TNF-alpha Protein Research Articles

Induction of systemic TNFα in Natalizumab‐treated multiple sclerosis

The mRNA expression of eight different cytokines in peripheral blood mononuclear cells in 19 individuals with multiple sclerosis was determined at baseline and after 6 months of open-label treatment with natalizumab. Cellular expression of tumor necrosis factor alpha (TNFalpha) mRNA and number of cells secreting TNFalpha and interferon gamma protein significantly increased over the 6 months. Kurtzke EDSS scores improved because of the resolution of relapses, but not fatigue severity scores. The observed increases in systemic proinflammatory cytokines by natalizumab treatment are discussed in relation to fatigue and systemic immunity.

β-Adrenergic Blockade Exacerbates Sepsis-Induced Changes in Tumor Necrosis Factor α and Interleukin-6 in Skeletal Muscle and is Associated With Impaired Translation Initiation

Sepsis stimulates the sympathetic nervous system. The resultant elevation in plasma catecholamines, both norepinephrine and epinephrine (Epi), might be expected to alter the expression of inflammatory cytokines, which may directly or indirectly influence muscle protein balance. The purpose of this study was twofold: (1) determine whether Epi per se increases cytokine expression in skeletal muscle, and (2) determine whether beta-adrenergic blockade alters the sepsis-induced expression of inflammatory cytokines and mediators of protein balance in skeletal muscle. In the first study, rats were infused with Epi for 2 hour to increase the circulating Epi concentration to levels seen in septic animals. In the second study, sepsis was induced by cecal ligation and puncture and a nonspecific beta-adrenergic blockade produced with a continuous infusion of propranolol (PP). Tissues were obtained 24 after induction of sepsis and analyzed for tumor necrosis factor (TNF)-alpha interleukin (IL)-1beta, IL-6 mRNA and protein content. In addition, the tissue content of insulin-like growth factor (IGF)-I and various regulators of protein synthesis were assessed. Epi acutely increased TNF-alpha IL-6 and IL-1beta mRNA content in muscle (3- to 40-fold). However, only the TNF-alpha and IL-6 protein content was increased in muscle by Epi. In the second study, beta-adrenergic blockade with PP exacerbated the sepsis-induced increase in muscle IL-6 and TNF-alpha mRNA but did not alter the increment in IL-1beta or HMGB1. Propranolol also accentuated the sepsis-induced increase in both IL-6 and TNF-alpha protein in muscle. The exaggerated muscle cytokine response in septic rats treated with PP was associated with a reduction in muscle IGF-I protein that was greater than detected in saline-infused septic rats. Finally, the combination of sepsis + PP also accentuated the sepsis-induced decrease in the phosphorylation of 4E-binding protein-1, ribosomal protein S6, and mTOR, which are key proteins controlling protein synthesis. These results demonstrate that although Epi is capable of increasing tissue cytokines in naive rats, inhibition of the beta-adrenergic effects of catecholamines exacerbates the sepsis-induced increase of selected inflammatory cytokines. This exaggerated tissue response is associated with alterations in muscle IGF-I protein and translation initiation, which would be expected to impair tissue protein synthesis.

Chlamydophila pneumoniaeInhibits Differentiation of Progenitor Adipose Cells and Impairs Insulin Signaling

Recent clinical studies have shown Chlamydophila pneumoniae seropositivity to be related to overweight status and inversely related to insulin sensitivity. The present study was performed to investigate the potential effects of C. pneumoniae infection of adipocytes. 3T3-L1 cells and primary epididymal preadipocytes were infected with C. pneumoniae either before or after induction of differentiation, and the effects on adipogenesis and insulin signaling were determined. Tumor necrosis factor (TNF)-alpha signaling was examined by assessing the effects of C. pneumoniae infection in preadipocytes isolated from epididymal adipose tissue of both wild-type and TNF-alpha(-/-) mice. C. pneumoniae successfully infected both undifferentiated and differentiated 3T3-L1 cells in vitro. The bacteria were also detected in adipose tissue of infected low-density lipoprotein receptor-deficient mice. TNF-alpha protein levels were significantly increased in cells infected with either live or heat-killed C. pneumoniae or treated with lipopolysaccharide or heat-shock protein 65; this increase was associated with inhibition of adipocyte differentiation and down-regulation of insulin-stimulated tyrosine-phosphorylated insulin receptor and its substrate. In contrast, C. pneumoniae infection in TNF-alpha(-/-) adipocytes produced no apparent changes, but addition of recombinant TNF-alpha reversed this effect. We demonstrate for the first time that C. pneumoniae can infect murine pre- and postdifferentiated adipocytes and, through a TNF-alpha-mediated inflammatory mechanism, can impair differentiation and insulin signaling.

Bronchiolar chemokine expression is different after single versus repeated cigarette smoke exposure

BackgroundBronchioles are critical zones in cigarette smoke (CS)-induced lung inflammation. However, there have been few studies on the in vivo dynamics of cytokine gene expression in bronchiolar epithelial cells in response to CS.MethodsWe subjected C57BL/6J mice to CS (whole body exposure, 90 min/day) for various periods, and used laser capture microdissection to isolate bronchiolar epithelial cells for analysis of mRNA by quantitative reverse transcription-polymerase chain reaction.ResultsWe detected enhanced expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) by bronchial epithelial cells after 10 consecutive days of CS exposure. This was mirrored by increases in neutrophils and KC, MIP-2, TNF-α, and IL-1β proteins in the bronchoalveolar lavage (BAL) fluid. The initial inhalation of CS resulted in rapid and robust upregulation of KC and MIP-2 with concomitant DNA oxidation within 1 hr, followed by a return to control values within 3 hrs. In contrast, after CS exposure for 10 days, this initial surge was not observed. As the CS exposure was extended to 4, 12, 18 and 24 weeks, the bronchiolar KC and MIP-2 expression and their levels in BAL fluid were relatively dampened compared to those at 10 days. However, neutrophils in BAL fluid continuously increased up to 24 weeks, suggesting that neutrophil accumulation as a result of long-term CS exposure became independent of KC and MIP-2.ConclusionThese findings indicate variable patterns of bronchiolar epithelial cytokine expression depending on the duration of CS exposure, and that complex mechanisms govern bronchiolar molecular dynamics in vivo.

Tumour necrosis factor‐α‐mediated human polymeric immunoglobulin receptor expression is regulated by both mitogen‐activated protein kinase and phosphatidylinositol‐3‐kinase in HT‐29 cell line

Human polymeric immunoglobulin receptor (pIgR) is present on the surface of glandular epithelium, and it plays a crucial role in the mucosal immune defence. pIgR expression in HT-29 cells is up-regulated by one of the proinflammatory cytokines, tumour necrosis factor (TNF)-alpha. However, the mechanism used by the TNF-alpha-mediated signalling pathway has not been examined exclusively. To elucidate this mechanism in detail, HT-29 cells were cotreated with TNF-alpha and mitogen-activated protein kinase kinase (MAPKK, also called MEK1) inhibitor, PD98059, and the amount of free secretory component (SC) secreted into the culture medium was measured. The amount of free SC stimulated by TNF-alpha was increased by addition of PD98059. This up-regulation occurred at the transcriptional level. The amount of SC was also up-regulated by addition of TNF-alpha with U0126, an inhibitor of MEK1 and MEK2. Nuclear factor (NF)-kappaB activity and NF-kappaB binding to the kappaB2 site localized upstream of the pIgR gene did not change after coincubation of HT-29 cells with TNF-alpha and PD98059. The expression level of pIgR by TNF-alpha was decreased by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), at the transcriptional level. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation and NF-kappaB binding to the kappaB2 site were not affected by LY294002 treatment. These data suggest that TNF-alpha-mediated pIgR expression is negatively regulated by ERK pathway, which is independent of NF-kappaB. In addition, decrease of SC production by Ly294002 suggests that the presence of PI3K mediated regulation of SC production.

Role of activator protein-1 in pathogenesis of silicosis: an in-vitro study

To study the role of activator protein-1 (AP-1) in the up-regulation expression of tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1(TGF-beta(1)) in silica-stimulated macrophage cells (RAW264.7). RAW264.7 cells were treated with AP-1 inhibitor Curcumin. The expression of c-jun and c-fos in nuclear protein was detected by western blotting. The level of TNF-alpha and TGF-beta(1) protein in the cell supernatant was measured using enzyme-linked immunoadsorbent assay (ELISA). Meanwhile the expression of TNF-alpha and TGF-beta(1) mRNA was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR). The nucleoprotein expression of c-jun and c-fos in 10 and 20 micromol/L Curcumin prevention group (1.150 +/- 0.020, 1.010 +/- 0.108, 80.430 +/- 0.023, 0.256 +/- 0.015) were lower than those in silica-stimulated group (1.550 +/- 0.029, 0.860 +/- 0.036) (P < 0.01). In 20 micromol/L Curcumin prevention group and silica stimulated group, the expression of TNF-alpha protein were 23.58 +/- 45.78 and 32.12 +/- 5.34, and the expression of TGF-beta(1) protein were 1582.18 +/- 437.52 and 55.60 +/- 5.51 (P < 0.05 =; the expression of TNF-alpha, TGF-beta(1) mRNA were 0.74 +/- 0.01, 0.22 +/- 0.04 and 2.27 +/- 0.33, 2.96 +/- 0.15 (P < 0.05 =. The expression of TNF-alpha, TGF-beta(1) mRNA and proteins is associated with activation of AP-1 in silica-stimulated macrophage cells.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Augments Acute Lung Injury via Its Neutrophil Priming Effects

Granulocyte macrophage-colony stimulating factor (GM-CSF) has immuno-stimulatory effects. We hypothesized that GM-CSF plays an important role both in lipopolysaccharide (LPS)- and hemorrhage-induced acute lung injury (ALI). We also postulated that GM-CSF augments LPS-induced inflammation by priming neutrophils. ALI was induced in GM-CSF-/- or control C57BL mice either by LPS injection or by hemorrhage. Lung inflammation (by lung expression for tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and keratinocyte-derived chemokine) and lung injury (by myeloperoxidase and evans blue dye assay) were evaluated after ALI. Incremental doses of LPS (0, 1, 10, and 100 ng/mL) and GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils. The expression of TNF-α, MIP-2, and IL-1β was evaluated with enzyme linked immunosorbent assay. The mRNA expression of three cytokines, and the nuclear translocation of nuclear factor kappa B (NFκ-B) were evaluated by reverse transcriptase-polymerase chain reaction and electropnoretic mobility shift assay, respectively. GM-CSF -/- mice showed decreased neutrophil infiltration, less leakage, and lower expression of cytokines in the lung after LPS or hemorrhage. GM-CSF augmented LPS-induced protein and mRNA expression of TNF-α, MIP-2 and IL-1β, which was mediated by increased intra-nuclear translocation of NF-κB. GM-CSF plays an important role in high-dose LPS and hemorrhage-induced ALI, which appears to be mediated by its priming effect on neutrophils.

Induction of arthritis by high mobility group box chromosomal protein 1 is independent of tumour necrosis factor signalling

IntroductionTNFα and high mobility group box chromosomal protein 1 (HMGB1) are two potent proinflammatory cytokines implicated as important mediators of arthritis. Increased levels of these cytokines are found in the joints of rheumatoid arthritis patients, and the cytokines trigger arthritis when applied into the joints of naïve mice. HMGB1 is actively released from immune cells in response to TNFα; once released, HMGB1 in turn induces production of several proinflammatory cytokines – including IL-6 and TNFα – by macrophages. Whether HMGB1-induced arthritis is mediated via the TNFα pathway, however, is unknown. The purpose of the present study was to investigate whether the arthritis-inducing effect of HMGB1 is dependent on TNFα expression in vivo and to assess whether TNFα deficiency affects a proinflammatory cytokine response to HMGB1 in vitro.MethodsTNFα knockout mice and backcrossed control animals on a C57Bl6 background were injected intraarticularly with 5 μg HMGB1. Joints were dissected 3 days after intraarticular injection and were evaluated histologically by scoring the frequency and severity of arthritis. For in vitro studies, mouse spleen cultures from TNFα knockout mice and from control mice were incubated with different doses of HMGB1, and cell culture supernatants were collected at different time points for analysis of IL-6.ResultsIntraarticular injection of HMGB1 into healthy mouse joints resulted in an overall frequency of 32% to 39% arthritic animals. No significant differences were found with respect to the severity and incidence of synovitis between mice deficient for TNFα (seven out of 18 mice with arthritis) in comparison with control TNFα+/+ animals (six out of 19). No significant differences were detected between spleen cells from TNFα+/+ mice versus TNFα-/- mice regarding IL-6 production upon stimulation with highly purified HMGB1 after 24 hours and 48 hours. Upon stimulation with a suboptimal dose of recombinant HMGB1, however, the splenocytes from TNFα+/+ animals released significantly more IL-6 than cells from the knockout mice (602 ± 112 pg/ml and 304 ± 50 pg/ml, respectively; P < 0.05).ConclusionOur data show that HMGB1-triggered joint inflammation is not mediated via the TNF pathway. Combined with our previous study, we suggest that HMGB1-triggered arthritis is probably mediated through IL-1 activation.

Preliminary clinical measurement of the expression of TNF‐related apoptosis inducing ligand in patients with ankylosing spondylitis

It has recently been reported that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) plays various roles in such autoimmune diseases as diabetes, multiple sclerosis (MS), and systemic lupus erythematosus (SLE). However, it has still remained unclear whether there is a close relationship between TRAIL and ankylosing spondylitis (AS). In this study, we investigated the association between the expression of TRAIL and AS. The specific messenger ribonucleic acid (mRNA) levels of TRAIL in peripheral blood mononuclear cells (PBMCs), serum sTRAIL, and TNF-alpha concentrations from 60 AS patients, 20 rheumatoid arthritis (RA) patients, and 30 healthy controls were determined by real-time fluorescent quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The results indicated that the expression levels of TRAIL mRNA, and serum sTRAIL were significantly elevated in AS patients, compared with RA patients and healthy controls, and there was a close association between TRAIL mRNA and sTRAIL levels. However, there was no significant difference between human leukocyte antigen (HLA)-B27-positive and -negative AS patients. In HLA-B27-positive patients, TRAIL mRNA and sTRAIL closely correlated with serum TNF-alpha and C-reactive protein (CRP), but did not correlate in HLA-B27-negative patients. In conclusion, upregulated expression of TRAIL might be somewhat specific for evaluation of AS.

Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-α in endobronchial biopsies in horses with recurrent airway obstruction

BackgroundThe aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-α, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission.ResultsDespite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls.In comparing real-time PCR analysis to results of immunohistochemistry only IL-10 mRNA and protein levels in RAO horses on pasture were significantly correlated (rs = 0.893, p = 0.007). Curiously, in controls examined on pasture the TNF-α protein level was positively correlated to IL-10 mRNA expression (rs = 0.967, p = 0.007) and negatively correlated to IL-6 mRNA expression (rs = -0.971, p = 0.001).ConclusionGiven the complementary relationship of assessing cytokines directly by immunohistochemistry, or indirectly by PCR to mRNA, the lack of significant changes in either mRNA or protein levels of IL-6, IL-10 or TNF-α mRNA in RAO horses in exacerbation suggests that these particular cytokines in bronchial tissue may not play a substantive role in the active inflammation of this disease. To support this contention further studies examining time dependency of expression of IL-6, IL-10 or TNF-α are needed, as is expansion of the range of cytokines to include other key regulators of airway inflammation.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNFalpha, LTA, TNFSF15 and fish TNFalpha proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamily molecules are present in marine invertebrates.

Increased severity of renal ischemia-reperfusion injury with venous clamping compared to arterial clamping in a rat model

Arterial inflow occlusion is a well-known mechanism of renal injury during major vascular surgery. In contrast, renal injury from venous outflow obstruction is poorly understood. The goal of this study was to examine the injury pattern of renal venous outflow obstruction, compare this with the traditional model of arterial occlusion, and examine possible mechanisms. Male Fisher rats were used for the renal warm ischemia model. Twenty-five minutes of renal ischemia was induced by selectively occluding either the renal artery or vein. After 24 h of reperfusion, whole blood and kidney tissue were collected for further analysis. Serum creatinine (SCr) concentrations taken 24 h after reperfusion were significantly greater in the venous occlusion group (V) when compared to the arterial group (A). While histology did not demonstrate significant differences in extent of necrosis between both groups, a stronger inflammatory response resulted from venous occlusion. Specifically, significantly greater MCP-1 mRNA and significantly greater MCP-1, TNF-alpha, and HO-1 protein levels were found in the venous group, while no differences in MIP-2, ICAM-1, and VCAM-1 mRNA expression existed between A and V. Further analysis demonstrated presence of increased cleaved caspase-3 protein in the artery group than in the venous group. Venous renal outflow obstruction results in more severe functional renal injury when compared to arterial inflow occlusion. Macrophage activation and neutrophilic infiltration appear to be exaggerated during venous occlusion.

Activated protein C, an anticoagulant polypeptide, ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases

Our aim was to investigate the changes of mitogen-activated protein kinases (MAPKs) by activated protein C (APC) treatment in rats with severe acute pancreatitis (SAP), and relate them to changes in SAP severity, thus providing evidence for developing clinical therapies. Sprague-Dawley rats were given an intravenous injection of saline (SAP group), APC (50 microg/kg or 10 microg/kg), or CNI1493 just before SAP induction. One group of rats underwent a sham operation (control group). Experimental samples were harvested 16 h after SAP induction. The gene expression of pancreatic MAPKs was evaluated by cDNA microarrays. The mRNA and protein/phosphorylated protein levels of p38 MAPK, extracellular signal-regulated protein kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK) and the protein levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta were determined in pancreatic tissue. The severity of disease was evaluated by pancreatic histology, the pancreatic wet/dry weight ratio, and the serum amylase level. In rats treated with APC (50 microg/kg) or CNI1493, the severity of pancreatitis and expression of pancreatic TNF-alpha and IL-1beta proteins were attenuated by the decreased expression and activity of p38 MAPK and JNK (vs. the SAP group, P < 0.01). The expression and activity of ERK1/2 were increased in APC-treated rats, especially in the group treated with APC 50 microg/kg (vs. the SAP or CNI1493-treated group, P < 0.01, respectively). Inhibition of expression of pancreatic p38 MAPK and JNK and upregulation of ERK1/2 expression by APC treatment may protect against pancreatic injury, thus ameliorating severity of the disease.

Role of β-Adrenergic Receptors in Inflammatory Marker Expression in Müller Cells

To determine whether beta-adrenergic receptors are involved in the modulation of inflammatory cytokines in Müller cells in a hyperglycemic environment. Rat Müller cells were grown in high (25 mM)- or low (5 mM)-glucose medium. Müller cells lysates were processed for real-time polymerase chain reaction to measure steady state mRNA expression for the following inflammatory markers: iNOS, TNF-alpha, IL-1B, and ICAM-1. Western blot analysis and ELISA assays were performed to determine the protein levels of these inflammatory markers and PGE2 content. Isoproterenol treatment significantly decreased protein levels of iNOS, TNF-alpha, and IL-1B, in rMC-1 cells cultured in high glucose as early as 1 hour, compared with cells receiving no treatment. PGE2 content was also reduced after isoproterenol treatment. There were no significant changes observed in protein levels of ICAM-1 production after isoproterenol treatment in high glucose. Steady state mRNA levels for iNOS were significantly decreased 1 hour after isoproterenol, whereas ICAM-1 gene expression was significantly increased after 1 hour. Isoproterenol significantly increased gene expression for IL-1B after 24 hours of treatment. These results suggest that stimulation of beta-adrenergic receptors with isoproterenol leads to decreased levels of PGE(2), TNF-alpha, and IL-1B protein content, and in both gene expression and protein levels of iNOS in Müller cells cultured in hyperglycemia. beta-Adrenergic receptor agonists had limited effects on ICAM-1 protein production. These results indicate that isoproterenol treatment reduces cytokine activation in cultured rat Müller cells.

Orthopedic Implant Particle‐Induced Tumor Necrosis Factor‐α Production in Macrophage–Monocyte Lineage Cells Is Mediated by Nuclear Factor of Activated T Cells

Wear particles produced from artificial joint prostheses are known to cause macrophage-monocyte lineage cells to produce proosteoclastogenic cytokines, including tumor necrosis factor (TNF)-alpha. The specific molecular mechanism, however, is not yet known. Bioinformatic analysis showed that the promoter region of TNF-alpha has several consensus sequences for NFAT binding. Consequently, we examined the role of nuclear factor of activated T cells (NFAT) in TNF-alpha production. Our investigation has shown that treatment with titanium nanoparticles increased TNF-alpha gene expression along with TNF-alpha protein secretion in murine macrophage-like RAW264.7 and primary monocyte-macrophage cells. Titanium particle-induced TNF-alpha induction was inhibited by VIVIT, a peptide inhibitor that targets the calcineurin/NFAT axis, which suggests that NFAT mediates metallic particle-induced TNF-alpha expression in monocyte-macrophage lineage cells.

Antiallergic Activities of Pigmented Rice Bran Extracts in Cell Assays

Using a panel of chemical, biochemical, and cell assays, we determined inhibitory effects of extracts of the pigmented black rice brans on in vitro allergic reactions. Ethanol-water (70% v/v) extracts from 5 pigmented brans were found to be more effective than an extract from a nonpigmented rice cultivar in suppressing the release of histamine and beta-hexosaminidase from basophilic RBL-2H3 cells stimulated with both Ionophore A23187 and immunoglobulin E (IgE)-antigen complexes. Suppression was also obtained with A23187-stimulated rat peritoneal mast cells. The extent of inhibition of these 2 markers of the immune response was accompanied by an influx of calcium ions. The inhibition of the immune process by the pigmented brans was confirmed by the observed modulation of the proinflammatory cytokine gene expressions and cytokine release, as indicated by the reduction in tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-4, and IL-6 mRNA expressions determined with the reverse transcription-polymerase chain reaction (RT-PCR). Reduction of TNF-alpha, IL-1beta, and IL-6 protein release from both the cultured cell line and peritoneal cells was further confirmed by enzyme-linked immunoadsorbent assays. Rice bran from the LK1-3-6-12-1-1 cultivar was the most effective inhibitor in all assays. This particular rice variety merits further evaluation as part of a human diet to ascertain its potential to protect against allergic diseases such as hay fever and asthma.

Neostigmine and pilocarpine attenuated tumour necrosis factor α expression and cardiac hypertrophy in the heart with pressure overload

The inflammatory cytokine tumour necrosis factor alpha (TNF alpha) is known to be a major factor contributing to cardiac remodelling and dysfunction. Parasympathetic nervous system cholinergic function can inhibit TNF alpha expression during systemic infection. In the present study, we tested the effects of a cholinesterase inhibitor, neostigmine, and a muscarinic cholinergic agonist, pilocarpine, on cardiac hypertrophy and TNF alpha levels during pressure overload. Rats with transverse aortic constriction exhibited elevated TNF alpha protein levels in the heart, increased heart weight to body weight ratios (an index of cardiac hypertrophy) and decreased left ventricular diastolic function. Two weeks of infusion with neostigmine (6 microg kg(-1) day(-1)) or pilocarpine (0.3 mg kg(-1) day(-1)) significantly reduced cardiac hypertrophy, reduced TNF alpha levels and elevated interleukin-10 levels in heart tissues, and improved ventricular function in rats with transverse aortic constriction. Neither of these treatments significantly changed ventricular pressure load. Furthermore, in primary cultured neonatal cardiac cells, treatment with pilocarpine attenuated adrenergic agonist phenylephrine-induced increased TNF alpha expression and [3H]leucine (a marker of protein synthesis) incorporation in the cells. Collectively, both cholinergic agents decreased TNF alpha levels and attenuated cardiac hypertrophy. Since both agents potentially enhanced cholinergic function, the anti-inflammatory action may be involved in the cardioprotective effect of the treatments with these agents.

Associations between insulin resistance and TNF-α in plasma, skeletal muscle and adipose tissue in humans with and without type 2 diabetes

Clear evidence exists that TNF-alpha inhibits insulin signalling and thereby glucose uptake in myocytes and adipocytes. However, conflicting results exist with regard to the role of TNF-alpha in type 2 diabetes. We obtained blood and biopsy samples from skeletal muscle and subcutaneous adipose tissue in patients with type 2 diabetes (n = 96) and healthy controls matched for age, sex and BMI (n = 103). Patients with type 2 diabetes had higher plasma levels of fasting insulin (p < 0.0001) and glucose (p < 0.0001) compared with controls, but there was no difference between groups with regard to fat mass. Plasma levels of TNF-alpha (p = 0.0009) and soluble TNF receptor 2 (sTNFR2; p = 0.002) were elevated in diabetic patients. Insulin sensitivity was correlated with quartiles of plasma TNF-alpha after adjustment for age, sex, obesity, WHR, neutrophils, IL-6 and maximum O(2) uptake (VO2/kg) in the diabetes group (p < 0.05). The TNF mRNA content of adipose or muscle tissue did not differ between the groups, whereas muscle TNF-alpha protein content, evaluated by western blotting, was higher in type 2 diabetic patients. Immunohistochemistry revealed more TNF-alpha protein in type 2 than in type 1 muscle fibres. After adjustment for multiple confounders, plasma TNF-alpha is associated with insulin resistance. This supports the idea that TNF-alpha plays a significant role in the pathogenesis of chronic insulin resistance in humans. However, findings on the TNF-alpha protein levels in plasma and skeletal muscle indicate that measurement of TNF mRNA content in adipose or muscle tissue provides no information with regard to the degree of insulin resistance.

Identification, affinity characterisation and biological interactions of lectin‐like peptide–carbohydrate complexes derived from human TNF‐α using high‐resolution mass spectrometry

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-alpha [TNF-alpha (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-alpha protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.

Aldose Reductase Inhibition Prevents Endotoxin-Induced Uveitis in Rats

The purpose of the present study was to elucidate the role of the polyol pathway enzyme aldose reductase (AR) in the mediation of ocular inflammation in a rat model of endotoxin-induced uveitis (EIU). EIU was induced by a subcutaneous injection of 200 microg lipopolysaccharide (LPS) in male Lewis rats treated with the AR inhibitor, zopolrestat (25 mg/kg body weight, intraperitoneally) or its carrier. The rats were killed 24 hours after LPS injection, the eyes were enucleated immediately, and aqueous humor (AqH) was collected. The number of infiltrating cells, protein concentration, and levels of nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and prostaglandin E(2) (PGE(2)) in the AqH were determined. Immunohistochemical analysis was performed in paraformaldehyde-fixed eye sections by staining with antibodies against iNOS, COX-2, TNF-alpha, NF-kappaB, and AR. The levels of reactive oxygen species (ROS) in rat eye sections were determined by dihydroethidium (hydroethidine) fluorescence staining. In the EIU rat eye AqH, both the number of infiltrating cells and protein concentrations of the inflammatory markers, TNF-alpha, NO, and PGE(2) were significantly higher than in the control rats, and inhibition of AR by zopolrestat suppressed the LPS-induced increases. The LPS-induced increased expression of AR, TNF-alpha, iNOS, and COX-2 proteins in the ciliary body, corneal epithelium, and retinal wall was also significantly inhibited by zopolrestat. Furthermore, AR inhibition prevented the LPS-induced increased levels of ROS and activation of NF-kappaB in the ciliary body, corneal epithelium, and retinal wall of the rat eye. AR inhibition also prevented the LPS-induced activation of NF-kappaB and expression of COX-2 and iNOS in the human monocyte cell line U-937. The results indicate that AR inhibition suppresses the inflammation in EIU by blocking the expression and release of inflammatory markers in ocular tissues, along with the attenuation of NF-kappaB activation. This finding suggests that AR inhibition could be a novel therapeutic target for the treatment of uveitis and associated ocular inflammation.