[The Thymic Hormone Thymosin-1α Reduces the Pro-Inflammatory Response of Raw 264.7 Cells Induced by Endotoxin].

The aim of this work was to study the effects of thymosin-1 alpha (Tα1) on the anti-inflammatory response of RAW 264.7 macrophages cultured in the presence of lipopolysaccharide (LPS) from the walls of gram-negative bacteria. As well, we evaluated production of pro-inflammatory cytokines and the activity of the NF-κB and SAPK/JNK signaling pathways. In addition, the level of expression of a number of genes that regulate cell apoptosis, as well as the activity of receptors involved in the pro-inflammatory response, was determined. First, the addition of Tα1 normalized the level of cytokine production to varying degrees, with a particularly noticeable effect on IL-1ß and IL-6. Second, the addition of Tα1 normalized the activity of the NF-κB and SAPK/JNK signaling cascades and the expression of the Tlr4 gene. Third, Tα1 significantly reduced p53 and the activity of the P53 gene, which is a marker of cell apoptosis. Fourth, it was shown that the increase in Ar-1 gene expression under the influence of LPS was significantly reduced using Tα1. Thus, it was found that the presence of Tα1 in the RAW 264.7 cell culture medium significantly reduced the level of the pro-inflammatory response of cells.

Protective effect of exogenous peroxiredoxin 6 and thymic peptide thymulin on BBB conditions in an experimental model of multiple sclerosis.

This study aimed to assess the effects of the immunomodulator thymulin, a thymic peptide with anti-inflammatory effects, and peroxiredoxin 6 (Prdx6), an antioxidant enzyme with dual peroxidase and phospholipase A2 activities, on the blood‒brain barrier (BBB) condition and general health status of animals with relapsing-remitting experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis in humans. Both thymulin and Prdx6 significantly improved the condition of the BBB, which was impaired by EAE induction, as measured by Evans blue dye accumulation, tight-junction protein loss in brain tissue, and lymphocyte infiltration through the BBB. The effect was associated with significant amelioration of EAE symptoms. Thymulin treatment was accompanied by a decrease in immune cell activation as judged by interleukin-6, -17, and interferon-gamma cytokine levels in serum and NF-kappaB cascade activation in splenocytes of mice with EAE. Prdx6 did not induce significant immunomodulatory effects but abruptly decreased EAE-induced NOX1 and NOX4 gene expression in brain tissue, which may be one of the possible mechanisms of its beneficial effects on BBB conditions and health status. The simultaneous administration of thymulin and Prdx6 resulted in complete symptomatic restoration of mice with EAE. The results demonstrate prospective strategies for multiple sclerosis treatment.

Geldanamycin Enhances the Radioprotective Effect of Peroxyredoxin 6 in Irradiated 3T3 Fibroblasts.

The aim of the study was to evaluate the possibility of increasing the radioprotective potential of peroxiredoxin 6 (Prdx6) and its mutant form S32A by their combined use with geldanamycin (GA) for 3T3 fibroblasts irradiated with X-rays at a dose of 6 Gy. The mutant enzyme S32A, which does not have phospholipase activity, exhibits a more pronounced radioprotective activity when combined with GA. The use of this combination of radioprotective drugs completely abolishes the peak of NF-κB activity in irradiated 3T3 cells. Another transcription factor, p53, which is an indicator of the level of cell apoptosis and increases upon irradiation, is also reduced by S32A in combination with GA. The low-molecular-weight protein p21, which is a marker of cell senescence and whose production increases upon irradiation, is also normalized when S32A is used in combination with GA. In addition, the use of this combination of radioprotective drugs significantly reduces the stress response of 3T3 cells to X-ray irradiation.

Participation of Hsp70 and Hsp90α Heat Shock Proteins in Stress Response in the Course of Type 1 Diabetes Mellitus.

The role of two heat shock proteins, Hsp70 and Hsp90α, on stress response in mice with severe diabetes mellitus induced by a high dose of alloxan (500 mg/kg body weight), as well as in RIN-m5F ß cells cultured in the presence of cytokines (IL-1 and TNF-α) was studied. Our results showed that severe type 1 diabetes mellitus (T1D) caused a higher expression of Hsp90α, but not Hsp70. Moreover, injections of the peroxiredoxin 6 antioxidant enzyme (PRDX6) did not affect the expression of these chaperones. Conversely, pro-inflammatory cytokines added to ß-cells caused a significant increase in the expression of Hsp90α and, substantially, Hsp70. Moreover, cells cultivated in the presence of PRDX6 were more susceptible to the cytokine effect. Thus, in the course of severe alloxan-induced T1D, no protective role of the heat shock proteins, was revealed, and their expression level was not increased by PRDX6. At the same time the protective potential of these proteins was shown in vitro with the use of RIN-m5F ß cells. Thus, the system of heat shock proteins was unable to prevent the devastating effects of severe T1D accompanied by high animal mortality.

Protective Effect of Peroxiredoxin 6 Against Toxic Effects of Glucose and Cytokines in Pancreatic RIN-m5F ß-Cells.

Taking into account a special role of pancreatic ß-cells in the development of diabetes mellitus, the effects of peroxiredoxin 6 (Prx6) on the viability and functional activity of rat insulinoma RIN-m5F ß-cells were studied under diabetes-simulating conditions. For this purpose, the cells were cultured at elevated glucose concentrations or in the presence of pro-inflammatory cytokines (TNF-α and IL-1) known for their special role in the cytotoxic autoimmune response in diabetes. It was found that the increased glucose concentration of 23-43 mM caused death of 20-60% ß-cells. Prx6 added to cells significantly reduced the level of reactive oxygen species and protected the RIN-m5F ß-cells from hyperglycemia, reducing the death of these cells by several fold. A measurement of insulin secretion by the RIN-m5F ß-cells showed a significant stimulatory effect of Prx6 on the insulin-producing activity of pancreatic ß-cells. It should be noted that the stimulatory activity of Prx6 was detected during culturing the cells under both normal and unfavorable conditions. The regulation of the NF-κB signaling cascade could be one of the mechanisms of Prx6 action on ß-cells, in particular, through activation of RelA/p65 phosphorylation at Ser536.

Immune response in the relapsing-remitting experimental autoimmune encephalomyelitis in mice: The role of the NF-κB signaling pathway.

Characteristics of the mouse model of relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) closely resemble manifestations of multiple sclerosis in humans. In the present study, we investigated the mechanisms of inflammatory response, focusing on NF-κB pathway activation. Cytokine response in rEAE mice was multiphasic: the early phase was characterized by the increase in interferon-γ level in plasma. In the later stage, the level of interleukin-17, but not of interferon-γ, was increased. The early phase of rEAE was also accompanied by increased RelA/p65 phosphorylation at Ser276 in spleen cells, whereas the rEAE maintenance phase was characterized by RelA/p65 phosphorylation at Ser536 and IKK phosphorylation. The IKKα/ß inhibitor reduced interleukin-17 and interferon-γ levels in plasma and alleviated rEAE symptoms. The IKKα/ß inhibitor decreased IKK and p65(Ser536) phosphorylation, but doubled p65(Ser276) phosphorylation in rEAE mice. The increased RelA/p65(Ser276) phosphorylation coincided in time with the production of interferon-γ, Hsp72, and the early phase of IL-17 generation, whereas increased RelA/p65(Ser536) phosphorylation coincided with the activation of IKK, SAPK/JNK, and p53, as well as the late phase of IL-17 production, indicating the role of the RelA/p65 phosphorylation events in the induction and maintenance of rEAE.

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide.

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1ß, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 µg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/ß, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.

Involvement of the p38 MAPK signaling cascade in stress response of RAW 264.7 macrophages.

The role of the p38 MAPK signaling cascade was studied in stress response of RAW 264.7 macrophages to extremely low-intensity centimeter microwaves. Irradiation stimulated production of a number of cytokines (IL-1, IL-6, TNF-α, INF-γ and IL-10), as well as induced activation of the signaling cascades NF- κB and p38 MAPK, and enhanced expression of Hsp72 heat shock protein. In the presence of the cascade p38 MAPK inhibitor (p38 MAP kinase inhibitor XI), the stimulating effects of electromagnetic waves were abrogated either completely (for NF-κB and Hsp72) or partially (for p38 MAPK and cytokines). The results obtained are indicative of a high sensitivity of the signaling cascade p38 MAPK to the effect of low-intensity physical fields.

The role of CK2 protein kinase in stress response of RAW 264.7 macrophages.

The role of casein kinase 2 (CK2) in the activation of the key NF-κB signaling cascade and other signaling and stress proteins during stress induced by exposure to nonthermal low-intensity electromagnetic radiation has been investigated. The ability of CK2 to regulate the activation of the NF-κB signaling cascade and upregulate the proinflammatory cytokine production and TLR4 receptor expression has been demonstrated. The existence of an alternative signaling pathway for NF-κB cascade activation directly involving protein kinase CK2 has been demonstrated using inhibition analysis in cells stressed by microwave irradiation.

Modulation of inflammatory response in mice with severe autoimmune disease by thymic peptide thymulin and an inhibitor of NF-kappaB signalling.

To investigate some cellular and molecular aspects of the autoimmune response and anti-inflammatory efficiency of potential therapeutic agents in a severe form of experimental autoimmune encephalomyelitis (sEAE), an inhibitor of NF-kappaB signalling, IKK Inhibitor XII, and/or thymic peptide thymulin, were injected intraperitoneally at 1.8 and 0.15mg/kg e.o.d, respectively, to C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein and several adjuvants. The immunization induced high lethality in three weeks. The biphasic cytokine response observed in earlier and delayed phases was attributed to the activity of Th1 and Th17 cells, respectively. Phosphorylation of RelA protein from the NF-kappaB family increased during the earlier phase and decreased in the delayed phase. SAPK/JNK signalling protein and heat shock protein Hsp72 significantly increased in lymphocytes. Both the IKK Inhibitor XII and thymulin reduced disease severity, attenuated immune imbalance, and increased mouse life-span. Co-administration of the agents produced no additive effect. Both the inhibitor and thymulin reduced the Th1 response but not the Th17 response. Therefore, RelA-associated Th1 activation and RelA-independent Th17 activation occurred in sEAE. Thymulin and the inhibitor demonstrate similar patterns of activity, potentially through the RelA pathway inhibition, resulting in a partial therapeutic effect on the animals' health status.

Anti-inflammatory effects of IKK inhibitor XII, thymulin, and fat-soluble antioxidants in LPS-treated mice.

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/ß kinases (IKK Inhibitor XII); with thymulin; with dietary coenzyme Q9, α-tocopherol, and ß-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.

Thymic peptides restrain the inflammatory response in mice with experimental autoimmune encephalomyelitis.

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 (15 µg 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.

[Effects of several inhibitors of intracellular signaling on production of cytokines and signal proteins in RAW 264.7 cells cultivated with low dose ammonium].

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.

Role of heat shock protein hsp90 in formation of protective reactions in acute toxic stress.

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.

[Stressful effects of chemical toxins at low concentrations].

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-alpha and interferon-gamma, as well as the expression of the inducible form of heat shock proteins (HSP72 and HSP90-alpha) and in the activation of signal cascades NF-kappaB 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.

[The role of protein kinase SAPK/JNK in cell responses to low-intensity nonionizing radiation].

The effect of low-intensity lases light (0.2 mW/cm2, 632.8 nm, exposure time 1 min) or centimeter waves (8.15-18 GHz, 1 W/cm2, exposure time 1 h) on PhosphoSAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of heat shock protein 90 (HSP90), were used in the experiments. A significant stimulation of PhosphoSAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that the activation of SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.

[Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice].

The effects of thymopentin, a synthetic analogue 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 lipopolysaccaride (LPS) from gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 microg/100 g of body weight, and chronic inflammation (sepsis) was modeled by a daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 microg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation in 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-gamma, 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-alpha production were observed after repeal of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.

[Effect of geldanamycin on the expression of signal proteins and heat shock proteins in normal mice lymphocytes].

The effects of geldanamycin, which is known as inhibitor of heat shock protein 90 activities, on expression of several signal and 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 geldanamycin resulted in decrease in transcription factor NF-kappaB amount, as well as decrease in its phosphorylated form, pNF-kappaB, and lowering in its suppressor, IkappaB-alpha. Besides, cells cultivated with geldanamycin demonstrated significant decrease in the amount of protein kinase SAPK(JNK). The modifications in signal pathways, which had been induced by geldanamycin, pointed to direct influence of the antibiotics on cellular stress response to damaging impact. This assumption was examined with the model of cellular stress response induced by low-level laser radiation. It was proved that Hsp90-binding drug, geldanamycin, significantly decreased in vitro stress response to laser light via lowering the production of heat shock proteins, Hsp70 and Hsp25, both in irradiated lymphocytes and in theirs intracellular structures. These findings show the prospect for using of geldanamycin in various therapies that are compromised with objectionable side effects manifested as heightened stress response in immune cells.

[The role of TLR4 receptor in the stress response of lymphocytes].

In vitro effects of low-level electromagnetic waves (8.18 GHz, frequency swings within 1 s, intensity 1 microW/cm, exposure for 1 h) and low-energy laser light (He-Ne laser with 632.8 nm, 0.2 mW/cm, dose 1.2 x 10(-2) J/cm2) on the expression of receptor protein TLR4, which is known as a part of the system for microbal toxin recognition, were studied in mouse lymphocytes. In addition, TLR4 expression was examined in situations when stress responses to low-level nonionizing radiation were modified by the antibiotic geldanamycin, which suppresses the activity of the heat shock protein Hsp90. It was found that low-level microwaves significantly raised the amount of TLR4; in contrast, laser light decreased the expression of the receptor in lymphocytes. In cells pretreated with geldanamycin, the TLR4 expression in irradiated cells was reduced to minimum levels, much lower than control values. The results showed that TLR4, which is involved in specific binding of toxin from gram-negative bacteria, can regulate cell responses to signals of other origin, in particular to nonionizig radiation, including low-level microwaves and laser light.