Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression

One of the major limitations to current gene therapy is the low-level and transient vector gene expression due to poorly defined mechanisms, possibly including promoter attenuation or extinction. Because the application of gene therapy vectors in vivo induces cytokine production through specific or nonspecific immune responses, we hypothesized that cytokine-mediated signals may alter vector gene expression. Our data indicate that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) inhibit transgene expression from certain widely used viral promoters/enhancers (cytomegalovirus, Rous sarcoma virus, simian virus 40, Moloney murine leukemia virus long terminal repeat) delivered by adenoviral, retroviral or plasmid vectors in vitro. A constitutive cellular promoter (beta-actin) is less sensitive to these cytokine effects. Inhibition is at the mRNA level and cytokines do not cause vector DNA degradation, inhibit total cellular protein synthesis, or kill infected/transfected cells. Administration of neutralizing anti-IFN-gamma monoclonal antibody results in enhanced transgene expression in vivo. Thus, standard gene therapy vectors in current use may be improved by altering cytokine-responsive regulatory elements. Determination of the mechanisms involved in cytokine-regulated vector gene expression may improve the understanding of the cellular disposition of vectors for gene transfer and gene therapy.     

Tumor necrosis factor-alpha and X-linked adrenoleukodystrophy

The two most common forms of X-linked adrenoleukodystrophy (X-ALD), the childhood cerebral form (CCER) and the adult form, adrenomyeloneuropathy (AMN), arise from the same mutations in the X-ALD gene at Xq28. These two forms are distinguished by the degree of cerebral inflammation. Segregation analysis suggests that an autosomal modifying gene may be a major determinant of phenotype in X-ALD. Thus, a modifying gene could be involved in initiating or promoting the inflammatory response. In this study we detected a difference in tumor necrosis factor-alpha (TNF-alpha) bioactivity, but not TNF-alpha protein levels, in serum from some advanced CCER patients. Early-stage CCER patients and AMN patients were in the normal range. Allelic differences in TNF-alpha or levels of soluble TNF receptor did not account for bioactivity differences or phenotypic heterogeneity in X-ALD.     

Tumor necrosis factor-alpha inversely regulates prostaglandin D2 and prostaglandin E2 production in murine macrophages. Synergistic action of cyclic AMP on cyclooxygenase-2 expression and prostaglandin E2 synthesis

Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNFalpha). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFalpha specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFalpha-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFalpha or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2 demonstrated that PGE2 synthase activity was increased after a TNFalpha pretreatment. Taken together, our results suggest that TNFalpha induced a switch from the PGD2 to PGE2 synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFalpha-induced increase in PGE2 through up-regulation of COX2 gene expression.     

Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.     

Tumor necrosis factor-alpha regulates in vivo expression of the rat UCP family differentially

A family of uncoupling proteins (UCPs), free fatty acid anion transporters, plays a crucial role in energy homeostatic thermoregulation. Tumor necrosis factor-alpha (TNF-alpha), a member of the cytokine family, is well known as an endogenous pyrogen. To evaluate the interaction of TNF-alpha with UCPs in thermogenesis, effects of TNF-alpha on rat UCP gene expression were examined in intrascapular brown adipose tissue (BAT), epididymal white adipose tissue (WAT) and soleus muscle (Muscle). Administration of TNF-alpha elevated rectal temperature by 0.7 degree C as well as serum leptin which peaked at 6 h, compared with saline controls. BAT UCP1 mRNA expression was increased by 1.2-fold at 6 h after the TNF-alpha treatment and decreased by 0.8-fold at 16 h after the treatment. In contrast to UCP1 expression in BAT, UCP2 mRNA expressions in BAT, WAT, and Muscle was increased to reach maximum levels of 1.3-, 1.6- and 1.8-fold, respectively, at 16 h after the treatment. UCP3 mRNA in Muscle, but not in BAT or WAT, was exclusively up-regulated by 1.7-fold at 16 h after the treatment. These results indicate that TNF-alpha up-regulates UCP gene expression differentially and tissue dependently, and add novel insights into thermogenesis under conditions of malignancy and inflammation.     

Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17beta-estradiol

OBJECTIVE: To determine the effects of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), dexamethasone, and 17beta-estradiol on the expression of cyclooxygenase-1 (COX-1) and COX-2 in bovine chondrocytes. METHODS: Northern blot analysis was used to quantify COX-1 and COX-2 mRNA expression in primary cultures of bovine chondrocytes and prostaglandin production to evaluate COX activity. RESULTS: IL-1alpha and TNF-alpha increased the expression of COX-2. This effect was independent of de novo protein synthesis and dependent on increased mRNA stability in the case of IL-1alpha. Dexamethasone inhibited the effects of both cytokines. 17beta-estradiol inhibited COX-2 mRNA expression in basal conditions, but had no effect on COX-2 expression induced by cytokines. The specific COX-2 inhibitor compound NS 398 prevented the increase in prostaglandin E2 (PGE2) production induced by the cytokines. COX-1 levels remained stable with all treatments. CONCLUSION: Increase in mRNA stability is a mechanism implicated in the induction of COX-2 by some cytokines. The effects of IL-1alpha and TNF-alpha on PGE2 production are mainly due to an increase in COX-2 activity as shown by the effect of compound NS 398. 17beta-estradiol inhibits COX-2 mRNA expression in basal conditions, suggesting that estrogens could be implicated in the control of cartilage metabolism.     

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.     

Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry

Abnormal expression of polypeptide growth factors and their receptors is closely associated with tumorigenic transformation. In this study tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were analyzed in polyps and proliferative lesions of endometrium as well as in low and high grade endometrial tumors by using in situ hybridization and immunocytochemistry. All samples contained products of the TNF-alpha gene. Histochemical scores (HS), which reflect the proportion of cells positive for TNF-alpha message or protein and the intensities of the signals, were higher for epithelial than for stromal cells. Benign lesions (endometrial polyps) contained little TNF-alpha mRNA or protein, whereas specific message was abundant in proliferative lesions (hyperplasia, adenofibroma). Although neoplastic cells in both low and high grade endometrial tumors contained TNF-alpha mRNA, two major differences were observed: HS for TNF-alpha mRNA were significantly less in low grade than in high grade neoplasms, and TNF-alpha message was restricted to the nucleus in low grade adenocarcinoma cells but was abundant in the cytoplasm of high grade tumor cells. In contrast to cells in benign and proliferative lesions, TNF-alpha protein scores in endometrial tumor cells were inversely rather than positively correlated with TNF-alpha mRNA scores. Collectively, the findings in this study are consistent with the postulate that TNF-alpha is useful to endometrial tumor cells and suggest that production may increase as cells diverge from normal.     

Tumor necrosis factor-alpha induces leukocyte recruitment by different mechanisms in vivo and in vitro

It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.     

Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs

The effects of both recombinant rat tumor necrosis factor-alpha (TNF-alpha) and an anti-TNF-alpha antibody were studied in isolated buffer-perfused rat lungs subjected to either 45 min of nonventilated [ischemia-reperfusion (I/R)] or air-ventilated (V/R) ischemia followed by 90 min of reperfusion and ventilation. In the I/R group, the vascular permeability, as measured by the filtration coefficient (Kfc), increased three- and fivefold above baseline after 30 and 90 min of reperfusion, respectively (P < 0.001). Over the same time intervals, the Kfc for the V/R group increased five- and tenfold above baseline values, respectively (P < 0.001). TNF-alpha measured in the perfusates of both ischemic models significantly increased after 30 min of reperfusion. Recombinant rat TNF-alpha (50,000 U), placed into perfusate after baseline measurements, produced no measurable change in microvascular permeability in control lungs perfused over the same time period (135 min), but I/R injury was significantly enhanced in the presence of TNF-alpha. An anti-TNF-alpha antibody (10 mg/rat) injected intraperitoneally into rats 2 h before the lung was isolated prevented the microvascular damage in lungs exposed to both I/R and V/R (P < 0.001). These results indicate that TNF-alpha is an essential component at the cascade of events that cause lung endothelial injury in short-term I/R and V/R models of lung ischemia.     

Tumor necrosis factor-alpha in sera of obese patients: fall with weight loss

In view of the recent demonstration that obesity in animals and humans is associated with an increase in tumor necrosis factor-alpha (TNFalpha) expression, that this expression falls with weight loss, and that TNFalpha may specifically inhibit insulin action, the possibility that TNFalpha may be a mediator of insulin resistance has been raised. We have undertaken this study to investigate whether serum TNFalpha concentrations are elevated in obese subjects, whether they fall after weight loss, and whether this fall parallels the fall in insulin release after glucose challenge. Obese patients (age range: 25-54, weight mean +/- SD: 96.4 +/- 13.8 kg, body mass index: 35.7 +/- 5.6 kg/m2) were started on a diet program. The mean weight fell to 84.5 +/- 11.3 (P < 0.0001) and body mass index to 31.3 +/- 4.9 (P < 0.0001). Plasma TNFalpha concentrations were markedly elevated in the obese (3.45 +/- 0.16 pg/mL), when compared with controls (0.72 +/- 0.28 pg/mL), and fell significantly (2.63 +/- 1.40 pg/mL) after weight loss (P < 0.02). The magnitude of insulin release after glucose (75 g) challenge (area under the curve) also fell significantly (P < 0.01) after weight loss. The magnitude of weight loss and fall in TNFalpha were related to basal body weight (r = 0.57, P < 0.001) and basal TNFalpha (r = 0.55, P < 0.001) concentrations, respectively, but not to each other or to the glucose-induced insulin release (area under the curve). We conclude that obesity is associated with increased plasma TNFalpha concentrations, which fall with weight loss. Because circulating TNFalpha may mediate insulin resistance in the obese, a fall in TNFalpha concentrations may contribute to the restoration of insulin resistance after weight loss, Thus, TNFalpha may be an important circulating cytokine, which may provide a potentially reversible mechanism for mediating insulin resistance.     

Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells

OBJECTIVE: Low serum levels of mannan binding lectin (MBL) are associated with increased risk of recurrent infections. We determined whether there was an association between serum MBL levels and the course and prognosis of rheumatoid arthritis (RA). METHODS: MBL was analyzed in sera from 99 patients with RA who were included in a longterm prospective study. RESULTS: Compared with controls, a high fraction of patients lacked detectable MBL in serum (11 vs 3%; p = 0.025). Comparing patients with MBL serum levels above and below the median revealed that those with levels below the median were younger at onset of RA (p = 0.043) and had higher erythrocyte sedimentation rate (p = 0.006), joint swelling score (p = 0.019), limitation of joint motion score (p = 0.027), and annual increase in radiographic destruction score (p = 0.053). CONCLUSION: MBL insufficiency may be a contributing pathogenetic factor in RA.     

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.     

Tumor necrosis factor-alpha causes insulin receptor substrate-2-mediated insulin resistance and inhibits insulin-induced adipogenesis in fetal brown adipocytes

Treatment of fetal brown adipocytes with 0.6 nM tumor necrosis factor (TNF)-alpha for 24 h resulted in a partial impairment in the expression of fatty acid synthase, glycerol-3-phosphate dehydrogenase, and glucose transporter (GLUT)-4 messenger RNAs (mRNAs), as well as in the enhancement in the cytoplasmic lipid content in response to insulin. However, the expression of the tissue-specific gene, uncoupling protein 1, is increased by the presence of TNF-alpha. The antiadipogenic effect of TNF-alpha was accompanied by a down-regulation of CCAAT/enhancer-binding protein-alpha and beta mRNAs and up-regulation of CCAAT/enhancer-binding protein-delta, with the expression of peroxisome proliferator-activated receptor-gamma remaining essentially unmodified. Moreover, TNF-alpha caused an insulin resistance on the insulin-induced glucose uptake in brown adipocytes. Pretreatment with TNF-alpha resulted in hypophosphorylation of the insulin receptor in response to insulin, without affecting the number of insulin receptors per cell or its molecular mass. However, insulin receptor substrate (IRS)-1 and IRS-2 signaling in response to insulin showed functional differences. Thus, TNF-alpha pretreatment induced a hypophosphorylation of IRS-2 but not of IRS-1. This effect leads to an impairment in the IRS-2-associated phosphatidylinositol (PI) 3-kinase activation due to a decreased association of alpha-p85 regulatory subunit of PI 3-kinase with IRS-2 but not in the IRS-1-associated PI 3-kinase activation in response to insulin. Our results indicate that TNF-alpha induced an IRS-2- but not IRS-1-mediated insulin resistance on glucose transport and lipid synthesis in fetal brown adipocytes.