Normalization of Epidermal Calcium Distribution Profile in Reconstructed Human Epidermis Is Related to Improvement of Terminal Differentiation and Stratum Corneum Barrier Formation
1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.1998.00251.x
ISSN1523-1747
AutoresJana Vičanová, Esther Boelsma, A. Mieke Mommaas, Johanna Kempenaar, B Forslind, Jan Pallon, Torbjörn Egelrud, Henk K. Koerten, Maria Ponec,
Tópico(s)Transgenic Plants and Applications
ResumoCalcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes. Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes. Nile red reconstructed epidermis stratum corneum stratum corneum chymotryptic enzyme stratum granulosum In vitro generated human epidermis provides an attractive alternative to animal cutaneous toxicity testing, a model for studying skin biology and pathology, and an abundant supply of a biologic material for a treatment of skin wounds. The definitive requirement for efficacy of cultured epidermal analogs is the formation of an effective stratum corneum (SC) barrier. During recent years, several various human skin substitutes have been developed showing a histologic resemblance to the native epidermis; however, these cultured analogs exhibited some abnormalities compared with the native skin. Maturation of the skin analog proceeded too rapidly and the number of living cell layers decreased as a function of the culturing time, whereas the thickness of the SC increased suggesting impaired desquamation (Ponec et al., 1988Ponec M. Weerheim A.M. Kempenaar J.A. Mommaas A.M. Nugteren D.H. Lipid composition of cultured keratinocytes in relation to their differentiation.J Lipid Res. 1988; 29: 949-962Abstract Full Text PDF PubMed Google Scholar). Most of the skin analogs showed a significantly deficient SC barrier function (Régnier et al., 1990Régnier M. Asselineau D. Lenoir M.C. Human epidermis reconstructed on dermal substrates in vitro: an alternative to animals in skin pharmacology.Skin Pharmacol. 1990; 3: 70-85Crossref PubMed Scopus (84) Google Scholar;Ponec et al., 1990Ponec M. Wauben-penris P.J.J. Burger A. Kempenaar J.A. Boddé H.E. Nitroglycerin and sucrose permeability as markers for reconstructed human epidermis.Skin Pharmacol. 1990; 3: 126-135Crossref PubMed Scopus (40) Google Scholar;Mak et al., 1991Mak V.H. Cumpstone M.B. Kennedy A.H. Harmon C.S. Guy R.H. Potts R.O. Barrier function of human keratinocyte cultures grown at the air–liquid interface.J Invest Dermatol. 1991; 96: 323-327Abstract Full Text PDF PubMed Google Scholar;Simonetti et al., 1995Simonetti O. Hoogstraate A.J. Bilaik W. Kempenaar J.A. Schrijvers A.H.G. Boddé H.E. Ponec M. Visualization of diffusion pathways across the stratum corneum of native and in vitro reconstructed epidermis by confocal laser scanning microscopy.Arch Dermatol Res. 1995; 287: 465-473Crossref PubMed Scopus (101) Google Scholar), impaired organization and desquamation of cornified layers (Vičanová et al., 1996Vičanová J. Mommaas A. Mulder A.A. Koerten H.K. Ponec M. Impaired desquamation in the in vitro reconstructed human epidermis.Cell Tissue Res. 1996; 286: 155-162Crossref Scopus (7) Google Scholar), and an abnormal profile and organization of SC lipids (Ponec, 1994Ponec M. Lipid biosynthesis.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 351-363Google Scholar;Fartasch and Ponec, 1994Fartasch M. Ponec M. Improved barrier structure formation in air-exposed human keratinocyte culture systems.J Invest Dermatol. 1994; 102: 366-374Abstract Full Text PDF PubMed Google Scholar;Bouwstra et al., 1995Bouwstra J.A. Gooris G.S. Weerheim A.M. Kempenaar J.A. Ponec M. Characterization of stratum corneum structure in reconstructed epidermis by X-ray diffraction.J Lipid Res. 1995; 36: 496-504PubMed Google Scholar). A recent study from our laboratory has described a marked improvement of the SC architecture and the barrier lipid profile of in vitro reconstructed epidermis (RE) generated in serum-free culture medium containing vitamin and lipid supplements (Ponec et al., 1997aPonec M. Weerheim A.M. Kempenaar J.A. Mulder A.A. Gooris G.S. Bouwstra J.A. Mommaas A.M. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.J Invest Dermatol. 1997 a; 109: 348-355Abstract Full Text PDF PubMed Scopus (243) Google Scholar;Gibbs et al., 1997Gibbs S. Vičanová J. Bouwstra J. Valstar I. Kempenaar J. Ponec M. Culture of reconstructed epidermis in a defined medium at 33°C shows a delayed epidermal maturation, prolonged lifespan and improved stratum corneum.Arch Dermatol Res. 1997; 289: 585-595Crossref PubMed Scopus (54) Google Scholar). The improvement of the lipid synthesis was accompanied by a significantly improved arrangement of the SC barrier lipids, as judged from the extensive production of lamellar bodies and the formation of multiple, broad lipid lamellar structures in the intercorneocyte space; however, small-angle X-ray diffraction analysis revealed differences in the SC lipid organization (Ponec et al., 1997aPonec M. Weerheim A.M. Kempenaar J.A. Mulder A.A. Gooris G.S. Bouwstra J.A. Mommaas A.M. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.J Invest Dermatol. 1997 a; 109: 348-355Abstract Full Text PDF PubMed Scopus (243) Google Scholar), and also the desquamation in these cultured skin analogs is still insufficient (unpublished observations). The maintaining deficiencies could be attributed to differences in the microenvironment. A lack of temperature, pH, and ion gradients can affect the activity of enzymes involved in the arrangement of lipid bilayers and in a disaggregation of the SC into individual squames. A great deal of evidence supports the role of calcium ions in the regulation of the normal epidermal differentiation and desquamation. The calcium cation has been shown to act in the late stage of the S phase, just prior to DNA synthesis (Egljo et al., 1986Egljo K. Hennings H. Clausen O.P.F. Altered growth kinetics proceed calcium-induced differentiation in mouse epidermal cells.In Vitro. 1986; 22: 332-336Google Scholar), and to be essential for the desmosome formation and cell adhesion (Hennings and Holbrook, 1983Hennings H. Holbrook K. Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture.Exp Cell Res. 1983; 143: 127-142Crossref PubMed Scopus (289) Google Scholar;Steinberg et al., 1987Steinberg M. Shida H. Giudice G.J. Shida M. Patel N.H. Blaschuk O.W. On the molecular organization, diversity and functions of desmosomal proteins.Ciba Found Symp. 1987; 125: 3-25PubMed Google Scholar). Ionic calcium probably plays the key role in the regulation and co-ordination of terminal differentiation. Calcium may be the trigger for several terminal events associated with cornification: it is required for activation of some enzyme systems (e.g., phospholipase A) involved in the initial activation of profilaggrin, in the dissolution of keratohyalin granules, and in the cross-linking of cornified envelope precursors via calcium dependent transglutaminase (reviewed in Dale et al., 1994Dale B.A. Resing K.A. Presland R.B. Keratohyalin granule proteins.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 323-350Google Scholar). In addition, it has been shown that the secretion of lamellar bodies is controlled by the concentration of calcium ions in the stratum granulosum (SG) (Menon et al., 1994aMenon G.K. Price L.F. Bommannan B. Elias P.M. Feingold K.R. Selective obliteration of the epidermal calcium gradient leads to enhanced lamellar body secretion.J Invest Dermatol. 1994 a; 102: 786-795Google Scholar), and calcium may also be involved in the initial formation of the lipid bilayers. In the inner SC, calcium has been suggested to be involved in a protecting mechanism of corneosomes (modified desmosomes) against premature proteolysis and/or in the inhibition of proteolytic enzymes involved in desquamation (Lundström and Egelrud, 1990Lundström A. Egelrud T. Cell shedding from human plantar skin in vitro: evidence that two different types of protein structures are degraded by a chymotrypsin-like enzyme.Arch Dermatol Res. 1990; 282: 234-237Crossref PubMed Scopus (47) Google Scholar). In an attempt to understand the physiology of cellular differentiation and desquamation in RE generated under various air-exposed conditions, we employed ion capture cytochemistry (the potassium oxalate-pyroantimonate method) and particle proton probe X-ray microanalysis for the analysis of calcium distribution over skin cross-sections. As the inefficient desquamation observed in RE might be caused by abnormalities in enzymatic activities in the SC, we examined expression of stratum corneum chymotryptic enzyme (SCCE), a human serine protease with chymotrypsin-like primary substrate specificity generally present in the human SC, as it is likely to be involved in the degradation of intercellular cohesive structures in the SC (Lundström and Egelrud, 1990Lundström A. Egelrud T. Cell shedding from human plantar skin in vitro: evidence that two different types of protein structures are degraded by a chymotrypsin-like enzyme.Arch Dermatol Res. 1990; 282: 234-237Crossref PubMed Scopus (47) Google Scholar,Lundström and Egelrud, 1991Lundström A. Egelrud T. Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation.Acta Derm Venereol (Stockh). 1991; 71: 471-474PubMed Google Scholar;Egelrud, 1993Egelrud T. Purification and preliminary characterization of stratum corneum chymotryptic enzyme: A proteinase that may be involved in desquamation.J Invest Dermatol. 1993; 101: 200-204Abstract Full Text PDF PubMed Google Scholar;Sondell et al., 1995Sondell B. Thornell L.-E. Egelrud T. Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies.J Invest Dermatol. 1995; 104: 819-823Crossref PubMed Scopus (87) Google Scholar). Moreover, as the maintenance of the epidermal calcium gradient in vivo has been shown to be dependent on a normal barrier function (Menon et al., 1992Menon G.K. Elias P.M. Lee S.H. Feingold K.R. Localization of calcium in murine epidermis following disruption and repair of permeability barrier.Cell Tiss Res. 1992; 270: 503-512Crossref PubMed Scopus (155) Google Scholar,Menon et al., 1994bMenon G.K. Elias P.M. Feingold K.R. Integrity of the permeability barrier is crucial for maintenance of the epidermal calcium gradient.Br J Dermatol. 1994 b; 130: 139-147Crossref PubMed Scopus (97) Google Scholar), we examined the barrier properties of the RE generated under serum-free culture conditions by investigating the penetration pathways of a model compound across the SC by means of confocal laser scanning microscopy. Cultures of normal human keratinocytes were prepared as described inPonec et al., 1997aPonec M. Weerheim A.M. Kempenaar J.A. Mulder A.A. Gooris G.S. Bouwstra J.A. Mommaas A.M. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.J Invest Dermatol. 1997 a; 109: 348-355Abstract Full Text PDF PubMed Scopus (243) Google Scholar. The keratinocytes were isolated from a split-thickness skin obtained from healthy adult donors undergoing surgical corrections. Reconstructed epidermis was generated by seeding of 2 × 105 second passage human keratinocytes per cm2 onto a de-epidermized dermis. The cells were incubated for 3 d submerged in the culture medium and then lifted to the air–liquid interface and incubated for another 14 d. When serum-containing medium was used, the keratinocyte cultures were incubated the entire culture period with a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (containing 1.6 mM calcium) supplemented with 5% HyClone serum (Greiner, Nürtingen, Germany), 1 μM hydrocortisone, 1 μM isoproterenol, 0.1 μM insulin with addition of 10 ng epidermal growth factor per ml when air exposed. This medium will be referred to as serum-containing medium. For the cultures grown in serum-free medium, the cells were first incubated overnight in culture medium consisting of a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 media (containing 1.6 mM calcium) supplemented with 1% HyClone serum (Greiner), 0.5 μM hydrocortisone, 1 μM isoproterenol, and 0.1 μM insulin. The second day, 10 μM carnitine, 10 mM serine, a mixture of fatty acids (25 μM palmitic acid, 25 μM oleic acid, 15 μM linoleic acid, 7 μM arachidonic acid, 1 μM α-tocopherol;Boyce and Williams, 1993Boyce S.T. Williams M.L. Lipid supplemented medium induces lamellar bodies and precursors of barrier lipids in cultured analogues of human skin.J Invest Dermatol. 1993; 101: 180-184Abstract Full Text PDF PubMed Google Scholar), and 50 μg ascorbic acid per ml (Ponec et al., 1997aPonec M. Weerheim A.M. Kempenaar J.A. Mulder A.A. Gooris G.S. Bouwstra J.A. Mommaas A.M. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.J Invest Dermatol. 1997 a; 109: 348-355Abstract Full Text PDF PubMed Scopus (243) Google Scholar) were added into the culture medium. When lifted to the air–liquid interface, the cultures were grown for 14 d at 37°C in serum-free medium with supplements as described above and an additional 1 ng epidermal growth factor per ml. This medium will be referred to as serum-free medium. When the effect of retinoic acid was examined, the medium was supplemented with 1 μM trans-retinoic acid. All additives were purchased from Sigma (St. Louis, MO). The culture medium was refreshed three times a week. Ultrastructural localization of calcium by ion-capture cytochemistry Cytochemical localization of calcium by oxalate-pyroantimonate precipitation was carried out as described by Menon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (368) Google Scholar. Keratinocyte cultures (n = 3) and freshly obtained biopsies (n = 6) were minced (0.5 mm3) and fixed overnight in ice-cold fixative containing 2% glutaraldehyde, 2% paraformaldehyde, 90 mM potassium oxalate, and 1.4% sucrose (adjusted to pH = 7.4 with potassium hydroxide). The tissue samples were post-fixed in 1% osmium tetroxide containing 2% potassium pyroantimonate for 2 h at 4°C, rinsed in cold distilled water (adjusted to pH = 10 with potassium hydroxide), dehydrated in 70% ethanol, and routinely embedded in Epon or Spurr. On ultrathin sections, calcium containing deposits appeared as electron-dense granules. To check the specificity of the reaction, unstained sections in Spurr were treated with 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (1 h, 60°C), as described by Ravazola, 1976Ravazola M. Intracellular localization of calcium in the chromaffin cells of the rat adrenal medulla.Endocrinology. 1976; 98: 950-953Crossref Scopus (27) Google Scholar). The calcium deposits disappeared after treatment with ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, whereas exposure to hot distilled water alone had no effect. The ultrathin sections were viewed unstained or double-stained with uranyl acetate and lead hydroxide in a Philips EM-410 transmission electron microscope operating at 80 kV. For quantitation of the calcium precipitates, a semiquantitative scale ranging from 0 (absence) to ++++ (the maximum density) was employed to facilitate comparisons of abnormal versus normal localization data. A fresh biopsy (n = 1) and RE generated in serum-free medium (n = 1) were frozen in liquid propane and subsequently cryo-sectioned to minimize ion movements. Freeze-sectioning was performed at –30°C in a conventional cryostat perpendicular to the skin surface with a nominal section thickness of 16 μm. The sections were collected on a carbon holder and dried at –30°C in the cryotome for 20 h. After freeze-drying, the sections were transferred to slot grids with no support film. The mounted specimens were stored in a sealed bag with drying material before being analyzed. The technical details of the particle induced X-ray emission analysis have been presented elsewhere (Pallon et al., 1992Pallon J. Knox J. Forslind B. Werner-linde Y. Pinheiro T. Applications in medicine using the new Lund microprobe.Nucl Instr Meth Phys Res B. 1992; 77: 287-293Crossref Scopus (23) Google Scholar;Forslind et al., 1997Forslind B. Lindberg M. Roomans G.M. Pallon J. Werner-linde Y. Aspects on the physiology of human skin: Studies using particle probe analysis.Microsc Res Tech. 1997; 38: 373-386Crossref PubMed Scopus (39) Google Scholar). The new Lund Nuclear Scanning Microprobe facility provided a 2.5 MeV proton beam and with the beam current of 0.5–1 nA on the specimen. A rectangular beam scan (nominal beam cross-section of 5 μm × 5 μm) was located over a selected area on each skin section so as to cover a cross-section of epidermis and dermis down to the reticular dermis that usually corresponds to a total skin depth of about 200 μm. To minimize the thermal load on the specimen a scanning mode for specimen excitation and data acquisition was used. Thus, the probe irradiates each specimen volume (pixel) on the average 5 ms in an iterate process. The pixel size was chosen so as to avoid overlap of the beam penumbra (i.e., total cross-section 5 + 2 μm in X- and Y-direction, respectively). Typical acquisition times for a pixel map (64 × 64 pixels) was about 2–3 h. The recorded complete X-ray data (the relative elemental content) and the mass cross-section data for corresponding pixels were merged into several pixel spectra. Pixels bands were chosen to produce cross-section distribution curves from the produced elemental maps. Three sections of each specimen were analyzed. Two different samples of RE generated in serum-free medium were examined for corneosome frequency, as described inVičanová et al., 1996Vičanová J. Mommaas A. Mulder A.A. Koerten H.K. Ponec M. Impaired desquamation in the in vitro reconstructed human epidermis.Cell Tissue Res. 1996; 286: 155-162Crossref Scopus (7) Google Scholar. Briefly, the SC was divided into a lower and an upper part (each consisting of seven layers of corneocytes), and 8–10 non-overlapping electron micrographs of each part were taken at a 12,000-fold magnification and reproduced at a 30,000-fold magnification. The total corneocyte envelope length and the total length of corneosomes were measured for all single electron micrographs using Vidas Rel.2.1 image program (Kontron Electronic, Echting, Germany). The corneosome frequency FC was calculated for each single electron micrograph as a percentage of the total corneocyte envelope length occupied by corneosomes as follows:FC=∑LC/∑LE×100(%)where LC is the corneosome length and LE is the corneocyte envelope length. The corneosome frequency was expressed as a mean ± SD for both parts of the SC (SPSS 6.1 for Windows). Two to three uppermost layers of the SC were not included for the quantitation of corneosome frequency, because these layers evidently represent an artefact of an imbalance in the differentiation process during the first days of culturing described in Results, Figure 3c). Reconstructed epidermis (RE) generated in both serum-containing and serum-free media was separated from the substrate, dried, and stored at –20°C until analyzed. Plantar SC was used as a control. Before the extraction, RE was minced with a pipette tip, and plantar SC was grinded with a mortar. RE or plantar SC (10 μg per ml) was suspended in Laemmli's sample buffer (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202762) Google Scholar) without mercaptoethanol and incubated for 1 h at room temperature. The suspension was homogenized in a glass homogenizer and then centrifuged for 10 min at 10,000 × g. The supernatant was analyzed by electrophoresis. Zymography in 10% sodium dodecyl sulfate-polyamide gels with 1% heat denatured casein was carried out as described (Egelrud and Lundström, 1991Egelrud T. Lundström A. A chymotrypsin-like proteinase that may be involved in desquamation in plantar stratum corneum.Arch Dermatol Res. 1991; 283: 108-112Crossref PubMed Scopus (103) Google Scholar), with extracts prepared at room temperature as above. Mercaptoethanol was added to the extracts from RE and plantar SC to give a final concentration of 5% (vol/vol). The extracts were then heated to 95°C for 2 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were carried out as described (Sondell et al., 1994Sondell B. Thornell L.-E. Stigbrand T. Egelrud T. Immunolocalization of stratum corneum chymotryptic enzyme in human skin and oral epithelium with monoclonal antibodies: evidence of a proteinase specifically expressed in keratinizing squamous epithelia.J Histochem Cytochem. 1994; 42: 459-465Crossref PubMed Scopus (68) Google Scholar) with an affinity purified polyclonal rabbit antibody to human SCCE as first antibody (Sondell et al., 1996Sondell B. Dyberg P. Anneroth G. Östman P.-O. Egelrud T. Association between expression of stratum corneum chymotryptic enzyme and pathological keratinization in human oral mucosa.Acta Derm Venereol (Stockh). 1996; 76: 177-181PubMed Google Scholar), followed by alkaline phosphatase goat anti-rabbit immunoglobulins. The alkaline phosphatase was detected according to Blake et al., 1984Blake M.S. Johnston K.H. Russel Jones G.J. Gotschlich E.C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.Anal Biochem. 1984; 136: 175-179Crossref PubMed Scopus (1616) Google Scholar. Confocal fluorescent images of diffusion pathways across the SC of freshly excised skin and RE grown in serum-free medium were obtained as described elsewhere (Simonetti et al., 1995Simonetti O. Hoogstraate A.J. Bilaik W. Kempenaar J.A. Schrijvers A.H.G. Boddé H.E. Ponec M. Visualization of diffusion pathways across the stratum corneum of native and in vitro reconstructed epidermis by confocal laser scanning microscopy.Arch Dermatol Res. 1995; 287: 465-473Crossref PubMed Scopus (101) Google Scholar). Ten microliters of 10 mM Nile red (NR, 9-diethyl amino-5H-benzo-α-phenox-azine-5-one; Sigma) in dimethyl sulfoxide was applied topically on the surface of the skin samples. After 10 min the solution was gently wiped off with a tissue. The skin samples were placed skin surface down on coverslip glasses, fixed in the sample holder, and placed into the confocal laser scanning microscopy system [BioRad MRC 600 confocal unit equipped with an argon laser (excitation lines at 488 and 514 nm) and mounted on a Zeiss IM-35 inverted microscope]. The NR was detected using the BioRad Green High Sensitivity filter block and the confocal images were obtained using a Zeiss PlanNeofluar lens. A stack of optical slices of the skin sample was gained by moving the plane of focus of the microscope along the Z-axis (perpendicular to the skin surface) with a stepper motor. In this way, a three-dimensional image was made. All images were the average of 10 scans and were obtained with the same laser intensity, filter block, lens, black level, and scan speed; the images were not enhanced after acquisition (Hoogstraate et al., 1994Hoogstraate A.J. Cullander C. Nagelkerke F. Senel S. Verhoef J. Junginger H.E. Boddé H.E. Diffusion rates and transport pathways of FITC-labeled model compounds through buccal epithelium.Pharm Res. 1994; 11: 83-89Crossref PubMed Scopus (56) Google Scholar). In the basal and spinous cells, the calcium containing deposits were localized intracellularly within some nuclei and mitochondrial matrix and to a variable extent extracellularly (not shown). Intracellular deposits were more abundant within the granular layer with the uppermost granular cells showing the highest density of the calcium precipitates localized free within the cytoplasm, within lamellar bodies, mitochondria, and nuclei. The SC contained intracellularly small quantities of precipitates in the lowest 1–2 layers, other layers were devoid of precipitates Figure 1a). In one of the six examined specimens, the calcium containing precipitates were found upper in the SC up to 4–5 layers Figure 1b). Occasionally, some labeled, probably not fully transformed desmosomes, were found in the lower SC Figure 1c). Similar observations were described previously byMenon et al., 1985Menon G.K. Grayson S. Elias P.M. Ionic calcium reservoirs in mammalian epidermis: Ultrastructural localization by ion-capture cytochemistry.J Invest Dermatol. 1985; 84: 508-512Abstract Full Text PDF PubMed Scopus (368) Google Scholar andMenon and Elias, 1991Menon G.K. Elias P.M. Ultrastructural localization of calcium in psoriatic and normal human epidermis.Arch Dermatol. 1991; 127: 57-63Crossref PubMed Scopus (162) Google Scholar. The living cell layers of the RE in general resembled the characteristic concentration gradient of calcium ions described in the native epidermis; however, at the level of the basal cells the extracellular domains contained slightly less calcium precipitate than that observed in the native tissue (not shown). The number of calcium precipitates progressively increased toward the upper SG, reaching about the same density as that seen in the native epidermis Figure 2a). The major difference compared with the native epidermis were the
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