Bradykinin (BK) is a major kinin with well-documented pharmacological properties including vascular leakage and induction of a variety of cytokines. However, the intracellular signalling mechanisms by which BK induced proinflammatory cytokine production have not been fully elucidated. This study investigated the role of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 mitogen-activated protein kinase (p38 MAPK) in the BK-induced interleukin (IL)-6 and IL-8 production by human lung fibroblasts.Lung fibroblasts were stimulated with BK in the presence or in the absence of PD98059, a specific MAPK/ERK kinase-1 inhibitor, or SB203580, a specific p38 MAPK inhibitor, and IL-6 or IL-8 production and their gene expression was examined. BK-induced ERK 1/2 or p38 MAPK phosphorylation was also analysed by Western blot analysis.BK at nanomolar concentrations stimulated lung fibroblasts to produce IL-6 and IL-8 along with increased ERK 1/2 and p38 MAPK phosphorylation. BK-induced IL-6 and IL-8 synthesis was inhibited by a B2-type BK receptor antagonist. Furthermore, PD98059 or SB203580 significantly suppressed BK-induced IL-6 and IL-8 production and their gene expression.These results indicate that bradykinin-induced interleukin-6 and interleukin-8 production are at least partly mediated through the extracellular signal-related protein kinase 1/2 and p38 mitogen-activated protein kinase pathway-dependent activation in human lung fibroblasts, and suggest that bradykinin appears to be involved in the inflammatory reaction leading to acute lung injury through stimulating interleukin-6 and interleukin-8 production by lung fibroblasts. Eur Respir J 2000; 16: 452±458.

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.

Fas mediates apoptosis following binding with Fas ligand. Fas is expressed in human airway epithelial cells and has a critical role in the pathophysiology of various pulmonary disorders. Hydrogen peroxide (H(2)O(2)) is an important mediator of airway epithelial injury. In this context, we hypothesized that H(2)O(2) would increase the expression of cell surface Fas in human airway epithelial cells. To test this hypothesis, the modulation of Fas expression with H(2)O(2) was assessed in normal human bronchial epithelial cells and A549 cells. The majority of Fas was cytoplasmic in both cell types without any stimulation. Hydrogen peroxide significantly increased Fas in the plasma membrane fraction, while decreasing Fas in the cytoplasmic fraction. Incubation with an agonistic antibody for Fas induced apoptosis in H(2)O(2)-treated cells in proportion to the level of surface Fas expression on those cells. Inhibitors of poly(ADP-ribose) polymerase abrogated the H(2)O(2)-induced Fas translocation to the plasma membrane and p53 activation. Expression of dominant-negative p53 also inhibited the Fas translocation induced by H(2)O(2) in A549 cells. These results indicate that H(2)O(2) induces Fas upregulation by promoting cytoplasmic transport of Fas to the cell surface in human airway epithelial cells, and that the activation of the poly(ADP-ribose) polymerase-p53 pathway may be involved in this mechanism.

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl2) and examined their apoptosis by DNA fragmentation assay, 4′,6-diamidino-2′-phenylindol dihydrochloride staining, and Western blot analysis. CoCl2 induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl2 inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl2 released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.

Reactive oxygen intermediates (ROIs) play an important role in the initiation and progression of lung diseases. In this study, we investigated whether ROIs were involved in the induction of interleukin (IL)-6 in human bronchial epithelial cells. We exposed normal human bronchial epithelial cells as well as a human bronchial epithelial cell line, HS-24, to ROIs. We measured the amount of IL-6 in the culture supernatants using ELISA and the IL-6 mRNA levels using RT-PCR. Superoxide anions ([Formula: see text]), but not hydrogen peroxide (H2O2), increased IL-6 production. To examine whether it is a cell type-specific mechanism of airway epithelial cells, the experiments were also performed in human lung fibroblasts, WI-38-40. In WI-38-40 cells, neither [Formula: see text] nor H2O2increased IL-6 production. In contrast, tumor necrosis factor (TNF)-α (200 U/ml) induced IL-6 at the protein and mRNA levels in both airway epithelial cells and lung fibroblasts. This cytokine-induced IL-6 production was significantly suppressed by several antioxidants, including dimethyl sulfoxide (DMSO), in airway epithelial cells. In WI-38-40 cells, DMSO was not able to suppress IL-6 production induced by TNF-α. Pretreatment with DMSO recovered the TNF-α-induced depletion of intracellular reduced glutathione in HS-24 cells. These findings indicate that oxidant stress specifically induces IL-6 production in human bronchial epithelial cells and that in these cells ROIs may be involved in IL-6 production after stimulation with cytokines such as TNF-α. Presumably, ROIs participate in the local immune response in lung diseases via IL-6 release from bronchial epithelial cells.