Regulation of HMG-1 Release in Endotoxemia

革兰氏阴性菌感染是危重患者中普遍存在的问题。 脓毒症的高死亡率部分由细菌内毒素（LPS）介导，其激活促分裂原活化蛋白（MAP）激酶（例如，p38、ERK 1/2和JNK），并刺激促炎细胞因子（例如，TNF和IL-1 β）、一氧化氮、血小板活化因子和其他产品。 巨噬细胞衍生的细胞因子与介导致死性内毒素血症有关，因为抑制它们的产生或活性可减弱动物模型中组织损伤的发展。 如果给予足够早，抗TNF可以是内毒素血症实验模型的有效治疗，但在临床上难以实现早期治疗。 另一种策略是确定“晚期”巨噬细胞介质，可能在临床上更容易获得。 我们最近鉴定了一种普遍存在的蛋白质，HMGB 1（以前称为HMG-1），作为内毒素致死性的晚期介质（Science 1999，285：248-251）。 HMGB 1被LPS刺激的巨噬细胞延迟释放，并且其血清水平在暴露于内毒素后16至32小时之间显著增加。 抗-HMGB 1抗体显著地保护免于致死性内毒素血症和LPS诱导的急性肺损伤，即使当抗体施用被延迟到早期TNF应答之后。 纯化的重组HMGB 1诱导多种细胞因子（例如，TNF、IL-1 β和IL-6），并促进组织损伤，甚至在给药小鼠时致死。 然而，HMG-1的释放和作用的调节机制仍然未知。 本提案中概述的研究的第一个目的是确定早期促炎细胞因子的作用（例如，TNF，IL-1 β）和MAP激酶（例如，p38和ERK 1/2）信号通路在调节LPS诱导的巨噬细胞/单核细胞培养物中HMG-1释放中的作用。 这将通过检查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HMGB 1释放的影响来实现。 该提议的第二个目的是检查HMGB 1受体的作用（例如，激酶）和MAP激酶（例如，p38、ERK 1/2和JNK）调节巨噬细胞/单核细胞培养物中HMGB 1诱导的细胞因子产生。 我们将研究HMGB 1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HMGB 1诱导的TNF释放。 这些问题的答案将有助于阐明HMGB 1释放和作用的潜在调节机制，并提高我们对内毒素血症中先天免疫应答调节机制的理解。

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.