Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.     

Distinct Contributions of TNF Receptor 1 and 2 to TNF-Induced Glomerular Inflammation in Mice

TNF is an important mediator of glomerulonephritis. The two TNF-receptors TNFR1 and TNFR2 contribute differently to glomerular inflammation in vivo, but specific mechanisms of TNFR-mediated inflammatory responses in glomeruli are unknown. We investigated their expression and function in murine kidneys, isolated glomeruli ex vivo, and glomerular cells in vitro. In normal kidney TNFR1 and TNFR2 were preferentially expressed in glomeruli. Expression of both TNFRs and TNF-induced upregulation of TNFR2 mRNA was confirmed in murine glomerular endothelial and mesangial cell lines. In vivo, TNF exposure rapidly induced glomerular accumulation of leukocytes. To examine TNFR-specific inflammatory responses in intrinsic glomerular cells but not infiltrating leukocytes we performed microarray gene expression profiling on intact glomeruli isolated from wildtype and Tnfr-deficient mice following exposure to soluble TNF ex vivo. Most TNF-induced effects were exclusively mediated by TNFR1, including induced glomerular expression of adhesion molecules, chemokines, complement factors and pro-apoptotic molecules. However, TNFR2 contributed to TNFR1-dependent mRNA expression of inflammatory mediators in glomeruli when exposed to low TNF concentrations. Chemokine secretion was absent in TNF-stimulated Tnfr1-deficient glomeruli, but also significantly decreased in glomeruli lacking TNFR2. In vivo, TNF-induced glomerular leukocyte infiltration was abrogated in Tnfr1-deficient mice, whereas Tnfr2-deficiency decreased mononuclear phagocytes infiltrates, but not neutrophils. These data demonstrate that activation of intrinsic glomerular cells by soluble TNF requires TNFR1, whereas TNFR2 is not essential, but augments TNFR1-dependent effects. Previously described TNFR2-dependent glomerular inflammation may therefore require TNFR2 activation by membrane-bound, but not soluble TNF.     

TNF receptors in Kupffer cells

Introduction: Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that Octreotide inhibits only LPS induced secretion of proinflammatory cytokines including TNFa by Kupffer cells (KC). We, therefore, tested the hypothesis that somatostatin modulates the expression of tumor necrosis factor alpha (TNF?) receptors and apoptosis of KC. Methods: Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after Octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments. TNFa mRNA expression was assessed by semiquantitative PCR and TNFa was measured in cell supernatants by ELISA. Results: TNFR1 and TNFR2 mRNA are constitutively expressed in KC. Octreotide incubation increased both receptors expression with a peak at 6?h and return to basal levels at 24?h. TNFR1 was mostly influenced. However, only increase in TNFR2 protein was identified, whereas a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNF? mRNA expression was inhibited by Octreotide and a significant inhibition was observed at 48?h. TNF had no effect on KC apoptosis, whereas Octreotide significantly increased their apoptosis, and this effect was not influenced by co-incubation with TNFa. Conclusion: TNFR1 and TNFR2 are constitutively expressed in KC and their expression is strongly increased by somatostatin. Moreover, somatostatin increases KC apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.     

Parenchymal,but not leukocyte,TNF receptor 2 mediates T cell-dependent hepatitis in mice

TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.     

TNF receptors in Kupffer cells

Introduction: Somatostatin is a mediator of immune functions and has been used as an antineoplastic agent in animal models and human neoplasias. We have demonstrated that Octreotide inhibits only LPS induced secretion of proinflammatory cytokines including TNFa by Kupffer cells (KC). We, therefore, tested the hypothesis that somatostatin modulates the expression of tumor necrosis factor alpha (TNFα) receptors and apoptosis of KC.

Methods: Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence before and after Octreotide pre-incubation. Apoptosis was assessed by quantitative measurement of cytoplasmic histone-associated DNA fragments. TNFa mRNA expression was assessed by semiquantitative PCR and TNFa was measured in cell supernatants by ELISA.

Results: TNFR1 and TNFR2 mRNA are constitutively expressed in KC. Octreotide incubation increased both receptors expression with a peak at 6?h and return to basal levels at 24?h. TNFR1 was mostly influenced. However, only increase in TNFR2 protein was identified, whereas a band at 90 kD was present instead of a band at 55 kD as expected for TNFR1. TNFα mRNA expression was inhibited by Octreotide and a significant inhibition was observed at 48?h. TNF had no effect on KC apoptosis, whereas Octreotide significantly increased their apoptosis, and this effect was not influenced by co-incubation with TNFa.

Conclusion: TNFR1 and TNFR2 are constitutively expressed in KC and their expression is strongly increased by somatostatin. Moreover, somatostatin increases KC apoptosis. These findings may in part explain the antineoplasmatic effect of somatostatin.     

Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappa B pathway

We have previously shown that two tumor necrosis factor (TNF) receptors (TNFR) exhibit antagonistic functions during neurodegenerative processes in vivo with TNFR1 aggravating and TNFR2 reducing neuronal cell loss, respectively. To elucidate the neuroprotective signaling pathways of TNFR2, we investigated glutamate-induced excitotoxicity in primary cortical neurons. TNF-expressing neurons from TNF-transgenic mice were found to be strongly protected from glutamate-induced apoptosis. Neurons from wild type and TNFR1(-/-) mice prestimulated with TNF or agonistic TNFR2-specific antibodies were also resistant to excitotoxicity, whereas TNFR2(-/-) neurons died upon glutamate and/or TNF exposures. Both protein kinase B/Akt and nuclear factor-kappa B (NF-kappa B) activation were apparent upon TNF treatment. Both TNFR1 and TNFR2 induced the NF-kappa B pathway, yet with distinguishable kinetics and upstream activating components, TNFR1 only induced transient NF-kappa B activation, whereas TNFR2 facilitated long term phosphatidylinositol 3-kinase-dependent NF-kappa B activation strictly. Glutamate-induced triggering of the ionotropic N-methyl-D-aspartate receptor was required for the enhanced and persistent phosphatidylinositol 3-kinase-dependent NF-kappa B activation by TNFR2, indicating a positive cooperation of TNF and neurotransmitter-induced signal pathways. TNFR2-induced persistent NF-kappa B activity was essential for neuronal survival. Thus, the duration of NF-kappa B activation is a critical determinant for sensitivity toward excitotoxic stress and is dependent on a differential upstream signal pathway usage of the two TNFRs.     

TNF receptor 1-dependent beta cell toxicity as an effector pathway in autoimmune diabetes

Autoimmune diabetes is characterized by a chronic progressive inflammatory autoimmune reaction that ultimately causes the selective elimination of pancreatic beta cells. To address the question of whether the cell death-inducing cytokines TNF and lymphotoxin alpha are involved in this process, we generated nonobese diabetic (NOD) mice that are deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in these mice similarly to that in normal control NOD mice, but progression to diabetes was completely abrogated. Since this was probably due to the complex immunomodulatory effects of TNF and lymphotoxin alpha signaled via TNFR1 on lymphohemopoietic cells, adoptive transfer experiments with spleen cells from diabetic NOD mice were conducted. It was found that the absence of TNFR1 in recipients delayed diabetes induced by normal control and precluded diabetes induced by perforin-deficient spleen cells. In a CD8+ T cell-mediated model of diabetes, however, diabetes induced by adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific CD8+ T cells was not delayed by the absence of TNFR1 in recipient mice. Together with the described expression patterns of perforin and TNF in the mononuclear islet infiltrates of NOD mice, these results indicate that two diabetogenic effector mechanisms are delivered by distinct cell populations: CD8+ T cells lyse beta cells via perforin-dependent cytotoxicity, whereas CD4+ T cells, macrophages, and dendritic cells contribute to diabetes development via TNFR1-dependent beta cell toxicity.     

Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability

We have investigated the role of p55 andp75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2,respectively) in TNF-induced alteration of endothelial permeability invitro and in vivo. Stimulation of TNFR1 with an agonist antibody or areceptor-selective TNF mutein increased the flux of¹²⁵I-albumin through endothelial cell monolayers. Anantagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1,but not TNFR2, induced cytoskeletal reorganization associated withincreased permeability. SB-203580, a p38 mitogen-activated proteinkinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation ofTNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibodypartially inhibited the effect of murine TNF on liver vessels,suggesting that TNFR2 also plays a role in the regulation ofTNF-induced vascular permeability in vivo.

     

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.     

TNF regulates thymocyte production by apoptosis and proliferation of the triple negative (CD3-CD4-CD8-) subset

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3(-)CD4(-)CD8(-) triple negative (TN) subset within a broad range of dosages (10(1)-10(5) pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44(-)CD25(+)) and TN4 (CD44(-)CD25(-)) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (10(4)-10(5) pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin alpha and beta triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.     

Cholera toxin‐sensitive GTP‐binding protein‐coupled activation of augmenter of liver regeneration (ALR) receptor and its function in rat kupffer cells

Mitogenic effect of augmenter of liver regeneration (ALR), a protein produced and released by hepatocytes, on hepatocytes in vivo but not in vitro suggests that the effect is mediated by nonparenchymal cells. Since mediators produced by Kupffer cells are implicated in hepatic regeneration, we investigated receptor for ALR and its functions in rat Kupffer cells. Kupffer cells were isolated from rat liver by enzymatic digestion and centrifugal elutriation. Radioligand ([¹²⁵I] ALR) receptor binding, ALR‐induced GTP/G‐protein association, and nitric oxide (NO), tumor necrosis factor (TNF)‐α, and interleukin‐6 (IL‐6) synthesis were determined. High‐affinity receptor for ALR, belonging to the G‐protein family, with K_d of 1.25 ± 0.18 nM and B_max of 0.26 ± 0.02 fmol/µg DNA was identified. ALR stimulated NO, TNF‐α, and IL‐6 synthesis via cholera toxin‐sensitive G‐protein, as well as p38‐MAPK activity and nuclear translocation of NFκB. While inhibitor of NFκB (MG132) inhibited ALR‐induced NO synthesis, MG132 and p38‐MAPK inhibitor (SB203580) abrogated ALR‐induced TNF‐α and IL‐6 synthesis. ALR also prevented the release of mediator(s) from Kupffer cells that cause inhibition of DNA synthesis in hepatocytes. Administration of ALR to 40% partially hepatectomized rats increased expression of TNF‐α, IL‐6, and inducible nitric oxide synthase (iNOS) and caused augmentation of hepatic regeneration. These results demonstrate specific G‐protein coupled binding of ALR and its function in Kupffer cells and suggest that mediators produced by ALR‐stimulated Kupffer cells may elicit physiologically important effects on hepatocytes. J. Cell. Physiol. 222: 365–373, 2010. © 2009 Wiley‐Liss, Inc.     

Apoptosis in mouse amniotic epithelium is induced by activated macrophages through the TNF receptor type 1/TNF pathway

The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis.     

Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.     

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-κB activation

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.     

Activation of platelet caspases by TNF and its consequences for kinetics

TNF is known to induce a thrombocytopenia, due to a reduced platelet life span. Injection of TNF (10 microg) to mice did markedly increase the number of platelet-derived microparticles in plasma, most pronounced 1h after injection. Injection of TNF induced a transient activation of platelet caspases, -1, -3, -6, -8, -9, as seen by the binding of caspases probes detected by flow cytometry, most pronounced 1h after injection. Activation of caspase-3 was also evidenced by antibodies. Injection of the caspases inhibitor ZVAD-fmk decreased TNF-induced generation of microparticles and thrombocytopenia, indicating a causal role of caspases in platelet fragmentation. Activation of platelet caspases was also evident in platelets exposed to TNF in vitro, indicating that TNF acts on platelets directly. Comparison of platelets from +/+, TNFR1 -/- and TNFR2 -/- mice showed that caspases are activated mainly by the TNFR1. These observations indicate that TNF activates platelet caspases via the TNFR1, which results in platelet fragmentation and thrombocytopenia.     

Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney

Bacterial endotoxin (LPS) is responsible for much of the widespread inflammatory response seen in sepsis, a condition often accompanied by acute renal failure (ARF). In this work we report that mice deficient in TNFR1 (TNFR1(-/-)) were resistant to LPS-induced renal failure. Compared with TNFR1(+/+) controls, TNFR1(-/-) mice had less apoptosis in renal cells and fewer neutrophils infiltrating the kidney following LPS administration, supporting these as mediators of ARF. TNFR1(+/+) kidneys transplanted into TNFR1(-/-) mice sustained severe ARF after LPS injection, which was not the case with TNFR1(-/-) kidneys transplanted into TNFR1(+/+) mice. Therefore, TNF is a key mediator of LPS-induced ARF, acting through its receptor TNFR1 in the kidney.     

Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function

During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.     

Membrane Tumor Necrosis Factor (TNF) Induces p100 Processing via TNF Receptor-2 (TNFR2)

Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFκB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFκB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFκB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFκB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFκB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.