Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung.

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1ß, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1ß and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

MAPKs and NF-κB differentially regulate cytokine expression in the diaphragm in response to resistive breathing: the role of oxidative stress.

Inspiratory resistive breathing (IRB) induces cytokine expression in the diaphragm. The mechanism of this cytokine induction remains elusive. The roles of MAPKs and NF-κB and the impact of oxidative stress in IRB-induced cytokine upregulation in the diaphragm were studied. Wistar rats were subjected to IRB (50% of maximal inspiratory pressure) via a two-way nonrebreathing valve for 1, 3, or 6 h. Additional groups of rats subjected to IRB for 6 h were randomly assigned to receive either solvent or N-acetyl-cysteine (NAC) or inhibitors of NF-κB (BAY-11-7082), ERK1/2 (PD98059), and P38 MAPK (SB203580) to study the effect of oxidative stress, NF-κB, and MAPKs in IRB-induced cytokine upregulation in the diaphragm. Quietly breathing animals served as controls. IRB upregulated cytokine (IL-6, TNF-α, IL-10, IL-2, IL-1ß) protein levels in the diaphragm and resulted in increased activation of MAPKs (P38, ERK1/2) and NF-κB. Inhibition of NF-κB and ERK1/2 blunted the upregulation of all cytokines except that of IL-6, which was further increased. P38 inhibition attenuated all cytokine (including IL-6) upregulation. Both P38 and ERK1/2 inhibition decreased NF-κB/p65 subunit phosphorylation. NAC pretreatment blunted IRB-induced cytokine upregulation in the diaphragm and resulted in decreased ERK1/2, P38, and NF-κB/p65 phosphorylation. In conclusion, IRB-induced cytokine upregulation in the diaphragm is under the regulatory control of MAPKs and NF-κB. IL-6 is regulated differently from all other cytokines through a P38-dependent and NF-κB independent pathway. Oxidative stress is a stimulus for IRB-induced cytokine upregulation in the diaphragm.

Clinical significance of tumor-associated antigen RCAS1 expression in human pancreatic ductal adenocarcinoma.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a human tumor-associated antigen that induces cell cycle arrest and/or apoptosis in RCAS1 receptor-expressing immune cells. The aim of the present study was to evaluate the clinical significance of RCAS1 expression in human pancreatic adenocarcinoma. Immunohistochemical analysis of RCAS1 expression was performed on paraffin-embedded tissue sections obtained from 76 pancreatic adenocarcinoma patients. RCAS1 positivity and overexpression and intensity of the staining were correlated with clinicopathological parameters, proliferative capacity and patient survival. Of the 76 adenocarcinoma patients, 65 (86%) tested positive for RCAS1; of these 65 RCAS1-positive cases, 36 (55%) showed RCAS1 overexpression. RCAS1 positivity was statistically significantly correlated with the histopathological grade of the tumor (P = 0.026), and it showed a trend to be correlated with tumor size (P = 0.071). RCAS1 intensity and overexpression of staining showed a trend to be correlated with the histopathological grade of the tumor (P = 0.061 and P = 0.089, respectively), whereas RCAS1 positivity and the overexpression and intensity of staining were not statistically significantly correlated with the proliferative capacity of the tumor or any other clinicopathological parameter examined nor with patients' survival. Our data provide evidence for the implication of RCAS1 in pancreatic neoplasia. However, the prediction of survival using RCAS1 expression as a marker seems uncertain for this type of cancer.