Responses of mouse macrophages to oxidative stress

Tissue injury induced by infections or xenobiotics is associated with oxidative stress and inflammation, which are thought to contribute to pathogenic response. We hypothesized that toll-like receptor 4 (TLR4) is important in macrophage responsiveness to oxidative stress. To test this, we compared the response of TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice to ozone and to bacterially-derived lipopolysaccharide (LPS). Exposure of C3H/HeOuJ mice to ozone (0.8 ppm for 3 h) resulted in increases in bronchoalveolar lavage lipocalin 24p3, 4-hydroxynonenal, surfactant protein-D, macrophage and protein content. Increased nuclear binding activity of NF-B and expression of TNF mRNA was also noted in lung macrophages. Findings that these responses to ozone were reduced in C3H/HeJ mice demonstrate that functional TLR4 contributes to ozone-induced injury, inflammation, and oxidative stress. We next determined if lung and liver macrophage responses to LPS are also mediated by TLR4. Treatment of control C3H/HeOuJ mice with LPS (3 mg/ml) resulted in increased numbers of macrophages in liver and lung after 48 h. In liver, but not lung macrophages, a rapid increase in mRNA expression of MnSOD and HO-1, as well as COX-2 and microsomal prostaglandin E synthase-1 was also observed. Conversely, macrophage COX-2 protein expression increased in both macrophage populations. The effects of LPS were significantly reduced in C3H/HeJ mice indicating TLR4 is also involved in LPS-induced oxidative stress, inflammation and macrophages activation in the liver and lung. To investigate mechanisms regulating macrophage responses, we evaluated the effects of hypoxia-induced oxidative stress on LPS-induced activation of macrophages using RAW 264.7 murine macrophages. Hypoxia augmented the effects of LPS on iNOS, COX-2, IL-1, GLUT-1 and VEGF-A mRNA expression. Hypoxia also upregulated LPS-induced protein expression of iNOS and COX-2, as well as MnSOD, lipocalin 24p3, and MMP-9. Some of these responses were dependent on p44/42 mitogen activated protein kinase signaling. Taken together, these studies demonstrate a key role of TLR4 in both sterile and infection driven inflammatory responses. These findings may be important in the development of effective therapeutics for treating diseases associated with prominent macrophages inflammatory responses.Ph. D.Includes bibliographical referencesIncludes vitaby Agnieszka Jankowska Conno