Increased level of c-erbB-2/neu/HER-2 protein in cutaneous squamous cell carcinoma

Overexpression of c-erbB-2/neu/HER-2 oncoprotein, a receptor tyrosine kinase, has been demonstrated in a variety of human cancers. To elucidate the involvement of c-erbB-2 in human skin carcinogenesis, we examined expression of the protein in skin samples from five cases of keratoacanthoma (KA), 10 of actinic keratosis (AK), 24 of squamous cell carcinoma (SCC) and 10 of basal cell carcinoma (BCC) and five samples of normal epidermis, using an immunohistochemical method on formalin-fixed, paraffin-embedded sections. Expression of c-erbB-2 was also examined in cultured SCC cell lines, a premalignant cell line and in cultured normal keratinocytes. Normal epidermal cells showed no or very little c-erbB-2 protein, but the covering epidermal layer of some tumours showed a few strongly positive cells. Samples of KA and AK showed barely detectable c-erbB-2 protein in only a few cases. Twenty of the 24 cases of SCC had elevated expression of c-erbB-2 protein with a tendency to more positive cells in metastatic lesions. Five of the 10 cases of BCC stained for c-erbB-2 but more weakly than those of SCC, Reaction products of the positive cells were seen in the cytoplasm. All three cultured SCC cell lines stained for c-erbB-2 protein more strongly than the premalignant HaCaT or normal keratinocytes. Our results indicate the possible involvement of c-erB-2 overexpression in the malignant conversion of keratinocytes.     

Distribution of an estrogen receptor-related protein (P29) in normal skin and in cultured human keratinocytes

A monoclonal antibody, ERD5, which recognizes a 29Kd phosphoprotein associated with human estrogen receptor of myometrium was used to study the expression of this protein in normal skin and in cultured human keratinocytes. By indirect immunofluorescence, both in vivo and in vitro keratinocytes showed a variable cytoplasmic staining which increased with cell differentiation. SDS gel electrophoresis of soluble extracts of cultured keratinocytes and normal epidermis showed that P29 was a minor protein. Immunoblot analysis demonstrated that ERD5 strongly reacted only with a 29Kd polypeptide band without any cross-reactivity. These data suggest that keratinocytes might be estrogen sensitive like other cells in which P29 has already been located. The exact role of this protein in the keratinocyte differentiation process and its relationship with estrogen receptors remain to be elucidated.     

Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve fibers and adnexal structures in human skin

BACKGROUND: Cannabinoid receptors mediate the psychopharmacological action of marijuana and have been localized in the central and peripheral nervous system as well as on cells of the immune system. OBJECTIVE: Up to now, two cannabinoid receptors (CB1 and CB2) have been cloned and recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in the skin. METHODS: In the present immunohistochemical investigation we determined the precise localization of CB1 and CB2 in sections of human skin and in one case of mastocytosis. RESULTS: CB1 and CB2 immunoreactivity was observed in cutaneous nerve fiber bundles, mast cells, macrophages, epidermal keratinocytes, and the epithelial cells of hair follicles, sebocytes and eccrine sweat glands. In epidermal keratinocytes, hair follicle and sebaceous glands, CB1 and CB2 were distributed in a complementary fashion. Double-immunostaining with an anti-CGRP antibody suggested the presence of cannabinoid receptors on small afferent peptidergic nerves. CONCLUSION: The abundant distribution of cannabinoid receptors on skin nerve fibers and mast cells provides implications for an anti-inflammatory, anti-nociceptive action of cannabinoid receptor agonists and suggests their putatively broad therapeutic potential.     

Expression and characterization of membrane co-factor protein (MCP) in human skin

Membrane co-factor protein (MCP; CD46) is an integral membrane protein with molecular weight (MW) of the two species of 63 kD and 55 kD, and regulates autologous complement activation, with the activity of factor I cofactor. The quantity of each species is genetically regulated, and two codominantly inherited allelic variants account for the three phenotypic patterns. By immunohistochemical study, MCP was found both in the intercellular spaces of the epidermis and on the endothelial cells in the dermis of normal human skin in vivo. The intensity of the staining pattern was higher in the basal layer than in the granular layer. By Western blot analysis with use of a monoclonal antibody, MCP in the epidermis appeared as several bands ranged from 60-50 kD, with a major band of 56 kD, which was different from those in either polymorphonuclear cells, platelets, and cultured keratinocytes. No other variants were found in the epidermis obtained from skin of 20 normal humans. Complement activation in human skin may be regulated at several steps, including DAF and HRF20, thereby protecting cells from autologous complement attack.     

Immunohistochemical localization of parathyroid hormone-related protein (PTHRP) in normal human skin

Human keratinocytes secrete large amounts of a parathyroid hormone-related peptide (PTHRP) in vitro. Because recent studies indicate that PTHRP could have a number of autocrine or paracrine functions in the skin, localization of this peptide in vivo is important. A monoclonal and two affinity-purified polyclonal antibodies were employed to locate PTHRP in normal human skin and cultivated human keratinocytes. PTHRP is present throughout the viable portion of the epidermis, in adnexal epithelial cells, and in all cultivated keratinocytes. These findings do not support the provocative suggestion that PTHRP is a marker for squamous differentiation.     

Localization of foetal antigen 2 (FA-2) in foetal and adult human skin

Foetal antigen 2 (FA-2) is a connective-tissue-associated antigen isolated from second trimester human amniotic fluid. FA-2 has an alpha-electrophoretic mobility and is a single-chain molecule with a molecular weight of 26 kDa as determined by polyacrylamide gel electrophoresis (PAGE). Using indirect immunofluorescence and the immunoperoxidase technique, FA-2 was found to be in the lamina densa/sublamina densa region of the basement membrane zone (BMZ) in adult as well as in foetal skin. FA-2 was found throughout the dermis in foetal skin, whereas in adult skin it was found to be associated with the BMZ and around the blood vessels, hair follicles and eccrine glands. Intracellular FA-2 antigen was demonstrated in proliferating fibroblasts by the indirect immunoperoxidase technique and immunoelectron microscopy of the fibroblasts revealed staining of the antigen in the cisternae of the rough endoplasmatic reticulum at the trans-side of the Golgi complex as well as in vesicles close to the plasma membranes. FA-2, a hitherto undescribed antigen associated with human BMZ, is probably being synthesized by proliferating fibroblasts.     

Distribution of metallothionein in normal and pathological human skin

The expression and distribution of metallothionein (MT) in frozen sections of normal and pathological human skin was studied using the monoclonal antibody L2E3 directed against MT derived from human fetal liver. Immunohistochemical staining of normal fetal and adult skin revealed strong reactivity in basal keratinocytes of epidermis and outer hair root sheath, hair matrix cells and the secretory coil, but not the exocrine portion of eccrine glands; myoepithelial cells around apocrine sweat glands were similarly stained. In epidermal hyperplasia, variable numbers of suprabasal keratinocytes were stained, whereas in interface dermatitis, interrupted staining was found in the basal layer. Weak or scattered staining was observed in squamous tumours, whereas basal cell carcinomas did not show consistent staining. The distribution of MT in normal skin was in line with the germinative role of basal keratinocytes and hair matrix cells, whereas its distribution in hyperplastic epidermis was in line with experimental animal data, and reflected the increase in the germinative pool in these conditions. It is concluded that monoclonal antibody L2E3 may serve as a valuable immunohistochemical marker in diagnostic cutaneous pathology since it labels basal keratinocytes selectively, and since it discriminates between eccrine and apocrine sweat glands.     

银屑病皮损c－erbB－1及c－erbB－2原癌基因表达及其意义

用抗ｃ－ｅｒｂＢ－１（ＥＧＦＲ）及ｃ－ｅｒｂＢ－２（Ｎｅｕ蛋白）原癌基因表达蛋白单抗及免疫组化技术，观察２０例银屑病皮损及１０例正常人皮肤。结果表明：（１）在正常人皮肤表皮中，ＥＧＦＲ基因表达蛋白主要分布在基底细胞层，Ｎｅｕ蛋白表达蛋白主要分布在棘层上部及颗粒细胞层。（２）银屑病活动性皮损表皮棘层及颗粒层ＥＧＦＲ表达增强，而Ｎｅｕ蛋白表达减弱。银屑病皮损表皮两种原癌基因表达异常对皮损形成可能起一定作用。     

Eosinophilic cationic protein (ECP) in skin disorders.

Eosinophil cationic protein (ECP) is exclusively secreted only by the eosinophilic leukocyte. In this study the ECP concentration in the serum was measured in patients (n = 155) with various skin disorders and compared with the number of circulating eosinophils. The presence of activated eosinophils in the skin was also studied immunohistochemically using the monoclonal antibody EG-2, which recognizes both the eosinophil protein X (EPX/EDN) and ECP. EG-2 distinctly revealed these proteins in the eosinophils and their granules. Non-activated eosinophils were studied with the monoclonal antibody EG-1. In most cases this did not disclose any more eosinophils and often it was located more diffusely and not seldom on collagen fibers. Elevated serum ECP but normal numbers of circulating eosinophils were found in half of the patients with progressive plaque psoriasis and long-standing daily chronic urticaria. In patients with prurigo nodularis, papular erythematous eruptions, vasculitis, purpura and toxic drug reactions, Wells' syndrome, porphyria cutanea tarda and persistent light reaction the serum ECP was increased, although in some cases the number of circulating eosinophils was normal. In these disorders an increased number of activated eosinophils was found in the skin. Both serum ECP and the number of activated eosinophils normalized when the patients' condition improved. In atopic dermatitis the serum ECP and the number of activated eosinophils in the skin were increased only during exacerbation of the disease. High serum levels of ECP and activated eosinophils in the skin are frequent findings in many skin disorders in spite of often normal blood eosinophil counts.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of c-erbB-2 protein in the keratinocytes of oral mucosal lichen planus

The expression of the c-crbB-2 protein was studied in the keratinocytes from patients with: (i) oral mucosal lichen planus (OLP) (n=2(S): (ii) oral mucosal squamous cell carcinoma (OMSCC) which had arisen in mucosa affected by OLP (n=5); and (iii) normal oral mucosa (n=5). C-erbB-2 protein was expressed on the cell membranes of the keratinocytes of nucleated epithelium in the stratum spinosum. The antigenic determinant recognized represents the cytoplasmic domain of a cell surface receptor which binds an as yet uncharacterized heparin binding ligand of unknown function.^1.2 The specimens from the live normal subjects showed positive immunohistochemical staining with the monoclonal c-erbB-2 protein antibody, the OMSCC specimens were negative, and 23 of 26 of the OLP specimens were positive. The lack of c-cerbB-2 expression in the three OLP and in the five OMSCC specimens may indicate a genetic alteration, or masking of the expression of c-cerbB-2. The absence of expression in OLP specimens might be an indicator of the possibility of future neoplastic transformation.     

Distribution density of intraepidermal nerve fibers in normal human skin

A total of 74 specimens was obtained from the normal human skin of patients from 3 to 90 years old. The specimens were roughly classified into 5 groups: 15 for the face group from the face; 15 for the abdomen group from the abdomen; 13 for the back group from the back; 14 for the arm group from the upper arm and forearm; and 17 for the leg group from the thigh and lower leg. They were all fixed in 4% paraformaldehyde and 14% saturated picric acid. Cryostat sections were examined by the immunoperoxidase method and indirect immunofluorescence (IF). Primary antibodies against neurofilament, neuron-specific enolase, protein gene product 9.5 (PGP 9.5) and S-100 protein were used. The most effective method was found to be the combination of IF with PGP 9.5; it visualized the intraepidermal nerve fibers easily and clearly. Of the 74 specimens, 32 (43%) had intraepidermal PGP 9.5-immunoreactive (or nerve) fibers (IPIF), and 42 (57%) did not have any. With reference to the different skin locations, the maximal rate of specimens having IPIF was 57% in the arm group, and the minimum was 23% in the back group. IPIF positive specimens had approximate surface lengths of 6 mm, in which the existence number of the IPIF was 1 to 75. Their distribution density per 1000 epidermal basal cells was highest at 9.63 in the arm group and lowest at 2.89 in the back group. Their thickness was 2.94 +/- 0.83 microns with no significant differences among the five groups. We concluded that intraepidermal nerve fibers may not be distributed evenly in the hairy portions of normal human skin, but they may be present focally. Physiologically, two-point discrimination of itch may be explained by the distribution mode of intraepidermal nerve fibers.     

Distribution of ATPase-positive Langerhans cells in normal adult human skin

The distribution of ATPase-positive Langerhans cells (LC) was investigated in 117 specimens of normal adult human skin and mucosa taken from different areas of the body. Although there were significant variations in the numbers of LC in each area examined, skin from the face and neck contained the highest density of cells (976 +/- 30.93/mm2). The densities of LC in trunk skin (740 +/- 28.97/mm2), scalp (693 +/- 69.56/mm2) and arm or leg skin (640 +/- 40.95/mm2) were similar. Buccal mucosa had significantly fewer LC (567 +/- 42.94/mm2) than trunk skin, and sacrococcyx skin and palm and sole skin displayed the smallest number of these cells (267 +/- 56.14/mm2 and, 189 +/- 19.15/mm2 respectively). No ATPase-positive LC were detected in the centre of two corneal specimens.     

Monoclonal anti-interleukin 2 (15-2) antibody binding to granular layer keratinocytes of human skin

Among several monoclonal antibodies (moABs) directed against human interleukin 2 (IL-2), the 15-2 moAB raised in our laboratory against unglycosylated recombinant IL-2 (produced in Escherichia coli) cross-reacted with a human skin epitope. This moAB gave a strong staining on the cell-surface membranes of keratinocytes from the granular layer of the epidermis. In addition, the 15-2 moAB stained 15% of epidermal cell suspensions obtained from suction blisters and reacted with cells from the spinous layer in parakeratosis and psoriasis, as well as with spinous epithelioma cells. Preincubation of the 15-2 moAB with pure human recombinant IL-2 abrogated skin binding, whereas a polyclonal antikeratin antiserum did not block 15-2 skin binding. Two other anti-IL-2 moABs, one directed against unglycosylated recombinant IL-2 (17-2 moAB) and one against glycosylated natural IL-2 (9B11 IE5 moAB), were unreactive on skin. Taken together, the data suggest that the 15-2 moAB binds to an epitope cross-reacting with, but different from, IL-2 which is located in the cell-surface membranes of granular layer cells. This cross-reactive epitope may provide a useful probe for the study of human epidermal cell differentiation.     

Characterization of decay-accelerating factor (DAF) in human skin.

Decay-accelerating factor (DAF) is a 70-kD membrane glycoprotein that regulates autologous complement activation, by preventing assembly of alternative or classical C3/C5 convertases, and has been shown to have a wide tissue distribution. In this study, DAF antigen has been demonstrated at the intercellular spaces of normal human epidermis with monoclonal antibody against DAF using the peroxidase-anti-peroxidase method. The amount of DAF was greater at the granular layer than the basal cell layer as judged by intensity of the staining. Western blot analysis of DAF in the epidermis showed a 55-kD band, whereas that of buffy coat cells was approximately 67 kD. When DAF of the epidermis was treated with neuraminidase, the molecular weight was reduced to 53 kD, whereas that of buffy coat cells was 56 kD. These results indicated that the content of sialic acid of DAF in the epidermis was different from that of buffy coat cells. In phosphatidylinositol-specific phospholipase C (PIPLC)-treated normal human skin, DAF was not demonstrated in the epidermis, whereas DAF remained unchanged on the elastic fibers. After the treatment of the epidermis by PIPLC, DAF was released into the buffer shown by Western blot analysis. These results suggested that DAF on the epidermis was anchored to keratinocyte via phosphatidylinositol (PI), whereas the anchoring mechanism of DAF on the elastic fibers was not through PI.     

Expression of estrogen-related receptor gamma (ERRgamma) in human skin

Skin is a non-classical target for estrogens. Despite evidence showing that estrogen receptors (ER) are expressed in skin, there are still extensive gaps in our understanding of how estrogens exert their action in non-reproductive tissues. Estrogen-related receptor gamma (ERRgamma), an orphan member of the nuclear receptor superfamily, shows a strong sequence homology with estrogen receptor alpha but it does not bind estradiol. Here, for the first time, we demonstrate the expression of ERRgamma in adult human skin. ERRgamma mRNA was detected in the keratinocytes and fibroblasts of 8 female donor skins using RT-PCR. The presence of the protein was confirmed using immunohistochemistry on 11 adult human skins and Western Blotting on monolayer-cultures of fibroblasts and keratinocytes from respectively 4 and 2 donors. This study shows that ERRgamma is expressed in human skin and could intervene in a potentially new estrogen signaling pathway in the skin.