Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells

Objective and design

Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear.

Material or subjects

RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control.

Methods

Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals.

Results

LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1β expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity.

Conclusions

Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.     

Role of HSP27 in tumor necrosis factor-α-stimulated interleukin-6 synthesis in osteoblasts

Heat shock protein 27 (HSP27) is known to act as a molecular chaperone. We have recently reported that HSP27 regulates osteocalcin synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of HSP27 in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 (IL-6) synthesis in MC3T3-E1 cells. The levels of IL-6 release and IL-6 mRNA stimulated by TNF-α in MC3T3-E1 cells transfected with HSP27 was significantly higher than those in the control cells. In addition, the levels of secreted IL-6 and IL-6 mRNA in the phospho-mimic HSP27-overexpressing cells were significantly higher than those in the non-phosphoryl-atable HSP27-overexpressing cells. Furthermore, we observed no significant differences in the phosphorylation levels of IκB/NFκB, Akt, and p44/p42 mitogen-activated protein kinase among the 4 types of transfected cells. Therefore, these results strongly suggest that HSP27 enhances TNF-α-stimulated IL-6 synthesis, and that the phosphorylation status of HSP27 is related to IL-6 synthesis in osteoblasts.     

Pneumocystis carinii stimulates in vitro production of tumor necrosis factor-α by human macrophages

The ability of Pneumocystis carinii to induce tumor necrosis factor (TNF)- release by monocytes/macrophages from immunocompetent humans was investigated. Monocytes and monocyte-derived macrophages from healthy individuals produced an increased amount of TNF- when exposed to P. carinii cysts obtained from rats with steroid-induced pneumocystosis. The cysts induced increased TNF- production in a dose-dependent manner; baseline TNF- production was restored after addition of an anti-P. carinii hyperimmune serum. Kinetics experiments showed that the secretion of TNF- occurs early and reachs a maximal peak after 8 h. Since TNF- is directly lethal to P. carinii in vitro, it is suggested that the production of this cytokine in response to the cysts may be one of the mechanisms for the control of this parasitic infection.     

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.     

Mechanisms of tumor necrosis factor-α-induced interleukin-6 synthesis in glioma cells

Background  

Interleukin (IL)-6 plays a pivotal role in a variety of CNS functions such as the induction and modulation of reactive astrogliosis, pathological inflammatory responses and neuroprotection. Tumor necrosis factor (TNF)-α induces IL-6 release from rat C6 glioma cells through the inhibitory kappa B (IκB)-nuclear factor kappa B (NFκB) pathway, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The present study investigated the mechanism of TNF-α-induced IL-6 release in more detail than has previously been reported.     

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.     

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.     

Synthesis of tumor necrosis factor α (TNF-α) by human monocytes in vitro

Research Institute of Hematology and Blood Transfusion, Ministry of Health of the Belorussian SSR, Minsk. Institute of Experimental Hematology and Biotechnology, All-Union Hematologic Scientific Center, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR E. A. Zotikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 9, pp. 287–289, September, 1991.     

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.     

Effects of clofazimine analogues and tumor necrosis factor-α individually and in combination on human polymorphonuclear leukocyte functions in vitro

In the present study the individual and interactive effects of clofazimine, or three analogues of this agent (selected on the basis of similar or superior pro-oxidative properties: B669, B746 and B4021) and human recombinant TNF-α on the generation of antimicrobial oxidants by human polymorphonuclear leukocytes (PMNL), as well as release of granule enzymes from these cells, were investigated in vitro. All four riminophenazines at the concentrations tested (0.5 and 1.0 μg/ml) significantly increased myeloperoxidase (MPO)-mediated iodination, superoxide (0₂·⁻) generation, oxygen (0₂) consumption and chemiluminescence (CL), as well as the release of both primary and secondary granule contents (measured as the release of MPO, lysozyme and vitamin B₁₂-binding protein) by stimulated PMNL. Similar, but less impressive effects were observed with TNF-α (0.4–50.0 ng/ml). When PMNL were preincubated with both TNF-α and clofazimine or its analogues, the observed stimulation of cellular oxidative metabolism and granule enzyme release was at least additive in many assays. These data demonstrate that the spectrum of effects of clofazimine and its analogues on PMNL closely resemble those of TNF-α. Furthermore, TNF-α potentiates the pro-oxidative effects of clofazimine and its analogues on PMNL. Among the riminophenazines tested, clofazimine and B669 appear to be the most potent pro-oxidative agents for PMNL.     

Determination of soluble tumor necrosis factor-α receptor type I (TNFRI) and II (TNFRII) in the urine of healthy Japanese subjects

We determined the urinary soluble tumor necrosis factor-α receptor type I (sTNFRI) and type II (sTNFRII) levels in healthy Japanese individuals to establish a reference value by means of specific ELISA. It was found that there were no significant differences between the urine sTNFRI and sTNFRII levels of children and adults. To demonstrate the usefulness of the reference value for children, we measured the urine sTNFRI and sTNFRII levels of children with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as a preliminary study. The urine sTNFRI and sTNFRII levels of the patients with HUS were markedly higher than those of healthy children from the onset of D?+?HUS. Our reliable reference value for healthy children will allow us to discriminate between normal and pathological conditions in future studies.     

The role of tumor necrosis factor (TNF)-α in the antitumor effect of intrapleural injection of Lactobacillus casei strain Shirota in mice

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1β, interferon (IFN)-γ, IL-12 and tumor necrosis factor (TNF)-α. Both anti-IFN-γ and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1β mAb did not influence the survival of the mice. However, anti-TNF-α mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-α into Meth A-bearing mice pretreated with anti-TNF-α mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-α induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo. Received: 22 February 1999     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.     

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.     

Determination of the safety and efficacy of therapeutic neutralization of tumor necrosis factor-α (TNF-α) using AZD9773, an anti-TNF-α immune Fab, in murine CLP sepsis

Objective and design

TNF-α neutralization is associated with increased mortality in mouse cecal ligation puncture (CLP) models. AZD9773 is an ovine polyclonal human TNF-α immune Fab, with pharmacological properties that differ from previously studied anti-TNF-α agents. We explored the safety and efficacy of therapeutically administered AZD9773 in mouse CLP sepsis.

Methods

A moderate/severe-grade CLP model resulting in 20–30 % 5-day survival and a mild-grade CLP model resulting in ~70 % 5-day survival were established in human TNF-α transgene/murine TNF null (Tg1278/?/?) mice.

Treatment

Mice received saline resuscitation and imipenem administration every 12 h (0–72 h post-CLP). AZD9773 (or DigiFab control) was dosed 24, 36, 48 and 60 h post-CLP.

Results

Therapeutic dosing of AZD9773 in moderate/severe-grade CLP resulted in significantly increased survival (>70 %) compared with DigiFab (27 %, P < 0.05). Therapeutic dosing of AZD9773 in mild-grade CLP did not significantly affect survival outcome compared with DigiFab or imipenem alone (~60–70 % survival).

Conclusions

These data demonstrate that TNF-α neutralization can improve survival in moderate/severe CLP sepsis. TNF-α suppression in mild-grade models was not associated with survival benefit and did not increase 5-day mortality. These findings suggest that therapeutic benefit following TNF-α attenuation in models of sepsis may depend on model severity.     

Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be 'trapped' on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.     

Monoclonal antibody to tumor necrosis factor-α modulates hepatocellular Ca2+ homeostasis during hemorrhagic shock in the rat

Tumor necrosis factor-α (TNF-α) is a key mediator of shock-induced cellular and humoral inflammatory cascades. The present study investigated the role of TNF-α in oxidative membrane injury and altered hepatocyte Ca²⁺ regulation, both of which are critical steps in cellular dysfunction during ischemia/reperfusion events. Hemorrhagic shock was induced by bleeding male Sprague-Dawley rats (200–250 g, n=6/group) to a mean arterial blood pressure of 40 mmHg for 60 min. Rats were resuscitated with 60% of shed blood and twice the shed blood volume as Ringers’ lactate. At the end of hemorrhage and 60 min after resuscitation, hepatocytes were isolated by liver collagenase perfusion. Hepatocyte Ca²⁺ uptake (Ca²⁺ _up) and Ca²⁺ membrane flux (Ca²⁺ _flux) were determined by ⁴⁵Ca²⁺ incubation techniques. Hepatocyte reduced/oxidized glutathione and lipid peroxidation were determined fluorometrically. Both hemorrhage and hemorrhage/resuscitation significantly increased hepatocyte Ca²⁺ _up and Ca²⁺ _flux. The monoclonal chimeric mouse γ₁ TNF-α antibody (TN3γ1.19.12; 20 mg/kg b.w.) given with resuscitation significantly decreased hepatocyte Ca²⁺ _up and Ca²⁺ _flux and prevented hepatocyte lipid peroxidation. These findings suggest that oxidative membrane injury could be the result of TNF-α modulation of hepatocellular Ca²⁺ regulation during hemorrhage/resuscitation. Received: 20 April 1998 / Accepted: 24 September 1998