoreactivity to IL-6, TNF-alpha and TGF-betawere detected on monocytes in LPS-stimulated PBMC. Some of lymphocytes showed positive immunoreactivity to TGF-beta. Double immunohistochemical stain showed that IL-1beta, IL-6, TNF-alpha expression was not associated with CD14 postivity on monocytes. IL-1beta, IL-6, TNF-alpha and TGF-/betamRNA expression worn same as observed in immunoreactivity for each cytokines. CONCLUISON: When monocytes are stimulted with LPS under serum-free conditions, IL-6 and TNF-alphaare secreted in early stage of inflammation. In contrast, the secretion of TGF-beta arise in the late stages and that N maintained after 96 his. The main cells releasing lL-1beta, IL-6, INF-alpha and TGF-beta are monocytes, but also lymphocytes can secret TGF-beta.

The Effects of Proinflammatory Cytokines and TGF-beta, on The Fibroblast Proliferation / 결핵

BACKGROUNDS: The injury of a tissue results in the infalmmation, and the imflammed tissue is replaced by the normal parenchymal cells during the process of repair. But, constitutional or repetitive damage of a tissue causes the deposition of collagen resulting in the loss of its function These lesions are found in the lung of patients with idiopathic pulmonary fibrosis, complicated fibrosis after diffuse alveolar damage (DAD) and inorganic dust-induced lung fibrosis. The tissue from lungs of patients undergoing episodes of active and/or end-stage pulmonary fibrosis shows the accumulation of inflammatory cells, such as mononuclear cells, neutrophils, mast cells and eosinophils, and fibroblast hyperplasia. In this regard, it appears that the inflammation triggers fibroblast activation and proliferation with enhanced matrix synthesis, stimulated by inflammatory mediators such as interleukin-1 (lL-1) and/or tumor necrosis factor (TNF). It has ken well known that TGF-beta enhance the proliferation of fibroblasts and the production of collagen and fibronectin, and inhibit the degradation of collgen. In this regard, It is likely that TGF-beta undergoes important roles in the pathogenesis of pulmonary fibrosis. Nevertheless, this single cytokine is not the sole regulator of the pulmonary fibrotic response. It is likely that the balance of many cytokines including TGF-beta, IL-1, IL-6 and TNF-alpha regulates the pathogenesis of pulmonary fibrosis. In this study, we investigate the interaction of 'PGF-beta, IL-1beta, IL-6 and TNF-alpha and their effect on the proliferation of fibroblasts. METHODS: We used a human fibroblast cell line, MRC-5 (ATCC). The culture of MRC-5 was confirmed by immunofluorecent staining. First we determined the concentration of serum in cuture medium, in which the proliferation of MRC-5 is supressed but the survival of MRC-5 is retained. Second, we measured optical density after staining the cytokine-stimulated cells with 0.5% naphthol blue black in order to detect the effect of cytokines on the proliferation of MRC-5. RESULT: In the medium containg 0.5% fetal calf serum, the proliferation of MRC-5 increased by 50%, and it was maintained for 6 days. IL-1beta, TNF-alpha and IL-6 induced the proliferation of MRC-5 by 45%, 160% and 120%, respectively. IL-1beta and TNF-a enhanced TGF-beta-induced proliferation of MRC-5 by 64% and 159%, but IL-6 did not affect the TGF-beta-induced proliferation. And TNF-alpha-induced proliferation of MRC-5 was reduced by IL-1beta in 50%. TGF-beta, TNF-alpha and both induced the proliferation of MRC-5 to 89%, 135% and 222 %, respectively. CONCLUSIONS: TNF-alpha TGF-beta and lL-1beta, in the order of the effectiveness, showed the induction of MRC-5 proliferation of MRC-5. TNF-alpha and IL-1beta enhance the TGF-beta-induced proliferation of MRC-5, but IL-6 did not have any effect. TNF-alpha-induced proliferation of MRC-5 is diminished by IL-i, and TNF-alpha and TGF-beta showed a additive effect.