S. B. Parfenyuk

Thymulin, a thymic peptide, prevents the overproduction of pro-inflammatory cytokines and heat shock protein Hsp70 in inflammation-bearing mice.

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 μg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1β, IL-2, IL-6, TNF-α, interferon-γ, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 μg/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-α 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.

Production of Heat Shock Proteins, Cytokines, and Nitric Oxide in Toxic Stress

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-α, TNF-β), interferon-γ (IFN-γ), interleukin-2,-3,-6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-α in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-α, TNF-β, and NO in mouse cells. The production of other cytokines (interleukins and IFN-γ) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.

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-α, IFN-γ, 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-κB 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-κB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.

Immunomodulatory effects of thymopentin under acute and chronic inflammations in mice

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.

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-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.

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 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-1β, 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/β, 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.

The NF-κB, IRF3, and SAPK/JNK signaling cascades of animal immune cells and their role in the progress of type 1 diabetes mellitus

255 The role of several signaling cascades related to activation of the TLRRfamily receptors was studied by means of modeling diabetes mellitus type 1 (T1D) in mice. T1D is an autoimmune disease that occurs when autoreactive T cell clones affect the insulinnproducing pancreatic βcells. A shortage of insulin leads to hyperglycemia, and, despite daily insulin injections, the disease results in numerous complications, mainly at the level of blood vessels of several organs, such as kidd neys, eyes, and legs. In the developed countries of Europe and North America, T1D prevalence grows dramatically (e.g., in Finland, 65.2 per 100 000 people [1]). The pathogenesis of human T1D is difficult to study because of a series of restrictions; therefore, experimental models of T1D are helpful in analysis of the molecular and cellular mechanisms underlying the development of this pathology. Several cell types of the immune system are involved in the T1D pathogenesis. Only 40% of T1D cases are the result of genetic prediss position; more frequently, various exogenous and endogenous factors cause the disease. Most researchers of T1D pathogenesis focus their attention mainly on pancreatic βcells undergoing an autoimmune attack. However, the immune system disorders leading to the appearance of T cell clones that affect the bodys own cells generally are the cause of the disease. This study is an attempt at clarifying the mechaa nism underlying the immune imbalance in mice injected with alloxan to induce T1D. We aimed mainly at studying the cascades of intracellular signaling within the immune cells. Note that, in general, in autoimmune diabetes, the role of the signaling cascades triggered by the trann scription factors NFFκB, IRF3, and SAPK/JNK kinase is still poorly studied upon autoimmune diabee tes. Despite a potential antiiapoptotic function of NFFκB in most cell populations, in the pancreatic β cells, NFFκB has prooapoptotic activity. On the other hand, the risk of type 2 diabetes mellitus in patients with obesity, which causes chronic microoinflammaa tion, is related to a direct NFFκB activation [2]. Therefore, it is expected that inhibition of NFFκB activation may prevent diabetes. However, NFFκB is believed to protect the pancreatic βcells from apoptoo sis caused by TNFFalpha [3, 4]. As for the signaling cascade SAPK/JNK, there is no published evidence of its role in T1D pathogenesis. To date, nothing is known about the role of interr feron regulatory factor 3 (IRF3) in T1D pathogenesis. At the same time, this is …

Dietary liposoluble antioxidants protect mouse immune cells from the toxic effects of atmospheric ammonia.

113 Antioxidative vitamins are necessary for the immune cell function, as well as formation of antibodd ies and signaling proteins participating in the immune response. The daily vitamin demand being small, vitaa mins nevertheless determine the normal functioning of the immune and other systems of vertebrates [1]. For instance, the lack of vitamin E (αtocopherol) leads to a decrease in the antibody level and lymphoo cyte activity. The deficiency of carotenoids causes the immune insufficiency by decreasing the level of antii body production in response to antigen stimulation [2]. Usually, antioxidants are classified as lipoo and waterrsoluble. Waterrsoluble antioxidants are shortt lived compounds that are quickly excreted from the body. Importantly, they are able to switch their properr ties from antii to proooxidant, causing considerable cell damage. Liposoluble antioxidants are easily bound by blood lipoproteins embedded in cell memm branes and may be deposited in liposomes staying active during a long period. Almost all known liposoll uble antioxidants or their precursors enter the body only with food. The examples are αtocopherol and carotenoids, which not only decrease the lowwdensity lipid level in blood plasma, but also defend them from oxidation [3]. The only exception is coenzymes Q (ubiquinones), because they can be synthesized in the body. Moreover, there are enzyme systems capable to reduction of the oxidized forms of ubiquinones [4]. It is known that exogenous antioxidants are succ cessfully used for treating a number of pathologies accompanied by oxidative stress as a result of deficiency of endogenous antioxidants. Vitamin E (αtocopherol), ascorbic acid, carotenoids, flavonoids, selenium, and zinc are among the most frequently used antioxii dants. Notably, reactive oxygen species (ROS) are espee cially damaging for immune cells, which are particuu larly sensitive to oxidative stress [5]. We have demonn strated earlier that antioxidant complex consisting of αtocopherol, βcarotene, and coenzyme Q 9 when present in a diet helps to lower the oxidative stress in cells of the mouse immune system during acute inflammation induced by an endotoxin [6]. The purpose of this work was to investigate the defensive effects of these antioxidants on the immune system of animals exposed to an ammoniaapolluted atmosphere threefold higher than the maximum allowable concentration (MAC) in the working area. Earlier, we found that the signaling cascades of NFFκB and SAPK/JNK, as well as expression of the TLR4 receptor, were activated in mouse lymphoid cells [7]. The finding indicated stress influence of atmospheric …

Effect of super-high frequency electromagnetic radiation on the immune status of mice in endotoxic shock

The influence of low-intensity centimeter electromagnetic waves (sweep 8.15–18 GHz, 1 μW/cm2, 1 h daily for 10 days) on the production of TNFα; interleukins 1α, 1β, 2, 6, and 10; IFNγ, NO; and Hsp 27, 72, and 90α was assessed in mice before and after acute intoxication with E. coli lipopolysaccharide. Irradiation after the endotoxic shock had no beneficial effect, whereas preliminary exposure improved the indices of organismic defense.