Elevated TNF-α mRNA Levels Research Articles

Reactive oxygen species up-regulates SOCS-3 in 3T3-L1 adipocytes

We investigated the effect of increased intracellular reactive oxygen species(ROS) on SOCS-3 expression in 3T3-L1 adipocytes. Increased intracellular ROS levels in 3T3-L1 adipocytes were achieved by two methods of exposure to H2O2 and the occurrence of oxidative stress in cells was assessed by flow cytometry . Expression of SOCS-3 mRNA and that of some adipokines were measured by real time PCR. The level of SOCS-3 protein was determined by western blot. The effect of the antioxidant alpha-lipoic acid was also investigated. Both the relatively mild increased intracellular ROS and the acute but transient increased ROS elevated the levels of SOCS-3 mRNA and protein in 3T3-L1 adipocytes, acompanied with elevated levels of TNF-α mRNA and resistin mRNA and the decreased levels of adiponectin mRNA and secretory adiponectin in culture medium. α-lipoic acid could attenuate the effects of ROS on 3T3-L1 adipocytes. We hypothesized that in mature 3T3-L1 adipocytes, SOCS-3 could be upregulated directly by the induction of increased intracellular ROS and to some extent which was up-regulated by adipokine modulation.

Probiotic Pediococcus acidilactici modulates both localised intestinal- and peripheral-immunity in tilapia (Oreochromis niloticus)

The application of probiotics in aquaculture has received concerted research efforts but the localised intestinal immunological response of fish to probiotic bacteria is poorly understood. Therefore, a study was conducted to evaluate the probiotic effect of Pediococcus acidilactici on Nile tilapia (Oreochromis niloticus) with specific emphasis on intestinal health and probiotic levels as well as system level responses such as growth performance, feed utilization and haemato-immunological parameters under non-challenged conditions. Fish (9.19 ± 0.04 g) were fed either a control diet or a P. acidilactici supplemented diet (at 2.81 × 106 CFU g−1) for six weeks. At the end of the study the probiotic was observed to populate the intestine, accounting for ca. 3% (1.59 × 105 CFU g−1) of the cultivable intestinal bacterial load. Real-time PCR indicated that the probiotic treatment may potentiate the immune-responsiveness of the intestine as up-regulation of the gene expression of the pro-inflammatory cytokine TNFα was observed in the probiotic fed fish (P < 0.05). Light microscopy observations revealed elevated intraepithelial leucocyte (IEL) levels in the intestine of P. acidilactici fed tilapia after six weeks (P < 0.05) of feeding and a trend towards elevated goblet cells was also observed after six weeks feeding (P = 0.08). Concomitantly at week six, along with elevated IELs and elevated TNFα mRNA levels in the intestine, an increased abundance of circulating neutrophils and monocytes were observed in fish fed the probiotic supplemented diet (P < 0.05). This haemopoietic expansion of innate immune cells could be reflective of an elevated state of immuno-readiness. Together these results suggest that the probiotic has a protective action on the intestinal mucosal cells, stimulating the innate immune response after feeding for a period of six weeks. These immunological modulations did not impair growth performance or the remaining haematological and zootechnical parameters compared to the control group (P > 0.05).

The autocrine TNFα signalling loop in keratinocytes requires atypical PKC species and NF-κB activation but is independent of cholesterol-enriched membrane microdomains

Tumor necrosis factor α (TNFα) is involved in the pathogenesis of many inflammatory skin diseases. Epidermal keratinocytes produce and respond to TNFα via the cognate type 1 receptor (TNFR1). Little is known about regulation of TNFα signalling in this cell type. In this study, we report that in keratinocytes TNFα upregulates its own mRNA synthesis in an autocrine manner. This response peaks at approximately 1 h of stimulation with TNFα but sustained elevated levels of TNFα mRNA are observed for up to 24 h after stimulation and are dependent on the presence of the soluble cytokine. This autocrine response is mediated by the signalling cascade comprising TNFR1, atypical protein kinase C (aPKC) species and the transcription factor NF-κB, but is not dependent on the integrity of cholesterol-enriched membrane microdomains (lipid rafts). TNFα-stimulated keratinocytes produced the membrane-bound form of TNFα. It is conceivable that the described autocrine signalling loop contributes to the proinflammatory TNFα effect in the skin. The discovery of the crucial roles of aPKC and NF-κB might have consequences for the development of more selective anti-TNFα therapies for inflammatory skin diseases.

Modulation of radiation-induced tumour necrosis factor α (TNF-α) expression in the lung tissue by pentoxifylline

Purpose: The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of inflammatory and fibrotic tissue reactions. Pentoxifylline (PTX) down-regulates the production of proinflammatory cytokines, particularly TNF-α, in response to noxious stimuli and may therefore provide protection against radiation-induced, cytokine-mediated cellular damage. The purpose of this study was to investigate the temporal and spatial release of TNF-α in the lung tissue after thoracic irradiation with 12 Gy. In addition, we evaluated the ability of PTX to reduce the radiation-induced TNF-α release in this animal model of thoracic irradiation. Materials and methods: C57BL/6J mice were exposed to either sham irradiation or single fraction of 12 Gy delivered to the thorax. Four study groups were defined: those that received neither irradiation nor PTX (NT group), those that received PTX but no irradiation (PTX group), those that underwent irradiation without PTX (XRT group) and those that received both PTX and irradiation (PTX/XRT group). Treated and sham-irradiated mice were sacrificed corresponding to the latent period and the pneumonic phase. The TNF-α mRNA expression in the lung tissue was quantified by ‘real-time’ quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical detection methods (alkaline phosphatase anti-alkaline phosphatase (APAAP)) and automated image analysis were used for objective quantification of TNF-α protein expression. Results: Following thoracic irradiation with a single dose of 12 Gy (XRT group), radiation-induced TNF-α mRNA release in the lung tissue was significantly increased during the acute phase of pneumonitis ( P<0.05). The elevated levels of TNF-α mRNA during the pneumonic phase correlate with a significant increase of positive inflammatory cells, predominantly macrophages, in the lung parenchyma ( P<0.05). In contrast to the radiation-only group (XRT-group), the lung tissue of the PTX-treated mice (PTX/XRT group) revealed only a minor radiation-mediated TNF-α response on mRNA and protein level. Conclusions: This study demonstrates a significant radiation-induced increase of TNF-α (on mRNA and protein level) in the lung tissue during the pneumonic phase. The predominant localisation of TNF-α in areas of inflammatory cell infiltrates suggests involvement of this cytokine in the pathogenesis of radiation-induced lung injury. In addition, we observed a pronounced reduction of the TNF-α mRNA and protein production in the study group that received both PTX and radiation (PTX/XRT group) as compared to the radiation-only group (XRT group). Therefore our results indicate that PTX down-regulates the TNF-α mRNA and protein production in the lung tissue in response to radiation.

Increased T cell cytotoxicity by Betathine™-induced upregulation of TNFα

Betathine™ (BT) is a low molecular weight disulfide that has previously been shown to exhibit in vivo antitumor activity in murine myeloma and melanoma models. We have shown that BT treatment of both human T cells and monocytes is associated with an increase in surface tumor necrosis alpha (TNFα) expression. Further, in T cells and monocytes that have been stimulated with PMA and ionomycin, the addition of BT results in a dose and time dependent increase in the percentage of high TNFα-expressing cells. Unlike TNFα upregulation produced by the commonly used thiol antioxidant N-acetyl- l-cysteine (NAC), the BT-induced increase in TNFα is observed consistently in different donors. This increase in surface TNFα is associated with elevated levels of TNFα mRNA. In addition, expression of TNFα receptor I is also significantly enhanced by BT treatment. The upregulation of surface TNFα by BT has functional consequences, in that, BT-treated T cells exhibit enhanced cytotoxic activity. Thus, increased TNFα expression may be one mechanism responsible for the antineoplastic activity of BT.

NEUTROPENIA ASSOCIATED WITH ELEVATED TNF-α IN COMBINED IMMUNODEFICIENCY(CID). • 54

PA was noted to have erythrodermatitis and alopecia soon after birth. These symptoms persisted and by age 2 months, lymphadenopathy, heptosplenomegaly, chronic neutropenia (0-100 cells/μl), and lymphopenia were present. Multiple hospitalizations due to infections ensued. Treatment with G-CSF(10-20 μg/kg/day) gave no definitive improvement in PMN counts (0-200 cells/μl), but precipitated thrombocytopenia. CID was diagnosed because of poor lymphocyte enumeration and proliferation studies, and absence of Ig production. Biopsy of skin, lymph node, and bone marrow (BM) revealed excessive numbers of histiocytes. On repeated studies, malignancy could not be detected and 4 separate chromosomal analyses were normal (nl). BM exhibited an apparent myeloid arrest. It was hypothesized that a serum inhibitory factor was suppressing PMN, and possibly lymphocyte, development. PA's serum enhanced patient and control lymphocyte proliferation to mitogens above that of nl serum. PA's serum inhibited both patient and control BM cell growth in soft agar progenitor assays, whereas patient colony formation was near-normal in the presence of nl serum. Serum TNF-α was 59 pg/ml (nl < 20 pg/ml) by ELISA assay. RT-PCR of RNA from BM mononuclear cells disclosed elevated levels of TNF-α mRNA. Normalizing the TNF-α levels with concurrently amplified β-actin by calculating ratios of densitometric readings gave 0.45±0.07 and 0.13±0.03 (p < 0.01) for PA and control samples, respectively. Comparisons with 20 other cytokines and growth factors revealed no differences between PA and controls. Previous studies have implicated that TNF-α is a potent inhibitor of myeloid development. We believe that this is the first report demonstrating that elevated TNF-α in the bone marrow is associated with severe neutropenia in a patient. The process leading to dysregulation of TNF-α production (from histiocytes?) could be responsible for both CID and neutropenia in PA, and perhaps plays a role in other conditions where neutropenia is a prominent feature.