Lower Spinous Layers Research Articles

105 The effect of oxygen on keratinocyte differentiation

A major function of the epidermis is maintaining good barrier integrity. The barrier is maintained by a constant renewal process where basal keratinocytes terminally differentiate to form corneocytes on the outermost layer of the epidermis. The structural and biochemical changes occurring during keratinocyte differentiation has been well studied. However the specific mechanisms triggering differentiation is less clear. Oxygen has been implicated as a driver of differentiation in other tissues such as the haematopoietic system. Oxygen concentration can be a powerful metabolic signal, and the proximity of the epidermis to atmospheric oxygen makes it a likely target for oxygen-induced differentiation. Immunohistochemistry staining of human epidermis with the oxygen-sensitive transcription factor Hif-2α revealed stabilization of the protein in the basal and lower spinous layers but not in the upper layers of the epidermis, suggesting that an oxygen gradient exists within the epidermis. Using 3-dimensional organotypic models constructed in varying oxygen levels, we show that lowering the culture oxygen results in decreased stratification, organization and expression of key differentiation markers of the epidermis.

Molecular cloning and characterization of a novel human papillomavirus, HPV 126, isolated from a flat wart-like lesion with intracytoplasmic inclusion bodies and a peculiar distribution of Ki-67 and p53

Infection with certain human papillomavirus types induces warts with specific macroscopic and microscopic features. We observed multiple flat wart-like lesions on the chest, neck and extremities of an adult T-cell leukemia patient. Histologically, atypical intracytoplasmic inclusion bodies currently known to be pathognomonic for genus gamma or mu papillomaviruses were disclosed in some cells of the epidermis showing histological features compatible with flat warts. In the present study, a novel human papillomavirus was identified and its whole genome, 7326bp in length, was cloned and characterized. Phylogenetic analysis showed the virus designated as HPV126 to be a novel type of genus gamma papillomavirus. Strikingly, Ki-67 and p53 expression was found to be increased in all layers of the epidermis except for horny layer, contrasting to expression restricted to the basal and lower spinous layers in ordinary flat warts.

Role for Protein Kinase C-α in Keratinocyte Growth Arrest

Multiple protein kinase C (PKC) isoforms have been associated with the epidermal keratinocyte (KC) granular layer differentiation program. Here we show PKCalpha membrane localization and substrate phosphorylation in the first suprabasal KCs of normal human epidermis, suggesting activation in vivo in the lower spinous layers where terminal differentiation-associated growth arrest occurs. To determine if PKCalpha is sufficient for KC growth arrest, we expressed a constitutively active PKCalpha (PKCalpha Delta22-28) in normal human KCs and observed growth arrest and accumulation of cells in G1. PKCalpha Delta22-28 inhibited DNA synthesis through the induction of the cyclin-dependent kinase inhibitor p21. Furthermore, downregulation of PKCalpha in an in vitro organotypic epidermis resulted in increased basal and suprabasal proliferation marker expression, decreased differentiation, and reduced epidermal stratification. Together these results indicate that PKCalpha activation is both necessary and sufficient to trigger irreversible growth arrest during human KC differentiation.

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Suprabasal overexpression of Beta-1 integrin is induced by bovine papillomavirus type 1

In a normal stratified squamous epithelium, β1-integrin is expressed in basal epithelial cells. In BPV-induced fibropapillomas β1-integrin is overexpressed and aberrantly localized, with uniform expression in the lower spinous layer, and sporadic expression within the mid-spinous region that co-localizes with expression of the viral E5 and E7 oncoproteins. In situ hybridization of fibropapillomas for β1-integrin RNA revealed sporadic hybridization in the spinous layer, indicating transcriptional induction. β1-integrin expression in cultured keratinocytes requires exogenous EGF in the media, but this requirement is lost if E7 is expressed, and E7 was able to abrogate the EGF-requirement of normal keratinocytes for the activation of ERK and DNA synthesis. Within fibropapillomas, suprabasal expression of E5 and E7 correlated with suprabasal expression of β1-integrin and PCNA, indicating that vegetative viral replication in the spinous layer correlated with the expression of E7 and β1 integrin. The ability of BPV-1 E7 to support β1-integrin expression and EGF independent DNA synthesis and the activation of ERK are the first biochemical correlates of its expression in keratinocytes.

Phenotypical and Molecular Profiling of the Extraneuronal Cholinergic System of the Skin

We present molecular and protein profiling of all acetylcholine receptors (ACh-R) in human scalp skin using PCR, in situ hybridization and double-labeling immunofluorescence. Within the epidermis, the nicotinic (n)ACh-R subunits, alpha3, alpha5, beta2, and beta4 were expressed in the basal cell layer (BCL) and in a single cell layer in the stratum granulosum; alpha9 was expressed in the basal and lower spinous layers. alpha7, alpha10, and beta1 were preferentially detected in the upper spinous and granular layers. Of the muscarinic (m)ACh-R, m1 and m4 were found in the suprabasal layers, whereas m2, m3, and m5 remained restricted to the lower layers. In the outer root sheath of the hair follicle, all ACh-R except alpha9, beta1, and m4 were found in the BCL whereas the alpha9, m4, and m5 ACh-R were restricted to the central cell layer. The alpha5, beta1, beta2, m1-m4 chains were strongly expressed in the inner root sheath. Undifferentiated sebocytes expressed the alpha3, alpha9, beta4, m3-m5 ACh-R whereas alpha7, beta2, beta4, m2, and m4 were found in mature sebocytes. In sweat glands, the alpha3*, alpha7, and m2-m5 ACh-R were most prominent in the myoepithelial cells whereas alpha9, beta2, m1, m3, and m4 ACh-R were present in the acinar cells. Taken together, our data result in a complete molecular map of the extraneuronal cholinergic system of the skin that may be translated into distinct functional reaction patterns.

Identity of TUNEL-positive cells in the oral buccal epithelium of normal mucosa and lichen lesions.

In situ detection of DNA fragmentation by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) is a widely used technique to identify apoptotic cells in the terminal phases of cell death. Several studies have shown that there are statistically increased numbers of TUNEL(+) cells within the epithelium of oral lichen (OL). It was suggested that this indicates an increased rate of apoptosis among basal and suprabasal keratinocytes in OL epithelium. The aim of this study was to identify the TUNEL(+) cells in the epithelium of erythematous (ERY) OL and normal oral mucosa (NOM). Sections of biopsies from NOM and ERY OL were processed for TUNEL combined with immunostaining for pan-cytokeratin or for cell markers specifically expressed by different leukocytes. In NOM, TUNEL(+) keratinocytes were almost exclusively seen in the outermost epithelial layers. This labeling was absent in ERY OL. In the basal and lower spinous layers, more TUNEL(+) cell nuclei were seen in ERY OL as compared with NOM, in accordance with previous studies. The present observations show, however, that only very few of these cells were keratinocytes, but rather were CD4(+) lymphocytes and CD68(+) macrophages. There was no difference between the numbers of TUNEL(+) keratinocytes in basal and lower spinous layers in ERY OL and NOM epithelium. No intraepithelial CD8(+) lymphocytes, Langerhans cells, or mast cells were found to be TUNEL(+). The findings indicate that the pathologic changes in ERY OL epithelium cannot be explained by increased prevalence of terminal keratinocyte cell death identified by TUNEL.

Immunohistochemical study on expression of alpha-defensin and beta-defensin-2 in human buccal epithelia with candidiasis.

It has been previously reported that alpha-defensin (HNPs) and beta-defensin-2 (HBD-2) peptides with antifungal and cytotoxic activities can be detected in oral carcinomas and the saliva of patients with oral carcinomas. The present study investigated the presence of HNPs and HBD-2 in oral epithelia with candidiasis. Tissue sections (4 microm) were prepared from biopsy and surgically removed specimens diagnosed as oral candidiasis (n = 10). The sections were examined immunohistochemically with antibodies directed against HNPs and HBD-2. Tissue sections of oral candidiasis were immunostained with antidefensin antibodies. Neutrophils in the inflamed lamina propria were positively immunostained with anti-HNPs antibody. The cytoplasm of cells in the upper spinous layer, in the lower spinous layer and in the parakeratinized layer of buccal epithelia with candidiasis was immunostained intensely with anti-HBD-2 antibody. In contrast, the expression of HBD-2 in the normal spinous layer was much weaker than that in oral candidiasis. No signals of HNPs were found in normal buccal epithelium. Buccal specimens from individuals with oral candidiasis show greater levels of expression of both HNPs and HBD-2. There might be a dual protection manner by defensins against fungal inflammation in infected buccal epithelia locally. Generally, HBD-2 signals have been found everywhere in the buccal epithelium; however, in an infected area, the signal intensity of HBD-2 has increased. HNPs signals have not been found in the normal buccal epithelium; however, HNPs signals have increased when the infection occurred.

Taurine levels and localisation in the stratified squamous epithelia.

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.

Immunohistochemical localization of large chondroitin sulphate proteoglycan in porcine gingival epithelia.

In this study, we report the immunohistochemical localization of versican in healthy porcine gingival epithelia. The monoclonal antibody (mAb), 5D5, specifically recognizes core proteins of large chondroitin sulphate proteoglycans such as versican, neurocan and brevican, but not the core protein of aggrecan. Because neurocan and brevican appear to be specific to nervous tissue, the large chondroitin sulphate proteoglycans examined in this study is most likely versican. In the keratinized layer of the attached gingival epithelium, the basal and spinous cell surfaces showed intense staining for mAb 5D5. In the parakeratinized layer of the sulcus epithelium, the localization was restricted to the basal and lower spinous layers. In the junctional epithelium, intense staining was observed in one or two cell layers near the enamel surface. Immunoelectron microscopy revealed high-density depositions of 5D5 immunoreactivity on epithelial cell surfaces. At the enamel surface, 5D5 immunoreactivity was localized to the dental cuticle of the junctional epithelium but was not present in the internal basal lamina. These results suggest that versican, a large chondroitin sulphate proteoglycan, is involved in epithelial differentiation and downgrowth.

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.

Stratification-related expression of isoforms of the desmosomal cadherins in human epidermis.

Desmosomal junctions are abundant in epidermis and contain two classes of transmembrane glycoprotein, the desmocollins and the desmogleins, which are members of the cadherin superfamily of Ca(2+)-dependent cell adhesion molecules. The desmocollin subfamily includes DGIV/V and DGII/III while the desmoglein subfamily includes DGI, HDGC and the autoantigen of the blistering skin disease pemphigus vulgaris (PVA). There are also several non-glycosylated proteins, including the desmoplakins and plakoglobin, present in the desmosomal plaque, which forms a link between the glycoproteins and the cytokeratin intermediate filaments. To provide a picture of the expression of the desmosomal genes and their products in epidermis, we have used in situ hybridisation and immunofluorescence staining on sections of human foreskin. We find that, as expected, desmoplakin DPI/II and plakoglobin are expressed throughout the epidermis, gradually accumulating during differentiation, which probably reflects the increased numbers of desmosomes. In contrast, while keratin 14 and the hemidesmosomal component bullous pemphigoid antigen I (BPAGI) are basal-specific, desmocollin DGIV/V is expressed only in the upper spinous/granular layers of the epidermis, whereas DGII/III expression is enriched in the basal layers. Amongst the desmogleins, expression of DGI appears similar to desmoplakin and plakoglobin; PVA is more prevalent in the lower spinous layers, whereas HDGC expression is detected basally but not suprabasally. The major desmosomal cadherin transcripts are desmocollin DGIV/V and desmoglein DGI. The resultant changes in desmosomal composition and structure may reflect the maturation of desmosomes, presumably being related to the need for changes in cell adhesion during stratification, terminal differentiation, and desquamation, and point to the desmosome being a key player in epidermal differentiation.

Effects of Topical Retinoids on Cytoskeletal Proteins: Implications for Retinoid Effects on Epidermal Differentiation

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Non-disulfide-linked cytoskeletal proteins, including keratins from the epidermal "living layers," were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins. The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation.

Papillomavirus-Infected Keratinous Cyst on the Sole

A 17-year-old boy had a keratinous cyst on the sole. The keratinous cyst and its overlying epidermis had solitarily scattered keratinocytes, which contained a peculiar intracytoplasmic inclusion body above the lower spinous layer. Immunohistochemistry and electron microscopy revealed that the nuclei of these cells had virions of papillomavirus. These virions appeared above the spinous layer. The inclusion bodies were highly eosinophilic masses in the viable layer, and slightly basophilic, fine granules in the cornified layer. Their number at any time was usually one. Their histologic and ultrastructural features and their N-(7-dimethylamino-4-methylcoumarinyl)-maleimide staining property were different from those of keratohyalin and from those of amyloid or hyaline body. Also the keratinous cyst was associated with colloid bodies, showing the lamellated figure in the subepithelial area.

Lectin binding of rat gingival epithelia

Oral gingival epithelium (OGE), oral sulcular epithelium (OSE) and junctional epithelium (JE) were examined histochemically by using different lectins as markers for epithelial differentiation. The staining pattern of gingival epithelia was compared with that of the buccal and palatal epithelia. Binding of WGA had a uniform distribution in all the epithelia examined. A positive reaction was found in all the basal and spinous layers, but not in the cornified layer of the epithelia. BPA binding was seen in the lower spinous layer of OGE, OSE, buccal and palatal epithelia, and in most of the JE. The basal layer and the cells directly attached to the tooth surface at the apical part of JE were nonreactive with BPA. GS-I reacted with the basal and suprabasal layers of each epithelium and with the cells attached to the tooth at the apical part of JE. UEA-I reacted with the upper spinous layer of OGE, OSE and epithelia of hard palate, but not with any of the cells of the JE. Our results agree with previous data suggesting that OGE and OSE exhibit squamous differentiation similar to that of the masticatory epithelium of hard palate. Furthermore, our results suggested that the JE cells undergo differentiation equivalent to that of the suprabasal and lower spinous cells of OGE. The cells along the tooth surface at the apical part of JE, however, form a distinct population of cells with basal nature.

Molecular cloning of mouse epidermal cystatin A and detection of regulated expression in differentiation and tumorigenesis

A gamma gt 11 cDNA expression library representing mouse epidermis mRNA was screened with polyclonal rabbit antiserum directed against 10-13 kDa epidermal antigens that had previously been shown to be regulated during epidermal differentiation. A cDNA clone was detected and isolated and its identity as the coding sequence for one of the antigens was confirmed by translation of hybrid-selected mRNA from mouse epidermis. The cDNA sequence predicted a peptide homologous to the reported sequence of rat epidermis cystatin A, a thiol proteinase inhibitor. This identification was confirmed by cross-reactivity of the gamma clone fusion protein with authentic antiserum to rat epidermis cystatin A. Southern gel analysis showed that mouse DNA may contain several closely related genes homologous to the cystatin probe. An mRNA of about 0.6 kb from epidermis and cultured mouse epidermal cells hybridized with the cystatin probe on northern analysis. The abundance of the message was high in cultured basal cells and was selectively diminished by inducing terminal differentiation in culture with an elevated Ca2+ concentration in the medium. Cystatin message was abundant in chemically induced mouse skin papillomas but reduced in carcinomas. In epidermis, mRNA was localized to the less differentiated basal and lower spinous layers by in situ hybridization. Regulation of expression of cystatin A in epidermis and tumors suggests that it may be important in the control of normal keratinocyte proliferation and differentiation and in malignant conversion.

へん平上皮由来しゅようにおける細胞骨格成分の免疫組織化学的研究

Cytoskeletal proteins; microtuble, microfilament, and intermediate-sized-filament proteins participate in mitosis, mobility, formation and maintenance of cells, as well as in malignant transformation. Immunohistochemical stainings of those proteins are available for differential diagnosis of tumor or investigation of several pathological lesions. Monoclonal antibodies to them have been used for more specifically detect to the subject.In the present study, total 246 cases of epithelial lesions and tumors; bening epithelial lesions (82), semimalignant lesions from epithelial origin (14), basal cell carcinomas (26) and squamous cell carcinomas (124) were examined by the use of immunohistochemical stainings for anti-total keratin (TK; 41-65 kDa), monoclonal anti-keratins (KL1; 55-57 kDa, PKK1; 41-56 kDa), vimentin, clesmin and actin.In normal stratified squamous epithelium, TK and KL1 showed strong staining in granular and upper spinous cell layer, moderate in lower spinous layer and negative in basal cell layer. PKK1 was limited to basal layer cells. Normal stratified squamous epithelia expressed regular distribution to keratin proteins. Almost all papillomas and other benign epithelial lesions indicated regular distribution of keratins as in normal epithelia. In Bowen's disease, the staining of TK and KL1 displayed an irregular pattern and that of PKK1 disappeared in basal layer cells. Basal cell carcinoma showed irregular distribution of TK and, KL1 and had negative to PKK1 staining. Squamous cell carcinoma showed irregular distribution to TK and KL1 with little decreased staining, and negative PKK1. Staining of vimentin, clesmin and actin were generally negative in benign lesions and tumors, however, these proteins existed in a few cases of squamous cell carcinoma. These findings suggested that cytoskeletal proteins in malignant tumors may change in their expression.

An unusual form of chronic gingivitis: An ultrastructural, histochemical, and immunologic investigation

Gingival tissue from a patient suffering from an unusual form of gingivitis, previously described as plasma cell gingivitis, has been subjected to ultrastructural, histochemical, and immunologic investigation. Macrophages and T-lymphocytes infiltrated the oral epithelium where a dissecting type of injury was produced, predominantly in the lower spinous layer. Interkeratinocyte junctions were disrupted and collections of isolated desmosomes were observed in the intercellular spaces. It is postulated that the primary lesion in this atypical gingivitis lies in the epithelium and that the plasma cell infiltrate in the connective tissue is secondary to this. No allergen could be identified, and over a period of 7 years the condition has shown a tendency to resolve spontaneously.