Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling.
OBJECTIVE
To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK).


METHODS
Prospective, experimental study.


METHODS
Research laboratory at a university hospital.


METHODS
Thermal injury models in the mice.


METHODS
In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye.


RESULTS
The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues.


CONCLUSIONS
Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.
Chen
Lee-Wei
LW
Department of Surgery, Kaohsiung Veterans General Hospital, National Yang-Ming Medical University, Taipei, Taiwan.
Chang
Wei-Jung
WJ
Wang
Jyh-Seng
JS
Hsu
Ching-Mei
CM
D000818:Animals; D001483:Base Sequence; D001707:Biopsy, Needle; D015153:Blotting, Western; D002056:Burns / Q000201:enzymology* / Q000503:physiopathology; D004195:Disease Models, Animal; D007150:Immunohistochemistry; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000378:metabolism; D008168:Lung / Q000201:enzymology* / Q000473:pathology*; D048670:MAP Kinase Kinase 4 / Q000032:analysis / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D020556:Neutrophil Infiltration; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D009195:Peroxidase / Q000032:analysis / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000032:analysis / Q000378:metabolism*; D011336:Probability; D012333:RNA, Messenger / Q000032:analysis; D011897:Random Allocation; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis
D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D030421:Peroxynitrous Acid; D009195:Peroxidase; D019001:Nitric Oxide Synthase; D048670:MAP Kinase Kinase 4