Khadaroo Rachel G, Kapus Andras, Powers Kinga A, Cybulsky Myron I, Marshall John C, Rotstein Ori D
Department of Surgery, University of Toronto and University Health Network, Toronto, Ontario M5G 2C4, Canada.
J Biol Chem. 2003 Nov 28;278(48):47834-41. doi: 10.1074/jbc.M302660200. Epub 2003 Aug 1.
Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-kappa B translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappa B translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappa B translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-kappa B translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-kappa B translocation in oxidant-treated macrophages, this strategy had no effect on NF-kappa B translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.
缺血/再灌注损伤期间产生的氧化应激已被证明会增强细胞反应性。虽然已知氧化剂本身会诱导多种细胞内信号级联反应，但其对由其他炎症刺激引发的信号通路的影响仍未得到充分阐明。先前的研究表明，氧化剂能够使肺泡巨噬细胞在受到脂多糖（LPS）处理时，增强核因子κB（NF-κB）的转位。由于已知氧化剂会刺激Src家族酪氨酸激酶，我们推测氧化剂可能通过Src家族激酶的参与，促进LPS诱导的NF-κB转位。为了在体外模拟巨噬细胞的致敏过程，首先将小鼠巨噬细胞系RAW 264.7与各种氧化剂孵育，然后暴露于低剂量LPS。这些研究表明，氧化应激能够增强巨噬细胞对LPS的反应性，早期和增强的NF-κB转位证明了这一点。发现通过使用PP2进行药理抑制或通过用Csk进行细胞转染的分子方法抑制Src家族激酶，可防止由氧化剂引起的LPS诱导的NF-κB转位增强。有趣的是，虽然Src激酶抑制能够阻止氧化剂处理的巨噬细胞中LPS诱导的NF-κB转位，但该策略对在没有氧化剂的情况下由LPS引起的NF-κB转位没有影响。这些发现表明，氧化应激可能使LPS信号沿着另一条信号通路传导。进一步的研究表明，氧化剂预处理诱导的Src依赖性途径涉及磷脂酰肌醇3激酶的激活。该途径的参与似乎独立于传统的LPS信号传导。总之，这些研究提供了一种新的潜在机制，通过该机制氧化剂可能使肺泡巨噬细胞对随后的炎症刺激反应性改变，并提示缺血/再灌注后免疫调节的不同细胞靶点。
