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), TNFα(tumour necrosis factor α), 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.
- dendritic cell
- heat-shock protein 70 (HSP70)
HSPs (heat-shock proteins or stress proteins) are intracellular chaperones of proteins. They are found both in mammalian cells and microorganisms, and are highly conserved . The 70 kDa HSP has acquired special significance in immunity, with the finding that CD40 is a receptor for both mHSP70 (microbial HSP70)  and human HSP70 .
HSP70 consists of a 44 kDa ATPase, an 18 kDa substrate-binding domain and a 10 kDa C-terminal fragment; crystal structures of these were determined separately [4–6]. The N-terminal ATPase domain has two lobes with a deep cleft that binds ATP. The C-terminal substrate-binding domain of the Escherichia coli HSP70 homologue, termed DnaK, consists of a β-sandwich and an α-helical subdomain. The β-sandwich subdomain is composed of two stacked anti-parallel four-stranded β-sheets. The upper sheet forms the substrate-binding site with loops L1,2 and L3,4, thus forming the sides of a channel, which is the primary site of interaction with the substrate. The outer loop L4,5 stabilizes L1,2 by forming hydrogen bonds and through hydrophobic interaction, whereas L5,6 forms hydrogen bonds that stabilize L3,4. The α-helical subdomain comprises five helices, with the first and second helices (αA and αB) forming hydrophobic side-chain contacts with the β-sandwich.
CD40 is a receptor for HSP70
CD40 is a 40–50 kDa glycoprotein, a member of the TNF (tumour necrosis factor) receptor superfamily, and is primarily expressed on B-cells, monocytes and DCs (dendritic cells) . CD40 can also be found on epithelial cells, some cancer cells and activated CD8+ T-cells [8,9] and plays an important role in T-cell-mediated immune responses. CD40 is crucial for T-cell-dependent B-cell activation, differentiation, immunoglobulin-class switching and germ-centre formation . CD40 is also involved in the activation of antigen-presenting cells, mediation of DC maturation, induction of CD8+ CTLs (cytotoxic T-lymphocytes) and generation of memory CD8 T-cells [9–11]. The natural ligand for CD40 is CD40 ligand (CD154), which is expressed by activated T-cells.
We reported previously that CD40 is a receptor for mHSP70  and this was confirmed and extended to human HSP70 . We postulated that the adjuvanticity of mHSP70 is accounted for by the stimulation of production of the CC chemokines CCL3, CCL4 and CCL5, which attract the entire repertoire of immunological cells . Since both the major co-stimulatory pathways CD80/86–CD28 and CD40–CD40L stimulate these CC chemokines [13–15], we explored the possibility that HSPs interact with one of the co-stimulatory molecules . Whereas antibodies to CD80 or CD86 had no effect, those to CD40 blocked the stimulation of CC chemokine production by HSP70. Furthermore, in depth investigation demonstrated that HSP70 stimulated the production of CC chemokines when HEK-293 cells (human embryonic kidney 293 cells) were transfected with human CD40, but not when transfected with control molecules. Immunoprecipitation studies revealed that HSP70 physically associates with cell-membrane CD40 when incubated with CD40-expressing cells, and surface plasmon resonance showed that HSP70 could directly bind to CD40 molecules . HSP70–peptide complexes binding CD40 deliver the peptide into the MHC class I pathway and this process is dependent on the ADP-loaded state of HSP70 .
CD40 mediates HSP70 stimulation of monocyte or monocyte-derived DCs, which are the principal antigen-presenting cells in priming CD4 and CD8 T-cell responses. Treatment of human monocyte-derived immature DC cultures for 2 days with mHSP70 induces significant changes in the cell-surface expression of MHC class II molecules, the co-stimulatory molecules CD80 and CD86 and the CD83, CCR7 maturation markers . The CC chemokines and Th1-polarizing cytokines IL-12 (interleukin-12) and TNFα are also produced, but the C-terminal portion (HSP70, amino acids 359–610) is more potent when compared with the full-length wild-type HSP70 in stimulating these cytokines and in DC maturation. In contrast, the N-terminal ATPase domain of HSP70 fails to up-regulate any of the DC phenotypes or cytokines. Indeed, there is evidence that the above function can be induced by the peptide-binding domain of mHSP70 (amino acids 359–494), and a stimulatory peptide epitope has been identified (Y. Wang, T. Whitall, E. McGowan, C.G. Kelly, L.A. Bergmeier, M. Singh and T. Lehner, unpublished work). Both human mHSP70 and mHSP70 bind the CD40 receptor. Surprisingly, the human ATPase domain of HSP70 binds one site, whereas a microbial C-terminal domain binds another site of the CD40 molecule [2,3,16]. It is not clear whether CD40L shares one of the two receptor sites on CD40 or whether it binds yet another site.
Despite the fact that HSP70 binds CD40, whereas LPS (lipopolysaccharide) binds CD14, it is important to exclude LPS contamination of HSP70 preparations, since some of the functional activities of the two molecules are similar. LPS activity can be abrogated or greatly reduced by polymixin B treatment [2,18], whereas HSP stimulation is decreased by heat denaturation [18,19]. Since the stimulating activity of HSP70 is calcium-dependent, the intracellular calcium chelator BAPTA-AM [where BAPTA stands for bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid] has been used to differentiate between LPS and HSP70 functions [2,18,20]. Furthermore, treatment with proteinase K also inhibits HSP70, but does not inhibit LPS-stimulating activity [17,20]. The C3H/HeJ and C57BL/10ScCr inbred mouse strains are homozygous for a mutant LPS allele (LPSd/d), which confers hyporesponsiveness to treatment with LPS  and provides a model to study the immunological functions of HSP. In studies of monocytes and DC using mHSP70, inhibition with antibodies to CD40 but not CD14 differentiates between HSP and LPS [2,16].
Role of HSP in innate immunity, adaptive immunity and vaccination
Adjuvants are essential components of vaccines against infections and vaccines against tumours and they elicit innate immune responses that drive specific immunity. mHSP70 has been used as a carrier molecule or adjuvant to enhance systemic immune responses when covalently linked to synthetic peptides [22–24]. Indeed, mHSP70 and HSP96 can be fused or covalently linked or loaded with peptides to elicit specific immunity to tumours or viruses [25–28]. The adjuvanticity of mHSP70 has been demonstrated not only by systemic immunization but also by mucosal immunization in non-human primates . Both systemic and mucosal adjuvanticities are dependent on the stimulation of production of three CC chemokines, CCL3, CCL4 and CCL5. CCL5 is a potent chemoattractant of monocytes, CD4 cells and activated CD8 cells [29–32]. CCL3 and CCL4 attract CD4+ T- and B-cells  and all three chemokines attract immature DC . Monocytes and DC internalize antigens, which are processed and presented on the cell surface. DC then undergo maturation and migrate to the regional lymph nodes and present the processed antigen to T- and B-cells, which elicit cellular and antibody responses.
Many important cytokines (IL-12, TNFα and NO) are also elicited by HSP70 and more efficiently by its C-terminal portion . Since IL-12 is one of the most potent cytokines inducing TH-1 polarization , it may be responsible for the TH-1-polarized adjuvanticity. The C-terminal portion of the HSP70-linked peptide elicits higher serum IgG2a and IgG3 subclasses of antibodies when compared with the native HSP70-bound peptide, consistent with a Th1-polarizing response . Furthermore, the Th2 type of cytokine (IL-4) was not produced in immunized macaques. Thus HSP70359–610 might be used as a microbial adjuvant that attracts the entire immunological repertoire of cells, by virtue of stimulating the production of CC chemokines and eliciting a Th1 response by generating IL-12.
The presence of HSP70 in most microorganisms [36,37] and their function in generating CC chemokines raises the possibility that the immunogenicity of whole organisms, which is well-recognized compared with that of a subunit antigen, is mediated by the CC chemokines generated by HSPs . This is consistent with the principle that the innate immune system may drive adaptive immunity [38,39]. HSP in microorganisms may function as a natural adjuvant generating CC chemokines and cytokines. This concept is also consistent with the ‘danger hypothesis’ of infection , with the HSP alerting the innate system to secrete CC chemokines and mobilizing the immune repertoire of cells to generate specific immune responses against the invading organism.
HSP70 may function as an alternative ligand to CD40L , stimulating the major co-stimulatory pathway CD40–CD40L. In mice lacking CD40 (CD40−/−), the production of IL-12 by bone marrow-derived DC was substantially reduced after HSP70 stimulation . In these CD40−/− mice, HSP70 failed to enhance DC function or to prime CD4+ and CD8+ T-cell antigen-specific responses, and protection from Mycobacterium tuberculosis infection depended on the alternative HSP70–CD40 co-stimulatory pathway . Furthermore, overexpression of HSP70 in M. tuberculosis resulted in impaired infection during the chronic phase  and this may also be interpreted as being due to the interaction between the increased amounts of HSP70 and CD40 on macrophages and DC, which enhance immunity to the organism. As reported in another study, co-administration of the tolerogenic LCMV peptide with human HSP70 leads to interaction with CD40 and may reverse tolerance and promote DC to induce autoimmune diabetes . Applications of HSP70 as a carrier of HIV gp120, SIVp27 and peptides derived from CCR5 in mucosal vaccination have been demonstrated recently in Rhesus macaques . Significant protection against SHIV 89.6P was associated with specific serum and secretory antibodies, IL-2 and interferon-γ stimulated by the vaccine components and an increased concentration of CC chemokines, which was inversely correlated with the proportion of CCR5+ cells .
CD8+ CTL can be generated by loading LCMV peptides on to human HSP70, which elicits protective anti-viral immunity in mice . Recently, human anti-influenza CTLs were generated by pulsing DC with mHSP70 loaded with peptides from influenza virus; the CTL response was significantly greater than pulsing DC with peptides alone . There is also evidence that, using human HSP70, NK cells (natural killer cells) can be stimulated to proliferate and this function resides in the C-terminal portion of HSP70 . Cell surface-bound HSP70 found on some tumour cells may induce migration of and cytolysis by CD56+CD94+ NK cells . A peptide (amino acids 450–463) that enhances NK cell activity has been identified within the sequence of human HSP70. A signal peptide derived from HSP60 has also been identified, which binds HLA-E, interferes with CD94/NKG2A recognition and enables NK cells to detect stressed cells . HSP70 up-regulates γδ+ T-cells both in vitro and in vivo in non-human primates and they generate CD8-suppressor factors and CC chemokines . Indeed, a significant increase in γδ+ T-cells was found in rectal mucosal tissue and in the draining lymph nodes of macaques immunized with SIVgp120 and p27 and protected from rectal mucosal challenge by SIV . Thus HSP70 plays an important role in eliciting innate immunity, generating chemokines and cytokines, maturation of DC and up-regulation of NK and γδ T-cells, all of which have significant effects on adaptive immunity.
Heat Shock Proteins and Modulation of Cellular Function: Focused Meeting held at Guy's Hospital, London, U.K. Organized by C. Kelly (King's College London) and I. Dransfield (MRC Centre for Inflammatory Research, Edinburgh). Edited By C. Kelly.
Abbreviations: CTL, cytotoxic T-lymphocyte; DC, dendritic cell; HSP, heat-shock protein; mHSP70, microbial HSP70; IL-12, interleukin-12; LPS, lipopolysaccharide; NK cell, natural killer cell; TNFα, tumour necrosis factor α
- © 2004 The Biochemical Society