Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages

Summary Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages.

Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.     

Differential expression of CD44s and CD4v10 proteins and syndecan in normal and irradiated mouse epidermis

 The role of the CD44s adhesion molecule, its epithelial isoforms and its relationship to epidermal proteoglycans such as syndecan was studied in normal and irradiated mouse skin. In normal mouse skin, only 10% of basal cells are strongly CD44s-immunopositive, with a cytoplasmic expression pattern. Double-label experiments with the basal cell marker keratin 14 confirmed the epithelial nature of the strongly CD44s-positive cell type in the basal layer. Some spinous keratinocytes and the majority of the remaining basal cells exhibited a weak membranous staining pattern. In contrast, the epithelial isoform, CD44v10, was strongly present in all basal and suprabasal epithelial cells of the epidermis, with a membranous staining pattern. Syndecan was found in the granular layer of the normal epidermis only. After 1 week of daily irradiation, the entire basal cell layer of the epidermis expressed CD44s in the membrane, but with a varying degree of staining intensity. This reactivity spread to the upper spinous layer after 3 weeks of treatment. In hyperproliferative epidermis, there was no difference in the staining patterns between CD44s and CD44v10. The expression of syndecan switched from the granular layer to the basal and lower spinous layers after 2 weeks of daily irradiation. Immunoreactivity for syndecan was also strongly enhanced in the dermis of irradiated samples. The results suggest an important role for syndecan and CD44 in proliferative processes during radiation-induced accelerated repopulation. Accepted: 30 September 1996     

CD44 variant isoforms are preferentially expressed in basal epithelia of non-malignant human fetal and adult tissues

CD44 is a transmembrane glycoprotein, which can exist in a multitude of isoforms due to alternative splicing of the pre-mRNA. We have generated monoclonal antibodies to several of these variant regions, which are encoded by 10 additional exons in the extracellular part of the molecule. CD44 variant isoforms have been reported to be involved in the malignant progression of rat and human tumours. The precise localization of CD44 variant isoforms in normal developmental and morphogenetic processes is essential for diagnostic studies of human tumorigenesis. Therefore, we have analysed a large number of different human tissues by immunohistochemistry for the expression of CD44 isoforms containing either exons 4v, 6v or 9v. Expression of exon 9v-isoforms was detected in almost all epithelia analysed, with a few exceptions. Exon 6v isoforms are expressed only in squamous and glandular epithelia, e.g. skin epidermis, sweat and sebaceous glands, oesophagus, ducts of the mammary gland, salivary and prostate glands. Detection of exon 4v-encoded isoforms was restricted to the epidermis and the oesophagus. Similar tissue distributions of CD44 variant isoforms were observed in 10-week-old fetal tissues. Since one of the ligands of CD44 is hyaluronic acid (HA), we also analysed the tissue distribution of HA synthetase. HA synthetase was detected in all tissues analysed, showing good correlation with the expression of the standard form of CD44, CD44s.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

Histology and cytochemistry of human skin. IX. The distribution of non-specific esterases

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

Characterization of metamorphic changes in anuran larval epidermis using lectins as probes

The skin of an adult frog of Xenopus laevis was characterized by the reactivity of 20 lectins. The lectins were classified into six groups in their binding to the epidermal cells: Lycopersicon esculentum lectin (LEL)-type which was positive for all epidermal cells; Pisum sativum agglutinin (PSA)-type for stratum germinativum; succinylated wheat germ agglutinin (sWGA)-type for strata spinosum, granulosum and corneum; Dolichos biflorus agglutinin (DBA)-type for strata germinativum and spinosum; peanut agglutinin (PNA)-type for stratum spinosum; and Ulex europaeus agglutinin (UEA-I)-type for strata granulosum and corneum. PSA and sWGA were utilized as markers of mitotically active germinative cells and the differentiated cells of the epidermis, respectively, to describe the metamorphic conversion of larval epidermal cells to adult type. PSA stained all epidermal cells of tadpoles before metamorphic climax. At the end of metamorphosis, PSA-positive cells were restricted to cells in the basal layer of body epidermis while all the tail epidermis remained PSA-positive. The other cell marker, sWGA, only stained apical cells in tadpole epidermis. During the metamorphic climax, sWGA-positive cells appeared in the cells beneath the stratum corneum of the body region, but not in the tail region. The present study demonstrates that PSA and sWGA are useful to investigate metamorphic changes in tadpole epidermal cells.     

HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN : IX. THE DISTRIBUTION OF NON-SPECIFIC ESTERASES

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

Expression of KIT receptor tyrosine kinase protein in normal human skin: preliminary observations

KIT proto-oncogene is a receptor tyrosine kinase critical for signal transduction in several cell types. An understanding of KIT distribution in normal skin may reveal new insights into its implication in cutaneous biology and its associated disease states. This study examines expression patterns of KIT protein in normal human skin. KIT protein expression was examined in 50 specimens of normal human skin using specific antibodies (CD117) and immunoperoxidase staining methods. KIT protein expression was seen in keratinocytes (stratum basale), melanocytes, mast cells, and in both sebaceous and sweat glands. In contrast, KIT protein expression was absent in stratum spinosum, stratum granulosum, stratum corneum, blood vessels and arrector pili muscle. Thus, the expression of KIT protein in normal human skin suggests its possible role in regulation of cutaneous development and function.     

Lectins as Markers of Human Epidermal Cell Differentiation

The expression of sugar residues on human epidermal cells was investigated by means of lectin binding, as a way of determining membrane structural changes occurring during the differentiation of the epidermis. Fourteen lectins of different sugar specificity were conjugated with fluorescein isothiocyanate (FITC-lectins) and tested in fluorescence microscopy on frozen sections of normal human epidermis. In parallel, FITC-lectins were tested on psoriatic-involved epidermis to visualize differences in the expression of sugar residues that might occur during abnormal epidermal differentiation. No labelling could be obtained with lectins from Bandeira simplicifolia I, Dolichos biflorus, Limulus polyhemus, Tetragonolobus purpureas, Ulex europeus I , and Triticum vulgaris (group 1 lectins). A "pemphigus-like" intercellular labelling of the whole epidermis, except the stratum corneum, was obtained with lectins from Canavalia ensiformis, Maclura pomifera, Phaseolus vulgaris , and Ricinus communis I (group 2 lectins). A selective intercellular labelling of the stratum spinosum and the stratum granulosum was seen in normal epidermis with lectins from Arachis hypogaea, Glycine max, Helix pomatia , and Sophora japonica (group 3 lectins). In psoriatic epidermis, not only the basal cell layer, but also cells from the adjacent lower stratum spinosum were found to be negative, using FITC-lectins of group 3. These data indicate that the expression of lectin binding sites in normal epidermis differs according to the maturation of the cell from the basal cell to the more mature keratinocyte in the stratum granulosum. They suggest that lectins may be used as markers of epidermal cells in various stages of normal and abnormal differentiation.     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Demonstration of β-glucan receptors in the skin of aquatic mammals—a preliminary report

Using immunohistochemistry, the study clearly demonstrates three important β-glucan receptors (Ficolin/P35, MBL, Dectin-1; members of the lectin-complement pathway of innate immunity) in the integument of six marine and freshwater aquatic mammals (Northern fur seal, Common seal, Walrus, Coypu, Capybara, Otter), but only weakly in two dolphin species. Most of the non-dolphin mammals exhibited strong reactions, especially with regard to the skin glands (tubular apocrine glands, sebaceous glands), for L-Ficolin/P35 and MBL. Distinct reaction staining could also be observed in the epidermis and the outer epithelial sheath of primary hair follicles. Positive Dectin-1 staining was limited to secretory cells of the apocrine tubular glands, and to peripheral and central cells of sebaceous glands of the seals. The Capybara was the only animal to show a clear Dectin reaction in the epidermis (stratum granulosum). The findings are discussed with regard to the constant and high microbial challenge of the skin in the aquatic medium, and variations in hair density of the animals.     

Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by interleukin-2-activated cytolytic lymphocytes

In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7.     

Biological features of the skin of the pika, Ochotona rufescens rufescens: histological structure and enzymatic histochemical reactivity

To examine the usefulness of the pika, Ochotona rufescens rufescens, as an experimental animal for skin irritability tests, the histological structure and enzymatic histochemical reactivity of pika skin were investigated. The pika had a hair cycle similar to that of the rabbit. The skin and epidermis of the pika trunk were 1.16mm and 29.5 microns thick, on the average, respectively. Both of them were the thickest in the dorsal region followed by the interscapular area, while they were the thinnest in the abdominal region. In the epidermis of the pika, the strata corneum, granulosum, spinosum and basale were rather clearly distinguished. The cell arrangement in the stratum basale was more compact than that in the rabbit. Dermal mast cells, which are distributed in the stratum reticulare in rabbits and guinea pigs, were distributed in the stratum papillare right beneath the epidermis. The mast cell of the pika in the TEM images had granules of low electron density and with relatively long microvilli and rather large mitochondria. The activities of the enzymes, SDH, MDH, LDH, beta HBDH, alpha GPDH, ALD, G6PDH and GPR, in the hair follicles and sebaceous glands of the pika were similar to those of the rabbit.     

Human 8-oxoguanine-DNA glycosylase 1 protein and gene are expressed more abundantly in the superficial than basal layer of human epidermis

Human 8-oxoguanine-DNA glycosylase 1 (hOGG1) repairs 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) which results from oxidation of guanine. Reactive oxygen species (ROS) formed in response to ultraviolet (UV) radiation cause this DNA damage, which is involved in pathological processes such as carcinogenesis and aging. The initiation of skin tumors probably requires penetration of UV to the actively dividing basal layer of the epidermis in order for acute damage to become fixed as mutations. Previously, the majority of UVB fingerprint mutations have been found in the upper layers of human skin tumors, while UVA mutations have been found mostly in the lower layer. Our aim was to determine whether this localization of UVA-induced DNA damage is related to stratification of the repair-enzyme hOGG1. Anti-hOGG1 immunohistochemical staining of frozen sections of human foreskin, adult buttock skin, and reconstructed human skin samples showed the highest expression of hOGG1 in the superficial epidermal layer (stratum granulosum). Study of the hOGG1 mRNA expression again showed the highest level in the upper region of the epidermis. This was not regulated by UV irradiation but by the differentiation state of keratinocytes as calcium-induced differentiation increased hOGG1 gene expression. UVA-induced 8-oxo-dG was repaired more rapidly in the upper layer of human skin compared to the lower layers. Our results indicate that weaker expression of the nuclear form of hOGG1 enzyme in the basal cells of the epidermis may lead to a lack of DNA repair in these cells and therefore accumulation of UVA-induced oxidative DNA mutations.     

Immunohistochemical localization of irisin in skin,eye, and thyroid and pineal glands of the crested porcupine (Hystrix cristata)

Irisin was first identified in muscle cells. We detected irisin immunoreactivity in various organs of the crested porcupine (Hystrix cristata). In the epidermis, irisin immunoreactivity was localized mainly in stratum basale, stratum spinosum and stratum granulosum layers; immunoreactivity was not observed in the stratum corneum. In the dermis, irisin was found in the external and internal root sheath, cortex and medulla of hair follicles, and in sebaceous glands. Irisin immunoreactivity was found in the neural retina and skeletal muscle fibers associated with the eye. The pineal and thyroid glands also exhibited irisin immunoreactivity.     

Changes in structure of ventral epidermis of Rana ridibunda during metamorphosis

Summary The organisation of the ventral epidermis organisation was followed throughout ontogenesis in Rana ridibunda. Epidermis of tadpoles with 2–3 limbs was organised into two layers: a stratum germinativum consisting of elongated columnar cells, and an outer stratum corneum consisting of two types of cuboid cells. Two types of cells can be distinguished; they are a light (clear) cell and a dark (dense) cell. In the 4-legged tadpoles the stratum corneum cells start to flatten and a replacement layer appeared underneath. A well-defined stratum germinativum is found and within it, epidermal glands. Moulting took place for the first time in tadpoles just before metamorphosis, and a well-organised stratum granulosum was formed still containing the two main types of epidermal glands. The flask cells appear in the juveniles for the first time, greatly increasing in numbers in the adult epidermis.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.