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Aberrant distribution patterns of corneodesmosomal components of tape-stripped corneocytes in atopic dermatitis and related skin conditions (ichthyosis vulgaris, Netherton syndrome and peeling skin syndrome type B)
Atopic dermatitis (AD), Netherton syndrome (NS) and peeling skin syndrome type B (PSS) may show some clinical phenotypic overlap. Corneodesmosomes are crucial for maintaining stratum corneum integrity and the components’ localization can be visualized by immunostaining tape-stripped corneocytes. In normal skin, they are detected at the cell periphery.
Objective
To determine whether AD, NS, PSS and ichthyosis vulgaris (IV) have differences in the corneodesmosomal components’ distribution and corneocytes surface areas.
Methods
Corneocytes were tape-stripped from a control group (n = 12) and a disease group (37 AD cases, 3 IV cases, 4 NS cases, and 3 PSS cases), and analyzed with immunofluorescent microscopy. The distribution patterns of corneodesmosomal components: desmoglein 1, corneodesmosin, and desmocollin 1 were classified into four types: peripheral, sparse diffuse, dense diffuse and partial diffuse. Corneocyte surface areas were also measured.
Results
The corneodesmosome staining patterns were abnormal in the disease group. Other than in the 3 PSS cases, all three components showed similar patterns in each category. In lesional AD skin, the dense diffuse pattern was prominent. A high rate of the partial diffuse pattern, loss of linear cell–cell contacts, and irregular stripping manners were unique to NS. Only in PSS was corneodesmosin staining virtually absent. The corneocyte surface areas correlated significantly with the rate of combined sparse and dense diffuse patterns of desmoglein 1.
Conclusion
This method may be used to assess abnormally differentiated corneocytes in AD and other diseases tested. In PSS samples, tape stripping analysis may serve as a non-invasive diagnostic test.
In vertically cut histology sections, the stratum corneum (SC) of the epidermis shows a basket-weave-like appearance. These corneocytes are attached only at the cell periphery by corneodesmosomes, which are modified desmosomes [
Bidimensional analysis of desmoglein 1 distribution on the outermost corneocytes provides the structural and functional information of the stratum corneum.
New non-invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape-stripped samples.
] reported that tape-stripped corneocytes from lesional skin of psoriasis vulgaris and lichen planus showed unique diffuse distribution patterns of Dsg1, but other skin diseases such as atopic dermatitis (AD) have not been tested. Distribution patterns of corneodesmosomal components other than Dsg1 have not been examined either.
Corneocyte surface areas vary not only by anatomic sites [
], and then measure the surface area with a light microscope. By tape-stripping, we could easily obtain horizontal images of corneocytes, and measure corneocyte surface areas directly from immunofluorescence images as performed by Mohammed et al. [
Diagnosis and treatment of atopic dermatitis in children and adults: European Academy of Allergology and Clinical Immunology/American Academy of Allergy, Asthma and Immunology/PRACTALL Consensus Report.
The spectrum of pathogenic mutations in SPINK5 in 19 families with Netherton syndrome: implications for mutation detection and first case of prenatal diagnosis.
] may show some overlap in clinical manifestations, especially in childhood. Patients with these diseases have red scaly skin lesions with severe pruritus. Laboratory tests often show increased serum IgE levels. Defective epidermal barrier function and recurrent skin infections are common complications. Diagnostic hair shaft abnormalities of NS may not always be apparent, particularly in childhood. The suggestion that PSS and NS are the same disease had been proposed [
], respectively. However, such causative gene analysis cannot be performed as a routine clinical diagnostic test. Immunohistochemical staining of LEKTI and Cdsn using patient skin could be a useful diagnostic test for NS [
In this study, we analyzed distribution patterns of corneodesmosome components and cell surface area in AD. We found that AD corneocytes showed different results depending on disease conditions: lesional or non-lesional. We also compared these results with those of NS, PSS and ichthyosis vulgaris (IV) which is a major predisposing condition for AD [
], in order to determine if there are any differences as compared with AD.
2. Materials and methods
All participants provided informed consent and the protocol was approved by the medical ethics committee of the Asahikawa Medical University. The study was conducted according to the principles of the Helsinki declaration.
2.1 SC samples
SC samples were obtained from 22 adult and 15 pediatric cases of AD, three adult cases of IV (two heterozygote for the filaggrin mutations (S2889X in both cases), one compound heterozygote (3321delA, S2554X)), two adult cases of NS (a heterozygote for the SPINK5 mutations R790X [
LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.
Clinical and laboratory evaluation of 4 pediatric cases with Netherton syndrome in the department of dermatology, Juntendo University School of Medicine.
Elevated human tissue kallikrein levels in the stratum corneum and serum of peeling skin syndrome-type B patients suggests an over-desquamation of corneocytes.
]) with tape stripping. Among the AD samples collected, 14 obtained from adults and 6 from children were collected from pretreated lesional skin of the forearms; the remaining samples (8 samples from adults and 6 from children) were collected from non-lesional skin of the arms and the lower legs. No AD cases showed ichthyosis clinically, and the filaggrin gene mutation analysis was not performed. In all IV cases, we collected samples from ichthyotic skin of the lower legs. In NS cases, samples were collected from the lichenified area of the upper arm. In PSS cases, we obtained them from both non-erosive and erosive areas. For normal controls, we obtained samples from the forearms of 7 adults and 5 children with no history of skin disease. We used an 18 mm wide double-sided plastic adhesive tape (Nichiban No TW-18SD; Nichiban, Tokyo, Japan). According to the methods described by Oyama et al. [
New non-invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape-stripped samples.
], we cut the tape into 18 mm × 5 mm pieces, and then fixed them onto a glass slide. The slide was pressed on the skin once for about 10 s and then stripped away slowly.
2.2 Antibodies
The following were used as primary antibodies: polyclonal rabbit antibodies raised against the central part of human Cdsn [
], monoclonal mouse antibodies against the extracellular part of human Dsg1 (Dsg1-P23, Progen Biotechnik GmbH, Heidelberg, Germany), and polyclonal goat antibody raised against extracellular domain of human Dsc1 (L-15, Santa Cruz). The following secondary reagents were used for immunofluorescence analysis: Alexa-Fluor 488 goat anti-mouse IgG highly cross-absorbed (Molecular Probes, Eugene, OR), Alexa-Fluor 546 donkey anti-goat IgG (Molecular Probes), and Cy3-labeled goat anti-rabbit IgG (Amersham Bioscience, Buckinghamshire, UK).
2.3 Immunofluorescence and microscopy
The tape-stripped corneocytes were rinsed with phosphate-buffered saline (PBS) for 10 min at room temperature and incubated with primary antibodies diluted in PBS overnight at 4 °C. After washing in PBS three times, fluorescent antibodies diluted in PBS were applied for 30 min at 37 °C. For double-labeling with antibodies raised in different animals, a mixture of primary antibodies (Dsg1 and Cdsn/Dsg1 and Dsc1) was applied and this was followed by incubation with a mixture of secondary antibodies-conjugated with different fluorescent dyes. The samples were covered with an aqueous-based mounting medium (PermaFluor, Thermo Fisher Scientific, Waltham, MA) and a cover glass. Fluorescence images were obtained using an Olympus BX50 microscope (Olympus, Tokyo, Japan) with a digital camera (DP71, Olympus). Imaging was performed using Lumina Vision software ver. 2.4.4 (Mitani Corporation, Fukui, Japan). Frequency of different staining patterns of corneodesmosome components in different diseases was compared using the Mann–Whitney U test.
2.4 Measurement of corneocyte surface area
Corneocyte surface area was measured on immunofluorescence images of Dsg1 stained corneocytes using Image-Pro Plus ver. 4.0 software (Media Cybernetics, USA). Data were expressed as median [range] of 10 corneocytes in each sample. Statistical significance of the differences between different samples was evaluated using Kruskal–Wallis test and Steel–Dwass test. We also tested whether the corneocyte surface areas were correlated with the dot distribution patterns of Dsg1 with Spearman's correlation.
3. Results
3.1 Classification of the distribution patterns of corneodesmosomal components
All three corneodesmosomal components were localized in discrete dots on the cell surface in immunofluorescent microscopy (Fig. 1). We classified the distribution patterns into four types and representative cells are shown in each panel of Fig. 1. (I) Peripheral (Fig. 1a): the stained dots were distributed in a linear fashion at the cell peripheral area. (II) Sparse diffuse (Fig. 1b): the dots were seen sparsely throughout the cell surface. (III) Dense diffuse (Fig. 1c): diffuse dots covered the entire cell surface. (IV) Partial diffuse (Fig. 1d): diffuse staining was seen in up to half of the cell surface, but the other surface areas showed different patterns. We randomly chose 50 corneocytes in each sample, classified them into the four patterns, and compared the rate of each pattern in each corneodesmosome component.
Fig. 1Classification of the distribution patterns of corneodesmosomal components. In all panels, green discrete dots on the corneocyte surface show the distribution of Dsg1 in immunofluorescent microscopy. Representative cells are marked with red dashed circles on each panel: (a) peripheral pattern; (b) sparse diffuse pattern; (c) dense diffuse pattern; and (d) partial diffuse pattern.
3.2 The distribution patterns of Dsg1, Cdsn and Dsc1 in normal and diseased skin
In normal skin, all three components showed essentially the same staining pattern. Peripheral pattern accounts for about 90% (Supplementary Figs. S1 and S3a). The corneocytes were stripped as a single-layered sheet and the cells maintained seamless contact with each other.
In AD, we obtained samples under two different conditions, one from untreated lesions of poorly controlled patients (AD lesion), and the other from clinically normal skin of well controlled patients (AD non-lesion). There was no apparent difference in immunofluorescence staining patterns among the three corneodesmosome components in the two conditions (Fig. 2 and Supplementary Fig. S3b and c). In AD lesion skin, a few layers of the corneocytes were stripped by single tape stripping and the corneodesmosomal components were distributed almost throughout the entire surface of the cells, as the dense diffuse pattern together with the sparse diffuse pattern accounted more than 90%. Small numbers of cells (about 2%) showed the partial diffuse pattern. In AD non-lesion skin, corneocytes were stripped off in a single layer and the corneodesmosomal components were arranged mainly in the peripheral pattern (60–65%), but the dense diffuse (about 10%) and the sparse diffuse patterns (26–31%) were more frequent than in the normal control.
Fig. 2Immunofluorescent images of corneodesmosomal components in tape-stripped atopic dermatitis (AD) lesional (a–c) and non-lesional (d–f) corneocytes. (a)–(c) Dsg1 (a), Cdsn (b) and Dsc1 (c) staining of tape-stripped corneocytes from the untreated lesion (AD lesion). The corneocytes are stripped with a few layers of the cells and dots of these components are distributed almost throughout the entire surface of the cells in the dense diffuse pattern. (d)–(f) Samples of non-lesional skin from well controlled AD patients (AD non-lesion). The corneocytes were stripped in a single layer and fluorescence dots of Dsg1 (d), Cdsn (e) and Dsc1 (f) show heterogeneous staining patterns.
In the icthyotic skin samples from IV patients (Supplementary Figs. S1 and S3d), the most frequent pattern was the peripheral pattern just as in normal skin samples, but the peripheral staining line was thicker than in normal samples. The rates of distribution patterns in IV and AD non-lesion skin samples resembled each other and distinguishing them was difficult by only examining the dot distribution patterns.
In NS patients (Fig. 3), some were stripped as a single layer of cells, but others were stripped as a block of several layers of cells. The linear cell–cell contacts were frequently lost, and some corneocytes were scattered individually. Loss of lateral cell–cell attachment was also observed in HE-stained vertical skin sections (Fig. 3d). The main dot distribution was the dense diffuse pattern (58–68%). About 20% of the corneocytes showed the sparse diffuse pattern, and the partial diffuse pattern accounted for 10% to 15% (Supplementary Fig. S3e). AD lesion data and NS data in the bar graph of Supplementary Fig. S3 appear to be similar. In order to determine whether differences between them exist, we compared the frequency of the partial diffuse pattern in each component of the corneodesmosomes. With the Mann–Whitney U test, we detected significant differences in the frequency of Dsg1 (p < 0.01), Cdsn (p < 0.01) and Dsc1 (p = 0.002). The partial diffuse pattern was more frequent in NS than in AD lesion skin.
Fig. 3Immunofluorescent images of corneodesmosomal components in tape-stripped Netherton syndrome (NS) corneocytes (a–c) and HE-stained histological (d) images. Cell–cell contacts are frequently lost and the staining patterns of corneodesmosomal components are heterogeneous in NS. The corneocytes were stripped from the lichenified area of the upper arm. (a)–(c) Examples of distribution patterns of Dsg1 (a), Cdsn (b) and Dsc1 (c). The major distribution pattern is the dense diffuse pattern (c), but the sparse diffuse pattern (b) and the partial diffuse pattern (a) were also present and (d) loss of lateral cell attachment in the stratum corneum (arrows) in a HE-stained vertical skin section.
We obtained PSS patients’ samples from two different areas: areas without erosion (PSS non-erosive) and areas with shallow erosion (PSS erosive) (Fig. 4g). The most characteristic and unique finding in PSS was the virtual absence of Cdsn staining (Fig. 4 and Supplementary Fig. S3f and g). 98% and 76% of the corneocytes did not show any indication of Cdsn in the non-erosive and erosive areas, respectively. The staining patterns of Dsg1 and Dsc1 were almost the same. In PSS non-erosive skin samples, the primary patterns seen were the peripheral and the sparse diffuse patterns. The line forming the peripheral pattern was thicker than that of normal skin. The sparse diffuse pattern accounted for about 45% in both molecules staining. In PSS erosive skin samples, the dense diffuse pattern was the most prominent.
Fig. 4Immunofluorescent images of corneodesmosomal components in tape-stripped peeling skin syndrome type B (PSS) non-erosive (a–c) and erosive (d–f) corneocytes and clinical image (g). Absence of Cdsn in PSS is clearly demonstrated in the tape-stripped corneocytes. Samples from a PSS patient (g). Corneocytes obtained from the skin without erosion (PSS non-erosive) (a–c), and those from shallow erosion (PSS erosive) (d–f), tape-stripped from the square areas on the right and left arms, respectively (g). Cdsn staining is absent in both PSS non-erosive (b) and PSS erosive (e) samples. For Dsg1 (a and d) and Dsc1 (c and f), the main patterns are the peripheral and the sparse diffuse patterns in the non-erosive skin, and the dense diffuse pattern in the erosive skin.
3.3 Corneocyte surface area in normal and diseased skin
Firstly, we measured surface areas of corneocytes obtained from the forearms of adults and children with normal skin. The surfaces were significantly larger in adults (median [range]) (1162.67 [1125.42–1308.89] μm2) than in children (992.68 [946.12–1048.73] μm2; p = 0.0027) (Fig. 5). We therefore compared the data from adult patients with those of normal adults, and the data from child patients with those of normal children in the present study.
Fig. 5Corneocyte surface areas (μm2) in normal and diseased skin from adult (a) and pediatric cases (b). Some diseased corneocytes are smaller than normal. The blue dots indicate the median for each case, and the black bars indicate the median of each category.
In adult samples (Fig. 5a), the corneocyte surface area of AD non-lesion skin and AD lesion skin were significantly smaller than in those of normal control. The corneocytes of AD non-lesion skin were significantly larger than those of AD lesion skin (p < 0.01). The corneocyte of NS skin tended to be smaller than those of all other skin categories, but results were not statistically significant. The results from IV patients did not differ significantly from those of normal skin or the two categories of AD skin.
In child samples (Fig. 5b), the corneocytes of child AD non-lesion skin, AD lesion skin, NS skin, PSS non-erosive skin, and PSS erosive skin tended to be smaller than those of normal child controls, but only the result of AD lesion skin was statistically significant. Additionally, the corneocytes of AD non-lesion skin were significantly larger than those of AD lesion skin (p < 0.05) of the children in the study.
3.4 The correlation between corneocyte surface areas and corneodesmosome component distribution patterns
In the scatter diagram, the corneocyte surface area of adults (Fig. 6a) and children (Fig. 6b) correlated significantly with the rate of combined sparse and dense diffuse pattern of Dsg1 (adult: rs = −0.82, p < 0.01, child: rs = −0.88, p < 0.01) (Fig. 6a and b). There was one overlapping area in adult scatter diagram, namely between IV skin samples and adult AD non-lesion skin samples. In the child scatter diagram, there were two overlapping areas, namely between AD lesion skin samples and PSS erosive skin samples, and between AD non-lesion skin samples and PSS non-erosive skin samples. All lesional AD and erosive PSS skin predominantly showed sparse or dense diffuse staining patterns, and the cell sizes in some lesional AD cases were as small as in erosive PSS cases. All NS skin samples showed lower rates of the combined sparse and dense diffuse staining patterns as compared with lesional AD and erosive PSS skin samples in both of adult and pediatric cases.
Fig. 6Scatter diagrams of corneocyte surface area and rate of combined sparse and dense diffuse pattern of Dsg1 staining dots in adult (a) and pediatric (b) cases. The corneocyte sizes show a negative correlation with the rate of combined sparse and dense diffuse pattern of Dsg1 staining dots in adult cases (a) (Spearman's correlation rs = −0.82, p < 0.01) and pediatric cases (b) (Spearman's correlation rs = −0.88, p < 0.01). Round dots indicate pediatric cases, rhomboidal dots indicate adult cases. Samples in the same category are showed in the same color.
In the present study, we reported that corneocytes of AD (lesion and non-lesion), IV, NS and PSS (erosive and non-erosive) skin showed different staining patterns from those of normal skin. In AD non-lesion corneocytes, while the major peripheral dot distribution pattern was similar to that in normal skin, the sparse diffuse pattern was more frequent than in normal samples in all three corneodesmosomal components (Fig. 2 and Supplementary Fig. S3). The corneocyte surface areas of AD non-lesion skin were significantly smaller than in those of normal skin in adults. These data suggest that subclinical abnormalities exist in AD non-lesion skin, supporting the data by Suárez-Fariñas et al. [
] showing broad terminal differentiation defects in non-lesional AD skin. A similar corneocyte staining pattern was observed in IV (Supplementary Figs. S2 and S3), suggesting that the corneodesmosome degradation process is abnormal in both conditions. This may hamper normal spreading of intracellular lipid layers inducing barrier dysfunction resulting in the development of dermatitis [
]. The corneocyte surface areas were significantly smaller in AD lesion skin than in AD non-lesion skin in adults and children (Fig. 5), as was reported by Kawai et al. [
]. This is thought to be because of deficient cornified cell envelope which cannot flatten corneocytes sufficiently, and this may also explain the smaller cell surface area of AD lesion skin. Suárez-Fariñas et al. [
] reported increased expression of immune-related genes in lesional skin compared with that in non-lesional skin in AD. These altered expressions of the genes involved in keratinization and immune response may relate to the differences in the staining patterns and cell sizes between lesional and non-lesional skin found in this study.
The distribution patterns of three corneodesmosomal components in AD lesions resembled those of NS most markedly among all conditions examined (Fig. 2, Fig. 3 and Supplementary Fig. S3), but rates of the partial diffuse pattern were notably higher in NS. Moreover, the loose lateral cell–cell contact that was also seen in HE vertical sections and the irregularly stripped patterns (Fig. 3) were frequently observed in NS. In NS corneocytes, we found that corneodesmosomes were split before degradation (Supplementary Fig. S4). This may represent a disorganized desquamation process [
LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.
], and explain the different staining patterns in NS and AD. In normal and most AD skin, adult corneocytes were larger than those in child samples. Contrary to that, no significant age-related difference was detected in NS. Similarly, some AD lesion skin also did not show significant age-related difference. This may indicate that continuous desquamation and inflammatory changes disturb physiological age-related alterations in corneocyte surface areas in NS and some severe AD lesions.
In PSS, distinct from any other conditions, there were very few Cdsn-positive corneocytes, even though all cells were positive for Dsg1 and Dsc1 (Fig. 4 and Supplementary Fig. S3). Although detection of Cdsn gene defects might be required for the definite diagnosis of PSS [
], the lack of Cdsn-immunostaining of the tape-stripped corneocytes was so conspicuous that it could possibly serve as a screening test for Cdsn abnormalities.
In both adult and child scatter diagrams, the corneocyte surface areas showed a strong negative correlation with the rate of combined sparse and dense diffuse patterns of Dsg1. The upper left corner characterized by smaller cell size and frequent sparse or dense diffuse Dsg1 staining is occupied by three clinically similar categories: AD lesion, NS, and PSS erosive. Among them, NS cases showed the lowest rates of sparse or dense diffuse staining patterns. This is because NS cases showed higher rates of partial diffuse staining pattern than the others; AD lesion:NS:PSS = 2.8%:15.5%:0% (mean of partial diffuse pattern rate in Dsg1 (%); p < 0.01 [AD lesion vs NS]).
There were two overlaps between AD lesion and PSS erosive and between AD non-lesion and PSS non-erosive in the child scatter diagram. This suggests that each of these two categories share abnormal keratinization processes despite their different pathogenesis. The positions of AD lesion and NS in pediatric cases are also very close. Mohammed et al. [
] reported corneocyte surface areas decreased with depth into the stratum corneum suggesting that more mature corneocytes were larger than less mature ones. The staining pattern and the cell surface area may link together irrespective of pathomechanism, and the results may indicate disease severity in each disease.
Another overlap was detected between adult AD non-lesion cases and IV cases. Since we did not perform filaggrin gene analysis in AD cases without clinical signs of ichthyosis in this study, we could not rule out the possibility that some of them had subclinical ichthyosis vulgaris. Recently, it was reported that filaggrin expression was reduced even in AD patients without major filaggrin gene mutations, and proinflammatory cytokines can down regulate filaggrin expression [
New non-invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape-stripped samples.
]. In the present study, apart from absence of Cdsn in PSS, there was no clear cut disease specific pattern in other diseases. A possible pathognomonic pattern was detected in NS, but more cases have to be examined to validate its specificity. In order to evaluate clinical usability of this test. Prospective studies of a wide range of skin diseases are needed as well.
Nevertheless, this method is non-invasive, is easily performed, and can provide vital information about corneodesmosomal components in various skin conditions including those with unknown etiology. In addition, this method may serve as an objective indicator of disease severity, because AD corneocytes showed different patterns depending on disease conditions. To validate this, repeated studies in the same individuals are needed in future studies. Since there are other molecules besides corneodesmosome components at the corneocyte surface, their analysis may widen the application range of this method even further.
Acknowledgments
A. Ishida-Yamamoto is supported by grants from the Ministry of Health, Labour and Welfare of Japan and the Ministry of Education, Culture, Sports, Science and Technology of Japan (24591620). H. Iizuka is supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21390323).
Appendix A. Supplementary data
The following are the supplementary data to this article:
Bidimensional analysis of desmoglein 1 distribution on the outermost corneocytes provides the structural and functional information of the stratum corneum.
New non-invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape-stripped samples.
Diagnosis and treatment of atopic dermatitis in children and adults: European Academy of Allergology and Clinical Immunology/American Academy of Allergy, Asthma and Immunology/PRACTALL Consensus Report.
The spectrum of pathogenic mutations in SPINK5 in 19 families with Netherton syndrome: implications for mutation detection and first case of prenatal diagnosis.
LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.
Clinical and laboratory evaluation of 4 pediatric cases with Netherton syndrome in the department of dermatology, Juntendo University School of Medicine.
Elevated human tissue kallikrein levels in the stratum corneum and serum of peeling skin syndrome-type B patients suggests an over-desquamation of corneocytes.
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