Endotoxin/lipopolysaccharide (LPS) is the major mediator that triggers the cellular and humoral responses of the shock induced by Gram-negative bacteria. The toxic responses of LPS are mediated by various factors and mainly by tumor necrosis factor a (TNFa). To study the role of TNF and to identify anti-TNF molecules in endotoxic/septic shock, numerous animal models have been utilized. The models described here are among the most widely used and are represented by LPS given at high dose, or at low dose in D-galactosamine-sensitized mice. The endpoints of these models are the survival, the organ toxicity, or the regulation of cytokines, and in particular of TNFa. An additional and more complex model of endotoxic/septic shock, the polymicrobial model of cecal ligation and puncture, where a synergistic interaction of several mediators occurs, is then described.
Key Words: TNF; LPS; endotoxic shock; polymicrobial sepsis; CLP. 1. Introduction
Activation of the immune system by Gram-negative bacteria, their products, or both can result in a cascade of events leading to endotoxic shock, a situation in which large amounts of cytokines, such as tumor necrosis factor a (TNFa), interleukin (IL)-1 and IL-6, as well as other inflammatory mediators, are produced. If unaltered, these events frequently lead to death. Endotoxin/ lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, was identified many years ago as a major mediator that triggers the massive cellular and humoral responses observed in shock induced by Gram-negative bacteria (1). The toxic effects of LPS are mediated by factors produced by host cells, mainly TNFa, as shown by the protective effects of TNF antibodies (2). There are two other points in favor of a role of TNFa in the pathogenesis of endotoxic shock: (1) endotoxin induces the production of TNFa, in vivo and in vitro, at an early time (3-5); (2) administration of TNFa to laboratory animals reproduces almost all the effects of LPS and in particular fever, hypertrigliceridemia, hypotension, and death (4). Furthermore, increased serum TNFa levels were measured in human volunteers treated with low doses of LPS, thus suggesting the relevance to man of the studies in animal models (6). TNFa has also been shown to be a mediator responsible for the initiation of the lethal toxicity of LPS in animals treated with a low dose of LPS and d-galactosamine, a liver-specific transcription inhibitor that increases the sensitivity to LPS itself (7,8). Unlike the response to high-dose LPS alone, death in this model is a direct result of hepatocyte apoptosis (9), which causes fulminant hepatitis, with TNFa and p55 TNF-receptor signaling playing an essential role.
Neutralizing monoclonal anti-TNF antibodies provided protection against shock during lethal bacteremia induced by Escherichia coli infusion in baboons, indicating that TNFa is also a mediator of fatal bacteremic shock (10). However, in other animal models of sepsis, such as the polymicrobial model of cecal ligation and puncture (CLP) or the intraperitoneal Gramnegative bacterial challenge, where the elevation of TNFa is only modest, inhibition of only TNFa failed to improve survival and even lessened it, suggesting that in these more complex models of sepsis a TNF requirement by the local defense mechanisms occurs (11-14). More recently it has been shown that a combination immunotherapy with soluble TNF receptors plus IL-1 receptor antagonist decreases sepsis mortality when provided for a sufficient amount of time (15). Moreover, drugs that inhibit the synthesis of multiple pro-inflammatory mediators, including TNFa, IL-6 and nitric oxide, were able to protect against death during polymicrobial sepsis, suggesting that the pathogenesis of this lethal syndrome is dependent upon the synergistic interaction of several mediators (16-18).
The models described here are among those most used to study anti-TNF molecules and TNF or TNFR knockout mice in sepsis, and include: (1) LPS at high dose, (2) LPS at low dose in d-galactosamine sensititized animals, and (3) polymicrobial sepsis induced by CLP. The endpoints of these models are either the survival or the immune regulation of cytokines and in particular of TNFa.
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