Different heat shock protein 60 species share pro-inflammatory activity but not binding sites on macrophages

In a study of seven different hsp60 species, we found that all mammalian and microbial proteins shared the property of eliciting an inflammatory response in mouse macrophages. In all cases, TNFalpha production was induced by 0.1 microM concentrations of hsp60. However, the different hsp60 preparations did not compete for the same binding site. The binding of fluorescence-labeled human hsp60 was inhibited by excess unlabeled human, rat or mouse hsp60, but not hamster, Escherichia coli, Chlamydia pneumoniae or Mycobacterium bovis hsp60. We conclude that phylogenetically separate hsp60 species interact with innate immune cells via different recognition pathways.     

Bacterial heat shock protein 60 may increase epithelial cell migration through activation of MAP kinases and inhibition of alpha6beta4 integrin expression

Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.     

Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis

Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.     

The receptor for heat shock protein 60 on macrophages is saturable, specific, and distinct from receptors for other heat shock proteins.

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4 degrees C was followed by endocytosis at 37 degrees C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the alpha(2)-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.     

Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae

AIMS: To determine the efficacy of solar disinfection (SODIS) for enteric pathogens and to test applicability of the reciprocity law. METHODS AND RESULTS: Resistance to sunlight at 37 degrees C based on F99 values was in the following order: Salmonella Typhimurium>Escherichia coli>Shigella flexneri>Vibrio cholerae. While F90 values of Salm. Typhimurium and E. coli were similar, F99 values differed by 60% due to different inactivation curve shapes. Efficacy seemed not to be dependent on fluence rate for E. coli stationary cells. Sensitivity to mild heat was observed above a temperature of 45 degrees C for E. coli, Salm. Typhimurium and Sh. flexneri, while V. cholerae was already susceptible above 40 degrees C. CONCLUSIONS: Salmonella Typhimurium was the most resistant and V. cholerae the least resistant enteric strain. The reciprocity law is applicable for stationary E. coli cells irradiated with sunlight or artificial sunlight. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli might not be the appropriate indicator bacterium to test the efficacy of SODIS on enteric bacteria and the physiological response to SODIS might be different among enteric bacteria. The applicability of the reciprocity law indicates that fluence rate plays a secondary role in SODIS efficacy. Stating inactivation efficacy with T90 or F90 values without showing original data is inadequate for SODIS studies.     

Human 60-kDa heat-shock protein: a danger signal to the innate immune system

Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-alpha; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-gamma in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation.     

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.     

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Evolution of the chaperonin families (HSP60, HSP 10 and TCP-1) of proteins and the origin of eukaryotic cells

The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ-Gram-negative purple bacteria —α-purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp-1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

Association of humoral immunity to human Hsp60 with the IL-6 gene polymorphism C-174G in patients with type 2 diabetes and controls.

The relationship between humoral immunity to hsp60 and type 2 diabetes along with other relevant metabolic, inflammatory and immunogenetic variables was studied in 76 non-diabetic and 74 diabetic persons aged 55-74 years selected from the population-based KORA Survey 2000. Antibodies to human hsp60 were measured in serum samples by ELISA. Hsp60 antibodies were detected in all but two individuals in a considerable range of titres (22-1,856 AU/ml). There was no significant association to age and sex, or to key clinical or metabolic parameters (BMI, WHR, HbA1c, total cholesterol, LDL cholesterol, HDL cholesterol, systolic and diastolic blood pressure, albumin, uric acid) or immunological parameters (CRP, IL-6, sIL-6R, TNFalpha, sTNFalpha R60, sTNFalpha R80). Analysis of antibody-positive individuals revealed an association between hsp60 antibodies and diabetes at borderline significance (p = 0.047), which was lost when the two antibody-negative individuals were included. Genetic analyses indicated that this association was significant in carriers of the C allele of the IL-6 promoter region polymorphism at nucleotide -174 (p = 0.02), but not in GG genotype carriers. We conclude that humoral immunity to human hsp60 may be enhanced in those diabetic patients carrying the -174C allele of the IL-6 gene. This finding may contribute to an understanding of the relationship between the -174C allele and increased risk of atherosclerosis.     

The promoter polymorphism of the IL-6 gene is associated with levels of antibodies to 60-kDa heat-shock proteins

Elevated levels of antibodies to 60 kDa heat shock proteins are associated with severe coronary heart disease and carotid atherosclerosis. The presence of self hsp60-reacting antibodies can only be partially explained by microbial infections and induction by bacterial hsp65 proteins, since important differences (including the epitope specificity and complement activating ability) between hsp60 and hsp65 reacting antibodies have been shown. The aim of this study was to investigate the possible effects of genetic polymorphisms of different genes of proinflammatory cytokines on anti-hsp60 autoantibody levels. One hundred and seventy-six male blood donors were recruited and antibody levels to human hsp60 and Mycobacterium bovis hsp65 were determined by ELISA. Also in these donors, polymorphisms of the promoter of the IL-6 gene at position -174, the biallelic base exchange of the IL-1 beta gene at the -511 position and the IL-1 alpha gene at position -889 were investigated by PCR. A strong association between IL-6 -174 polymorphism and anti-hsp60 antibody levels was seen; the effect on anti-hsp65 antibody was less marked. Carriers of allele C at this position had significantly lower levels of anti-hsp60 and anti-hsp65 antibodies. A lack of associations between IL-1 beta and IL-1 alpha gene polymorphisms and antibody levels was detected. This is the first study in which associations between genetic polymorphisms and autoantibody levels have been described in healthy subjects. Further studies are needed to gain insight into the detailed mechanism of how the IL-6 gene polymorphism at position -174 influences anti-hsp60 autoantibody levels.     

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

Heat shock protein 60 (hsp60) is a highly conserved stress protein and target of self-reactive T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance between T helper cells type 2 and regulatory T cells, it is of interest to know whether hsp60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether hsp60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expression of hsp60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with hsp60 and proliferative responses, cytokine secretion, surface markers, and functional assays were compared to responses of PBMC of healthy controls (HC). Hsp60 was detected more in lesional skin of AD patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to hsp60 compared to HC. hsp60-reactive T cells of atopic children produced high levels of IFNγ and low levels of IL-10. In vitro activation with hsp60 leads to the induction of CD4⁺CD25^bright T cells expressing FOXP3 in both HC as well as in atopic children. However, despite their regulatory phenotype, hsp60-induced CD4⁺CD25^brightCD127⁻FOXP3⁺ T cells of AD patients were incapable of suppressing effector T cells in vitro. hsp60 is recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that hsp60-specific T cell responses contribute to local inflammation in AD.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0361-3) contains supplementary material, which is available to authorized users.     

Loss of mitochondrial hsp60 function: nonequivalent effects on matrix-targeted and intermembrane-targeted proteins.

We have created yeast strains in which the mitochondrial chaperonin, hsp60, can be either physically depleted or functionally inactivated. Cells completely depleted of hsp60 stop growing but retain for awhile the capacity to reaccumulate hsp60. While this newly made hsp60 is targeted to and processed correctly within the mitochondrion, assembly of a functional hsp60 complex does not occur. Rather, the hsp60 monomers are localized in different-size soluble complexes containing another mitochondrial chaperone, the mitochondrial form of hsp70. A number of other mitochondrial matrix-targeted proteins synthesized in the absence of functional hsp60 are imported into mitochondria but often show some buildup of precursor forms and, unlike hsp60, accumulate as insoluble aggregates. By contrast, several mitochondrial proteins normally targeted to the intermembrane space show normal processing in the complete absence of a functional hsp60 complex. Similar and complementary results were obtained when we examined the metabolism of matrix- and intermembrane space-localized proteins in cells expressing three different temperature-sensitive alleles of HSP60. In all cases, matrix-targeted proteins synthesized at nonpermissive (i.e., hsp60-inactivating) temperatures were correctly targeted to and processed within mitochondria but accumulated predominantly or totally as insoluble aggregates. The metabolism of two intermembrane space proteins, cytochrome b2 and cytochrome c1, was unaffected at the nonpermissive temperature, as judged by the correct processing and complete solubility of newly synthesized forms of both proteins. These findings are discussed with regard to current models of intermembrane targeting.     

Function of the maize mitochondrial chaperonin hsp60: specific association between hsp60 and newly synthesized F1-ATPase alpha subunits.

Mitochondria contain a protein, hsp60, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp60, we looked for association between hsp60 and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp60. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp60 was the alpha subunit of the multicomponent complex F1-ATPase. We have not identified the other protein. These results indicate that hsp60 can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.     

Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex

Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-alpha and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.     

Heat shock protein 60 elicits abnormal response in macrophages of diabetes-prone non-obese diabetic mice

Heat shock protein 60 (hsp60) is a target antigen in autoimmune diabetes and injections of human hsp60 for tolerance induction were found to protect non-obese diabetic (NOD) mice, an animal model of human type 1 diabetes, from disease development. We tested whether innate immune cells of NOD mice exhibit an abnormal response to extracellular hsp60. Bone marrow derived macrophages (BMM) were grown from NOD, C57BL/6J, non-obese non-diabetic (NON) mice, and NOD-related congenic variants differing in the Idd-3, Idd-10/18, or major histocompatibility complex (MHC) region. Hsp60-stimulated BMM of NOD mice were found to produce high levels of interleukin (IL)-12(p70). The addition of IL-10 downregulated, whereas cyclooxygenase inhibitors elevated, IL-12(p70) production of activated BMM. BMM of NON, NON-NOD-H-2(g7) as well as of NOD-NON-H-2(nbl) mice produced significantly less IL-12(p70) than BMM of NOD mice, indicating that an interaction between the MHC haplotype and non-MHC genes of the NOD mouse is required for hyperresponsiveness to hsp60.