Regulation of HMG-1 Release in Endotoxemia

革兰氏阴性细菌感染是危重病人普遍存在的问题。脓毒症的高死亡率部分是由细菌内毒素（LPS）介导的，细菌内毒素（LPS）激活有丝分裂原活化蛋白（MAP）激酶（如p38、ERK 1/2和JNK），刺激促炎细胞因子（如TNF和il -1 β）、一氧化氮、血小板活化因子等产物的释放。巨噬细胞来源的细胞因子与介导致死性内毒素血症有关，因为在动物模型中，抑制它们的产生或活性可以减轻组织损伤的发展。如果早期给药，抗tnf在内毒素血症的实验模型中可以成为一种有效的治疗方法，但在临床中很难实现早期治疗。另一种策略是确定临床更容易获得的“晚期”巨噬细胞介质。我们最近发现了一种普遍存在的蛋白质，HMGB1（以前称为HMG-1），作为内毒素致死的晚期介质（Science 1999, 285: 248-251）。lps刺激的巨噬细胞释放HMGB1较晚，暴露于内毒素后16 ~ 32小时，其血清水平显著升高。抗hmgb1抗体对致死性内毒素血症和lps诱导的急性肺损伤具有显著的保护作用，即使抗体给药延迟到早期TNF反应之后。纯化的重组HMGB1在巨噬细胞/单核细胞培养中诱导多种细胞因子（如TNF、il -1 β和IL-6）的释放，并在给药小鼠时促进组织损伤甚至致死。然而，调控HMG-1释放和作用的机制尚不清楚。本建议概述的研究的第一个目的是确定巨噬细胞/单核细胞培养中，早期促炎细胞因子（如TNF、il -1 β）和MAP激酶（如p38和ERK1/2）信号通路在lps诱导的HMG-1释放调控中的作用。这将通过检测TNF-或il -1 β特异性中和抗体，以及MAP激酶特异性抑制剂或反义寡核苷酸对lps诱导的HMGB1释放的影响来完成。本研究的第二个目的是研究HMGB1受体（如RAGE）和MAP激酶（如p38、ERK1/2和JNK）在巨噬细胞/单核细胞培养中调控HMGB1诱导的细胞因子产生中的作用。我们将研究HMGB1是否会激活MAP激酶，以及rage特异性中和抗体或反义寡核苷酸是否会阻止HMGB1诱导的TNF释放。这些问题的答案将揭示HMGB1释放和作用的调控机制，并提高我们对内毒素血症先天免疫反应调控机制的理解。

Gram negative bacterial infection is a widespread problem in critically ill patients. The high mortality of sepsis is in part mediated by bacterial endotoxin (LPS), which activates mitogen-activated protein (MAP) kinases (e.g., p38, ERK 1/2, and JNK), and stimulates the release of proinflammatory cytokines (e.g., TNF and IL-1beta), nitric oxide, platelet-activating factor, and other products. Macrophage-derived cytokines have been implicated in mediating lethal endotoxemia, because inhibition of their production or activity attenuates the development of tissue injury in animal models. If delivered early enough, anti-TNF can be an effective therapy in experimental models of endotoxemia, but early treatment is difficult to achieve in the clinic. An alternative strategy would be to identify "late" macrophage mediators that may be clinically more accessible. We recently identified a ubiquitous protein, HMGB1 (formerly known as HMG-1), as a late mediator of endotoxin lethality (Science 1999, 285: 248-251). HMGB1 is released late by LPS-stimulated macrophages, and its serum levels increase significantly between 16 to 32 hours after exposure to endotoxin. Anti-HMGB1 antibodies significantly protect against lethal endotoxemia and LPS-induced acute lung injury, even when antibody administration is delayed until after the early TNF response. Purified recombinant HMGB1 induced the release of multiple cytokines (e.g., TNF, IL-1beta and IL-6) in macrophage/monocyte cultures, and promoted tissue injury and even lethality when administered into mice. However, the mechanisms underlying the regulation of HMG-1 release and action are still unknown. The first aim of the studies outlined in this proposal is to determine the roles of early pro-inflammatory cytokines (e.g., TNF, IL-1beta) and MAP kinase (e.g., p38 and ERK1/2) signaling pathways in regulation of LPS-induced HMG-1 release in macrophage/monocyte cultures. This will be accomplished by examining the effect of TNF- or IL-1beta-specific neutralizing antibodies, as well as MAP kinase-specific inhibitors or anti- sense oligonucleotides on LPS-induced HMGB1 release. The second aim of this proposal is to examine the role of HMGB1 receptor (e.g., RAGE) and MAP kinases (e.g., p38, ERK1/2, and JNK) in regulation of HMGB1-induced cytokine production in macrophage/monocyte cultures. We will examine whether HMGB1 will activate MAP kinases, and whether RAGE-specific neutralizing antibodies or anti-sense oligonucleotides will prevent HMGB1- induced TNF release. Answers to these questions will shed light on the mechanisms underlying regulation of HMGB1 release and action, and improve our understanding of mechanisms underlying regulation of the innate immune response in endotoxemia.