Increased lipopolysaccharide-induced tumour necrosis factor-alpha, interferon-gamma and interleukin-10 production in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is based on a genetic predisposition, but environmental factors may trigger skin inflammation. According to the hygiene hypothesis, decreased exposure to microbial products in early childhood does not allow sufficient maturation of the immune system that is associated with an increased risk of atopic sensitization. OBJECTIVES: The effect of lipopolysaccharide (LPS) on the cytokine production of peripheral blood mononuclear cells (PBMC) of AD patients and nonatopic controls was studied. PATIENTS AND METHODS: PBMC were isolated from heparinized blood of 10 patients with AD and 10 nonatopic individuals, suspended in culture medium and stimulated with LPS. Cytokine levels in the supernatants were measured by immunoassays. Results Upon stimulation with LPS, PBMC from AD patients produced significantly higher amounts of tumour necrosis factor-alpha, interferon-gamma and interleukin (IL)-10 compared with control PBMC. LPS stimulation blocked the increased spontaneous production of IL-4 and IL-5 by PBMC from AD patients, but had no effect on IL-13 production. CONCLUSIONS: These results demonstrate that the effects of LPS stimulation depend on both the type of cytokine and the origin of PBMC. Endotoxin exposure is suggested to modulate the disease course of AD.

Expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in the brain during experimental group B streptococcal meningitis

We performed mRNA in situ hybridization for TNF-alpha and IL-1beta from infant rats with group B streptococcal meningitis. Induction of both cytokines was seen in the ependyma and the meninges at 4 h. Both cytokines were expressed in the brain parenchyma at 12 h. Induction of IL-1beta mRNA was seen in vessels within the brain cortex. Neutrophilic infiltrate at all time points examined was minimal and could not account for the observed cytokine expression.

Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis

Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) converting enzyme (TACE) contribute synergistically to the pathophysiology of bacterial meningitis. TACE proteolytically releases several cell-surface proteins, including the proinflammatory cytokine TNF-alpha and its receptors. TNF-alpha in turn stimulates cells to produce active MMPs, which facilitate leucocyte extravasation and brain oedema by degradation of extracellular matrix components. In the present time-course studies of pneumococcal meningitis in infant rats, MMP-8 and -9 were 100- to 1000-fold transcriptionally upregulated, both in CSF cells and in brain tissue. Concentrations of TNF-alpha and MMP-9 in CSF peaked 12 h after infection and were closely correlated. Treatment with BB-1101 (15 mg/kg subcutaneously, twice daily), a hydroxamic acid-based inhibitor of MMP and TACE, downregulated the CSF concentration of TNF-alpha and decreased the incidences of seizures and mortality. Therapy with BB-1101, together with antibiotics, attenuated neuronal necrosis in the cortex and apoptosis in the hippocampus when given as a pretreatment at the time of infection and also when administration was started 18 h after infection. Functionally, the neuroprotective effect of BB-1101 preserved learning performance of rats assessed 3 weeks after the disease had been cured. Thus, combined inhibition of MMP and TACE offers a novel therapeutic strategy to prevent brain injury and neurological sequelae in bacterial meningitis.

Tumor necrosis factor-alpha contributes to apoptosis in hippocampal neurons during experimental group B streptococcal meningitis

To evaluate the role of tumor necrosis factor-alpha (TNF-alpha) in neuronal injury in experimental group B streptococcal meningitis, infected neonatal rats were treated with a monoclonal antibody against TNF-alpha (20 mg/kg intraperitoneally) or saline given at the time of infection. Histopathology after 24 h showed necrosis in the cortex and apoptosis in the hippocampal dentate gyrus. Treated animals had significantly less hippocampal injury than did controls (P < .001) but had similar cortical injury and cerebrospinal fluid (CSF) inflammation. The antibody was then administered directly intracisternally (170 microg) to test whether higher CSF concentrations reduced inflammation or cortical injury. Again, hippocampal apoptosis was significantly reduced (P < .01), while cortical injury and inflammation were not. Thus, TNF-alpha played a critical role in neuronal apoptosis in the hippocampus, while it was not essential for the development of inflammation and cortical injury in this model.

Tumor necrosis factor-alpha upregulates 11beta-hydroxysteroid dehydrogenase type 1 expression by CCAAT/enhancer binding protein-beta in HepG2 cells

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of inactive to active glucocorticoids. 11beta-HSD1 plays a crucial role in the pathogenesis of obesity and controls glucocorticoid actions in inflammation. Several studies have demonstrated that TNF-alpha increases 11beta-HSD1 mRNA and activity in various cell models. Here, we demonstrate that mRNA and activity of 11beta-HSD1 is increased in liver tissue from transgenic mice overexpressing TNF-alpha, indicating that this effect also occurs in vivo. To dissect the molecular mechanism of this increase, we investigated basal and TNF-alpha-induced transcription of the 11beta-HSD1 gene (HSD11B1) in HepG2 cells. We found that TNF-alpha acts via p38 MAPK pathway. Transient transfections with variable lengths of human HSD11B1 promoter revealed highest activity with or without TNF-alpha in the proximal promoter region (-180 to +74). Cotransfection with human CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPbeta-LAP expression vectors activated the HSD11B1 promoter with the strongest effect within the same region. Gel shift and RNA interference assays revealed the involvement of mainly C/EBPalpha, but also C/EBPbeta, in basal and only of C/EBPbeta in the TNF-alpha-induced HSD11B1 expression. Chromatin immunoprecipitation assay confirmed in vivo the increased abundance of C/EBPbeta on the proximal HSD11B1 promoter upon TNF-alpha treatment. In conclusion, C/EBPalpha and C/EBPbeta control basal transcription, and TNF-alpha upregulates 11beta-HSD1, most likely by p38 MAPK-mediated increased binding of C/EBPbeta to the human HSD11B1 promoter. To our knowledge, this is the first study showing involvement of p38 MAPK in the TNF-alpha-mediated 11beta-HSD1 regulation, and that TNF-alpha stimulates enzyme activity in vivo.

Transmembrane tumor necrosis factor alpha is required for enteropathy and is sufficient to promote parasite expulsion in gastrointestinal helminth infection

To study the specific role of transmembrane tumor necrosis factor (tmTNF) in protective and pathological responses against the gastrointestinal helminth Trichinella spiralis, we compared the immune responses of TNF-alpha/lymphotoxin alpha (LTalpha)(-/-) mice expressing noncleavable transgenic tmTNF to those of TNF-alpha/LTalpha(-/-) and wild-type mice. The susceptibility of TNF-alpha/LTalpha(-/-) mice to T. spiralis infection was associated with impaired induction of a protective Th2 response and the lack of mucosal mastocytosis. Although tmTNF-expressing transgenic (tmTNF-tg) mice also had a reduced Th2 response, the mast cell response was greater than that observed in TNF-alpha/LTalpha(-/-) mice and was sufficient to induce the expulsion of the parasite. T. spiralis infection of tmTNF-tg mice resulted in significant intestinal pathology characterized by villus atrophy and crypt hyperplasia comparable to that induced following the infection of wild-type mice, while pathology in TNF-alpha/LTalpha(-/-) mice was significantly reduced. Our data thus indicate a role for tmTNF in host defense against gastrointestinal helminths and in the accompanying enteropathy. Furthermore, they also demonstrate that TNF-alpha is required for the induction of Th2 immune responses related to infection with gastrointestinal helminth parasites.

Hypoxia inducible factor-1 alpha induction by tumour necrosis factor-alpha, but not by toll-like receptor agonists, modulates cellular respiration in cultured human hepatocytes

BACKGROUND/AIMS: Genes encoding for some of the mitochondrial proteins are under the control of the transcriptional factor hypoxia inducible factor-1 alpha (HIF-1 alpha), which can accumulate under normoxic conditions in inflammatory states. The aim of this study was to evaluate the effects of cobalt chloride (CoCl(2), a hypoxia mimicking agent), tumour necrosis factor-alpha (TNF-alpha) and toll-like receptor (TLR) -2, -3 and -4 agonists on HIF-1 alpha accumulation, and further on HIF-1 alpha-mediated modulation of mitochondrial respiration in cultured human hepatocytes. METHODS: The human hepatoma cell line HepG2 was used in this study. Cells were treated with CoCl(2), TNF-alpha and TLR-2, -3 and -4 agonists. HIF-1 alpha was determined by Western blotting and mitochondrial respiration in stimulated cells by high-resolution respirometry. RESULTS: CoCl(2), TNF-alpha and TLR agonists induced the expression of HIF-1 alpha in a time-dependent fashion. TNF-alpha and CoCl(2), but not TLR agonists, induced a reduction in complex I-, II- and IV-dependent mitochondrial oxygen consumption. TNF-alpha-associated reduction of cellular oxygen consumption was abolished through inhibition of HIF-1 alpha activity by chetomin (CTM). Pretreatment with cyclosporine A prevented CoCl(2)-induced reduction of complex I- and II-dependent mitochondrial oxygen consumption and TNF-alpha-induced reduction of complex-I-dependent respiration, implicating the involvement of the mitochondrial permeability transition pore openings. TNF-alpha and TLR-2, -3 and -4 agonists induced the expression of vascular endothelial growth factor, which was partially abolished by the blockage of HIF-1 alpha with CTM. CONCLUSIONS: The data suggest that HIF-1 alpha modulates mitochondrial respiration during CoCl(2) and TNF-alpha stimulation, whereas it has no effect when induced with TLR-2, -3 and -4 agonists.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures.

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^

Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.

Binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production.

In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.