Demonstration of allospecific differences in human stratum corneum autoantibodies by blocking immunofluorescence

Stratum corneum antibody titers of six normal human sera were determined by indirect immunofluorescence on three different epidermal substrates with and without preincubation of the substrates with one monkey serum. In three out of six human sera the reaction of stratum corneum antibodies was inhibited on all three substrates, in one serum the inhibition was noted on two out of three substrates and no effect was observed in two sera. These findings provide further evidence for the existence of allospecific stratum corneum antibodies in human sera and for the presence of both species-specific and species-nonspecific glycoprotein stratum corneum antigens in human epidermis.     

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.     

High-quality Fluorescence Imaging of the Human Acrosyringium Using a Transparency: Enhancing Technique and an Improved,Fluorescent Solvatochromic Pyrene Probe

Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.     

The rheumatoid arthritis-associated autoantibodies to filaggrin label the fibrous matrix of the cornified cells but not the profilaggrin-containing keratohyalin granules in human epidermis.

Since they were first described, serum IgG antibodies to the stratum corneum of rat oesophagus epithelium, highly specific for rheumatoid arthritis (RA), have been consensually called antikeratin antibodies (AKA). However, we recently demonstrated that they actually recognize three new proteins of rat oesophagus epithelium distinct from cytokeratins, and also human epidermal filaggrin. In this work we provided further evidence that AKA and RA-associated anti-filaggrin autoantibodies are the same antibodies. Moreover, analysing by indirect immunofluorescence on human skin a large series of 212 well characterized RA sera and anti-filaggrin autoantibodies purified from RA sera by affinity chromatography, we demonstrated the specific binding of AKA to the stratum corneum of human epidermis and the absence of any staining of the granular keratinocytes. This binding was confirmed and the AKA antigen precisely localized in human epidermis by immunoelectron microscopy. The antigen was found to be restricted to the filaggrin-containing intracellular fibrous matrix of the corneocytes, up to the desquamating cells. In contrast, MoAbs directed to human filaggrin and to profilaggrin, its precursor, not only stained the intracellular matrix of the lower corneocytes but also the keratohyalin granules of the granular cells, where profilaggrin is stored. These results reinforced by the absence of immunoblotting reactivity of RA sera to profilaggrin suggest that the epitopes recognized by AKA are absent from profilaggrin. Their identification may provide more insight into the pathogenesis of RA.     

Thymic stromal lymphopoietin expression is increased in the horny layer of patients with atopic dermatitis

Thymic stromal lymphopoietin (TSLP) is known for its capacity to induce CD11c⁺ myeloid dendritic cells to promote T helper type 2 (Th2)‐skewed inflammatory responses. Although increased expression of TSLP was reported in the lesional skin of limited numbers of patients with atopic dermatitis (AD), the relationships between the degree of TSLP expression in the skin and the severity of AD, epidermal barrier function and eruption type remain to be elucidated. The aim of this study was to examine the relationships between the degree of TSLP expression in the skin and the severity of AD, eruption type and epidermal barrier function using a non‐invasive method in a sizeable group of the patients. Stratum corneum tissue was obtained from AD patients by tape stripping, and the stratum corneum TSLP (scTSLP) expression level was evaluated using a TSLP‐specific antibody followed by image analysis. The correlations between the scTSLP intensity and the severity scoring of AD (SCORAD) index and epidermal barrier function, such as stratum corneum hydration and transepidermal water loss (TEWL), were analysed. The changes in the scTSLP level induced by the application of moisturizer were also examined. The scTSLP expression level was increased in AD patients compared with healthy subjects and was correlated with SCORAD, especially with the dry skin score, and stratum corneum hydration. Moisturizer application resulted in reduced scTSLP levels. The scTSLP level can be used as a biomarker of AD severity and particularly epidermal barrier status.     

Acute modulations in permeability barrier function regulate epidermal cornification: role of caspase-14 and the protease-activated receptor type 2

Stratum corneum comprises corneocytes, derived from outer stratum granulosum during terminal differentiation, embedded in a lipid-enriched extracellular matrix, secreted from epidermal lamellar bodies. Permeability barrier insults stimulate rapid secretion of preformed lamellar bodies from the outer stratum granulosum, regulated through modulations in ionic gradients and serine protease (SP)/protease-activated receptor type 2 (PAR2) signaling. Because corneocytes are also required for barrier function, we hypothesized that corneocyte formation could also be regulated by barrier function. Barrier abrogation by two unrelated methods initiated a wave of cornification, assessed as TdT-mediated dUTP nick end-labeling-positive cells in stratum granulosum and newly cornified cells by electron microscopy. Because cornification was blocked by occlusion, corneocytes formed specifically in response to barrier, rather than injury or cell replacement, requirements. SP inhibitors and hyperacidification (which decreases SP activity) blocked cornification after barrier disruption. Similarly, cornification was delayed in PAR2(-/-) mice. Although classical markers of apoptosis [poly(ADP-ribose)polymerase and caspase (Casp)-3] remained unchanged, barrier disruption activated Casp-14. Moreover, the pan-Casp inhibitor Z-VAD-FMK delayed cornification, and corneocytes were structurally aberrant in Casp14(-/-) mice. Thus, permeability barrier requirements coordinately drive both the generation of the stratum corneum lipid-enriched extracellular matrix and the transformation of granular cells into corneocytes, in an SP- and Casp-14-dependent manner, signaled by PAR2.     

Epidermal and papillary dermal characteristics of the bowhead whale (Balaena mysticetus)

Skin samples from most body regions of the bowhead whale were examined. The epidermis is 2.7 to 50 times thicker than that reported in other cetaceans with both regional and individual variations in thickness. The thinnest areas examined (1 mm) occur on the eyelid margins and the thickest (25 mm) occur on the lower jaw. A distinctive parakeratotic stratum corneum with a thick underlying stratum spinosum (without a stratum granulosum) extends over the entire body surface. From a few dozen to several hundred epidermal lesions are present on all whales studied. A typical stratum basale of germinative keratinocytes (with melanocytes in pigmented areas) rests upon a well-defined basal lamina. Epidermal rod arrays arise from the basal keratinocytes which cover highly elongated dermal papillae and extend to the epidermal surface through the distal stratum spinosum and the stratum corneum. At least four diatom genera occur on and in the stratum corneum and lesion areas of different whales. The superficial dermis consists of a papillary layer with long (up to 13 mm) dermal papillae interdigitating with the epidermis from a basal area that is 2-4 mm in thickness. The number of dermal papillae per mm2 varies inversely with the thickness of the epidermis. Large diameter, sensory papillae packed with tortuous, highly elongated, encapsulated nerve end organs also interdigitate with the thin epidermal areas of the ventral surface of the rostrum, the upper and lower lip margins, and the upper and lower eyelid margins. Scattered, single, stiff hairs emerge from the skin only in specific, pigmented regions of the head.     

The characterization of two monoclonal anti-keratin antibodies and their use in the study of epithelial disorders

The present paper describes the production and characterization of two monoclonal antibodies, K20 and K92. Immunohistological staining showed these two antibodies to be specific for keratinizing epithelium. However, whereas K20 stained all layers of the epidermis K92 reacted with only the suprabasal epidermal layers. Immunoblotting studies with preparations of keratins from both the non-cornified (i.e. the basal, spinous and granular layers) and cornified (stratum corneum) layers of epidermis showed that K20 recognized the 46, 48, 50, 55, 56, 56.5, 59, 61, 62, 64, 65, 66 and 67 kd bands, of which the 50 and 46 kd bands appeared to be masked in tissue sections. In contrast, antibody K92 was more restricted in its activity, recognizing only the 55 and 56 kd bands strongly. These antibodies were used in the study of various epithelial disorders and revealed alterations in the epithelial intermediate filament expression in both benign and malignant disease processes.     

New monoclonal antibodies recognizing epidermal differentiation-associated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10 expression in carcinoma of the vulva

Two monoclonal antibodies (MAb) specific for differentiation-related epidermal keratins have been developed. They represent specific molecular probes for different stages of epidermal differentiation. Antibody DE-K10 is chain-specific for cytokeratin polypeptide no. 10 (56.5 kD) expressed in all suprabasal layers of the epidermis. Antibody DE-SCK is specific for modified stratum corneum keratins and thus represents a marker for the terminal step of epidermal differentiation. Since the epitopes identified by both antibodies are preserved in formalin-fixed, paraffin-embedded tissue sections, these antibodies can be used for retrospective studies of differentiation in various pathological processes. We have used antibody DE-K10 to study the cytokeratin 10 expression in 26 stage II or III vulvar squamous cell carcinomas. Preliminary data suggest an increased risk of recurrence in cytokeratin 10 negative tumours.     

Cutaneous lipids: from physiology to the clinical

The primary function of the epidermis is to produce the waterimpermeable stratum corneum that permits terrestrial life. Extracellular glycosylceramides are the main precursors of the stratum corneum ceramides which play a role in the cohesion of corneocytes and the establishment of the barrier function. The physiological desquamative process depends upon the transformation of glycosylceramides to ceramides and of cholesterol sulfate to cholesterol. Intracellular ceramides participate to the terminal differentiation process of the epidermis. Abnormalities in the synthesis and/or the secretory mechanisms of the epidermal lipids induce various skin disorders. Such diseases may have a genetic background or result from the influence of environmental factors.     

Studies in immunodermatology. VI. IF studies of autoantibodies to the stratum corneum and of in vivo fixed IgG in stratum corneum of psoriatic lesions.

Indirect immunofluorescent (IF) tests on sections of normal human skin reveal the presence of antibodies to the stratum corneum in most normal human sera. Sera absorbed with hyperkeratotic scales gave negative reactions. These reactions appeared to correspond to those of the stratum corneum antibodies which were first detected by Krough and Tonder by immune adherence, though this remains to be documented since the conjugate alone stained the stratum granulosum in a pattern comparable to that of the IF staining reported by Krough. The staining was caused by conjugates with high but not by those with low fluorescein to protein ratios. In indirect IF tests, comparable titers of stratum corneum antibodies were found in sera of psoriasis patients and control subjects. Since they reacted with the stratum corneum of the antibody producer they are referred to as autoantibodies. Direct IF tests of psoriatic lesions revealed the presence of in vivo bound IgG as well as other immunoglobulins and complement in the stratum corneum. To differentiate staining with conjugated protein from direct and indirect IF staining of the stratum corneum a four-compartment test was devised. Skin sections were treated with saline or with stratum corneum antibodies and then with conjugates without (compartments 1 or 2) or with (compartments 3 or 4) anti-IgG antibodies. Sections were read by the method of Kawamura and his associates with both UV and BV illumination. Compartment 3 is a direct IF test and compartment 4 is an indirect IF test. In four-compartment tests performed on both biopsies of psoriatic lesions or scales the difference between compartments 1 or 2 and 3 affords a measure of in vivo bound IgG and the difference between compartments 3 and 4 provides information on the presence of free stratum corneum antigen. With this four-compartment test system it was found that in vivo fixation of IgG occurs in the intercellular areas of the stratum corneum of psoriatic lesions or scales and that this corresponds to the site of binding of the stratum corneum autoantibodies. This in vivo binding of IgG in the stratum corneum of psoriatic lesions resembles somewhat the intercellular fixation of IgG in the stratum spinosum in pemphigus. In the materials examined in these preliminary studies most, if not all of the stratum corneum antigen appeared to be covered with in vivo bound IgG.     

Hyaluronan participates in the epidermal response to disruption of the permeability barrier in vivo

Hyaluronan (hyaluronic acid, HA) is a glycosaminoglycan in the extracellular matrix of tissues that plays a role in cellular migration, proliferation and differentiation. Injury to the stratum corneum elicits an epidermal hyperproliferative response, a pathogenic feature in many cutaneous diseases including eczema and psoriasis. Because HA is abundant in the matrix between keratinocytes, we asked whether the presence of HA is required for epidermal hyperplasia to occur in response to barrier injury. Disruption of the stratum corneum, by acetone application on the skin of hairless mice, led to a marked accumulation of HA in the matrix between epidermal basal and spinous keratinocytes, and also within keratinocytes of the upper epidermis. To test whether HA may have a functional role in epidermal hyperplasia, we used Streptomyces hyaluronidase (StrepH), delivered topically, to degrade epidermal HA and blunt the accumulation of epidermal HA after acetone. StrepH signficantly reduced epidermal HA levels, and also significantly inhibited the development of epidermal hyperplasia. This reduction in epidermal thickness was not attributable to any decrease in keratinocyte proliferation, but rather to an apparent acceleration in terminal differentiation (ie, increased keratin 10 and filaggrin expression). Overall, the data show that HA is a significant participant in the epidermal response to barrier injury.     

Unusual cell ultrastructure in ventral epidermis of the African reed frog Hyperolius viridiflavus, (Anura; Hyperoliidae)

The stratum corneum of the epidermis of Hyperolius viridiflavus contains several replacement layers. The outer layer is covered by mucopolysaccharide secretion. H. viridiflavus in their dry phase do not moult the sloughed off layers; these remain attached to the stratum corneum. Long and slender pillar-like cells situated under the stratum corneum extend through the stratum granulosum, stratum germinativum, and the basement membrane into the dermis. These cells abound in tonofilaments. Flask-shaped cells rich in mitochondria, reaching under the stratum corneum, extend into the stratum granulosum. They show delicate, membranous infoldings in their neck-like apical part. Granule-cells, arranged in 2 or 3 layers are situated in the stratum granulosum between the stratum corneum and germinativum. The germinative cells are large and separated from each other by wide intercellular spaces. ATPase activity was localized cytochemically in the baso-lateral cell membranes bordering with the intercellular spaces under the stratum corneum. Accepted: 25 May 1999     

FITC-insulin binding to normal and psoriatic epidermis

FITC-insulin was prepared and applied to frozen human skin sections. In normal and nonlesional psoriatic epidermis, the binding was cytoplasmic in suprabasal cells. In psoriatic lesions, fairly stained or even unstained cells were littered in suprabasal epidermis indicating the expansion of selected basal cell qualities into the stratum spinosum.     

Ultrastructural characterization of the spontaneous papilloma of Japanese newts

The spontaneous epithelioma of the Japanese newt, Cynops pyrrhogaster, is a useful animal model for study of development and regression of papillomata of suspected viral origin. Therefore, ultrastructural studies of this cutaneous tumor were undertaken in order to characterize this model further. The ultrastructure of normal Cynops pyrrhogaster epidermis resembled teleost more than anuran epidermis. Surface cells of the bilayered stratum corneum were covered by micro-ridges and contained prominent nuclei, unlike the stratum corneum of mammals. Capillary endothelial cells contained unique small dense granules. Principal changes in tumors consisted of hyperplasia of stratum granulosum cells, increased melanin bodies in corneal cells, distorted configurations and aberrant location of outer corneal cells, which also demonstrated irregularity of micro-ridges and increased mucin and vacuole content. Intercellular spaces were enlarged between all cell types. Virus-like particles were observed in approximately 40 percent of tumors, particularly in the middle of the tumor.     

A distinct stratum corneum antigen in psoriasis and its reactions with stratum corneum autoantibodies.

Stratum corneum antibodies are ubiquitous and can be detected by various immunological methods. Of these, the ones detected by hemagglutination undergo changes in antibody titers and have been implicated in psoriasis. The purpose of our study was to examine if differences exist in the activities of the antigens isolated from psoriatic scales in comparison to normal callus. Stratum corneum antigens were prepared by trypsin-phenol-water extraction. The water phase, which contains the stratum corneum antigen, was used to sensitize the red blood cells in the hemagglutination assay. The antibody activity in human sera was determined before and after absorption with antigens isolated from callus, psoriatic scales, and cell envelopes. We found notable differences in the antigens obtained from callus and psoriatic scales. These include higher antibody titers to the antigens of the scales, the presence of unique antigenic determinants on psoriatic scales and the localization of the antigen on cell envelopes. These immunological differences were corroborated by the marked biochemical differences of certain amino acids, most notably glycine and proline, and these differences were unique to psoriatic scales as they were not shared with other hyperproliferative disorders.     

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.     

Avian sebokeratocytes and marine mammal lipokeratinocytes: Structural,lipid biochemical,and functional considerations

In terrestrial mammals, stratum corneum lipids derive from two sources: deposition of lamellar body lipids in stratum corneum interstices and excretion of sebaceous lipids onto the skin surface, resulting: in a two-compartment (bricks and mortar) system of lipid-depleted cells surrounded by lipid-enriched intercellular spaces. In contrast, intracellular lipid droplets, normally not present in the epidermis of terrestrial mammals, are prominent in avian and marine mammal epidermis (cetaceans, manatees). We compared the transepidermal water loss, ultrastructure, and lipid biochemistry of the viable epidermis and tratum corneum of pigeon apterium, fledgling (featherless) zebra finches, painted storks, cetaceans, and manatees to those of humans and mice. Marine mammals possess an even more extensive lamellar-body secretory system than do terrestrial mammals; and lamellar-body contents, as in terrestrials, are secreted into the stratum corneum interstices. In cetaceans, however, glycolipicls, but not ceramides, persist into the stratum corneum; whereas in manatees, glycolipids are replaced by ceramides, as in terrestrial mammals. Acylglucosylceramides, thought to be critical for lamellarbody deposition and barrier function in terrestrial mammals, are present in manatees but virtually absent in cetaceans, a finding that indicates that they are not obligate constituents of lamellar-body-derived membrane structures. Moreover, cetaceans do not elaborate the very long-chain, saturated N-acyl fatty acids that abound in terrestrial mammalian acylglucosylceramides. Furthermore, cold-water marine mammals generate large, intracellular neutral lipid droplets not found in terrestrial and warm-water marine mammals; these lipid droplets persist into the stratum corneum, suggesting thermogenesis, flotation, and/or cryoprotectant functions. Avians generate distinctive multigranular bodies that may be secreted into the intercellular spaces under xerotic conditions, as in zebra fledglings; ordinarily, however, the internal lamellae and limiting membranes deteriorate, generating intracellular neutral lipid droplets. The sphingolipid composition of avian stratum corneum is intermediate between terrestrials and cetaceans (= 50% glycolipids), with triglycerides present in abundance. In the midstratum corneum of avians, neutral lipid droplets are released into the interstices, forming a large extracellular, lipid-enriched compartment, surrounding wafer-thin corneocytes, with a paucity of both iipid and keratin (“platesand-platesand-mortar” rather than the “bricks-and-mortar” of mammals). These studies demonstrate remarkable structural and biochemical heterogeneity of epidermal lipids in various homeotherms and provide new insights into the functions of specific lipid-enriched organelles in these organisms.     

Morphology of ventral epidermis of Rana catesbeiana during metamorphosis

A detailed morphological examination of the bullfrog tadpole ventral epidermis and changes in structure that occur during metamorphosis has not been done. Knowledge of this is crucial to interpretation of physiological studies such as those dealing with development of transepithelial Na+ transport. Examination of tadpole epidermis with light microscopy reveals the presence of three different cell types: apical, basal, and skein. This epidermal morphology is constant until Taylor and Kollros (Anat. Rec. 94:7-23, 1946) stage 19 when degeneration of apical cells is noted. Stages 20 and 21 are characterized by rapid proliferation of basal cells and development of a true stratum germinativum together with the disappearance of other tadpole cell types. By stage 22, epidermal morphology is similar to that of the adult frog. Studies with the electron microscope reveal that as the proliferation proceeds during metamorphosis, the skein cells, at stage 20, differentiate to form the apical border of the skin. The development of the adult frog cell phenotype appears to mimic the cellular differentiation that occurs in the adult epidermis with the cells first developing into progranular cells in the intermediate stratum of the skin and then progressing to granular cells in the outermost living cell layer. The granular cells then undergo cornification to form the stratum corneum. Mitochondria rich cells are not seen in the developing epidermis until stage 21. These observations, when considered with previous results from Na+ transport studies (Hillyard et al.: Biochim. Biophys. Acta 692:455-461, 1982), suggest that both the physiological differentiation and morphological differentiation are simultaneous events.     

Ultrastructure of psoriatic epidermis

The ultrastructure of human affected and unaffected psoriatic epidermis was studied in skin biopsies from 5 patients and 3 normal controls. Transmission electron microscopic investigations revealed abnormalities in all cell layers of the affected epidermis. Common to psoriatic keratinocytes from affected epidermis was the reduction of tonofilaments. The essential ultrastructural changes were located in the stratum granulosum and stratum corneum. Thus, absence of the fusion between the keratohyalin granules and the tonofilaments was found in stratum granulosum. The keratinocytes of the stratum corneum showed a large accumulation of ribosomes and vesicles resembling lipid vesicles.