Heat shock proteins (Hsps) can be found in two forms, intracellular and extracellular. The intracellular Hsps are induced as a result of stress and have been found to be cytoprotective in many instances due to their chaperone functions in protein folding and in protein degradation. The origin and role of extracellular Hsps is less clear. Although they were suspected originally to be released from damaged cells (necrosis), their presence in most normal individuals rather suggests that they have regulatory functions in circulation. As immunodominant molecules, Hsps can stimulate the immune system, leading to the production of autoantibodies recognizing epitopes shared by microbial and human Hsps. Thus, extracellular Hsps can influence the inflammatory response as evidenced by the production of inflammatory cytokines. Antibodies to Hsps have been found under normal conditions but seem to be increased in certain stresses and diseases. Such antibodies could regulate the inflammatory response positively or negatively. Here, we review the literature on the findings of antibodies to Hsps in situations of environmental or occupational stress and in a number of diseases and discuss their possible significance for the diagnosis, prognosis, or pathogenesis of these diseases.

Toxoplasma gondii –derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow–derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-β mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.

Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.

Heat shock protein (Hsp)–peptide complexes purified from tumors can prime the immune system against tumor antigens, but how they contribute to the generation of immune responses against naturally occurring tumors is unknown. Murine tumors expressing high amounts of Hsp70 are preferentially rejected by the immune system, suggesting that low Hsp70 expression is advantageous for tumor growth in the host. To determine whether Hsp70 was differentially expressed in human tumors, inducible Hsp70 expression was quantitatively (by Western blot) and qualitatively (by immunohistology) analyzed in 53 biopsies of tumor and normal breast tissue. The mean expression of inducible Hsp70 was significantly higher in tumor compared with normal tissue (U = 899.0; P = 0.0033). However, a significant negative association of the amount of Hsp70 expressed by tumor tissue was found with metastasis (r² = −0.309; P = 0.05). After 3 years, follow-up analysis determined that 7 of the 53 patients relapsed, and 5 died. Hsp70 expression in tumor (but not normal) cells was significantly lower in relapse patients and patients with metastatic disease than in patients with no relapse or metastasis. Together, these observations support the hypothesis that Hsp70 plays a role in tumor expansion in vivo, and tumors that downregulate it may be able to evade immunosurveillance and grow.

Expression of heat shock proteins is a cellular response to a variety of stressors. HSP70, the major stress-induced heat shock protein, is involved in repair and protection after the insult. However, the prolonged presence of this protein is detrimental. Consequently, Hsp70 expression must be tightly regulated. We have previously shown an increase in the degradation of Hsp70 messenger ribonucleic acid (mRNA) paralleling the accumulation of HSP70. Incubation of cells with transcriptional and translational inhibitors after heat shock resulted in a significant reduction in Hsp70 mRNA degradation. These observations suggest that newly synthesized, stress-induced factors might be involved in the decay of Hsp70 mRNA. We found that HSP70 binds directly to Hsp70 mRNA, as demonstrated by immunoprecipitation. This observation was confirmed by RNA gel-shift assays. These results are evidence for a novel and likely direct interaction between HSP70 and Hsp70 mRNA in cells after stress. This interaction may be part of a self-limiting mechanism to reduce HSP70 production, thus avoiding potential toxic effects of this protein in the absence of stress.

The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42°C, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.

The human genome codes for 10 so-called mammalian small heat shock or stress proteins (sHsp) with the various tissues expressing characteristic sets of sHsps. Most sHsps interact with each other and form homo- and hetero-oligomeric complexes. Some of the sHsps are phosphoproteins in vivo, and phosphorylation has been implicated in the regulation of complex size and composition. In this study, we analyze, by the 2-hybrid method, the reporter gene activation pattern of several sHsp pairs that previously have been demonstrated to interact. We show that pseudophosphorylation (mimicry of phosphorylation) of the homologous phosphorylation sites Ser15 and Ser16 in Hsp27 and Hsp20, respectively, modulates characteristics of these sHsps that can be detected by their ability to activate reporter genes in suitable 2-hybrid assays. Pseudophosphorylation of the separated N-terminus of Hsp27 alone is not sufficient for the activation of the reporter genes, whereas the separated C-terminus is sufficient. We conclude that pseudophosphorylation of Hsp27 and Hsp20 at their N-termini results in conformational changes that can be detected by their interaction with other sHsps. Pseudophosphorylation of αB-crystallin at Ser19, in contrast, had no detectable consequences.

p26, an abundantly expressed small heat shock protein, is thought to establish stress resistance in oviparously developing embryos of the crustacean Artemia franciscana by preventing irreversible protein denaturation, but it might also promote survival by inhibiting apoptosis. To test this possibility, stably transfected mammalian cells producing p26 were generated and their ability to resist apoptosis induction determined. Examination of immunofluorescently stained transfected 293H cells by confocal microscopy demonstrated p26 is diffusely distributed in the cytoplasm with a minor amount of the protein in nuclei. As shown by immunoprobing of Western blots, p26 constituted approximately 0.6% of soluble cell protein. p26 localization and quantity were unchanged during prolonged culture, and the protein had no apparent ill effects on transfected cells. Molecular sieve chromatography in Sepharose 6B revealed p26 oligomers of about 20 monomers, with a second fraction occurring as larger aggregates. A similar pattern was observed in sucrose gradients, but overall oligomer size was smaller. Mammalian cells containing p26 were more thermotolerant than cells transfected with the expression vector only, and as measured by annexin V labeling, Hoescht 33342 nuclear staining and procaspase-3 activation, transfected cells effectively resisted apoptosis induction by heat and staurosporine. The ability to confer thermotolerance and limit heat-induced apoptosis is important because Artemia embryos are frequently exposed to high temperature in their natural habitat. p26 also blocked apoptosis in transfected cells during drying and rehydration, findings with direct relevance to Artemia life history characteristics because desiccation terminates cyst diapause. Thus, in addition to functioning as a molecular chaperone, p26 inhibits apoptosis, an activity shared by other small heat shock proteins and with the potential to play an important role during Artemia embryo development.

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42°C or to 15dPGJ₂ for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ₂ treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ₂ are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ₂ is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ₂ up to a concentration of 1 μg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

Heat stress (HS) reduces the many sequelae of lipopolysaccharide (LPS)-induced endotoxemia. Without HS, endotoxins have been shown to induce a transcriptional down-regulation of hepatocyte transport proteins for bile acids and organic anions. We performed experiments in isolated perfused rat livers at various times after LPS administration with and without HS pretreatment to determine whether HS would correct deficient transport of bromosulfophthalein (BSP). Possible mechanisms involved were investigated in livers from intact animals. In isolated perfused livers, LPS injection reduced BSP excretion to 48% compared with saline-injected controls (P < 0.01). When HS was applied 2 hours prior to LPS, BSP excretion increased to 74% of controls (P < 0.05 vs LPS and controls). Expression of the basolateral (Oatp1a1) and canalicular (Mrp2) organic anion transporter involved in the transport of BSP recovered more rapidly when HS preceded LPS application. Recovery of mRNA levels of these transporters occurred also earlier. Coimmunoprecipitation experiments and immunoelectron microscopy using a double immunogold labeling of heat shock protein 70 (HSP70) and various hepatocyte transporters suggested colocalization with HSP70 for the canalicular bile acid transporter (Bsep) in the subcanalicular space. In contrast, no colocalization was shown for Ntcp and anion transporters. In conclusion, we could show that HS enhances recovery of organic anion transporters and bile acid transporters following endotoxemia. Faster recovery of mRNA seems to be a key mechanism for anion transporters, whereas physical interaction with HSP70 plays a role in preservation of bile acid transporters. This interaction of HSP70 and canalicular transporters occurs only in pericanalicular vesicles but not when the protein is integrated into the plasma membrane.

It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223–406) μg/mL compared to 150 (88–207) μg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.