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Abstract
Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.
Highlights
Tumor necrosis factor (TNF)1 ␣ is a potent cytokine produced by many cell types that exerts pleiotropic functions, mediating inflammation, immunity, and modulation of cell death, differentiation, and survival [1, 2]
TNF␣-triggered activation of neutral sphingomyelinase is mediated by the adapter protein FAN, which binds a short motif of the intracellular part of CD120a termed the neutral sphingomyelinase domain [24, 25]
Because liver injury is viewed as the primary factor responsible for LPS-induced lethality and because hepatic damage is due to apoptosis of hepatocytes [35, 36], we analyzed histologically and biochemically the livers of FAN-deficient and wildtype mice challenged with D-galactosamine and LPS
Summary
Cytokines and Antibodies—Recombinant human and murine TNF␣s were purchased from PeproTech (Tebu-Bio, Le Perray-en-Yvelines, France). Animals and Treatments—FAN-deficient (FAN Ϫ/Ϫ) mice were obtained from FAN ϩ/Ϫ embryos IL-6-deficient mice, maintained in C57BL/6 background [31], were obtained from Drs M. At the age of 7–10 weeks, mice (weighing ϳ20 –25 g) were injected intraperitoneally with a single dose of D-galactosamine (20 mg; Sigma) followed by either intraperitoneal injection of LPS (0.1–10 g; Salmonella minnesota; Sigma) or intravenous injection of murine recombinant TNF␣ (1–10 g/kg of body weight) in a total volume of 0.1 ml of PBS alone or PBS containing 1% bovine serum albumin, respectively. Mice received intraperitoneally only LPS (100 g) or anti-CD95 antibody (0.36 g/g of body weight) in PBS. Determination of Caspase Activity—Effector caspase activity was measured on liver lysates using the fluorogenic substrate Ac-AspGlu-Val-Asp-aminomethylcoumarin (Bachem, Voisins-Le-Bretonneux, France) as described [32]. The differences in 75% survival times of the two animal groups were statistically studied by actuarial analysis
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