The role of tumour necrosis factor-α and tumour necrosis factor receptor signalling in inflammation-associated systemic genotoxicity

Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1(-/-)TNFR2(-/-) mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1β, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators.     

Colocalization of substance P with tumor necrosis factor-α in the lymphocytes and mast cells in gastritis in experimental rats

Objective  

Substance P (SP) elicits numerous potent neuroimmunomodulatory effects, increasing the release of tumor necrosis factor alpha (TNF-α). The study aimed to investigate immunoneural communication in experimentally-induced gastritis in rats.     

Role of tumor necrosis factor-α in the pathogenesis of indomethacin-induced small intestinal injury in mice

The pathogenesis of small intestinal damage caused by non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is still unclear. For this reason, there is currently no therapeutic strategy for ameliorating such damage. On the other hand, molecular treatment strategies targeting tumor necrosis factor (TNF)-α exert beneficial effects on intestinal lesions in patients with inflammatory bowel disease (IBD). To clarify the participation of TNF-α in NSAID-induced small intestinal damage, we investigated the effects of indomethacin administration in mice with targeted deletion of the TNF-α gene. Indomethacin (10?mg/kg) was administered subcutaneously to male C57BL/6 (wild-type: WT) mice and TNF-α-deficient (TNF-α-/-) mice to induce small intestinal damage. The ulcer score, the tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the expression of keratinocyte chemoattractant (KC) mRNA in the small intestinal mucosa were measured. In addition, we performed a TUNEL assay to evaluate indomethacin-induced apoptosis of intestinal epithelial cells and measured the expression of caspase-3 protein and Bcl-2 mRNA. The ulcer score, MPO activity, and expression of KC mRNA were significantly increased after indomethacin administration. These increases were significantly inhibited in TNF-α-/- mice compared with WT mice. Apoptotic cells were observed by the TUNEL assay in the area of the ulcerative lesion, and they were significantly fewer in TNF-α-/- mice compared with WT mice. The expression of cleaved caspase-3 protein was induced by indomethacin administration, and significantly inhibited in TNF-α-/- mice compared with that of WT mice. The expression level of Bcl-2 mRNA in indomethacin-treated TNF-α-/- mice was significantly higher than that in WT mice. TNF-α plays an important role in the pathogenesis of indomethacin-induced small intestinal damage. These results suggest that TNF-α could become a new therapeutic target for NSAID-induced small intestinal damage.     

The effect of tumour necrosis factor-α and insulin on equine digital blood vessel function in vitro

Objective and design

Insulin and inflammatory cytokines may be involved in equine laminitis, which might be associated with digital vascular dysfunction. This study determined the effects of TNF-α and insulin on the endothelial-dependent relaxant responses of equine digital blood vessels and on equine digital vein endothelial cell (EDVEC) cGMP production.

Material

Isolated rings of equine digital arteries (EDAs) and veins (EDVs) were obtained and EDVECs were cultured from horses euthanized at an abattoir.

Methods

The effect of incubation with TNF-α (10 ng/ml) and/or insulin (1,000 μIU/ml) for 1.5 h or overnight under hyperoxic and hypoxic conditions on carbachol (endothelium-dependent) induced relaxation was assessed. The time course and concentration dependency of the effect of TNF-α and the effect of insulin (1,000 μIU/ml) on EDVEC cGMP production was determined.

Results

Incubation of EDAs overnight with TNF-α under hypoxic conditions resulted in endothelial-dependent vascular dysfunction. EDVs produced a more variable response. TNF-α increased EDVEC cGMP formation in a time- and concentration-dependent manner. Insulin had no significant effects.

Conclusions

There is a mismatch between the results obtained from isolated vessel rings and cultured endothelial cells suggesting TNF-α may reduce the biological effect of NO by reducing its bioavailability rather than its formation, leading to endothelial cell dysregulation.     

Tumor necrosis factor-α mediates the early pathology in Salmonella infection of the gastrointestinal tract

Salmonella infection of the intestinal tract results in damage to the gut epithelium. While it is generally believed that bacteria and/or bacterial products account for this pathology, the role of host factors has not been explored. Using a ligated intestinal loop model, we investigated whether tumor necrosis factor-α (TNF-α) could contribute to the tissue pathology associated with Salmonella infection. Intestinal segments infected with Salmonella typhimurium had high levels of fluid secretion as early as 6 h post bacterial infection. At this time point, low levels of TNF activity were also present in the fluid obtained from infected segments. At 20 h post-infection, high levels of TNF activity were present in fluids obtained from infected intestinal segments and was characterized as TNF-α by neutralization experiments using rabbit antisera to TNF-α. TNF-α production was further verified by Northern blot analysis using RNA obtained from cells eluted from the infected intestinal segments. In contrast, no TNF activity was found in fluid obtained from intestinal segments challenged with cholera toxin, which induces fluid secretion with little to no inflammatory response. Double labeling by in situ hybridization and immunocytochemistry revealed that macrophages in the lamina propria were producing the TNF-α mRNA. To investigate what role TNF-α might play in Salmonella-induced inflammation, intestinal segments were injected with recombinant mouse TNF-α (rTNF-α) or mice were pretreated with antibody to TNF-α or a control antibody prior to Salmonella infection. The histological profile of intestinal segments injected with rTNF-α appeared identical to segments infected with S. typhimurium. Further, pathology was completely eliminated in infected mice pretreated with antibody to TNF-α. These results document the production of TNF-α in the intestinal tract following S. typhimurium infection and show that the early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms.     

Efficacy of Kelamidium® in the prevention and treatment of Trypanosoma brucei brucei infection in albino rats

The efficacy of Kelamidium® in the prevention and treatment of experimental Trypanosoma brucei brucei infection of albino rats was studied. Adult albino rats (55) weighing between 147 and 240 g were used for the study. The rats were kept in metal cages in a fly-proof house and were adequately fed and given water ad libitum. Two experiments were carried out. In experiment I (chemotherapy), 30 adult albino rats were divided into six groups of five rats each, whereas in experiment II (chemoprophylaxis), 25 adult albino rats were divided into five groups of five rats each. In both experiments, groups I and II were uninfected control and infected untreated control, respectively. In experiment I, rats in groups III and V were each infected with 5.0?×?10⁵ trypanosomes and were later treated with 0.5 mg/kg of Kelamidium® (low-dose treatment), and rats in groups IV and VI were infected with 5.0?×?10⁵ trypanosomes and treated with 1.0 mg/kg of Kelamidium® (high-dose treatment). Treatment was given to rats in groups III and IV at day 7 postinfection (PI; early treatment), whereas groups V and VI were treated at day 10 PI (late treatment). In experiment II, rats in groups III, IV, and V were each treated with 2.0 mg/kg of Kelamidium® at day 0 and were later infected at days 14, 28, and 42 PI, respectively, with 5.0?×?10⁵ trypanosomes. Parasites were detectable in the blood of the infected rats in all the infected groups in experiment I and in group II in experiment II, 4–7 days PI. Parasitemia, however, was not recorded in the remaining groups in experiment II. The drug cleared the parasites from the blood of the infected rats in experiment I, 2–7 days posttreatment (PT). Relapse of infection, however, occurred in all the infected treated groups. It was thus concluded that Kelamidium® may be more useful as a prophylactic agent than as a chemotherapeutic agent in the management of animal trypanosomosis.     

Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis.     

New method for quantification of Trypanosoma cruzi in animal’s tissue in the chronic phase of experimental Chagas' disease

We developed a new method for the quantification of parasites in tissue. Trypanosoma cruzi strain CL parasites were genetically engineered to express the Escherichia coli β-galactosidase gene, lacZ and this enzyme is able to catalyze a colorimetric reaction with chlorophenol red β-d galactopyranoside (CPRG) as the substrate. The animals were infected with clone CL Brener strain B5 of T. cruzi and treated with benznidazole in order to verify the reduction in the number of parasites in tissue study by quantifying the enzyme β-galactosidase. The assay demonstrates a reduction in the number of parasites in the groups treated. Thus, this test can be used to test other substances with the aim of verifying the effectiveness in the chronic phase of experimental Chagas' disease.     

Continuous recruitment, co-expression of tumour necrosis factor-α and matrix metalloproteinases, and apoptosis of macrophages in gout tophi

Gout tophi are characterised by foreign body granulomas consisting of mono- and multinucleated macrophages surrounding deposits of monosodium urate microcrystals. After primary formation, granulomas grow associated with degradation of the extracellular matrix. Based on this background, we have sought (1) to investigate whether during granuloma's growth new macrophages are recruited into the tophi, (2) to find in situ evidence for macrophages' active role in matrix degradation and (3) to examine whether shrunk cells seen within gout tophi are apoptotic. Immunohistochemistry showed that perivascular localised mononuclear cells are CD68+, S100A8+, S100A9+, 25F9-, representing freshly migrated monocytes/macrophages. In contrast, almost all CD68+ mono- and multinucleated cells arranged within granulomas were S100A8-, S100A9-, 25F9+, representing mature (non-migrating) macrophages. Serial sections revealed that macrophages co-express tumour necrosis factor (TNF)-alpha and matrix metalloproteinases (MMPs) 2 and 9. In situ end-labelling of fragmented DNA demonstrated that CD68+ macrophages undergo apoptosis within gout tophi. Our data show that macrophages are continuously recruited into the gout tophi. These macrophages co-produce the proinflammatory cytokine TNF-alpha and two TNF-alpha inducible lytic enzymes, MMP-2 and MMP-9, suggesting that TNF-alpha may induce MMP production followed by matrix degradation within foreign body granulomas. In parallel, macrophages undergo apoptosis, a phenomenon that may restrict the destructive potential of inflammatory macrophages.     

Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-α antibody

The role of tumor necrosis factor- in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor- and in eight chimpanzees after intravenous administration of endotoxin in the absence or presence of concurrent treatment with a neutralizing anti-tumor necrosis factor- monoclonal antibody. Injection of tumor necrosis factor- in humans elicited a significant, instantaneous (after 15 min) increase in the plasma concentrations of both types of soluble tumor necrosis factor receptors. In chimpanzees, treatment with the anti-tumor necrosis factor- antibody completely neutralized endotoxin-induced tumor necrosis factor- activity. The release of soluble tumor necrosis factor receptors was strongly (80–90%) inhibited in the presence of the neutralizing antibody. Our results indicate that tumor necrosis factor- is a prime mediator of endotoxin-induced release of its own soluble receptors.     

Role of tumor necrosis factor-α in the regulation of T-type calcium channel current in HL-1 cells

AIM: Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation( AF). Although tumor necrosis factor( TNF)-α levels are increased in patients with AF,the role of TNF-α in the pathogenesis of AF remains unclear. Recent research has revealed that T-type Ca~(2+)currents( ICa,T) play an important role in the pathogenesis of AF. METHODS: In this study,we used the whole-cell voltage-clamp technique and biochemical assays to explore the role of TNF-α in the regulation of ICa,Tin atrial myocytes. RESULTS: We found that compared with sinus rhythm( SR) controls,T-type calcium channel( TCC)subunit m RNA levels were decreased,while TNF-α expression levels were increased,in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells,after cultured for 24 h,12. 5,25 and 50 μg / L TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12. 5 or 25μg / L TNF-α in a concentration-dependent manner [from(- 15. 08 ± 1. 11) p A / p F in controls to(- 11. 89 ± 0. 83) p A / p F and(- 8. 54 ± 1. 55) p A/p F in 12. 5 and 25 μg/L TNF-α groups,respectively]. TNF-α application also inhibited voltage-dependent inactivation of I~(Ca,T)shifted the inactivation curve to the left. CONCLUSION: These results suggest that TNF-α is involved in the pathogenesis of AF,probably via decreasing ICa,Tfunction in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF.     

The role of Gr-1+ cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice

The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1⁺ cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1⁺ cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1⁺ cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.     

Association of CTLA4 exon-1 polymorphism with the tumor necrosis factor-α in the risk of systemic lupus erythematosus among South Indians

Cytotoxic T lymphocyte associated-antigen (CTLA4) is a potential negative regulatory molecule of T-cells and associated with several autoimmune diseases. Several reports from different ethnic groups showed that the polymorphisms of the CTLA4 gene have been associated with autoimmune diseases including SLE. Therefore, we aimed to investigate the +49 A/G polymorphism in South Indian SLE patients and its association with disease aetiology and serological markers. A total of 534 samples were genotyped for the +49 A/G polymorphism in exon 1 of the CTLA-4 gene through PCR-RFLP method. We found significant association of genotype and allele frequencies with +49 A/G polymorphism in SLE patients. The frequency of the +49 A/G polymorphism rs231775 ‘GG’ genotype was significantly higher in patients with SLE (12.32%) than those in healthy control subjects (4.6%) (OR: 1.797; 95% CI 1.264–2.554; p = 0.001). The frequency of mutant allele ‘G’ also found to be significantly higher in cases (36.01%) than controls (24.92%) (OR: 1.695, 95% CI: 1.298–2.214, p < 0.001). We observed significant increase in serum TNF-α, interferon-α, IL-10 and IL-12 in SLE cases compared to controls. We also found a significant association of serum TNF-α, interferon-α, IL-10 and IL-12 with SLE phenotypes. In addition there was a significant increase in serum TNF-α level in “GG” genotype SLE subjects suggesting that it might play a major role in the advancement of SLE disease.     

Nonhypotensive dose of β-adrenergic blocker ameliorates capillary deficits in the hearts of rats with moderate renal failure

Renal failure causes sympathetic overactivity and inadequate capillary growth in response to cardiomyocyte hypertrophy in experimental renal failure, as well as in uremic patients. In nonuremic animals, sympathetic overactivity was shown to suppress capillary growth. The purpose of this study was to examine whether blockade with α- and β-adrenoblockers ameliorates the capillary deficit that was documented in the hearts of rats with moderate renal failure. Male Sprague–Dawley rats, 3 days after surgical ablation [subtotal nephrectomy (SNX)] or sham operation (sham), were treated with phenoxybenzamine, metoprolol, or a combination of both: After 12 weeks, the hearts were investigated using morphometric and stereologic techniques. The length density of myocardial capillaries was lower (p<0.05) in untreated SNX than in sham (2,786±372 vs 3,397±602 mm/mm³); the decrease was abrogated by metoprolol (3,305±624 mm/mm³), but not by phenoxybenzamin (2,628±480 mm/mm³). The intercapillary distance increased (p<0.05) in SNX (20.5±1.5 μm) and tended to be lower after metoprolol treatment (19.0±1.9 μm). The media area of intramyocardial arterioles was significantly higher in untreated SNX (1,158±1,343 vs 686±771 μm² in sham). Metoprolol in nonhypotensive doses prevents the capillary deficit in the hearts of rats with moderate renal failure and presents an argument for an important role of sympathetic overactivity in the genesis of the capillary deficit in moderate chronic renal insufficiency.     

Changes in the oncolytic properties of the serum of rats with Guérin's carcinoma undergoing treatment with sarcolysin

Summary An attempt was made to employ the oncolytic reaction to evaluate the efficacy of sarcolysin treatment in Guérin's rat carcinoma. The oncolytic properties of the sera were periodically investigated in rats suffering from this tumor and treated with sarcolysin. The results obtained were compared with the changes in the oncolytic properties of the serum of animals with tumors untreated with this perparation. It was demonstrated that the rise of the serum oncolytic activity in the treated animals is associated with the reduction of the tumor size. Conversely, the activity fall in the serum oncolytic activity in rats suffering from tumors not treated with sarcolysin is associated with the continuous growth of the tumor. It appears that determination of the oncological properties of the serum during sarcolysin treatment of animals suffering from tumors would enable the efficacy of this therapy on the tumor to be evaluated.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

Role of tumor necrosis factor-α in the regulation of keratinocyte cell cycle and DNA repair after ultraviolet-B radiation

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced in the skin in response to ultraviolet B radiation (UVB). TNF-α facilitates UVB-induced apoptosis and probably contributes to removal of damaged cells. Surprisingly, murine TNF-α-knockout models have demonstrated that TNF-α is necessary for the early stages of skin carcinogenesis and development of squamous cell carcinoma. In the present PhD thesis, we examined the effects of TNF-α on DNA repair and cell cycle regulation in UVB-irradiated keratinocytes. In the model of premalignant keratinocytes (HaCaT), TNF-α abolished the UVB-induced G2/M checkpoint and diminished the DNA repair despite induction of apoptosis. TNF-α activated the protein kinase B/Akt and regulation of its downstream targets, mTOR, Bad and FoxO3a. This effect was dependent on atypical protein kinase C species (aPKC) since a specific peptide blocking the activity of the PKCξ and ι/λ abrogated the activation of Akt by TNF-α. The aPKC-Akt axis was likely to be responsible for the TNF-α-induced decrease in DNA repair since blocking of Akt activity restored DNA repair. Since anti-TNF-α approaches are increasingly used in the therapy of autoimmune diseases and one of the safety concerns is the potential enhancement of skin carcinogenesis, we investigated the effect of the chimeric monoclonal anti-TNF-α antibody infliximab on UVB-irradiated HaCaT cells. Cells treated with infliximab had significantly increased levels of DNA damage despite enhanced G2/M checkpoint arrest, increased apoptosis and inhibition of Akt. In conclusion, we identified a possible novel mechanism by which TNF-α promotes UVB-induced skin carcinogenesis. This depends on aPKC-Akt activation and inhibition of DNA repair. TNF-α-treated cells are prone to escape checkpoint control and are possibly more likely to accumulate mutations, which may constitute a relevant mechanism enhancing tumor development. The effect of anti-TNF-α therapy on skin carcinogenesis warrants further investigation as our study indicates that, in contrast to what had been expected, infliximab may impair DNA repair.