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      Expression and ultrastructural localization of plasmin(ogen) in the terminally differentiated layers of normal human epidermis

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          Abstract

          Objective

          Plasmin, a relatively unspecific trypsin‐like serine protease, is involved in many physiological and pathological conditions, particularly in dermatoses with barrier impairment. It is secreted as the inactive zymogen plasminogen and is activated to plasmin by plasminogen activators, such as urokinase. There still exists a paucity of data on the precise localization of epidermal plasmin(ogen) within the epidermis and the stratum corneum. The aim of the present study was to get information about its origin and ultrastructural localization within normal human epidermis.

          Method

          We performed immunoelectron transmission electron microscopy immunogold labelling in normal abdominal human skin.

          Result

          Plasmin was only observed in the terminally differentiated cell layers of the epidermis and was largely associated with the corneocyte envelopes and to some extent with the intercellular lipid matrix in the stratum corneum.

          Conclusion

          Our results indicate that in normal human skin, plasmin(ogen) is synthesized by differentiated epidermal keratinocytes of the stratum granulosum and is not serum‐born.

          Abstract

          There exists a paucity of data on the subcellular localization of plasmin(ogen) within normal human epidermis. Immunoelectron transmission electron microscopy labelling revealed that the inflammatory serine protease is located in the terminally differentiated keratinocytes and was largely associated with the corneocyte envelopes and to some extent with the intercellular lipid matrix in the stratum corneum.

          Résumé

          Objectif

          Plasmine, une relativement peu spécifique ‘ trypsin‐like’ protéase sérine, participe aux plusieurs processus physiologiques et pathologiques et, plus particulièrement, à la physiopathologie des dermatoses caractérisées par l’altération de la barrière de perméabilité. Elle est sécrétée sous forme d’un zymogene inactif, plasminogène, et devient activée par les activateurs du plasminogène, telle urokinase. A l’heure actuelle, on manque de précision quant à la localisation de plasmine (ou son précurseur) dans l’épiderme et le stratum corneum. Le but du présent travail a été de d’apporter l’information sur la provenance et la localisation ultrastructurale de plasmine/plasminogène présents dans l’épiderme humain.

          Méthode

          L’étude ultrastructurale de l’épiderme humain normal (plastie abdominale) a fait appel à l’immunomarquage à l’or colloïdal sur coupes ultrafines des tissus inclus à froid dans des résines acryliques.

          Résultat

          L’anticorps monoclonal anti –plasmine/plasminogène a détecté l’antigène situé exclusivement dans la partie la plus différenciée de l’épiderme et persistant dans la couche cornée. Il n’y a pas eu de réactivité dans les couches épineuse et basale. Le marquage a été prédominant sur les enveloppes cornifiées des kératinocytes granuleux et cornéocytes. Des foyers du marquage ont été également présents dans le cytoplasme et les espaces intercellulaires de la couche granuleuse, ainsi que dans la matrice lipidique de la couche cornée profonde.

          Conclusion

          Nos résultats indiquent la production de novo de plasmine/plasminogène dans les kératinocytes le plus différenciés et ne suggèrent pas l’origine sérique de cette enzyme dans l’épiderme.

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          Most cited references24

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          Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients.

          Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.
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            Increased stratum corneum serine protease activity in acute eczematous atopic skin.

            Atopic dermatitis (AD) is a chronic inflammatory disease associated with changes in stratum corneum (SC) structure and function. The breakdown of epidermal barrier function in AD is associated with changes in corneocyte size and maturation, desquamation, lipid profiles, and some protease activities. The purpose of this study was: (i) to examine physiological changes in lesional (L) skin of acute eczematous AD, compared with nonlesional (NL) AD skin and healthy (H) skin, using sequential tewametry and SC protein analysis to estimate SC thickness; and (ii) to assess which serine proteases might be involved in pathogenesis. Six subjects with H skin, six AD patients with NL skin and six AD patients with mild to moderate eczema (L skin) were enrolled. Skin was assessed using several noninvasive techniques but SC thickness was estimated using tewametry and SC protein content of D-Squame strippings. SC integrity was determined by sequential tape stripping (D-Squame) and infrared densitometry. Kallikreins, plasmin, urokinase and leucocyte elastase protease activities together with a novel SC tryptase-like enzyme activity were quantified. Transepidermal water loss (TEWL) levels after D-Squame stripping were elevated in L compared with NL and H skin at all sampling points (P < 0.05). Conversely, the amount of SC removed by sequential tape stripping was decreased in L skin, indicating increased intracorneocyte cohesion (P < 0.05). By correlating 1/TEWL values and SC removed as an estimate of SC thickness, a significantly thinner SC was observed in L compared with NL and H skin (P < 0.05). Elevated extractable serine protease activity was measured in AD skin in the order: SC tryptase-like enzyme (45x), plasmin (30x), urokinase (7.1x), trypsin-like kallikreins (5.8x) and chymotrypsin-like kallikreins (3.9x). Leucocyte elastase activity was not detected in H and NL skin but was observed in AD SC samples (L skin). All enzymes were elevated in the deeper layers of L SC compared with NL and H SC samples. All consistently elevated SC protease activities were significantly correlated with the bioinstrumental data. We report increased serine protease activities in acute eczematous AD, especially in deeper layers of the SC, including SC tryptase-like enzyme, plasmin, urokinase and leucocyte elastase activities. These elevations in protease activities were associated with impaired barrier function, irritation, and reduced skin capacitance. Increased SC cohesion was apparent despite elevated TEWL during tape stripping, which would indicate reduced SC thickness in acute eczematous lesions of AD. Indeed, this was observed using an estimate of SC thickness.
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              Serine protease signaling of epidermal permeability barrier homeostasis.

              Evidence is growing that protease-activated receptor-2 (PAR-2) plays a key role in epithelial inflammation. We hypothesized here that PAR-2 plays a central role in epidermal permeability barrier homeostasis by mediating signaling from serine proteases (SP) in the stratum corneum (SC). Since the SC contains tryptic- and chymotryptic-like activity, we assessed the influence of SP activation/inhibition on barrier function. Acute barrier disruption increases SP activity and blockade by topical SP inhibitors (SPI) accelerates barrier recovery after acute abrogation. This improvement in barrier function is due to accelerated lamellar body (LB) secretion. Since tryptic SP signal certain downstream responses through PAR-2, we assessed its potential role in mediating the negative effects of SP on permeability barrier. Firstly, PAR-2 is expressed in the outer nucleated layers of the epidermis and most specifically under basal condition to the lipid raft (LR) domains. Secondly, tape stripping-induced barrier abrogation provokes PAR-2 activation, as shown by receptor internalization (i.e. receptor movement from LR to cytolpasmic domains). Thirdly, topical applications of PAR-2 agonist peptide, SLIGRL, delay permeability barrier recovery and inhibit LB secretion, while, conversely, PAR-2 knockout mice display accelerated barrier recovery kinetics and enhanced LB secretion, paralleled by increased LR formation and caveolin-1 expression. These results demonstrate first, the importance of SP/SPI balance for normal permeability barrier homeostasis, and second, they identify PAR-2 as a novel signaling mechanism of permeability barrier, that is, of response linked to LB secretion.
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                Author and article information

                Contributors
                rainer.voegeli@dsm.com
                Journal
                Int J Cosmet Sci
                Int J Cosmet Sci
                10.1111/(ISSN)1468-2494
                ICS
                International Journal of Cosmetic Science
                John Wiley and Sons Inc. (Hoboken )
                0142-5463
                1468-2494
                20 November 2019
                December 2019
                : 41
                : 6 ( doiID: 10.1111/ics.v41.6 )
                : 624-628
                Affiliations
                [ 1 ] DSM Nutritional Products Ltd. Kaiseraugst Switzerland
                [ 2 ] AVR Consulting Ltd. Northwich UK
                [ 3 ] CNRS UMR5305 LBTI Lyon France
                Author notes
                [*] [* ] Correspondence: Rainer Voegeli, DSM Nutritional Products Ltd., P.O. Box 2676, Bldg. 205/315, CH‐4303, Kaiseraugst, Switzerland. Tel.: +41 61 815 92 98; fax: +41 61 815 80 50; e‐mail: rainer.voegeli@ 123456dsm.com

                Author information
                https://orcid.org/0000-0002-3951-0329
                Article
                ICS12585
                10.1111/ics.12585
                6899821
                31604367
                ef3ee364-e144-462a-a151-62336c288597
                © 2019 The Authors. International Journal of Cosmetic Science published by John Wiley & Sons Ltd on behalf of Society of Cosmetic Scientists and the Société Française de Cosmétologié.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                History
                : 25 July 2019
                : 08 October 2019
                Page count
                Figures: 3, Tables: 0, Pages: 5, Words: 6347
                Categories
                Research Communication
                Research Communications
                Custom metadata
                2.0
                December 2019
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.7.2 mode:remove_FC converted:05.12.2019

                Medicine
                skin physiology/structure,immunogold labelling,transmission electron microscopy,corneocyte envelope,lamellar body

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