M. O. Khrenov

Naturally occurring antioxidant nutrients reduce inflammatory response in mice.

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.

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.

Involvement of NF-κB transcription factor in the antiinflammatory activity of thymic peptides

484 The thymus is known to play an important role in the normal functions and development of mammals; its state is especially important for the immune system of humans and animals. A number of disturbing fac tors, such as infections, poisons, and ionizing radia tion, have been demonstrated to lead to acute atrophy of the thymic tissue. Accelerated thymolysis is a side effect of some drugs, e.g., for tuberculosis and cancer, which complicates the treatment. Note that all physi ological stress factors, including malnutrition, are supposed to lead to acute thymolysis [1].

The role of the NF-κB, SAPK/JNK, and TLR4 signalling pathways in the responses of RAW 264.7 cells to extremely low-intensity microwaves

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. Results: 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. Conclusions: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.

Thymus peptides regulate activity of RAW 264.7 macrophage cells: inhibitory analysis and a role of signal cascades

Objectives: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation. Methods: 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. Results: 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. Conclusions: 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.

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.

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

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.

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

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.