Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ∼3 nM, which is ∼10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37°C to 32°C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32°C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.

Radiation induces apoptosis of crypt intestinal epithelial cells (IEC) through a pathway that is largely dependent on p53. However, exactly how p53 mediates IEC apoptosis is unclear. Studies in vitro suggest that one mechanism by which p53 mediates apoptosis is through its ability to transactivate members of the TNF receptor family of`Death Receptors'. Here, we examined the role of one of its member, TNF receptor type 1 (TNFR1), in an in vivo model of p53-dependent radiation-induced IEC apoptosis. We demonstrate that mice genetically engineered to be de®cient in TNF receptor type 1 (TNFR1 7/7) and mice injected with TNFR1-fusion chimeric protein (TNFR1-Fc; a competitive inhibitor of TNFR1) were partially protected (30 ± 40%) from p53-dependent radiation-induced IEC apoptosis. However, we found no evidence to support the possibility p53 transcriptionally regulates the expression of TNFR1 nor increases the susceptibility of IEC to TNF-mediated apoptosis. Interestingly, we found that injection of TNF readily induced IEC apoptosis and that radiation induced a p53-dependent increase in the intestinal level of TNF. Furthermore, injection of a neutralizing anti-TNF mAb reduced p53-dependent radiation-induced IEC apoptosis by approximately 60%. Overall, these results suggest that p53-dependent radiation-induced IEC apoptosis is mediated in part through ability of p53 to regulate TNF, which subsequently induces IEC apoptosis through TNFR1. Oncogene (2001) 20, 812 ± 818.

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Graft-vs-host disease (GVHD) is a devastating, frequently fatal, pathological condition associated with lesions in specific target organs, including the intestine, liver, lung, and skin, as well as pancytopenia and alopecia. Bone marrow (BM) atrophy is observed in acutely diseased animals, but the underlying mechanisms of hemopoietic stem cell depletion remained to be established. We used an experimental mouse model of acute GVHD in which parental cells were injected into F1 hosts preconditioned by sublethal irradiation. The resulting graft-vs-host response was kinetically consistent, resulting in lethality within 3 wk. We observed disease pathology in the liver and small intestine, and consistent with previous observations, we found BM atrophy to be a factor in the onset of acute disease. The product of the protooncogene, p53, is known to be a key player in many physiological examples of apoptosis. We investigated the role of p53 in the apoptosis of BM cells (BMC) during the development of acute disease and found that at least one copy of the p53 gene is necessary for depletion of BM and subsequent lethality in host animals. BM depletion was preceded by induction of the death receptor, Fas, on the surface of host stem cells, and induction of Fas was coincidental with the sensitization of BMC to Fas-mediated apoptosis. Our data indicate that BM depletion in acute GVHD is mediated by p53-dependent up-regulation of Fas on BMC, which leads to Fas-dependent depletion and subsequent disease.