[Show abstract][Hide abstract] ABSTRACT: Purpose:
To investigate the role of the toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), and stress activated protein kinases/Jun N-terminal kinase (SAPK/JNK) signalling pathways in the responses of RAW 264.7 macrophages to low-intensity microwaves (MW).
Materials and methods:
Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment.
MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells.
The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.
International Journal of Radiation Biology 12/2014; 91(4):1-24. DOI:10.3109/09553002.2014.996261 · 1.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To explore the effect of the spaceflight environment on immunity in animals, C57/BL6 mice flown on a 30-day space high-orbit satellite mission (BION-M1) were analysed. Cytokine response in mice was measured in tandem with the following parameters: the synthesis of inducible forms of the heat shock proteins HSP72 and HSP90α; activity of the NF-κB, IFR3, and SAPK/JNK signalling pathways; and TLR4 expression. In addition, apoptosis in the thymus was measured by caspase-3 and ph-p53/p53 ratio testing. In response to flight environment exposure, mice had a reduction in spleen and thymus masses and decreased splenic and thymic lymphocyte counts. Plasma concentration of IL-6 and IFN-γ but not TNF-α was decreased in C57BL6 mice. The NF-κB activity in splenic lymphocytes through the canonical pathway involving IκB degradation was significantly increased at 12 h after landing. One week after landing, however, the activity of NF-κB was markedly decreased below even the control values. Non-canonical NF-κB activity increased during the whole observation period. The activities of SAPK/JNK and IRF-3 were invariable at 12 h but significantly increased seven days after landing. The expression of Hsp72 and Hsp90α was somewhat increased 12 h (Hsp72) and seven days (Hsp90α). TLR4 expression in splenic cells was significantly increased only at 12 h, returning to normal seven days after landing. To assess the apoptosis in thymus lymphocytes, caspase-3 and levels of p53 protein along with its phosphorylated form were measured in thymic lymphocytes. The results indicated that the high-orbit spaceflight environment caused an increase in the level of p53 but more notably in the activated, phosphorylated form of the p53 protein. The calculated ratio of the active to inactive forms of the protein (ph-53/p53) 12 h after landing increased by more than twofold, indicating the apparent induction of apoptosis in thymus cells. Interestingly, seven days after the landing, this ratio was not restored, but rather increased: the specified ratio was four times higher compared to the ground-based control. Measurements of caspase-3 in thymic cells indicated more expressive increase in apoptosis. Taken together, the results of the present study indicate that spaceflight induces an imbalance in the immunity of mice, showing variation in signalling, apoptosis and stress response that are not restored by seven days after landing. These changes are distinguished from classic stress-related alterations usually caused by conventional stressors.
[Show abstract][Hide abstract] ABSTRACT: The present study was designed to compare the anti-inflammatory effects of several agents applied in vivo, namely, a synthetic inhibitor of the NF-κB cascade, fat-soluble antioxidants, and the thymic peptide thymulin. Cytokine response in LPS-treated mice was analysed in tandem with the following parameters: the synthesis of inducible forms of the heat shock proteins HSP72 and HSP90α; activity of the NF-κB and SAPK/JNK signalling pathways; and TLR4 expression. Inflammation-bearing Balb/c male mice were pretreated with an inhibitor of IKK-α/β kinases (IKK Inhibitor XII); with thymulin; with dietary coenzyme Q9, α-tocopherol, and β-carotene; or with combinations of the inhibitor and peptide or antioxidants. Comparable anti-inflammatory effects were observed in inflammation-bearing mice treated separately with thymulin or with dietary antioxidants administered daily for two weeks before LPS treatment. When LPS-injected mice were treated with the inhibitor and antioxidants together, neither plasma cytokines, signal proteins, nor heat shock proteins recovered more efficiently than when mice were treated with these agents separately. In contrast to antioxidant diet, the thymulin was shown to increase the effect of IKK Inhibitor XII in preventing IKK activation in LPS-treated mice.
Mediators of Inflammation 06/2014; 2014:724838. DOI:10.1155/2014/724838 · 3.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro and in vivo effects of some inhibitors of the activity of signal cascades NF-κB and SAPK/JNK, and the TLR4 receptor on the immune cells activity were studied. To evaluate in vitro effects, the macrophage-like RAW 264.7 cells were cultured with each of the inhibitors, namely IKK inhibitor XII, SP600125, CLI-095, and OxPAPK (the first two are the inhibitors of NF-κB, SAPK/JNK cascades, and the last two compounds are the inhibitors of the TLR4 receptor activity). On the whole, all of the used inhibitors did not induce pro-inflammatory response in RAW 264.7 cells. On the contrary, the inhibitor of SAPK/JNK cascade, and, especially, the inhibitor of NF-κB cascade significantly decreased production of the TNF-α, IL-1, IL-6, IFN-γ, and IL-10 in RAW 264.7 cells. In these cells, the inhibitors substantially decreased “back-ground stress response” of macrophages, differently reducing a production of heat shock proteins, HSP72 and HSP90-α, and diminishing phosphorylation of signal proteins from NF-κB and SAPK/JNK cascades. Results of in vitro experiments suggest that the inhibitor of NF-κB activity was the most effective. It was this inhibitor that was intraperitonealy injected in Balb/C male mice in the in vivo experiments in order to study its effect on the activity of immune cells. Results showed that IKK Inhibitor XII applied in vivo did not induce pro-inflammatory response in mice, but decreased the activity of NF-κB cascade, and lowered HSP90-α expression in mouse splenic lymphocytes. So, among the studied compounds, IKK Inhibitor XII seems to be a very effective inhibitor that may be used to decrease cytokine and stress response in various pathologies.
[Show abstract][Hide abstract] ABSTRACT: The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90α; activities of NF-κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF-κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF-α, IL-1, and IFN-γ production. In addition, inhibitors of the NF-κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF-κB and SAPK/JNK proteins, thereby preventing the activation NF-κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90-α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF-κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors.
Journal of Immunotoxicology 07/2012; 10(2). DOI:10.3109/1547691X.2012.700652 · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modulation of autoimmune inflammation by the thymic peptides thymulin and thymopentin was studied in mice with acute experimental autoimmune encephalomyelitis (EAE), which resembles multiple sclerosis in humans. EAE was induced in NZW mice by a single immunisation with myelin basic protein coupled with adjuvants. Visible signs of pathology appeared on days 12-14 after the immunisation, peaked on days 20-25, were retained up to day 45, and then reverted. A biphasic cytokine response was also detected. In the "early" phase, which started at day 35, increased levels of interferon-gamma and interleukin-6 in the blood were observed; during the "delayed" phase, which started at day 48, the levels of plasma interleukin-17 and tumour necrosis factor-alpha were also raised. In addition, the phosphorylation of NF-kappaB signalling proteins and the production of heat shock protein Hsp72 were significantly increased in splenic lymphocytes from EAE-bearing mice. When applied intraperitoneally every other day for 30 days, either thymulin or thymopentin (15μg per 100g of body weight) significantly reduced the disease severity compared to untreated EAE mice. The effect of thymulin but not thymopentin remained after its withdrawal. Thymulin reduced the cytokine response in both the early and the delayed phases, whereas thymopentin only reduced the "early phase cytokines" (IL-6 and interferon-gamma). Both peptides significantly reduced the level of phosphorylation of the NF-kappaB signalling protein IKK and the production of Hsp72 protein. The data presented here indicate the presence of time-dependent immune responses in EAE-bearing mice, which may be associated with the Th1 and Th17 subpopulations of T-cells. Thymulin and thymopentin demonstrated different patterns of activity, most likely via mechanisms involved in NF-kappa B signalling and Hsp72 expression.
[Show abstract][Hide abstract] ABSTRACT: Effects of four inhibitors of NF-kappaB, SAPK/JNK and TLR4 signaling, namely, inhibitor XII, SP600125, CLI-095 and Oxpapc on a macrophage response to low dose ammonium were studied in RAW 264.7 cells. Low dose ammonium induced pro-inflammatory response in cells as judged from enhanced production of TNF-alpha, IF-gamma, and IL-6, and by activation of signal cascades. The increase in production of cytokines, namely TNF, IFN, and IL-6, demonstrated that low-dose ammonium induced a pro-inflammatory cellular response. In addition, an activation of NF-kappaB and SAPK/JNK cascades, as well as enhancement of TLR4 expression was shown. Each of used inhibitors reduced to a variable degree the pro-inflammatory response of RAW 264.7 cells on chemical toxin by decreasing cytokine production. The inhibitor of NF-kappaB cascade, IKK Inhibitor XII, was more effective, and not only prevented the development of pro-inflammatory response induced by ammonium, but also decreased cytokine production below control values. The inhibitor of extra cellular domains of TLR2 and TLR4 (OxPAPC) had almost the same anti-inflammatory effect, and an addition of the inhibitor of JNK cascade (SP600125) to cell culture practically neutralized effect of ammonium ions by decreasing cytokine production to control level. Inhibitory analysis showed that activation of RAW 264.7 cells induced by chemical toxin coincide incompletely with intracellular signaling pathways that were early determined regarding macrophage's response to toxin from gram-negative bacteria. Nevertheless, application of the inhibitors defended RAW 264.7 from toxic effect of the low dose ammonium.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation.
The effects of two thymic peptides- thymulin and thymopentin were studied in cultured RAW 264.7 macrophages (lipopolysaccharide-stimulated or unstimulated) by measuring cytokine production and signal protein levels.
Both peptides increased proinflammatory cytokine secretion by unstimulated RAW 264.7 macrophages and these effects were blocked by the NF-κB cascade inhibitor, stress-activated protein kinase (SAPK)/JNK cascade inhibitor and, to a lesser extent, Toll-like 4 receptor activity inhibitor. In macrophages stimulated by bacterial lipopolysaccharide, peptides alone did not affect cytokine secretion, but significantly enhanced effects of each of the inhibitors. Thymopentin increased activation of both NF-κB and SAPK/JNK cascades in unstimulated macrophages, while thymulin significantly decreased activation of the SAPK/JNK but not NF-κB cascade in LPS-stimulated macrophages. Thymulin and thymopentin increased production of the heat shock protein HSP72 both in LPS-stimulated and unstimulated cells.
Thymulin and thymopentin are effective anti-inflammatory modulators with direct actions on innate immune cells; the effects involve multiple signal cascades, including NF-κB and SAPK/JNK pathways. Since signaling cascades are now considered to be targets for new therapies, thymic peptides may be prospective modulators of signaling cascades, acting alone or in combination with other agents.
[Show abstract][Hide abstract] ABSTRACT: The involvement of heat shock protein Hsp90 in pro-inflammatory response in male NMRI mice under conditions of acute toxic stress, caused by lipopolysaccharide from Gram negative bacteria, was studied using geldanamycin, a specific blocker of the activity of this protein. It is shown that the introduction of geldanamycin lowers total intoxication of the organism upon acute toxic stress caused by endotoxin. Thus, a decrease in cytokine TNF-alpha, IFN-gamma, IL-1, and IL-10 concentrations in blood serum of the geldanamycin-treated animals with acute toxic stress was found along with normalization of functional activity of nitric oxide producing peritoneal macrophages. Studying expression of receptor protein Tlr-4 as well of proteins of two signal cascades, NF-kappaB and SAPK/JNK, has shown that mechanisms of the geldanamycin protective effect are realized at the level of inhibition of Tlr-4 receptor expression, which provides for endotoxin-to-cell binding, and due to lowering the endotoxin-stimulated activation of signal cascades NF-kappaB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.
[Show abstract][Hide abstract] ABSTRACT: Effects of three chemical compounds: ammonia, diethyl ether, and acetic acid, known as common environmental contaminants in
technogenic accidents, were investigated in vivo and in vitro in low concentrations. When added in cultivation media, each
of the chemicals has affected peritoneal macrophages and spleen lymphocytes isolated from male NMRI mice and led to a rise
in the production of several cytokines, particularly the tumor necrosis factor-α and interferon-γ, as well as the expression
of the inducible form of heat shock proteins (HSP72 and HSP90-α) and in the activation of signal cascades NF-κB and SAPK/JNK.
The increase of the nitric oxide (NO) production in macrophages has been observed only when ammonia was added in cultivation
media. Also, low concentrations of all compounds investigated led to the activation of the expression of receptor protein
TLR4. When mice were exposed to airborne toxic contaminants in a hermetically sealed experimental chamber, an increase in
the concentrations of cytokines, heat shock proteins, and signal proteins in immune cells was also observed in response to
low concentrations of all chemicals investigated. Similarly to in vitro experiments, the NO production was augmented only
in the presence of the airborne ammonia. The results indicate the environmental hazard of chemical contaminants even in rather
low concentrations, which nevertheless lead to the stress response.
Key wordsammonia-diethyl ether-acetic acid-cytokines-heat shock proteins-NF-κB-SAPK/JNK
[Show abstract][Hide abstract] ABSTRACT: The effects of mixed dietary coenzyme Q(9), alpha-tocopherol, and beta-carotene on immune cell activity and blood cytokine profile were studied in peritoneal macrophages, spleen lymphocytes, and blood plasma from mice with acute inflammation induced by lipopolysaccharide (LPS). The activity of each fat-soluble antioxidant was also investigated separately in several model systems, both in vivo and in vitro. NMRI male mice were fed a diet supplemented with fat-soluble antioxidants for 15 days prior to LPS injection. LPS-induced inflammation resulted in induction of cellular production of pro-inflammatory cytokines such as TNF-alpha, IL-1alpha, IL-1beta, IL-2, IL-6, IFN-gamma, and also IL-10, an anti-inflammatory cytokine, and subsequent accumulation of these cytokines in blood plasma. In animals fed the antioxidant-rich diet, the inflammatory response to LPS injection was significantly reduced. The production of anti-inflammatory cytokine IL-10 in response to toxic stress and its accumulation in plasma were not modified by the diet. In addition, the expression of the inducible form of heat-shock protein 70 in mice treated with endotoxin was reduced in the animals pretreated with the antioxidant-rich diet. We showed that the diet suppressed phosphorylation of NF-kappaB, I kappaB kinase and SAPK/JNK proteins, thereby preventing the activation of the NF-kappaB kinase and SAPK/JNK signaling pathways in LPS-treated mice. In this report we demonstrate the potential effectiveness of naturally occurring antioxidant nutrients in the reduction of the inflammatory response. Therefore, it may be possible to develop novel therapeutic combinations, containing coenzyme Q(9), alpha-tocopherol, and beta-carotene, which promote immune stimulation.
European journal of pharmacology 09/2009; 615(1-3):234-40. DOI:10.1016/j.ejphar.2009.05.004 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of thymopentin, a synthetic analog of the active center of the thymus hormone thymopoietin, on the immune status
of mice with two different models of inflammation induced by injection of lipopolysaccharide (LPS) from Gram-negative bacteria
were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 μg/100 g of body weight, and chronic
inflammation (sepsis) was modeled by daily injection of LPS for 11 days with a gradual increase in the dose range from 25
to 250 μg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional
stimulation of cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized
by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus an increase in the
production of nitric oxide, interferon-μ, and Hsp70 was demonstrated. In addition, a more effective restoration of the number
of thymus cells, as well as an increase in macrophage tumor necrosis factor-α production were observed after cessation of
LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell
activity under acute and chronic inflammation.
[Show abstract][Hide abstract] ABSTRACT: The effect of low-intensity laser light (He-Ne, 0.2 mW/cm2, 632.8 nm, exposure time 1 min) or centimeter waves (8.15–18 GHz, 1 μW/cm2, exposure time 1 h) on Phospho-SAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes
or cells incubated previously with geldanamycin, an inhibitor of Hsp90, were used in the experiments. Significant stimulation
of Phospho-SAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models.
It was proposed that activation of the SAPK/JNK signal pathway plays one of the central roles in cellular stress response
to low-power nonionizing radiation.
[Show abstract][Hide abstract] ABSTRACT: Effects of geldanamycin (GA), a known inhibitor of the heat shock protein 90 (Hsp90) activity, on the expression of some signal
proteins and various heat-shock proteins were studied by Western blot analysis in cultivated spleen lymphocytes isolated from
male NMRI mice. It has been revealed that cultivating the cells with GA results in a significant decrease in the amount of
transcription factor NF-κB, as well as in its phosphorylated form pNF-κB and the protein suppressing its activity, IκB-α.
Furthermore, the presence of GA in the cell-cultivation medium produced a significant decrease in the amount of protein kinase
SAPK/JNK. Such a GA-produced modification in the signal protein system suggests the participation of GA in the development
of a stress response in an animal cell to damaging action. This suggestion was checked on a model of a cell’s stress response
to the action of low-intensity laser radiation. It was proven that the Hsp90-binding agent, GA, significantly decreased in
vitro the level of the stress response to laser radiation by decreasing the production of heatshock proteins, Hsp70 and Hsp25,
both in irradiated isolated lymphocytes and in their intracellular structures. These findings open prospective uses for geldanamycin
in various therapies that are accompanied by undesired side effects connected with increased levels of stress response in
immune system cells.
Cell and Tissue Biology 07/2008; 2(4):366-372. DOI:10.1134/S1990519X08040044
[Show abstract][Hide abstract] ABSTRACT: In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production
of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male
NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide,
and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received
intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather
than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is
most pronounced at the NF-κB phosphorylation stage.
[Show abstract][Hide abstract] ABSTRACT: The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 microg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1 beta, IL-2, IL-6, TNF-alpha, interferon-gamma, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 microg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-alpha production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.