M. G. Vinokurov

Exogenous mammalian extracellular HSP70 reduces endotoxin manifestations at the cellular and organism levels

In this study, we checked whether HSP70 preparations of different origins are able to protect model animals (rats) from endotoxic shock and modify the response of myeloid cells to lipopolysaccharide (LPS) challenge. It was shown that HSP70 preparations can effectively protect organisms from endotoxic shock by strongly decreasing mortality and restoring both homeostasis and various hemodynamic characteristics. At the cellular level, HSP70 preparations significantly inhibit LPS‐induced reactive oxygen species production in various myeloid cells and decrease NO expression in macrophages, which is enhanced after LPS priming. In parallel, HSP70 preconditioning partially normalizes neutrophil apoptosis, which is disturbed as a result of LPS stimulation. These results suggest that the antiseptic actions of HSP70 preparations are probably realized at the level of receptor membrane complexes of myeloid cells, which represent the major target of LPS action. Taken together, our findings show that extracellular mammalian HSP70 may play an important role in innate immunity modulation and stimulation of endogenous protective mechanisms, both at the cellular and organism levels, which make this protein a promising base for the development of efficient antiseptic drugs.

Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels

It has been previously reported that pretreatment with exogenous heat shock protein 70 (Hsp70) is able to protect cells and animals from the deleterious effects of bacterial lipopolysaccharide (LPS) produced by Gram-negative bacteria. However, the effects of Hsp70 pretreatment on lipoteichoic acid (LTA) challenge resulted from Gram-positive bacteria infection have not been fully elucidated. In this study, we demonstrated that preconditioning with human recombinant Hsp70 ameliorates various manifestations of systematic inflammation, including reactive oxygen species, TNFα, and CD11b/CD18 adhesion receptor expression induction observed in different myeloid cells after LTA addition. Therefore, exogenous Hsp70 may provide a mechanism for controlling excessive inflammatory responses after macrophage activation. Furthermore, in a rat model of LTA-induced sepsis, we demonstrated that prophylactic administration of exogenous human Hsp70 significantly exacerbated numerous homeostatic and hemodynamic disturbances induced by LTA challenge and partially normalized the coagulation system and multiple biochemical blood parameters, including albumin and bilirubin concentrations, which were severely disturbed after LTA injections. Importantly, prophylactic intravenous injection of Hsp70 before LTA challenge significantly reduced mortality rates. Thus, exogenous mammalian Hsp70 may serve as a powerful cellular defense agent against the deleterious effects of bacterial pathogens, such as LTA and LPS. Taken together, our findings reveal novel functions of this protein and establish exogenous Hsp70 as a promising pharmacological agent for the prophylactic treatment of various types of sepsis.

[Heat-shock protein HSP70 protects neuroblastoma cells SK-N-SH from the neurotoxic effects hydrogen peroxide and the β-amyloid peptide].

Neuronal cell death in Alzheimers disease is associated with the development of oxidative stress caused by the reactive oxygen species (ROS), which can be generated as a result of the effect of beta-amyloid peptides. One of the sources of ROS is hydrogen peroxide, inducing the apoptosis and necrosis of neural tissue cells. The mechanism of hydrogen peroxide apoptotic action includes launching signaling pathways that involve protein kinases PI3K, p38MAPK, JNK and ERK. Oxidative stress leads to increased synthesis of heat-shock proteins in the cells including HSP70. It was shown that the exogenous HSP70 could reduce generation of ROS in cells. In this study, we determined how HSP70 affected apoptosis and necrosis in human neuroblastoma cells SK-N-SH, induced by hydrogen peroxide and β-amyloid peptide Aβ(1-42). It was shown that HSP70 reduces the cytotoxic effects of hydrogen peroxide and beta-amyloid, and protein kinases PI3K and JNK play an important role in the mechanism of HSP70 protective effect on the peroxide induced apoptosis in SK-N-SH cells.

Dynamics of the Fas-and stress-induced apoptosis of human neutrophils under the action of endotoxins

355 Endotoxin interaction with neutrophils that play the key role in innate immunity is extremely important at the initial stages of Gram-negative sepsis and endotoxin shock. Endotoxins (lipopolysaccharides, LPS) released from the bacterial cell walls enter the blood, where they form a complex with LPS-binding protein. Afterwards, this complex interacts with CD14 receptor and activates the assembly of the membrane receptor complex of neurtrophils, which includes the Tool-like receptor (TlR4), MD2, MyD88, and other proteins and receptors. The complex assembly is followed by a regulatory signal for the transcription factors, which results in the synthesis of various antiinflammatory cytokines [1]. Neutrophils are terminally differentiated short-lived cells, which normally circulate in blood for 10 h and then migrate into tissues, where they undergo spontaneous apoptosis and phagocytosis by macrophages [2]. During sepsis and in vitro, endotoxins inhibit apoptosis of neutrophils [3, 4]. The LPS-dependent regulation of neutrophil apoptosis depends on mitogen-activated protein kinases, including p38-mitogen-activated protein kinases ( 38åÄêä ) and extracellular signalregulated kinases (ERK). Interaction between the 38åÄêä and ERK signal pathways may influence sepsis development [3]. Phosphatidylinositol-3 kinase (PI-3K) also participates in neutrophil activation and regulation of their apoptosis [5]. Targeted control of neutrophil apoptosis during inflammation is now considered a promising approach to the recovery of cell homeostasis in treatment of sepsis and other pathological states [6]. The Fas-receptor-dependent mechanism plays an important role in the induction of neutrophil apoptosis. Interaction between monoclonal anti-Fas-antibodies and Fas receptor leads to trimerization of this receptor and the caspase-dependent transduction of the apoptosis-specific signal to the transcription factors of the cell [2]. It is known that p38MAPK are involved in the regulation of the Fas-induced apoptosis [7]. Various stress factors, in particular, ultraviolet radiation within the C region (254 nm, UVC) are also capable of inducing neurtrophil apoptosis [8]. The stress-induced apoptosis of neutrophils is also activated with involvement of 38MAPK [9]. UVC causes trimerization of Fas receptor [10].

Amyloid-β with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation

Neuronal dysfunction and loss associated with the accumulation of amyloid-β (Aβ) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer’s disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aβ42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aβ42 with isomerised Asp7 (isoAβ42) which is one of the most abundant Aβ isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAβ42 rather than intact Aβ42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAβ42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAβ42, being to date the only known synthetic Aβ species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAβ42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

Heat-shock protein HSP70 reduces the secretion of TNFα by neuroblastoma cells and human monocytes induced with beta-amyloid peptides

The progress of neurodegeneration in Alzheimer’s disease is closely associated with inflammatory processes in the brain tissues induced by beta-amyloid peptides (Aβ). In this paper, we showed that Aβ(1-42) and isoAβ(1-42) in human neuroblastoma cells SK-N-SH and promonocyte THP-1 activated the production of tumor necrosis factor (TNFα). Notably, isoAβ(1-42) had the strongest effect on the increase in the level of TNFα. The addition of recombinant heat-shock protein HSP70 reduces TNFα production induced by Aβ, which leads to a decrease in neuronal cell damage at the organism level.

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.

Exogenous heat shock protein HSP70 modulates lipopolysaccharide-induced macrophage activation

320 Innate immunity cells, including macrophages, play an important role in the defense of a mammalian body against pathogenic bacteria. Lipopolysacchaa rides (LPS) produced by Grammnegative bacteria actii vate macrophages. They begin to produce reactive oxygen species (ROS), synthesize and secrete proinn flammatory cytokines, and raise the production of heat shock proteins (HSP), including HSP70 [1]. After entering the bloodstream, LPSs interact with LPSSbinding protein and, subsequently, with the CD14 receptor of target cells. Then, LPSs are transs ferred from CD14 to lymphocyte antigen 96 (MDD2) to form two identical complexes including Tollllike receptor 4 (TLR4), MD2, and LPS. Then, a hett erodimeric receptor complex is formed in target cell membranes through interaction of the phosphate moii eties of LPS from one complex with TLR4 of the other complex [2]. The next step is the transduction of the signal from the receptor to transcription factors of tarr get cells to elicit the cell response. Early steps of the response are characterized by elevated ROS producc tion and adhesion factor expression. Then, proinflamm matory cytokines are produced. The first of them to be secreted is TNFFα [3]. Most enterobacteria E. coli contain the S form of LPSs. It includes hydrophobic lipid A, an oligosaccha ride core, and OOantigen. In addition to the S form, enterobacteria produce coreedeficient LPS molecules (R chemotypes) containing lipid A and cores of differr ent lengths. The molecules with abnormal cores are designated as Rb–Re chemotypes, and molecules with a fulllsize external core, as the Ra chemotype [4]. It is supposed that the LPS receptor complex can also bind heat shock proteins with M r = 70 kDa (HSP70) [5]. Our earlier results indicate that administration of exogenous recombinant HSP70 increases the survival of animals in the septic shock model and suppresses the activation of neutrophils and macrophages induced by the S form of LPS [6]. By now, the effect of S LPSs on myeloid cells has been studied in detail, but little is known about the action of R forms. Previously, we found an effect of various LPS chemotypes on ROS production and apoo ptosis in neutrophils [7]. Here, we studied the effect of exogenous HSP70 on ROS and TNFα production by macrophages exposed to various LPS chemotypes. We also studied the effect of exogenous HSP70 and LPSs on the TLR2 and TLR4 expression in macrophages. Earlier studies showed that administration of exogg enous HSP70 reduced …

The role of the TLR-dependent signaling pathway in the mechanism of phagocyte protection by exogenous heat shock protein HSP70 from the endotoxin action

305 Endotoxins play an important role in the developp ment of a Grammnegative sepsis, as well as in some diss eases, such as metabolic syndrome and coronary heart disease [1]. The effect of endotoxins (lipopolysacchaa rides, LPS) on the target cells is realized via Tollllike receptors 4 (TLR4) which they contain. Entering the bloodstream, LPS first interact with the LPSSbinding protein and then with CD14 receptors of the target cells (including blood phagocytes). Then, LPS pass from CD14 to MDD2 (lymphocyte antigen 96) with subsequent formation of two LPS–MDD2–TLR4 complexes, which further form a heterodimeric comm plex in the target cell membrane [2]. Signal transducc tion from this complex activates transcription factors (in particular, NFFκB, nuclear factor kappa B) and triggers the cell response, characteristic of which is an increase in adhesion factors and synthesis of proinn flammatory cytokines, TNFFα being the first to be synthesized [3]. In inflammatory diseases (in particular, sepsis), the synthesis and secretion of heat shock proteins (HSP70) are elevated, which plays an important role in the mechanism providing protection from heat shock and other types of stress. It is assumed that HSP70 can bind to TLRs [4]. Application of exogee nous recombinant HSP70 decreases the animal morr tality rate in the septic shock model, as well as inhibits endotoxinninduced activation of neutrophils and macrophages [5]. Study of the effects of HSP70 and LPS on TLR expression has yielded ambiguous results, suggesting both an increase and a decrease in TLR expression induced by HSP70 and LPS [6, 7]. The effect of intraa cellular HSP70 has been rather comprehensively studd ied; however, the mechanisms underlying the action of extracellular HSP70 are still unclear. We have studied the role of the TLRRdependent signaling pathway in the mechanism underlying the protection of blood phagocytes from endotoxins by the exogenous heat shock protein HSP70. We have earlier demonstrated that exogenous HSP70 protects innate immunity cells from endotoxins [5]; however, the mechanism of its protective action is unclear. Here, we have shown that HSP70 increases the numm ber of TLR2 and TLR4 on the cell membrane of neuu trophils and monocytes and insignificantly elevates TNFFα production by these cells predominantly with involvement of TLR44dependent signal transduction. On the other hand, exogenous HSP70 decreases the LPSSinduced increase in TLR2 and TLR4 expression in these cells. MATERIALS AND METHODS The following reagents were used in this study: E. The gene of recombinant human …

Exogenous heat shock protein HSP70 protects human blood phagocytes at the action of different chemotypes of lipopolysaccharide.

392 It is known that lipopolysaccharides (LPS) play the key role in the development of Gram negative sepsis. They also have important implications for develop ment of certain socially significant diseases [1]. The majority of the enterobacterium Escherichia coli have S form of LPS, which includes hydrophobic lipid A, an oligosaccharide core, and O antigen. In addition to the LPS S form, enterobacteria also synthetize core defect molecules of LPS (R chemotypes), which have lipid A and a core part of different lengths. Core defect structures are marked Rb–Re chemotypes; and structures that have a complete external core, Ra chemotype [2]. Entering blood stream, LPS first inter act with lipopolysaccharide binding protein (LBP), then with CD14 receptor of target cells. After that, LPS are transferred from CD14 to MD 2 (lymphocyte antigen 96), and subsequent formation of two com plexes LPS MD 2 TLR4 occurs; then, they form a heterodimer receptor complex in the membrane of target cells [3]. Then, a signal is transferred from the receptor to transcription factors in target cells, and the cell response is developed. At the early stages of cell response, reactive oxygen species (ROS) production, as well as adhesion factors expression, increase. In a later period, pro inflammatory cytokines are synthe tized, TNF α being the first of them to be secreted [4].