Fumitaka Ogushi

Chymase is a potent chemoattractant for human monocytes and neutrophils.

Chymase is a major chymotrypsin‐like serine protease expressed in the secretory granules of mast cells in many mammalian species. In this study, we revealed the chemotactic activity of chymase for human mononuclear cells and neutrophils with a 48‐well microchemotaxis chamber technique. Human chymase showed the potent chemotactic activity for monocytes and neutrophils dose‐dependently in a concentration range from 0.1 to 10 μg/mL, corresponding to about 4–400 μM. The activity was as potent as that of N‐formyl‐methionyl‐leucyl‐phenylalanine. Chymase also stimulated cell migration of lymphocytes and purified T cells, but checkerboard analysis revealed that the effect was chemokinetic rather than chemotactic. Inhibition of chymase activities with chymase inhibitors, such as antileukoprotease and Bowman‐Birk soybean trypsin inhibitor, significantly inhibited the chemotactic activity of chymase, suggesting that the proteolytic activity of chymase participates in the chemotactic activity. Our results suggest that mast cell chymase acts as a chemoattractant, and may play a role in the accumulation of inflammatory cells in development of the chronic inflammatory responses of allergic and nonallergic diseases. J. Leukoc. Biol. 67: 585–589; 2000.

Decreased prostaglandin E2 synthesis by lung fibroblasts isolated from rats with bleomycin-induced lung fibrosis.

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)‐1α, 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.

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.

Comparison of suppressive effects of a new anti-inflammatory compound, FR167653, on production of PGE2 and inflammatory cytokines, human monocytes, and alveolar macrophages in response to endotoxin.

FR167653 {1‐[7‐(4‐fluorophenyl)‐1, 2, 3, 4‐tetrahydro‐8 (4‐pyridyl) pyrazoro [5‐1‐c] [1, 2, 4] triazin‐2‐yl]‐2‐phenylethanedion sulfate monohydrate} was developed to inhibit proinflammatory cytokine production. However, the effects of FR167653 on prostanoid production are unclear. We investigated the effect of FR167653 on proinflammatory cytokine and prostaglandin (PG) production by lipopolysaccharide (LPS)‐stimulated human peripheral blood monocytes and alveolar macrophages (AM) from the same individuals, and compared the effects in monocytes and AM. FR167653 inhibited interleukin‐1β and tumor necrosis factor a production. The effect on PGE2 production was assessed by four parameters. FR167653 inhibited PGE2 synthesis and LPS induction of cyclooxygenase activity. Western and Northern blot analyses revealed that LPS induction of cyclooxygenase‐2 expression was attenuated by this compound. The suppression in monocytes was greater than that in AM. We concluded that the reduction of LPS‐induced PGE2 synthesis by FR167653 was due to inhibition of cyclooxygenase‐2 production. These results show that FR167653 may be therapeutically useful for inhibiting production of both inflammatory cytokines and PG production in inflammatory diseases. J. Leukoc. Biol. 65: 80–86; 1999.

Increased binding and chemotactic capacities of PDGF-BB on fibroblasts in radiation pneumonitis.

Abstract Tada, H., Ogushi, F., Tani, K., Nishioka, Y., Miyata, J., Sato, K., Asano, T. and Sone, S. Increased Binding and Chemotactic Capacities of PDGF-BB on Fibroblasts in Radiation Pneumonitis. Radiat. Res. 159, 805–811 (2003). Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of 125I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that 125I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.

Thrombin promotes fibroblast proliferation during the early stages of experimental radiation pneumonitis

Abstract Huang, L., Ogushi, F., Tani, K., Ogawa, H., Kawano, T., Endo, T., Izumi, K., Ueno, J., Nishitani, H. and Sone, S. Thrombin Promotes Fibroblast Proliferation during the Early Stages of Experimental Radiation Pneumonitis. Radiat. Res. 156, 45–52 (2001). To clarify the role of thrombin in the pathogenesis of radiation-induced pneumonitis, we measured the thrombin activity and fibroblast growth-inducing activity in bronchoalveolar lavage fluid obtained from the irradiated lungs of rats at 1, 2, 4, 8 and 18 weeks after irradiation. Thrombin activity was not detected in the bronchoalveolar lavage fluid from unirradiated rats, but the bronchoalveolar lavage fluid from irradiated rats showed significantly increased thrombin activity which reached a maximum at 4 weeks after treatment. Higher fibroblast growth-inducing activity was detected in the bronchoalveolar lavage fluid from irradiated rats at 4 and 18 weeks than in fluid from unirradiated rats. Bronchoalveolar lavage fluid from irradiated rats that were pretreated with the thrombin inhibitors antithrombin III and argatroban showed significantly inhibited fibroblast growth-inducing activity and thrombin activity at 4 weeks. However, these thrombin inhibitors did not inhibit fibroblast growth-inducing activity in bronchoalveolar lavage fluid from irradiated rats at 18 weeks. Purified rat thrombin similarly induced proliferation of fibroblasts derived from irradiated and unirradiated rats. These findings suggest that thrombin may play an important role as a fibroblast growth-inducing factor during the early stages of radiation pneumonitis.

TOLUENE DIISOCYANATE (TDI) INDUCES PRODUCTION OF INFLAMMATORY CYTOKINES AND CHEMOKINES BY BRONCHIAL EPITHELIAL CELLS VIA THE EPIDERMAL GROWTH FACTOR RECEPTOR AND p38 MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS

Toluene diisocyanate (TDI) is known as one of causes of occupational asthma and hypersensitivity pneumonitis. To investigate the stimulatory effect on bronchial epithelial cells in response to TDI, the authors examined production of cytokines by the bronchial epithelial cell line BEAS-2B and intercellular signal transduction stimulated by TDI–human serum albumin (HSA) conjugate. The production of interleukin (IL)-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation normal T cell expressed and secreted (RANTES) from the bronchial epithelial cells were augmented by the TDI-HSA conjugate. Extracellular signal-regulated kinase (Erk) 1/2 and p38 mitogen-activated protein kinase (MAPK) were phosphorylated by the TDI-HSA conjugate. AG1478, SB203580, and dexamethasone prevented augmentation of these cytokine production. TDI-HSA conjugate did not augment release of epidermal growth factor (EGF) ligands from BEAS-2B. These results suggest that TDI directly induces production of proinflammatory cytokines and chemokines through p38 MAPK and EGF receptor (EGFR)-Erk pathway without an autocrine mechanism. Thus, TDI was shown to have a stimulatory effect on bronchial epithelial cells, suggesting the potent role of bronchial epithelial cells in TDI-induced asthma.

Asymptomatic primary tuberculous pleurisy with intense 18-fluorodeoxyglucose uptake mimicking malignant mesothelioma

BackgroundThe pathogenesis of primary tuberculous pleurisy is a delayed-type hypersensitivity immunogenic reaction to a few mycobacterial antigens entering the pleural space rather than direct tissue destruction by mycobacterial proliferation. Although it has been shown that pulmonary tuberculosis induces 18-fluorodeoxyglucose (FDG) uptake in active lesions, little is known about the application of FDG positron emission/computed tomography (FDG PET/CT) to the management of primary tuberculous pleurisy.Case presentationWe report a case of asymptomatic primary tuberculous pleurisy presenting with diffuse nodular pleural thickening without distinct pleural effusion and parenchymal lung lesions mimicking malignant mesothelioma. An initial FDG PET/CT scan demonstrated multiple lesions of intense FDG uptake in the right pleura and thoracoscopic biopsy of pleural tissue revealed caseous granulomatous inflammation. The patient received antituberculous therapy for 6 months, with clearly decreased positive signals on a repeated FDG PET/CT scan.ConclusionFDG PET/CT imaging may be useful for evaluating disease activity in tuberculous pleurisy patients with an unknown time of onset.

Increased macrophage inflammatory protein-1alpha and -1beta in BAL fluid of bronchiolitis obliterans organizing pneumonia.

Objective:  CC chemokines are mainly chemotactic for monocytes and lymphocytes. The aim of this study was to evaluate the involvement of the CC chemokines, macrophage inflammatory protein (MIP)‐1α and MIP‐1β, in the pathogenesis of bronchiolitis obliterans organizing pneumonia (BOOP).

Increased incidence of autoantibodies to interleukin-1α in rheumatoid arthritis with interstitial lung disease

To clarify the clinical significance of autoantibodies (auto‐Ab) to interleukin‐1α (IL‐1α) in rheumatoid arthritis (RA) with interstitial lung disease (ILD), we examined the IL‐1α auto‐Ab level in serum of patients with RA with/without ILD.