Individual Heat Shock Proteins Research Articles

Heat Shock Proteins and HSF1 in Cancer.

Fitness of cells is dependent on protein homeostasis which is maintained by cooperative activities of protein chaperones and proteolytic machinery. Upon encountering protein-damaging conditions, cells activate the heat-shock response (HSR) which involves HSF1-mediated transcriptional upregulation of a group of chaperones – the heat shock proteins (HSPs). Cancer cells experience high levels of proteotoxic stress due to the production of mutated proteins, aneuploidy-induced excess of components of multiprotein complexes, increased translation rates, and dysregulated metabolism. To cope with this chronic state of proteotoxic stress, cancers almost invariably upregulate major components of HSR, including HSF1 and individual HSPs. Some oncogenic programs show dependence or coupling with a particular HSR factor (such as frequent coamplification of HSF1 and MYC genes). Elevated levels of HSPs and HSF1 are typically associated with drug resistance and poor clinical outcomes in various malignancies. The non-oncogene dependence (“addiction”) on protein quality controls represents a pancancer target in treating human malignancies, offering a potential to enhance efficacy of standard and targeted chemotherapy and immune checkpoint inhibitors. In cancers with specific dependencies, HSR components can serve as alternative targets to poorly druggable oncogenic drivers.

Implication of a lysosomal antigen in the pathogenesis of lupus erythematosus

Naturally-occurring autoantibodies to certain components of autophagy processes have been described in a few autoimmune diseases, but their fine specificity, their relationships with clinical phenotypes, and their potential pathogenic functions remain elusive. Here, we explored IgG autoantibodies reacting with a panel of cytoplasmic endosomal/lysosomal antigens and individual heat-shock proteins, all of which share links to autophagy. Sera from autoimmune patients and from MRL/lpr and NZB/W lupus-prone mice reacted with the C-terminal residues of lysosome-associated membrane glycoprotein (LAMP)2A. No cross-reaction was observed with LAMP2B or LAMP2C variants, with dsDNA or mononucleosomes, or with heat-shock protein A8. Moreover, administering chromatography-purified LAMP2A autoantibodies to MRL/lpr mice accelerated mortality. Furthermore, flow cytometry revealed elevated cell-surface expression of LAMP2A on MRL/lpr B cells. These findings reveal the involvement of a new class of autoantibodies targeting the C-terminus of LAMP2A, a receptor for cytosolic proteins targeted for degradation via chaperone-mediated autophagy. These autoantibodies could affect the autophagy process, which is abnormally upregulated in lupus. The data presented support a novel connection between autophagy dysregulation, autoimmune processes and pathophysiology in lupus.

Heat shock protein genes in the green alga Tetraselmis suecica and their role against redox and non-redox active metals

Microalgae are capable of tolerating variations in water temperature and sudden exposures to toxic substances, and cellular heat shock proteins (HSPs) help to protect cells from such stress. Here, we determined the complete open reading frames (ORF) of small TsHSP20 and large TsHSP70 and 100 in the chlorophyte Tetraselmis suecica, and examined the expression levels of these genes after exposure to thermal stressors, redox-active metals, and non-redox-active metals. Putative TsHSP20, TsHSP70, and TsHSP100 proteins had conserved HSP-family motifs with different C-terminus motifs. Phylogenetic analyses of individual HSPs showed that T. suecica clustered well with other chlorophytes. Real-time PCR analysis showed that thermal stress did not significantly change the expression of all the tested TsHSPs. In addition, TsHSP20 showed little gene expression after being exposed to copper, whereas TsHSP70 and 100 genes greatly responded to the redox-active metals in CuSO4 followed by CuCl2, but not to the non-redox active metals. Redox-active metals strongly affected the physiology of the cells, as judged by cell counting, reactive oxygen species imaging and photosynthetic efficiency. These findings suggest that small and large HSPs are differentially involved in the response against environmental stressors. Moreover, metal toxicity may be specifically controlled by the anions in the metal compounds.

Mesenchymal stem cells maintain their defining stem cell characteristics after treatment with cisplatin.

Mesenchymal stem cells (MSCs) aid the regeneration of tissues damaged by treatment with cisplatin. However, the effects of this cytotoxic drug on the stem cells have been largely unknown. Here we demonstrate that human bone marrow-derived MSCs are relatively resistant to cisplatin treatment and show resistance levels comparable to these of differentiated fibroblasts. Cisplatin did not affect cellular morphology, adhesion or induction of apoptosis in MSCs. The potential for differentiation was preserved after exposure to cisplatin, and established MSC surface markers were observed to be stably expressed irrespective of cisplatin treatment. Cytoskeletal rearrangements and high expression levels of individual heat shock proteins were detected in MSCs and may be partly responsible for the observed cisplatin resistance. The cisplatin-resistant phenotype of human MSCs supports the concept of further investigating these stem cells as a potential treatment option for cisplatin-induced tissue damage.

Mitochondrial and Axonal Abnormalities Precede Disruption of the Neurofilament Network in a Model of Charcot-Marie-Tooth Disease Type 2E and Are Prevented by Heat Shock Proteins in a Mutant-Specific Fashion

Mutations in NEFL encoding the light neurofilament subunit (NFL) cause Charcot-Marie-Tooth disease type 2E (CMT2E), which affects both motor and sensory neurons. We expressed the disease-causing mutants NFL and NFL in motor neurons of dissociated spinal cord-dorsal root ganglia and demonstrated that they are incorporated into the preexisting neurofilament network but eventually disrupt neurofilaments without causing significant motor neuron death. Importantly, rounding of mitochondria and reduction in axonal diameter occurred before disruption of the neurofilament network, indicating that mitochondrial dysfunction contributes to the pathogenesis of CMT2E, as well as to CMT caused by mitofusin mutations. Heat shock proteins (HSPs) are involved in the formation of the neurofilament network and in protecting cells from misfolded mutant proteins. Cotransfection of HSPB1 with mutated NEFL maintained the neurofilament network, axonal diameter, and mitochondrial length in motor neurons expressing NFL, but not NFL. Conversely, HSPA1 cotransfection was effective in motor neurons expressing NFL, but not NFL. Thus, there are NFL mutant-specific differences in the ability of individual HSPs to prevent neurofilament abnormalities, reduction in axonal caliber, and disruption of mitochondrial morphology in motor neurons. These results suggest that HSP inducers have therapeutic potential for CMT2E but that their efficacy would depend on the profile of HSPs induced and the type of NEFL mutation.

HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector-mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector-mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with approximately 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector-mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.

Lentivirus Mediated HO-1 Gene Transfer Enhances Myogenic Precursor Cell Survival After Autologous Transplantation in Pig

Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation.

Hsf1-Mediated Stress Response can Transiently Enhance Cellular Radioresistance

To elucidate how the heat-shock transcription factor 1 (Hsf1)-mediated stress response affects cellular radioresistance, mouse embryo fibroblasts with Hsf1-gene knockout (Hsf1(-/-) cells) or with normal wild-type Hsf1 expression (Hsf1 wild-type cells) were preconditioned by heating (43 degrees C, 30 min) without or with quercetin (an inhibitor of Hsf1) and then exposed to gamma radiation (4 or 6 Gy). Some cell samples were infected with virus-based vectors to overexpress the constitutively active (mutant) form of Hsf1 or individual heat-shock proteins (Hsps). The heat preconditioning transiently up-regulated the Hsp levels in Hsf1 wild-type cells and significantly improved their postirradiation survival; these effects could be abolished by quercetin or simulated (without preheating) by the Hsf1 overexpression. In contrast, no enhanced radioresistance was found in heat-preconditioned Hsf1(-/-) cells that were unable to trigger Hsf1-mediated Hsp induction after heating. However, when the constitutively active Hsf1 was overexpressed in Hsf1(-/-) cells, the latter accumulated stress-inducible Hsps and became more radioresistant like heat-preconditioned Hsf1 wild-type cells. The overexpression of Hsp70 or/ and Hsp27 also enhanced radioresistance of both cell cultures. Thus the preirradiation stress response resulting in the intracellular Hsp accumulation can improve survival of severely irradiated mammalian cells.

Proteomic Analysis of Primary Cultures of Human Adipose-derived Stem Cells: Modulation by Adipogenesis

Adipogenesis plays a critical role in energy metabolism and is a contributing factor to the obesity epidemic. This study examined the proteome of primary cultures of human adipose-derived adult stem (ADAS) cells as an in vitro model of adipogenesis. Protein lysates obtained from four individual donors were compared before and after adipocyte differentiation by two-dimensional gel electrophoresis and tandem mass spectroscopy. Over 170 individual protein features in the undifferentiated adipose-derived adult stem cells were identified. Following adipogenesis, over 40 proteins were up-regulated by > or = 2-fold, whereas 13 showed a > or = 3-fold reduction. The majority of the modulated proteins belonged to the following functional categories: cytoskeleton, metabolic, redox, protein degradation, and heat shock protein/chaperones. Additional immunoblot analysis documented the induction of four individual heat shock proteins and confirmed the presence of the heat shock protein 27 phosphoserine 82 isoform, as predicted by the proteomic analysis, as well as the crystallin alpha phosphorylated isoforms. These findings suggest that the heat shock protein family proteome warrants further investigation with respect to the etiology of obesity and type 2 diabetes.

Ectopic expression of Hsp70 confers resistance and silencing its expression sensitizes human colon cancer cells to curcumin-induced apoptosis

We have shown earlier that heat shock renders human colon cancer cells resistant to curcumin-induced apoptosis, but the contribution of individual heat shock proteins (hsps) to this resistance has not been tested. High expression of hsp27 and hsp70 in breast, endometrial and gastric cancers has been associated with metastasis, poor prognosis and resistance to chemo- or radiotherapy. In this study, SW480 cells were transfected with hsp70 cDNA in either the sense or antisense orientation and stable clones were selected and tested for their sensitivity to curcumin. The cells were protected from curcumin-induced cell death by hsp70 while cells harboring antisense hsp70 (Ashsp70) were highly sensitive to curcumin. Curcumin-induced nuclear condensation was less in hsp70 but more in Ashsp70 cells when compared with control vector-transfected cells. Loss of mitochondrial transmembrane potential induced by curcumin was further accelerated by antisense hsp70 expression and hsp70 restored it partly. Ashsp70 cells released more cytochrome c, AIF and Smac from mitochondria upon curcumin treatment than control cells. hsp70 partly prevented the release of AIF but not the other proteins. Activation of caspases 3 and 9 induced by curcumin was also inhibited by hsp70, whereas more activation could be seen in Ashsp70 cells, although caspase 8 activation was unaffected by changes in hsp70 expression. Curcumin-induced cleavage of PARP and DFF45 was inhibited by hsp70 but enhanced in Ashsp70 cells. The present study demonstrates the potential of hsp70 in protecting SW480 cells from curcumin-induced apoptosis and highlights that silencing the expression of hsp70 is an effective approach to augment curcumin-based therapy in cancers that are resistant due to hsp70 expression.

Expressional pattern of chaperones in neuronal, glial, amnion, mesothelial, and bronchial epithelial cell lines

Although literature is abundant on expression of individual heat shock proteins (HSPs) and molecular chaperones, no comprehensive information is given on their expressional pattern. The aim of our study was therefore to study expressional differences between several cell types that may provide evidence for the types of HSPs and chaperones that may be operating in the corresponding lineages. For this purpose neuronal (HCN-2), glial (SVG-p12), amnion, mesothelial (Met-5A), and bronchial epithelial (16HBE14o −) cell lines were grown, harvested, and protein was separated on two-dimensional electrophoresis with subsequent in-gel digestion and identification of protein spots by MALDI-MS and specific software. A series of 29 high abundance HSPs and chaperones were unambiguously identified altogether. We observed distinct expressional patterns and although overlapping, there was an apparent paucity of HSPs and chaperones in bronchial epithelial and mesothelial cells. We learn from this study that individual cell lines express and may use different HSP and chaperones systems and strategies. Specific functions of cells may be responsible as well as the presence of protein specific chaperones, although we cannot rule out that cell culture conditions were at least in part responsible for the different expressional patterns.

Expression of heat shock proteins in a linear rodent wound.

Heat shock proteins (hsps) are ubiquitous and known to be expressed in all organisms. These stress proteins are likely to be induced in the wound environment and may play a critical role in the overall process of wound repair. Linear incisions were made in Sprague-Dawley rats. Serial skin biopsies were taken, the dermis and epidermis were separated and a protein lysate made. The expression of hsp 72, 47, and 32 were analyzed by Western blotting and immunohistochemistry. There were distinct patterns of expression of hsp 72, 47, and 32 in the wound. In unwounded dermis, there was no constitutive expression of any of the heat shock proteins studied. In the epidermis, there was constitutive expression of hsp 32 and 72, but not hsp 47. With wounding, all hsps exhibited increased expression both in the dermis and epidermis. These patterns of protein expression are suggestive of the individual heat shock proteins' molecular function, such as hsp 72's role as an indicator of cellular stress and injury, hsp 47's role in collagen synthesis, and hsp 32's role as an antioxidant.

Molecular chaperones in the kidney: distribution, putative roles, and regulation.

Molecular chaperones are intracellular proteins that prevent inappropriate intra- and intermolecular interactions of polypetide chains. A specific group of highly conserved molecular chaperones are the heat shock proteins (HSPs), many of which are constitutively expressed but most of which are inducible by diverse (in some cases specific) stress factors. HSPs, either alone or in cooperation with "partner" chaperones, are involved in cellular processes as disparate as correct folding and assembly of proteins, transport of proteins to specific intracellular locations, protein degradation, and preservation and restructuring of the cytoskeleton. The characteristic distribution of individual HSPs in the kidney, and their response to different challenges, suggests that a number of HSPs may fulfill specific, kidney-related functions. HSP72 and the osmotic stress protein 94 (Osp94) appear to participate in the adaptation of medullary cells to high extracellular salt and urea concentrations; the small HSPs (HSP25/27 and crystallins) may be involved in the function of mesangial cells and podocytes and contribute to the volume-regulatory remodeling of the cytoskeleton in medullary cells during changes in extracellular tonicity. HSP90 contributes critically to the maturation of steroid hormone receptors and may thus be a critical determinant of the aldosterone sensitivity of specific renal epithelial cells. Certain HSPs are also induced in various pathological states of the kidney. The observation that the expression of individual HSPs in specific kidney diseases often displays characteristic time courses and intrarenal distribution patterns supports the idea that HSPs are involved in the recovery but possibly also in the initiation and/or maintenance phases of these disturbances.

Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis.

Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.

Identification of acquired thermotolerance deficiency within the ditelosomic series of `Chinese Spring' wheat

The relative contribution of individual heat shock proteins to acquired thermotolerance was evaluated through analysis of chromosomal deletions in a ditelosomic series of the hexaploid wheat ( Triticum aestivum L.) cultivar `Chinese Spring'. This study describes the identification of a line within this ditelosomic series that exhibited a reduced level of acquired thermotolerance. Changes in the temperature sensitivity of chlorophyll accumulation were used as an indicator of acquired thermotolerance. The temperature providing maximum chlorophyll accumulation was 30 °C in leaves under continuous light. A 30-min challenge temperature of 48 °C prior to the light exposure was shown to inhibit subsequent chlorophyll accumulation. Preincubation at 40 °C for 4 h before the 30-min 48 °C challenge triggered the acquired thermotolerance system of the plant resulting in chlorophyll accumulation upon exposure to light. Evaluation of the ditelosomic series revealed reductions in acquired thermotolerance levels in the DT7DS line relative to controls. Two-dimensional SDS polyacrylamide gel analysis was used to identify reduction in the level of two low molecular mass heat shock proteins in DT7DS.

Primary Structure and MHC Restriction of Peptide-Defined T Cell Epitopes from Recombinant Mycobacterial Protein Antigens

By combining DNA subclone and synthetic peptide approaches, immunogenic epitopes have been mapped from the mycobacterial heat shock proteins (HSP) of 18, 65 and 70 kD, as well as from a novel non-HSP antigen of 24 kD recognized by antigen-specific human T cells. In addition the HLA-DR molecules used in antigen presentation of the individual peptide-defined epitopes have been identified. The donor groups used for establishing antigen-specific CD4+ T cell clones and lines were primarily healthy subjects immunized with Mycobacterium bovis BCG and killed Mycobacterium leprae. The results showed that HSP18 contains one epitope (aa 38-50) presented by HLA-DR4; HSP65 contains 4 epitopes presented by DR1 and DR2 and 5 epitopes presented by DR4; whereas HSP70 contains multiple epitopes presented by DR2, DR3, DR5, DR7, and DRw53. The 24-kD non-HSP antigen also displayed one HLA-DRw53-restricted T cell epitope. In conclusion, the individual HSP T cell epitopes defined here are exclusively presented by only one HLA-DR molecule. However, at the protein level the results suggest that HSP65 and HSP70 are relevant to the subunit vaccine design, since they contain multiple T cell epitopes which can be presented by a spectrum of different HLA-DR molecules.

Trigeminal ganglion neurons are protected by the heat shock proteins hsp70 and hsp90 from thermal stress but not from programmed cell death following nerve growth factor withdrawal.

A prior mild thermal stress (heat shock) can protect neuronal cells against a subsequent exposure to either severe thermal stress or the induction of programmed cell death (apoptosis). By micro-injecting trigeminal ganglion neurons with expression constructs we show that over-expression of the individual heat shock proteins hsp70 and hsp90 can protect these cells against severe thermal stress but not against apoptosis. However, the protective effect of prior heat shock against subsequent apoptosis is dependent upon its ability to induce heat shock protein (hsp) synthesis rather than, for example, the inhibition of other protein synthesis associated with heat shock. The significance of these effects is discussed in terms of the role of different hsps in protecting neuronal cells from distinct stresses.

HSP27 and HSP70 increase the survival of WEHI-S cells exposed to hyperthermia.

Exposure of cells to hyperthermia and various other stress conditions induces synthesis of a small group of proteins, the heat shock proteins (HSPs). Synthesis of HSPs correlates with the development of thermotolerance, but little is known about the role of individual HSPs in this phenomenon. Using stably transfected WEHI-S murine fibrosarcoma cells we show that overexpression of either HSP27 or HSP70 clearly protects these cells from the toxic effect of elevated temperatures. Moreover, a clone expressing HSP70 mRNA in antisense orientation, and thereby reduced levels of endogenous HSP70 protein, is more thermosensitive than transfection control cells. Using indirect immunofluorescence we show that following heat treatment exogeneous HSP27 and HSP70 are relocated from the cytoplasm to the nucleus and nucleoli respectively. A similar pattern of localization was seen for the endogenous HSPs. Taken together, these results indicate that both HSP27 and HSP70 protect cells from heat mediated killing.

The Heat-Shock Response and Expression of Heat-Shock Proteins in Wheat Under Diurnal Heat Stress and Field Conditions

The occurrence of heat-shock proteins (HSPs) in response to high temperature stress is a universal phenomenon in higher plants and has been well documented. However, in agriculturally important species, less is known about the expression of HSPs under natural environments. A review of the heat-shock response in wheat (Triticum aestivum L.) is presented and recent results on the expression of wheat HSPs under diurnal stress and field conditions are reported. In the field experiment, flag leaf blade temperatures were obtained and leaf blades collected for northern blot analysis using HSP 16.9 cDNA as a probe. Temperatures of leaf blades ranged from 32 to 35�C under the tested field conditions at New Deal near Lubbock, Texas. Messenger RNAs encoding a major class of low molecular weight HSPs, HSP 16.9, were detected in all wheat genotypes examined. The results suggested that HSPs are synthesised in response to heat stress under agricultural production, and furthermore, that HSPs are produced in wheats differing in geographic background. In the controlled growth chamber experiment, HSP expression in two wheat cultivars, Mustang (heat tolerant) and Sturdy (heat susceptible) were analysed to determine if wheat genotypes differing in heat tolerance differ in in vitro HSP synthesis (translatable HSP mRNAs) under a chronic, diurnal heat-stress regime. Leaf tissues were collected from seedlings over a time-course and poly (A)+RNAs were isolated for in vitro translation and 2-D gel electrophoresis. The protein profiles shown in the 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between these two wheat lines, but also some unique HSPs which were only found in the heat tolerant line. This data provides evidence of a correlation between HSP synthesis and heat tolerance in wheat under a simulated field environment and suggests that further genetic analysis of HSPs in a segregating population is worthy of investigation. In conclusion, the results of this study provide an impetus for the investigation of the roles of HSP genes in heat tolerance in wheat.

Induction of Heat-Shock Proteins by Bacterial Toxins, Lipid Mediators and Cytokines in Human Leukocytes

We studied the influence of a lipid mediator (12-hydroxyeicosatetraenoic acid, 12-HETE), cytokines (IL-6 and TNF-alpha) and different bacterial toxins (alveolysin; exfoliative toxin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human leucocytes. 12-HETE induces the expression of individual heat shock proteins (65- and 83 kDa) protein in human leukocytes (lymphocytes, monocytes, basophilic granulocytes; LMBs). As was shown by Western blotting (anti-hsp72), IL-6 or TNF-alpha induced hsps preferentially in human LMBs and PMNs, respectively. Among the toxins, ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml), no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as determined by PCR analysis. In contrast, exfoliative toxin led to a reduction of the hsp signal in PMNs as determined by Western blotting. Finally, it was demonstrated that PMNs which had been pretreated with TNF-alpha and therefore expressed intracellular hsps were more resistant to cytolytic attack by leukocidin than untreated cells.