The Effect Of Transmembrane TNF-α On Acute Liver Failure And Endotoxin Shock

Transmembrane tumor necrosis factor-alpha (tmTNF-a) is known to be the precursor of soluble form TNF-α (TNF-a) that is released after processing by TNF--converting enzyme (TACE), both forms of TNF-α display bioactivities via TNF receptors, TNFRI and TNFRII. sTNF-α is a pleiotropic proinflammatory cytokine which plays a critical role in both of acute liver failure (ALT) and endotoxic shock. However, it’s not clear whether native tmTNF-α is involved in ALT and entotoxic shock. Although there are tmTNF transgenic animal experimental reports in these two diseases, the transfected tmTNF in these reports is an TACE enzyme site deficient mutant rather then wild-type tmTNF-α. Our previous research results showed that there are big difference between this mutant and wild-type tmTNF-α, and other different labs have different results.Since these reports cannot reflect the function of native tmTNF-a in these two diseases, our study aim to observe the effect and mechanism of endogenous tmTNF-α on ALT and endotoxic shock.The main results are showed as follow:Part I Effects of tmTNF-α on acute liver failureⅠ. Feature of liverdamage in ALTALT was induced in mouse by intraperitoneal injection with LPS(10μg/kg)/D-gal(500mg/kg), The results of HE showed that hepatocytes became slightly swollen at2h and heavily swollen at4h after LPS/D-gal injection. There were significant hepatic sinus congestion, hepatic cords fracture and inflammatory cell infiltration6h after LPS/D-gal treatment. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) also increased significantly6h after LPS/D-gal treatment. It indicated that liver damage was remarkable6h after LPS/D-gal treatment.Ⅱ. Relationship between level of endogenous TNF-α and liver damage1. Feature of sTNF-α changes:The ELISA results showed that serum sTNF-α levels increased rapidly and peaked at2h, while it declined sharply4h after LPS/D-gal treatment. The level of sTNF-a in the supernatant of KCs culture isolated from mice liver also increased and peaked2h after treated with LPS/D-gal injection, and then decreased gradually, but the sTNF-a level at4h and6h in LPS/D-gal group is higher than normal mice. This results indicated that the sTNF-a level in circulation didn’t represent the change of sTNF-α level in local inflammatory site.2-Feature of tmTNF-α changes:The results of FCM showed that level of tmTNF-a on KCs and PMs isolated from ALT mice was low at2h and4h after LPS/D-gal injection, while it was much more higher at6h.3. Relationship between level of endogenous TNF-a and aminotransferase:In acute liver failure mice, level of serum sTNF-a was negative correlated with serum transaminase activity, but levels of tmTNF-a expressed on KCs-6h or PMs-6h were positively correlated with serum transaminase activity. The results indicated that tmTNF-a, instead of sTNF-a, was involved in liver damage in ALT mice.Ⅲ. Imbalance between pro-and anti-inflammatory cytokines secreted by KCs-6h and PMs-6h1. Ratio of serum pro-inflammatory cytokine IL-6and anti-inflammatory cytokine IL-10:In ALT mice, the result of ELISA showed that serum level of IL-6remarkably increased and peaked at4h, and then decreased at6h, the serum IL-6level at6h was87.8%of that at4h; While serum level of IL-10peaked at2h, and then decreased gradually, the serum IL-10level at6h was only23.6%of that at6h. It was showed that IL-6/IL-10ratios significantly increased at6h, and were consistent with liver injury, which suggested that the increase of IL-6/IL-10ratios would predict severe liver injury better.2. Imbalance between proinflammatory and anti-inflammatory cytokines secretion of KCs-6h and PMs-6h:Kupffer cells (KCs) and peritoneal macrophages (PMs) isolated from LPS-D-gal-treated mice at various time-points were cultured respectively in vitro, and it was found that the changes of the level of IL-6and IL-10in the supernatant of cell culture were consistent with that in serum; IL-6/IL-10ratio increased significantly in ALT mice6h after LPS/D-gal treatment. The increase of IL-6/IL-10ratio at this two time point is positively related with the high expression of tmTNF-α. The above results suggested the pro-and anti-inflammatory cytokines secreted by KCs-6h and PMs-6h, which were isolated from mice treated with LPS-D-gal-treated for6h, were severely imbalanced and then worsen the liver damage.Ⅳ. tmTNF-α high-expressing KCs induced hepatocyte apoptosis1. KCs-6h induced hepatocyte apoptosis via cell-cell contact:KCs-2h or KCs-6h were co-cultured with hepatocytes in vitro, it was found that KC-6h expressed high level of tmTNF-α induced hepatocyte apoptosis rapidly via cell-cell contact, and resulted in high level of AST in culture supernatant, but KCs-2h expressed low level of tmTNF-α couldn’t induce hepatocyte apoptosis; KCs-6h couldn’t induce hepatocyte apoptosis when KCs-6h were separated from hepatocyte by transwell memberane.2. KCs-6h induced hepatocytes apoptosis by tmTNF-o:KCs-6h were pre-treated with anti-TNF-α which blocked the tmTNF-α on cell surface, and then were cocultured with hepatocytes, it was found that apoptosis of hepatocytes decreased significantly. The results indicated that KCs-6h induced hepatocytes apoptosis by tmTNF-a.3. tmTNF-α high-expressing KCs expressed high level of FasL:Although the apoptosis of hepatocyte was reduced remarkably by pre-treated with anti-TNF-α in KCs-6h, apoptosis still remained, this indicated that the apoptosis of hepatocyte was induced by some other apoptotic pathway. Real time-PCR and western blot results showed that mRNA and protein level of FasL were significantly higher in KCs-6h than that in KCs-2h. It indicated that tmTNF-α can induce hepatocyte apoptosis through enhancement of FasL expression.V. tmTNF-α high-expressing KCs adoptive transfer accelerated liver damage induced by LPS/D-gal1. KCs-6h expressed tmTNF-α continuosly:In order to ensure the effectiveness of KCs adoptive transfer, we checked first whether expression was stable. The results showed that mTNF-α expression remained for6days at high levels when KCs isolated from6h LPS/D-gal-treated mice were cultured in vitro.2. KCs-6h adoptive transfer aggravated damage of liver tissue:The HE results showed that adoptive transfer of KCs-6h aggravated damage of liver tissue in mice treated with LPS/D-gal for4h. Compared with LPS/D-gal group, there were significant severe hemorrhage(indicated by black arrow) and inflammatory cell infiltration (indicated by red arrow) after adoptive transfer.3. KCs-6h adoptive transfer promoted hepatocyte apoptosis:The TUNEL stain result of liver tissue showed that KCs-6h adoptive transfer promoted hepatocyte apoptosis, the level of AST serum was significantly higher in KCs-6h adoptive transfer mice than that in KCs-2h adoptive transfer mice. It suggested that tmTNF-α high-expressing KCs-6h aggravated liver damage in vivo.4. KCs-6h adoptive transfer increased IL-6/IL-10ratio in mice serum:The ELISA results showed that the levels of pro-inflammatory cytokine IL-6and IL-1β in serum of mice adoptive transferred with KCs-6h were much higher than that transferred with KCs-2h, but the level of anti-inflammatory IL-10was much lower compared with mice transferred with KCs-2h. The IL-6/IL-10ratio increased. It suggested that the imbalance of pro-and anti-inflammatory from tmTNF-α high-expression KCs-6h was involved in ALT.Part Ⅱ The effect of tmTNF-α towards endotoxic shockI. The preparation and identification of specific tmTNF-α Ab1. The preparation and identification of specific tmTNF-α Ab:The rabbit anti-mice tmTNF-α Ab was successfully prepared against the single epitope of tmTNF-α. The ELISA results showed that the rabbit serum contains high titer of Ab against tmTNF-α, which only bind with the peptide fragment of tmTNF-α, and does not has the cross-reaction towards sTNF-α. 2. tmTNF-α Ab can bind with tmTNF-α expressed on cell surface:FCM result showed that the prepared tmTNF-α Ab can bind with the cell surface of TNF-α positive NIH3T3cells, wheares it cannot bind with the TNF-α negative CT26cells, suggesting that tmTNF-α Ab can recognize the intact tmTNF-α molecule on cell surface.Ⅱ. tmTNF-α Ab can block the convert of tmTNF-α into sTNF-α effectively1. tmTNF-α Ab can block the convert of tmTNF-α into sTNF-α effectively:To stimulate RAW264.7Macrophage and PMs primary cell with100ng/ml LPS and tmTNF Ab for4h, ELISA and FCM result showed that tmTNF-α Ab can inhibit the secretion of sTNF-α, and increase tmTNF-α expression significantly, suggesting that tmTNF-α Ab can block the convert of tmTNF-α into sTNF-α effectively.2. tmTNF-α Ab can inhibit the cytotoxicity of sTNF-α in the supernatant of RAW264.7cells:Biological activity test result showed that the supernatant of RAW264.7cells induced by LPS stimulation has significant cytotoxicity effect towards the TNF-α sensitive target L929cells, after pre-neutralized with tmTNF-α Ab, this cytotoxicity effect can be blocked. This demonstrated again that tmTNF-α Ab can inhibit the cleavage of tmTNF-a, so that the sTNF-α secretion was reduced, thus the cytotoxicity effect was inhibited.Ⅲ. The effect of tmTNF-α Ab on the production of pro-inflammatory and anti-inflammatory cytokines1. tmTNF-α Ab can inhibit NO production of macrophage induced by LPS:To stimulate RAW264.7Macrophage and PMs cell with LPS and tmTNF Ab, the results showed that tmTNF-α Ab can inhibit significantly the the transcription of iNOS and secretion of NO of these two kinds of macrophages.2. tmTNF-α Ab can inhibit the production of pro-inflammatory cytokines, e.g IL-6and IL-1β:To stimulate RAW264.7Macrophage and PMs cell with LPS and tmTNF Ab, the results showed that tmTNF-α Ab can inhibit significantly the transcription and secretion of pro-inflammatory cytokines, e.g IL-6and IL-1β. 3.tmTNF-a Ab has no effect on the production of anti-inflammatory cytokine IL-10:To stimulate RAW264.7Macrophage and PMs cell with LPS and tmTNF Ab, the results showed that tmTNF-a Ab has no effect on the transcription and secretion of anti-inflammatory cytokine IL-10.IV. tmTNF-α Ab can protect the mice to be resistant to endoxemia1. tmTNF-α Ab can block the convert of tmTNF-α into sTNF-α in endoxemia mice model:The pre-treatment of tmTNF-α Ab can incease significantly the tmTNF-α expression of peritoneal macrophages4h after the injection of LPS, and inhibit the serum TNF-α level at that time point(p<0.01), indicating that tmTNF-α Ab can block the convert of tmTNF-α into sTNF-α in vivo.2. tmTNF-α Ab can improve the clinical symptoms of endotoxic shock, and increase the mice suvival rate:The treatment of tmTNF-a Ab can relieve the lip cyanosis of endotoxic shock, improve the clinical score, increase the mice suvival rate significantly.3. tmTNF-α Ab can inhibit the production of pro-inflammatory cytokines in endoxemia mice model:When treated with LPS at2h, the production of pro-inflammatory cytokines, e.g IL-6and IL-1β, NO and anti-inflammatory cytokine IL-10start to increase, peaked at6h, and then decrease. The treatment of tmTNF-a Ab can inhibit the production of pro-inflammatory cytokines significantly, but slightly increase the level of anti-inflammatory cytokine IL-10without significance.In conclusion, tmTNF-a exert different function in different pathological status. In the acute hepatic failure induced by LPS/D-gal, the expression of KCs and PMs can induce the apoptosis of hepatic apoptosis directly, increase the hepatic inflammatory damage through the imbalance of secretion of pro-inflammatory cytokines and anti-inflammatory cytokine. On the contrary, the treatment of tmTNF-α Ab can increase the expression of tmTNF-α and decrease the secretion of sTNF-α, it can inhibit the response of macrophage towards LPS in vitro, and protect the mice to be resistant against endoxemia in vivo. The study demonstrate not only the different function of tmTNF-α in different disease, but also provide the novel strategy in the treatment of TNF related diseases...