Nuclear Translocation of NF-κB in Lipopolysaccharide-Treated Macrophages Fails To Correspond to Endotoxicity: Evidence Suggesting a Requirement for a Gamma Interferon-Like Signal

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-κB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-κB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, using d-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations from Escherichia, Salmonella, Klebsiella, Bacteroides, Pseudomonas, Neisseria, and Rhodobacter species as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by >160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-α) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice, Rhodobacter capsulatus LPS stimulated RAW cell NF-κB-like DNA binding protein mobilization and TNF-α production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-κB activation nor TNF-α production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability of R. capsulatus LPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-γ) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-γ-like signal that accounts for enhanced toxicity. In sum, the activation of NF-κB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-κB alone as a signal transduction factor necessary for LPS-mediated toxicity.

阐明脂多糖（lipopolysaccharide, LPS）诱导的巨噬细胞激活所必需的信号转导通路，可为败血症休克的治疗提供全新靶点。有鉴于此，学界普遍认为转录因子NF-κB的激活对于LPS刺激下巨噬细胞炎症介质的生成至关重要，但部分免疫学、遗传学与分子生物学证据表明，还存在其他参与调控的因素。为解答这一问题，我们提出假说：LPS诱导的NF-κB活化程度，应与体外实验中所用LPS亚型的小鼠内毒素毒性水平相关。为此，我们采用d-半乳糖胺致敏小鼠，检测了来自埃希菌属、沙门菌属、克雷伯菌属、拟杆菌属、假单胞菌属、奈瑟菌属以及红细菌属的8种LPS制剂，以及内毒素亚结构脂质X（lipid X）的致死效价。这些LPS制剂的致死效价差异超过160倍。用上述相同LPS制剂处理RAW 264.7细胞后，所诱导的肿瘤坏死因子α（tumor necrosis factor alpha, TNF-α）与一氧化氮（nitric oxide, NO）生成水平，与对应LPS的半数致死剂量呈显著相关。对RAW细胞中这两种介质的响应水平进行联合分析，可有效预测小鼠的内毒素致死性。值得注意的是，荚膜红细菌（Rhodobacter capsulatus）来源的LPS虽在小鼠体内毒性较弱，但其刺激RAW细胞产生的NF-κB样DNA结合蛋白活化程度与TNF-α生成水平，可与毒性更强的LPS亚型相当，却无法诱导RAW细胞生成NO。这些数据表明，仅依靠NF-κB激活或TNF-α生成，均无法可靠预测LPS的致死性。此外，用强效炎症介质二磷酸腺苷（adenosine diphosphate, ADP）与RAW细胞共处理，对荚膜红细菌LPS诱导NO生成的能力无影响，但可显著增强毒性LPS亚型对NO生成的诱导作用。与之相反，用γ干扰素（gamma interferon, IFN-γ）与RAW细胞或腹膜巨噬细胞共处理，可使毒性与非毒性LPS制剂诱导NO生成的能力均恢复正常，这提示部分LPS制剂可优先产生类似IFN-γ的信号，从而增强其毒性。总而言之，NF-κB的激活与LPS的致死性并不对应，这使得仅将NF-κB作为LPS介导毒性所必需的信号转导因子的巨噬细胞激活模型变得更为复杂。