Localization of Ceramide and Glucosylceramide in Human Epidermis by Immunogold Electron Microscopy
2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês
10.1046/j.0022-202x.2001.01527.x
ISSN1523-1747
AutoresGabriele Vielhaber, Stephan Pfeiffer, Lore Brade, Buko Lindner, Torsten Goldmann, Ekkehard Vollmer, Ulrich Hintze, K. P. Wittern, Roger Wepf,
Tópico(s)Advancements in Transdermal Drug Delivery
ResumoCeramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound ω-hydroxy ceramides and one covalently bound ω-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis. Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound ω-hydroxy ceramides and one covalently bound ω-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis. ceramide glucosylceramide O-acyl-N-(ω-hydroxyacyl)-sphingosine N-acyl-sphingosine N-acyl-4-hydroxysphinganine O-acyl-N-(ω-hydroxyacyl)-6-hydroxysphingosine N-(2-hydroxyacyl)-sphingosine N-(2-hydroxyacyl)-4-hydroxysphinganine N-(2-hydroxyacyl)-6-hydroxysphingosine N-(ω-hydroxyacyl)-sphingosine N-(ω-hydroxyacyl)-6-hydroxysphingosine endoplasmic reticulum 1-O-(β-D-glucopyranosyl)-N-acyl-sphingosine N-(2-hydroxypalmitoyl)-4,8-sphingadienine lamellar body matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Among sphingolipids, outstanding interest has been dedicated to the biologic functions of ceramides (Cer) and in the recent past also to those of glucosylceramides (GlcCer). Cer are involved in numerous cellular signaling processes in response to extracellular stress, such as apoptosis, terminal differentiation, or cell cycle arrest and growth suppression (reviewed inGeilen et al., 1997Geilen C.C. Wieder T. Orfanos C.E. Ceramide signalling. regulatory role in cell proliferation, differentiation and apoptosis in human epidermis.Arch Dermatol Res. 1997; 289: 559-566Crossref PubMed Scopus (104) Google Scholar;Perry and Hannun, 1998Perry D.K. Hannun Y.A. The role of ceramide in cell signalling.Biochim Biophys Acta. 1998; 1436: 233-243Crossref PubMed Scopus (289) Google Scholar). Cer analogs with natural fatty acid chain lengths (C16–C24) activate several protein kinases (Müller et al., 1995Müller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (459) Google Scholar;Huwiler et al., 1996Huwiler A. Brunner J. Hummel R. Vervoordeldonk M. Stabel S. van den Bosch H. Pfeilschifter J. Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.Proc Natl Acad Sci USA. 1996; 93: 6959-6963https://doi.org/10.1073/pnas.93.14.6959Crossref PubMed Scopus (180) Google Scholar;Zhang et al., 1997Zhang Y. Yao B. Delikat S. et al.Kinase suppressor of Ras is ceramide-activated protein kinase.Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), protein phosphatases (Chalfant et al., 1999Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid.J Biol Chem. 1999; 274: 20313-20317https://doi.org/10.1074/jbc.274.29.20313Crossref PubMed Scopus (259) Google Scholar), and the protease cathepsin D (Heinrich et al., 1999Heinrich M. Wickel M. Schneider-Brachert W. et al.Cathepsin D targeted by acid sphingomyelinase.EMBO J. 1999; 18: 5252-5263https://doi.org/10.1093/emboj/18.19.5252Crossref PubMed Scopus (286) Google Scholar). The fate of intracellular Cer has been followed using synthetic Cer short-chain analogs (C2–C6) (Rosenwald and Pagano, 1993Rosenwald A.G. Pagano R.E. Intracellular transport of ceramide and its metabolites at the Golgi complex: Insights form short-chain analogs.Adv Lipid Res. 1993; 26: 101-118PubMed Google Scholar) or radiolabeled biosynthetic precursors (Van Echten-Deckert et al., 1997Van Echten-Deckert G. Klein A. Linke T. Heinemann T. Weisgerber J. Sandhoff K. Turnover of endogenous ceramide in cultured normal and Farber fibroblasts.J Lipid Res. 1997; 38: 2569-2579Abstract Full Text PDF PubMed Google Scholar), but the sites of Cer compartmentalization are still obscure. Cer de novo synthesis takes place at the cytoplasmic leaflet of the endoplasmic reticulum (ER). The lipid can be further metabolized to GlcCer at the cytosolic surface of the cis-Golgi cisternae or converted into sphingomyelin, most probably in the Golgi lumen (Jeckel et al., 1992Jeckel D. Karrenbauer A. Burger K.N. van Meer G. Wieland F. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.J Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (249) Google Scholar). Another source of Cer generation is the hydrolysis of sphingomyelin at the plasma membrane in response to extracellular stimuli, such as tumor necrosis factor-α, interleukin-1, and endotoxin (reviewed inLevade and Jaffrézou, 1999Levade T. Jaffrézou J.-P. Signalling sphingomyelinases. which, where, how and why?.Biochim Biophys Acta. 1999; 1438: 1-17Crossref PubMed Scopus (280) Google Scholar). Cell permeable short chain analogs of Cer accumulate in the Golgi apparatus (Lipsky and Pagano, 1983Lipsky N.G. Pagano R.E. Sphingolipid metabolism in cultured fibroblasts. microscopic and biochemical studies employing a fluorescent ceramide analogue.Proc Natl Acad Sci USA. 1983; 80: 2608-2612Crossref PubMed Scopus (153) Google Scholar,Lipsky and Pagano, 1985Lipsky N.G. Pagano R.E. A vital stain for the Golgi apparatus.Science. 1985; 228: 745-747Crossref PubMed Scopus (233) Google Scholar), but the various sites of Cer action also suggest a localization near the plasma membrane and the mitochondria (Perry and Hannun, 1998Perry D.K. Hannun Y.A. The role of ceramide in cell signalling.Biochim Biophys Acta. 1998; 1436: 233-243Crossref PubMed Scopus (289) Google Scholar). GlcCer are important intermediates in glycosphingolipid biosynthesis and markers for multidrug resistant tumors that express enhanced GlcCer levels (Lavie et al., 1996Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. Accumulation of glucosylceramides in multidrug-resistant cancer cells.J Biol Chem. 1996; 271: 19530-19536Crossref PubMed Scopus (302) Google Scholar;Lucci et al., 1998Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Glucosylceramide: a marker for multiple-drug resistant cancers.Anticancer Res. 1998; 18: 475-480PubMed Google Scholar). Moreover, they play an essential part in mitogenesis, epidermal differentiation, and neuronal growth (reviewed inIchikawa and Hirabayashi, 1998Ichikawa S. Hirabayashi Y. Glucosylceramide synthase and glycosphingolipid synthesis.Trends Cell Biol. 1998; 8: 198-202Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar); however, enzyme targets for GlcCer have not yet been identified. As with Cer, the subcellular distribution of endogenous GlcCer is not yet clear. A part of newly synthesized GlcCer is translocated from the cytosolic to the luminal site of the cis-Golgi for further glycosylation; however, a considerable portion of GlcCer is directly transported to the plasma membrane, where the main fraction (up to 45%) of cellular GlcCer is found (Klenk and Choppin, 1970Klenk H.-D. Choppin P.W. Glycosphingolipids of plasma membranes of cultured cells and an enveloped virus (SV5) grown in these cells.Proc Natl Acad Sci USA. 1970; 66: 57-64Crossref PubMed Scopus (114) Google Scholar;Jeckel et al., 1992Jeckel D. Karrenbauer A. Burger K.N. van Meer G. Wieland F. Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.J Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (249) Google Scholar;Warnock et al., 1993Warnock D.E. Roberts C. Lutz M.S. Blackburn W.A. Young W.W. Baenzinger J.U. Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradients magnetic affinity chromatography.J Biol Chem. 1993; 268: 10145-10153Abstract Full Text PDF PubMed Google Scholar). In the epidermis, Cer and GlcCer play a pivotal role in the maintenance of the epidermal permeability barrier. This barrier consists of a compact lipid matrix of Cer, free fatty acids and cholesterol embedded between the corneocytes in the stratum corneum (SC). GlcCer are the major precursors of epidermal Cer (Holleran et al., 1993Holleran W.M. Takagi Y. Menon G.K. Legler G. Feingold K.R. Elias P.M. Processing of epidermal glucosylceramide is required for optimal mammalian cutaneous permeability function.J Clin Invest. 1993; 91: 1656-1664Crossref PubMed Scopus (226) Google Scholar). Both lipid families are transported to the SC by specialized secretory organelles, the lamellar bodies (LB). These are enriched in a polar lipid mixture of GlcCer, phospholipids, and sterols, and contain several acid hydrolases (Freinkel and Traczyk, 1985Freinkel R.K. Traczyk T.N. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.J Invest Dermatol. 1985; 85: 295-298Crossref PubMed Scopus (122) Google Scholar). At the transition of the stratum granulosum (SG) into the SC the LB fuse with the plasma membrane of the uppermost granular cell and extrude their contents into the intercellular space of the SC. Concomitantly, the polar lipids are enzymatically cleaved into the unpolar barrier lipids. The latter are arranged into multiple intercellular lipid lamellae, forming an efficient barrier against transcutaneous water loss (Elias and Menon, 1991Elias P.M. Menon G.K. Structural and lipid biochemical correlates of the epidermal permeability barrier.Adv Lipid Res. 1991; 33: 301-313Google Scholar;Forslind et al., 1997Forslind B. Engström S. Engblom J. Norlén L. A novel approach to the understanding of human skin barrier function.J Dermatol Sci. 1997; 14: 115-125https://doi.org/10.1016/s0923-1811(96)00559-2Abstract Full Text PDF PubMed Scopus (0) Google Scholar). The epidermis is marked by a unique diversity of Cer and GlcCer species. To date, eight Cer and six GlcCer classes are known, the structures of which vary in the degree of hydroxylation of the sphingosine backbone and of the fatty acid Figure 1. Among them there are two Cer and two GlcCer species that contain an ω-hydroxy long chain fatty acid (C28–C34) (Wertz and Downing, 1983Wertz P.W. Downing D.T. Glucosylceramides of pig epidermis: structure determination.J Lipid Res. 1983; 24: 1135-1139Abstract Full Text PDF PubMed Google Scholar;Hamanaka et al., 1989Hamanaka S. Asagami C. Suzuki M. Inagaki F. Suzuki A. Structure determination of Glcβ-1-N-(ω-O-linoleoyl)-acylsphingosines of human epidermis.J Biochem. 1989; 105: 684-690Crossref PubMed Scopus (41) Google Scholar;Robson et al., 1994Robson K.J. Stewart M.E. Michelsen S. Lazo N.D. Downing D.T. 6-Hydroxy-4-sphingenine in human epidermal ceramides.J Lipid Res. 1994; 35: 2060-2068Abstract Full Text PDF PubMed Google Scholar;Stewart and Downing, 1999Stewart M.E. Downing D.T. A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.J Lipid Res. 1999; 40: 1434-1439Abstract Full Text Full Text PDF PubMed Google Scholar). The Cer with ω-hydroxyacyl (ω-OH-acyl) residues are known to be covalently bound to proteins of the cornified envelope of the corneocytes in the SC, thereby connecting the lipid matrix and the surrounding corneocytes (Marekov and Steinert, 1998Marekov L.N. Steinert P.M. Ceramides are bound to structural proteins of the human foreskin epidermal cornified envelope.J Biol Chem. 1998; 273: 17763-17770https://doi.org/10.1074/jbc.273.28.17763Crossref PubMed Scopus (170) Google Scholar). A corresponding covalently bound form of ω-OH-acyl GlcCer has recently been identified in the epidermis of mice accumulating these lipids due to a defect in GlcCer catabolism (Doering et al., 1999aDoering T. Proia R.L. Sandhoff K. Accumulation of protein-bound epidermal glucosylceramides in β-glucocerebrosidase deficient type 2 Gaucher mice.FEBS Lett. 1999; 447: 167-170https://doi.org/10.1016/s0014-5793(99)00274-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). As the amounts of the corresponding Cer were significantly reduced in these transgenic mice, it had been proposed that protein-bound GlcCer are at least in part direct precursors of the protein-bound Cer; however, protein-bound GlcCer have not yet been identified in normal human epidermis. Considering the multiple targets of Cer action and the essential role of GlcCer and Cer in epidermal homeostasis, it is of increasing interest to identify the subcellular localization of these lipids at the ultrastructural level. Recently, specific anti-sera against GlcCer and Cer have become commercially available. First applications of these sera in immunofluorescence staining of human epidermis have been reported (Brade et al., 2000Brade L. Vielhaber G. Heinz E. Brade H. In vitro characterization of anti-glucosylceramide rabbit antisera.Glycobiology. 2000; 10: 629-636Crossref PubMed Scopus (24) Google Scholar;Vielhaber et al., 2001Vielhaber G. Brade L. Lindner B. et al.Mouse anti-ceramide antiserum. A specific tool for the detection of endogenous ceramide.Glycobiology. 2001; 11: 451-457Crossref PubMed Scopus (34) Google Scholar), but proof for the localization of Cer and GlcCer in epidermis by immunoelectron microscopy is still lacking. Here we show that the anti-sera react with both free and acylated ω-OH-GlcCer and ω-OH-Cer, respectively, and report on the ultrastructural localization of GlcCer and Cer in normal human epidermis. The anti-GlcCer rabbit anti-serum and the IgM-enriched mouse anti-Cer anti-serum were a gift from GlycoTech Produktions- und Handelsgesellschaft mbH, Kuekels, Germany. Cer-2 (ceramide type III) and Cer-5 (ceramide type IV), GlcCer-2, cholesterol, N-palmitoyl-D-sphingomyelin, phosphatidyl (Taufkirchen, Germany). Cer-3 was obtained from Cosmoferm (Delft, the Netherlands). Cer-1 was chemically synthesized by R.R. Schmidt (University of Konstanz). Soybean GlcCer was isolated as described previously (Brade et al., 2000Brade L. Vielhaber G. Heinz E. Brade H. In vitro characterization of anti-glucosylceramide rabbit antisera.Glycobiology. 2000; 10: 629-636Crossref PubMed Scopus (24) Google Scholar). In the same publication we had erroneously designated GlcCer-sb as GlcCer-3; however, MALDI-TOF mass spectrometric analysis (positive ion mode) of GlcCer-sb showed a major quasimolecular ion of m/z 736.2 [M + Na]+, corresponding to a GlcCer containing a 2-hydroxypalmitic acid and a 4,8-sphingadienine backbone, which represents the main GlcCer species in soybean (Sullards et al., 2000Sullards M.C. Lynch D.V. Merrill A.H. Adams J. Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry.J Mass Spectrom. 2000; 35: 347-353Crossref PubMed Scopus (97) Google Scholar). All substances were dissolved in chloroform/methanol (3:1, vol/vol). Epidermal lipid extracts from human breast skin were prepared as described byDoering et al., 1999aDoering T. Proia R.L. Sandhoff K. Accumulation of protein-bound epidermal glucosylceramides in β-glucocerebrosidase deficient type 2 Gaucher mice.FEBS Lett. 1999; 447: 167-170https://doi.org/10.1016/s0014-5793(99)00274-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar. For isolation of covalently bound Cer and GlcCer, the residual epidermal fragments were subjected to a further extraction, washed twice with 100% MeOH at room temperature and incubated in 95% MeOH for 1 h at 60°C. The pellet was repeatedly washed and incubated in warm MeOH until the supernatant was free of lipid [as judged from thin-layer chromatography (TLC) staining] before it was treated with 1 M KOH in 95% MeOH for 2 h at 40°C. Liberated lipids were recovered by two subsequent extractions of the supernatant. MALDI-TOF-MS of isolated soybean GlcCer (see Lipids section) and of covalently bound Cer and GlcCer was performed with a Bruker-Reflex III (Bruker-Franzen Analytik, Bremen, Germany) in linear and reflector TOF configuration at an acceleration voltage of 20 kV and delayed ion extraction. The compounds were dissolved in CHCl3/CH3OH (50:50 vol/vol) at a concentration of 1 µg per µl and vigorously vortexed. One microliter of the sample was then mixed with 1 µl 0.5 M matrix solution of 2,5-dihydroxybenzoic acid (gentisic acid, DHB, Aldrich, Steinheim, Germany) in a 2:1 mixture of 0.1% trifluoroacetic acid/acetonitrile and aliquots of 0.5 µl were deposited on a metallic sample holder and dried in a stream of air. The mass spectra shown are the average of at least 50 single analyses. Mass scale calibration was performed externally with similar compounds of known chemical structure. Lipids were separated twice on silica gel 60 TLC plates with a solvent system of CHCl3/MeOH/CH3COOH (190:9:1 vol/vol/vol) and visualized by spraying with 10% CuSO4 and 8% H3PO4 in water and heating at 180°C (Imokawa et al., 1991Imokawa G. Abe A. Kumi J. Higaki Y. Kawashima M. Hidano A. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?.J Invest Dermatol. 1991; 96: 523-526Abstract Full Text PDF PubMed Scopus (0) Google Scholar). For separation of GlcCer, a third run in CHCl3/MeOH/NH4OH (65:35:5 vol/vol/vol) was performed. TLC immunostaining was performed as described previously (Brade et al., 2000Brade L. Vielhaber G. Heinz E. Brade H. In vitro characterization of anti-glucosylceramide rabbit antisera.Glycobiology. 2000; 10: 629-636Crossref PubMed Scopus (24) Google Scholar;Vielhaber et al., 2001Vielhaber G. Brade L. Lindner B. et al.Mouse anti-ceramide antiserum. A specific tool for the detection of endogenous ceramide.Glycobiology. 2001; 11: 451-457Crossref PubMed Scopus (34) Google Scholar). Briefly, the plates were blocked with 0.1% nonfat dry milk/1% polyvinyl pyrrolidone in washing buffer (50 mM Tris/HCl, pH 7.4, 200 mM NaCl) and subsequently incubated overnight with rabbit-anti-GlcCer, diluted 1:1000 in blocking solution. After five washings bound antibody was detected by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG, diluted 1:1000 in blocking solution and following treatment with a mixture of 4-chloro-1-naphthol and hydrogen peroxide as the substrate. For immunostaining with anti-Cer the TLC plates were first incubated with 0.1% saponin in washing buffer, blocked with 5% nonfat dry milk in washing buffer and subsequently incubated with mouse serum against ceramide (1:25) in blocking solution. After five washings bound antibody was detected by incubation with horseradish peroxidase-conjugated goat anti-mouse IgM, diluted 1:1500 in blocking solution and developed as described for anti-GlcCer. Normal human skin biopsies obtained from human forearm were used in this study. Cryopreparation by high-pressure freezing and freeze substitution was carried out as described byPfeiffer et al., 2000Pfeiffer S. Vielhaber G. Vietzke J.-P. Wittern K.-P. Hintze U. Wepf R. High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study of human epidermis by cryoimmobilization.J Invest Dermatol. 2000; 114: 1030-1038Crossref PubMed Scopus (61) Google Scholar. Briefly, the skin specimens were placed in the cavity (100 µm in depth) of a standard aluminum platelet filled with 1-hexadecene. The sandwich was inserted in the specimen holder and frozen in a high-pressure freezer (HPM 010 Bal-Tec, Balzers, FL;Müller and Moor, 1983Müller M. Moor H. Cryofixation of thick specimens by high-pressure freezing.in: Revel J.P. Barnad T. Haggis G.H. The Science of Biological Specimen Preparation for Microscopy and Microanalysis. Scanning Electron Microscopy Inc., AFM O'Hara, Dr Om Jahari, Chicago, IL1983: 131-138Google Scholar). The subsequent freeze substitution (at -90°C for 30 h, -70°C for 8 h, -50°C for 8 h) was performed using acetone as substitution medium and uranyl acetate as fixing agent in a conventional freeze-substitution unit (FSU 010 Bal-Tec, Balzers, FL). Before cooling down to -90°C, the substitution medium was saturated with uranyl acetate at room temperature. The specimens were transferred directly to transfer vessels (Hohenberg et al., 1994Hohenberg H. Mannweiler K. Müller M. High-pressure freezing of cell suspensions in cellulose capillary tubes.J Microscopy. 1994; 175: 34-43Crossref PubMed Scopus (207) Google Scholar) and placed in 1.5 ml microfuge tubes (Eppendorf) filled with the substitution medium. After freeze substitution the skin specimens were infiltrated with methacrylate (HM20, Polysciences, Eppelheim, Germany) by the following protocol (at -50°C). Specimens were washed in pure acetone for 30 min before infiltration with 30% (vol/vol) HM20/70% (vol/vol) acetone, and 70% HM20/30% acetone for 2 h, followed by three incubations for 2 h each in pure HM20. The ultraviolet polymerization was carried out at -50°C for 48 h followed by a postpolymerization at room temperature for at least 3 d. The following steps were performed at room temperature and all dilutions were prepared in washing buffer (phosphate-buffered saline/0.5% bovine serum albumin/0.2% fish gelatin). For immunofluorescence, semithin sections (200 nm) were placed on glass coverslips, treated with normal goat serum (3% in phosphate-buffered saline) for 1 h, washed twice and incubated with anti-GlcCer-3 anti-serum (1:50) or with anti-Cer-anti-serum (1:2) for 1 h. Subsequently, the sections were washed four times and incubated with a Cy3-conjugated goat anti-rabbit IgG or goat anti-mouse IgM secondary antibody (Dianova, Hamburg, Germany) 1:800 for 1 h. The samples were washed six times, fixed on slides with MOWIOL and investigated with a Axioskop 35 fluorescence microscope (Zeiss, Jena, Germany). Image documentation was done with a Kodak Ektachrome 400 film. Immunogold staining was performed on ultrathin sections (70 nm) as described for immunofluorescence except that the number of washing steps was doubled. For the double labeling experiments the primary and secondary antibodies were applied simultaneously. As secondary antibodies a goat anti-rabbit IgG (F(ab′)2 fragment) conjugated with 10 nm gold (anti-GlcCer) or a goat anti-mouse IgM (µ-chain specific) conjugated with either 5 nm, 15 nm, or (in the double labeling experiments) 20 nm gold was used (each 1:50). Finally, the sections were poststained with uranyl acetate and investigated with a TEM912Ω (LEO, Oberkochen, Germany). Digital image acquisition was performed with a CCD camera (ProScan, München, Germany). Image processing and statistical analysis were performed with the software AnalySIS 2.01 (Soft Imaging System, Münster, Germany). In previous studies it had been demonstrated that the anti-sera against Cer and GlcCer used here for immunohistochemistry react with several epidermal Cer and GlcCer species, respectively, in TLC immunostaining (Brade et al., 2000Brade L. Vielhaber G. Heinz E. Brade H. In vitro characterization of anti-glucosylceramide rabbit antisera.Glycobiology. 2000; 10: 629-636Crossref PubMed Scopus (24) Google Scholar;Vielhaber et al., 2001Vielhaber G. Brade L. Lindner B. et al.Mouse anti-ceramide antiserum. A specific tool for the detection of endogenous ceramide.Glycobiology. 2001; 11: 451-457Crossref PubMed Scopus (34) Google Scholar). Anti-Cer reacts strongly with Cer-2, Cer-5, and Cer-6, and to a lesser extent with Cer-1, Cer-3, and Cer-7 (Figure 2, lanes 1and 3). A reaction with Cer-4 could not be detected, probably due to the low amounts of the lipid present in the epidermal extract. Anti-GlcCer reacts preferentially with GlcCer from soybean (GlcCer-sb), but also with human GlcCer-2 and some yet unidentified epidermal GlcCer species (Figure 3, lanes 1and 3); however, the reactivity with very long chain ω-OH-Cer and ω-OH-GlcCer that occur ester-linked to proteins of the cornified envelope had not so far been investigated. To ensure that the anti-sera are able to detect also these lipid species, the following TLC and ITLC experiments were done.Figure 3Anti-GlcCer detects a covalently bound epidermal GlcCer species in TLC immunostaining. Aliquots of a standard lipid mixture (lane 1), lipids released by base hydrolysis from pre-extracted epidermis (0.5 mg, lane 2) and an epidermal lipid extract (1.0 mg, lane 3) were separated by TLC and visualized with a spray reagent (TLC) or by immunostaining with rabbit anti-GlcCer (ITLC), diluted 1: 1000. The standard lipid mixture was composed of cholesterol (Chol), Cer-2, GlcCer-2, GlcCer from soybean (GlcCer-sb), sphingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylserine (PS) (5 nmol lipid each). The bracket indicates the covalently bound GlcCer. In addition to the specific staining of GlcCer species, a cross-reaction with cholesterol occurred due to the high amounts of cholesterol present in the epidermal extract (lane 3). The arrow indicates the origin.View Large Image Figure ViewerDownload (PPT) Human epidermis was subjected to exhaustive lipid extraction and subsequently treated with alkaline methanol to cleave off ester-linked lipids (Wertz et al., 1989Wertz P.W. Madison K.C. Downing D.T. Covalently bound lipids of human stratum corneum.J Invest Dermatol. 1989; 92: 109-111Abstract Full Text PDF PubMed Google Scholar). The liberated lipids were separated by TLC in parallel with a mixture of purified lipids and an untreated epidermal lipid extract and checked for lipid species reacting with anti-Cer Figure 2 or anti-GlcCer Figure 3. Anti-Cer reacted with two lipids of the fraction released by alkaline hydrolysis (Figure 2, lane 2), the molecular weight of which corresponded to the known protein-bound ω-OH-Cer species, Cer A and Cer B, as determined by MALDI-TOF-MS Figure 4a, b. In TLC immunostaining with anti-GlcCer one major and one minor band with Rf values similar to those of GlcCer-2 were stained in the extract of released covalently bound lipids (Figure 3, lane 2). The mass spectrometric analysis revealed that both bands represented a GlcCer with a ceramide portion corresponding to Cer A with fatty acid chain lengths from C30 up to C37 Figure 4c; however, in accordance withDoering et al., 1999aDoering T. Proia R.L. Sandhoff K. Accumulation of protein-bound epidermal glucosylceramides in β-glucocerebrosidase deficient type 2 Gaucher mice.FEBS Lett. 1999; 447: 167-170https://doi.org/10.1016/s0014-5793(99)00274-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar we could not detect a GlcCer with a ceramide portion corresponding to Cer B. Taken together, with the reaction of the two anti-sera with the majority of known epidermal Cer and GlcCer species, respectively, including very long chain ω-OH-Cer and ω-OH-GlcCer, the prerequisite for a representative immunohistochemical investigation on the ultras
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