DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis: identification of a regulatory HSP60 peptide

Adjuvant arthritis (AA) is induced by immunizing Lewis rats with Mycobacterium tuberculosis suspended in adjuvant. The mycobacterial 65-kDa heat shock protein (HSP65) contains at least one epitope associated with the pathogenesis of AA: T cell clones that recognize an epitope formed by aa 180-188 of HSP65 react with self-cartilage and can adoptively transfer AA. Nevertheless, vaccination with HSP65 or some of its T cell epitopes can prevent AA by a mechanism that seems to involve cross-reactivity with the self-60-kDa HSP60. We recently demonstrated that DNA vaccination with the human hsp60 gene can inhibit AA. In the present work, we searched for regulatory epitopes using DNA vaccination with HSP60 gene fragments. We now report that specific HSP60 DNA fragments can serve as effective vaccines. Using overlapping HSP60 peptides, we identified a regulatory peptide (Hu3) that was specifically recognized by the T cells of DNA-vaccinated rats. Vaccination with Hu3, or transfer of splenocytes from Hu3-vaccinated rats, inhibited the development of AA. Vaccination with the mycobacterial homologue of Hu3 had no effect. Effective DNA or peptide vaccination was associated with enhanced T cell proliferation to a variety of disease-associated Ags, along with a Th2/3-like shift (down-regulation of IFN-gamma secretion and enhanced secretion of IL-10 and/or tumor growth factor beta1) in response to peptide Mt176-190 (the 180-188 epitope of HSP65). The regulatory response to HSP60 or its Hu3 epitope included both Th1 (IFN-gamma) and Th2/3 (IL-10/tumor growth factor beta1) secretors. These results show that regulatory mechanisms can be activated by immunization with relevant self-HSP60 epitopes.     

DNA vaccination with heat shock protein 60 inhibits cyclophosphamide-accelerated diabetes

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-gamma secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of beta cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.     

Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-gamma by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.     

Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa heat shock protein autoimmunity

Nonobese diabetic (NOD) mice develop insulitis and diabetes through a process involving autoimmunity to the 60-kDa heat shock protein (HSP60). Treatment of NOD mice with HSP60 or with peptides derived from HSP60 inhibits this diabetogenic process. We now report that NOD diabetes can be inhibited by vaccination with a DNA construct encoding human HSP60, with the pcDNA3 empty vector, or with an oligonucleotide containing the CpG motif. Prevention of diabetes was associated with a decrease in the degree of insulitis and with down-regulation of spontaneous proliferative T cell responses to HSP60 and its peptide p277. Moreover, both the pcDNA3 vector and the CpG oligonucleotide induced specific Abs, primarily of the IgG2b isotype, to HSP60 and p277, and not to other islet Ags (glutamic acid decarboxylase or insulin) or to an unrelated recombinant Ag expressed in bacteria (GST). The IgG2b isotype of the specific Abs together with the decrease in T cell proliferative responses indicate a shift of the autoimmune process to a Th2 type in treated mice. These results suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.     

Modulatory effects of the human heat shock protein 70 on DNA vaccination

DNA vaccination with the plasmid expressing Japanese encephalitis virus (JEV) nonstructural protein 1 (pJNS1) has been shown to induce effective immunity against JEV infection. To further increase the efficacy of pJNS1 DNA vaccination, we coinjected pJNS1 with a plasmid that expresses heat shock protein 70.1 (pHSP70.1) into mice. We found that coinjection of pHSP70.1 enhanced both T cell proliferation and cytotoxic effects, but not the antibody response to JEV. Moreover, mice immunized with both pHSP70.1 and pJNS1 were resistant to lethal challenges of JEV, indicating that the protective immunity against JEV is not decreased, in spite of the low antibody titer via the immunization of pHSP70.1. Since DNA vaccination administered by pJNS1 did not elicit strong cellular immunity in our previous study, the administration of pHSP70.1 apparently could be used as an adjuvant to enhance cell-mediated immunity in this model system. Thus, coadministration of pHSP70.1 DNA with plasmid DNA encoding tumor- or virus-specific antigens might be very useful in the treatment of cancers and other infectious diseases.     

Human 60-kDa heat shock protein is a target autoantigen of T cells derived from atherosclerotic plaques

Epidemiological studies suggest the potential importance of an inflammatory component in atherosclerosis and support the hypothesis that immune responses to Ags of pathogens cross-react with homologous host proteins due to molecular mimicry. Protein candidates involved may be the stress-induced proteins known as heat shock proteins (HSP). In this study, we report that atherosclerotic plaques harbor in vivo-activated CD4(+) T cells that recognize the human 60-kDa HSP. Such in vivo-activated 60-kDa HSP-specific T cells are not detectable in the peripheral blood. In patients with positive serology and PCR for Chlamydia pneumoniae DNA, but not in patients negative for both, most of plaque-derived T cells specific for human 60-kDa HSP also recognized the C. pneumoniae 60-kDa HSP. We characterized the submolecular specificity of such 60-kDa HSP-specific plaque-derived T cells and identified both the self- and cross-reactive epitopes of that autoantigen. On challenge with human 60-kDa HSP, most of the plaque-derived T cells expressed Th type 1 functions, including cytotoxicity and help for monocyte tissue factor production. We suggest that arterial endothelial cells, undergoing classical atherosclerosis risk factors and conditioned by Th type 1 cytokines, express self 60-kDa HSP, which becomes target for both autoreactive T cells and cross-reactive T cells to microbial 60-kDa HSP via a mechanism of molecular mimicry. This hypothesis is in agreement with the notion that immunization with HSP exacerbates atherosclerosis, whereas immunosuppression and T cell depletion prevent the formation of arteriosclerotic lesions in experimental animals.     

Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats.

The present study examined the expression of 73-kDa of heat shock cognate protein (HSC70), 72-kDa of heat shock protein (HSP70) and 47-kDa of HSP (HSP47) observed in the ulcer healing process in rats. Gastric ulcers were induced by a luminal application of acetic acid in male Donryu rats. During the ulcer healing process, the expression of HSPs in the ulcerated tissue was determined. A high level of HSC70 expression was observed both in the normal mucosa and ulcerated tissue, but the level did not change upon ulceration and ulcer healing. While HSP70 and HSP47 were markedly expressed in the ulcer base during ulceration, and decreased with ulcer healing. HSP70 expression in the ulcer margin was gradually increased with ulcer healing. Omeprazole accelerated the healing of gastric ulcers with strong inhibition of gastric acid secretion, while indomethactin delayed in ulcer healing despite slight inhibition of gastric acid secretion. Omperazole enhanced the expression of HSP70 both in the ulcer margin and base, but it reduced HSP47 expression in the ulcer base Indomethacin markedly enhanced HSP47 expression only in the ulcer base. In conclusion, the expression of HSP70 and HSP47 is changed during ulcer healing. Furthermore, it was suggested that the enhanced expression of HSP70 is involved in acceleration of ulcer healing, but overexpression of HSP47 is involved in delayed ulcer healing.     

Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Abstract Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti- Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.     

The regulatory C-terminal determinants within mycobacterial heat shock protein 65 are cryptic and cross-reactive with the dominant self homologs: implications for the pathogenesis of autoimmune arthritis

The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in the Lewis rat (RT.1(l)) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD, but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Stress-induced release of HSC70 from human tumors

In this study, we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFN-gamma) induces the active release of the constitutive form of the 70-kDa heat shock protein (HSC70) from K562 erythroleukemic cells. Treatment of K562 cells with IFN-gamma induced the upregulation of the inducible form of the 70-kDa heat shock protein (HSP70), but not the constitutive form of HSC70 within the cytosol, in a proteasome-dependent manner. In addition, IFN-gamma induced the downregulation of surface-bound HSC70, but did not significantly alter surface-bound HSP70 expression. These findings indicate that HSC70 can be actively released from tumor cells and is indicative of a previously unknown mechanism by which immune modulators stimulate the release of intracellular HSC70. This mechanism may account for the potent chaperokine activity of heat shock proteins recently observed during heat shock protein-based immunotherapy against a variety of cancers.     

Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.     

Immunization with heat shock protein 105-pulsed dendritic cells leads to tumor rejection in mice

Recently, we reported that heat shock protein 105 (HSP105) DNA vaccination induced anti-tumor immunity. In this study, we set up a preclinical study to investigate the usefulness of dendritic cells (DCs) pulsed with mouse HSP105 as a whole protein for cancer immunotherapy in vivo. The recombinant HSP105 did not induce DC maturation, and the mice vaccinated with HSP105-pulsed BM-DCs were markedly prevented from the growth of subcutaneous tumors, accompanied with a massive infiltration of both CD4+ T cells and CD8+ T cells into the tumors. In depletion experiments, we proved that both CD4+ T cells and CD8+ T cells play a crucial role in anti-tumor immunity. Both CD4+ T cells and CD8+ T cells specific to HSP105 were induced by stimulation with HSP105-pulsed DCs. As a result, vaccination of mice with BM-DCs pulsed with HSP105 itself could elicit a stronger tumor rejection in comparison to DNA vaccination.     

Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles.

Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.     

HSP60 as a Target of Anti-Ergotypic Regulatory T Cells

The 60 kDa heat shock protein (HSP60) has been reported to influence T-cell responses in two ways: as a ligand of toll-like receptor 2 signalling and as an antigen. Here we describe a new mechanism of T-cell immuno-regulation focused on HSP60: HSP60 is up-regulated and presented by activated T cells (HSP60 is an ergotope) to regulatory (anti-ergotypic) T cells. Presentation of HSP60 by activated T cells was found to be MHC-restricted and dependent on accessory molecules - CD28, CD80 and CD86. Anti-ergotypic T cells responded to T-cell HSP60 by proliferation and secreted IFNγ and TGFβ1. In vitro, the anti-ergotypic T cells inhibited IFNγ production by their activated T-cell targets. In vivo, adoptive transfer of an anti-ergotypic HSP60-specific T-cell line led to decreased secretion of IFNγ by arthritogenic T cells and ameliorated adjuvant arthritis (AA). Thus, the presentation of HSP60 by activated T cells turns them into targets for anti-ergotypic regulatory T cells specific for HSP60. However, the direct interaction between the anti-ergotypic T regulators (anti-HSP60) and the activated T cells also down-regulated the regulators. Thus, by functioning as an ergotope, HSP60 can control both the effector T cells and the regulatory HSP60-specific T cells that control them.     

T cell and antibody reactivity with the Borrelia burgdorferi 60-kDa heat shock protein in Lyme arthritis.

The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes.