The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.     

Chromosome assignments of the human TNF p55 and p75 receptor genes.

At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.     

Tumor necrosis factor (TNF)-alpha induces leptin production through the p55 TNF receptor

Tumor necrosis factor (TNF)-alpha acts directly on adipocytes to increase production of the lipostatic factor, leptin. However, which TNF receptor (TNFR) mediates this response is not known. To answer this question, leptin was measured in plasma of wild-type (WT), p55, and p75 TNFR knockout (KO) mice injected intraperitoneally with murine TNF-alpha and in supernatants from cultured WT, p55, and p75 TNFR KO adipocytes incubated with TNF-alpha. Leptin also was measured in supernatants from C3H/HeOuJ mouse adipocytes cultured with blocking antibodies to each TNFR and TNF-alpha as well as in supernatants from adipocytes incubated with either human or murine TNF-alpha, which activate either one or both TNFR, respectively. The results using all four strategies show that the induction of leptin production by TNF-alpha requires activation of the p55 TNFR and that although activation of the p75 TNFR alone cannot cause leptin production, its presence affects the capability of TNF-alpha to induce leptin production through the p55 TNFR. These results provide new information on the interplay between cells of the immune system and adipocytes.     

Circulating IL-6 mediates lung injury via CXCL1 production after acute kidney injury in mice

Serum IL-6 is increased in patients with acute kidney injury (AKI) and is associated with prolonged mechanical ventilation and increased mortality. Inhibition of IL-6 in mice with AKI reduces lung injury associated with a reduction in the chemokine CXCL1 and lung neutrophils. Whether circulating IL-6 or locally produced lung IL-6 mediates lung injury after AKI is unknown. We hypothesized that circulating IL-6 mediates lung injury after AKI by increasing lung endothelial CXCL1 production and subsequent neutrophil infiltration. To test the role of circulating IL-6 in AKI-mediated lung injury, recombinant murine IL-6 was administered to IL-6-deficient mice. To test the role of CXCL1 in AKI-mediated lung injury, CXCL1 was inhibited by use of CXCR2-deficient mice and anti-CXCL1 antibodies in mice with ischemic AKI or bilateral nephrectomy. Injection of recombinant IL-6 to IL-6-deficient mice with AKI increased lung CXCL1 and lung neutrophils. Lung endothelial CXCL1 was increased after AKI. CXCR2-deficient and CXCL1 antibody-treated mice with ischemic AKI or bilateral nephrectomy had reduced lung neutrophil content. In summary, we demonstrate for the first time that circulating IL-6 is a mediator of lung inflammation and injury after AKI. Since serum IL-6 is increased in patients with either AKI or acute lung injury and predicts prolonged mechanical ventilation and increased mortality in both conditions, our data suggest that serum IL-6 is not simply a biomarker of poor outcomes but a pathogenic mediator of lung injury.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.     

Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P 相似文献   

Signaling for myocardial depression in hemorrhagic shock: roles of Toll-like receptor 4 and p55 TNF-alpha receptor

Hemorrhagic shock causes myocardial contractile depression. Although this myocardial disorder is associated with increased expression of tumor necrosis factor-alpha (TNF-alpha), the role of TNF-alpha as a myocardial depressant factor in hemorrhagic shock remains to be determined. Moreover, it is unclear which TNF-alpha receptor mediates the myocardial depressive effects of TNF-alpha. Toll-like receptor 4 (TLR4) regulates cellular expression of proinflammatory mediators following lipopolysaccharide stimulation and may be involved in the tissue inflammatory response to injury. The contribution of TLR4 signaling to tissue TNF-alpha response to hemorrhagic shock and TLR4's role in myocardial depression during hemorrhagic shock are presently unknown. We examined the relationship of TNF-alpha production to myocardial depression in a mouse model of nonresuscitated hemorrhagic shock, assessed the influence of TLR4 mutation, resulting in defective signaling, on TNF-alpha production and myocardial depression, and determined the roles of TNF-alpha and TNF-alpha receptors in myocardial depression using a gene knockout (KO) approach. Hemorrhagic shock resulted in increased plasma and myocardial TNF-alpha (4.9- and 4.5-fold, respectively) at 30 min and induced myocardial contractile depression at 4 h. TLR4 mutation abolished the TNF-alpha response and attenuated myocardial depression (left ventricular developed pressure of 43.0 +/- 6.2 mmHg in TLR4 mutant vs. 30.0 +/- 3.6 mmHg in wild type, P < 0.05). TNF-alpha KO also attenuated myocardial depression in hemorrhagic shock, and the p55 receptor KO, but not the p75 receptor KO, mimicked the effect of TNF-alpha KO. The results suggest that TLR4 plays a novel role in signaling to the TNF-alpha response during hemorrhagic shock and that TNF-alpha through the p55 receptor activates a pathway leading to myocardial depression. Thus TLR4 and the p55 TNF-alpha receptor represent therapeutic targets for preservation of cardiac mechanical function during hemorrhagic shock.     

TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.     

Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants.

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Identification of TNF-alpha binding peptides from a D-amino acid hexapeptide library that specifically inhibit TNF-alpha binding to recombinant p55 receptor

Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine with pleiotropic activity that binds to two transmembrane receptors. Its role in mediating the inflammatory response to injury or infection has been well documented and it has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Using synthetic peptide libraries composed exclusively of D-amino acids, two distinct hexapeptide families that block the binding of TNF-alpha to its receptors were identified. In the deconvolution of the library, activity increased from submillimolar to the low micromolar range with the most active compound having an IC50 of 0.33 microM. With the aid of biotinylated constructs of these hexapeptides it was possible to demonstrate that their antagonistic effect is due to specific binding to TNF-alpha and not to its receptor.     

Kupffer cell-expressed membrane-bound TNF mediates melphalan hepatotoxicity via activation of both TNF receptors

Isolated hepatic perfusion of nonresectable liver cancer using the combination of TNF and melphalan can be associated with a treatment-related hepatotoxicity. We investigated whether, apart from TNF, also melphalan is cytotoxic in primary murine liver cells in vitro and investigated mediators, mode of cell death, and cell types involved. Melphalan induced a caspase-dependent apoptosis in hepatocytes, which was not seen in liver cell preparations depleted of Kupffer cells. Neutralization of TNF prevented melphalan-induced apoptosis and liver cells derived from mice genetically deficient in either TNFR 1 or 2, but not from lpr mice lacking a functional CD95 receptor, were completely resistant. Cell-cell contact between hepatocytes and Kupffer cells was required for apoptosis to occur. Melphalan increased membrane-bound but not secreted TNF in Kupffer cells and inhibited recombinant TNF-alpha converting enzyme in vitro. Melphalan induced also severe hepatotoxicity in the isolated recirculating perfused mouse liver from wild-type mice but not from TNFR 1 or 2 knockout mice. In conclusion, this study shows that melphalan elicits membrane TNF on Kupffer cells due to inhibition of TNF processing and thereby initiates apoptosis of hepatocytes via obligatory activation of both TNFRs. The identification of this novel mechanism allows a causal understanding of melphalan-induced hepatotoxicity.     

Dissociation of TNF-alpha cytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants.

Human tumour necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine capable of killing mammalian tumour cells in vitro and in vivo, and of enhancing the proinflammatory activity of leucocytes and endothelium, the latter effects limiting its usage as an antitumour agent in humans. Using TNF-alpha mutants with a selective capacity to bind to the TNF p55 receptor (TNFR55) or to the p75 receptor (TNFR75) we show here that these two major activities of TNF-alpha can be dissociated. The TNFR55-selective mutants (R32W, E146K and R32W-S86T) which bind poorly to TNFR75 displayed similar potency to wild-type TNF in causing cytotoxicity of a human laryngeal carcinoma-derived cell line (HEp-2) and cytostasis in a human leukaemic cell line (U937). However, these TNFR55-selective mutants exhibited lower proinflammatory activity than wild-type TNF. Specifically, TNF-alpha's priming of human neutrophils for superoxide production and antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis and adhesion to endothelium were reduced by up to 170-fold. Activation of human endothelial cell functions represented by human umbilical venular endothelial cell (HUVEC) adhesiveness for neutrophils, E-selectin expression, neutrophil transmigration and IL-8 secretion were also reduced by up to 280-fold. On the other hand, D143F, a TNFR75-selective mutant tested either alone or in combination with TNFR55-selective mutants, did not stimulate these activities despite being able to cause cytokine production in TNFR75-transfected PC60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A selective role for the TNF p55 receptor in autocrine signaling following IFN-gamma stimulation in experimental autoimmune uveoretinitis

IFN-gamma stimulates macrophage activation and NO production, which leads to destruction of the retina in experimental autoimmune uveoretinitis. In this study, we investigate the mechanism of disease resistance in TNF p55 receptor-deficient animals. We show that although T cell priming is relatively unaffected, macrophages lacking the TNF p55 receptor fail to produce NO following IFN-gamma stimulation because of a requirement for autocrine TNF-alpha signaling through the TNF p55 receptor. In contrast to the impaired activation of NO synthesis, MHC class II up-regulation was indistinguishable in wild-type and TNFRp55-/- mice stimulated with IFN-gamma. These defects could be overcome by stimulating macrophages with LPS. Together, these results show that selected aspects of IFN-gamma activation are controlled by autocrine secretion of TNF-alpha, but that this control is lost in the presence of signals generated by pathogen-associated molecular patterns recognizing receptors.     

TAK1 mediates excessive autophagy via p38 and ERK in cisplatin‐induced acute kidney injury

The ability of cisplatin (cis‐diamminedichloroplatinum II) toxicity to induce acute kidney injury (AKI) has attracted people's attention and concern for a long time, but its molecular mechanisms are still widely unknown. We found that the expression of transforming growth factor‐β (TGF‐β)‐activated kinase 1 (TAK1) could be increased in kidneys of mice administrated with cisplatin. Autophagy is an evolutionarily conserved catabolic pathway and is involved in various acute and chronic injuries. Moreover, p38 MAPK (mitogen‐activated protein kinase) and ERK regulate autophagy in response to various stimuli. Therefore, our hypothesis is that cisplatin activates TAK1, which phosphorylates p38 and ERK, leading to excessive autophagy of tubular epithelial cells and thus exacerbating kidney damage. Here, BALB/c mice were intraperitoneally injected with a TAK1 inhibitor and were then administrated with sham or cisplatin at 20 mg/kg by intraperitoneal injection. Compared with mice in the vehicle cisplatin group, mice intraperitoneally injected with a TAK1 inhibitor were found to have lower serum creatinine and less tubular damage following cisplatin‐induced AKI. Furthermore, inhibition of TAK1 reduced p38 and Erk phosphorylation, decreased expression of LC3II and reversed the down‐regulation of P62 expression induced by cisplatin. The hypothesis was verified with tubular epithelial cells administrated with cisplatin in vitro. Finally, p38 inhibitor or ERK inhibitor abated autophagy activation and cell viability reduction in tubular epithelial cells treated with cisplatin plus TAK1 overexpression vector. Taken together, our results show that cisplatin activates TAK1, which phosphorylates p38 and ERK, leading to excessive autophagy of tubular epithelial cells that exacerbates kidney damage.