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.     

Selective induction of tumor necrosis factor receptor type II gene expression by tumor necrosis factor-alpha in C6 glioma cells

The aim of this study, in rabbit tibia, was an evaluation of the early reactions of the tissues to the insertion of polylactic membranes, used in connection with titanium implants. The specimens were retrieved after 1-4 weeks, and a histological analysis was performed. It was possible to see that, in the early implantation phases, no degradation of the macrostructure of the membrane was present. On the outer portion of the membrane many multinucleated giant cells (MGC) were present and membrane fragments were present inside the cytoplasm of these cells. These cells could explain the inflammatory processes reported, in some reports, with the use of materials made by polylactic and polyglycolic acid. We did not observe detrimental effects in the bone tissue around the membrane, and the membrane appeared to have a mechanical stability for the time necessary for bone regeneration.     

Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium

The myocardium is a highly adaptive tissue, as evidenced by phenotypic alterations throughout development and under conditions of altered hemodynamic load. With pressure overload, the myocardium displays adult-to-fetal transitions in expression of contractile and non-contractile proteins. Most intriguing is the fact that many of these transitions are also observed in the senescent heart. The purpose of this work was to establish if the thin filament regulatory proteins, troponin I and troponin T, exhibit reexpression of early developmental isoforms, suggestive of coordinate reprogramming of contractile protein isoform expression. As a functional index of reexpression of the early isoform of troponin I, slow skeletal troponin I, myofibrils were isolated from 12 and 24-month-old Fischer 344 rat ventricles and assayed for myofibrillar ATPase activity at pH 7.0 and 6.5. Both preparations displayed rightward shifts in Ca-ATPase relationships with no differences between groups. SDS-PAGE and Western blot analysis showed that whereas myosin heavy chain expression underwent a transition to predominance of the early development isoform, beta-myosin heavy chain, there was no reexpression of the fetal isoforms of either troponin I or troponin T in the rat heart at 24 months of age. Northern blot analysis using cDNA probes specific for cardiac or slow skeletal troponin I also confirmed the lack of slow skeletal reexpression in the 24-month ventricle. These results are significant in that they demonstrate a lack of coordinate expression of contractile protein isoforms under myocardial adaptation to the aging process.     

Chronic alcohol ingestion enhances tumor necrosis factor-alpha expression and salivary gland apoptosis

We investigated the extent of induction in sublingual salivary gland cells apoptosis and tumor necrosis factor-alpha (TNF-alpha) expression with chronic ethanol ingestion. The experiments were conducted on rats pair-fed for 8 weeks with alcohol-containing and control liquid diet. The animals were killed, their sublingual glands dissected, and the glandular tissue used for quantization of TNF-alpha expression and the assays of acinar cells apoptosis employing sandwich enzyme immunoassay for histone-associated DNA fragments. The mean value for TNF-alpha in sublingual gland of the control group was 22.3 pg/mg of protein and showed a 1.6-fold increase in the chronic ethanol diet group to 36.5 pg/mg of protein. In comparison with the controls, the sublingual gland of the chronic ethanol diet group also exhibited a 3.4-fold enhancement in acinar cell apoptosis. These findings suggest that chronic ethanol ingestion causes the enhancement in TNF-alpha expression and leads to the induction in salivary gland acinar cells apoptosis. Thus, the diminished secretion of saliva in alcoholics may be a direct result of increased salivary gland apoptosis.     

Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression

High concentrations of bile acids have been reported as injurious to hepatocytes. We report the influence of various combinations of bile acids on the liver-specific function of cultured rat hepatocytes. Using 4 bile acids (glycocholate [GC], taurocholate [TC], glycohenodeoxycholate [GCDC], and taurochenodeoxycholate [TCDC]), we obtained 6 bile-acid mixtures, each containing equal amounts of 2 bile acids (total bile acids [TBA], 2 mM). Changes in gluconeogenesis, ureagenesis, DNA contents, medium alanine aminotransferase, and morphologies were compared among the paired bile acid compositions by measuring the C/CDC ratio ([GC + TC]/[GCDC + TCDC]) of each. In terms of their relative impairments of ureagenesis from greatest to least, the acids were GCDC, TCDC, and GC, which was almost the same as TC. When the C/CDC ratio was 0, the values of all parameters measured deteriorated. When the C/CDC ratio was 1 in the presence of 1 mM GCDC, only the rate of ureagenesis was diminished. When the C/CDC ratio was infinite, no hepatocellular injury was observed. GCDC and TCDC, together or separately, showed significant hepatocellular injury when the TBA concentration was 2 mM.     

TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.     

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.     

Obstructive jaundice in rats results in exaggerated hepatic production of tumor necrosis factor-alpha and systemic and tissue tumor necrosis factor-alpha levels after endotoxin

BACKGROUND: Obstructive jaundice (OJ) predisposes patients to postoperative sepsis. We determined whether OJ led to an increased endotoxin stimulated tumor necrosis factor-alpha (TNF-alpha) production by macrophage-rich organs and whether a lack of intraluminal gut bile contributed to this increased sensitivity. METHODS: Rats underwent laparotomy and common bile duct ligation and division (CBDL) or sham operation after they were given low-dose endotoxin or saline solution (NS). TNF-alpha levels in plasma, perfusate from the isolated perfused rat liver, and tissue from lung, spleen, and liver were measured 90 minutes later. An additional group underwent creation of a choledochal-vesical fistula and endotoxin administration. RESULTS: The plasma TNF-alpha, liver perfusate TNF-alpha, and the tissue TNF-alpha levels in liver, lung, and spleen were significantly elevated in the CBDL + endotoxin (CBDL + ETX) group compared with the SHAM + ETX and CBDL + NS groups (p < 0.05). The choledochal-vesical fistula group after endotoxin had plasma TNF-alpha levels only 27% that of the CBDL + ETX group (p < 0.05). CONCLUSIONS: OJ sensitizes macrophage-rich organs to produce larger amounts of TNF-alpha in response to endotoxin. This sensitization is not solely due to decreased intraluminal gut bile.     

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.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-alpha gene promoter

To obtain insight into the basic mechanisms controlling endocytosis, we tested the effects of different perfusates containing the cytological stain light green on endocytosis and ultrastructure of the vacuolar apparatus in renal proximal tubule cells. Rat proximal tubules were microinfused in vivo for 2 min in the presence or absence of light green with the following solutions: (A) perfusates containing inorganic salts and (B) perfusates with organic components or protein. In other experiments, the tubules were first microinfused for 2 min with 0.9% NaCl in the presence or absence of light green, then 15 min later further microinfused with or without light green using either group A or B solutions in order to either aggravate or reverse possible changes. All infused tubules were fixed after 5 min with 1% glutaraldehyde and examined by electron microscopy. In tubules microinfused without light green, the endocytic vacuolar apparatus in the apical cytoplasm showed a normal ultrastructure. However, microinfusion of solutions containing light green in either inorganic salts or a low concentration of protein caused significant changes in the apical endocytic apparatus. Large endocytic vacuoles were absent, and invaginations and small endocytic vacuoles were decreased in frequency. On the other hand, the amount of dense apical tubules was significantly increased, and in some cells dense apical tubules had transformed into a cisternalike network. These changes were aggravated in tubules which received a second microinfusion of NaCl and reversed in tubules that received a second infusion of protein. Furthermore, in the tubules microinfused with light green using perfusates containing organic components or protein, the apical cytoplasm of proximal tubule cells showed an essentially normal endocytic apparatus. The present study demonstrates that microinfusion of renal proximal tubules with light green disrupted normal endocytic membrane trafficking and recycling. These changes could be prevented or reversed by microinfusion of solutions containing protein or organic components.     

Tumor necrosis factor-alpha gene polymorphism in chronic bronchitis

Airway inflammation is an important pathologic feature in chronic bronchitis, and we hypothesized that individuals with greater inflammatory responses may be more likely to acquire the disease. A polymorphism at -308 position of the tumor necrosis factor-alpha (TNF-alpha) gene has been described, with the rarer allele, TNF2, demonstrated to have higher inducibility in vitro. We investigated the distribution of this polymorphism in a case-control study. The genotype was determined in 42 male patients with chronic bronchitis, 42 sex-, age-, and smoking index-matched control subjects, and 99 random-sampled schoolchildren. We report here that the TNF2 allele is overrepresented in the patient group. The allele frequency of TNF2 is 5.1% in the schoolchildren, 2.4% in the control group, and 19% in the bronchitis group (p < 0.01). Carriage of the TNF2 allele confers a higher risk to the development of chronic bronchitis (odds ratio = 11.1, 95% CI = 2.89-42.57). The results demonstrate the important pathologic role of TNF-alpha in chronic bronchitis and suggest that greater inflammatory response may predispose an individual to this disease.     

Contribution of tumor necrosis factor-alpha to pulmonary cytokine expression and lung injury after hemorrhage and resuscitation

OBJECTIVE: To examine the role of tumor necrosis factor-alpha (TNF-alpha) in producing acute inflammatory lung injury after hemorrhage and resuscitation. DESIGN: Prospective, controlled animal study. SETTING: Research laboratory. SUBJECTS: Male BALB/c mice. INTERVENTIONS: Treatment with rat antimouse monoclonal anti-TNF-alpha antibodies or control rat immunoglobulin G 1 hr after 30% blood volume hemorrhage and resuscitation. MEASUREMENTS AND MAIN RESULTS: Therapy with monoclonal anti-TNF-alpha antibodies prevented the posthemorrhage increases in pulmonary TNF-alpha and interferon-gamma protein levels that normally occur after blood loss. Administration of monoclonal anti-TNF-alpha antibodies also diminished the increases in interleukin-1 beta, interleukin-6, and interleukin-10 mRNA, but not the increases in TNF-alpha and interferon-gamma mRNA, which are found in the lungs following hemorrhage. In addition, therapy with monoclonal anti-TNF-alpha antibodies was associated with significant improvement in the histologic parameters of posthemorrhage lung injury, particularly intra-alveolar hemorrhage and pulmonary vascular congestion. CONCLUSIONS: These results indicate that TNF-alpha has an important role in the development of acute inflammatory lung injury after blood loss. Blockade of TNF-alpha with monoclonal antibodies significantly reduces hemorrhage-induced lung injury.     

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.     

The locus of tumor necrosis factor-alpha action in lung inflammation

The pulmonary host response to infection and inflammation appears, at least in part, to be compartmentalized from the systemic host response. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in lung inflammation and injury, but its site(s) of action has not been clearly defined. To investigate this, transgenic mice (surfactant apoprotein C promotor/soluble TNF receptor type II-Fc fusion protein ([SPCTNFRIIFc] mice) were generated in which TNF-alpha was selectively antagonized in the distal lung through tissue-specific expression of sTNFRIIFc, a soluble TNF inhibitor. The lung inflammatory response in these mice to pulmonary challenge with Micropolyspora faeni antigen or lipopolysaccharide (LPS) was compared with the response of wild-type mice, wild-type mice treated with recombinant sTNFRIIFc intravenously, and type I TNF-receptor knockout mice. Recruitment of polymorphonuclear leukocytes (PMN) to the lung after challenge with M. faeni antigen was essentially abolished in the TNFRI knockout mice and markedly reduced in the SPCTNFRIIFc mice. Wild-type mice given sTNFRIIFc intravenously in amounts resulting in lung concentrations similar to those in SPCTNFRIIFc mice also showed significantly reduced lung PMN recruitment, whereas those given doses that achieved such concentrations in the blood but low levels in the lung did not. In contrast, PMN recruitment to the lung following aerosol challenge with LPS was reduced significantly in the TNFRI knockout mice and in mice given high-dose sTNFRIIFc intravenously, but was not reduced significantly in SPCTNFRIIFc mice. Thus, inhibition of PMN recruitment in response to M. faeni antigen correlated largely with the extent of intrapulmonary inhibition of TNF-alpha, whereas the response to LPS correlated best with the extent of extrapulmonary inhibition of TNF-alpha. These studies indicate that TNF-alpha may act at different loci to mediate lung inflammation, with the site of action depending in part on the nature of the inflammatory stimulus, and that SPCTNFRIIFc mice provide a tool by which the locus of TNF action can be addressed.