Originally published online as doi:10.1189/jlb.0905523 on December 30, 2005
Published online before print December 30, 2005
(Journal of Leukocyte Biology. 2006;79:425-434.)
© 2006
by Society for Leukocyte Biology
Releasing signals, secretory pathways, and immune function of endogenous extracellular heat shock protein 72
John D. Johnson and
Monika Fleshner1
Department of Integrative Physiology and the Center for Neuroscience, University of Colorado, Boulder
1 Correspondence: Campus Box 354, Department of Integrative Physiology, University of Colorado, Boulder, CO 80309-0354. E-mail: Fleshner{at}colorado.edu
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ABSTRACT
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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.
Key Words: stress proteins • danger signals • adrenergicreceptors • catecholamines • inflammation • cytokines
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INTRODUCTION
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Heat shock proteins (Hsp) consist of several families of highly conserved proteins that play a role in a number of important cellular functions [1
]. The first demonstrations of cellular induction of Hsp in response to environmental stressors were reported in 1962 when Ritossa [2
] noted that Drosophila exposed to temperature shock exhibited an unusual gene expression profile. In 1974, the term Hsp was first coined [3
]. The focus of the current review is on one member of the 70-kDa Hsp (Hsp70) family of proteins, Hsp72. The Hsp70 family of proteins includes the Hsp constitutive 73-kDa protein and a highly stress-inducible 72-kDa protein (Hsp72) [1
, 4
]. Hsp72 is found in nearly every cell of the body and can be up-regulated after exposure to a variety of cellular and whole organism stressors [1
, 4
]. Basal concentrations of Hsp72 are low in many tissues; high concentrations of intracellular Hsp72 can be found in the absence of overt environmental stressors in some tissues such as the frontal cortex [5
], pituitary [6
], adrenal [6
], and brown fat [7
, 8
]. Although a great deal is understood about intracellular Hsp72 protein induction signals and function, only recently has it become clear that Hsp72 may have unique releasing signals and immunomodulatory functions when expressed in an extracellular context such as on the cell surface or in the circulation.
In this article, we review our evidence that 
-adrenergic receptors (ADRs) regulate in vivo release of endogenous extracellular Hsp72 (eHsp72) into the blood and that eHsp72 can have in vivo immunomodulatory function(s). Furthermore, we emphasize the need to approach, with caution, in vitro immunological assessments of Hsp72 function.
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INTRACELLULAR Hsp72 INDUCTION
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The gene for Hsp72 contains at least two regulatory elements that interact with heat shock transcription factors (HSFs) [9
, 10
]. Specifically, the induction of Hsp72 protein requires HSF1 binding to the heat shock element in the promoter region of the Hsp70 gene [11
]. Hsp70 mRNA is transcribed, resulting in the synthesis and accumulation of cytosolic Hsp72 protein [12
]. Many factors induce transcription and translation of intracellular Hsp72 protein, and these vary depending on the tissue examined. Among the various signals for the induction of intracellular Hsp72 are adrenocorticotropin hormone [13
, 14
], corticosterone [15
16
17
], glycogen deprivation [18
, 19
], norepinephrine (NE) or epinephrine (E) [5
, 7
, 8
, 20
21
22
23
], heat or hyperthermia [15
, 24
25
26
27
], and oxidative stress [28
29
30
]. Clearly, a wide array of physiological signals is capable of stimulating intracellular Hsp72 synthesis, making this a ubiquitous response to cellular stress.
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INTRACELLULAR AND CELL SURFACE Hsp72 FUNCTION
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The cellular functions of intracellular Hsp72 have been studied thoroughly and include limiting protein aggregation, facilitating protein refolding, and chaperoning proteins [1
, 4
]. These cellular functions en masse serve to improve cell survival in the face of a broad array of cellular stressors [1
, 4
]. There are many examples from the literature that demonstrate convincingly that induction of Hsp72 is not simply a consequence of cellular stress, but it clearly improves resistance to death after cellular insult. It has been reported recently, for example, that Hsp72 induction protects neurons [31
, 32
], heart [33
34
35
36
37
], kidney [38
, 39
], intestine [40
], and liver [12
, 41
] from heat, endotoxin, and/or ischemia-induced apoptosis and cell death. Furthermore, intracellular Hsp72 could play an important therapeutic role in the treatment of various neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis, which are associated with aggregating intracellular proteins (e.g., -synuclein, ß-amyloid peptide, huntingtin, superoxide dismutase) [42
].
In addition to basic cell survival functions, the induction of intracellular Hsp72 in monocytes can impact immune function directly. The induction of Hsp72 gene transcription can suppress inflammatory cytokine gene transcription [43
44
45
] and contribute to "cross-priming" [46
, 47
]. Intracellular Hsp72 may be a negative regulator of inflammatory cytokines [48
49
50
51
]. Constraint of inflammatory cytokines will protect the cell from high levels of inflammatory cytokines that could result in "innocent bystander" injury or death during inflammation [52
53
54
]. One example of the importance of this function can be found in HSF1-deficient mice. Mice deficient in HSF1 lack the ability to up-regulate intracellular Hsp72 and have reduced survival and excessive tumor necrosis factor (TNF-) after a single injection of lipopolysaccharide (LPS) compared with wild-type controls [55
]. In addition to limiting inflammatory cytokine transcription, another immunological function of intracellular Hsp72 is the chaperoning of peptides for immune presentation in major histocompatibility class I (MHC I) molecules [56
]. This function is believed to be especially important for cross-priming or the loading of vesicular peptides into MHC I molecules [47
].
More recently, it has been established that cell surface Hsp also modulate immunity. For example, Hsp can be recognized by natural killer cells and cytotoxic T lymphocyte cells when expressed on the surface of stressed cells [57
58
59
60
61
62
63
64
]. This recognition appears to be direct and does not involve the MHC I pathway. The immunological function of Hsp in this context has been suggested to stimulate anti-cancer immunity [65
, 66
], exacerbate chronic inflammatory disease states [67
, 68
], and/or inhibit chronic inflammation [69
70
71
]. In addition, there is evidence that cell surface expression of Hsp, especially Hsp60, can contribute to autoimmune responses as a result of infection-induced molecular mimicry or cross-recognition between mammalian and microbial Hsp [72
, 73
]. Thus, the immunological function of intracellular and cell surface Hsp72 is complex.
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INTRODUCTION TO ENDOGENOUS eHsp72
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The first reports that eHsp72 was detectable in the circulation of humans were published by Pockley and colleagues [74
] in 2000. This group reported that people suffering from a variety of disease states such as renal disease [74
], hyptertension [75
], and atherosclerosis [76
] have chronically elevated basal levels of eHsp72 relative to healthy, aged-matched controls. In addition to elevated basal eHsp72 associated with disease pathology, Dybdahl et al. reported that patients with coronary artery disease have an acute increase in eHsp72 in response to the stress of coronary bypass surgery. Not long after these reports, we [77
78
79
] and Febbraio et al. [80
, 81
] reported that organisms in the absence of clinical disease states also rapidly increase the concentration of eHsp72 in blood after exposure to acute physical and/or psychological stressors. These papers were the first to demonstrate that an increase of eHsp72 in the blood occurs in healthy organisms after exposure to acute stressors and led us to suggest that stress-induced eHsp72 release may be a previously unrecognized feature of the normal stress response [82
].
The current review will focus on endogenous eHsp72, as stress-induced release of eHsp72 into the blood has only recently been documented, and little is understood about its unique mechanism(s) of induction/release and its powerful in vivo immunological function(s). Understanding more about the stress-induced eHsp72 response is important because the function of in vivo endogenous eHsp72 is likely context-dependent, such that in a normal physiological state, eHsp72 facilitates immunity [82
], whereas in a pathophysiological state, eHsp72 exacerbates inflammatory diseases (e.g., atherosclerosis, Alzheimer’s, inflammatory bowel disease) [83
]. This idea requires additional investigation because depending on the circumstances, eHsp72 can also stimulate anti-inflammatory cytokines [59
, 67
, 69
].
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RELEASING SIGNALS AND SECRETORY PATHWAYS OF eHsp
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As a result of the vast immunomodulatory effects of Hsp72, it is critical to understand its regulation and the mechanisms involved in the extracellular expression/release of eHsp72. In addition to increasing our knowledge of a newly recognized immunomodulatory pathway, understanding eHsp72 regulation will allow for manipulation of eHsp72 levels for clinical purposes. For example, one may wish to up-regulate eHsp72 during certain vaccinations to stimulate cross-presentation or during the treatment of various tumors and cancers to enhance their detection and elimination.
It was first suggested that eHsp72 is only released as a result of necrotic/lytic cell death [84
], and although it is true that necrotic cell death can cause the release of eHsp72 [85
86
87
], it is now recognized that elevated eHsp72 may be found in the absence of necosis. In fact, glial cells [88
], B cells [89
], tumor cells [64
], and human peripheral blood mononuclear cells (PBMC) [90
] have been shown to exocytotically release eHsp72 in the absence of detectable cell death. Furthermore, numerous whole organism stressors have been observed to elevate circulating eHsp72 [82
, 91
], often within 10–25 min of stressor onset, a speed that suggests the classic protein induction/necrosis release pathway is not likely. Circulating eHsp72 also increases in response to stressors, which are unlikely to result in large necrotic cell death such as following conditioned contextual fear, predatory stress, and exercise stress (described in ref. [92
]). Recent data from our laboratory suggest hormone receptor-mediated exocytotic pathways may exist, leading to increases in circulating eHsp72 during times of stress.
Our laboratory has demonstrated that exposure to 90 min of tail shock stress elevates circulating eHsp72 in Fischer 344 male rats and that pretreatment with a nonselective ADR antagonist (i.e., labetalol) or a selective 1-ADR antagonist (i.e., prazosin), but not a selective ß-ADR antagonist (i.e., propranolol), prior to stressor exposure, blocks the rise in circulating eHsp72 compared with stressed rats given a vehicle injection (Fig. 1A
) [93
]. In addition, administration of a selective 1-ADR agonist (i.e., phenylephrine), but not a selective ß-ADR agonist (i.e., isoproterenol), to nonstressed rats was sufficient to elevate circulating eHsp72 (Fig. 1B)
[93
]. As NE binds with higher affinity than E to 1-ADRs [94
], and adrenalectomy, which depletes 95–99% of E [95
, 96
], has been shown to have no effect on stress-induced eHsp72 release after tail shock stress [93
], we hypothesize that the increase in circulating eHsp72 during stressor exposure is a result of sympathetic nervous system activation and the release of NE, which acts at 1-ADR to increase the concentration of circulating eHsp72. Thus, although necrotic cell death can result in the extracellular release of cytoplasmic Hsp72, there are accumulating data that suggest other factors, such as NE, may stimulate a receptor-mediated exocytotic pathway of eHsp72 release (Fig. 2
).
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Figure 2. Semantic diagram of mechanisms of eHsp72 release. It is believed that Hsp72 can be released from cells via necrosis or exocytosis. Necrotic release of Hsp72 results when tissue damage occurs, and cytosolic Hsp72 is suddenly dumped into the extracellular space. Exocytotic release of Hsp72 does not involve tissue damage or cell death. We propose that activation of the sympathetic nervous system, such as during stressor exposure, results in the release of NE and the subsequent activation of 1-ADR. Stimulation of 1-ADR results in the increase in intracellular Ca2+, which may stimulate the release of exosomes containing Hsp72. In addition, 1-ADR activation may increase intracellular levels of Hsp72, which may raise the concentration of Hsp72 within each exosome. In either case, both pathways result in the increase of eHsp72, which can then modulate immune responses via interaction with various surface receptors expressed on specific immune cells. MVB, Multivesicular bodies; TLR-2, Toll-like receptor 2; TLR-4, Toll-like receptor 4.
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It has been recently reported that Hsp72 may be contained within exosomes that are released from various cell types [64
, 89
, 90
, 97
]. Exosomes are membranous vesicles that form within MVB and are secreted from cells when the membrane of the MVB fuses with the plasma membrane, and the internal vesicles become extracellular [98
]. It should be noted that membrane vesicles within MVB could have other fates besides being released as exosomes. For example, they may fuse with a lysosome to result in the degradation of proteins within the vesicle or fuse with the plasma membrane to result in the surface expression of proteins contained in the vesicle membrane [98
]. The surface expression of Hsp72 is thought to play an important role in cell-to-cell interactions involved in the recognition of tumors [56
]. In fact, tumor cell lines with higher surface expression of Hsp72 are more readily killed by immune cells [97
]. Conversely, exosomes are small vesicles that can diffuse and interact with distal cells. In addition to Hsp72, exosomes have been shown to contain other immunomodulatory molecules such as classic antigen-presenting molecules (MHC I and II) [99
100
101
], costimulatory molecules (CD80 and CD86) [101
, 102
], and adhesion molecules (CD54) [101
]. This provides exosomes with the necessary factors to stimulate potent immune responses [103
].
The association of Hsp72 with exosomes has been widespread across various cell types including B cells [89
], tumors [97
], and human PBMC [90
], although there appears to be some cellular specificity of Hsp72 expression/release within exosomes. For example, Hsp72 was only detected within the lumen of exosomes in B cells [89
] but was expressed on the exosome surface and lumen in tumor cells [97
]. Although there is agreement in the literature that release of Hsp72-containing exosomes occurs via a nonclassical protein transport pathway [90
, 97
, 104
, 105
], the exact mechanism may be cell type-specific, as disruption of lipid rafts prevents interferon-
-induced excretion of exosomes in tumor cells [97
] but has no effect on heat stress-induced exosome release in PBMC [90
]. Furthermore, there appears to be at least two mechanisms by which eHsp72 levels can increase via exosome release. First, a stimulus may increase the quantity of Hsp72-containing exosomes that are released, as observed in B lymphoblastoid cells following heat stress [89
]. Second, a stimulus may have no effect on the quantity of exosomes released but increases the concentration of Hsp72 within each exosome, as observed in PBMC following heat stress [90
]. This is clearly an early stage of our knowledge about the regulation of exosomes, and additional experiments are necessary to determine the critical factors (e.g., cell type, stimulus) involved in exosomal Hsp72 expression, release, and regulation.
Currently, it is unknown whether stress-induced elevations in circulating eHsp72 are associated with exosomes. As described previously, our laboratory has demonstrated that 1-ADRs play a critical role in triggering the elevation of circulating eHsp72 during times of stress [93
]. It has been well characterized that 1-ADR stimulation results in a Ca2+ influx in cells [106
], which is particularly interesting, as Ca2+ is an intracellular signal that has been observed to trigger the release of exosomes [107
]. In addition, 1-ADR stimulation is known to increase intracellular Hsp72 levels in many cell types [7
, 108
, 109
]; thus, it is possible that activation of 1-ADR stimulates exosome release and increases the concentration of Hsp72 stored within each exosome. Our current hypothesis is that the increase in circulating eHsp72 during stressor exposure is a result of the release of NE, which acts at 1-ADR to increase the concentration of Hsp72 contained within exosomes and to stimulate exosomal release from various cell types (Fig. 2)
.
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eHsp AND IMMUNITY
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Hsp, when expressed extracellularly, can modulate immunological responses [92
, 110
111
112
]. For example, eHsp72 can robustly stimulate inflammatory cytokine production and other innate immune responses [63
, 112
, 113
]. We and others have reported that eHsp72 in vitro stimulates inducible nitric oxide (NO) synthase [114
], NO [79
], TNF- [79
, 113
], interleukin (IL)-1ß [79
, 113
], and IL-6 [79
, 113
] production from macrophages and neutrophils. Finally, eHsp can lead directly to activation of the C1q and the complement cascade, independent of antibody [115
]. Thus, Hsp72 may play an important immunomodulatory role during times of stress, and the ability to manipulate levels of Hsp72 may be advantageous in the clinical setting when immunoenhancement or immunosuppression is desired.
There is evidence that TLR2 and/or TLR4 act as cell surface receptors for eHsp72, which transduce an inflammatory signal to innate immune cells {macrophages/dendritic cell (DC)/neutrophils [113
, 116
117
118
]}. Mammalian TLRs are transmembrane proteins that are evolutionarily conserved between primitive organisms (such as insects) and humans [119
]. Via stimulation of TLRs, eHsp72 exerts it effects on innate immune cells by stimulating the inflammatory myeloid differentiation primary-response protein 88/IL-1 receptor-associated kinase/nuclear factor (NF)-
B signal transduction pathway [117
]. Within 10 s of eHsp72 binding to monocytes or macrophages, there is an increase in intracellular Ca2+ [113
]. This is important, as it distinguishes eHsp72 signaling from LPS signaling, which does not induce Ca2+ flux [120
]. Based on work by Asea and colleagues [113
, 116
, 121
], eHsp72 induction of NF-B and inflammatory cytokines requires the expression of CD14, in addition to TLR2 and TLR4; thus, CD14 could function as a coreceptor for eHsp72 [113
]. It is interesting that binding CD14 plus TLR2 and/or TLR4 with selective receptor agonists (Pam3Cys binds TLR2, or Taxol binds TLR4) results in synergistic increases in NF-B [116
], suggesting that eHsp72 released into the blood after exposure to psychological and/or physical stressors may result in optimal stimulation of the inflammatory cascade only in the presence of CD14 activation.
Thus, facilitation of innate immune responses by eHsp72 after exposure to stress may be restricted to cells that express CD14 and/or are binding bacteria or LPS via CD14. We have previously proposed that acute stress-induced release of eHsp72 may act to prepare the immune system for possible, subsequent pathogenic challenge [78
]. Should a pathogenic challenge ensue, eHsp72 may potentiate a NO and/or cytokine response resulting in facilitated bacterial killing. If no pathogenic challenge occurs, then eHsp72 levels subside with minimal impact on innate immune cell production of NO and/or inflammatory cytokines. One exception may be if the host suffers from chronic inflammatory disease (e.g., atherosclerosis, Alzheimer’s, Crohn’s); then, stress-induced eHsp72 may exacerbate the disease state.
In a series of elegant studies by Lehner and colleagues, unique, immunological consequences of Hsp72 can be localized to specific domains of the Hsp72 molecule [58
, 59
, 122
, 123
]. For example, the C-terminal portion of Hsp72 [amino acids (aa) 359–610] stimulates production of chemokines, IL-12, TNF-, and NO, induces T helper cell type 1 polarization, and stimulates the maturation of DC. The N-terminal ATPase portion (aa 1–358) largely lacks these functions [58
, 59
, 122
, 123
]. The C-terminal portion of several species of Hsp72 (microbial and human) binds to CD14, TLR4, and CD40 on antigen-presenting cells (APC) [124
]. CD40-CD40 ligand interactions made between APC and T cells serve an important costimulatory role. Thus, Hsp72 may function to stimulate innate immunity via CD14 and TLRs and facilitate T cell responses via activation of CD40+ APC. This makes Hsp72 a molecule positioned to play an important role at the interface between innate and adaptive immunity [59
].
It is important to note that the immune effects of Hsp vary depending on several factors including the specific Hsp family (e.g., Hsp60, Hsp70, Hsp90), the cellular source of the Hsp (e.g., normal self, cancerous self, viral-infected self, bacterial), the cellular location of the Hsp (e.g., intracellular, cell surface, circulating), and the physiological circumstances of the Hsp expression (oxidative stress, bacterial infection, viral infection, psychological stress, physical stress, whole organism vs. localized cellular stress). Thus, the specific physiological context of these proteins greatly impacts their function, preventing any global statements about the in vivo, immunological functions of Hsp.
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IN VITRO IMMUNOLOGICAL ASSESSMENTS OF Hsp72 FUNCTION: PROCEED WITH CAUTION
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One issue that is a source of debate in this field is whether in vitro macrophage activation and/or inflammatory cytokine release induced by Hsp72 are instead actually a result of LPS contamination inherent in the recombinant Hsp72 (rHsp72). Gao and Tsuan [125
126
127
] have published several articles warning that researchers must consider the contribution of LPS contamination, which is inherent in rHsp when testing the immune function of these proteins in vitro. One observation that is reported to support this idea is that endotoxin-free recombinant human Hsp72 (free-rhHsp72) does not stimulate inflammatory cytokines from murine macrophages in vitro [125
]. We have now also measured levels of endotoxin in three commercially available rHsp72 products: low endotoxin rhHsp72 [low-rhHsp72; Stressgen #ESP-555, Stressgen Biotechnologies, Victoria, BC, Canada); rhHsp72 (Stressgen #NSP-555); and recombinant rat Hsp72 (rrHsp72; Stressgen #SPP-758). Although the low-rhHsp72 had undetectable levels of endotoxin as measured by the Limulus amebocyte lysate (LAL) assay (Cambrex, Walkersville, MD), the rhHsp72 and rrHsp72 had detectable levels that increased in a dose-dependent manner (Fig. 3A
). When the various rHsp72 products were added to cells collected from the peritoneal cavity of Fischer 344 male rats (95% macrophage) and cultured for 24 h at 37°C, it was found that the rhHsp72 and rrHsp72 stimulated the production of NO, TNF-, and IL-1ß in a dose-dependant manner, and low-rhHsp72 had no immunostimulatory effect (Fig. 3B 3C
3D)
. Cytokines were measured using ELISAs (R&D Systems, Minneapolis, MN), and nitrite was measured using the Griess reagent. Furthermore, preincubation of rhHsp72 and rrHsp72 for 30 min with polymyxin-B (Sigma Chemical Co., St. Louis, MO), a substance that neutralizes endotoxin by binding to the lipid A region, eliminated the induction of proinflammatory cytokines and NO by rhHsp72 and rrHsp72 (data not shown). Thus, we have replicated the observations by Gao and Tsuan [125
126
127
] and agree with their conclusion that in vitro testing of the immunological function of rhHsp72 or rrHsp72 must be conducted with caution.
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Figure 3. Relative endotoxin levels and cytokine induction of various rHsp72. (A) Endotoxin activity measured by the LAL assay of three commercially available Hsp72: low-rhHsp72 (Stressgen #ESP-555), rhHsp72 (Stressgen #NSP-555), and rrHsp72 (Stressgen #SPP-758). Peritoneal macrophages (1.0x106/ml) were cultured (37°C) with culture media (unstimulated) or three different concentrations of low-rhHsp72, rhHsp72, or rrHsp72; supernatants were collected 24 h later for measurement of (B) nitrite, (C) TNF-, and (D) IL-1ß. Bars represent means ± SE (n=6). N.D., Not determined.
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These results, however, do not diminish the results of many studies by researchers who have carefully isolated and purified Hsp72 and demonstrated that the intracellular signaling pathways used by LPS versus Hsp72 are unique and distinguishable [79
, 113
, 114
]. In addition, the immunomodulatory functions of endogenous eHsp72 released in vivo remain valid and need further investigating. In fact, the findings that rHsp72 may be associated with endotoxin peptides have stimulated a new series of studies designed to elucidate which peptides are associated with the Hsp72 released in vivo and if those peptides work in concert with the Hsp72 to stimulate immune function. It seems clear that endogenous Hsp72 released into the blood is likely not "naked." In fact, it has been suggested previously that considering the complex-forming ability and chaperoning function of Hsp72, in vivo free-Hsp is probably nonexistent [68
]. If it is the case that particular Hsp72/peptide complexes are actually necessary for immune stimulation, then it is possible, as suggested by Seong and Matzinger [128
], that the process of purifying Hsp72 may remove the fraction of Hsp72 which is immunostimulatory. In addition, as exposure to a variety of physical and psychological stressors stimulates bacterial translocation from the gut [129
130
131
132
133
134
], we speculate that perhaps some or all of the endogenous eHsp72 released by acute stress into the blood may, in fact, be associated with LPS released from endogenous gut bacterial flora. Quintana and Cohen [135
] offer the intriguing idea that eHsp72/LPS complexes may augment immunostimulatory responses by facilitating the transfer of LPS to the TLR4-MD2, leading to improved signal transduction and inflammatory cytokine responses. The immunological effects of eHsp are not restricted to innate immunity. Hsp72 can also stimulate DC antigen-presenting functions, thus facilitating antigen-specific T cell activity [56
, 58
, 65
, 111
, 136
, 137
]. Therefore, one beneficial aspect of the Hsp72/LPS complex may be its ability to stimulate innate and acquired immunity. Studies have begun in our laboratory to determine if elevated eHsp72 during times of stress stimulates innate and acquired immune responses.
Our laboratory has published a series of studies demonstrating that rats exposed to tail shock stress and then challenged with subcutaneous (s.c.) Escherichia coli have increased eHsp72 at the site of inflammation and that eHsp72 administered to the site of inflammation in the absence of stress-improved recovery from bacterial challenge [78
]. We hypothesize that the eHsp72 in the blood only interacts with immune cells if vasodilation and extravasation of circulating Hsp72 into an inflammatory site occur. In our experimental model, we produce an inflammatory site via the s.c. injection of live E. coli. We hypothesize that increased vascular leaking at the site of inflammation as a result of histamine release from mast cells allows eHsp72 to extravasate from the blood vessels into the tissue. In fact, we have tested this hypothesis by pretreating rats with vehicle or 40 mg/kg ketotifen (a selective histamine type 1 receptor antagonist that prevents vascular leaking) [138
, 139
] 30 min before exposure to stress (100, 5 s, 1.6 mA tail shocks) or no stress. Immediately after stressor exposure, all rats were injected s.c. with E. coli (2.5x108 colony-forming units), and the inflammatory sites were removed 90 min later. Inflammatory sites were homogenized and eHsp72 concentration measured by ELISA, and the number of live E. coli in the s.c. tissue was assessed by growth on McConkey agar plates. Replicating our previous work [78
], eHsp72 tissue concentrations were increased, and the number of E. coli decreased only in animals that were exposed to tail shock stress (and hence, had elevated blood eHsp72) and were injected with vehicle (and hence, had vascular leaking). In vivo ketotifen blocked the stress-induced increase of eHsp72 and the stress-induced decrease in bacterial load at the inflammatory site [82
]. These data support our hypothesis that eHsp72 can extravasate from the blood into inflamed tissue and suggest that something in the blood of stressed animals is necessary for the decrease in bacterial load at the inflammatory site.
Additional studies tested if it is the extravasation of eHsp72 at the site of inflammation that is necessary for stress-induced facilitation of innate immunity. In this study, rats were exposed to no stress or tail shock stress and immediately after stressor termination, injected with s.c. E. coli and anti-Hsp70-Ab46 or nonspecific control antibody. Anti-Hsp70-Ab46, generously provided by Dr. Asea, will immunoneutralize the inflammatory and NO-stimulatory function of eHsp72 when tested in vitro. Bacterial inflammation was monitored daily until resolution. Once again, stress reduced the number of days required to resolve the inflammatory site, and immunoneutralization of eHsp72 by anti-Hsp70-Ab46 at the site of inflammation prevented the beneficial effect of tail shock stress on bacterial inflammation resolution [82
]. Studies are currently being conducted to examine the bacterial load and cytokine response at the inflammatory site following immunoneutralization of eHsp72.
There is growing evidence to suggest that eHsp72 can enhance aspects of innate and acquired immunity. Although one must be careful of endotoxin contamination when working with recombinant proteins, there appear to be in vivo and in vitro data to support the fact that eHsp72 has immunomodulatory effects; however, more research is needed to determine if these effects are dependent on the peptides associated with the Hsp. Furthermore, the lack of a commercially available, selective Hsp72 antagonist and knowledge of the regulation of eHsp72 prevent studies, which could be designed to manipulate eHsp72 levels during immunological responses.
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SUMMARY AND CONCLUSIONS
|
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With accumulating data demonstrating the immunomodulatory effects of eHsp72 and thus, its potential for therapeutic use, it is important to understand its endogenous regulation. Beyond the fact that eHsp72 can be released during necrotic cell death and in the absence of detectable cell death, little is known about the signals that stimulate the release of in vivo eHsp72 or the cell types that release it. Research in our laboratory has focused on the in vivo releasing signals during times of stressor exposure. Our studies demonstrate that activation of 1-ADR is critical for elevating circulating eHsp72 after exposure to tail shock stress, as an 1-ADR but not a ß-ADR antagonist administered prior to stressor exposure blocked the stress-induced increase in circulating eHsp72. Furthermore, stimulation of 1-ADR but not ß-ADR is sufficient to elevate circulating eHsp72 in nonstressed animals, suggesting 1-ADR is necessary and sufficient to elevate eHsp72 levels. The cellular source of eHsp72 during times of stress is currently unknown.
In vitro studies have demonstrated that heat stress and/or cytokine stimulation can induce the release of Hsp72 from numerous cell types (e.g., tumor cells, B cells, PBMC, and glial cells) without measurable cell death. It is interesting that eHsp72 is often detected in association with exosomes, small vesicles formed within MVB that become extracellular when the MVB fuse with the outer cell membrane. The mechanism of exosomal release does not appear to use the classical protein transport pathway, as several studies have now demonstrated that the blockade of this pathway in different cell types has no effect on exosome release. However, numerous cellular differences have been reported regarding the association of Hsp72 with exosomes. For example, researchers examining B cells report that Hsp72 exists only in the lumen of exosomes, and Hsp72 is observed on the exosome surface of tumor and PBMC. In addition, there appear to be cellular differences regarding the packaging and release of Hsp72-containing exosomes. Upon stimulation, some cells exhibit increasing rates of exosomal release, and others have constant rates of exosomal release but increased concentrations of Hsp72 within each exosome. Clearly, future studies are necessary to better understand the mechanisms involved in eHsp72 regulation and release. This work will be critical for determining if manipulation of eHsp72 expression can be used for clinical purposes.
The immunomodulatory properties of eHsp72 are an exciting, new area of research, which has spurred a flurry of papers over the last several years. This review reiterates the warning voiced by Gao and Tsan [125
126
127
] that in vitro immunological tests of Hsp72 function must precede with caution as a result of endotoxin contamination in some recombinant forms of Hsp72. These data present interesting questions about whether it is eHsp72 itself or Hsp72 associated with various peptides that has immunomodulatory properties. Furthermore, we present in vivo data, which largely did not use rHsp72, to examine the immunoenhancing properties of endogenous eHsp72.
Exposure to a variety of stressors has been reported to increase endogenous, circulating eHsp72, and we propose that this circulating eHsp72 can extravasate into inflammatory sites within the organism, resulting in enhanced immunological responses. Our laboratory has demonstrated that animals exposed to tail shock stress have elevated, circulating levels of eHsp72 and that following a s.c. E. coli challenge, stressed animals have increased concentrations of eHsp72, increased levels of NO, and decreased numbers of live bacteria at the inflammatory site compared with nonstressed controls. In addition, stressed animals show a restricted, inflammatory response, as measured by the diameter of the inflammatory site, and a more rapid recovery compared with nonstressed controls. Data to date demonstrate that preventing extravasation of eHsp72 into the inflammatory site via preventing vascular leaking (i.e., administration of a histamine receptor antagonist) prevents the stress-induced decrease in bacteria number at the inflammatory site. Furthermore, immunoneutralizing eHsp72 at the site of inflammation blocks the restricted inflammatory response and enhances the time to recovery normally observed in stressed animals. In addition, s.c. administration of Hsp72 to nonstressed rats restricts the inflammatory response and facilitates the time to recovery following bacterial challenge. These data support the hypothesis that endogenous eHsp72 facilitates innate immune responses and has led us to suggest that the release of eHsp72 during times of stress is an adaptive feature of the acute stress response.
Received September 21, 2005;
revised October 10, 2005;
accepted October 14, 2005.
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ABSTRACT
INTRODUCTION