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J Appl Physiol 99: 1789-1795, 2005. First published July 21, 2005; doi:10.1152/japplphysiol.00390.2005
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Adrenergic receptors mediate stress-induced elevations in extracellular Hsp72

John D. Johnson, Jay Campisi, Craig M. Sharkey, Sarah L. Kennedy, Molly Nickerson, and Monika Fleshner

Department of Integrative Physiology and Center for Neuroscience, University of Colorado, Boulder, Colorado

Submitted 7 April 2005 ; accepted in final form 14 July 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}1-adrenergic receptor-mediated signaling pathway. Activation of {alpha}1-adrenoceptors results in a rapid increase in circulating Hsp72, and blockade of {alpha}1-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.

catecholamine; rat; norepinephrine; immune; circulating; heat shock protein

HEAT SHOCK PROTEINS, particularly those in the 70-kDa family, are released by various cell types, including tumor cells (4), macrophages (27), human peripheral blood mononuclear cells (29), and glial cells (24), after in vitro challenge with cytokines or heat stress. It has been proposed that extracellular heat shock protein serves to enhance immunologic responses (6, 35, 47), as demonstrated by the ability of heat shock proteins to act as powerful adjuvants for eliciting antigen-specific and antitumor immunity (31); stimulating dendritic cell maturation and antigen-specific cytotoxic T-cell proliferation (30, 31, 49), phagocytosis (26), and release of reactive oxygen/nitrogen species (e.g., nitric oxide) (1–3, 7, 9, 41); and enhancing cytokine-stimulated natural killer cytolysis of tumor cells (36, 37). Extracellular heat shock protein may also stimulate the release of proinflammatory cytokines via a calcium-dependent pathway (2, 7). Such findings remain controversial because of suggestions that endotoxin contamination of commercially available recombinant heat shock protein may be responsible for induction of proinflammatory cytokines in some in vitro studies (21, 22).

Elevated plasma levels of endogenous heat shock protein 72 (Hsp72) have been reported in laboratory animals and humans after exposure to stressful events, such as predatory stress (16), tail-shock stress (7, 9), hand-shock stress (13), trauma (42), and exercise stress (15, 17, 48). During stress, elevated Hsp72 may act as a "danger signal" to prime and/or enhance immunologic responses and improve recovery (17, 19, 20). In humans, higher levels of circulating Hsp72 after trauma correlate with improved survival (42); in laboratory animals, elevated levels of circulating Hsp72 after tail-shock stress correlate with elevated levels of Hsp72 and nitric oxide at an inflammatory site (i.e., subcutaneous Escherichia coli) (9) and inversely correlate with levels of viable bacteria, size of the inflammatory site, and time to full recovery (8).

The numerous immunostimulatory effects of extracellular Hsp72 have made it a widely studied molecule with potential importance in vaccinations, tumor treatment, autoimmunity, and stress-induced exacerbation of inflammatory conditions. Prior research has demonstrated that interferon-{gamma} can stimulate the release of extracellular heat shock protein during immunologic challenge (4), but the endogenous signal(s) that mediate(s) the elevation of extracellular Hsp72 during stress in the intact organism remain(s) unknown. The experiments presented here examined the hypothesis that elevated levels of stress hormones/neurohormones (i.e., catecholamines, ACTH, and glucocorticoids) mediate the increase in plasma Hsp72 during stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals.   Adult male viral-free Fischer 344 rats (200–250 g; Harlan, Indianapolis, IN) were housed in pairs in Plexiglas cages (60 x 30 x 24 cm), with food and water available ad libitum. The animal colonies were maintained in a pathogen-free barrier facility with a 12:12-h light-dark cycle (lights on from 0600 to 1800). Rats were given ≥2 wk to habituate to the colonies and were handled daily for ≥4 days before experimentation. Care and use of the animals were in accordance with protocols approved by the University of Colorado Institutional Animal Care and Use Committee.

Adrenalectomy.   Animals were anesthetized with isoflurane, and adrenal glands were removed via the dorsal approach, as previously described (18). Sham animals underwent the same surgery, except the adrenal glands were left intact. Basal corticosterone was maintained in adrenalectomized animals by corticosterone supplementation (0.25% cortisosterone, 0.2% ethanol, and 0.5% NaCl) of their drinking water. Sham animals were given unsupplemented water (vehicle). Animals were allowed to recover for 1 wk before stressor exposure. Animals were killed immediately after stressor termination for measurement of circulating Hsp72. Corticosterone was also measured from plasma samples for verification of adrenalectomy.

Hypophysectomy.   Hypophysectomized animals (Harlan) were maintained on 5% {beta}-D-glucose for 10 days as recommended. Sham animals were anesthetized with isoflurane, and a small incision was made on the top of the skull. The incision was sutured, and all animals were allowed to recover for 2 wk before stressor exposure. Animals were killed immediately after stressor termination for measurement of circulating Hsp72. ACTH was measured from plasma samples for verification of hypophysectomy.

Adrenergic antagonist.   In studies examining the role of stress-induced catecholamines on the elevation of extracellular Hsp72, various adrenergic receptor antagonists (Sigma, St. Louis, MO) specific to different subclasses of adrenergic receptors were administered intraperitoneally 30 min before stressor exposure: labetalol (30.0 mg/kg, a nonspecific adrenoceptor antagonist), propranolol (10.0 mg/kg, a specific {beta}1/{beta}2-adrenoceptor antagonist), prazosin (2.0 mg/kg, a specific {alpha}1-adrenoceptor antagonist), or vehicle. Labetalol and propranolol were dissolved in sterile endotoxin-free saline; prazosin was dissolved in sterile endotoxin-free water and subjected to heat. Immediately after stressor termination, animals were killed by decapitation, and trunk blood was collected in EDTA-containing tubes. Plasma was collected and frozen at –20°C until time of assay.

Tyrosine administration.   Depletion of tissue norepinephrine by exposure to tail-shock stress (23) can be prevented by pretreatment with the catecholamine precursor tyrosine (unpublished observation). Thus, to examine the effects of maintaining high-stress levels of tissue norepinephrine on circulating Hsp72, rats were injected intraperitoneally with tyrosine (catalog no. T-8566, Sigma) or vehicle 30 min before the onset of stress and killed immediately or 2 h later for measurement of plasma Hsp72. Tyrosine, dissolved in sterile, endotoxin-free saline at 400 mg/kg, has previously been shown to increase tissue norepinephrine concentrations (50).

Adrenergic agonist.   To determine whether activation of adrenergic receptors is sufficient to increase circulating extracellular Hsp72, animals were injected intraperitoneally with vehicle, phenylephrine (1 mg/kg), or isoproterenol (5 mg/kg) and killed after 15, 30, or 60 min for measurement of plasma Hsp72. Phenylephrine and isoproterenol (both from Sigma), selective {alpha}1- and {beta}-adrenergic receptor agonists, respectively, were dissolved in sterile, endotoxin-free saline.

Stress procedure.   Animals remained in their home cages as controls or were placed in Plexiglas tubes (23.4 x 7 cm) and exposed to 100 tail shocks (5 s, 1.5 mA), with an average intertrial interval of 60 s. In most studies, animals were killed immediately after stressor termination, which, according to previous studies that characterized the kinetics of plasma Hsp72, is the time of maximally elevated plasma Hsp72 (7, 9). This stress procedure has previously been shown to reliably and consistently elevate circulating Hsp72 and enhance immune responses after endotoxin or bacterial challenge (7, 9).

Measurement of Hsp72.   Hsp72 was measured using a commercially available ELISA (Stressgen, Victoria, BC, Canada) according to the manufacturer's instructions. The detection limit is ~540 pg/ml, and intra-assay variability is <10%.

Measurement of plasma corticosterone.   Blood for corticosterone measurement was collected in EDTA-coated tubes and kept on ice. Blood was centrifuged (10 min, 4,000 rpm), and plasma was stored at –20°C until time of assay. Plasma levels of corticosterone were measured using a commercially available RIA kit (ICN Biomedicals, Costa Mesa, CA) according to the manufacturer's instructions.

Measurement of plasma ACTH.   Blood for ACTH measurement was collected in EDTA-coated tubes and kept on ice. Blood was centrifuged (10 min, 4,000 rpm) within 1 h of collection, and plasma was stored at –80°C until time of assay. Plasma levels of ACTH were measured using a commercially available RIA kit (Peninsula Laboratories, San Carlos, CA) according to the manufacturer's instructions.

Statistics.   Data were analyzed using a 2 x 2 ANOVA between stress condition (tail shock vs. home cage control) and treatment condition (surgery vs. sham or drug vs. vehicle), a 2 x 2 x 2 ANOVA between stress condition (tail shock vs. home cage control), treatment condition (tyrosine vs. vehicle), and time (immediately after vs. 2 h after stress), or a 2 x 3 ANOVA between treatment condition (drug vs. vehicle) and time (15, 30, and 60 min). Post hoc analyses were done using Fisher's least significant difference test. In all cases, P < 0.05 was used for the level of confidence for acceptance of significance to exclude the null hypothesis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of glucocorticoids on extracellular Hsp72.   Stressor exposure results in high levels of circulating glucocorticoids (mainly corticosterone in the rat), which are known to augment necrotic cell death (10), a possible mechanism by which endogenous Hsp72 becomes extracellular. To examine the role of corticosterone in the elevation of plasma Hsp72 during stress, rats underwent adrenalectomy or sham surgery and 1 wk later were exposed to tail-shock stress. Rats were killed immediately after 100 min of tail-shock exposure, and plasma corticosterone and Hsp72 were measured. Adrenalectomy completely blocked the stress-induced rise in circulating corticosterone (Fig. 1A; P < 0.0001) but had no effect on plasma Hsp72 levels compared with sham controls (Fig. 1B; P = 0.681). This suggests that corticosterone does not mediate the elevation of plasma Hsp72 during stressor exposure and that the in vivo levels of corticosterone during tail-shock stress are not sufficient to result in necrotic cell death to an extent that would contribute to plasma Hsp72.



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  		Fig. 1. Effects of adrenalectomy on stress-induced plasma heat shock protein 72 (Hsp72) levels. Adrenalectomized (ADX) and sham-operated rats were exposed to 100 min of tail-shock stress and killed immediately after stressor termination. Plasma was collected and measured for circulating corticosterone (CORT) by RIA (A) and circulating Hsp72 by ELISA (B). Values are means ± SE (n = 4). *P < 0.05 vs. control.

 
Effect of ACTH on extracellular Hsp72.   It has been proposed that ACTH contributes to the induction of intracellular Hsp72 during stressor exposure (5). To examine the possible role of ACTH in mediating the elevation of plasma Hsp72 after stressor exposure, rats underwent hypophysectomy or sham surgery and 2 wk later were exposed to tail-shock stress. Rats were killed immediately after stressor termination, and plasma ACTH and Hsp72 were measured. Hypophysectomy completely blocked the stress-induced rise in plasma ACTH (Fig. 2A; P < 0.0001) but had no effect on the elevation of plasma Hsp72 (Fig. 2B; P = 0.294). This suggests that high levels of ACTH during stressor exposure are not necessary for the elevation of plasma Hsp72.



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  		Fig. 2. Effects of hypophysectomy (HPX) on stress-induced plasma Hsp72. Hypophysectomized and sham-operated rats were exposed to 100 min of tail-shock stress and killed immediately after stressor termination. Plasma was collected and measured for circulating ACTH by RIA (A) and circulating Hsp72 by ELISA (B). Values are means ± SE (n = 9). *P < 0.05 vs. control.

 
Effect of adrenergic receptors on extracellular Hsp72.   Catecholamines have often been demonstrated to induce intracellular Hsp72 during stressor exposure via activation of {alpha}1-adrenergic receptors (12, 25, 34). To determine whether catecholamines may also mediate the elevation of plasma Hsp72 during stress, rats were pretreated with labetalol (30.0 mg/kg), propranolol (10.0 mg/kg), prazosin (2.0 mg/kg), or vehicle 30 min before tail-shock exposure and killed immediately thereafter for measurement of plasma Hsp72. Tail-shock exposure consistently resulted in an increase in plasma Hsp72 in control animals; however, pretreatment with labetalol or prazosin completely blocked the stress-induced increase in plasma Hsp72 (Fig. 3, A and B; P = 0.011 and 0.015, respectively). Conversely, pretreatment with propranolol significantly augmented stress-induced plasma Hsp72 (Fig. 3C; P < 0.0001). Taken together, these data suggest that adrenergic receptors play an important role in mediating stress-induced levels of circulating Hsp72 and indicate that activation of {alpha}1-adrenoceptors elevates circulating Hsp72, whereas activation of {beta}-adrenoceptors inhibits circulating Hsp72 during tail-shock stress.



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  		Fig. 3. Effects of adrenoceptor blockade on stress-induced plasma Hsp72. Rats were injected intraperitoneally with vehicle, labetalol (30 mg/kg; A), prazosin (2 mg/kg; B), or propranolol (10 mg/kg; C) 30 min before exposure to 100 min of tail-shock stress. Plasma was collected, and circulating Hsp72 was measured by ELISA. Values are means ± SE (n = 7–8). *P < 0.05 vs. control. #P < 0.05 vs. vehicle during stress.

 
To further examine the role of adrenergic receptors in mediating circulating Hsp72, rats were injected with phenylephrine (1 mg/kg), isoproterenol (5 mg/kg), or vehicle and killed after 15, 30, or 60 min for measurement of plasma Hsp72. Phenylephrine significantly increased plasma Hsp72 over time (Fig. 4A; P = 0.042) compared with vehicle-injected controls, whereas isoproterenol had no effect (Fig. 4B; P = 0.419). This further supports an important role for adrenergic receptors in mediating levels of plasma Hsp72 and demonstrates that not only are {alpha}1-adrenoceptors necessary during stressor exposure to elevate plasma Hsp72 but also activation of {alpha}1-adrenoceptors in the absence of stress is sufficient to increase plasma Hsp72.



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  		Fig. 4. Effects of adrenoceptor activation on circulating Hsp72. Rats were injected intraperitoneally with vehicle, phenylephrine (1 mg/kg; A), or isoproterenol (5 mg/kg; B) and killed 15, 30, or 60 min later. Plasma was collected, and circulating Hsp72 was measured by ELISA. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 
Effects of tyrosine pretreatment on stress-induced Hsp72.   Exposure to stressors often results in depletion of tissue norepinephrine content because of the slow rate of synthesis compared with release. Pretreatment of laboratory animals with tyrosine, the precursor to catecholamine synthesis, prevents the depletion of tissue norepinephrine during stressor exposure (50). Thus we pretreated rats intraperitoneally with tyrosine (400 mg/kg) or vehicle 30 min before tail-shock exposure and measured plasma Hsp72 levels immediately after and 2 h after stressor termination. Again, exposure to tail-shock stress resulted in a significant rise in plasma Hsp72 (Fig. 5; P < 0.0001). Tyrosine pretreatment significantly augmented the stress-induced rise in plasma Hsp72 (Fig. 5; P = 0.005) compared with vehicle-injected controls. As a control for tissue norepinephrine content, spleens were also collected, and norepinephrine content was measured by HPLC. Stressor exposure depleted levels of norepinephrine in the spleen immediately after and 2 h after tail-shock stress (Fig. 5B; P = 0.003), and although there was no significant interaction between stressor exposure and tyrosine pretreatment (P = 0.19), tyrosine pretreatment significantly elevated levels of splenic norepinephrine (P = 0.003) compared with vehicle-injected controls. These data suggest that increasing available tissue norepinephrine during times of stress augments stress-induced plasma Hsp72.



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  		Fig. 5. Effects of tyrosine pretreatment on stress-induced circulating Hsp72. Rats were injected intraperitoneally with vehicle or tyrosine (400 mg/kg) 30 min before 100 min of tail-shock stress. Spleens were collected for measurement of splenic norepinephrine (NE) content by HPLC (A), and plasma was collected for measurement of circulating Hsp72 by ELISA (B). Values are means ± SE (n = 7–8). *P < 0.05 vs. saline.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The studies presented here demonstrate that {alpha}1-adrenergic receptors, at least in part, mediate plasma levels of Hsp72. Administration of an {alpha}1-adrenoceptor, but not a {beta}-adrenoceptor, agonist elevated plasma Hsp72, and pharmacological blockade of {alpha}1-adrenoceptors, but not {beta}-adrenoceptors, has been shown to block the increase in plasma Hsp72 after tail-shock stress. Although activation of {alpha}1-adrenoceptors has previously been observed to induce intracellular Hsp72, these are the first studies that demonstrate a role for {alpha}1-adrenoceptors in mediating circulating levels of endogenous extracellular Hsp72.

Exposure to stressors, including tail-shock stress, stimulates sympathetic postganglionic neurons, resulting in norepinephrine release onto peripheral tissues (spillover can be measured in plasma) and sympathetic preganglionic neurons innervating the adrenal medulla to result in epinephrine release into the blood (14). Norepinephine has a significantly higher affinity to bind to {alpha}1-adrenoceptors than epinephrine (44), and adrenalectomy (which removes the primary source of circulating epinephrine) has no effect on stress-induced plasma Hsp72; therefore, it appears that norepinephrine, and not epinephrine, is the critical mediator of circulating endogenous extracellular Hsp72. One might have predicted that adrenalectomy would at least attenuate levels of extracellular Hsp72 after tail shock, as observed in adrenalectomized rats exposed to predator stress (16). The reason for differences between the studies is unclear, but one explanation may be the differences in intensity between the two stressors. Tail-shock stress results in a much greater corticosterone response (35 µg/dl) than was reported after predator stress (22 µg/dl) (16). Because catecholamine levels typically also reflect differences in stressor intensity (39, 40, 43), it is possible that higher levels of norepinephrine after tail-shock stress compensate for the elimination of epinephrine after adrenalectomy, whereas during predator stress high levels of norepinephrine and epinephrine may be required for a comparable Hsp72 response. Furthermore, there is evidence that different stressors result in different patterns of sympathetic activation (28); thus the contribution of norepinephrine and epinephrine to the increase in extracellular Hsp72 levels may depend on the individual stressor.

Although these data suggest that catecholamines mediate levels of circulating extracellular Hsp72 via {alpha}1-adrenoceptors, the cellular source of Hsp72 remains unknown. Norepinephrine appears to be the primary mediator of plasma Hsp72 (see above); thus it is plausible that plasma Hsp72 is released from cells in tissues innervated by postsynaptic sympathetic neurons and that plasma levels of Hsp72 are due to spillover into the blood similar to that observed with norepinephrine. It was demonstrated that tyrosine, the amino acid precursor to norepinephrine, prevents the depletion of norepinephrine in tissues during stressor exposure (50), and this significantly augments the stress-induced increase in plasma Hsp72. In addition, Febbraio et al. (15) used plasma arterial-venous differences to suggest that Hsp72 is released from hepatosplanchnic tissues during exercise stress in humans. Because many cell types express {alpha}1-adrenoceptors and contain basal levels of Hsp72, whether extracellular Hsp72 is released from a single tissue/cell type or from multiple sources is unknown.

The mechanism by which catecholamines mediate plasma Hsp72 levels also remains unknown. Catecholamines have previously been shown to upregulate intracellular Hsp72 via an {alpha}1-adrenoceptor pathway; thus it is possible that elevated circulating Hsp72 depends on the induction of intracellular Hsp72 (12, 25, 34). If this is the case, {alpha}1-adrenoceptor blockade may prevent stress-induced plasma Hsp72 by blocking the upregulation of intracellular Hsp72. However, this is unlikely, because circulating Hsp72 increases within 20–30 min of stressor onset (7, 48) or administration of an {alpha}1-adrenoceptor agonist, which is too soon for upregulation of intracellular Hsp72 protein. These data suggest that elevated levels of extracellular Hsp72 are probably not dependent on de novo production of intracellular Hsp72 but that preformed pools of Hsp72 are released on {alpha}1-adrenoceptor stimulation. Many tissues have high basal levels of intracellular Hsp72 that could be released during times of stress (32, 38). Exosomes, small membrane vesicles secreted by various cell types, including antigen-presenting cells, B cells, and T cells of the immune system (11), are among the possible mechanisms by which Hsp72 is released during stress. Exosomes contain numerous costimulatory and antigen-presenting molecules, including Hsp70 (29), and are released in a calcium-dependent fashion on stimulation of the cell (45). Because activation of {alpha}1-adrenoceptors results in an increase in intracellular calcium (46), the release of exosomes is one potential mechanism by which catecholamines trigger the rise in extracellular Hsp72.

It has been proposed that extracellular Hsp72 acts as a "danger signal" during times of stress to stimulate or enhance immune function (17). As described in the introduction, innate and acquired immune cells express surface receptors that bind Hsp72 and enhance various immunologic responses. Thus increase in circulating extracellular Hsp72 during times of stress may help protect an organism during increased risk of infection and/or injury. Elevated levels of extracellular Hsp72 during stress may also exacerbate chronic inflammatory responses, such as arthritis, arthrosclerosis, multiple sclerosis, or inflammatory bowel syndrome. High levels of extracellular Hsp72 have recently been found in the synovial fluid of an arthritic joint bound to local immune cells (33). Data presented here add to the understanding of the regulation of extracellular Hsp72 and could have potential importance when treatments to enhance or suppress immunologic responses are considered. Although the potential function of endogenous circulating extracellular Hsp72 remains unknown, it is clear that extracellular Hsp72 is elevated after exposure to physical and psychological stressors (7, 9, 13, 15–17, 42, 48) in humans and laboratory animals and depends on stimulation of {alpha}1-adrenergic receptors. Thus increased levels of extracellular Hsp72 should be considered a normal and potentially adaptive feature of the acute-stress response.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Johnson, Center for Neuroscience, Dept. of Integrative Physiology, Univ. of Colorado at Boulder, Boulder, CO 80309-0354 (e-mail: john.johnson{at}colorado.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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