Reduced Expression Of TNF-α Research Articles

TP508, a thrombin‐derived peptide, modulates TLR‐4 dependent cytokine expression in differentiated THP‐1 cells

Accelerated wound healing by TP508, a thrombin‐derived synthetic peptide, may involve inflammatory mediators. The mechanism of action for TP508 is hypothesized to be integrin binding at its RGD site. THP‐1 monocytes were differentiated to an adherent macrophage phenotype with 50 ng/mL phorbol 12‐myristate 13‐acetate (PMA). Cells were pretreated with TP508 for 30 minutes, followed by lipopolysaccharide (LPS) for 3.5 hours. TNFα, IL‐8 and IL‐1 β were analyzed via ELISA. TP508 decreased expression of TNFα and IL‐8 and increased IL‐1 β expression independently of LPS. When heparin‐sulfate was used to stimulate TLR4 receptors, TP508 again reduced TNFα expression.Given the peptide sequence of TP508, direct blocking of the TLR4 receptor is improbable, so the TP508 RGD site was explored for potential interaction with TLR4‐dependent cytokine stimulation. Variants of TP508 were used to test whether the RGD‐integrin binding was responsible for the reduction in cytokine expression in these cells. An αV β3 integrin blocking antibody was as effective in reducing LPS‐stimulated cytokine expression as the TLR4 activity‐blocking antibody. Treatment of cells with αV β3 integrin‐binding peptide GRGDSP also significantly reduced TNFα expression in response to LPS. This preliminary evidence suggests that TP508 may modulate cytokine expression through an integrin dependent mechanism.

Metallic gold reduces TNFα expression, oxidative DNA damage and pro-apoptotic signals after experimental brain injury

Brain injury represents a major health problem and may result in chronic inflammation and neurodegeneration. Due to antiinflammatory effects of gold, we have investigated the cerebral effects of metallic gold particles following a focal brain injury (freeze-lesion) in mice. Gold particles 20–45 μm in size or the vehicle (placebo) were implanted in the cortical tissue followed by a cortical freeze-lesioning. At 1–2 weeks post-injury, brains were analyzed by using immunohistochemistry and markers of inflammation, oxidative stress and apoptosis. This study shows that gold treatment significantly reduces the cerebral levels of tumor necrosis factor alpha (TNFα), oxidative DNA damage (as judged by 8-oxoguanine levels), and pro-apoptotic markers (cleaved caspase-3, cytochrome c leakage), when compared to those of controls. The data presented here points toward gold particles as a tool to modulate the cerebral response to injury.

Voluntary exercise training in mice increases the expression of antioxidant enzymes and decreases the expression of TNF-α in intestinal lymphocytes

Acute exercise in mice induces intestinal lymphocyte (IL) apoptosis. Freewheel running reduces apoptosis and forced exercise training increases splenocyte antioxidant levels. The purpose of this study was to examine the effect of freewheel running and acute exercise on mouse IL numbers and concentrations of apoptosis and antioxidant proteins and pro-inflammatory cytokines in IL. Female C57BL/6 mice had access to in-cage running wheels (RW) or cages without wheels (NRW) for 16 weeks and were randomized at the end of training to no exercise control (TC) or to treadmill exercise with sacrifice after 90 min of running (TREAD; 30 min, 22 m min −1; 30 min, 25 m min −1; 30 min, 28 m min −1; 2° slope). IL were analyzed for pro-(caspase 3 and 7) and anti-(Bcl-2) apoptotic proteins, endogenous antioxidants (glutathione peroxidase: GPx; catalase: CAT) and the pro-inflammatory cytokine, TNF-α. RW mice had higher cytochrome oxidase ( p < 0.001) and citrate synthase ( p < 0.01) activities in plantaris and soleus muscles and higher GPx and CAT expression in IL ( p < 0.05) (indicative of training) compared with NRW mice. TNF-α expression was lower ( p < 0.05) and IL numbers higher ( p < 0.05) in RW vs. NRW mice. No training effect was observed for apoptotic protein expression, although TREAD resulted in higher caspase and lower Bcl-2. These results suggest that freewheel running in mice for 16 weeks enhances antioxidant and reduces TNF-α expression in IL but does not reduce pro-apoptotic protein expression after acute exercise. Results are discussed in terms of implications for inflammatory bowel diseases where apoptotic proteins and TNF-α levels are elevated.

TIMP-3 and MMP-3 contribute to delayed inflammation and hippocampal neuronal death following global ischemia

Hippocampal neuronal death following transient global ischemia in the mouse takes days to occur, providing a potential timeframe for therapeutic intervention. Since matrix metalloproteinase-3 (MMP-3) enhances inflammation and tissue inhibitor of metalloproteinases-3 (TIMP-3) promotes apoptosis in ischemia, we hypothesized that they are involved in neuronal death secondary to transient global ischemia. Timp-3 knockout (T3KO) and wild type (T3WT) mice underwent 30 min bilateral carotid artery occlusion (BCAO), which causes hippocampal neuronal death 7 days after reperfusion. Mice lacking the Timp-3 gene have significantly less astrocytosis, microglial reactivity, MMP-3 activity and neuronal cell death. In addition, T3KO mice had decreased tumor necrosis factor (TNF) receptor-1 (TNFR1) expression and increased TNF-α converting enzyme (TACE) activity. Mmp-3 KO mice with a similar BCAO showed significantly fewer microglial cells, reduced TNF-α expression, and less neuronal death than the Mmp-3 WT. To see if TIMP-3 and MMP-3 cell death pathways were independent, we blocked MMPs with the broad-spectrum MMP inhibitor, BB-94, on days 3 through 6 of reperfusion in T3WT and T3KO mice. BB-94 rescued hippocampal neurons at 7 days in both T3WT and T3KO mice, but significantly fewer neurons died in T3KO mice treated with BB-94. Our results indicate a novel additive role for TIMP-3 and MMP-3 in delayed neuronal death, and show that delayed treatment with MMP inhibitors can be used to reduce hippocampal death.

Acute Pulmonary Lipopolysaccharide Tolerance Decreases TNF-α without Reducing Neutrophil Recruitment

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-alpha. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-alpha were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-alpha mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-alpha was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-alpha expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-alpha levels, while still maintaining the antimicrobial capacity of the lung.

Abstract 1948: Over-expression of HSP27 Provides Chronic Atheroprotection: Reductions in Foam Cell Content and Inflammatory Responses

Introduction: Heat Shock Protein 27 (HSP27) was recently shown by our laboratory to be a biomarker of atherosclerosis and after 4 weeks of a high fat diet (HFD) apoE −/− mice that over-express HSP27 (or apoE −/− HSP27 o/e mice) show a marked attenuation of aortic lesion size compared to apoE −/− mice - but only in females (ATVB 20005; Circ Res in press). Moreover, we demonstrated that serum HSP27 levels were inversely related to atherosclerotic burden, estrogens mediated the release of HSP27 from cells and extracellular HSP27 bound to Scavenger Receptor-A where it could competitively inhibit acLDL uptake. Purpose/Methods: The goal of this study was to determine if and how HSP27 over-expression persistently attenuates atherogenesis in apoE −/− mice fed a HFD for 12 weeks. Results: Compared to female apoE −/− mice, female apoE −/− HSP27 o/e mice showed persistent reductions in aortic (en face) lesion area and aortic sinus cross-sectional lesion area at 12 weeks (e.g., 38% and 39% respectively vs. 35% and 40% after 4 weeks of HFD). Although our original studies revealed that male apoE −/− HSP27 o/e mice had no protection from atherogenesis after 4 weeks of a HFD, in the current study we noted that 12 weeks of a HFD resulted in a 17% reduction in aortic (en face) lesion area and a 24% attenuation in aortic sinus cross-sectional lesion area with HSP27 over-expression. While lesions observed after 4 weeks of HFD consisted mainly of lipid-laden foam macrophages, after 12 weeks of HFD the histopathology was different and included a distinct foam cell layer (or FCL) luminal to a deeper necrotic layer (e.g., with cholesterol crystals). The FCL area was reduced in size in apoE −/− HSP27 o/e vs. apoE −/− mice (e.g., −63% in females, p=0.03; and −48% in males, p=0.02). In vitro, macrophages from apoE −/− HSP27 o/e mice showed a reduction in TNF-α expression compared to apoE −/− mice (e.g., 40% at baseline, and 55% after LPS provocation). Conclusions: Over-expression of HSP27 provides a persistent atheroprotective effect that benefits both sexes. Chronic lesions are not only smaller in size with HSP27 over-expression but show fewer foam cells and macrophages are less pro-inflammatory. These results highlight a potential clinical role for chronic HSP27 modulation for atheroprotection.

Mao-to Prolongs the Survival of and Reduces TNF-α Expression in Mice with Viral Myocarditis

Goal of this study was to evaluate effects of Mao-to on development of myocarditis induced by encephalomyocarditis (EMC) virus in mice. Mice were randomly divided into five groups. Group N included uninfected controls (n = 18), while group A, B and C underwent intraperitoneal injection of EMC virus. Group A was administered oral saline from day 0 to day 4. Group B was administered oral Mao-to (500 mg−1 kg−1 day−1) from day 0 to day 4. Group C was administered Mao-to from day 2 to day 6. Group D was administered Mao-to from day 5 to day 10. Treated mice were followed for survival rates during 2 weeks after infection. Body weight (BW) and organ weights including heart (HW), lungs, thymus and spleen were examined on days 4, 6 and 14. Survival rate of group C (36.4%) was significantly improved compared with group A, B or D (0% of each, P < 0.05). HW and HW/BW ratio in group C was significantly (P < 0.05) lower than those in group A, B or D. Viral titers of hearts were significantly different among groups A, B and C. Cardiac expression in tumor necrosis factor-α (TNF-α) was significantly reduced in group C in comparison with group A, B or D on day 6 by immunohistochemical study. Administration of Mao-to starting on day 2 improves mortality resulting from viral myocarditis in mice with reduced expression of cardiac TNF-α. These findings suggest that timing of Mao-to is crucial for preventing cardiac damage in mice with viral myocarditis.

Cisplatin Increases TNF-α mRNA Stability in Kidney Proximal Tubule Cells

Cisplatin induces acute renal injury in part by increasing the production of TNF-α. However, the mechanism by which cisplatin increases renal TNF-α expression is not known. The transcription, translation, and stability of TNF-α mRNA are sites of regulation of TNF-α production. This study investigated the effects of cisplatin on TNF-α mRNA stability and the role of MAP kinases in this process in cultured renal proximal tubule cells. Cisplatin increased the expression of TNF-α mRNA by proximal tubule cells in a time- and dose-dependent manner, as well as activated p42/44 ERK kinase, p38 MAP kinase, and JNK in a dose-dependent manner. The inhibition of these pathways reduced TNF-α expression significantly. Cisplatin also increased the stability of TNF-α mRNA, but this effect was not mediated by MAP kinases and did not require the synthesis of a new protein. The treatment of cells with cisplatin induced the formation of complexes of cytosolic proteins and the AU-rich region of the TNF-α 3′UTR. These results are consistent with the view that cisplatin increases TNF-α mRNA stability in a MAP kinase-independent manner. The stabilization of TNF-α mRNA by cisplatin may involve the binding of certain proteins to AU-rich regions in the 3′UTR.

Ischemic Preconditioning Ameliorates Microcirculatory Disturbance through Downregulation of TNF-Alpha Production in a Rat Cremaster Muscle Model

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IĸB-α-NF-ĸB-TNF-α (tumor necrosis factor-α) pathway in IPC’s ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-α expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-α protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IĸB-α phosphorylation and NF-ĸB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-ĸB activation and subsequently reduced TNF-α expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.

Pioglitazone, a Specific Ligand of the Peroxisome Proliferator-Activated Receptor Gamma Reduces Gastric Mucosal Injury Induced by Ischaemia/Reperfusion in Rat

Background: The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor that has been implicated in the control of metabolism and numerous cellular processes, including cell cycle control, carcinogenesis, and inflammation. The present study was designed to investigate the effect of the specific PPARγ ligand, pioglitazone, on the mucosal lesions induced by ischaemia and reperfusion (I/R) in rats. Methods: I/R lesions were induced in Wistar rats by applying a small clamp to the coeliac artery for 30 min (ischaemic phase), followed by the removal of the clamp for 3 h (reperfusion phase). Vehicle (saline) or increasing doses of pioglitazone (2.5, 10, and 30 mg/kg i.g.) were given 30 min before exposure to I/R. The animals were killed immediately after the end of the reperfusion phase (time 0) and at 12 and 24 h after I/R. The area of gastric lesions was measured by planimetry, and the gastric blood flow was determined by the H[Formula: See Text] gas clearance method. The gastric mucosal gene expressions of PPARγ, interleukin-1beta (IL-1β), tumour necrosis factor alpha (TNF-α), leptin, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were examined by RT-PCR. In addition, protein expression of COX-2 and leptin was assessed by Western blot. Results: The pretreatment with pioglitazone reduced in a dose-dependent manner the mean lesion area induced by I/R, and this effect was accompanied by a significant increase in the gastric blood flow. The decrease in gastric ulcerations by pioglitazone was also observed 12 and 24 h after the I/R. The PPARγ mRNA was weakly expressed in the intact gastric mucosa, but significantly up-regulated after exposure to I/R at each time interval studied. The expression of IL-1β was not changed significantly after pioglitazone applied i.g. at doses 2.5 and 10 mg/kg, but it was down-regulated at the dose 30 mg/kg. TNFα mRNA was strongly increased after the exposure to I/R, but it was down-regulated after pioglitazone pretreatment. In contrast, both leptin and COX-2 mRNA and protein expression were increased in the gastric mucosa after exposure to I/R. The pretreatment with pioglitazone caused a significant up-regulation of mRNA and protein expression of leptin, reaching its peak at the dose 30 mg/kg i.g. In contrast, COX-2 expression did not change significantly after the 2.5 and 10 mg/kg of pioglitazone, but it significantly decreased after pioglitazone at dose 30 mg/kg given to rats before exposure to I/R. Conclusions: Pioglitazone reduces the acute erosions and deeper gastric lesions induced by I/R. The beneficial effect of this PPARγ ligand on I/R-induced gastric damage may be due to its anti-inflammatory properties, especially to the reduction in TNF-α expression and to up-regulation of leptin mRNA in the gastric mucosa. The inhibition of COX-2 expression by pioglitazone may reflect the anti-inflammatory properties of this compound.

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages.

The APO-1/Fas ligand (FasL) and tumor necrosis factor-alpha (TNF-alpha) are two functionally related molecules that induce apoptosis of susceptible cells. Although the two molecules have been reported to induce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-alpha, raising the possibility that TNF-alpha may be involved in FasL-induced apoptosis. Because TNF-alpha gene expression is under the control of nuclear factor-kappaB (NF-kappaB), we investigated whether FasL can induce NF-kappaB activation and whether such activation plays a role in FasL-mediated cell death in macrophages. Gene transfection studies using NF-kappaB-dependent reporter plasmid showed that FasL did activate NF-kappaB promoter activity. Gel shift studies also revealed that FasL mobilized the p50/p65 heterodimeric form of NF-kappaB. Inhibition of NF-kappaB by a specific NF-kappaB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-kappaB inhibitory subunit IkappaB caused an increase in FasL-induced apoptosis and a reduction in TNF-alpha expression. However, neutralization of TNF-alpha by specific anti-TNF-alpha antibody had no effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-kappaB and is independent of TNF-alpha activation, suggesting the antiapoptotic role of NF-kappaB and a separate death signaling pathway mediated by FasL.

Hydroxy acids and retinoids in cosmetics

In this study, we developed a water-in-oil microemulsion containing vitamin A (retinol) and vitamin E (α-tocopherol), which serves as a multifunctional nanosystem that co-delivers antioxidants and displayed additive effect against acute skin inflammation. Microemulsion (ME) was prepared by mixing a surfactant blend (Tween 80 and propylene glycol, 5:1) with isopropyl myristate and water (ratio of 50:40:10, respectively). Vitamin A (0.05% w/w concentration) and/or vitamin E (0.1% w/w concentration) were incorporated into the surfactant mixture of ME by stirring with a magnetic stirrer for 30 min. This multifunctional ME displayed physical stability, with low cytotoxicity in 3T3 cell line, as well as cellular internalization into the cytosol. In vivo treatments using ME delivering α-tocopherol reduced dermal expression of TNF-α by 1.3-fold (p < 0.01), when compared to unloaded ME treatment group. When retinol was added into the ME containing α-tocopherol, it further reduced TNF-α expression by 2-fold (p < 0.001), suggesting the additive effect of vitamin E and vitamin A in the treatment against skin inflammation. In conclusion, we successfully developed the use of water-in-oil ME to pack both vitamin E and vitamin A, and demonstrated for the first time its anti-inflammatory potential when applied topically to TPA-induced inflamed skin.

Antisense oligodeoxynucleotide inhibition of tumor necrosis factor-alpha expression is neuroprotective after intracerebral hemorrhage.

Tumor necrosis factor-alpha (TNF-alpha) expression is increased in brain after cerebral ischemia, although little is known about its abundance and role in intracerebral hemorrhage (ICH). A TNF-alpha-specific antisense oligodeoxynucleotide (ORF4-PE) was used to study the extent to which TNF-alpha expression influenced neurobehavioral outcomes and brain damage in a collagenase-induced ICH model in rat. Male Sprague-Dawley rats were anesthetized, and ICH was induced by intrastriatal administration of heparin and collagenase. Immediately before or 3 hours after ICH induction, ORF4-PE was administered directly into the site of ICH. TNF-alpha mRNA and protein levels were measured by reverse transcriptase-polymerase chain reaction and immunoblot analyses. Cell death was measured by terminal deoxynucleotidyl transferase-mediated uridine 5'triphosphate-biotin nick end labeling (TUNEL). Neurobehavioral deficits were measured for 4 weeks after ICH. ICH induction (n=6) elevated TNF-alpha mRNA and protein levels (P:<0.01) at 24 hours after the onset of injury compared with sham controls (n=6). Immunohistochemical labeling indicated that ICH was accompanied by elevated expression of TNF-alpha in neutrophils, macrophages, and microglia. Administration of ORF4-PE (2.0 nmol) directly into striatal parenchyma, 15 minutes before (n=4) or 3 hours after (n=6) ICH, decreased levels of TNF-alpha mRNA (P:<0.001) and protein (P:<0. 01) in the brain tissue surrounding the hematoma compared with animals treated with saline alone (n=6). Mean+/-SEM striatal cell death (cells per high-powered field) was also reduced in animals receiving ORF4-PE (34.1+/-5.0) compared with the saline-treated ICH group (80.3+/-7.50) (P:<0.001). ORF4-PE treatment improved neurobehavioral deficits observed at 24 hours (P:<0.001) after induction of ICH (n=6) compared with the untreated ICH group (n=6). This improvement was maintained at 28 days after hemorrhage induction (P:<0.001). These results indicate a pathogenic role for TNF-alpha during ICH and demonstrate that reducing TNF-alpha expression using antisense oligodeoxynucleotides is neuroprotective.

Methylprednisolone inhibition of TNF-α expression and NF-kB activation after spinal cord injury in rats

Post-traumatic inflammatory reaction has been implicated in the secondary injury after SCI. TNF-α is a key inflammatory mediator, which plays a pathogenetic role in cell death in inflammatory disorders and traumatic brain injury. TNF-α exerts its effector actions, at least partially, through the activation of a pro-inflammatory transcription factor, NF-kB, which in turn upregulates such genes as iNOS, cytokines, adhesive molecules, and others. Consistent with a post-traumatic inflammatory reaction after SCI, we noted an increase in TNF-α expression by Western blotting (4.5-fold increase at 1 day after SCI, P<0.01) and immunohistochemistry in a rat SCI model. Post-traumatic TNF-α expression was accompanied by an increase in NF-kB binding activity in nuclear proteins isolated from the injured cord (3.9-fold increase, P<0.01). MP is the only drug proven effective in improving neurological function in patients with acute SCI. The mechanism of action of MP is not fully understood, but is thought to be related to its antioxidant effects. MP is also a potent anti-inflammatory agent, which has been recently shown to inhibit NF-kB binding activity. MP (30 mg/kg, i.v.) given immediately after SCI reduced TNF-α expression by 55% ( P<0.01) and NF-kB binding activity. These findings suggest that post-traumatic inflammatory activity that is mediated by the TNF-α-NF-kB cascade can be suppressed by MP.