Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany, 2Analytical Research Department, Beiersdorf AG, Unnastrasse 48, D-20245 Hamburg, Germany, and 3Institute of General Botany, University of Hamburg, Ohnhornstrasse 18, D-22609 Hamburg, Germany
Received on November 10, 1999; revised on December 20, 1999; accepted on December 20, 1999.
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Abstract |
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Key words: glycolipid antibody/thinlayer immunostaining/immunohistochemistry/epidermal lipids
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Introduction |
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We were especially interested in the localization of GlcCer in human epidermis since GlcCer represent the major precursors of the epidermal ceramides which are essential components of the skin permeability barrier (Holleran et al., 1993). The GlcCer content increases from the basal cell layer up to the outer stratum granulosum about 2-fold, whereas in the stratum corneum only traces of GlcCer are detectable (Lampe et al., 1983
; Yardley, 1983
; Cox and Squier, 1986
). From lipid biochemical and morphological investigations, it has been concluded that GlcCer are transported to the stratum corneum via specialized organelles, the lamellar bodies (LB). The LB are enriched in polar lipids like GlcCer, phospholipids and sterols and contain several acid hydrolases (Freinkel and Traczyk, 1985
; Grayson et al., 1985
). They first appear in the stratum spinosum and accumulate with ongoing differentiation until they fill up to 25% of the cytosol of the granular cells (Elias and Friend, 1975
). Upon reaching the interface of the stratum corneum and stratum granulosum, the LB fuse with the plasma membrane of the uppermost granulocytes and extrude their contents into the intercellular space of the stratum corneum. Concomitantly, the lipids are enzymatically processed into an unpolar mixture of ceramides, fatty acids, and cholesterol which are arranged into multiple lamellae. Together with the surrounding corneocytes, the intercellular lipid lamellae constitute the epidermal permeability barrier (Elias and Menon, 1991
; Forslind et al., 1997
).
From studies with knock out mice it is evident that the conversion of GlcCer into ceramides at the interface of the stratum corneum and stratum granulosum is of decisive importance for epidermal permeability barrier construction: accumulation of GlcCer within the lower parts of the stratum corneum due to deficiency of the processing enzyme ß-glucocerebrosidase (Holleran et al., 1994; Sidransky et al., 1996
) or the related sphingolipid activator protein C (Döring et al., 1999a
) results in impaired barrier function. Nevertheless, the immunohistochemical proof for the localization of GlcCer in the epidermis is still lacking.
Here we report on commercially available rabbit sera claimed to be specific for GlcCer. We determined their in vitro binding properties, their specificity and sensitivity and demonstrate their use for the identification of GlcCer by EIA, on TLC plates by immunostaining, and for the immunohistochemical localization in human epidermis.
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Results |
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Discussion |
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The manifold applications of these reagents are evident and cannot be discussed here in all aspects but the role of GlcCer-2 and GlcCer-3 in the epidermis of mammalians for maintaining the barrier function of the skin is one well established example. Therefore, we proved the high quality and the specificity of these reagents by immunohistochemistry of human skin biopsies. Moreover, it was possible for the first time to visualize the distribution of a direct precursor of an epidermal barrier lipid in human epidermis. The observed immunofluorescence staining pattern for anti-GlcCer with concentration of GlcCer in the granular and upper spinous layers corresponds well to the known lipid analytical data of GlcCer distribution in human epidermis (Lampe et al., 1983; Yardley, 1983
). However, the lack of signal in the lower epidermis points out that the concentration of GlcCer has to exceed a critical threshold to cause a signal in immunohistochemistry. This threshold is reached by the increasing accumulation of GlcCer in LB with ongoing epidermal differentiation as seen from our light microscopical data. In addition, preliminary immunoelectron microscopy experiments support the general assumption that GlcCer are transported via LB to the stratum corneum.
The samples used in this study have been prepared by a protocol developed for transmission electron microscopy with the aim of an optimal lipid and protein antigen preservation (Pfeiffer et al., forthcoming). However, we detected a similar antigenic staining pattern in chemically fixed skin biopsies which had been dehydrated stepwise by a graded ethanol series with simultaneous lowering of the temperature from 273 K to 223 K (data not shown). If the dehydration was performed continuously at 273 K, the staining blurred and a remarkable background was obtained. These findings suggest that one should be careful of completely dehydrating the tissue with ethanol at 273 K or higher temperatures for anti-GlcCer immunohistochemistry.
Since it was not the primary aim of the present study to focus on the function of GlcCer in epidermis, we did not determine the identity of all epidermal cerebroside species reacting with the GlcCer-3 antiserum. At least six different glucosylceramide species of porcine epidermis (Wertz and Downing, 1983) and of human keratinocyte cell cultures (Hamakana et al., 1993
), respectively, have been structurally characterized. Among these
-hydroxylated GlcCer are of special interest, two of which have been also identified in human epidermis (Hamakana et al., 1989
). They are discussed as precursors of corresponding ceramides which are covalently bound to protein components of the cornified cell envelope (Swartzendruber et al., 1987
; Wertz et al., 1989
). The latter represents a rigid polymer structure that is composed of protein and lipid and coats the surface of the corneocytes (Nemes and Steinert, 1999
). Recent work provided evidence that at least a portion of
-hydroxylated GlcCer is first covalently bound to the cornified cell envelope (Döring et al., 1999a
,b), before they are catabolized to protein-bound ceramides. Therefore, a reactivity with
-hydroxylated glucosylceramides would be helpful to elucidate the ultrastructural relations of lipids and proteins in the cornified envelope. We are currently investigating this possibility.
Anti-glycolipid antibodies are potential tools for the diagnosis and therapy of diseases, e.g., cancer (Alfonso and Zeuthen, 1996; Hakomori and Zhang, 1997
) and neurological disorders (Fredman and Lekman, 1997
). The anti-GlcCer antibodies described herein may be a useful tool for the rapid follow-up of enzyme replacement therapy of patients with Gaucher disease type 3 (Gornati et al., 1998
) by EIA instead of glycolipid analysis. Moreover, it will help in the diagnosis of multidrug-resistant cancer cells which are characterized by high cellular levels of GlcCer (Lavie et al., 1996
, 1997).
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Materials and methods |
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Steryl ß-D-glucopyranoside was obtained from the same lecithin fraction. After exhaustive elution with 18% acetone in chloroform (for removal of galactosyldiacylglycerol), steryl ß-D-glucopyranoside was eluted with 25% acetone in chloroform. A final purification by preparative TLC in chloroform/methanol (85:15, vol/vol) and recovery by phase partitioning yielded pure steryl glucoside.
Other glycolipids
GlcCer-2, N-stearoyl-DL-dihydro-glucocerebroside, N-stearoyl-DL-dihydro-galactocerebroside, galactosylceramide type I and II, lactosylceramide, cholesterol, phosphatidylethanolamine, phosphatidylserine, dimyristoylphosphatidylcholine and ceramide-2 (Ceramide Typ III) were purchased from Sigma. Ceramide-3 was obtained from Cosmoferm (Delft, The Netherlands). All substances were dissolved in chloroform/methanol (3:1, vol/vol).
Antisera
Rabbit antisera against purified GlcCer-2 and GlcCer-3 were purchased from GlycoTech Produktions und Handelsgesellschaft mbH, Kuekels, Germany. Samples of preimmune sera were also provided.
Preparation of natural lipid extracts
Epidermal lipid extracts from human breast skin were prepared as described by Döring et al. (1999a).
TLC and TLC-immunostaining
Glycolipids were separated on silica gel 60 TLC plates with aluminum support (Merck) with a solvent system of CHCl3/MeOH/25% aqueous NH4OH (65:35:5, vol/vol/vol) and visualized by spraying with -naphthol in H2SO4 (Figure 4) or 10% CuSO4 and 8% H3PO4 in water (Figure 5) and heating at 180°C (Imokawa et al., 1991). For TLC-immunostaining the plates were incubated with blocking buffer (1% polyvinylpyrrolidone and 0.1% nonfat dry milk in 50 mM TrisHCl pH 7.4, 200 mM NaCl) for 1 h at room temperature and then incubated with rabbit antisera against GlcCer-3 or GlcCer-2 at a dilution of 1:1000 and 1:500, respectively, overnight at room temperature. After 5 washings (5 min each) in washing buffer (50 mM TrisHCl, pH 7.4, 200 mM NaCl), the plates were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (heavy and light chain specific, Dianova), diluted 1:1000 in blocking buffer for 2 h at room temperature, washed four times as before, and a fifth time in substrate buffer (0.1 M sodium citrate buffer, pH 4.5). Bound antibody was then detected by incubation with substrate solution (10 ml) which was freshly prepared and composed of 8.33 ml substrate buffer, 1.67 ml of 4-chloro-1-naphthol (3 mg/ml in MeOH) and hydrogen peroxide (3.3 µl of a 30% solution).
Dot-blot
Antigens were dissolved (1 mg/ml) in CHCl3/MeOH (3:1, vol/vol) and aliquots (1 µl) were dotted onto uncharged nylon membrane (Qiagen 60010) and air dried. All following steps were done at room temperature. After blocking in blotting buffer (50 mM TrisHCl, 0.2 M NaCl, pH 7.4, supplemented with 10% nonfat dry milk) for 1 h, serial dilutions of rabbit antisera in the same buffer were added, incubated over night and washed 5 times (5 min each) in blotting buffer. Alkaline phosphatase-conjugated goat anti-rabbit IgG (heavy and light chain specific, Dianova) was added (diluted 1:1,000 in blotting buffer) and incubation was continued for another 2 h. After washing as before, 5-bromo-4-chloro-3-indoylphosphate and p-toluidine p-nitroblue tetrazolinum chloride (Bio-Rad) were added as a substrate according to the suppliers instruction. The reaction was stopped after 15 min by the addition of distilled water.
Enzyme-immunoassay (EIA)
PolySorp microtiter plates (U-bottom, Nunc) were coated with glycolipid antigens in methanol and dried under a hood. Unless stated otherwise, 50 µl volumes were used. Plates were blocked with 200 µl of PBS supplemented with 1% bovine serum albumin (PBS-BSA) for 1 h at room temperature followed by removal of the blocking solution by gentle trashing on paper towels. Appropriate antiserum dilutions in PBS-BSA were added and incubated at 4°C overnight. After three washings in PBS-BSA, peroxidase-conjugated goat anti-rabbit IgG (heavy and light chain specific, Dianova) was added (diluted 1:1000) and incubation was continued for 2 h at room temperature. After four washings in PBS (10 mM), the plates incubated with freshly prepared substrate solution, which was composed of azino-di-3-ethylbenzthiazolinsulfonic acid (1 mg) dissolved in substrate buffer (0.1 M sodium citrate, pH 4.5, 1 ml), with sonication in an ultrasound water bath for 3 min followed by the addition of hydrogen peroxide (25 µl of a 0.1% solution). After 30 min at 37°C on a rocking platform, the reaction was stopped by the addition 2% aqueous oxalic acid and the plates were read by a microplate reader (Dynatech MR 700) at 405 nm. All tests were done in quadruplicates.
Tissue preparation and immunofluorescence
Normal human skin biopsies obtained from the forearms of three different individuals were used in this study. The biopsies were high-pressure frozen and freeze-substituted with temperature steps from 183 K to 223 K with acetone saturated with uranyl actetate. Subsequently, the specimens were embedded in HM20 at 223 K. A detailed description of the procedure and its use for immunoelectron microscopy will be published separately (Pfeiffer et al., forthcoming). The following steps were performed at room temperature and all dilutions were prepared in washing buffer (PBS/0.1% Tween 20). Semithin sections (200 nm) were placed on glass cover slips, treated with normal goat serum (3% in PBS) for 1 h, washed twice, and incubated with anti-GlcCer-3 antiserum (1:50) for 1 h. Subsequently, the sections were washed four times and incubated with a Cy3-conjugated goat anti-rabbit IgG secondary antibody (Dianova, Hamburg, Germany) (1:800) for 1 h. The samples were washed six times, stained for nuclei with 4,6 diamidino-2-phenylindole (5 µg/ml PBS) (Sigma, Deisenhofen, Germany) for 15 min, washed twice with H2O, fixed on slides with MOWIOL, and analyzed with a Zeiss Axioskop 35 fluorescence microscope.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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