Tumor necrosis factor alpha-induced osteoclastogenesis requires tumor necrosis factor receptor-associated factor 6.

Although tumor necrosis factor receptor-associated factor 6 (TRAF6) is required in receptor activator of NF-kappaB-receptor activator of NF-kappaB ligand (RANK-RANKL) signaling for osteoclastogenesis, it has remained unclear whether TRAF6 is crucial in tumor necrosis factor alpha (TNF-alpha)-induced osteoclastogenesis. We examined TRAF6 function in the TNF-alpha-induced osteoclastogenesis by using osteoclast progenitor cells from TRAF6-deficient mice. The results indicated that TNF-alpha did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c-jun N-terminal kinase (JNK) and TNF-alpha activation was observed in osteoclast progenitor cells. Thus, we have provided the first evidence showing that TRAF6 is involved in TNF-alpha-induced osteoclastogenesis.     

Tumor necrosis factor and cardiac function.

The direct effect of tumor necrosis factor (TNF), a product of activated macrophages, on myocardial performance was determined using an isolated papillary muscle technique and a modified Langendorff preparation. Papillary muscle was obtained from male adult rats 4-5 hours after they received either 100 ng/kg TNF (group A), or 100 micrograms/kg TNF (group B) or saline (control). Group B animals exhibited significantly greater peak tension development and velocity of contraction compared with controls (p less than 0.05). In group A animals these variables were not significantly different from those of the controls (p greater than 0.05). Electrophysiologic measurements revealed a significant decrease in resting membrane potential in both group A and group B animals compared with the controls (p less than 0.05). Whole hearts perfused with serum from animals treated with TNF 18-22 hours earlier exhibited significant impairment of contractility, decreased rate of systolic pressure development, and decreased rate of relaxation compared with the controls (p less than 0.05). Coronary flow and myocardial water content were similar for both groups of perfused hearts. These data suggest that tumor necrosis factor stimulates an early beneficial effect on myocardial function, which 18-22 hours later is associated with impairment of myocardial performance. This effect appears to be serum transferable.     

Tumor necrosis factor and wound healing.

Tumor necrosis factor alpha (TNF), 1 to 500 ng in saline (PBS) or collagen, was applied to the wounds of normal and Adriamycin-impaired mice and wound disruption strength (WDS) and histology were examined. Also wounded mice were administered TNF 25 to 75 micrograms/kg IP daily and WDS was determined. Wound histology was examined 6 months after wounding and local TNF application. Local TNF 5 to 500 ng in PBS did not significantly affect WDS. Local TNF 5 to 50 ng in collagen increased WDS 33% to 65% in Adriamycin-impaired animals (p = 0.05 to p less than 0.02). Local TNF 50 to 500 ng in collagen increased WDS 23% to 49% in normal animals (p = 0.08 to p less than 0.01). Adriamycin-impaired animals demonstrated improved wound histology with local TNF in collagen. Systemic TNF did not significantly affect WDS. Local TNF in collagen did not induce histologic pathology at 6 months. TNF may modulate macrophage function and local TNF in collagen can improve WDS in normal and Adriamycin-impaired animals.     

Tumor necrosis factor alpha disrupts tight junction assembly

BACKGROUND: We have previously shown an increase in intestinal permeability and a corresponding decrease in the expression of tight junction (TJ) proteins in the in testines of patients with Crohn's disease (CD). Tumor necrosis factor-alpha (TNFalpha) has been implicated in the inflammatory process of CD and its suppression has therapeutic benefit. ZO-1, occludin, and the claudins are key proteins in the TJ. Hypothesis: TNFalpha disrupts the TJ. METHODS: MDCK cells were incubated with TNFalpha (0-100 ng/ml) for 5 days. Qualitative evaluation of the TJ was done with monoclonal antibody to ZO-1 detected by an immunofluorescence. Duplicate cells were lysed and ZO-1, occludin, and claudin-1 amount determined by western blot. RESULTS: Immunofluorescent staining of MDCK cells for ZO-1 showed TJ structural disruption with increasing amount of TNFalpha characterized by fragmented staining of ZO-1. There were no significant differences in quantitation of ZO-1 or occludin in the MDCK cells for all TNFalpha concentrations. There was a significant decrease in the amount of claudin-1 with increasing concentration of TNFalpha. CONCLUSIONS: (1) MDCK TJs are qualitatively disrupted by TNFalpha. (2) This disruption is not because of a decrease in cell number, lack of cell layer confluency, or a decrease in the amount of ZO-1 or occludin. (3) The amount of claudin-1 present in the cell is decreased with increasing amounts of TNFalpha suggesting that the lack of claudin-1 may cause a relocation of ZO-1 away from the TJ. (4) This rearrangement may play a role in the increased intestinal permeability seen in CD and other diseases.     

肿瘤坏死因子与人工关节无菌性松动

人工关节无菌性松动是影响关节置换术长期疗效的重要因素.许多研究表明无菌性松动与肿瘤坏死因子的关系密切.肿瘤坏死因子可募集并直接或间接激活破骨细胞,影响其他细胞因子的释放,刺激核因子-κB受体活化因子配体的表达,使成骨细胞凋亡并影响其分泌细胞因子.该文就近年肿瘤坏死因子与人工关节无菌性松动机制的研究进展作一综述.     

Tumor necrosis factor and interleukin-1 protection against the lethal effects of tumor necrosis factor

Based on the hypothesis that tumor necrosis factor (TNF) causes the lethality of gram-negative sepsis and previous work of tolerance to the lethal effects of TNF induced by repetitive exposure to sublethal intraperitoneal doses of human recombinant (r) TNF, we studied the protective role of a single sublethal intravenous dose of either rTNF (100 micrograms/kg) or recombinant interleukin-1 (rIL-1; 10(5) units/kg) or both before a subsequent lethal intravenous dose of rTNF (800 to 1000 micrograms/kg) in C3H/HEN mice. Mice were treated with a single intravenous dose of saline, rTNF, rIL-1 or both cytokines and challenged within 2 hours to 10 days with a lethal dose of rTNF. Mice treated with rTNF showed significant protection against the lethal effects of TNF when the treatment dose was given only 2 hours before the lethal dose, but maximal protection required a 24-hour interval and lasted as long as 8 days. The treatment dose of rTNF was toxic, and it resulted in occasional treatment deaths. Mice treated with rIL-1 showed maximal protection when treatment was given only 2 hours before challenge and protection lasted for 8 days. No toxicity was apparent secondary to IL-1 treatment. The combination of rIL-1 and rTNF was not as effective as either cytokine alone. The results suggest that rTNF or rIL-1 may be clinically useful in the prevention and treatment of sepsis lethality by the induction of tolerance to the lethal effects of TNF. The more promising cytokine appears to be rIL-1 because it has less toxicity and more rapid induction of full therapeutic effectiveness.     

Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration.

Although direct cytotoxicity is a well-established phenomenon of tumor necrosis factor alpha (TNF alpha)-induced tissue damage, the intracellular events leading to cell death are still poorly understood. To study the cytotoxic effects of TNF alpha on normal parenchymal cells, rat hepatocytes were purified and incubated with various concentrations of TNF alpha. Mitochondrial respiration, total protein synthesis, and enzyme release were measured to assess metabolic performance and cell integrity. Treatment with TNF alpha suppressed mitochondrial respiration in a concentration-dependent fashion, resulting in a reduction of the activity of complex I of the respiratory chain to 67.0 +/- 3.5% of that of untreated hepatocytes by 2000 U/mL TNF alpha. Under these conditions protein synthesis and the release of intracellular enzymes were significantly increased. Both hepatocellular enzyme release and inhibition of mitochondrial respiration appear to be associated with the generation of reactive oxygen intermediates by the hepatocyte itself, because oxygen radical scavengers prevented these adverse effects of TNF alpha. Inhibition of protein synthesis by cycloheximide as well as addition of cyclic adenosine monophosphate synergistically enhanced the suppression of mitochondrial respiration by TNF alpha, resulting in complex I activity of 6.9 +/- 1.6% and 24.9 +/- 2.9% of that of untreated cells. These data indicate that inhibition of mitochondrial respiration is one of the mechanisms by which TNF alpha induces tissue injury.     

Tumor necrosis factor induces adult respiratory distress syndrome in rats

To evaluate the effect of tumor necrosis factor (TNF), a major mediator of sepsis, on lung structure and function, we infused 200-g male Wistar rats with TNF (0, 2 x 10(5), or 4 x 10(5) U/kg of TNF) for 24 hours. Volume-pressure measurements were determined in the excised lungs using both air and saline, which eliminated surface tension forces. Total lung wet and dry weight, nitrogen level, and DNA and protein content were measured. Lungs of the rats that received TNF accepted significantly smaller volumes of air and saline at all pressures compared with the control group. Both the lung wet and dry weights increased with TNF. Lung DNA and protein content also increased, suggesting increased cellularity in the TNF-infused lungs. Thus, the lungs of the TNF-treated rats were stiffer, with reduced compliance values, and heavier due to increased water content and increased cellularity. These data indicate that sublethal administration of TNF in this rat model induces the adult respiratory distress syndrome and increases the work potential of respiration.     

Tumor necrosis factor depresses myocardial contractility in endotoxemic swine.

BACKGROUND: Depression of myocardial contractility occurs in septic shock. METHODS: Fourteen pigs were instrumented to measure cardiopulmonary dynamics after a challenge of Escherichia coli endotoxin (lipopolysaccharide endotoxin, LPS). A volumetric Swan-Ganz catheter was placed via the jugular vein, and a carotid arterial line was placed into the aortic root. Eight pigs received LPS alone and six pigs received tumor necrosis factor monoclonal antibody (TNF MAb) 15 minutes before the administration of LPS. Pulmonary artery and aortic root blood were sampled for amounts of TNF. Ninety minutes after LPS administration, thoracotomy was performed to biopsy the right and left ventricles for TNF levels. Contractility was determined from the end systolic pressure-volume relationships of pressure-volume diagrams. RESULTS: Right ventricular end diastolic volume index nearly doubled and myocardial contractility decreased by 40% from baseline in the pigs receiving only LPS. Pigs that received TNF MAb had no change in myocardial contractility or right ventricular end diastolic volume index from baseline. There was a higher level of TNF in the aortic sample than in the pulmonary samples at 60 minutes. Right ventricular tissue TNF levels were significantly higher in the LPS-alone group. There was no such difference in left ventricular tissue. CONCLUSION: The left and right ventricles have different susceptibilities to TNF MAb. TNF may decrease myocardial contractility in sepsis. Blockade of TNF with TNF MAb reverses the depression of myocardial contractility and the right ventricular dilatation associated with septic shock.     

Tumor necrosis factor gene polymorphism and cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) limits survival after cardiac transplantation. Tumor necrosis factor-alpha (TNF-alpha) may be a key factor in the development of CAV. Two bi-allelic polymorphisms associated with high TNF-alpha production have been identified in the TNF gene locus, TNFA1/2, at position -308 and TNFB1/2 at +252. We hypothesized that recipient TNFA2 and TNFB2 homozygosity is associated with the development of CAV after heart transplantation. METHODS: TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase mini-sequencing in 70 cardiac transplant recipients. Recipients homozygous for TNFA2 or TNFB2 (Group A, n = 29) were compared with recipients heterozygous or homozygous for TNFA1 and TNFB1 (Group B, n = 41). Coronary arteriography was performed annually or when indicated. Cumulative freedom from CAV and survival was calculated according to the Kaplan-Meier test. RESULTS: Mean follow-up was 3.8 +/- 0.3 years. In Group A, 11 of 29 recipients (38%) developed CAV compared with 9 of 41 (22%) in Group B (p = 0.12). Cumulative freedom from CAV at 3 years was 42% in Group A and 80% in Group B (p = 0.043). In Group A, 11 of 29 recipients (38%) died during follow-up compared with 4 of 41 (10%) in Group B (p = 0.006). Cumulative survival at 3 years was 72% in Group A and 93% in Group B (p = 0.003). CONCLUSIONS: The results suggest that TNFA2 and TNFB2 allele homozygosity is associated with cardiac allograft vasculopathy and mortality in heart transplant recipients.     

Tumor necrosis factor alpha inhibits wound healing in the rat.

This work was undertaken to study the effects of tumor necrosis factor-alpha (TNF-alpha/cachectin) on developing granulation tissue in rats. Cylindrical hollow sponge implants were used as an inductive matrix for the growth of granulation tissue. In the two test groups the implants were injected daily for 4 days with a solution containing either 50 or 200 ng of TNF-alpha, while the implants of the control group were treated correspondingly with phosphate-buffered saline solution only. Analyses of granulation tissue and would fluid in the sponge implants were carried out 7, 14 and 21 days after implantation. In histological specimens the ingrowth rate of granulation tissue into the sponge was significantly lower after 7 days in the group treated with 200 ng of TNF-alpha. No such an effect was observed after 14 or 21 days. After 7 days, the mean amounts of nucleic acids reflecting cellularity in the granulation tissue decreased dose-dependently, but nonsignificantly, in the groups treated with TNF-alpha. Simultaneously, the accumulation of collagen hydroxyproline of the sponge was significantly lower in the group treated with 200 ng of TNF-alpha than in the controls (-30%, one-way analysis of variance). This effect was not observed after 14 or 21 days. No significant differences were detected in the amounts of nitrogen, hexosamines and uronic acids between the groups, reflecting unchanged accumulation of glycosaminoglycans in the developing granulation tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor inhibitor gene transfer ameliorates lung graft ischemia-reperfusion injury

OBJECTIVE: Tumor necrosis factor is an important mediator of lung transplant ischemia-reperfusion injury, and soluble type I tumor necrosis factor receptor binds to tumor necrosis factor and works as a tumor necrosis factor inhibitor. The objectives of this study were to demonstrate that gene transfer of type I tumor necrosis factor receptor-IgG fusion protein reduces lung isograft ischemia-reperfusion injury and to compare donor endobronchial versus recipient intramuscular transfection strategies. METHODS: Three donor groups of Fischer rats (n = 6/group) underwent endobronchial transfection with either saline, 2 x 10(7) plaque-forming units of control adenovirus encoding beta-galactosidase, or 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein. Left lungs were harvested 24 hours later. Two recipient groups (n = 6/group) underwent intramuscular transfection with 2 x 10(7) plaque-forming units or 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein 24 hours before transplantation. All donor lung grafts were stored for 18 hours before orthotopic lung transplantation. Graft function was assessed 24 hours after reperfusion. Transgene expression was evaluated by means of enzyme-linked immunosorbent assay and immunohistochemistry of type I tumor necrosis factor receptor. RESULTS: Endobronchial transfection of donor lung grafts with 2 x 10(7) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein significantly improved arterial oxygenation compared with the saline and beta-galactosidase donor groups (366.6 +/- 137.9 vs 138.8 +/- 159.9 and 140.6 +/- 131.4 mm Hg, P =.009 and.010, respectively). Recipient intramuscular transfection with 1 x 10(10) plaque-forming units of adenovirus encoding type I tumor necrosis factor receptor-IgG fusion protein improved lung graft oxygenation compared with that seen in the low-dose intramuscular group (2 x 10(7); 320.3 +/- 188.6 vs 143.6 +/- 20.2 mm Hg, P =.038). Type I tumor necrosis factor receptor-IgG fusion protein was expressed in endobronchial transfected grafts. In addition, intramuscular type I tumor necrosis factor receptor-IgG fusion protein expression was dose dependent. CONCLUSIONS: Donor endobronchial and recipient intramuscular adenovirus-mediated gene transfer of type I tumor necrosis factor receptor-IgG fusion protein improved experimental lung graft oxygenation after prolonged ischemia. However, donor endobronchial transfection required 500-fold less vector. Furthermore, at low vector doses, it does not create significant graft inflammation.     

Tumor necrosis factor in benign and malign tissue of the kidney

Summary The use of tumor necrosis factor (TNF) in immunotherapy of tumor diseases has attracted increasing interest. Since the direct antitumor effect of the TNF is mediated by receptor-bound TNF, we immunohistologically stained both benign and malignant tissue from 35 tumor-bearing human kidneys for TNF. Using a polyclonal anti-TNF-antiserum, paraffin sections were tested in the presence and absence of in vitro preincubation with TNF. Futhermore, all specimens were stained immunohistologically for Tamm-Horsfall protein (THP) because this renospecific glycoprotein can bind TNF in a lectin-like manner. In the absence of TNF preincubation, malignant tissue was TNF-positive in 34 specimens, as was benign tissue from the same tumor-bearing kidneys in 35 cases. In several specimens the staining was so intense that preincubation with TNF did not enhance the reaction. Whereas TNF staining in tumor tissue was relatively homogenous, that in benign tissue was intensive in distal tubuli, moderate in proximal tubuli, and negative in glomeruli. THP staining was negative in malignant kidney tissue but positive in the distal tubuli of benign tissue, i. e., in the regions in which TNF staining was most intense. These results indicate that TNF binds not only to membrane, most likely in a receptor-mediated manner, but also to THP both in vivo and in vitro. In vivo binding of TNF to THP was confirmed in animal experiments in which pigs were given injections of TNF. Immunohistological staining of the animals' kidneys revealed positive reactions for both TNF and THP at the distal tubuli, indicating TNF binding to THP after in vivo TNF administration. The presence of TNF in human kidney tumors implies that renal-cell carcinoma cells in situ are resistant to the direct cytotoxic effect of TNF. This resistance should be taken into account when TNF is considered for use as a possible immunotherapeutic agent in renal-cell carcinoma.     

Tumor necrosis factor induces contraction of mesangial cells and alters their cytoskeletons

Cultures of human mesangial cells (MC) were established from the renal cortex of surgical specimen. The characteristic spindle-shaped or stellate appearance of MC was altered after treatment with tumor necrosis factor (TNF). After two hours, the MC retracted and lost reciprocal contacts. Furthermore, this treatment altered the cytoskeletal organization of MC, since a peripheral band of actin and stress fibers disappeared while the streaks of vinculin at focal contacts decreased. These changes were reversible when the MC were cultured in fresh medium. After five minutes of treatment with platelet activating factor (PAF), changes similar to those induced by TNF were observed. Inhibitors of PAF synthesis, such as plasma alpha 1-proteinase inhibitor and an anti-inflammatory peptide, blocked changes induced by TNF, PAF receptor antagonists inhibited changes induced by PAF and also by TNF. These results and the finding that MC are stimulated to produce PAF by TNF suggest that PAF is a secondary mediator of the changes in cell shape and cytoskeletal organization induced by this cytokine.     

Tumor necrosis factor alone does not explain the lethal effect of lipopolysaccharide

Lethality and tumor necrosis factor production induced by different types of lipopolysaccharide were studied in naive (non-primed) rats during the late phase of endotoxin tolerance. The correlation with antilipopolysaccharide antibodies was also analyzed. No correlation was found between tumor necrosis factor levels and lipopolysaccharide-induced mortality in naive animals. Low-toxicity lipopolysaccharide preparations induced levels of tumor necrosis factor similar to those induced with more toxic types of lipopolysaccharide. Late tolerance was associated with progressively lower levels of lipopolysaccharide-induced tumor necrosis factor and increasing titers of antilipopolysaccharide antibodies after repeated injections of homologous lipopolysaccharide. During late endotonxin tolerance, a direct correlation between the lipopolysaccharide dose and peak tumor necrosis factor serum levels was found. We conclude that since tumor necrosis factor serum levels do not correlate with mortality, tumor necrosis factor alone cannot explain the lethal effect of lipopolysaccharide.