Chaperokine-Induced Signal Transduction Pathways

Abstract

A turning point in understanding the function of heat shock proteins (HSP) on components of the immune system has now begun. From their original description as intracellular molecular chaperones of naïve, aberrantly folded or mutated proteins and primarily involved in cytoprotection in response to stressful stimuli, in recent years, new functions of HSP have been revealed. Strong evidence now exists demonstrating that the seventy-kDa heat shock protein (HSP70) exits mammalian cells not only following necrotic cell death but also by a process involving its active release in response to stresses including cytokines, acute psychological stress and exercise. The released extracellular HSP70 interacts with cells of the immune system and exerts immunoregulatory effects - known as the chaperokine activity of HSP70. The chaperokine activity of HSP70 is mediated in part by utilizing surface receptors for both Toll-like receptor-2 (TLR2; receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) in a CD14-dependent fashion. These findings suggest an important role for heat shock proteins in host protection against pathogenic infection. This review will briefly discuss chaperokine-induced signaling and its relevance to infection and exercise.

Introduction

There is now a clear dichotomy between the previously ascribed functions of the seventy kilo-Dalton heat shock protein (HSP70), which can be accounted for due to its localization: intracellular or extracellular. Intracellular HSP70 is involved in protein chaperoning, transport and folding which are induced when cells are exposed to a variety of stresses most notably heat shock itself (17, 26). The upregulation of the expression of intracellular HSP70 protects cells from proteotoxic stress by a variety of “holding and folding” pathways that prevent the formation of denatured proteins and the progression of lethal aggregation cascades in cells and prevents cell death through both necrotic and apoptotic pathways (17). In contrast, extracellular HSP70 has been shown to specifically bind with high affinity to the plasma membrane of antigen presenting cells (APC), elicit a rapid intracellular [Ca²⁺] flux, activate NF-κB, and up-regulate the expression of pro-inflammatory cytokines including TNF-α, interleukin-1 beta (IL-1β) and IL-6 by antigen presenting cells, in a CD14-dependent fashion (3–5), by utilizing both Toll-like receptor-2 (TLR2; receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria).

The first line of defense against infectious agents including bacterial and viral infections are cells of the innate immune system, which are adorned with recognition structures called pattern recognition receptors (PRRs) (28, 29). PRRs such as Toll-Like Receptors (TLRs), CD14, β2-intergrins (CD11/CD18), complement receptors (CR1/CD35) and C-type lectins are expressed either as soluble proteins or plasma membrane-bound proteins that recognize invariant molecular structures called pathogen-associated molecular patterns (PAMPs) (28). Recent studies on the recognition of microbial PAMPs have highlighted the central role played by one group of PRRs, the TLR, in pathogen recognition and host defense (see review in (1, 22, 45)).

Pathogenic infections are a major problem for athletes undergoing intensive training (18, 23). The exact mechanism by which this occurs is hitherto unknown; however, exercise-induced alteration in numbers and effector functions of circulating neutrophils and monocytes (38), and in cytokine concentrations and kinetics (42) result in the increased susceptibility of individuals to infections. This review briefly discusses role of chaperokine-induced signaling and its relevance to infection and exercise.

Chaperokine-Induced Signaling

A variety of cells have been studied for their ability to bind HSP70, in particular natural killer (NK) cells (19, 31, 32) and APC including dendritic cells (5, 40), macrophages, peripheral blood monocytes (3, 4, 41), and B lymphocytes (2) efficiently bind HSP70. In contrast, T lymphocytes do not bind HSP70 (2). To date, a number of cell surface proteins have been described as the receptor for HSP70 including TLR2 and TLR4 with their cofactor CD14 (5), the scavenger receptor, CD36 (14, 36), and the co-stimulatory molecule, CD40 (7).

We recently addressed the steps involved in HSP70-induced signal transduction cascade and revealed that HSP70 binds with high affinity to the plasma membrane of APC and within 10 seconds elicits a rapid intracellular Ca²⁺ ([Ca²⁺]_I) flux (4). This is an important signaling step that distinguishes HSP70- from LPS-induced signaling, since treatment of APC with LPS does not result in [Ca²⁺]_i flux (27). The possibility that endotoxin contamination might confound our results was addressed by using Polymyxin B and Lipid IVa (LPS inhibitor) which abrogates LPS-induced but not HSP70-induced cytokine expression, boiling the proteins at 100°C for 1 h abrogates HSP70-induced but not LPS-induced cytokine expression. We noted that rapid HSP70-induced [Ca²⁺]_i flux is followed by the phosphorylation of I-κBα (4). Activation of NF-κB is regulated by its cytoplasmic inhibitor, I-κBα via phosphorylation at Serine 32 (Ser-32) and 36 (Ser-36) which targets it for degradation by the proteosome and releases NF-κB to migrate to the nucleus and activate the promoter of target genes (6). As early as 30 minutes post-exposure to exogenous HSP70, I-κBα was phosphorylated at Serine 32 (Ser-32) and 36 (Ser-36) resulting in the release and nuclear translocation of NF-κB (4). Mechanistic studies using the HEK293 model system revealed that HSP70-induced NF-κB promoter activity is MyD88-dependent, CD14-dependent and is transduced via both TLR2 and TLR4 (5). Further, the presence of both TLR2 and TLR4 synergistically stimulates HSP70-induced cytokine production (5). Interestingly, we found that the synergistic activation of NF-κB promoter by co-expression of both TLR2 and TLR4 is MyD88-independent, suggesting an alternative pathway by which exogenous HSP70 stimulates cells of the immune system. As early as 2–4 hours post-exposure of APC to exogenous HSP70, there is significant release of TNF-α, IL-1β, IL-6 and IL-12 (4, 5). Human monocytic cells, THP1 transfected with the dominant negative MyD88 plasmid or a combination of both dominant negative TLR2 and TLR4 inhibited HSP70-induced IL-1β expression (Fig. 1). However, only a combination of dominant negative MyD88/TLR2/TLR4 completely inhibited HSP70-induced IL-1β expression (Fig. 1). By 3–5 days post-exposure there is significant increase in proliferation of immature dentritic cells and augmentation of co-stimulatory molecules, MHC class II and CD86 (5).

Figure 1.

Chaperokine-induced IL-1β expression. Human monocytic cells THP1 were grown on Falcon CultreSlides (BD Labware, Franklin Lakes, NJ) overnight and co-transfected for 24 h with vector alone or dominant negative MyD88 (MyD88-DN) or dominant negative TLR2 (TLR2-DN) or dominant negative TLR4 (TLR4-DN) or a combination of TLR2-DN + TLR4-DN or TLR2-DN + TLR4-DN + MyD88-DN. Empty vector (Vector), was used as a control. Cells were then stimulated with 100 ng/ml HSP70 or control (not shown) at 37ºC for 4 h in the presence of 10 μM Brefeldin A (Sigma). Following this incubation period, cells were simultaneously fixed and permeabilized using Cytofix/Cytoperm™ kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions and counter stained with anti-human IL-1β-FITC (BD Biosciences). One drop of mounting media containing DAPI stain to visualize nuclear staining (Oncogene, Boston, MA) was placed onto the glass slide before the cover slip was sealed with nail polish. Results show fluorescence microscope pictograms of an overlay of a phase contrast image (PhC) and DAPI stain showing nuclear morphology in blue (PhC + DAPI; upper panel), and an overlay of a FITC stain showing intracellular IL-1β expression in green (IL-1β), and nuclear morphology (IL-1β + DAPI; bottom panel). Results are a representative of two independently performed experiments with similar results.

Another co-stimulatory molecule, CD40 expressed on APC and found to play an important role in B lymphocyte function and autoimmunity (8), has recently been demonstrated to bind HSP70-peptide complexes via its exoplasmic domain (7). These authors showed that the HSP70-CD40 interaction is mediated by the NH₂-terminal ATPase domain of HSP70 in its ADP-bound state and is strongly augmented by the presence of substrate peptides in the COOH-terminal domain of HSP70. This interaction was suppressed by Hip, a co-chaperone that is known to stabilize the HSP70 ATPase domain in the ADP state (7). Using the HEK293 cell model system, specific HSP70-CD40 binding was shown to stimulate signal transduction via the phosphorylation of p38 (previously shown to induce the release of TNF-α and secretion of IFN-γ (39)), and resultant activation of NF-κB and uptake of peptide (7).

The scavenger receptor, CD36 is another protein that has recently been shown to bind HSP70 (14). Specifically LOX-1, on human dendritic cells was shown to bind HSP70, while a neutralizing anti-LOX-1 monoclonal antibody (mAb) inhibited HSP70 binding to dendritic cells and abrogated HSP70-induced antigen cross-presentation (14). GST pull-down assays and immunoprecipitation analyses showed that HSP70, HSP90 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bind to the cytoplasmic domain of scavenger receptors (36). Furthermore, in vivo studies have shown that pre-treatment of animals with anti-LOX-1 mAb induces antitumor immunity (14).

Biological Significance

Although the exact nature of the HSP70 receptor is still being elucidated, studies on the expression of HSP70 on the surface of cells has been well defined. In comparison with immunocompetent cells, malignant tumor cells express high levels of surface bound HSP70 (9, 11, 21, 31, 33). This HSP70 expression on tumors correlates with an increased sensitivity to natural killer (NK)-mediated cytolysis following cytokine stimulation (10, 34, 35). Recent studies have shown that the cytolytic activity of HSP70 can also be transduced by specific fragments of the HSP70 protein. Both the full-length HSP70 protein and the C-terminal domain of HSP70 stimulates the cytolytic activity of naive NK cells against HSP70-positive tumor target cells (19). In addition, tumor growth in mice with severe combined immunodeficiency was shown to be the inhibited by HSP70-peptide-activated, CD94 positive natural killer cells (30). Recent work from the Multhoff laboratory demonstrates that a 14 amino acid sequence of the HSP70 protein, termed TKD (TKDNNLLGRFELSG, aa_450–463) is the extracellular recognition site for NK cells (35). These authors demonstrated that granzyme B specifically binds to portions of the HSP70 expressed on the plasma surface of tumors but not normal cells (20), thus demonstrating a hitherto unknown mechanism by which cytolytic effector cells eliminate HSP70 expressing tumors in a perforin-independent, granzyme B-dependent manner. These studies are in agreement with recent findings that immunization with the peptide binding C-terminal portion of HSP70 (aa_359–610) (HSP70_359–610) is responsible for stimulating Th1-polarizing cytokine (IL-12 and TNF-α), C-C chemokine release, and acts as an adjuvant (44). Immunization of nonhuman primates with HSP70_359–610 induced the production of RANTES and IL-12, and acted as an adjuvant when loaded with CC5-peptide (44), suggesting a possible alternative vaccine strategy for HIV infection (24, 25).

Recent studies have demonstrated that both acute psychological stress (12, 13, 37) and physical exercise (43) result in a marked increase in circulating serum HSP70 levels. A comparison of arterio-venous concentration differences showed that the source of HSP70 is not from the contracting skeletal muscle (16), but from the hepatosplanchnic tissues (15). It is hypothesized that the released circulating HSP70 serves as a chaperokine and enhances the host defense against pathogens during exercise.

Conclusion

Although the exact nature of the surface bound receptor for HSP70 is still under intense investigation, it is clear that immunocompetent cells bind exogenous HSP70 and this binding results in the chaperokine-induced transduction of signals important for the survival of the host against microbial pathogens (Fig. 2). This is important since both exercise and acute psychological stress stimulate the release of HSP70 into the serum where it might play an important role in host defense against pathogens. Examining the complete spectrum of the chaperokine activity of exogenous HSP70 is thus essential and will enable the design and development of highly effective pharmacological or molecular tools that could be used to either upregulate or suppress chaperokine-induced functions. This could include conditions where potentiation of chaperokine-induced pro-inflammatory responses is desirable (exercise, psychological stress, infectious diseases and cancer) or undesirable (arthritis and cardiovascular disease).

Figure 2.

Chaperokine-induced signal transduction pathways. Schematic representation of the main steps involved in chaperokine-induced signal transduction. Exogenous HSP70 binds to either TLR2 or TLR4 (characterized by a cytoplasmic Toll-/IL-1 receptor (TIR) domain and extracellular leucine-rich repeats), or CD40 or CD36 or a yet unknown receptor (?) at the surface of APC. Upon engagement of HSP70 with TLR2 or TLR4, the TLRs activate a downstream MyD88/IRAK signaling cascade which bifurcates at TRAF6 and results in NF-κB and c-Jun/ATF2/TCF activation (solid lines). Engagement of both TLR2 and 4 by exogenous HSP70 synergistically activates a signaling cascade that results in the MyD88-independent activation of NF-κB and c-Jun/ATF2/TCF, via TRAF6 (dashed lines). Engagement of HSP70 to CD40 activates the MyD88-independent activation p38 (dashed lines). Key; MyD88, myeloid differentiation factor 88; IRAK, IL-1R-associated kinase; TRAF, TNF-associated factor, MAPK, mitogen activated protein kinase; ERK, extracellular-signal regulated kinase; JNK, Jun N-terminal kinase.

Abbreviations used in this paper

APC

antigen presenting cells

ER

endoplasmic reticulum

HSC70

constitutive form of the seventy-kilo Dalton heat shock protein

HSP70

inducible form of the seventy-kilo Dalton heat shock protein

IFN-γ

interferon-gamma

IL

interleukin

TLR

Toll-Like Receptors