Releasing signals, secretory pathways, and immune function of endogenous extracellular heat shock protein 72

Heat shock proteins (Hsp) were first characterized as intracellular proteins, which function to limit protein aggregation, facilitate protein refolding, and chaperone proteins. During times of cellular stress, intracellular Hsp levels increase to provide cellular protection. Recently, it has been recognized that Hsp, particularly Hsp72, are also found extracellularly (eHsp72), where they exhibit potent immunomodulatory effects on innate and acquired immunity. Circulating eHsp72 levels also greatly increase during times of stress (i.e., when an organism is exposed to a physical/psychological stressor or suffers from various pathological conditions). It has been proposed that elevated eHsp72 serves a protective role by facilitating immunological responses during times of increased risk of pathogenic challenge and/or tissue damage. This review focuses on the in vivo releasing signals and immunomodulatory function(s) of endogenous eHsp72. In addition, we present data that emphasize the importance of caution when conducting in vitro immunological tests of Hsp72 function.

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title: Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs.
abstract: The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [35S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified. 
 
Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [3H]uridine labelling. By correlating the amount of [3H]uridine in some puffs with the level of [35S]methionine in some bands a tentative relation is suggested in a few instances. 
 
The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.

title: Molecular chaperones in cellular protein folding.
abstract: The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.

title: Habitual physical activity facilitates stress-induced HSP72 induction in brain, peripheral, and immune tissues.
abstract: The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.

title: Adrenergic regulation of the heat shock response in brown adipose tissue.
abstract: One of several ways that cells respond to damage or stress is by the expression of a set of highly conserved proteins termed, heat shock proteins (HSP). Induction of the heat shock response has been positively correlated with adaptation or protection of cells and tissues from the destructive effects of various types of stressors. Although heat can induce a generalized HSP response in most cells, the selective induction of HSP in specific cell populations by pharmacological agents may prove therapeutically useful for the protection of organs or tissues at risk for damage. Results from our studies suggest that the HSP response is integrated with fundamental physiological stress responses and demonstrate that distinct regulatory events couple neurotransmitter/hormone-receptor interactions with HSP expression in mammalian tissues. We demonstrate that the adrenergic receptor agonist, phenylephrine, induces HSP expression in brown adipose tissue (BAT). Apparently, this response is mediated by alpha-adrenergic receptors in BAT because prazosin, but not propranolol, blocks HSP induction and hexamethonium is without effect. Based on the transcripts induced and the magnitude of heat shock element-binding activity, phenylephrine appears to induce HSP expression through unique transcriptional regulatory mechanisms. The phenylephrine-induced HSP response is not unique to BAT as we have found that HSP are induced in other tissues as well. In BAT, HSP may facilitate the thermogenic function of this tissue, however, their function in other tissues remains unclear. The results of this study characterize a model system where the heat shock response is differentially evoked by a specific pharmacological agent and may aide in the development of treatment strategies to selectively target HSP expression in vivo.

title: Cold-Induced Heat Shock Protein Expression in Rat Aorta and Brown Adipose Tissue
abstract: Recent reports indicate that Heat Shock Proteins (HSPs) are induced in mammalian tissues as part of a homeostatic response to environmental stressors. Administration of sympathomimetic drugs and neuroendocrine stress hormones has been shown to evoke an HSP response in unstressed animals indicating that cell signaling events exists that couple specific neurotransmitter/hormone-receptor interactions with HSP expression in mammalian tissues. Herein, we demonstrate that exposure of rats to a cold ambient temperature (6 degrees C) results in increased expression of constitutive and inducible members of the HSP70 gene family in association with increased expression of the mitochondrial uncoupling protein in brown adipose tissue (BAT). Increased HSP70 expression was not restricted to BAT because HSP70 was also induced in the aorta. This cold-induced HSP response is characterized by a transient increase in HSP70 protein and mRNA in both tissues during continued exposure. Ganglionic blockade prevented cold-induced HSP70 expression in BAT and aorta, indicating that sympathetic activity is requisite to this response. Administration of the alpha 1-adrenergic receptor antagonist, prazosin, also blocked expression, further delineating possible signaling mechanisms mediating this response. Apparently, cells in some mammalian tissues have adopted unique cellular regulatory mechanisms to support HSP induction that have been incorporated into the physiological response of the entire organism to an environmental stressor.

title: Human HSP70 promoter contains at least two distinct regulatory domains.
abstract: The expression of the human HSP70 gene is induced by a wide range of physiological stresses, including exposure to heat shock and heavy metals, or under nonstress conditions, such as in response to serum stimulation. We have previously demonstrated that in either case the regulated expression is at the primary level of transcription. To determine whether transcription is mediated through a single or multiple genetic elements, we have dissected the sequences upstream of the transcription start site of the human HSP70 gene by constructing chimeric genes retaining variable amounts of 5' flanking regions fused to the bacterial gene encoding chloramphenicol acetyltransferase. Transcription from the chimeric genes was determined by S1 nuclease analysis of separate stable transfectants. The sequences required for heat shock and cadmium induction lie between -107 and -68. Within this region is the sequence CTGGAATATTCCCG, which is identical in 12/14 positions with the heat shock element of Drosophila heat shock genes, and a separate sequence, CGNCCCGG, which is homologous to the core of the human metallothionein II metal-responsive element. The sequences required for serum-stimulated transcription are distinct from the heat shock element. The sequence CCAAT at -68 is required for high levels of correctly initiated transcripts, and a purine-rich sequence, GAAGGGAAAAG, at -58 is required for serum stimulation. The human HSP70 promoter contains at least two regulatory domains--a distal domain responsive to heat shock or cadmium and a proximal domain responsive to stimulation by serum.

title: Heat shock transcription factors: structure and regulation.
abstract: Organisms respond to elevated temperatures and to chemical and physiological stresses by an increase in the synthesis of heat shock proteins. The regulation of heat shock gene expression in eukaryotes is mediated by the conserved heat shock transcription factor (HSF). HSF is present in a latent state under normal conditions; it is activated upon heat stress by induction of trimerization and high-affinity binding to DNA and by exposure of domains for transcriptional activity. Analysis of HSF cDNA clones from many species has defined structural and regulatory regions responsible for the inducible activities. The heat stress signal is thought to be transduced to HSF by changes in the physical environment, in the activity of HSF-modifying enzymes, or by changes in the intracellular level of heat shock proteins.

title: Roles of the heat shock transcription factors in regulation of the heat shock response and beyond
abstract: The heat shock response, characterized by increased expression of heat shock proteins (Hsps) is induced by exposure of cells and tissues to extreme conditions that cause acute or chronic stress. Hsps function as molecular chaperones in regulating cellular homeostasis and promoting survival. If the stress is too severe, a signal that leads to programmed cell death, apoptosis, is activated, thereby providing a finely tuned balance between survival and death. In addition to extracellular stimuli, several nonstressful conditions induce Hsps during normal cellular growth and development. The enhanced heat shock gene expression in response to various stimuli is regulated by heat shock transcription factors (HSFs). After the discovery of the family of HSFs (i.e., murine and human HSF1, 2, and 4 and a unique avian HSF3), the functional relevance of distinct HSFs is now emerging. HSF1, an HSF prototype, and HSF3 are responsible for heat‐induced Hsp expression, whereas HSF2 is refractory to classical stressors. HSF4 is expressed in a tissue‐specific manner;similar to HSF1 and HSF2, alternatively spliced isoforms add further complexity to its regulation. Recently developed powerful genetic models have provided evidence for both cooperative and specific functions of HSFs that expand beyond the heat shock response. Certain specialized functions of HSFs may even include regulation of novel target genes in response to distinct stimuli.—Pirkkala, L., Nykanen, P, Sistonen, L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond.—Pirkkala, L., Nykäen, P, Sistonen, L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J. 15, 1118–1131 (2001)

title: Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance.
abstract: Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). The precise functions of HSPs are unknown, but there is considerable evidence that these stress proteins are essential for survival at both normal and elevated temperatures. HSPs also appear to play a critical role in the development of thermotolerance and protection from cellular damage associated with stresses such as ischemia, cytokines, and energy depletion. These observations suggest that HSPs play an important role in both normal cellular homeostasis and the stress response. This mini-review examines recent evidence and hypotheses suggesting that the HSPs may be important modifying factors in cellular responses to a variety of physiologically relevant conditions such as hyperthermia, exercise, oxidative stress, metabolic challenge, and aging.

title: Stress-induced heat shock protein 70 expression in adrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependent response.
abstract: The induction of heat shock proteins (HSP) by cellular stress and the activation of the hypothalamic-pituitary-adrenal axis by physiologic stress are biological responses that aid in the maintenance of cellular and organismal homeostasis, respectively. In this report, restraint stress, known to activate the hypothalamic-pituitary-adrenal axis, is shown to induce expression of HSP70 mRNA selectively in the adrenal cortex of the rat. Restraint-induced HSP70 expression in the adrenals is rapid and is preceded by the activation of a protein factor capable of binding to the heat shock transcriptional control element. The ability of restraint to induce HSP70 expression in the adrenal is virtually eliminated in hypophysectomized rats but can be restored by the exogenous administration of adrenocorticotropic hormone. The magnitude of this induction declines as a function of increasing age, which may contribute to a reduced stress tolerance by aged animals. These results support a role for HSP70 in the physiologic stress response mediated by the hypothalamic-pituitary-adrenal axis.

title: Neural and endocrine mechanisms of cocaine-induced 70-kDa heat shock protein expression in aorta and adrenal gland.
abstract: Cocaine has properties of a physiologic stressor that are reflected by its ability to activate both the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system. We have previously reported that activation of the hypothalamic-pituitary-adrenal axis and sympathetic nervous system by restraint and pharmacologic agents induces expression of a set of highly conserved cellular stress response proteins (heat shock proteins, HSP) in the adrenal gland and aorta. In the adrenal gland, HSP expression appears to be mediated by stress-induced increases in adrenocorticotropic hormone whereas expression in the aorta involves noradrenergic neurotransmission. In this report we capitalize on the ability of cocaine to stimulate physiologic stress responses to define further mechanisms regulating HSP70 expression in these tissues. We report the novel observation that cocaine administration induces both adrenal and vascular HSP70 mRNA expression. Elevated HSP70 mRNA was preceded by activation of factors capable of binding to the heat shock transcriptional control element and was followed by an elevation in HSP70 protein. Cocaine significantly increased plasma adrenocorticotrophic hormone whereas hypophysectomy eliminated cocaine-induced expression in the adrenal gland suggesting that in this tissue, the effect of cocaine on HSP70 expression is also mediated via adrenocorticotrophic hormone. In the aorta, depletion of catecholamines by reserpine pretreatment paradoxically augmented cocaine-induced HSP70 expression. Based on these results, it appears that HSP70 expression in the aorta occurs through direct actions of cocaine on vascular cells that are ultimately transduced to activation of the HSP70 gene rather than indirectly through alterations in catecholamine reuptake and release.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor.
abstract: Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 degrees C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive--Hsc70 and inducible--Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.

title: Activation of HSF and selective increase in heat-shock proteins by acute dexamethasone treatment.
abstract: Heat-shock proteins (HSPs) are an important family of endogenous protective proteins, which increase in response to myocardial ischemia and other stresses. Overexpression of HSP72 is cardioprotective. We were interested in the regulation of heat-shock factor (HSF), the transcription factor for HSP genes. Previously we have observed that the inflammatory cytokine tumor necrosis factor-alpha increases HSP72 levels and postulated that dexamethasone might effect the heat shock response. In the adult rat cardiac myocyte we found that treatment with either low (10 microM)- or high (100 microM)-dose dexamethasone activated HSF by 2-6 h as determined by gel shift assay without evidence of cytotoxicity. Although HSF activation is a key step in expression of HSP72, this may not result in an increase in HSP72. We found that 10 microM dexamethasone increased HSP72 38%, and 100 microM dexamethasone increased HSP72 62% (P < 0.05). HSP27 and HSP60 were unchanged. The selective increase in HSP72 was associated with protection of the cardiac myocytes from hypoxia and reoxygenation. We conclude that dexamethasone is a novel inducer of the heat shock response.

title: Glucocorticoid pretreatment protects cardiac function and induces cardiac heat shock protein 72.
abstract: Acute administration of glucocortiocoids reduces inflammation. Increasing knowledge of the mechanisms of action indicate that pretreatment with glucocorticoids could have organ-protective effects. We investigated whether pretreatment with methylprednisolone (MP) protected the heart against ischemia-reperfusion dysfunction, and we hypothetized that this protection might be due to induction of the cardioprotective heat shock protein 72 (HSP72). Rats were given vehicle or MP-40 mg/kg im as a double injection starting either 24 or 120 h (5 days) before their hearts were excised for Langendorff perfusion (n = 6-11 hearts in each group). MP improved left ventricular function and coronary flow during reperfusion after 30 min of global ischemia and reduced infarct size. Cardiac HSP72 gradually increased in a 24-h time course after MP treatment, and the increase was sustained 5 days afterward (immunoblotting). HSP72 mRNA was either reduced or unchanged, indicating a posttranscriptional regulation. Pretreatment with hydrocortisone or dexamethasone (n = 7-8 hearts of each) similarily increased cardiac HSP72 24 h afterward. This paper demonstrates that glucocorticoids increase cardiac HSP72 and protect organ function against ischemia-reperfusion injury.

title: Heat shock factors and the control of the stress response.
abstract: Living cells are continually challenged by conditions which cause acute and chronic stress. To adapt to environmental changes and survive different types of injuries, eukaryotic cells have evolved networks of different responses which detect and control diverse forms of stress. One of these responses, known as the heat shock response, has attracted a great deal of attention as a universal fundamental mechanism necessary for cell survival under a variety of unfavorable conditions. In mammalian cells, the induction of the heat shock response requires the activation and translocation to the nucleus of one or more heat shock transcription factors which control the expression of a specific set of genes encoding cytoprotective heat shock proteins. The discovery that the heat shock response is turned on under several pathological conditions and contributes to establish a cytoprotective state in a variety of human diseases, including ischemia, inflammation, and infection, has opened new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear as possible candidates for novel cytoprotective drugs. This article focuses on the regulation and function of the heat shock response in mammalian cells and discusses the molecular mechanisms involved in its activation by stress and bioactive cyclopentenone prostanoids, as well as its interaction with nuclear factor kappaB, a stress-regulated transcription factor with a pivotal role in inflammation and immunity.

title: Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle
abstract: To test the hypothesis that a decrease in intramuscular glycogen availability may stimulate heat shock protein expression, seven men depleted one leg of muscle glycogen the day before performing 4–5 h of exhaustive, two‐legged knee extensor exercise at 40 % of leg peak power output. Subjects then rested for a further 3 h. Muscle biopsies were obtained from the depleted and control leg before, immediately after and 3 h into recovery from exercise. These samples were analysed for muscle glycogen, and HSP72 gene and protein expression. In addition, catheters were placed in one femoral artery and both femoral veins and blood was sampled from these catheters prior to exercise and at 1 h intervals during exercise and into recovery for the measurement of arterial‐venous differences in serum HSP72. Plasma creatine kinase (CK) was also measured from arterial blood samples. Pre‐exercise muscle glycogen content was 40 % lower in the depleted compared with the control leg and this difference was maintained throughout the experiment (P < 0.05; main treatment effect). Neither HSP72 gene nor protein expression was different pre‐exercise. However, both HSP72 gene and protein increased (P < 0.05) post‐exercise in the depleted leg, but not in the control leg. Exercise did not increase plasma CK concentrations and we were unable to detect HSP72 in the serum of any samples. These results demonstrate that while acute, concentric exercise is capable of increasing HSP72 in human skeletal muscle, it does so only when glycogen is reduced to relatively low levels. Hence, our data suggest that HSP72 protein expression is related to glycogen availability. In addition, because CK did not increase and we found no evidence of HSP72 in the venous effluent, our data suggest that skeletal muscle is impermeable to HSP72.

title: Elevated levels of circulating heat shock protein 70 (Hsp70) in peripheral and renal vascular disease
abstract: Heat shock proteins (Hsp) are families of phylogenetically conserved molecules that have a range of cytoprotective and intracellular functional roles. Reactivity to heat shock proteins has been implicated in the development of autoimmune disease and tissue expression of heat shock proteins and increased levels of anti-Hsp antibodies have also been reported in vascular disease. This study compared circulating levels of Hsp60 and Hsp70 and antihuman Hsp60, antihuman Hsp70, and antimycobacterial Hsp65 antibodies in peripheral (PVD) and renal (RVD) vascular disease with those in age- and sex-matched controls. Levels of Hsp70 were higher in both PVD (median 580 vs 40; P < 0.01) and RVD (median 160 vs 0; P < 0.03), whereas there were no differences in Hsp60 levels. Anti-Hsp60 antibody levels were elevated in PVD (146 vs 81 arbitrary units/ml; P < 0.04), but not RVD. This is the first study to demonstrate increased levels of circulating Hsp70 in pathological disease states; however, its physiological role remains to be determined.

title: Circulating heat shock protein and heat shock protein antibody levels in established hypertension
abstract: Serum Hsp60 and anti-Hsp65 antibody levels are raised in subjects with borderline hypertension, and there is an association between circulating Hsp60 levels and early atherosclerosis. Given the recognized relationship between hypertension and atherosclerosis, this study determined heat shock protein and heat shock protein antibody levels in subjects with established hypertension.  Samples from 111 men with hypertension were obtained from the European Lacidipine study on Atherosclerosis and samples from 75 normotensive controls were taken from a population-screening programme (diastolic pressure, > 95 and < 80 mmHg, respectively). Hsp60, Hsp70 and anti-human Hsp60, anti-human Hsp70 and anti-mycobacterial Hsp65 antibody levels were measured by enzyme immunoassay. Intima–media thickness (I–M) and the presence of carotid atherosclerosis were determined by ultrasonography. Results Hsp60, Hsp70 and anti-Hsp60 antibody levels in hypertension were similar to those in normotensive controls, whereas anti-Hsp70 and anti-Hsp65 antibody levels were elevated (P < 0.001). Hsp60 levels and atherosclerosis were not associated. Anti-Hsp70 and anti-Hsp65 antibody levels were both associated with hypertension, independently of age, smoking habits and blood lipids.  This study demonstrates elevated levels of selected heat shock protein antibodies in subjects with hypertension. Although the association between heat shock protein antibody levels and human cardiovascular stress/disease appears to be robust, the relationship of the latter with heat shock protein levels is more complex. Further studies are required before the factors inducing, and the clinical significance of, circulating heat shock proteins can be evaluated.

title: Serum Heat Shock Protein 70 Levels Predict the Development of Atherosclerosis in Subjects With Established Hypertension
abstract: Although heat shock proteins (Hsp’s) are present in the sera of healthy individuals and at elevated levels in subjects with early cardiovascular disease, their physiologic role in and value for predicting the development and/or progression of atherosclerosis have not been evaluated. Serum was obtained from 218 subjects with established hypertension (diastolic pressure >95 mm Hg) before their enrollment in the European Lacidipine Study on Atherosclerosis. Hsp60 and Hsp70, and anti-human Hsp60, anti-human Hsp70, and anti-mycobacterial Hsp65 antibody levels were measured by enzyme immunoassay. As an indicator of the presence/progression of atherosclerosis, the means of the maximum intima-media (I-M) thicknesses in the far walls of common carotid arteries and bifurcations (CBMmax) were determined by ultrasonography at the time of enrollment and 4 years afterward. Increases in I-M thicknesses at follow-up were less prevalent in subjects having high serum Hsp70 levels (75th percentile) at the time of enrollment (odds ratio, 0.42; 95% confidence interval [CI], 0.22 to 0.8, P =0.008). Although a similar trend was observed for serum Hsp60 levels, this was not statistically significant (odds ratio, 0.6; 95% CI, 0.32 to 1.11, P =0.10). There was no relation between anti-Hsp antibody levels and changes in I-M thicknesses. The relation between Hsp70 levels and changes in I-M thickness was independent of age, atenolol or lacidipine treatment, smoking habits, and blood lipid levels. These findings indicate that circulating Hsp70 levels predict the development of atherosclerosis in subjects with established hypertension, and an intriguing possibility is that Hsp70 protects against or modifies the progression of atherosclerosis in this subject group.

title: Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system.
abstract: Extracellular heat-shock proteins (eHsp) such as those belonging to the 70-kDa family of Hsp (eg, Hsp72) have been hypothesized to act as a "danger signal" to immune cells, promote immune responses, and improve host defense. The current study tested this hypothesis. Adult male F344 rats were exposed to an acute laboratory stressor (100, 5-second, 1.6-mA inescapable tail shocks) and challenged with Escherichia coli. The number of colony-forming units (CFU) of bacteria at the site of injection, the levels of eHsp72, the immune response to eHsp72 and E. coli-derived lipopolysaccharide (LPS), and the amount of time required to recover from in vivo bacterial challenge were measured. CFUs were reduced 2, 4, and 6 hours after injection of E. coli in rats exposed to stress. Rats exposed to stress had elevated eHsp72 that was elevated rapidly (25 minutes) and remained elevated in the circulation and at the inflammatory site (2 hours after stressor termination). Both stressor exposure and eHsp72 administration in the absence of stress resulted in a facilitated pattern of recovery after bacterial inflammation induced by subcutaneous E. coli injection. Rats exposed to acute restraint (100 minutes) did not demonstrate elevated circulating eHsp72 or a facilitated pattern of recovery after bacterial challenge. In vitro stimulation of rat splenocytes and macrophages with eHsp72 elevated nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, and this effect was specific to eHsp72 because it was not diminished by polymyxin B and was reduced by earlier heat-denature treatment. Stimulation of cells with eHsp72 combined with LPS resulted in a greater NO and cytokine response than that observed after stimulation with eHsp72 or LPS alone. In vivo, at the inflammatory site, the bacterial-induced NO response was potentiated by stress, and NO inhibition (L-NIO) reduced the stress-induced facilitation but had no effect on the control kinetics of bacterial inflammation recovery. Thus, these results lend support to the hypothesis that intense stressor exposure increases eHsp72, which acts as a danger signal to potentiate the NO response to bacterial challenge and facilitate recovery from bacterial inflammation.

title: Exercise increases serum Hsp72 in humans
abstract: Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87 ± 0.24 and 1.02 ± 0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

title: Exercise induces hepatosplanchnic release of heat shock protein 72 in humans
abstract: Physical exercise results in the appearance of heat shock protein (HSP) 72 in the circulation that precedes any increase in gene or protein expression in contracting skeletal muscle. In rodents, exercise increases liver HSP72 expression and the hepatosplanchnic viscera are known to release many acute phase proteins. In the present study, we tested the hypothesis that the splanchnic tissue beds release HSP72 during exercise. Seven male subjects performed 120 min of semi‐recumbent cycling at 62 ± 2 % of maximal oxygen uptake. Blood samples were obtained simultaneously from a brachial artery, a femoral vein and the hepatic vein prior to and at 30, 60 and 120 min of exercise. Leg blood flow (LBF) was measured by thermodilution in the femoral vein, and hepatosplanchnic blood flow (HBL) was measured using indocyanine green dye. Net leg and net hepatosplanchnic HSP72 balance were calculated as the product of LBF and femoral venous‐arterial HSP72 difference and the product of HBF and hepatic venous‐arterial HSP72 difference, respectively. Arterial plasma HSP72 was only detected in one subject at rest but progressively appeared in the arterial samples throughout exercise such that at 120 min it was detected in all subjects (0.88 ± 0.35 pg l−1; P < 0.05 compared with rest). The contracting muscle did not, however, contribute to this increase since there was no difference in the femoral venous‐arterial HSP72 concentration at any time. Rather, the increase in arterial HSP72 was accounted for, at least in part, by release from the hepatosplanchnic viscera with values increasing (P < 0.05) from undetectable levels at rest to 5.2 ± 0.2 pg min−1 after 120 min. These data demonstrate that the splanchnic tissues release HSP72 during exercise and this release is responsible, in part, for the elevated systemic concentration of this protein during exercise.

title: Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells 1
abstract: The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407–426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457–496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457–496 but enhanced by p407–426.

title: Heat shock proteins as regulators of the immune response
abstract: Until recently, heat shock proteins (also known as heat stress proteins) have mostly been regarded as intracellular molecules that mediate a range of essential housekeeping and cytoprotective functions. However, interest in their role as intercellular signalling molecules has been fuelled by the observations that these molecules can be released and are present in the extracellular environment under physiological conditions. They can elicit cytokine production by, and adhesion molecule expression of, a range of cell types, and they can deliver maturation signals and peptides to antigen presenting cells through receptor-mediated interactions. These functions suggest that heat shock proteins could be immunoregulatory agents with potent and widely-applicable therapeutic uses. Furthermore, the induction of self heat shock protein immune reactivity can attenuate autoimmunity and delay transplant rejection, and heat shock proteins derived from tumours and pathogens can elicit specific, protective immunity. This review will focus on this rapidly evolving area of heat shock protein biology.

title: Natural adjuvants: Endogenous activators of dendritic cells
abstract: Dendritic cells, the most potent antigen-presenting cells, need to be activated before they can function to initiate an immune response. We report here that, in the absence of any foreign substances, dendritic cells can be activated by endogenous signals received from cells that are stressed, virally infected or killed necrotically, but not by healthy cells or those dying apoptotically. Injected in vivo with an antigen, the endogenous activating substances can function as natural adjuvants to stimulate a primary immune response, and they may represent the natural initiators of transplant rejection, spontaneous tumor rejection, and some forms of autoimmunity.

title: Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway.
abstract: Dendritic cells (DC) are key components of innate and adaptive immune responses. The identity of endogenous signals that activate DC is a crucial and unresolved question. We report here that heat shock proteins (HSP), the most abundant and conserved mammalian molecules, constitute such an internal signal. Necrotic but not apoptotic cell death leads to release of HSP gp96, calreticulin, hsp90 and hsp70. HSP stimulate macrophages to secrete cytokines, and induce expression of antigen-presenting and co-stimulatory molecules on the DC. The HSP gp96 and hsp70 act differentially, and each induces some but not all molecules. HSP interact with these antigen-presenting cells through the highly conserved NF-kappa B pathway. As HSP are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen-presenting cells (APC) constitutes an excellent mechanism for response to cell death. As HSP are conserved from bacteria to mammals, the ability of HSP to activate APC provides a unified mechanism for response to internal and external stimuli.

title: Consequences of Cell Death
abstract: Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4+ and CD8+ T cells. Cross-presentation of antigens to CD8+ T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.

title: Virally Induced Lytic Cell Death Elicits the Release of Immunogenic GRP94/gp96*
abstract: Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8+ T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVAMet258–265, a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVAMet258–265-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

title: In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance
abstract: Glial cells release a variety of molecules that support neuronal function. Because heat shock proteins (Hsps) are important in the survival of neurons subjected to metabolic stress, the possibility that glia can release the inducible form of the 70 kDa Hsp (Hsp70) was examined. Additionally, the ability of neuronal cells to show increased stress tolerance by taking up a mixture of constitutive and inducible forms of Hsp70 (Hsc/Hsp70) added to the extracellular fluid was tested. Human T98G glioma cells and differentiated LA-N-5 neuroblastoma cells were used as model glia and neurons to investigate these points. Hsp70 was analyzed using affinity chromatography, Western blotting, and immunofluorescence microscopy. The glioma cells were shown to export Hsp70 into the culture medium whether under normal conditions or subjected to heat shock. The amount of glial Hsp70 released ranged from 5 to 15 pg per 10(6) cells per day, being greater following heat shock. Neuroblastoma cells took up biotinylated Hsc/Hsp70 within 1 h after it was added to the culture medium and it made them more resistant to heat shock (44 degrees C) and to staurosporine-induced apoptosis. This increased stress tolerance was especially important in neuroblastoma cells induced to differentiate with phorbol ester because those 'mature neurons' showed a 10-fold decline in endogenous Hsp70, which was accompanied by increased susceptibility to heat shock and staurosporine-induced apoptosis. These results suggest that extracellular Hsp70 may provide a means by which glia can affect neuronal function, perhaps enhancing neuronal stress tolerance.

title: Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.
abstract: Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

title: Exosome-dependent Trafficking of HSP70
abstract: The heat shock proteins (HSPs) are a family of intracellular proteins found in all eukaryotes and prokaryotes. Their functions are well characterized and are central to maintaining cellular homeostasis and in promoting cell survival in response to stressful cellular conditions. However, several studies provide evidence that specific members of the HSP family might be secreted via an unidentified exocytotic pathway. Here we show that exosomes, small membrane vesicles that are secreted by numerous cell types, contribute to the release of HSP70 from human peripheral blood mononuclear cells (PBMCs) in both basal and stress-induced (heat shock at 40 or 43 °C for 1 h) states. HSP70 release from PBMCs is independent of the common secretory pathway because Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP70 release. Furthermore, we show that HSP70 release from PBMCs does not occur via a lipid raft-dependent pathway, because treatment with methyl-β-cyclodextrin, a raft-disrupting drug, had no affect on HSP70 release. To examine whether exosomes contributed to HSP70 release from PBMCs, exosomes were purified from PBMC cultures, and exosomal number and HSP70 content were determined. We demonstrate that although heat shock does not influence the exosomal secretory rate, the HSP70 content of exosomes isolated from heat shocked PBMCs is significantly higher than control. These data identify a novel secretory pathway by which HSP70 can be actively released from cells in both the basal and stress-induced state.

title: Serum levels of Hsp 72 measured early after trauma correlate with survival.
abstract: Experimental studies have shown that hemorrhagic shock is associated with the expression of inducible heat proteins, especially heat shock protein (Hsp) 72, in liver, brain, heart, and kidney. Moreover, induction of Hsp 72 by various stressors before the onset of shock has been associated with the attenuation of organ injury caused by hemorrhage. However, it is not known whether Hsp 72 is expressed after severe trauma in humans. The purpose of this study was therefore to determine whether Hsp 72 could be detected in the serum of patients early after severe trauma and whether serum levels of Hsp 72 might correlate with survival of trauma patients or the severity of the postinjury inflammatory response.



Clinical data were collected prospectively over a 3-year period for trauma patients mechanically ventilated for more than 2 days who met the following inclusion criteria: Injury Severity Score > or = 16, and age > 18 years. Physiologic data for quantitative assessment of organ dysfunction were collected for each patient. Hsp 72 and nitrate and nitrite levels were measured in the serum of trauma patients collected at or 12 to 48 hours after the admission to the emergency department.



Sixty-seven patients with severe trauma were enrolled in the study. Hsp 72 was detected in the serum of all trauma patients. All patients with high initial serum levels of Hsp 72 (serum levels > 15 ng/mL) survived, whereas 29% of the patients with low Hsp 72 serum levels died from their traumatic injuries (p = 0.01). The overall mortality was 21%, occurring within 5 to 7 days. Patients who died were older (mean age, 54 +/- 15 years) than those who survived (mean age, 36 +/- 15 years) (p < 0.0.05). The cause of death was attributable to head injury in 79%, although the severity of head injury (Abbreviated Injury Scale score) was not statistically different between survivors with high serum values of Hsp 72 and patients who died. There was no correlation between the initial serum Hsp 72 values and the severity of organ dysfunction or clinical indicators of the inflammatory response.



Hsp 72 can be detected in the serum of severely traumatized patients within 30 minutes after injury. Elevated initial serum levels of Hsp 72 (serum levels > 15 ng/mL) are associated with survival after severe trauma, but are not related to the incidence or severity of the postinjury inflammatory response or organ dysfunction.

title: Endogenous extra-cellular heat shock protein 72: Releasing signal(s) and function
abstract: Exposure to acute physical and/or psychological stressors induces a cascade of physiological changes collectively termed the stress response. The stress response is demonstrable at the behavioural, neural, endocrine and cellular levels. Stimulation of the stress response functions to improve an organism's chance of survival during acute stressor challenge. The current review focuses on one ubiquitous cellular stress response, up-regulation of heat shock protein 72 (Hsp72). Although a great deal is known about the function of intra-cellular Hsp72 during exposure to acute stressors, little is understood about the potential function of endogenous extra-cellular Hsp72 (eHsp72). The current review will develop the hypothesis that eHsp72 release may be a previously unrecognized feature of the acute stress response and may function as an endogenous ‘danger signal’ for the immune system. Specifically, it is proposed that exposure to physical or psychological acute stressors stimulate the release of endogenous eHsp72 into the blood via an α1-adrenergic receptor-mediated mechanism and that elevated eHsp72 functions to facilitate innate immunity in the presence of bacterial challenge.

title: Adrenergic receptors mediate stress-induced elevations in extracellular Hsp72.
abstract: Heat-shock protein concentrations in the blood increase after exposure to a variety of stressors, including trauma and psychological stress. Although the physiological function of extracellular heat shock protein remains controversial, there is evidence that extracellular heat shock protein 72 (Hsp72) can facilitate immunologic responses. The signal(s) that mediate(s) the in vivo elevation of extracellular Hsp72 in the blood after stressor exposure remain(s) unknown. Here we report that Hsp72 increases in the circulation via an alpha1-adrenergic receptor-mediated signaling pathway. Activation of alpha1-adrenoceptors results in a rapid increase in circulating Hsp72, and blockade of alpha1-adrenoceptors prevents the stress-induced rise in circulating Hsp72. Furthermore, our studies exclude a role for beta-adrenoceptors, glucocorticoids, and ACTH in mediating stress-induced elevations in circulating extracellular Hsp72. Understanding the signals involved in elevating extracellular Hsp72 could facilitate the use of extracellular Hsp72 to bolster immunity and perhaps prevent exacerbation of inflammatory diseases during stress.

title: Effect of adrenal demedullation on urinary excretion of catecholamines in thermal trauma in rats.
abstract: The urinary excretions of adrenaline and noradrenaline were determined before and after a moderate thermal trauma in rats with intact adrenal glands, after adrenal demedullation and after adrenalectomy with grafting of adrenal cortical tissue. The basal excretions of adrenaline were 0.28 mug, 0.06 mug and 0.04 mug/24 h/rat, respectively. In rats with intact adrenal glands the adrenaline excretion was greatly increased already in the first 24 hours after the burn. In both the demedullated and adrenalectomized rats only a very slight increase of the adrenaline excretion was noted, but this may indicate a slight reaction of the extramedullary chromaffin tissue to the trauma. The basal excretion of noradrenaline was not affected either by demedullation or adrenalectomy, indicating that it consisted mainly in noradrenaline released from peripheral sympathetic nerve endings. From the second day after the burn there was an increase in the noradrenaline excretion, of equal magnitude in all experimental groups, indicating an increased liberation of peripheral adrenergic transmittor substances.

title: The role of the adrenal medulla in neural control of blood pressure in rats.
abstract: The role of the adrenal medulla in the regulation of blood pressure was assessed in rats. In conscious intact (SHAM) and adrenal demedullated (DEMED) animals, baseline blood pressures and heart rates were not different. Moreover, equivalent pressor and depressor response curves to norepinephrine and sodium nitroprusside were obtained. Plasma concentration and 24 hr urinary excretion of norepinephrine were not altered by demedullation. However, epinephrine was undetectable in the urine of the DEMED rats. The effectiveness of the demedullation was further evidenced by a greater than 99% decrease in the epinephrine and norepinephrine content of the adrenal glands of DEMED animals. In pithed SHAM rats, electrical stimulation of preganglionic sympathetic neurons (.5 to 16 hz) elicited marked increases in plasma epinephrine indicating adrenal stimulation, whereas in DEMED rats there were no increases. Furthermore, increments in plasma norepinephrine were significantly reduced in DEMED rats, yet the blood pressure increases were greater than in SHAM rats. Thus, adrenal catecholamine release did not contribute to the blood pressure responses, but in fact, may have had an opposite action due to the vasodilatory actions of epinephrine. The results demonstrate that the adrenal medulla is not essential for reflex compensation to a hypotensive challenge or to the blood pressure increases produced to direct neural stimulation.

title: Alternative Mechanism by which IFN-γ Enhances Tumor Recognition: Active Release of Heat Shock Protein 721
abstract: IFN-γ exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-γ enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-γ triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-γ-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl β-cyclodextrin completely abrogated IFN-γ-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-γ promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.

title: The Biogenesis and Functions of Exosomes
abstract: Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. They correspond to the internal vesicles of an endosomal compartment, the multivesicular body and are released upon exocytic fusion of this organelle with the plasma membrane. Intracellularly, they are formed by inward budding of the endosomal membrane in a process that sequesters particular proteins and lipids. The unique composition of exosomes may confer specific functions on them upon secretion. Although their physiological role in vivo is far from being unraveled, it is apparent that they function in a multitude of processes, including intercellular communication during the immune response. Exosomes may have evolved early in the evolution of multicellular organisms and also seem to be important for tissue developmental processes.

title: Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses.
abstract: Heat shock proteins are abundant soluble intracellular proteins, present in all cells. Members of the heat shock protein family bind peptides including antigenic peptides generated within cells. Heat shock proteins also interact with antigen presenting cells through CD91 and other receptors, eliciting a cascade of events including re-presentation of heat shock protein-chaperoned peptides by MHC, translocation of NF kappa B into the nuclei and maturation of dendritic cells. These consequences point to a key role of heat shock proteins in fundamental immunological phenomena such as activation of antigen presenting cells, indirect presentation (or cross-priming), and chaperoning of peptides during antigen presentation. Heat shock proteins appear to have been involved in innate immune responses since the emergence of phagocytes in early multicellular organisms and to have been commandeered for adaptive immune responses with the advent of specificity. These properties of heat shock proteins also allow them to be used for immunotherapy of cancers and infections in novel ways.

title: Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.
abstract: We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

title: Selective Enrichment of Tetraspan Proteins on the Internal Vesicles of Multivesicular Endosomes and on Exosomes Secreted by Human B-lymphocytes*
abstract: Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50–60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.

title: Selective release from cultured mammalian cells of heat‐shock (stress) proteins that resemble glia‐axon transfer proteins
abstract: Cultured rat embryo cells were stimulated to rapidly release a small group of proteins that included several heat‐shock proteins (hsp110, hsp71, hscp73) and nonmuscle actin. The extracellular proteins were analyzed by two‐dimensional polyacrylamide gel electrophoresis. Heat‐shocked cells released the same set of proteins as control cells with the addition of the stress‐inducible hspllO and hsp71. Release of these proteins was not blocked by either monensin or colchicine, inhibitors ofthe common secretory pathway. A small amount of the glucose‐regulated protein grp78 was externalized by this pathway. The extracel‐lular accumulation of these proteins was inhibited after they were synthesized in the presence of the lysine analogue aminoethyl cysteine. It is likely that the analogue‐substituted proteins were misfolded and could not be released from cells, supporting our conclusion that a selective release mechanism is involved. Remarkably, actin and the squid heat‐shock proteins homologous to rat hsp71 and hsp110 are also among a select group of proteins transferred from glial cells to the squid giant axon, where they have been implicated in neuronal stress responses (Tytell et al.:Brain Res., 363:161‐164, 1986). Based in part on the similarities between these two sets of proteins, we hypothesized that these proteins were released from labile cortical regions of animal cells in response to perturbations of homeostasis in cells as evolutionarily distinct as cultured rat embryo cells and squid glial cells.

title: Expression of the Molecular Chaperone Hsp70 in Detergent-resistant Microdomains Correlates with Its Membrane Delivery and Release*
abstract: Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-β-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.

title: Recent advances in the molecular pharmacology of the α1-adrenergic receptors
abstract: This review is intended to discuss recent developments in the molecular pharmacology of the α 1 -adrenergic receptor ( α 1 -AR) subtypes. After a brief historical development will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the α 1 -AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the α 1 -AR subtypes in regulating physiologic processes.

title: Exosome Release Is Regulated by a Calcium-dependent Mechanism in K562 Cells*
abstract: Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells.

title: Noradrenaline and α-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells
abstract: Expression of heat shock proteins (hsp) is a homeostatic mechanism induced in both prokaryotic and eukaryotic cells in response to metabolic and environmental insults. A growing body of evidence suggests that in mammals, the hsp response is integrated with physiological responses through neuroendocrine signaling. In the present study, we have examined the effect of noradrenaline (NA) on the hsp70 response in mollusc immune cells. Oyster and abalone hemocytes transfected with a gene construct containing a gastropod hsp70 gene promoter linked to the luciferase reporter-gene were exposed to physiological concentrations of NA, or to various α- and β-adrenoceptor agonists and antagonists. Results show that NA and α-adrenergic stimulations induced the expression of luciferase in transfected mollusc immunocytes. Furthermore, exposure of hemocytes to NA or to the α-adrenoceptor agonist phenylephrine (PE) resulted in the expression of the inducible isoform of the hsp70 protein. Pertussis toxin (PTX), the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor calphostin C, the Ca 2+ -dependent PKC inhibitor Go 6976 and the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 blocked the PE-mediated induction of the hsp70 gene promoter. These results suggest that α-adrenergic signaling induces the transcriptionnal upregulation of hsp70 in mollusc hemocytes through a PTX-sensitive G-protein, PLC, Ca 2+ -dependent PKC and PI 3-kinase. Thus, a functional link exists between neuroendocrine signaling and the hsp70 response in mollusc immune cells.

title: Norepinephrine induces cardiac heat shock protein 70 and delayed cardioprotection in the rat through alpha 1 adrenoceptors.
abstract: Previous studies have demonstrated that norepinephrine (NE) confers immediate cardioprotection against ischemia and reperfusion injuries. The present study tests the hypothesis that in vivo treatment with NE induces delayed cardioprotection against postischemic dysfunction in the rat heart which is associated with expression of c-fos and heat shock protein (HSP) 70 in the myocardium.



Rats were treated with NE (3.1 mumol/kg, ip) and hearts isolated and perfused with a modified Langendorff technique 2 or 24 h after injection. Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded during 25 min normothermic global ischemia and 40 min reperfusion. Total RNA was extracted from left ventricular tissue at various time points and Northern hybridization was applied to detect c-fos and HSP70 mRNAs. Expression and distribution of c-fos and HSP72 proteins in the myocardium were examined by immunohistochemical staining.



In vivo NE treatment improved postischemic recovery of both LVDP and LVEDP in the hearts isolated at 24 h after treatment but not in those isolated at 2 h. LVDP was 68.9 +/- 4.8 mmHg in NE 24 h group at the end of reperfusion in comparison to 43.6 +/- 2.9 mmHg in saline control group (P < 0.01). Pretreatment with prazosin abolished NE-induced cardioprotection while propranolol pretreatment had no effect. Northern analysis demonstrated rapid and transient increases in c-fos and HSP70 mRNAs in the myocardium after NE treatment. Accumulation of c-fos protein was observed at 3 h and increased amount of HSP72 protein was demonstrated at 24 h in the myocardium. Pretreatment of rats with prazosin eliminated NE-induced increase in cardiac HSP70 mRNA.



The results suggest that in vivo treatment with NE upregulates the expression of c-fos and HSP70 in the myocardium and induces delayed protection against postischemic myocardial dysfunction in the isolated rat heart. Induction of both the expression of cardiac HSP70 and the delayed cardioprotection by NE appears to be mediated by alpha 1 adrenoceptors.

title: Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of natural killer cells.
abstract: We previously demonstrated that lysis of tumor cells that express Hsp70, the highly stress-inducible member of the HSP70 family, on their plasma membrane is mediated by natural killer (NK) cells. Here, we studied the effects of different proteins of the HSP70 family in combination with interleukin 2 (IL-2) on the proliferation and cytotoxic activity of human NK cells in vitro. Proliferation of NK cells was significantly enhanced by human recombinant Hsp70 (rHsp70) and to a lesser extent by rHsp70homC, the recombinant C-terminal peptide-binding domain derived from Hsp70hom, but not by the constitutive Hsc70 or DnaK, the Escherichia coli analogue of human Hsp70. Even rHsp70 protein alone moderately enhances proliferation and cytolytic activity of NK cells, thus indicating that the stimulatory effect is not strictly dependent on IL-2. NK cells stimulated with rHsp70 protein also exhibit an increased secretion of interferon gamma (IFN-gamma). The phenotypic characterization of NK cells with specificity for Hsp70-expressing tumor cells revealed a CD16dim/CD56bright and increased CD57 and CD94 expression. The cytolytic activity of NK cells also was significantly reduced when a CD94-specific antibody or rHsp70 was added directly before the cytotoxicity assay, whereas other antibodies directed against CD57 and major histocompatibility complex class I molecules or Hsp70 proteins, including Hsc70 and DnaK, did not affect the NK-mediated killing. However, long-term incubation of NK cells with rHsp70 protein enhances not only the proliferative but also the cytolytic response against Hsp70-expressing tumor cells. Our results indicate that the C-terminal domain of Hsp70 protein affects not only the proliferative but also the cytolytic activity of a phenotypically distinct NK cell population with specificity for Hsp70-expressing tumor cells. 1999 International Society for Experimental Hematology.

title: Heat shock proteins as "danger signals": eukaryotic Hsp60 enhances and accelerates antigen‐specific IFN‐γ  production in T cells
abstract: The heat shock proteins (HSP) gp96, Hsp70 and Hsp60 activate professional antigen‐presenting cells (APC) to secrete proinflammatory cytokines and to express costimulatory molecules. Here, we analyze the impact of Hsp60 as a hypothetical danger signal on the antigen‐specific activation of T cells derived from DO11.10 TCR‐transgenic mice. The release of IFN‐γ, induced by the antigenic OVA323–339‐peptide, is increased and accelerated dramatically by the addition of Hsp60 to ex vivo purified populations of T cells and peritoneal macrophages (PEC), while the antigen‐specific IL‐2 production or proliferation of the T cells remain unchanged. In contrast, "effector" T cells, undergoing secondary stimulation, displayed almost unchanged activation kinetics in the presence of Hsp60. The presence of Hsp60 induces IFN‐γ and up‐regulation of CD69 in T cell/PEC cocultures even in the absence of antigenic peptide and this induction of IFN‐γ is strictly dependent on the ability of the macrophages to produce IL‐12. Taken together, our data strongly suggest that the presence of eukaryotic mitochondrial Hsp60 allows antigen‐specific IFN‐γ secretion under conditions when an antigenic stimulus alone is not sufficient to activate T cells.

title: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine
abstract: Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-κB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1β, IL-6 and TNF-α; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-α but not IL-1β or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

title: Heat Shock Proteins gp96 and hsp70 Activate the Release of Nitric Oxide by APCs1
abstract: NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-γ; stimulation of macrophages with mixtures of IFN-γ and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.

title: Heat shock protein 70 is a potent activator of the human complement system
abstract: According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

title: Toll-like receptors: critical proteins linking innate and acquired immunity
abstract: Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R–associated protein kinase and tumor necrosis factor receptor–activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.

title: HSP70 as Endogenous Stimulus of the Toll/Interleukin-1 Receptor Signal Pathway*
abstract: Human heat-shock protein (HSP)70 activates innate immune cells and hence requires no additional adjuvants to render bound peptides immunogenic. Here we tested the assumption that endogenous HSP70 activates the Toll/IL-1 receptor signal pathway similar to HSP60 and pathogen-derived molecular patterns. We show that HSP70 induces interleukin-12 (IL-12) and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) promoters in macrophages and that this is controlled by MyD88 and TRAF6. Furthermore, HSP70 causes MyD88 relocalization and MyD88-deficient dendritic cells do not respond to HSP70 with proinflammatory cytokine production. Using the system of genetic complementation with Toll-like receptors (TLR) we found that TLR2 and TLR4 confer responsiveness to HSP70 in 293T fibroblasts. The expanding list of endogenous ligands able to activate the ancient Toll/IL-1 receptor signal pathway is in line with the “danger hypothesis” proposing that the innate immune system senses danger signals even if they originate from self.

title: Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide
abstract: To determine the role of intracellular calcium ([Ca2+]i) in the priming of monocytes (MΦ) by bacterial lipopolysaccharide (LPS), the membrane expression of two functional proteins and phagocytosis and respiratory burst were examined by microfluorimetry. LPS induced a significant increase in HLA-DR and C3bi receptor (CR3) expression within 2 h of its addition to whole blood. The enhanced expression of both antigens by LPS was dose-dependent, with concentrations as low as 0.1 ng/ ml producing a response. The involvement of [Ca2+]i was demonstrated by loading isolated MΦ with the intracellular calcium chelator quin-2 or the inhibitor of intracellular calcium redistribution TMB-8 prior to addition of LPS. Both compounds inhibited the LPS-induced increase in HLA-DR and CR3 expression. No role for extracellular calcium, for calcium slow channel flux, or for the calcium-calmodulin complex in LPS priming was demonstrated when LPS was added in the presence of EGTA, trifluperazine (TFP), or verapamil. The addition of the calcium ionophores A23187 or ionomycin failed to increase expression of either antigen. Prior exposure to LPS primed MΦ for enhanced phagocytosis and respiratory burst activity. These functions were inhibited by TMB-8, but not by TFP or verapamil. Addition of LPS to isolated MΦ increased [Ca2+]i by 23% at 30 sec and 42% at 5 min, as measured by the calcium-sensitive, intracellular probe indo-1. These results suggest that intracellular Ca2+ mobilization is necessary, but not sufficient, for LPS-induced priming of human peripheral blood monocytes.

title: Novel Signal Transduction Pathway Utilized by Extracellular HSP70
abstract: Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca2+ flux, activate NF-κB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-κB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).

title: HSP70 peptide-bearing and peptide-negative preparations act as chaperokines
abstract: We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-κB activity and subsequent augmentation of proinflammatory cytokine TNF-α, IL-1β, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

title: Functional domains of HSP70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity.
abstract: Microbial HSP70 (heat-shock protein 70) consists of three functionally distinct domains: an N-terminal 44 kDa ATPase portion (amino acids 1-358), followed by an 18 kDa peptide-binding domain (amino acids 359-494) and a C-terminal 10 kDa fragment (amino acids 495-609). Immunological functions of these three different domains in stimulating monocytes and dendritic cells have not been fully defined. However, the C-terminal portion (amino acids 359-610) stimulates the production of CC chemokines, IL-12 (interleukin-12), TNFalpha(tumour necrosis factor alpha), NO and maturation of dendritic cells and also functions as an adjuvant in the induction of immune responses. In contrast, the ATPase domain of microbial HSP70 mostly lacks these functions. Since the receptor for HSP70 is CD40, which with its CD40 ligand constitutes a major co-stimulatory pathway in the interaction between antigen-presenting cells and T-cells, HSP70 may function as an alternative ligand to CD40L. HSP70-CD40 interaction has been demonstrated in non-human primates to play a role in HIV infection, in protection against Mycobacterium tuberculosis and in conversion of tolerance to immunity.

title: Stimulation of Th1-Polarizing Cytokines, C-C Chemokines, Maturation of Dendritic Cells, and Adjuvant Function by the Peptide Binding Fragment of Heat Shock Protein 701
abstract: The peptide binding C-terminal portion of heat shock protein (HSP)70 (aa 359–610) stimulates human monocytes to produce IL-12, TNF-α, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP701–358) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70359–610 stimulation of chemokine production is mediated by the CD40 costimulatory molecule. Maturation of dendritic cells was induced by stimulation with native HSP70, was not seen with the N-terminal HSP701–358, but was enhanced with HSP70359–610, as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70359–610 enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70359–610 in mice induced higher serum IgG2a and IgG3 Abs than the native HSP70-bound peptide. This study suggests that the C-terminal, peptide-binding portion of HSP70 is responsible for stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant function.

title: Heat shock proteins generate β‐chemokines which function as innate adjuvants enhancing adaptive immunity
abstract: Heat shock proteins (HSP) are widely distributed and highly immunogenic molecules. A novel property reported here is that stimulation with HSP70 of CD8‐enriched T cells derived from naive non‐human primates caused a dose‐dependent increase in concentrations of the β‐chemokines RANTES, macrophage inflammatory protein (MIP)‐1α or MIP‐1β. However, the concentrations of these β‐chemokines were greatly increased when the CD8 T cells derived from HSP70‐immunized non‐human primates were stimulated with HSP70. HSP linked to peptides or proteins combined generation of β‐chemokines with an adjuvant function by enhancing specific T cell proliferative responses and IgG and IgA antibodies. The β‐chemokine and adjuvant functions were also elicited by topical mucosal administration of HSP linked to an antigen. We postulate that microbial HSP can stimulate β‐chemokine production which may be responsible for innate adjuvanticity, as was found in cells eluted from normal rectal mucosal tissue, and constitutes a significant component of the mucosal‐associated lymphoid system. Furthermore, stimulation of innate immunity may drive adaptive immunity and account for the protective effects of HSP against tumors and viruses.

title: CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC-chemokines.
abstract: The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.

title: Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages*
abstract: Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor α (TNFα) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70–1) with a LPS content of 1.4 pg/μg was unable to induce TNFα release by RAW264.7 murine macrophages at concentrations up to 5 μg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70–2) at 1 μg/ml with a LPS content of 0.2 ng/μg was able to induce TNFα release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70–1 to induce TNFα release was not because of defective physical properties since rhHsp70–1 and rhHsp70–2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70–2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFα-inducing activity of rhHsp70–2. The addition of LPS at the concentration found in rhHsp70–2 to rhHsp70–1 resulted in the same TNFα-inducing activity as observed with rhHsp70–2. The TNFα-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70–1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFα release from murine macrophages and that the observed TNFα-inducing activity in the rhHsp70–2 preparation is entirely due to the contaminating LPS.

title: Recombinant Human Heat Shock Protein 60 Does Not Induce the Release of Tumor Necrosis Factor α from Murine Macrophages*
abstract: Recent studies have shown that commercially available recombinant human heat shock protein 60 (rhHSP60) could induce tumor necrosis factor α (TNF-α) release from macrophages and monocytes in a manner similar to that of lipopolysaccharide (LPS), e.g. via CD14 and Toll-like receptor 4 complex-mediated signal transduction pathway. In this study, we demonstrated that a highly purified rhHSP60 preparation with low endotoxin activity (designated rhHSP60-1) was unable to induce TNF-α release from murine macrophages at concentrations of up to 10 μg/ml. In contrast, a less purified rhHSP60 preparation (designated rhHSP60-2) was able to induce a marked TNF-α release at concentrations as low as 1 μg/ml. Failure of rhHSP60-1 to induce TNF-α release was not due to defective physical properties because rhHSP60-1 and rhHSP60-2 contained a similar amount of HSP60 as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHSP60 antibody. Both rhHSP60 preparations also had similar enzymatic activities as judged by their ability to hydrolyze ATP. Polymyxin B added in the incubation media abolished the endotoxin activity but inhibited only about 50% of the TNF-α-inducing activity of rhHSP60-2. However, both the endotoxin activity and the TNF-α-inducing activity of rhHSP60-2 were essentially eliminated after passing through a polymyxin B-agarose column that removes LPS and LPS-associated molecules from the rhHSP60 preparation. The TNF-α-inducing activities of both rhHSP60-2 and LPS with equivalent endotoxin activity present in rhHSP60-2 were equally sensitive to heat inactivation. These results suggest that rhHSP60 does not induce TNF-α release from macrophages. Approximately 50% of the observed TNF-α-inducing activity in the rhHSP60-2 preparation is due to LPS contamination, whereas the rest of the activity was due to the contamination of LPS-associated molecule(s).

title: Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages*
abstract: Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor α (TNFα) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70–1) with a LPS content of 1.4 pg/μg was unable to induce TNFα release by RAW264.7 murine macrophages at concentrations up to 5 μg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70–2) at 1 μg/ml with a LPS content of 0.2 ng/μg was able to induce TNFα release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70–1 to induce TNFα release was not because of defective physical properties since rhHsp70–1 and rhHsp70–2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70–2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFα-inducing activity of rhHsp70–2. The addition of LPS at the concentration found in rhHsp70–2 to rhHsp70–1 resulted in the same TNFα-inducing activity as observed with rhHsp70–2. The TNFα-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70–1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFα release from murine macrophages and that the observed TNFα-inducing activity in the rhHsp70–2 preparation is entirely due to the contaminating LPS.

title: Immunological aspects of heat-shock proteins-the optimum stress of life.
abstract: This review summarizes the complex role of heat-shock proteins (Hsp) in immune reactions, especially the cellular effects of heat-shock proteins during the recognition processes by innate immunity. The role of heat-shock proteins in the pathogenesis of two multifactorial diseases, i.e. inflammatory bowel disease (IBD) and atherosclerosis is highlighted. A new hypothesis on "immunodeficiency burden" is presented. According to this hypothesis, susceptibility to any multifactorial disease in any given subject and in the presence of specific environmental factors is the aggregate effect of polymorphisms resulting in the failure of protective immunity with consequent disease.

title: Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses
abstract: It is currently thought that immune responses are initiated by pathogen-associated molecular patterns or by tissue-derived danger/alarm signals. Here, we propose that these two groups of molecules might not be mutually exclusive. Many of them might be part of an evolutionarily ancient alert system in which the hydrophobic portions of biological molecules act, when exposed, as universal damage-associated molecular patterns to initiate repair, remodelling and immunity.

title: Heat Shock Proteins as Endogenous Adjuvants in Sterile and Septic Inflammation1
abstract: Heat shock proteins (HSPs) have been reported to stimulate the immune system via innate receptors. However, the role of HSPs as endogenous adjuvants has been challenged by reports claiming that pure HSPs are not innate ligands; it is only the bacterial molecules trapped by the HSPs that can signal the innate immune system. In this review, we discuss data suggesting that both views, in essence, are correct; pure HSPs are indeed innate immunostimulators, but HSPs can also function as transducers of pathogen signals. In other words, HSPs perform diverse functions in two alternative modes of inflammation: sterile inflammation, which results from endogenous stimuli and is necessary for body maintenance, and septic inflammation, which protects us from environmental pathogens. Endogenous HSPs are key players in the modulation of these two modes of inflammation, and as such, they are potential targets for new and more efficient therapies for cancer, infections, and autoimmunity.

title: Tumor-specific immunogenicity of stress-induced proteins: convergence of two evolutionary pathways of antigen presentation?
abstract: Stress-induced proteins (hsps) elicit tumor-specific immunity to a number of murine tumors. Specificity of this immunity is puzzling in view of the fact that no tumor-specific DNA sequence polymorphisms have been identified in stress-induced genes, nor is there evidence for tumor-specific posttranslational modification of hsps. In this light, the possibility that hsps may not be antigenic per se, but may be carriers of antigenic moieties such as peptides, and may be accessory antigen-presenting molecules, is considered. A model where an hsp molecule such as gp96 acts as an intermediate in presentation of peptides to MHC is discussed and it is suggested that the hsp and MHC antigens are mediators of independent but functionally convergent phylogenetic pathways.

title: Roles of heat-shock proteins in innate and adaptive immunity
abstract: Heat-shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins. In single-cell organisms, invertebrates and vertebrates, they perform a multitude of housekeeping functions that are essential for cellular survival. In higher vertebrates, their ability to interact with a wide range of proteins and peptides — a property that is shared by major histocompatibility complex molecules — has made the HSPs uniquely suited to an important role in organismal survival by their participation in innate and adaptive immune responses. The immunological properties of HSPs enable them to be used in new immunotherapies of cancers and infections.

title: Heat shock proteins transfer peptides during antigen processing and CTL priming
abstract: Recently emerging evidence indicates that the heat shock proteins (HSPs) gp96, hsp90, and hsp70 associate with antigenic peptides derived from cellular proteins. This evidence forms the basis of the following two hypotheses: 1) that HSPs constitute a relay line in which the peptides, after generation in the cytosol by the action of proteases, are transferred from one HSP to another, until they are finally accepted by MHC class I molecules in the endoplasmic reticulum, and 2) that the binding of peptides by HSPs constitutes a key step in the priming of cytotoxic T lymphocytes (CTLs) in vivo. The following chain of events is suggested: HSPs are released from virus-infected cells or tumor cells in vivo during lysis of cells during infection or by the action of antibodies or nonspecific effectors. The HSPs, which are now complexed with antigenic peptides derived from the cognate cells, are taken up by macrophage or other specialized antigen-presenting cells, possibly by a receptor-mediated mechanism. The HSP-borne peptide is then routed to the endogenous presentation pathway in the antigen-presenting cell and is displayed in the context of that cell's MHC class I, where it is finally recognized by the precursor CTLs. Thus it is suggested that, as with antigen presentation by MHC class II molecules, presentation by MHC class I molecules is also carried out primarily by the host antigen-presenting cells. This mechanism explains the phenomenon of cross-priming and has implications for the development of immunological strategies against cancer and infectious diseases.

title: Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world.
abstract: During the analysis of representation of HSP-peptide complexes by APCs, another facet of HSP-APC interaction came to light. Exposure of APCs to gp96 preparations was observed to lead to stimulation of APCs to secrete low levels of cytokines regardless of the peptides associated with gp96 (Suto and Srivastava 1995xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995;  1585–1588Crossref | PubMedSee all ReferencesSuto and Srivastava 1995; . This was surprising but consistent with the observation that immunization with cancer-derived HSP-peptide complexes elicits, in addition to the CD8+ and CD4+ T cell response, an NK response that is crucial for eliciting protective immunity (Tamura et al. 1997xImmunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Tamura, Y, Peng, P, Kang, L, Daou, M, and Srivastava, P.K. Science.  117–120Crossref | PubMed | Scopus (572)See all ReferencesTamura et al. 1997). In light of the fact that HSPs are abundant components of pathogens as well as hosts, this observation may indicate a basic role for HSP-APC interaction in innate immunity in addition to the crucial role of such interactions in adaptive, peptide-dependent immunity discussed thus far (73xHeat shock proteins transfer peptides during antigen processing and CTL priming. Srivastava, P.K, Udono, H, Blachere, N.E, and Li, Z. Immunogenetics. 1994; 39: 93–98Crossref | PubMed | Scopus (456)See all References, 81xCellular requirements for tumor-specific immunity elicited by heat shock proteins (tumor rejection antigen gp96 primes CD8+ T cells in vivo) . Udono, H, Levey, D.L, and Srivastava, P.K. Proc. Natl.  91: 3077–3081Crossref | PubMedSee all References, 75xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995;  1585–1588Crossref | PubMedSee all References). Contemplation of these ideas leads to some interesting insights. HSPs are among the most primitive proteins in living systems, and macrophage-like cells of one kind or another are present in the earliest multicellular living systems (Beck and Habicht 1996xImmunity and invertebrates. Beck, G and Habicht,  60–63Crossref | PubMedSee all ReferencesBeck and Habicht 1996). It is conceivable that in a less polymorphic era, when adaptive immune response was but a distant gleam in the evolutionary eye, the interaction of HSPs with macrophage-like cells, leading to stimulation of the macrophage-like cells to secrete IL-1 and other messengers, was the primary “innate” defense mechanism. As HSPs are the most abundant soluble molecules in cells, they can be reasonably expected to be the most reliable messengers of cell death or, at any rate, cell lysis, which would result if the organism were in stress or danger, e.g., being consumed by a predator. This chain of events would also explain the otherwise strange presence of a surface receptor for HSPs, which are normally intracellular. In this view, the HSPs would have been transformed from being protectors of cells from cellular stress in the single-celled organisms to being messengers of stress and protectors against such stress in the first multicellular organisms or, at any rate, among those in which a differentiated macrophage-like cell had evolved. By all accounts, this happened quite early, as phagocytes of echinoderms, mollusks, annelids, and tunicates had already acquired the ability to secrete IL-1 like molecules (seeOttaviani and Franceschi 1997xThe invertebrate phagocytic immunocyte (clues to a common evolution of immune and neuroendocrine systems) . Ottaviani, E and Franceschi, C. Immunol. Today. 1997; 18: 169–174Abstract | Full Text PDF | PubMed | Scopus (130)See all ReferencesOttaviani and Franceschi 1997). From this initial point, it is relatively straightforward to see how the HSP-APC interaction could have been incorporated into future versions of the developing defense mechanisms until, with the arrival of specific immunity and its paraphernalia of specificity of peptide-binding and T cell receptors, the primal peptide-binding proteins, i.e., the HSPs, would become accessories in antigen presentation by MHC molecules. The evolutionary implications of a convergence of the peptide-binding functions of the HSPs and the MHC molecules has been discussed in detail elsewhere (Srivastava and Heike 1991xTumor-specific immunogenicity of stress-induced proteins (convergence of two evolutionary pathways of antigen presentation?) . Srivastava, P.K and Heike, M. Semin. Immunol. 1991; 3: 57–64PubMedSee all ReferencesSrivastava and Heike 1991).In the proposed role of HSPs as harbingers of cell death and, therefore, of danger to an organism (seeMatzinger 1994xTolerance, danger, and the extended family. Matzinger, P. Annu.  991–1045Crossref | PubMedSee all ReferencesMatzinger 1994), a kind of death that results in an encounter of HSPs with the immune system will be more productive, i.e., communicative of danger, and protective than a “silent death” that does not lead to HSP-APC interaction. It is therefore our belief that immunogenicity of cells, infectious agents, and cancers is defined to a significant degree upon the extent to which cell death leads to induction of HSPs and consequent HSP-APC interaction. Studies that show a cosegregation of immunogenicity of cancers with the expression of molecules of the hsp60 and hsp70 family (39xCo-segregation of tumor immunogenicity with expression of inducible but not constitutive hsp70 in rat colon carcinomas. Menoret, A, Patry, Y, Burg, C, and Le Pendu,  740–747PubMedSee all References, 85xRestoration of MHC class I surface expression and endogenous antigen presentation by a molecular chaperone. Wells, A.D, Rai, S.K, Salvato, M.S, Band, H, and Malkovsky, M. Scand.  605–612Crossref | PubMedSee all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile,  1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References) are consistent with this belief. It may be tempting in this context to imagine that immunologically unproductive and productive modes of death will correlate neatly with apoptotic and necrotic types of death; however, recent results indicate that this is not necessarily so (7xProcessing of engulfed apoptotic bodies yields T cell epitopes. Bellone, M, Iezzi, G, Rovere, P, Galati, G, Ronchetti, A, Protti, M.P, Davoust, J, Rugarli, C, and Manfredi,  159: 5391–5399PubMedSee all References, 1xDendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Albert, M.L, Sauter, B, and Bhardwaj, N.  1998;  86–89Crossref | PubMed | Scopus (1839)See all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile,  1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References). Other factors yet to be discovered must influence the immunological productivity of dying cells. In any case, it is safe to suggest that the recent fascination of immunologists with death has atavistic origins in the evolutionary past, when the presence of extracellular HSPs signaled physical disintegration.*To whom correspondence should be addressed (e-mail: srivastava@nso2.uchc.edu).

title: Proconvulsant effect of ketotifen, a histamine H1 antagonist, confirmed by the use of d-chlorpheniramine with monitoring electroencephalography.
abstract: Ketotifen produced seizures in a 5-year-old boy with secondary generalized epilepsy (SGE) and allergic rhinitis. To confirm that the seizures were due to histamine H1 receptors blockade, d-chlorpheniramine was administered with monitoring electroencephalography (EEG). Administration of d-chlorpheniramine significantly increased the number of epileptic discharges in the patient, compared with those before administration. Plasma noradrenaline and dopamine levels were not affected by treatment with d-chlorpheniramine. These findings indicate that histamine H1 receptors blockade produced convulsions and increased epileptic discharges on EEG. Recently, several experimental reports have shown that histamine has an inhibitory role on convulsions through histamine H1 receptors. Experimental findings and present results show that histamine H1 antagonists have proconvulsant effects, especially in the developmental period. Thus, it is recommended that centrally-acting histamine H1 antagonists should be avoided in epileptic patients, especially in children of pre-school age.