In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide

To examine the role of tumor necrosis factor-alpha (TNF alpha) in mediating leptin secretion during an immunological challenge, we studied the effects of lipopolysaccharide (LPS) and TNF alpha on leptin secretion in endotoxin-sensitive C3H/HeOuJ (OuJ) mice, endotoxin-insensitive C3H/HeJ (HeJ) mice, and primary adipocytes cultured from both. Intraperitoneal injection of LPS increased plasma concentrations of TNF alpha and leptin in OuJ mice, but not in HeJ mice, suggesting a causal relationship between the induction of TNF alpha and leptin. Consistent with this idea, i.p. injection of recombinant murine TNF alpha increased plasma leptin in both OuJ and HeJ mice. To determine whether TNF alpha induces leptin secretion by acting directly on fat cells, primary adipocytes from OuJ and HeJ mice were cultured in the presence of TNF alpha or LPS. Whereas LPS was without effect on leptin secretion by adipocytes, TNF alpha induced a marked increase in the cell supernatant leptin concentration. These data demonstrate that TNF alpha plays a role in regulating the increase in leptin caused by LPS. Moreover, they show that TNF alpha can act directly on adipocytes to stimulate leptin secretion. Our results are consistent with the emerging view that leptin is a key hormone coupling immune system activity to energy balance.     

Increased tumor necrosis factor-alpha (TNF-alpha) gene expression in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis

Increased airway resistance and airway hyperresponsiveness induced in rats by infection with parainfluenza type I (Sendai) virus is associated with bronchiolar fibrosis. To determine whether increased tumor necrosis factor (TNF)-alpha gene expression is an important regulatory event in virus-induced bronchiolar fibrosis, pulmonary TNF-alpha mRNA and protein expression was assessed in rat strains that are susceptible (Brown Norway; BN) and resistant (Fischer 344; F344) to virus-induced bronchiolar fibrosis. Virus-inoculated BN rats had increased TNF-alpha pulmonary mRNA levels (P < 0.05) and increased numbers of bronchiolar macrophages and fibroblasts expressing TNF-alpha protein compared with virus-inoculated F344 rats (P < 0.05). Virus inoculation also induced elevated TNF-alpha mRNA and protein levels (P < 0.05) in cultured rat alveolar macrophages (NR8383 cells). A 55-kd soluble TNF receptor-immunoglobulin G fusion protein (sTNFR-IgG) was used to inhibit TNF-alpha bioactivity in virus-inoculated BN rats. Treated rats had fewer proliferating bronchiolar fibroblasts, as detected by bromodeoxyuridine incorporation, compared with virus-inoculated control rats (P < 0.05). There was also increased mortality in p55sTNFR-IgG-treated virus-inoculated rats associated with increased viral replication and decreased numbers of macrophages and lymphocytes in bronchoalveolar lavage fluid (P < 0.05). The results of this study indicate that 1) Sendai virus can directly up-regulate TNF-alpha mRNA and protein expression in macrophages, 2) TNF-alpha is an important mediator of virus-induced bronchiolar fibrosis, and 3) TNF-alpha has a critical role in the termination of Sendai viral replication in the lung.     

Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.     

Autocrine regulation of matrix metalloproteinase-9 gene expression and secretion by tumor necrosis factor-alpha (TNF-alpha) in NB4 leukemic cells: specific involvement of TNF receptor type 1

Matrix metalloproteinases have been reported to be involved in tumor cell invasion and metastasis. Dissemination of malignant cells in acute myeloid leukemia (AML) may be mediated by similar mechanisms. Here, we report, that the t(15/17)+ acute promyelocytic leukemia (APL) cell line NB4 constitutively expresses and releases the proenzyme form of matrix metalloproteinase-9 (MMP-9, 92 kDa type IV collagenase/gelatinase, gelatinase B), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1). Both proteins were identified by N-terminal amino acid sequence analysis after purification using gelatin Sepharose affinity chromatography. Whereas 12-O-tetradecanoylphorbol-13 acetate (TPA) increased both MMP-9 and TIMP-1 mRNA levels, tumor necrosis factor-alpha (TNF-alpha) stimulated only MMP-9 gene expression in a dose- and time-dependent manner. Neutralizing monoclonal antibodies (MoABs) to TNF-alpha (anti-TNF-alpha) decreased the constitutive and TPA-dependent expression of MMP-9 but did not influence TIMP-1 expression, either in unstimulated or in TPA-treated NB4 cells. FACS analyses showed that NB4 cells express both TNF receptor 1 (TNF-R1) and TNF-R2 to a similar extent. Blocking MoABs against TNF-R 1 (anti-TNF-R1) decreased the constitutive expression of MMP-9, whereas anti-TNF-R2 had almost no effect. Our results show, that in NB4 cells the expression of MMP-9 but not of TIMP-1 is maintained by autocrine stimulation with TNF-alpha. Thus, leukemic cells may be enabled to leave the bone marrow and infiltrate peripheral tissues by a dysfunction in the regulation of the MMP-9:TIMP-1 equilibrium, possibly triggered through autostimulation by TNF-alpha.     

Influence of the anti-allergic agent, azelastine, on tumor necrosis factor-alpha (TNF-alpha) secretion from cultured mouse mast cells

The survival of infants with trisomy 14 mosaicism has been scarcely reported in the literature, with only 15 cases being documented up to 1992. We present a case of a dichorionic twin pregnancy in which prenatal sonography at 24 weeks' gestation showed that one of the twins had several anomalies including intrauterine growth restriction, alobar holoprosencephaly, a cleft lip and palate, a recessive chin, a small stomach, overlapping fingers, a ventricular septal defect, and polyhydramnios. Twin 2 was structurally normal. In view of the lethal condition and associated polyhydramnios affecting one of the twins, prenatal surveillance for signs of tense polyhydramnios and premature labour was undertaken. The pregnancy proceeded uneventfully until 37 weeks, at which time a Caesarean section was performed. At birth, neonatal blood from the abnormal twin confirmed trisomy 14 mosaicism in 12 per cent of lymphocytes. The infant died on day 36 of life.     

In vitro mechanism(s) of ultraviolet-induced tumor necrosis factor-alpha release in a human keratinocyte cell line

It has been demonstrated that ultraviolet (UV) irradiation is able to induce both in vivo and in vitro, tumor necrosis factor-alpha (TNF) release. The purpose of the present study was to evaluate, using a human keratinocyte cell line NCTC 2544, the mechanism(s) of UV-induced TNF release and the ability of commonly used sunscreens to modulate UV-induced TNF release. TNF release can be partially prevented both by adding an anti-human IL-1 alpha antibody after irradiation, suggesting an autocrine effect of IL-1 alpha in inducing TNF release, and by adding antioxidants indicating also a role of oxidant species. TPCK, a I kappa-B alpha protease inhibitor, was able to virtually abolish UV-induced TNF release, indicating that UV-induced TNF release requires NF-kappa B activation. Anti-human IL-1 beta antibody was ineffective as expected, considering that keratinocytes are unable to process pre-IL-1 beta to its active form. To evaluate the sunscreen's modulation on UV-induced TNF release, confluent cells were irradiated, in the presence or absence of the tested sunscreens (Uvinul MS40, Uvinul P25 and Uvinul DS49). Different IC50 values could be calculated, which may be related to different UV absorption spectrums: Uvinul MS40 offers great protection by virtue of its broader absorption spectrum, closely followed by Uvinul P25 and finally by Uvinul DS49.     

Functional analysis of tumor necrosis factor (TNF) receptors in TNF-alpha-mediated insulin resistance in genetic obesity

Although obesity has become the most common metabolic disorder in the developed world and is highly associated with insulin resistance and noninsulin-dependent diabetes mellitus, the molecular mechanisms underlying these disorders are not clearly understood. Tumor necrosis factor-alpha (TNF-alpha) is overexpressed in obesity and is a candidate mediator of obesity-induced insulin resistance. Complete lack of TNF-alpha function through targeted mutations in TNF-alpha gene or both of its receptors results in significant improvement of insulin sensitivity in dietary, chemical, or genetic models of rodent obesity. In this study, we have analyzed the in vivo role of TNF signaling from p55 [TNF receptor (TNFR) 1] and p75 (TNFR 2) TNFR in the development of insulin resistance by generating genetically obese mice (ob/ob) lacking p55 or p75 TNFRs. In the ob/ob mice, the absence of p55 caused a significant improvement in insulin sensitivity. p75 deficiency alone did not affect insulin sensitivity but might potentiate the effects of p55 deficiency in animals lacking both TNFRs. These results indicate that TNF-alpha is a component of insulin resistance in the ob/ob model of murine obesity and p55 TNFR is the predominant receptor mediating its actions.     

Potential mechanism(s) involved in the regulation of glycogen synthesis by insulin

The aim of this study was to elucidate the significance of aberrant DNA methylation in gastric carcinogenesis. The DNA methylation status at the D17S5 locus, at which a candidate tumor suppressor gene, HIC-1, was identified, of gastric cancers and non-cancerous gastric mucosae from 42 gastric cancer patients was examined by Southern blotting using a methylation-sensitive restriction enzyme. DNA hypermethylation was observed in 15, 13, 25 and 45% of the tissues showing no remarkable histological findings, chronic gastritis without intestinal metaplasia, intestinal metaplasia and gastric cancer, respectively. The incidence of DNA hypermethylation was significantly higher in gastric cancers than in non-cancerous gastric mucosae (P < 0.05). DNA hypermethylation was often accompanied by allelic loss at the same locus in gastric cancers. DNA hypermethylation at the D17S5 locus, which was even detected in precancerous conditions, including intestinal metaplasia, may play a role in gastric carcinogenesis.     

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities in the sera and milk of cows with naturally occurring coliform mastitis

The presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) activities was determined in milk and serum of cows with naturally occurring coliform mastitis (CFM). TNF-alpha was detected in the sera from 26 of 32 cows with CFM. TNF-alpha levels were higher in the sera than in the milk. IL-6 was high in the sera of surviving CFM animals, but was low in animals that died and in healthy controls. Furthermore, the mean level of IL-6 was 20-fold higher in the milk than in the sera of mastitic cows. The level of IL-6 in the serum was correlated to that in the milk in individual animals. The presence of IL-6 and TNF-alpha in the sera appears to relate to severe clinical condition of CFM, in the milk whereas they may play a role in generating inflammation of the mammary gland.     

Responses of human glioblastoma cells to human natural tumor necrosis factor-alpha: susceptibility, mechanism of resistance and cytokine production studies

Responses and susceptibility of 14 human glioblastoma cell lines to human natural tumor necrosis factor-alpha (TNF) were studied in vitro. Susceptibility of glioblastoma cells to TNF varied in experimental conditions applied. Most of glioblastoma cell lines were resistant to cytotoxic activity of TNF in a MTT assay at concentrations below 16 U/ml for 72 h exposure. However, TNF at higher dose, in prolonged exposure and against low density of target cells was antiproliferative for certain glioblastoma cultures. TNF exposure at 10 U/ml for 48 h suppressed DNA synthesis in 9 of 14 glioblastoma cultures, but increased in 3 cultures. In addition, colony forming assay showed anti-clonogenic activity of TNF in 5 of 6 glioblastoma cell lines tested. In spite of their low susceptibility to TNF, glioblastoma cells well responded to TNF stimulation at low dose (10 U/ml) for a short period in the absence of cell damage. Productions of Interleukin-6 (IL-6), IL-8-like activity, granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2 (PGE2) and manganous superoxide dismutase (Mn-SOD) were enhanced or induced by the low-dose TNF stimulation. Mn-SOD, a protein protective against oxidative cell damage, was well induced in time- and dose-dependent manner, however did not correlate with TNF resistance. Whereas the levels of PGE2 in TNF-susceptible cell lines, H-4 and SF-188, were higher than those of other lines. In conclusion, most of glioblastoma cells are resistant to TNF cytotoxic effects, but highly responsive to TNF stimulation. Its effect on glioblastoma cells appears to modulate cell differentiation rather than to kill the cells.     

Effect of morphine on lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo: involvement of the sympathetic nervous system

The unicellular flagellate Euglena gracilis was used in order to assess the inhibition of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) mutagenicities, which induce white mutants due to the irreversible loss of chloroplasts. All tested compounds, including o-aminobenzoic acid and p-aminobenzoic acid, salicylic acid, acetylsalicylic acid, sodium salicylate and p-aminosalicylic acid, were not mutagenic per se and inhibited MNNG mutagenicity by at least 50%. The last two compounds inhibited by at least 50% also MNU mutagenicity.     

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.     

Intestinal epithelial cells down-regulate macrophage tumor necrosis factor-alpha secretion: a mechanism for immune homeostasis in the gut-associated lymphoid tissue

BACKGROUND: The gut lumen contains more than 10(6) organisms per gram of luminal contents. The mechanisms that limit the response of macrophages in the lamina propria to these microbial antigens are unknown, although an intrinsic defect in this mechanism may contribute to the development of inflammatory bowel disease. Intestinal epithelial cells (IEC) may play an important role in mediating tonic down-regulation of local immune cell activation. The purpose of this study was to discern whether IEC might modulate macrophage activation in response to a variety of microbial stimuli. METHODS: Thioglycollate-elicited murine peritoneal macrophages were activated by endotoxin, zymosan, Escherichia coli, and Candida albicans in the presence or absence of IEC from the rat intestinal epithelial cell line IEC-6. Macrophage tumor necrosis factor-alpha (TNF-alpha) secretion was determined by enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide or zymosan-activated macrophages in coculture with IEC secreted significantly less TNF-alpha than macrophages cultured alone. The inhibitory effect of the IEC was dependent on their activation by lipopolysaccharide. Interleukin-1 alpha production was not affected. IEC-mediated suppression of macrophage TNF-alpha secretion was reversed by indomethacin but not by neutralizing antibody to TGF-beta. CONCLUSIONS: Lipopolysaccharide-activated IEC down-regulate macrophage TNF-alpha secretion in response to microbial stimuli through a prostanoid-mediated mechanism. IEC may mediate tonic down-regulation of immune cell activation in the gut-associated lymphoid tissue and may thereby regulate local and systemic inflammatory responses.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.     

Complete regression of anaplastic astrocytoma by intravenous tumor necrosis factor-alpha (TNF alpha) after recurrence: a case report

A 61-year-old male patient had a recurrence of anaplastic astrocytoma after initial chemoradiotherapy. After histologic confirmation of the recurrent tumor, he was treated with intravenous administration of human natural tumor necrosis factor-alpha (TNF alpha). Morphometric analysis of the tumor volumes demonstrated that the tumor continued to regress for 8 months during the course of intravenous TNF alpha therapy, and finally disappeared (total dose of intravenous TNF alpha, 785,000 JRU). The regression of tumor paralleled the neurologic improvement. He has had no signs of recurrence for 2 years. The present experience indicates that at least a certain population of malignant gliomas can be successfully treated by a systemic administration of TNF alpha without a major adverse effect.     

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.