Heat shock proteins (HSP): dermatological implications and perspectives

In recent years, several studies have demonstrated the protective effect of Heat Shock Proteins (HSP) on different organs and tissues under stressful conditions. However, most research explores the performance of those molecular chaperones during immune responses or pathological conditions like cancer, whereas the number of studies related to the performance of HSPs in the skin during diverse natural or physiopathological conditions is very low. Therefore, the aim of this article was to summarize the main concepts concerning the expression and performance of HSPs, from analysis of current medicine and cosmetics publications, as well as exploring the importance of these proteins in the dermatological area in physiological events such as cutaneous aging, skin cancer and wound healing and to present final considerations related to biotechnology performance in this area.     

Differential expression of heat shock protein 70 (HSP70) and heat shock cognate protein 70 (HSC70) in human epidermis

Autoimmunity and microbial agents have been suggested as playing a pathogenetic role in psoriasis. Since immune responses to microbial infections are often directed towards heat shock proteins (HSP), we investigated the expression of three HSP families in normal and inflamed human skin. Specimens from ten patients with psoriasis and three patients with positive patch tests for nickel and from five healthy volunteers were analysed by means of immunohistochemistry. The patterns observed were qualitatively similar in these conditions showing only minor quantitative differences. Psoriatic epidermis exhibited the highest level of expression. HSP27, HSP70 and heat shock cognate protein 70 (HSC70) were readily detectable. HSP27 was homogeneously distributed throghout the epidermis, whereas HSP70 was restricted to the basal layer and HSC70 primarily to the suprabasal layers. Other HSPs were detected to a lesser degree and showed a more irregular pattern. Thus, the qualitative expression pattern of HSPs seems to be constant between different skin conditions, but the expression of constitutive and inducible HSP70 depends on the differentiation state of keratinocytes.Abbreviations BiP immunoglobulin heavy chain binding protein - GrP glucose regulated protein - HSC70 heat shock cognate protein 70 - HSP heat shock protein - HSP70 heat shock protein 70     

Analysis of 70 KD Heat Shock Protein (HSP70) Expression in the Lesional Skin of Lupus Erythematosus (LE) and LE Related Diseases

Biopsied specimens from skin lesions of SLE were studied for expression of 70 KD heat shock protein (HSP70). The pattern of HSP70 expression in SLE was diffuse in whole epidermis, hair follicles, and sweat gland cells and rather more intense than that in other control groups or normal skin. No significant differences in HSP70 expression were observed between sun-exposed and protected areas of SLE skin lesions. Unlike SLE, reduced or no expression of HSP70 was observed in skin lesions of DLE. In tissue culture, UVB radiation in vitro induced relatively intense expression of HSP70 in the nuclear area of keratinocytes. A few γδT cell receptor positive cells which might respond to HSP70 expressing cells were detected in the basal layer of skin lesions of diseases. These studies suggest that abberant expression of HSP70 in skin lesions of SLE might contribute to both skin lesions and antibody formation in SLE.     

Heat (shock) and the skin

Whereas hyperthermia has long been used in dermatology for the therapy of diseases as diverse as syphilis, gonorrhea, psoriasis or melanoma, the understanding of the biological effects of heat shock on the skin attracts new interests to an old field. The proteins induced by heat (stress, or heat shock proteins) appear to play a general role in protection from cellular injury and eventually in the natural defences from solar radiation. On the other hand, these ubiquitous proteins may also be involved in the immunopathology of diseases such as systemic lupus erythematosus or leprosy.     

A histologic study of epithelial dysplasia in oral lichen planus

One hundred cases of oral lichen planus were reviewed together with 100 nonspecific oral mucosal inflammatory lesions as a control group. The presence of dysplasia was noted, using well-established histologic criteria. Mild dysplasia was found in 57% of cases, moderate dysplasia in 9%, and severe dysplasia in 2% of cases. In the control group, mild dysplasia was observed in 32% of cases, moderate in 10%, and severe dysplasia was not present. It is suggested that, while mild or moderate dysplasia may not indicate precancerous potential, severe dysplasia in lichen planus may signify the development of a precancerous lesion.     

Expression of the small heat shock protein HSP 27 in developing human skin

The 27 kDa heat shock protein (HSP 27) is expressed in keratinocytes of the upper epidermal layers, and recent evidence suggests that this protein is involved in the regulation of epidermal differentiation. The expression of HSP 27 was investigated in developing human skin by immunohistochemistry utilizing a specific monoclonal antibody. We used formalin-fixed, paraffin-embedded tissue of abdominal skin obtained from 34 human fetuses ranging between 13 and 30 weeks estimated gestational age (EGA). We found that HSP 27 is not expressed in keratinocytes until week 14 EGA. At this stage staining is observed in the periderm and the upper intermediate cells but not in hair germs. During further development, HSP 27 expression correlates with increasing epidermal differentiation, i.e. shedding of the periderm and beginning of keratinization. HSP 27 expression is confined to the upper cell layers and sparse basal cells. In hair follicles, HSP 27 can be detected in the innermost cell layer of the outer root sheath and in keratinocytes of the bulge identical to what is observed in adult skin. The hair papilla, matrix cells and sebaceous glands are negative for HSP 27 and remain so during further development. In eccrine sweat glands of the 24th week EGA, HSP 27 is confined to the superficial cell layer of the sweat ducts. In the present report we demonstrate differentiation-related expression of HSP 27 in developing human skin. Further in vitro studies will address the molecular function of HSP 27 in epidermal differentiation and development.     

Heat shock protein HSP 27 is expressed in all types of basal cell carcinoma in low and high risk UV exposure groups.

HSP 27, a marker of differentiation and proliferation, helps the cell in repair processes after environmental stress such as heat, UV-irradiation and oxidative stress. So far, its role on carcinogenesis is not yet understood. HSP 27 was studied immunohistochemically in different types of primary, untreated basal cell carcinoma (BCC) in two populations with different UV exposure habits: descendents from Germany, "Pommern", living in Brazil (Pommeranos, n = 15), rural workers with high UV exposure and Germans from the region Baden-Württemberg with indoor jobs (n = 14). Age distribution and type of BCC were similar between the Pommeranos and the Germans. Specimens of BCC (n = 15 solid, n = 6 keratotic, n = 4 adenoid, n = 4 fibrotic) were evaluated in cryostat and paraffin sections for HSP 27, HSP 72 and bcl-2. In Pommeranos in Brazil (UV high risk group) versus (vs) inhabitants from Baden-Württemberg (UV low risk group), HSP 27 was expressed in 93% vs 79% in all histological subclasses, HSP 72 in 20% vs 21% and bcl-2 in 93 % in both groups. Antibodies against HSP 27, but not HSP 72, labeled BCC of all types. In contrast to the lack of HSP 27 in squamous cell carcinoma reported in the literature, we found HSP 27 and bcl-2 positive cells in BCC which might characterize the tumour as relatively benign and slow progressing.     

Heat shock proteins in cultured human keratinocytes and fibroblasts

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.     

Heat shock proteins 60 and 70 expression of cutaneous lichen planus: comparison with normal skin and psoriasis vulgaris

BACKGROUND: Heat shock proteins (HSPs) are expressed by most living cells and play fundamental roles in many biological processes. Their synthesis increases by a variety of stresses in order to enable cellular survival. Although it is known that they play an important role in immune and inflammatory responses of the skin, the role of HSPs in the pathogenesis of skin diseases has been studied in only limited skin diseases. Lichen planus (LP) is a relatively common papulosquamous dermatosis, and cell-mediated immunity plays an important role in its pathogenesis. Although an altered expression of certain HSPs was reported in oral LP lesions, the expression of HSPs in cutaneous lesions of LP has not been investigated. In this immunohistochemical study, we aimed at investigating the role of HSPs in the pathogenesis of LP by studying whether there is any difference in HSP expression in cutaneous lesions of LP when compared to normal skin and psoriasis vulgaris (PV). METHODS: Formalin-fixed paraffin-embedded skin biopsy specimen blocks from LP patients (n = 39), patients with psoriasis (n = 20), and normal skin controls (n = 20) were used in the study. Antibodies to HSPs 60 and 70 were applied immunohistochemically by using streptavidin-biotin-horseradish peroxidase complex. An immunoreactivity intensity distribution index (IRIDI) was calculated to express the proportion of the immunoreactive cells as well as the staining intensity in different layers of the epidermis. RESULTS: The mean IRIDI scores for HSP60 expression in the basal, suprabasal, and superficial epidermal layers of cutaneous LP were moderately higher than those of normal skin, but not different from those of PV skin. These scores for HSP70 in lesions of LP were moderately lower than those for normal skin in the basal layer, but not significantly different from normal in the other two layers. Scores for HSP70 in PV lesions were markedly lower in all three layers. In the cells of the inflammatory infiltrates (mostly lymphocytes), HSP60 scores for LP were moderately higher, compared to those for PV, whereas scores for HSP70 were much lower for LP and very much lower for PV. CONCLUSIONS: Significantly altered levels of HSP proteins were found in cutaneous LP lesions in comparison with normal skin and psoriasis, suggesting the role of HSPs in the pathogenesis of LP.     

Human keratinocytes respond to osmotic stress by p38 map kinase regulated induction of HSP70 and HSP27.

Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity.     

Ultraviolet B-mediated phosphorylation of the small heat shock protein HSP27 in human keratinocytes

Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.     

Interferon alpha-inducible protein 27 (IFI27) is upregulated in psoriatic skin and certain epithelial cancers

IFI27 is an interferon alpha-inducible protein found to be upregulated in lesional and non-lesional psoriatic skin in a gene array study. To further characterize its function, we studied by in situ hybridization whether IFI27 is expressed in psoriasis, other inflammatory skin diseases, and wound repair in vivo. We also examined its regulation by different growth factors and anti-psoriatic agents using quantitative RT-PCR (TaqMan). IFI27 mRNA expression was highly upregulated in lesional psoriatic epidermis and also detected in non-lesional keratinocytes. It was also expressed in lichen planus, chronic eczema, cutaneous squamous cell cancers, and during normal wound repair when IFI27 was found in the proliferating subpopulation of keratinocytes. A 3-17-fold upregulation of IFI27 mRNA expression was observed when keratinocytes were stimulated with IFN-gamma, TNF-alpha, or TGF-beta1 while retinoids and vitamin D downregulated its expression. Our results suggest that IFI27 is a novel marker of epithelial proliferation and cancer.     

Heat shock protein 27 is expressed in normal and malignant human melanocytes in vivo

BACKGROUND: Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously in mammalian cells, believed to be regulators of normal cell physiology and the cellular stress response. In addition, the small 27-kDa heat shock protein (HSP27) has previously been found to be a differentiation marker for keratinocytes and a prognostic marker associated with increased survival in certain cancerous tumors. METHODS: Using immunohistochemistry on routinely processed paraffin sections, we examined skin biopsies from 15 invasive melanomas, 13 intradermal nevi, and two compound nevi immunostained with a mouse monoclonal antibody to HSP27. In addition, cultured melanocytes were heat stressed at 45 degrees C for 1 h and then fixed and immunostained in order to localize HSP27 expression intracellularly. RESULTS: We found cytoplasmic and strong perinuclear staining of HSP27 in melanocytes in normal skin, in melanomas, and in nevi. Nuclear reactivity was absent. In addition, in cultured non-malignant melanocytes, HSP27 expression relocated from the cytoplasm to the nucleus with heat stress. CONCLUSIONS: To our knowledge, this investigation is the first to demonstrate that HSP27 is expressed in melanocytes in normal skin, in nevi, and in non-malignant cultured melanocytes.     

Selected eosinophil proteins as markers of inflammation in atopic dermatitis patients

Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic and recurrent course, beginning primarily in early childhood. The etiopathogenesis of AD has not yet been fully understood, although various types of inflammatory cells including eosinophils may be involved in its pathomechanism. The basic aim of the study was to evaluate the usefulness of selected eosinophil proteins in serum and urine of AD patients, as markers of disease severity. The study also aimed to analyze correlations between the level of examined proteins and parameters such as skin prick test (SPT) results, serum concentration of total IgE, and coexistence of symptoms of other atopic diseases. The study included 30 AD patients and two control groups: 30 patients suffering from chronic urticaria and 30 healthy individuals. The mean level of eosinophil proteins measured in serum and urine of AD patients was higher than that in controls, although a significant difference was only recorded for serum and urine level of eosinophil protein X (EPX). Patients with very severe/severe AD presented higher levels of eosinophil proteins than patients presenting with mild/moderate AD, although no significant difference was found between these two groups. AD patients with positive SPT results and detectable specific IgE in serum, and with coexisting symptoms of other atopic diseases presented with higher mean levels of serum and urine eosinophil proteins than AD cases with negative SPT results and without any symptoms of other atopic diseases. In children suffering from AD, serum eosinophil cationic protein level, EPX level and urine EPX level were higher than those in healthy children, however, without statistical significance. Study results suggested a significant role of eosinophils in the etiopathogenesis of AD. Serum and urine levels of selected eosinophil proteins may serve as an important part of diagnostic approach to AD patients, especially in differentiation of allergic and non-allergic forms of AD. The results are also promising for the usefulness of selected eosinophil proteins in the diagnosis of AD in children, however, thorough analysis on a larger group of patients is needed.     

A quantitative assessment of Langerhans cells in oral mucosal lichen planus and leukoplakia

Quantitative analysis of Langerhans cells stained with OKT6 in the epithelium of 42 patients with oral mucosal lichen planus and 14 patients with oral mucosal leukoplakia showed significantly greater numbers of Langerhans cells in lichen planus. This may reflect the role of Langerhans cells in the pathogenesis of this condition and their identification could help in distinguishing lichen planus from leukoplakia.     

Effects of thermal shocks on interleukin-1 levels and heat shock protein 72 (HSP72) expression in normal human keratinocytes

Interleukin-1 expression is reported to be modified under a number of cell conditions including physiological stress, injury and activation. We report the effects of the physiological stresses cold and heat shock on IL-1 levels in keratinocytes. Having observed that normal human skin obtained from plastic surgery, usually stored at 4C for a few hours, highly expressed HSP72, a constant feature of stressed human keratinocytes, we wondered whether this induction could be linked to a cold shock and to modification of IL-1 levels in keratinocytes. Cultured keratinocytes were incubated at 4, 37, 40 and 43C for 1.5, 4, 8 and 16 h in a defined medium. HSP72 expression was studied by immunohistochemistry and immunoblot and IL-1 was quantified using specific and sensitive radio-immunoassay. Our findings showed that intracellular IL-1 and IL-1 levels are not significantly modified by thermal shock. HSP72 is only induced after cell exposure to 43C and is not a cold-shock protein. These results demonstrate that thermal stress is not an inductive signal for IL-1 modification in keratinocytes.