Tumor Necrosis Factor-alpha mRNA Levels Research Articles

In vitro and in vivo burn healing study of standardized propolis: Unveiling its antibacterial, antioxidant and anti-inflammatory actions in relation to its phytochemical profiling.

Natural propolis has been used since decades owing to its broad-spectrum activities. Burn injuries are a global health problem with negative impacts on communities. Bacterial infections usually accompany burns, which demand implementation of antibiotics. Antibiotics abuse led to emergence of microbial drug resistance resulting in poor treatment outcomes. In such instances, the promising alternative would be natural antimicrobials such as propolis. Full chemical profiling of propolis and evaluation of in vitro antibacterial, antioxidant and anti-inflammatory activities as well as in vivo burn healing properties. Chemical profiling of propolis was performed using Liquid chromatography (UHPLC/MS-PDA and HPLC-PDA). In vitro assessment was done using Disc Diffusion susceptibility test against Staphylococcus aureus and infected burn wound mice model was used for in vivo assessment. In vitro antioxidant properties of propolis were assessed using DPPH, ABTS and FRAP techniques. The anti-inflammatory effect of propolis was assessed against lipopolysaccharide/interferon-gamma mediated inflammation. UHPLC/MS-PDA results revealed identification of 71 phytochemicals, mainly flavonoids. Upon flavonoids quantification (HPLC-PDA), Pinocembrin, chrysin and galangin recorded high content 21.58±0.84, 22.73±0.68 and 14.26±0.70 mg/g hydroalcoholic propolis extract, respectively. Propolis showed concentration dependent antibacterial activity in vitro and in vivo burn healing via wound diameter reduction and histopathological analysis without signs of skin irritation in rabbits nor sensitization in guinea pigs. Propolis showed promising antioxidant IC50 values 46.52±1.25 and 11.74±0.26 μg/mL whereas FRAP result was 445.29±29.9 μM TE/mg. Anti-inflammatory experiment results showed significant increase of Toll-like receptor 4 (TLR4), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) mRNA levels. Nitric oxide and iNOS were markedly increased in Griess assay and western blot respectively. However, upon testing propolis against LPS/IFN-γ-mediated inflammation, TLR4, IL-6 and TNF-α expression were downregulated at transcriptional and post-transcriptional levels. Propolis proved to be a promising natural burn healing agent through its antibacterial, antioxidant and anti-inflammatory activities.

Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-\u03b3-mediated inflammation in doxorubicin-treated macrophages

Doxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

Can aprepitant used for nausea and vomiting be good gastrointestinal complaints?

Aprepitant is a selective SP/NK-1 receptor antagonist and used in postoperative and chemotherapeutics induced emesis and vomiting. The aim of our study is to show aprepitant may have beneficial effects on gastrointestinal complaints in cancer patients undergoing chemotherapeutics by indomethacin-induced gastric ulcer model. A total of 48 rats were fasted 24h for ulcer experiment. Aprepitant doses of 5, 10, 20, and 40mg/kg were evaluated for their antiulcer activity. Omeprazole (20mg/kg) was used as a positive control group. Six hours after 25mg/kg indomethacin administration, all stomachs were dissected out. After macroscopic analyses, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), COX-1, and COX-2 mRNA levels and SOD activity, and GSH and MDA levels of stomachs were determined. Histopathological examinations were evaluated. Aprepitant administration exerted 48.14%, 49.62%, 65.92%, and 76.77% ulcer inhibition effects at 5, 10, 20, and 40mg/kg, respectively. Aprepitant administration decreased oxidative stress and inflammatory parameters in stomach tissues dose dependently. Aprepitant administration increased stomach COX-2 mRNA levels at 20 and 40mg/kg doses. Although aprepitant appears to be disadvantageous in terms of treating gastric ulcer due to COX enzyme inhibition according to the previous studies, aprepitant has been shown to have ulcer healing effect in our study. When aprepitant is given as an anti-nausea and vomiting drug to cancer patients undergoing chemotherapy, we can argue that it will not be necessary to add a new gastric protective agent as it also shows beneficial effects in gastrointestinal complaints.

Roman chamomile inhalation combined with clomipramine treatment improves treatment-resistant depression-like behavior in mice

It is well known that chamomile is one of the oldest known medicinal herbs and has been used to treat various disorders, but it is mainly German chamomile. The effects of Roman chamomile on depression still unclear. In this study, we used chronically stressed mice to investigate whether inhalation of Roman chamomile essential oil affects depression-like behavior. We previously reported that restraint and water immersion stress produce depression-like behavior and a blunted response to the tricyclic antidepressant clomipramine. Each mouse was exposed to restraint and water immersion stress for 15 days, and resistance to the effect of clomipramine was induced in a behavioral despair paradigm. In the present study, we found that cotreatment with clomipramine and inhalation of Roman chamomile attenuated depression-like behavior in a forced swim test. Next, we examined the hippocampal mRNA levels of two cytokines, tumor necrosis factor (TNF) alpha and interleukin-6 (IL-6); a neurotrophic factor, brain derived-neurotrophic factor (BDNF); and nerve growth factor (NGF). TNF alpha, IL-6 and BDNF mRNA levels did not change in the hippocampus of stressed mice. However, the NGF mRNA level was significantly decreased, and this decrease was not attenuated by treatment with clomipramine or inhalation of Roman chamomile alone. We also examined whether Roman chamomile combined with clomipramine treatment affects hippocampal neurogenesis and serum corticosterone levels. Stressed mice had fewer doublecortin (DCX)-positive cells in the subgranular zone of the dentate gyrus, but this was significantly attenuated by Roman chamomile and clomipramine treatment. In addition, the serum corticosterone level was also significantly decreased by treatment with Roman chamomile and clomipramine. These results suggest that Roman chamomile inhalation may enhance the antidepressant effect of clomipramine by increasing hippocampal neurogenesis and modulating corticosterone levels in patients with treatment-resistant depression.

Physiological Responses and Gene Expression in Ultrasound-Guided Supraclavicular Brachial Plexus Block: a Comparative Study

Background/Aims: Ultrasound-guided supraclavicular brachial plexus block (BPB) has come into wider use as a regional anesthetic during upper limb operations. This study assessed the neurological and hemodynamic changes and gene expression after co-administration of midazolam or neostigmine with bupivacaine during supraclavicular BPB. Methods: The study involved 90 adults divided into three groups: control (bupivacaine), midazolam (bupivacaine plus midazolam), and neostigmine (bupivacaine plus neostigmine). Blood samples were taken and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) mRNA levels were measured by real-time PCR, and oxidative stress markers were identified. In addition to the hemodynamic variables, the onset and duration of sensory and motor blockades, duration of analgesia, pain scores, time of first request for an analgesic, and amounts of analgesics ingested were evaluated. Results: Compared with the control and neostigmine groups, the midazolam group experienced longer sensory and motor blockades, prolonged analgesia, lower pain scores at 12 h and 24 h, and lower need for postoperative analgesics. Moreover, the midazolam group exhibited lower oxidative stress markers with a higher fold change in IL-6 and TNF-α mRNA levels. Conclusion: Midazolam co-administered with bupivacaine provided better analgesic quality than did neostigmine with bupivacaine. This might be due to its superior antioxidant and anti-inflammatory effects.

Tumor necrosis factor‐alpha (TNF) regulates NKCC2 and AQP2 expression and adaptation to high NaCl intake

We previously showed that ambient urine osmolality was higher in TNF deficient mice compared with age‐ and sex‐matched wild type (WT) mice. As the role of TNF in the kidney is still being defined, effects of renal‐specific TNF silencing on renal Na(+)‐K(+)‐2Cl(‐) cotransporter (NKCC2) and aquaporin‐2 (AQP2) expression was determined. Six days after intrarenal injection of lentivirus construct EGFP‐TNF‐ex4 into both kidneys of WT mice, renal but not splenic TNF mRNA levels were significantly reduced. Injection of control lentivirus (U6) did not alter TNF mRNA in either kidney or spleen. EGFP‐TNF‐ex4, but not U6, also increased NKCC2A mRNA levels in renal outer medulla and inner medullary AQP2 mRNA and protein expression. Urine osmolality increased in mice injected with EGFP‐TNF‐ex4 compared with those injected with U6, suggesting that the increase in NKCC2 and AQP2 due to renal silencing of TNF contributed to an increase osmolality. As TNF production by the TAL was induced by administration of 1% NaCl in the drinking water for 3 days, the effects of TNF silencing on urine volume were determined. Mice were acclimated in metabolic cages for 3 days after receiving intrarenal injections of either EGFP‐TNF‐ex4 or U6. Food, body weight, water intake, and urine output was then measured in mice given 1% NaCl or tap water for 3 days. Both water intake and urine output were reduced in mice injected with EGFP‐TNF‐ex4 compared with mice given U6 lentivirus. The decrease in urine volume was associated with an increase in NKCC2A and AQP2 mRNA accumulation. Collectively, these findings suggest that TNF produced by the kidney inhibits transporter expression and regulates responses to high NaCl intake.

Eradication of Helicobacter pylori Increases Ghrelin mRNA Expression in the Gastric Mucosa

It has been suggested that Helicobacter pylori eradication may influence production of some peptides in the stomach, which can affect appetite. This hypothesis is controversial. To verify the hypothesis, we conducted this randomized controlled trial using H. pylori infected subjects without any gastrointestinal symptoms. The treatment group received triple H. pylori eradication therapy for 7 days and the control group received no medication. We measured ghrelin, obestatin and the tumor necrosis factor-α (TNF-α) mRNA levels in endoscopic biopsy specimens and the changes from baseline to follow-up. The plasma active n-octanoyl ghrelin and obestatin levels were measured in both groups. The ghrelin/obestatin ratios in plasma and gastric mRNA expression were calculated at baseline and follow-up. Ghrelin mRNA expression in the fundic mucosa after H. pylori eradication increased significantly compared to the control group (4.47±2.14 vs. 1.79±0.96, P=0.009), independent of inflammatory changes. However, obestatin mRNA expression decreased in the antral mucosa (-0.57±1.06 vs. 0.41±0.72, P=0.028). The treatment group showed a marginal increase (P=0.060) in plasma ghrelin/obestatin ratio. The TNF-α mRNA expression also decreased significantly with treatment. This randomized controlled trial demonstrates that H. pylori eradication increases ghrelin mRNA expression, independent of inflammatory cell changes.

Transient early expression of TNF-α in sciatic nerve and dorsal root ganglia in a mouse model of painful peripheral neuropathy

The proinflammatory cytokine tumor necrosis factor-alpha (TNF) is an important mediator in neuropathic pain. We investigated the temporal pattern of TNF mRNA expression in the sciatic nerve, in dorsal root ganglia (DRG) and spinal cord in the mouse chronic constriction injury model of neuropathy with quantitative real-time polymerase chain reaction. Neuropathic pain-like behaviour was monitored by evaluating thermal hyperalgesia and mechanical allodynia. Pain-related behaviour and TNF expression were evaluated 6 h, 1, 3, 7 and 14 days after injury. Naive animals and sham-operated mice were used as controls. We found an early upregulation of sciatic nerve TNF mRNA levels in chronic constriction injury (CCI) and sham-operated animals 6 h after surgery: 1 day later TNF overexpression was present in CCI mice only and disappeared 3 days after injury. The mRNA cytokine levels were elevated in DRG 1 and 3 days after surgery in CCI animals only, while the cytokine was not modulated in the spinal cord. A significant hyperalgesia was present in CCI and sham-operated mice at 6 h and 1 day, while at later time point only CCI mice presented lower thresholds. Mechanical allodynia was already present only in CCI animals 6 h from surgery and remained constant up to the 14 th day. The results indicate that a transient early TNF upregulation takes place in peripheral nervous system after CCI that can activate a cascade of proinflammatory/pronociceptive mediators.

Effect of Bakumijiogan, an Herbal Formula in Traditional Chinese Medicine, on Atopic Dermatitis-Like Skin Lesions Induced by Mite Antigen in NC/Jic Mice

We evaluated the effectiveness of bakumijiogan (BJG), an herbal formula in traditional Chinese medicine used to treat atopic dermatitis (AD), using a NC/Jic mouse model of AD. AD symptoms were induced by repeated injections of Dermatophagoides farinae antigen (Df-antigen) into the ear auricle at 2- to 3-d intervals for 16 d. Ear thickness dramatically increased up to 16 d after the first injection of Df-antigen. Daily oral administration of BJG from 7 d before to 16 d after the first injection significantly reduced ear swelling. Serum concentrations of total immunoglobulin (Ig)E and Df-antigen-specific IgG1 were augmented when assayed 17 d after the first injection of Df-antigen, and these increases were slightly suppressed by BJG administration. Serum interferon (IFN)-gamma and lesional IFN-gamma mRNA levels were significantly higher, whereas lesional IL-1alpha and tumor necrosis factor-alpha mRNA levels were lower in BJG-treated mice than those in control mice. These results suggest that BJG suppressed AD-like symptoms by correcting the Th1/Th2 imbalance skewed toward Th2. Evaluation of herbal constituents in BJG revealed that the combination of two herbal ingredients, ophiopogon tuber and schisandra fruit, mainly contributed to the effects of BJG.

Effects of Low Concentrations of Arsenic on the Innate Immune System of the Zebrafish (Danio Rerio)

Arsenic has been associated with a multitude of human health problems; however, its impact on host resistance to infection has not been extensively researched. In vertebrates, the innate immune response is vital for potentiating the adaptive immune response. Therefore, dampening of the innate immune response results in an immunocompromised host. In this present study, effects of low concentrations of arsenic on zebrafish resistance to infection are evaluated. Exposure to 2 and 10 ppb arsenic, both considered safe levels in drinking water, resulted in a greater than 50-fold increase in viral load and at least a 17-fold increase in bacterial load in embryos. To determine the cause of this amplified pathogen load, important components of the innate immune system were analyzed. Presence of arsenic dampened the overall innate immune health of the fish as evidenced by reductions in respiratory burst activity. Viral infection, after arsenic exposure, showed decreases of up to 13- and 1.5-fold changes in interferon and Mx mRNA expression, respectively. Bacterial infection, post arsenic exposure, demonstrated at least 2.5- and 4-fold declines in interleukin-1beta and tumor necrosis factor-alpha mRNA levels, respectively. Maximum expression of these essential cytokines was also delayed upon arsenic exposure. Our data indicate that arsenic exposure, at concentrations deemed safe in drinking water, suppresses the overall innate immune function in zebrafish and present the zebrafish as a unique model for studying immunotoxicity of environmental toxicants. To our knowledge, this is the first report describing the effects of such low levels of arsenic on host resistance to infection.

Early cytokine expression in mouse sciatic nerve after chronic constriction nerve injury depends on calpain

Nerve injury initiates Wallerian degeneration with subsequent alterations of cytokine expression contributing to neuropathic pain. To investigate the very early temporal pattern of cytokine regulation we studied 140 mice of C57Bl/6J background after chronic constriction injury (CCI) of the right sciatic nerve and measured the relative mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1β) and of the anti-inflammatory cytokines IL-4 and IL-10 with quantitative real-time polymerase chain reaction (qRT-PCR). The measurements were performed in ipsi- and contralateral sciatic nerves and dorsal root ganglia (DRG) 1, 3, 6, 9, 12, 24 h, and 3 and 7 days after CCI. We found an ipsilateral upregulation of TNF, IL-1β and IL-10 mRNA levels as early as one hour after CCI. To investigate upstream regulatory mechanisms, we used inhibitors to the N-methyl- d-aspartate (NMDA) receptor ((+)-MK-801) and to calpain (MDL-28170). MDL-28170, but not (+)-MK-801 inhibited TNF and IL-1β upregulation one hour after CCI. This leads us to suggest that calpain is one of the earliest mediators of cytokine upregulation in injured peripheral nerves.

Conjugated linoleic acid preserves muscle mass in mice bearing the Lewis lung carcinoma, but not the B16 melanoma

Conjugated linoleic acid (CLA), which is found in dairy products, reduces synthesis of tumor necrosis factor-alpha (TNFa), a pro-inflammatory cytokine that plays a major role in tumor-induced skeletal muscle wasting (SMW). The B16 melanoma expresses TNFa mRNA, and induced SMW with no change in muscle levels of TNFa type 1 receptor (TNFR1) protein. A diet containing .5% CLA had no effect on SMW or TNFR1 in mice bearing B16 tumors. In contrast, the Lewis lung carcinoma expresses low levels of TNFa mRNA, induced SMW, and increased muscle levels of TNFR1. A diet containing .5% CLA reduced SMW, but had no effect on muscle levels of TNFR1. We conclude that that tumor-induced SMW can occur independent of muscle levels of TNFR1. Further study is needed before CLA can be tested in persons with cancer cachexia.

Conjugated Linoleic Acid Deteriorates Insulin Resistance in Obese/Diabetic Mice in Association with Decreased Production of Adiponectin and Leptin

Dietary supplementation of conjugated linoleic acids (CLA) is known to have some beneficial effects such as anti-carcinogenic and anti-obesity effects in several animal species, while it also induces insulin resistance and fatty liver, especially in mice. To explore the possible factors responsible for the CLA-induced insulin resistance, we examined the plasma and mRNA expression levels of several adipocytokines, which are likely involved in the regulation of insulin sensitivity, in normal C5 7BL, mildly obese/diabetic KK and morbidly obese/diabetic KKAy mice. Feeding a diet supplemented with 0.5%, CLA oil consisting of 30.5/% c9, t11-CLA and 28.9% t10, c12-CLA for 4 wk resulted in a decrease in white adipose tissue (WAT), an increase in liver weight with excess accumulation of triglyceride, and insulin resistance associated with hyperglycemia and hyperinsulinemia. The plasma and WAT mRNA levels of leptin were higher in KK and KKAy mice than C57BI. mice, whereas those of adiponectin were higher in C5 7BL mice. CLA-feeding decreased the levels of leptin, adiponectin and resistin, especially in KK and KKAy mice. In contrast, tumor necrosis factor-alpha (TNFalpha) mRNA levels were higher in KK and KKAy mice than C57BL mice, and were increased by CLA feeding. The present results thus indicate that CLA feeding promotes insulin resistance in obese/diabetic mice by at least inverse regulation of leptin and adiponectin, and TNFalpha, adipocytokines known to either ameliorate or deteriorate insulin sensitivity, respectively.

In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumour necrosis factor-α and transforming growth factor-β genes

This study investigated the effects of the natural polyphenol mangiferin (MA) on superoxide anion (O 2 −) production, xanthine oxidase (XO) activity, vascular contractility, inducible nitric oxide synthase (iNOS) mRNA levels, tumour necrosis factor-alpha (TNF-α) mRNA levels, and tumour growth factor-beta (TGF-β) mRNA levels. O 2 − was generated by the hypoxanthine–xanthine oxidase (HX–XO) and phenazine methosulphate (PMS)–NADH systems. XO activity was determined by measurement of uric acid production with xanthine as substrate. Vascular contraction experiments were performed with intact rat aortic rings. iNOS, TNF-α and TGF-β gene expression in rat macrophages stimulated in vivo with 3% thioglycollate and in vitro with 100 ng/mL lipopolysaccharide and 10 U/mL of interferon-gamma were evaluated semiquantitatively by the retrotranscriptase–polymerase chain reaction. MA at 10–100 μM, like the known O 2 − scavenger superoxide dismutase (1 U/mL), scavenged O 2 − produced by the HX/XO and PMS–NADH systems. By contrast MA at 1–100 μM, unlike allopurinol (10 μM), was unable to inhibit XO activity. MA at 1–100 μM did not modify resting tone or the contractile responses elicited by 1 μM phenylephrine or 1 μM phorbol 12-myristate 13-acetate in rat aorta. MA at 1–100 μM, like dexamethasone (100 μM), decreased iNOS mRNA levels in activated macrophages. At 100 μM, MA also reduced TNF-α mRNA levels, but increased TGF-β mRNA levels. These results thus indicate that MA is an O 2 − scavenger and that it inhibits expression of the iNOS and TNF-α genes, suggesting that it may be of potential value in the treatment of inflammatory and/or neurodegenerative disorders. In addition, the finding that MA enhances TGF-β gene expression suggests that this polyphenol might also be of value in the prevention of cancer, autoimmune disorders, atherosclerosis and coronary heart disease.

Tumor necrosis factor expressed by primary hippocampal neurons and SH-SY5Y cells is regulated by alpha(2)-adrenergic receptor activation.

Neuron expression of the cytokine tumor necrosis factor-alpha (TNF), and the regulation of the levels of TNF by alpha(2)-adrenergic receptor activation were investigated. Adult rat hippocampal neurons and phorbol ester (PMA)-differentiated SH-SY5Y cells were examined. Intracellular levels of TNF mRNA accumulation, as well as TNF protein and that released into the supernatant were quantified by in situ hybridization, immunocytochemistry and bioanalysis, respectively. Both neuron cultures demonstrated constitutive production of TNF. Activation of the alpha(2)-adrenergic receptor increased intracellular levels of TNF mRNA and protein in SH-SY5Y cells after addition of graded concentrations of the selective agonist, Brimonidine (UK-14304) to parallel cultures. Intracellular levels of mRNA were increased in a concentration-dependent fashion within 15 min of UK-14304 addition and were sustained during 24 hr of receptor activation. In addition, the levels of TNF in the supernatant were increased in both types of neuron cultures within 15 min of alpha(2)-adrenergic receptor activation. Furthermore, levels of TNF significantly increased in the supernatants of both neuron cultures after potassium-induced depolarization. A reduction in this depolarization-induced release occurred in hippocampal neuron cultures after exposure to the sympathomimetic tyramine with media replacement to deplete endogenous catecholamines. This finding reveals a role for endogenous catecholamines in the regulation of TNF production. Potassium-induced depolarization resulted in the release of TNF in hippocampal neuron cultures within 15 min but not until 24 hr in SH-SY5Y cultures demonstrating a temporally mediated event dependent upon cell type. Neuron expression of TNF, regulated by alpha(2)-adrenergic receptor activation demonstrates not only how a neuron controls its own production of this pleiotropic cytokine, but also displays a normal role for neurons in directing the many functions of TNF.

Iron overload impairs pro-inflammatory cytokine responses by Kupffer cells.

The aim of the present study was to determine the effect of chronic iron overload on Kupffer cell cytokine production. Kupffer cells were isolated from rats that were fed either a control or iron-supplemented diet for 12 months. Cytokine mRNA and protein levels were determined by using a ribonuclease protection assay and ELISA, respectively. Baseline levels of tumor necrosis factor-alpha, transforming growth factor-beta1, interleukin-6 and granulocyte macrophage colony stimulating factor were similar in iron-loaded and control Kupffer cells. Following the addition of lipopolysaccharide to control cells, tumor necrosis factor-alpha, interleukin-1alpha and interleukin-6 mRNA levels increased. Tumor necrosis factor-alpha mRNA and protein levels were reduced by 40 and 60%, respectively, in iron-loaded cells compared with controls following the addition of lipopolysaccharide. Interleukin-6 mRNA levels in iron-loaded Kupffer cells were also reduced. Granulocyte macrophage colony stimulating factor mRNA levels remained unchanged in controls, but were significantly elevated in iron-loaded cells. Tumor growth factor-beta1 mRNA and protein levels were similar in control and iron-loaded cells. Deposition of iron in Kupffer cells in chronic dietary iron overload results in an impaired pro-inflammatory cytokine response to lipopolysaccharide. Our observations may have relevance to the altered immune function observed in chronic iron-overload syndromes.

Early activation of pulmonary nuclear factor kappaB and nuclear factor interleukin-6 in polymicrobial sepsis.

Transcription factor activation may be a pivotal step in the pathophysiology of sepsis syndrome and adult respiratory distress syndrome. This study investigated the activation of lung nuclear factor kappaB (NFkappaB) and nuclear factor interleukin-6 (NF-IL6) and how they correlate to proinflammatory cytokine expression and mortality in a murine model of cecal ligation and puncture (CLP). Polymicrobial sepsis was induced by CLP. Transcription factor activation was assessed at 0, 1, 2, 3, 4, 5, 6, 8, and 24 hours after CLP by the electrophoretic mobility-shift assay. Lung cytokine mRNA levels were established by reverse transcriptase-polymerase chain reaction. CLP induced pulmonary NFkappaB activation at 3, 4, and 8 hours (p < 0.05). Lung NFkappaB activation peaked at 3 hours (533% vs. no surgery, 2,900% vs. sham treatment) after CLP. Supershift analysis revealed a predominance of p50 subunits in the lung nuclear extracts of septic mice 3 hours after CLP, indicating the presence of p50 homodimer. In contrast, liver nuclear extracts from septic mice indicated the presence of both p65 and p50 subunits at 3 hours. Lung NF-IL6 activation (p < 0.05) was observed at 4 hours (649% vs. no surgery, 296% vs. sham treatment) and 6 hours after CLP. Lung tumor necrosis factor-alpha mRNA levels were increased (p < 0.05) at all time intervals after CLP. Lung IL-6 mRNA levels were increased at 3, 6, and 8 hours after CLP. Early activation of lung NFkappaB and NF-IL6 and lung cytokine mRNA expression correlated with mortality in polymicrobial sepsis. Although IL-6 mRNA levels correlated with NFkappaB and NF-IL6 activation, tumor necrosis factor-alpha mRNA levels did not, in that they preceded transcription factor activation. These data suggest a potential role for NFkappaB and NF-IL6 activation in the initiation and propagation of acute lung injury.

Regulation of Nucleoside Transport by Lipopolysaccharide, Phorbol Esters, and Tumor Necrosis Factor-α in Human B-lymphocytes

Nucleoside transport systems and their regulation in human B-lymphocytes have been characterized using the cell lines Raji and Bare lymphoma syndrome-1 (BLS-1) as experimental models. These cells express at least three different nucleoside transport systems as follows: a nitrobenzylthioinosine-sensitive equilibrative transport system of the es-type, which appears to be associated with hENT1 expression, and two Na+-dependent transport systems that may correspond to N1 and to the recently characterized N5-type, which is nitrobenzylthioinosine-sensitive and guanosine-preferring. B cell activators such as phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) up-regulate both concentrative transport systems but down-regulate the equilibrative es-type transporter, which correlates with lower hENT1 mRNA levels. These effects are dependent on protein kinase C activity. Phorbol 12-myristate 13-acetate and LPS also induce an increase in tumor necrosis factor-alpha (TNF-alpha) mRNA levels, which suggest that this cytokine may mediate some of the effects triggered by these agents, since addition of TNF-alpha alone can increase N1 and N5 transport activities by a mechanism that also depends on protein kinase C activation. Interestingly, TNF-alpha down-regulates es activity, but this effect cannot be abolished by inhibiting protein kinase C. This study reveals differential regulation of nucleoside transport systems following activation of human B-lymphocyte cell lines by agents of physiological relevance such as TNF-alpha and LPS. Moreover, it indicates that the recently characterized N5 transport system can also be regulated following B cell activation, which may be relevant to lymphocyte physiology and to the treatment of lymphocyte malignancies.

Sublethal gamma irradiation increases IL-1alpha, IL-6, and TNF-alpha mRNA levels in murine hematopoietic tissues.

The effects of sublethal (7.75 Gy) 60Co gamma radiation exposure on endogenous bone marrow and splenic interleukin-1alpha (IL-1alpha), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein levels were assayed in B6D2F1 female mice. Bone marrow and spleen were harvested from normal and irradiated mice on days 2, 4, 7, 10, and 14 postexposure, and cytokine mRNA levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis. IL-1alpha mRNA levels were significantly increased in bone marrow at days 2 and 4 postirradiation and at day 7 in spleen compared with controls. Postirradiation IL-6 mRNA levels showed a significant increase at day 2 in bone marrow and at days 7 and 10 in spleen. TNF-alpha mRNA levels exhibited a significant increase at day 2 postirradiation in bone marrow, but in spleen no difference between control and irradiated samples was observed on any day postirradiation. Interestingly, there were no significant differences in the cytokine protein levels in postirradiation bone marrow, spleen, or serum when compared with normal controls.

Surfactant protein A activates NF-kappa B in the THP-1 monocytic cell line.

The expression of many genes for which products are involved in inflammation is controlled by the transcriptional regulator nuclear factor (NF)-kappa B. Because surfactant protein (SP) A is involved in local host defense in the lung and alters immune cell function by modulating the expression of proinflammatory cytokines as well as surface proteins involved in inflammation, we hypothesized that SP-A exerts its action, at least in part, via activation of NF-kappa B. We used gel shift assays to determine whether SP-A activated NF-kappa B in the THP-1 cell line, a human monocytic cell line. Activation of NF-kappa B in THP-1 cells by SP-A doses as low as 1 microgram/ml occurred within 30 min of SP-A treatment, peaked at 60 min, and then declined. This activation is inhibited by known inhibitors of NF-kappa B or by simultaneous treatment of the cells with surfactant lipids. Moreover, the NF-kappa B inhibitors blocked SP-A-dependent increases in tumor necrosis factor-alpha mRNA levels. These observations suggest a mechanism by which SP-A plays a role in the pathogenesis of some lung conditions and point to potential therapeutic measures that could be used to prevent SP-A induced inflammation in the lung.