The role of Src & ERK1/2 kinases in inspiratory resistive breathing induced acute lung injury and inflammation.

BACKGROUND: Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury. METHODS: Anaesthetized, tracheostomized adult rats breathed spontaneously through a 2-way non-rebreathing valve. Resistance was added to the inspiratory port to provide a peak tidal inspiratory pressure of 50% of maximum (inspiratory resistive breathing). Activation of Src and ERK1/2 in the lung was estimated during IRB. Following 6 h of IRB, respiratory system mechanics were measured by the forced oscillation technique and bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. IL-1b and MIP-2a protein levels were measured in lung tissue samples. Wet lung weight to total body weight was measured and Evans blue dye extravasation was estimated to measure lung permeability. Lung injury was evaluated by histology. The Src inhibitor, PP-2 or the inhibitor of ERK1/2 activation, PD98059 was administrated 30 min prior to IRB. RESULTS: Src kinase was activated 30 min after the initiation of IRB. Src inhibition ameliorated the increase in BAL cellularity after 6 h IRB, but not the increase of IL-1ß and MIP-2a in the lung. The increase in BAL total protein and lung injury score were not affected. The increase in tissue elasticity was partly inhibited. Src inhibition blocked ERK1/2 activation at 3 but not at 6 h of IRB. ERK1/2 inhibition ameliorated the increase in BAL cellularity after 6 h of IRB, blocked the increase of IL-1ß and returned Evans blue extravasation and wet lung weight to control values. BAL total protein and the increase in elasticity were partially affected. ERK1/2 inhibition did not significantly change total lung injury score compared to 6 h IRB. CONCLUSIONS: Src and ERK1/2 are activated in the lung following IRB and participate in IRB-induced lung injury.

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.

Inspiratory resistive breathing induces acute lung injury.

RATIONALE: Resistive breathing is associated with large negative intrathoracic pressures. Increased mechanical stress induces high-permeability pulmonary edema and lung inflammation. OBJECTIVES: To determine the effects of resistive breathing on the healthy lung. METHODS: Anesthetized rats breathed through a two-way nonrebreathing valve. The inspiratory line was connected to a resistance setting peak inspiratory tracheal pressure at 50% of maximum (inspiratory resistive breathing), while 100% oxygen was supplied to prevent hypoxemia. Quietly breathing animals (100% oxygen) served as controls. Lung injury was evaluated after 3 and 6 hours of resistive breathing. MEASUREMENTS AND MAIN RESULTS: After both 3 and 6 hours of resistive breathing, lung permeability was increased, as assessed by (99m)Tc-diethylenetriaminepentaacetic acid scintigraphy and Evans blue dye extravasation. Tissue elasticity, measured on the basis of static pressure-volume curves and by the low-frequency forced oscillation technique, was also increased. After both 3 and 6 hours of resistive breathing, gravimetric measurements revealed the presence of pulmonary edema and analysis of bronchoalveolar lavage showed increased total protein content, whereas the total cell count was elevated only after 6 hours of resistive breathing. Cytokine levels were assessed in bronchoalveolar lavage fluid and lung tissue by ELISA and were increased after 6 hours compared with controls. Western blot analysis showed early activation of Src kinase via phosphorylation (at 30 min), and Erk1/2 and IκBα (nuclear factor-κB inhibitor) were phosphorylated at 3 and 6 hours. Pathology revealed the presence of lung injury after resistive breathing. CONCLUSIONS: Resistive breathing induces acute lung injury and inflammation.

The complement inhibitor CD59 and the lymphocyte function-associated antigen-3 (LFA-3, CD58) genes possess functional binding sites for the p53 tumor suppressor protein.

p53 is an oncosuppressor protein, which acts via transcriptional and non-transcriptional mechanisms. The transcriptional function of p53 is mediated by specific responsive elements. In the present study we found active responsive elements, specific for the p53 within the 5'flanking region and within the first intron of the gene encoding for the CD59 membrane inhibitor of reactive lysis, and within the first intron of the gene encoding for the CD58 membrane protein (LFA-3). The results suggest that p53 may enhance the transcription of both CD59 and CD58 and imply a novel role for p53 as a direct regulator of the immune response.

Nitric oxide stimulates interleukin-6 production in skeletal myotubes.

Strenuous exercise leads to the up-regulation of interleukin-6 (IL-6) production and enhanced nitric oxide (NO) release within the contracting skeletal muscles. In this study, we investigated whether NO regulates IL-6 production in C2C12 myotubes. These cells exhibited a concentration-dependent increase in IL-6 production upon stimulation with NO donors (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate), (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NONOate), and sodium nitroprusside (SNP). This treatment did not alter cGMP levels nor did the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one(ODQ), alter this response. The NO-independent sGC activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY41-2272) and cyclic guanosine monophosphate (cGMP) analog 8Br-cGMP failed to induce IL-6 production. Upon exposure to NO donors, we observed an increase in Erk1/2 and p38 MAPK phosphorylation but not in SAPK/JNK. In addition, NO-induced IL-6 release was inhibited in a concentration-dependent fashion by the MEK1/2 inhibitor PD98059 and the p38 MAPK inhibitor SB203580 but not by the SAPK/JNK inhibitor SP600125. We conclude that NO-stimulated IL-6 production in differentiated C2C12 myotubes is cGMP-independent and mediated by activation of MAPK pathways.