Song Grace Y, Chung Chun-Shiang, Jarrar Doraid, Cioffi William G, Ayala Alfred
Division of Surgical Research, Department of Surgery, Brown University School of Medicine, and Rhode Island Hospital, Providence, Rhode Island 02903, USA.
J Trauma. 2002 Aug;53(2):276-82; discussion 282-3. doi: 10.1097/00005373-200208000-00015.
After the onset of sepsis, there is a marked dysfunction in cell-mediated immunity that contributes to the morbidity and mortality seen in this condition. Although both nitric oxide (NO) from inducible NO synthase (iNOS) and the activation of p38 mitogen-activated protein kinase (p38 MAPK) appear to contribute to this immune dysfunction, the extent to which NO regulates p38 MAPK activity in sepsis remains unknown.
To examine this, we induced sepsis by cecal ligation and puncture (CLP) in iNOS knockout (iNOS -/-) or C57BL/6 control mice. Twenty-four hours after CLP or sham operation, splenic T cells and macrophages were isolated and then stimulated with monoclonal antibody against the T-cell marker CD3 (anti-CD3) or lipopolysaccharide. At 4 or 24 hours after stimulation, cytokine release was determined by enzyme-linked immunosorbent assay, and p38 MAPK phosphorylation (activation) was determined by immunoblotting with antibody specific to phosphorylated p38 MAPK.
Splenic T-cell p38 MAPK activation and interleukin (IL)-10 release was increased by CLP, whereas Th1 cytokine (IL-2, interferon-gamma) release was depressed. iNOS gene deficiency inhibited p38 MAPK activation in splenic T cells taken from septic mice, and also suppressed IL-10 release in both sham and septic mice. Interestingly, although deficiency of iNOS restored IL-2 release after CLP, both sham and CLP T cells remained depressed in their ability to release interferon-gamma. Septic insult markedly suppressed C57BL/6 splenic macrophage release of proinflammatory agents tumor necrosis factor, IL-12, and IL-1, while augmenting the release of IL-10. However, although deficiency of iNOS concomitantly restored the ability to produce tumor necrosis factor while suppressing the rise in IL-10 release and p38 MAPK activation, it only partially restored IL-1 release and had no effect on IL-12 production seen after CLP.
These data suggest that NO release from iNOS regulates aspects of sepsis-induced immune dysfunction by the activation of p38 MAPK.
脓毒症发作后，细胞介导的免疫功能出现明显障碍，这是导致该病症发病和死亡的原因之一。尽管诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）以及p38丝裂原活化蛋白激酶（p38 MAPK）的激活似乎都与这种免疫功能障碍有关，但在脓毒症中NO调节p38 MAPK活性的程度仍不清楚。
为了研究这一问题，我们通过盲肠结扎和穿刺（CLP）在iNOS基因敲除（iNOS -/-）小鼠或C57BL/6对照小鼠中诱导脓毒症。CLP或假手术后24小时，分离脾T细胞和巨噬细胞，然后用抗T细胞标志物CD3的单克隆抗体（抗CD3）或脂多糖刺激。刺激后4或24小时，通过酶联免疫吸附测定法测定细胞因子释放，并使用针对磷酸化p38 MAPK的特异性抗体通过免疫印迹法测定p38 MAPK磷酸化（激活）。
CLP增加了脾T细胞p38 MAPK激活和白细胞介素（IL）-10释放，而Th1细胞因子（IL-2、干扰素-γ）释放受到抑制。iNOS基因缺陷抑制了脓毒症小鼠脾T细胞中的p38 MAPK激活，并且也抑制了假手术和脓毒症小鼠中的IL-10释放。有趣的是，尽管iNOS缺陷在CLP后恢复了IL-2释放，但假手术和CLP T细胞释放干扰素-γ的能力仍然受到抑制。脓毒症损伤显著抑制了C57BL/6脾巨噬细胞促炎因子肿瘤坏死因子、IL-12和IL-1的释放，同时增加了IL-10的释放。然而，尽管iNOS缺陷在抑制IL-10释放增加和p38 MAPK激活的同时恢复了产生肿瘤坏死因子的能力，但它仅部分恢复了IL-1释放，并且对CLP后观察到的IL-12产生没有影响。
这些数据表明，iNOS释放的NO通过激活p38 MAPK调节脓毒症诱导的免疫功能障碍的各个方面。
