Changes in the expression of tumor necrosis factor (TNF) alpha,TNFalpha receptor (TNFR) 2, and TNFR-associated factor 2 in granulosa cells during atresia in pig ovaries

Tumor necrosis factor (TNF) alpha can induce both cell death and cell proliferation and exerts its effects by binding to either TNF receptor (TNFR) 1 or 2. When TNFalpha-bound TNFR2 interacts with TNFR-associated factor 2 (TRAF2), expression of survival/antiapoptotic genes is up-regulated. In the present study we determined the changes in localization of TNFalpha and TRAF2 and their mRNAs and the expression of TNFR2 in granulosa cells during follicular atresia in pig ovaries. In healthy follicles, intense signals for TNFalpha and TRAF2 and their mRNAs were demonstrated in the outer zone of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased or trace staining for TRAF2 and its mRNA and decreased expression of TNFR2 were observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in pig ovaries.     

Characterisation of receptor-specific TNFalpha functions in adipocyte cell lines lacking type 1 and 2 TNF receptors

Tumour necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that exerts a myriad of biological actions in numerous different tissues including adipocytes through its two distinct cell surface receptors. To address the role of each TNF receptor in the biological actions of TNFalpha in adipocytes, we have developed four new preadipocyte cell lines. These were established from wild type controls (TNFR1(+/+)R2(+/+)) and from mice lacking TNFR1 (TNFR1(-/-)), TNFR2 (TNFR2(-/-)) or both (TNFR1(-/-)R2(-/-)). All four new cell lines can fully differentiate to form mature adipocytes, under appropriate culture conditions, as judged by cell morphology, expression of multiple adipogenic markers and the ability to mediate agonist-stimulated lipolysis and insulin-stimulated glucose transport. In wild type (TNFR1(+/+)R2(+/+)) and TNFR2(-/-) adipocytes, TNFalpha stimulated lipolysis and inhibited insulin-stimulated glucose transport as well as insulin receptor autophosphorylation. In contrast, these activities were completely lost in the TNFR1(-/-)R2(-/-) and TNFR1(-/-) cells. Taken together, these studies demonstrate that TNFalpha-induced lipolysis, as well as inhibition of insulin-stimulated glucose transport are predominantly mediated by TNFR1 and that the presence of TNFR2 is not necessary for these functions. This new experimental system promises to be useful in dissecting the molecular pathways activated by each TNF receptor in mediating the biological functions of TNFalpha in differentiated adipocytes.     

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.     

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.     

Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons

Localized tumor necrosis factor-alpha (TNFalpha) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFalpha mRNA levels. TNFp55R (55 kDa type-1 TNFR) and TNFp75R (75 kDa type-2 TNFR) immunoreactivity in AC3(+) neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFalpha antibody and in TNFp55R-/-p75R-/- mice that showed higher constitutive mRNA levels for interleukin (IL-1alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), TNFalpha, transforming growth factor beta1, Fas, and TNFRSF6-assoicated via death domain (FADD). TNFp75R-/- mice showed exacerbated injury and elevated mRNA levels for IL-1alpha, MIP-1alpha, and TNFalpha. In TNFp55R-/- mice, constitutive mRNA levels for TNFalpha, IL-6, caspase 8, FADD, and Fas-associated phosphatase were higher; IL-1alpha, MIP-1alpha, and transforming growth factor beta1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co-expression of TNFp75R in AC3(+) neurons. The data demonstrate TNFR-mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R-mediated apoptosis in the absence of normal TNFp55R activity.     

A TNF receptor 2 selective agonist rescues human neurons from oxidative stress-induced cell death

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.     

Elevated CSF levels of TACE activity and soluble TNF receptors in subjects with mild cognitive impairment and patients with Alzheimer's disease

We recently reported that expression levels of tumor necrosis factor (TNF) receptors, TNFR1 and TNFR2, are significantly changed in the brains and cerebrospinal fluid (CSF) with Alzheimer's disease (AD). Moreover, we also found that, in an Alzheimer's mouse model, genetic deletion of TNF receptor (TNFR1) reduces amyloid plaques and amyloid beta peptides (Aβ) production through β-secretase (BACE1) regulation. TNF-α converting enzyme (TACE/ADAM-17) does not only cleave pro- TNF-α but also TNF receptors, however, whether the TACE activity was changed in the CSF was not clear. In this study, we examined TACE in the CSF in 32 AD patients and 27 age-matched healthy controls (HCs). Interestingly, we found that TACE activity was significantly elevated in the CSF from AD patients compared with HCs. Furthermore, we also assayed the CSF levels of TACE cleaved soluble forms of TNFR1 and TNFR2 in the same patients. We found that AD patients had higher levels of both TACE cleaved soluble TNFR1 (sTNFR1) and TNFR2 (sTNFR2) in the CSF compared to age- and gender-matched healthy controls. Levels of sTNFR1 correlated strongly with the levels of sTNFR2 (rs = 0.567-0.663, p < 0.01). The levels of both sTNFR1 and sTNFR2 significantly correlated with the TACE activity (rs = 0.491-0.557, p < 0.05). To examine if changes in TACE activity and in levels of cleaved soluble TNFRs are an early event in the course of AD, we measured these molecules in the CSF from 47 subjects with mild cognitive impairment (MCI), which is considered as a preclinical stage of AD. Unexpectedly, we found significantly higher levels of TACE activity and soluble TNFRs in the MCI group than that in AD patients. These results suggest that TACE activity and soluble TNF receptors may be potential diagnostic candidate biomarkers in AD and MCI.     

Expression of tumor necrosis factor alpha and its receptors during cellular differentiation

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine also involved in cellular differentiation processes. TNFalpha and both of its receptors (TNFR1 and TNFR2) can be co-expressed on the same cell, allowing for local signaling. This study has examined the expression of all components necessary for autocrine cytokine regulation during human hematopoietic, epithelial, and mesenchymal models of cellular differentiation. Macrophage and dendritic differentiation of human peripheral blood monocytes decreased their TNFalpha and TNFR2 expression while increasing the TNFR1 mRNA. In colon epithelial cell lines (HT-29 and Caco-2) TNFalpha-, TNFR1-, and TNFR2-expression was decreased upon differentiation. No changes, however, were seen during human skin keratinocyte differentiation. TNFR1 expression was unchanged in all three mesenchymal lineages (adipogenesis, chondrogenesis, osteogenesis) tested. Differentiation decreases the TNFalpha message in adipocytes and the TNFR2 mRNA in adipocytes and osteocytes. Our results demonstrate that there is no general principle for TNFalpha signaling during conversion of cells from progenitor to a more differentiated phenotype. Paracrine signaling by TNFalpha to orchestrate different cell types during tissue development and remodeling, therefore, probably overrides the autocrine regulation of differentiation by TNFalpha. Non-signaling TNF-receptors may protect chondrocytes and osteocytes from the anti-differentiation effects of local TNFalpha production.     

In situ localization of TNFalpha/beta,TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue

Tumor necrosis factor (TNF) has been implicated in several infectious and inflammatory lung diseases. Two closely related variants, TNFalpha and TNFbeta, elicit various cellular responses via two distinct TNF receptors, the 55-kDa TNF-R1 and the 75-kDa TNF-R2. Recently, a TNFalpha-converting enzyme (TACE) was described, which cleaves and releases the membrane-bound TNFalpha. In the present study in normal rat and human lung tissue, the constitutive expression of TNFalpha/beta, TACE and TNF-R1/R2 was investigated by immunohistochemical techniques. In addition, TNFalpha and TNFbeta mRNA were localized by in situ hybridization. Both TNFalpha and TNFbeta were detected in various lung cell types. Expression of TNFalpha was particularly prominent in bronchial epithelial cells and vascular smooth muscle cells, next to alveolar macrophages. Both in situ hybridization for TNFalpha message and TACE immunostaining matched this expression profile. TNFbeta-so far only known to be produced by lymphocytes-was demonstrated in alveolar macrophages, bronchial epithelial cells, vascular smooth muscle cells and endothelial cells at the protein and the message level. Both TNF receptors were detected, with TNF-R1 being prominent on bronchial epithelial cells and endothelial cells, and TNF-R2 being expressed by nearly all cell types. Following LPS stimulation in isolated rat lungs TNFalpha/beta signal intensity was largely reduced due to liberation of stored TNFalpha/beta, while TACE immunoreactivity remained unchanged or was enhanced, demonstrating increased TNF generation.We conclude that both TNFalpha and TNFbeta are constitutively expressed by several non-leukocytic cell types in the human and rat lung. In concert with the expression of TACE and the TNF receptors R1 and R2, this finding suggests in addition to the known role of the TNF system in inflammation physiological functions of the TNF system in different compartments of the adult lung, with the vasculature and the bronchial tissue being of particular interest in addition to the leukocyte/macrophage populations.     

Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1

The TNF signaling pathway is a valuable target in the therapy of autoimmune diseases, and anti-TNF drugs are successfully used to treat diseases such as rheumatoid arthritis, Crohn's disease and psoriasis. By their ability to interfere with inflammatory processes at multiple levels, these TNF blockers have become invaluable tools to inhibit the inflammation induced damage and allow recovery of the affected tissues. Unfortunately this therapy has some drawbacks, including increased risk of infection and malignancy, and remarkably, the onset of new auto-immune diseases. Some of these effects are caused by the unwanted abrogation of beneficial TNF signaling. More specific targeting of the pathological TNF-induced signaling might lead to broader applicability and improved safety. Specificity might be increased by inhibiting the soluble TNF/TNFR1 axis while leaving the often beneficial transmembrane TNF/TNFR2 signaling untouched. This approach looks promising because it inhibits the pathological effects of TNF and reduces the side effects, and it opens the way for the treatment of other diseases in which TNFR2 inhibition is detrimental. In this review we give an overview of in vivo mouse studies of TNF mediated pathologies demonstrating that the blockade or genetic deletion of sTNF or TNFR1 is preferable over total TNF blockade.     

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.     

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.     

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.     

TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm weakness

Doxorubicin, a common chemotherapeutic agent, causes respiratory muscle weakness in both patients and rodents. Tumor necrosis factor-α (TNF), a proinflammatory cytokine that depresses diaphragm force, is elevated following doxorubicin chemotherapy. TNF-induced diaphragm weakness is mediated through TNF type 1 receptor (TNFR1). These findings lead us to hypothesize that TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm muscle weakness. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via intravenous injection. Three days later, we measured contractile properties of muscle fiber bundles isolated from the diaphragm. We tested the involvement of TNF/TNFR1 signaling using pharmaceutical and genetic interventions. Etanercept, a soluble TNF receptor, and TNFR1 deficiency protected against the depression in diaphragm-specific force caused by doxorubicin. Doxorubicin stimulated an increase in TNFR1 mRNA and protein (P < 0.05) in the diaphragm, along with colocalization of TNFR1 to the plasma membrane. These results suggest that doxorubicin increases diaphragm sensitivity to TNF by upregulating TNFR1, thereby causing respiratory muscle weakness.