3Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany, and 4Analytical Research Department, Beiersdorf AG, Unnastr. 48, D-20245 Hamburg, Germany
Received on October 23, 2000; revised on January 5, 2001; accepted on January 11, 2001.
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Abstract |
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Key words: sphingolipids/thin-layer immunostaining/epidermis/immunohistochemistry/glucosylceramide/antibodies
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Introduction |
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Besides its importance in signal transduction processes, ceramide displays an essential role as a structural component of the epidermal permeability barrier against transcutaneous water loss, which is located in the stratum corneum. Here, ceramide, together with cholesterol and free fatty acids, forms multiple intercellular lipid lamellae, which are embedded in a rigid matrix of corneocytes (Elias and Menon, 1991; Forslind et al., 1997
). Specialized secretory organelles, the lamellar bodies, deliver the barrier lipids to the stratum corneum. They are enriched in a polar lipid mixture of glucosylceramides, phospholipids, and sterols and contain several acid hydrolases (Freinkel and Traczyk, 1985
). At the stratum granulosumstratum corneum interface the lamellar bodies fuse with the plasma membrane of the uppermost granulocyte and secrete their contents into the intercellular space of the stratum corneum. Concomitantly, the lipids are enzymatically hydrolyzed into ceramide, cholesterol, and free fatty acids.
To date, eight epidermal ceramide species are known, varying in the hydroxylation of the sphingosine backbone and of the fatty acid moiety (Figure 1; Robson et al., 1994; Stewart and Downing, 1999
). Among them, two ceramide species exist that possess an
-hydroxyacyl moiety (Swartzendruber et al., 1987
; Wertz et al., 1987
). They are covalently bound to proteins of the cornified envelope, a compact polymer structure composed of protein and lipid that coats the surface of the corneocytes (Marekov and Steinert, 1998
). Glucosylceramide and, to a minor extent, sphingomyelin are the major precursors of epidermal barrier ceramide, as demonstrated by studies on mice deficient in the respective acid hydrolases, ß-glucocerebrosidase and acid sphingomyelinase (Holleran et al., 1993
, 1994; Jensen et al., 1999
; Schmuth et al., 2000
).
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Results |
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Enzyme-linked immunosorbent assay
The antiserum was also tested by enzyme-linked immunosorgent assay (ELISA). Figure 6 shows binding curves of serially diluted antiserum with graded amounts of Cer-5 ranging from 0.032 nmol/well (open diamonds) to 4 nmol/well (filled circles) as a solid-phase antigen. Over a broad range of antigen concentrations the binding curves showed similar slopes. With very low concentrations of 0.063 and 0.032 nmol antigen per well, the steepness of the curves started to decrease. Also the ranges of confidence values were dependent on the ceramide concentrations applied to the plate. With 4.0 and 2.0 nmol per well the confidence values did not exceed 10%, with 1.0 and 0.5 nmol per well they remained below 15%, whereas with lower antigen concentrations the confidence values increased up to a range of 20%. No specific binding was observed with a control mouse serum, which had not been enriched for anti-ceramide antibody.
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Discussion |
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Recently, two reagents with claimed specificity for ceramide became commercially available: a monoclonal mouse IgM antibody and an IgM-enriched polyclonal mouse serum. However, already in the first set of experiments (dot blot assays) the monoclonal antibody turned out to be specific not for ceramide but for sphingomyelin. Because our study was aimed at the characterization of anti-ceramide reagents, the monoclonal antibody was omitted from further experiments.
In all methods applied, the antiserum specifically reacted with purified and natural Cer-2 and Cer-5. Comparing the results of the dot blot assays (Figure 2A) and the TLC immunostaining experiments (Figures 3 and 4), the strong reaction with purified Cer-2 and Cer-5 was highly reproducible in all experiments and with both methods, whereas the weaker reactions of anti-Cer with purified Cer-1 and Cer-3 exhibited some variability, which was more pronounced in TLC immunostaining than in dot blot assays. This suggests that the adherence of the ceramides on the silica gel TLC support was more critical than on the nylon membrane used for the dot blot assay. Because overlay immunostaining of TLC plates is known to vary in intensity from one experiment to another, the different staining shown in Figures 3 and 4 was not surprising. Since N-(-hydroxyacyl)-6-hydroxysphingosine (ceramide 4), liberated from protein-bound ceramides of the cornified envelope, was well detected with the anti-ceramide serum (Vielhaber et al., unpublished data), the long acyl chain and the
-hydroxy group cannot be the only reason for the negative outcome of the experiment shown in Figure 3. However, the complete unreactiveness of Cer-1 in dot blot (Figure 2) was not expected. It seems that Cer-1 adopts a different conformation on nylon membranes than after chromatography on TLC plates. Further experiments using chemically synthesized analogues of ceramide may help answer this question.
In the ELISA system, a higher variability in the weaker reactions was also seen. With decreasing antigen concentrations (1 nmol per well and less) the confidence value increased. On the other hand, ELISA is highly sensitive, allowing the detection of ceramides in the low nanomolar range and has the advantage of being a quantitative assay. Based on these results, an ELISA inhibition test can be envisaged in which liposome-incorporated lipids could be tested and compared quantitatively for their inhibitory capacity.
In addition, the antiserum recognized all epidermal ceramides (Figure 4). However, the reaction with epidermal Cer-1 was weak, and the reactivity against epidermal Cer-4 could not be judged from these experiments due to the low amounts of these lipids present in the epidermal extract. Remarkably, there was a strong immune reaction with epidermal Cer-6 taking into account the low amounts of Cer-6 present in the epidermal lipid extract, whereas the reaction with epidermal Cer-7 was only moderate. Thus, the proximity of the additional hydroxyl group to the hydrophilic "head" of the ceramide molecule seems to decisively support the antigenic recognition.
In some experiments the antiserum also reacted to some extent with cholesterol. However, because the reaction with cholesterol was only observed at concentrations that were 100 times higher than with ceramide (Figure 3B) and because cholesterol and ceramide share no structural similarity, this faint reaction is most likely due to nonspecific binding of IgM antibodies to hydrophobic compounds.
The specificity for ceramide was further confirmed by the results of immuno-light microscopy of cryoprocessed human skin biopsies (Figure 5), which showed a separate staining pattern of anti-ceramide and anti-glucosylceramide in the epidermis with only poor colocalization of the named lipids in the uppermost stratum granulosum. Furthermore, the concentration of ceramide staining at the corneocyte cell boundaries correlated well with the general assumption that ceramides are localized intercellularly in the stratum corneum and in part covalently bound to the protein matrix of the corneocytes. The intense ceramide fluorescence in the cells of the lower epidermis and of the dermis underlines the high sensitivity of the ceramide antiserum, because the ceramide content per cell is about 30 times lower in the lower epidermis than in the stratum corneum (Yardley, 1983). The fact that no vesicular staining pattern in the stratum granulosum was obtained with anti-ceramide suggests that, in contrast to glucosylceramide (Wertz et al., 1984
; Freinkel and Traczyk, 1985
; Elias and Menon, 1991
; Brade et al., 2000
), ceramide is not localized within the lamellar bodies. Immuno-gold electron microscopical investigations have resolved the subcellular distribution of ceramide in the cells of the lower epidermis and will be published elsewhere.
Conclusions
With the aid of a polyclonal antiserum specific for ceramide it is now possible to visualize the distribution of endogenous ceramide in subcellular compartments of human tissue. This reagent will help to further elucidate the structural diversity and biological function of distinct ceramide species.
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Materials and methods |
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Antibodies
The mouse antiserum against ceramide and the rabbit antiserum against GlcCer were purchased from GlycoTech Produktions- und Handelsgesellschaft mbH (Kuekels, Germany). The monoclonal antibody against Cer was purchased from Alexis Deutschland GmbH (Grünberg, Germany).
Preparation of natural lipid extracts
Epidermal lipid extracts from human breast skin were prepared as described (Doering et al., 1999). For further purification of the epidermal ceramide fraction, 40 mg of epidermal lipids were separated twice on a preparative TLC plate (2 mm silica gel) in CHCl3/MeOH/CH3COOH (190:9:1; v/v/v). The ceramide fraction was scraped off, eluted from the silica gel with chloroform/methanol (2:1; v/v) by shaking overnight, and the organic phase sucked off and dried under nitrogen.
Dot blot
The dot blot procedure was exactly performed as described (Brade et al., 2000) with amounts of antigen and antiserum dilutions as indicated in Figure 2.
TLC and TLC-immunostaining
Ceramides were separated twice on silica gel 60 TLC plates with aluminum support (Merck) with a solvent system of CHCl3/MeOH/CH3COOH (190:9:1 v/v/v) and visualized by spraying with 10% CuSO4 and 8% H3PO4 in water and heating at 180°C (Imokawa et al., 1991). For TLC-immunostaining the plates were first incubated with 0.1% saponin in washing buffer (50 mM TrisHCl, pH 7.4, 200 mM NaCl) for 30 min, then blocked with 5% nonfat dry milk in washing buffer for 1 h at room temperature and subsequently incubated with mouse serum against ceramide at the indicated dilution overnight at room temperature. After five washings (5 min each) in washing buffer, the plates were incubated with peroxidase-conjugated goat anti-mouse IgM (heavy and light chain specific, Dianova), diluted 1:1500 in blocking solution for 2 h at room temperature, washed 4 times, 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).
ELISA
ELISA was performed as described elsewhere (Brade et al., 2000).
Immunohistochemistry
For immunohistochemistry, high-pressure frozen and freeze-substituted human skin biopsies embedded in HM20 were used (Pfeiffer et al., 2000). Immunofluorescence staining was performed according to Brade et al. (2000)
, but with 0.5% bovine serum albumin/0.2% fish gelatin in phosphate-buffered saline as washing buffer. The primary and secondary antibodies were applied simultaneously (dilutions: anti-Cer 1:2; anti-GlcCer 1:50, Rhodamine Red-X-conjugated goat anti-(mouse IgM) 1:200; Alexa488-conjugated goat anti-(rabbit IgG) 1:800). The images were acquired with a CLSM Leica TCS SP.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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