TNF-α Toxicity Research Articles

Measurement of cytotoxicity by propidium iodide staining of target cell DNA: Application to the quantification of murine TNF-α

A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-α), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane becomes permeable due to TNF-α-induced cell damage. The analytical range for the proposed method is 0.3–200 pg/ml of TNF-α after 5 h of incubation. The optimal number of target cells was found to be 4–5 × 10 4/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-α. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-release assays that kinetic studies of TNF-α toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-α.

Interleukin-1 and nitric oxide protect against tumor necrosis factor α-induced liver injury through distinct pathways

Mice sensitized with d-galactosamine (GalN) and challenged with recombinant murine tumor necrosis factor a (TNFα) developed severe apoptotic and secondary necrotic liver injury as assessed by histology, measurement of cytosolic DNA fragments, and determination of liver specific enzymes in plasma. Pretreatment with recombinant human interleukin-1β (IL-1) rendered mice insensitive to this TNFα toxicity. Coadministration of the liver-specific transcriptional inhibitor GaIN with IL-1 prevented the development of tolerance, implicating de novo synthesis of liver specific proteins in the induction of tolerance. Pretreatment of mice with IL-1 resulted in elevated levels of nitrite/nitrate in serum and in enhanced nitric oxide synthase (NOS) activity in liver cells isolated from these animals. In addition, pharmacological doses of the nitric oxide (NO) donor sodium nitroprusside conferred complete protection against TNFα induced liver injury in galactosamine-sensitized mice, suggesting a possible link between IL-1 and NO-induced protection. However, prevention of NO-synthesis by N G-monomethyl- l-arginine (NMMA) did not abolish IL-1-induced tolerance to TNFα in vivo. Cytotoxicity of TNFα to isolated hepatocytes sensitized with actinomycin D (ActD) was not significantly altered by inhibition of endogenous nitrite release. Also, enhanced NO production elicited in vitro by glycerol trinitrate or ex vivo by pretreatment with IL-1 had no significant effect in this system. We conclude that IL-1- and NO-induced protection of mice against TNFα-mediated liver damage follow distinct pathways.

Tumor necrosis factor α differentially modulates the cellular response of rat hepatocytes in periportal- and pericentral-equivalent cultures

Alterations of cellular functions induced by recombinant human tumor necrosis factor α (TNFα) were compared in rat hepatocytes cultured under either periportal-equivalent (10 nM insulin; 10 nM glucagon; 13% O 2) or perivenous-equivalent conditions (10 nM insulin; 1 nM glucagon; 4% O 2). TNFα induced a time- and dose-dependent increase in nitric oxide (NO) production and an acute phase response (inhibition of albumin secretion and elevation of α 2-macroglobulin production) under both culture conditions. NO production was more pronounced in periportal cultures, while the acute phase response was stronger in pericentral cultures. This suggests that NO production and the acute phase response are controlled by different pathways. After exposure to TNFα, DNA content was measured fluorimetrically and biochemically. A marked decrease in nuclear DNA content was found exclusively in pericentral cultures after an 8-h exposure, followed by an elevation of lactic dehydrogenase (LDH) release after a 12-h exposure. Aurintricarboxylic acid (100 μM), an inhibitor of endonuclease, significantly inhibited the TNFα-induced decrease in nuclear DNA content but only partially inhibited the LDH release. This indicates that the loss of nuclear DNA content in pericentral cultures is due to an activation of endonuclease and the resulting DNA fragmentation and does not correlate with NO production. Furthermore, the release of LDH seems to be only partially associated with DNA damage. Dexamethasone (100 nM) completely inhibited both TNFα-induced DNA fragmentation and the elevation of LDH release. The results clearly indicate that the toxicity of TNFα is influenced by the metabolic state of hepatocytes. Accordingly, the preferential perivenous cell injury observed after exposure to endotoxins in vivo seems to be due to a higher sensitivity of the pericentrally localized hepatocytes towards TNFα rather than a TNFα concentration gradient.