Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome

The interaction of TNF-alpha with TNF receptor 1 (TNFR1) activates several signal transduction pathways that lead to apoptosis or NF-kappa B-dependent inflammation and immunity. We hypothesized that host TNFR1 expression contributes to noninfectious lung injury and inflammation commonly observed after bone marrow transplantation (BMT), termed idiopathic pneumonia syndrome (IPS). C57BL/6 TNFR1-sufficient (TNFR1(+/+)) and -deficient (TNFR1(-/-)) mice were total body irradiated with or without cyclophosphamide conditioning and were given bone marrow plus IPS-inducing donor spleen T cells from B10.BR wild-type mice. TNFR1(-/-) recipient mice exhibited improved early post-BMT survival associated with decreased permeability edema. In addition, the low lung compliance measured in anesthetized, ventilated TNFR1(+/+) mice on day 7 after BMT was restored to baseline during TNFR1 deficiency. Importantly, bronchoalveolar lavage fluid (BALF) inflammatory cells from TNFR1(-/-) vs. TNFR1(+/+) mice generated less nitric oxide (.NO) and nitrating species and exhibited suppressed programmed cell death as assessed using flow cytometry. However, cellular infiltration and levels of proinflammatory cytokines and chemokines were generally higher in BALF collected on day 7 after BMT from TNFR1(-/-) compared with TNFR1(+/+) recipient mice. Our results support a major role of host TNFR1 in regulation of .NO production and lung dysfunction after allogeneic BMT.     

Modulation of allergic inflammation in mice deficient in TNF receptors

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.     

Effects of theophylline on insulin receptors and insulin action in the adipocyte

Summary The effects of theophylline on insulin receptors and insulin action in isolated rat adipocytes were studied. Theophylline reduced insulin binding by a decrease of receptor affinity. As concentration-response curves revealed, the effect was paralleled by a reduction of the cellular ATP content. Basal as well as insulin-stimulated glucose transport (2-deoxyglucose and 3-O-methylglucose uptake) were inhibited by much smaller theophylline concentrations (0.15–0.6 mM ) than those necessary to reduce insulin binding and to lower ATP levels (1–4.8 mM), or to stimulate lipolysis (0.3-2.4 mM). Insulin fully antagonized the effect of theophylline on lipolysis but failed to reverse the inhibition of glucose transport completely. The results suggest that (a) theophylline impairs insulin action at a post-receptor level and, at higher concentrations, by a decrease of receptor binding, (b) the reduction of insulin receptor affinity probably reflects ATP depletion of the adipocyte, and (c) the xanthine inhibits glucose transport independently from its effects on lipolysis.     

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.     

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.     

Impaired insulin-mediated inhibition of lipolysis and glucose transport with aging

Regulation of hormone action with aging has been extensively studied; adipocytes provide an interesting model for some of these questions. We have compared the ability of insulin to stimulate glucose uptake and suppress lipolysis in adipocytes isolated from two month and twelve month-old rats. The ability of insulin to stimulate maximal glucose transport was decreased in adipocytes from the older rats (P less than 0.001); as well, insulin's EC50 was also higher (P less than 0.01) in these cells. Furthermore, these defects were present when insulin-stimulated glucose transport was measured in the presence or absence of adenosine deaminase which metabolizes endogenously released adenosine. Endogenously released adenosine is a stimulator of glucose transport and an inhibitor of lipolysis. Maximal suppression of isoproterenol-induced lipolysis by insulin was similar when adipocytes isolated from the two age groups were incubated in the absence of adenosine deaminase. However, maximal insulin-mediated suppression of lipolysis was found to be significantly decreased (P less than 0.001) in adipocytes isolated from older rats when the experiments were done in the presence of adenosine deaminase; also, insulin's EC50 was increased in these cells under these conditions (P less than 0.001). These results emphasize the importance of the adenosine receptor in modulating the response of isolated adipocytes to insulin, particularly for lipolysis, and document the presence of age-associated defects in insulin regulation of both glucose transport and lipolysis.     

Signaling pathways utilized by tumor necrosis factor receptor 1 in adipocytes to suppress differentiation

Tumor necrosis factor-alpha (TNFalpha) has profound effects on cultured adipocytes, one of which is the inhibition of terminal differentiation. Previous studies in TNF receptor (TNFR)-deficient preadipocytes have demonstrated that the anti-adipogenic effect of both secreted and transmembrane TNFalpha is mediated solely by TNFR1. In this study, we performed a structure-function analysis of the intracellular domains of TNFR1 and investigated the signaling pathway(s) involved in TNFR1-mediated inhibition of adipocyte differentiation. Our results show that repression of adipogenesis required the juxtamembrane and death domains and was independent of the pathways involving nuclear factor kappaB and neutral sphingomyelinase.     

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.     

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.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

Genetic disregulation of TNF alpha and TNF alpha type II receptors in colon cancer at the II and III stage of disease

The expression of the genes coding TNFalpha and TNF RII receptors (TNF RII: TNFR2 membrane and soluble domain, TNFR2/R7 soluble domain) was analysed in colon cancer at the II and III stage of disease, by estimation of mRNA expression. The study included 80 patients with histopathologically confirmed adenocarcinoma. The number of TNFalpha mRNA, TNFR2 mRNA and TNFR2/R7 mRNA copies were estimated in tumour and healthy tissue. The highest number of mRNA TNF-alpha copies were investigated in all samples of tissue and independently of the stage of disease. Simultaneously, we noticed the largest number of mRNA copies for TNFalpha and TNF R2/R7 in healthy cells at stage III of the disease. It is possible to draw a hypothetical line separating the anti-cancer activity of TNFalpha and its influence on cancer progression.     

Curcumin is a direct inhibitor of glucose transport in adipocytes

Curcumin has been reported to inhibit insulin signaling and translocation of GLUT4 to the cell surface in 3T3-L1 adipocytes. We have investigated the effect of curcumin on insulin signaling in primary rat adipocytes. Curcumin (20 μM) inhibited both basal and insulin-stimulated glucose transport (2-deoxyglucose uptake), but had no effect on insulin inhibition of lipolysis. Dose–response experiments demonstrated that curcumin (0–100 μM) inhibited basal and insulin-stimulated glucose transport, but even at the highest concentration tested did not affect lipolysis. Inhibition was equal in cells that had been pre-incubated with curcumin and in cells to which curcumin was added immediately before the glucose transport assay. Similarly, time-course experiments revealed that the inhibitory effect of curcumin was evident at the earliest time point tested (30 s). Thus it is unlikely that inhibition of insulin signaling or of translocation of GLUT4 to the cell surface is involved in the inhibitory effect of curcumin. Curcumin did not affect the stimulatory action of insulin on phosphorylation of Akt at serine 473. We conclude that curcumin is a direct inhibitor of glucose transporters in rat adipocytes.     

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.     

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.     

Structure-Function Relationship of Tumor Necrosis Factor (TNF) and Its Receptor Interaction Based on 3D Structural Analysis of a Fully Active TNFR1-Selective TNF Mutant

Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR.Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function.To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.     

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.