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Elafin, a Secretory Protein, is Cross-Linked into the Cornified Cell Envelopes from the Inside of Psoriatic Keratinocytes

2002; Elsevier BV; Volume: 119; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.2002.01803.x

1523-1747

Hiroshi Nakane, Akemi Ishida‐Yamamoto, Hidetoshi Takahashi, Hajime Iizuka,

Contact Dermatitis and Allergies

Elafin is a serine proteinase inhibitor highly expressed in psoriatic epidermal keratinocytes, but expressed scarcely, if at all, in normal skin. In addition to the proteinase inhibiting domain, elafin contains multiple transglutaminase substrate domains and has been identified as a constituent of the epidermal cornified cell envelope. It also contains a signal peptide sequence, and previous immunoelectron microscopy studies detected elafin in lamellar granules and also in the intercellular spaces. It has not been explained, however, how and when elafin molecules stored in the granules are cross-linked into the cell envelope. In order to elucidate this issue, we performed pre-embedding and postembedding immunoelectron microscopy of elafin and involucrin, another cell envelope constituent, using psoriatic epidermis. Postembedding double immunoelectron microscopy revealed that elafin was within the secretory (lamellar) granules and released into the intercellular spaces when the cell envelope was not formed. In the cells with involucrin-positive cell envelope, elafin immunolabels were localized diffusely within the cells and also along the cell envelope. Pre-embedding immunoelectron microscopy of purified cell envelope from psoriatic scale samples detected involucrin and elafin colocalizing on the cytoplasmic side of the cell envelope. These findings strongly suggest that elafin-containing granules are disintegrated upon the initiation of cell envelope formation, and that elafin is cross-linked on to the involucrin-positive cell envelope from the inside of keratinocytes. It seems that psoriatic keratinocytes utilize elafin as a major component of the cell envelope, consistent with the previously proposed "precursor availability hypothesis". Elafin is a serine proteinase inhibitor highly expressed in psoriatic epidermal keratinocytes, but expressed scarcely, if at all, in normal skin. In addition to the proteinase inhibiting domain, elafin contains multiple transglutaminase substrate domains and has been identified as a constituent of the epidermal cornified cell envelope. It also contains a signal peptide sequence, and previous immunoelectron microscopy studies detected elafin in lamellar granules and also in the intercellular spaces. It has not been explained, however, how and when elafin molecules stored in the granules are cross-linked into the cell envelope. In order to elucidate this issue, we performed pre-embedding and postembedding immunoelectron microscopy of elafin and involucrin, another cell envelope constituent, using psoriatic epidermis. Postembedding double immunoelectron microscopy revealed that elafin was within the secretory (lamellar) granules and released into the intercellular spaces when the cell envelope was not formed. In the cells with involucrin-positive cell envelope, elafin immunolabels were localized diffusely within the cells and also along the cell envelope. Pre-embedding immunoelectron microscopy of purified cell envelope from psoriatic scale samples detected involucrin and elafin colocalizing on the cytoplasmic side of the cell envelope. These findings strongly suggest that elafin-containing granules are disintegrated upon the initiation of cell envelope formation, and that elafin is cross-linked on to the involucrin-positive cell envelope from the inside of keratinocytes. It seems that psoriatic keratinocytes utilize elafin as a major component of the cell envelope, consistent with the previously proposed "precursor availability hypothesis". Cornified cell envelope (CE) is a covalently cross-linked layer of protein that replaces the plasma membrane and is covalently attached to the extracellular lipid envelopes in the differentiated keratinocytes (Ishida-Yamamoto and Iizuka, 1998Ishida-Yamamoto A. Iizuka H. Structural organization of cornified cell envelopes and alterations in inherited skin disorders.Exp Dermatol. 1998; 7: 1-10Crossref PubMed Scopus (101) Google Scholar;Nemes and Steinert, 1999Nemes Z. Steinert P.M. Bricks and mortar of the epidermal barrier.Exp Mol Med. 1999; 31: 5-19Crossref PubMed Scopus (415) Google Scholar). CE provides the tissue with a protective barrier against the environment and also prevents water loss from the body surface. The importance of CE for physiologic functions of the integument is reflected by the association of a severe form of ichthyosis, lamellar ichthyosis with decreased activities of transglutaminases that cross-link CE-precursor molecules (Huber et al., 1995Huber M. Rettler I. Bernasconi K. et al.Mutations of keratinocyte transglutaminase in lamellar ichthyosis.Science. 1995; 267: 525-528Crossref PubMed Scopus (406) Google Scholar;Parmentier et al., 1995Parmentier L. Blanchet-Bardon C. Nguyen S. Prud'homme J-F Dubertret L. Weissenbach J. Autosomal recessive lamellar ichthyosis. identification of a new mutation in transglutaminase 1 and evidence for genetic heterogeneity.Hum Mol Genet. 1995; 4: 1391-1395Crossref PubMed Scopus (81) Google Scholar;Russell et al., 1995Russell L.J. DiGiovanna J.J. Rogers G.R. Steinert P.M. Hashem N. Compton J.G. Bale S.J. Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis.Nat Genet. 1995; 9: 279-283Crossref PubMed Scopus (308) Google Scholar). The composition of CE varies considerably among different epithelia and various pathologic conditions. Among many CE precursor proteins, involucrin is ubiquitously synthesized by all stratified squamous epithelia and cross-linked into CE at the early stage serving as a scaffold on to which other proteins are cross-linked (Steinert and Marekov, 1999Steinert P.M. Marekov L.N. Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia.Mol Biol Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (121) Google Scholar;Steinert, 2000Steinert P.M. The complexity and redundancy of epithelial barrier function.J Cell Biol. 2000; 151: F5-F7Crossref PubMed Google Scholar). By contrast, expression of elafin, another CE protein, is negligible, if any, in normal adult epidermis, but is high in some pathologic conditions, including psoriatic epidermis and wounded epidermis (Schalkwijk et al., 1993Schalkwijk J. van Vlijmen IMJJ Alkemade J.A.C. de Jongh G.J. Immunohistochemical localization of SKALP/elafin in psoriatic epidermis.J Invest Dermatol. 1993; 100: 390-393Abstract Full Text PDF PubMed Google Scholar;Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar;van Bergen et al., 1996van Bergen B.H. Andriessen M.P.M. Spruijt K.I.J. van de Kerkhof P.C.M. Schalkwijk J. Expression of SKALP/elafin during wound healing in human skin.Arch Dermatol Res. 1996; 288: 458-462Crossref PubMed Scopus (50) Google Scholar). Elafin (elastase specific inhibitor, SKALP; skin-derived anti-leukopeptidase) is a small serine proteinase inhibitor derived from the precursor preproelafin (Saheki et al., 1992Saheki T. Ito F. Hagiwara H. Saito Y. Kuroki J. Tachibana S. Hirose S. Primary structure of the human elafin precursor preproelafin deduced from the nucleotide sequence of its gene and the presence of unique repetitive sequences in the prosegment.Biochem Biophys Res Commun. 1992; 185: 240-245Crossref PubMed Scopus (68) Google Scholar;Molhuizen et al., 1993Molhuizen H.O.F. Alkemade H.A.C. Zeeuwen PLJM de Jongh G.J. Wieringa B. Schalkwijk J. SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.J Biol Chem. 1993; 268: 12028-12032Abstract Full Text PDF PubMed Google Scholar;Nara et al., 1994Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin".J Biochem. 1994; 115: 441-448PubMed Google Scholar). This precursor protein contains a signal peptide, and the consensus transglutaminase motifs, in addition to the proteinase inhibiting domain. (Pro)elafin fragments and their cross-linkage with other CE components have been detected from the fractions of proteolytically digested CE (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar). As the elafin gene translation product has a signal peptide sequence, it is stored within secretory granules and is later extruded from the cells (Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar). All other known CE precursor proteins do not have signal sequences and appear to be cross-linked from the inside of the plasma membrane. It remains unknown how and when a secretory protein (pro)elafin, is incorporated into CE. To address this question, we performed pre-embedding and postembedding immunoelectron microscopy of elafin together with involucrin in psoriatic epidermis. Biopsies from chronic skin lesions of six patients with psoriasis vulgaris were taken after obtaining informed consent. Scales were also collected by scraping the psoriatic plaques of six patients with a scalpel. Anti-human elafin rabbit serum was purchased from Peptide Institute (Osaka, Japan). It does not cross-react with endothelin-1 (human), atrial natriuretic peptide (human), corticotropin releasing factor (CRF) (human), substance P, and neuropeptide Y (human). With enzyme immunoassay using β-D-galactosidase as a label, IC50 was 0.4 pmol per ml. Human elafin (0.2 µg; Ala-Gln-Glu-Pro-Val-Lys-Gly-Pro-Val-Ser-Thr-Lys-Pro-Gly-Ser-Cys-Pro-Ile-Ile-Leu-Ile-Arg-Cys-Ala-Met-Leu-Asn-Pro-Pro-Asn-Arg-Cys-Leu-Lys-Asp-Thr-Asp-Cys- Pro-Gly-Ile-Lys-Lys-Cys-Cys-Glu-Gly-Ser-Cys-Gly-Met-Ala-Cys-Phe-Val-Pro-Gln, MW 5999.1, Peptide Institute) was loaded on to a lane of 15% sodium dodecyl sulfate–polyacrylamide slab gels 1 mm thick and subjected to electrophoresis as described byLaemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202763) Google Scholar. As molecular weight markers, ovalbumin (45,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), lysozyme (14,300), and aprotinin (6500) (Amersham, Buckinghamshire, U.K.) were also loaded. After electrophoresis, gel slabs containing separated proteins were electro phoretically transferred to polyvinylidene difluoride membrane (Immobilon-p, Nihon Millipore, Tokyo, Japan) in transfer buffer containing 25 mM Tris and 192 mM glycine. Nonspecific binding sites were blocked by immersing the membranes in 5% skim milk in Tris–HCl buffered saline (TBS) pH 7.6 for 1 h at room temperature. After rinsing in TBS, the membrane was incubated with anti-elafin anti-serum (in 5000 dilution in TBS) overnight at 4°C. After rinsing in TBS, the membrane was incubated with horseradish peroxidase linked F(ab′)2 fragment against rabbit immunoglobulin from donkey (Amersham; 1:5000 dilution in TBS). ECL plus Western blotting detection system (Amersham) was used for immunodetection. The experiments were performed in duplicate, with the same results. Cryostat sections of psoriatic skin were incubated with a mixture of rabbit anti-elafin anti-serum (in 1:100 dilution) and anti-involucrin mouse monoclonal antibodies (SY5, NeoMarkers, Fremont, CA; 1:100 dilution) for 30 min at 37°C. This was followed by incubation with a mixture of fluorescein isothiocyanate-conjugated swine anti-rabbit IgG antibodies (1:20 dilution; DAKO, Glostrup, Denmark) and Texas Red-conjugated sheep anti-mouse immunoglobulins (1:10 dilution; Amersham) for 30 min at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Nacalai Tesque, Kyoto, Japan). Fluorescence immunolabeling was observed using an Olympus BX-FLA-1 system (Tokyo, Japan). Digital images were captured using an electric-cooled CCD camera (SenSys, Photometrics, Tucson, AZ). The camera and image processing were controlled using IP Laboratory Spectrum software (Scanalytics, Fairfax, VA). For immunohistochemistry, a standard streptavidin–biotin method using diaminobenzidine as a substrate for peroxidase was employed on formalin-fixed and paraffin-embedded sections of psoriatic skin with anti-elafin antibody. For postembedding immunoelectron microscopy, skin tissue samples were either unfixed or prefixed in 1% glutaraldehyde and 0.2% picric acid in phosphate-buffered saline (PBS) for 3 h at 4°C, and washed in PBS. Unreacted aldehyde groups were quenched by incubating with 100 mM glycine in PBS for 1 h. The tissues were then cryoprotected in 15% glycerol–PBS, rapidly frozen in liquid propane at -190°C, subjected to cryosubstitution in methanol -80°C and embedded in Lowicryl K11M (at -60°C) or HM20 resin (at -50°C) according to the manufacturer's protocol (Chemische Werke Lowi, Waldkraiburg, Germany). Ultrathin sections were prepared, collected on formvar-coated nickel grids, and immunostained as described previously (Ishida-Yamamoto et al., 1996Ishida-Yamamoto A. Eady R.A.J. Watt F.M. Roop D.R. Hohl D. Iizuka H. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.J Histochem Cytochem. 1996; 44: 167-175Crossref PubMed Scopus (74) Google Scholar). As the primary antibodies, anti-elafin anti-serum and anti-involucrin mouse monoclonal antibodies were used. As labels, 10 nm gold-conjugated goat anti-rabbit IgG and 5 nm gold-conjugated goat anti-mouse IgG (Amersham, 1:10 dilution) were used. For contrast, the sections were stained with uranyl acetate alone or with uranyl acetate and lead citrate. For pre-embedding immunoelectron microscopy, psoriatic stratum corneum samples were obtained by scraping the lesions with a scalpel. The samples were minced with scissors, homogenized, and treated with four 10 min cycles of boiling in fresh washes (10 ml) of 10 mM Tris–HCl buffer, pH 7.4, containing 2% sodium dodecyl sulfate and 1% β-mercaptoethanol under rigorous agitation. The insoluble remnants composed of CE were spun down for 30 min at 22,000 g, resuspended in 10 mM Tris–HCl buffer. The purified CE were then rinsed twice in large volumes of PBS and sequentially immunolabeled with anti-human elafin antibody, 5 nm gold-conjugated goat anti-rabbit IgG, anti-involucrin antibody, and finally 10 nm gold-conjugated goat anti-mouse IgG. The samples were fixed with 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon812 resin. For all immunohistochemistry, negative controls included incubation in the presence of a secondary antibody alone, and incubation with unrelated primary antibodies. Specificity of anti-elafin antibody was confirmed by immunoblot experiments that showed a band of 6 kDa matched to the size of human elafin (Figure 1). In immunohistochemical studies, all samples from typical psoriatic plaques showed essentially the same result. As has been reported previously (Molhuizen et al., 1993Molhuizen H.O.F. Alkemade H.A.C. Zeeuwen PLJM de Jongh G.J. Wieringa B. Schalkwijk J. SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.J Biol Chem. 1993; 268: 12028-12032Abstract Full Text PDF PubMed Google Scholar;Schalkwijk et al., 1993Schalkwijk J. van Vlijmen IMJJ Alkemade J.A.C. de Jongh G.J. Immunohistochemical localization of SKALP/elafin in psoriatic epidermis.J Invest Dermatol. 1993; 100: 390-393Abstract Full Text PDF PubMed Google Scholar;Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar), elafin was highly expressed in the psoriatic epidermis (Figure 2). Double immuno fluorescent staining of cryostat sections of frozen samples demonstrated that elafin was expressed later than involucrin in the differentiation process of keratinocytes (Figure 2a). In elafin staining of formalin-fixed paraffin-embedded samples, which showed better tissue preservation, polarity was detected within each spinous cell. Namely, stronger staining was noted in the apical half than in the basal half of the cells (Figure 2b). Cell-peripheral staining was noted in the more differentiated cells.Figure 2Elafin and involucrin were expressed in the psoriatic epidermis.(a) Double immunofluorescent microscopy of elafin and involucrin. Involucrin (red) is expressed from the lower spinous cells, whereas elafin (green) is expressed from the upper spinous cells. Nuclei are stained with 4′,6-diamidino-2-phenylindole dihydrochloride (blue). (b) Immunoperoxidase staining of elafin. Cytoplasmic elafin staining is stronger in the apical half than in the basal half in the spinous cells. It is associated with cell membrane in the cells just under the stratum corneum. Scale bar: 50 µm.View Large Image Figure ViewerDownload (PPT) Subcellular localization of elafin was further analyzed by immunoelectron microscopy. Using the postembedding method with Lowicryl K11M resin-embedded samples of both unfixed and prefixed materials, elafin labeling was detected in the perinuclear Golgi apparatus (Figure 3a) and within the granules close to the apical surface of the keratinocytes (Figure 3b), confirming previous observations (Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar). As ultrastructures of lamellar granules were not well preserved in these samples, another preparation, fixation with 1% glutaraldehyde and embedding in Lowicryl HM20 resin, was performed that disclosed that elafin-positive granules were with laminated internal structures (Figure 3c). Although CE-associated elafin immunostaining was not detected in a previous report (Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar), considerable elafin labels along the CE were found as well as in the intercellular spaces in the Lowicryl K11M samples (Figure 4). As CE-associated staining was poor in the Lowicryl HM20 samples, we used K11M for the rest of this study. It was also noted that in the cells with elafin-positive CE, considerable elafin immunolabels were observed within the cytoplasm, where most of the labels were not associated with granular structures (Figure 4b).Figure 4Elafin molecules are detected along the CE. (a) Large arrowheads denote elafin-positive granules that have migrated toward the cell periphery in the upper spinous cell layer. Some are accumulated at the apical side of the cells (arrows) to be released extracellularly. Some labels are also seen along the CE (small arrowheads) in the upper cell. Note that the cell membrane (*) of the lower cell without CE is undulated compared with that of the upper cell with CE. (b) Elafin labels are detected along the CE (arrows) as well as intracellular and extracellular (*) spaces in the cornified layer. d, desmosome. Scale bar: 100 nm.View Large Image Figure ViewerDownload (PPT) Subcellular localization of elafin and involucrin was compared by double immunoelectron microscopy staining. The timing of CE formation was also assessed. It was revealed that where involucrin was not deposited along the plasma membrane, elafin molecules were within the secretory granules (Figure 5a). In contrast, in the cells with CE, involucrin and elafin labels were colocalized along the CE and also within the cytoplasm (Figure 5b). Although involucrin and elafin labels were colocalized very closely together along CE in postembedding immunoelectron microscopy samples, it was not certain whether these two proteins were on the same side of the CE or on the opposite side of it. Pre-embedding immunoelectron microscopy of purified CE from psoriatic epidermal scale samples was then performed to address this issue. In this method, the brushy cytoplasmic side of CE could be easily distinguished from the relatively smooth extracellular surface. It was clearly demonstrated that involucrin and elafin were colocalized on the cytoplasmic side of the CE (Figure 6). From these observations, the process of elafin cross-linking into CE as schematically shown in Figure 7 was proposed. In the psoriatic epidermis, elafin-containing lamellar granules derived from Golgi apparatus move to the apical side of keratinocytes and elfin is released extracellularly before the formation of CE. In the cells with CE, elafin-containing granules, which have not been secreted, are disintegrated and some elafin molecules are cross-linked into the cytoplasmic side of the CE.Figure 7Diagram of elafin granules and CE formation.★, elafin; ♦, involucrin.View Large Image Figure ViewerDownload (PPT) Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar were the first to identify elafin as a component of CE. They performed proteolytic digestion of CE purified from human foreskin epidermis. Following fractionation, they detected various peptides that contained sequences cross-linked by isodipeptide bonds. Elafin sequences were detected forming cross-bridges with loricrin. Differing from all other known CE components, elafin is a secretory protein and it has been left unexplained how and when it is cross-linked into CE. Immunoelectron microscopic demonstration of CE components has been done for various molecules (Haftek et al., 1991Haftek M. Serre G. Mils V. Thivolet J. Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.J Histochem Cytochem. 1991; 39: 1531-1538Crossref PubMed Scopus (58) Google Scholar;Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar;Ishida-Yamamoto et al., 1996Ishida-Yamamoto A. Eady R.A.J. Watt F.M. Roop D.R. Hohl D. Iizuka H. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.J Histochem Cytochem. 1996; 44: 167-175Crossref PubMed Scopus (74) Google Scholar;Steinert and Marekov, 1999Steinert P.M. Marekov L.N. Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia.Mol Biol Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (121) Google Scholar), but not for elafin. Previous postembedding immunoelectron microscopy of elafin in psoriatic epidermis fixed with 2% paraformaldehyde and embedded in Lowicryl HM20 resin detected elafin labels in the intercellular spaces of the stratum corneum, over the lamellar granules, small vesicular structures, and Golgi apparatus, but not on CE (Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar). In this study, we detected elafin signals on CE using postembedding and pre-embedding immunoelectron microscopy, confirming the notion that elafin is a component of CE. In addition, it was demonstrated that elafin-containing granules are disintegrated during CE assembly and elafin is cross-linked to CE from the inside of cells. Previous pre-embedding immunoelectron microscopy of purified CE has shown epitopes of various CE components on the cytoplasmic side (Haftek et al., 1991Haftek M. Serre G. Mils V. Thivolet J. Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.J Histochem Cytochem. 1991; 39: 1531-1538Crossref PubMed Scopus (58) Google Scholar;Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar;Steinert and Marekov, 1999Steinert P.M. Marekov L.N. Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia.Mol Biol Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (121) Google Scholar). These include loricrin, keratin, desmoplakin, envoplakin, periplakin, involucrin, and SPR1. Two major transglutaminases that cross-link various components to form CE are transglutaminases 1 and 2. The bulk of former enzyme is bound to the plasma membranes from the cytoplasmic side by its N- and S-fatty acylated terminal part. The latter enzyme is a cytoplasmic protein. When transglutaminases are activated various cytoplasmic proteins are cross-linked to the cytoplasmic side of the plasma membrane. Interestingly it has been shown that extracellular ceramides secreted from lamellar granules are also covalently cross-linked into involucrin, periplakin, and envoplakin of CE (Marekov and Steinert, 1998Marekov L.N. Steinert P.M. Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope.J Biol Chem. 1998; 273: 17763-17770Crossref PubMed Scopus (170) Google Scholar). Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation was mediated by transglutaminase 1 (Nemes et al., 1999Nemes Z. Marekov L.N. Fesus L. Steinert P.M. A novel function for transglutaminase 1. attachment of long-chain omega-hydroxyceramides to involucrin by ester bond formation.Proc Natl Acad Sci USA. 1999; 96: 8402-8407Crossref PubMed Scopus (207) Google Scholar). Cross-linking of CE with extracellular molecule has also been suggested for corneodesmosin, another component of lamellar granules (Haftek et al., 1991Haftek M. Serre G. Mils V. Thivolet J. Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.J Histochem Cytochem. 1991; 39: 1531-1538Crossref PubMed Scopus (58) Google Scholar). Although the mechanisms for cross-linking of these molecules into the extracellular side of CE remain to be determined, it seems that CE formation processes progress on both intracellular and extracellular sides. There is a possibility that some elafin molecules are cross-linked from the extracellular side and the epitopes were lost or masked and undetectable in our pre-embedding immunoelectron microscopy method. Epitope masking can be problematic in the postembedding method as well. We detected rich elafin labeling on CE in Lowicryl K11M-resin embedded samples, but not in Lowicryl HM20 samples, as has been noted in a previous study (Pfundt et al., 1996Pfundt R. van Ruissen F. van Vlijmen-Willems IMJJ et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (135) Google Scholar). Although no detailed comparative studies of antigen preservation in these two resins for embedding biologic specimens have been performed, polarity of the resins might be relevant; Lowicryl K11M is polar, whereas HM20 is apolar. Polar resin gives increased surface irregularity that correlates with increased ability to detect antigen on the sections (Kellenberger et al., 1987Kellenberger E. Durrenberger M. Villiger W. Carlemalm E. Wurtz M. The efficiency of immunolabel on Lowicryl sections compared to technical predictions.J Histochem Cytochem. 1987; 35: 959-969Crossref PubMed Scopus (144) Google Scholar). Temperature used for tissue embedding might also be critical. It has been suggested that embedding at lower temperature gives better antigen preservation (Hobot, 1989Hobot J.A. Lowicryls and low-temperature embedding for colloidal gold methods.in: Colloidal Gold: Principles, Methods, and Applications. Vol. 2. San Diego, Academic Press1989: 75-115Google Scholar). Specimens can be embedded at lower temperatures in K11M resin (-60°C) than in HM20 resin (-50°C). As far as structural preservation of lamellar granules is concerned, the latter resin was far superior to the former. This might be related to the extent of lipid extraction during tissue preparation because the laminated structure of lamellar granules represents a stack of lipid lamellae. In the study of extent of membrane lipid extraction during tissue preparation, HM20 gave much less extraction than a polar resin, Lowicryl K4M (Weibull et al., 1983Weibull C. Christiansson A. Carlemalm E. Extraction of membrane lipids during fixation, dehydration and embedding of Acholeplasma laidlawii-cells for electron microscopy.J Microsc. 1983; 129: 201-207Crossref PubMed Scopus (65) Google Scholar). In normal epidermis, secretion of contents of lamellar granule is completed just before the cornification. Whereas in psoriatic epidermis, corneocytes contain remnants of lamellar granules, indicating incomplete secretion (Mottaz and Zelickson, 1975Mottaz J.H. Zelickson A.S. Keratinosomes in psoriatic skin.Acta Derm Venereol. 1975; 55: 81-85PubMed Google Scholar;Ghadially et al., 1996Ghadially R. Reed J.T. Elias P.M. Stratum corneum structure and function correlates with phenotype in psoriasis.J Invest Dermatol. 1996; 107: 558-564Crossref PubMed Scopus (155) Google Scholar). It has previously been shown that in psoriatic epidermis, CE was formed precociously in the spinous cells (Ishida-Yamamoto and Iizuka, 1995Ishida-Yamamoto A. Iizuka H. Differences in involucrin immunolabeling within cornified cell envelopes in normal and psoriatic epidermis.J Invest Dermatol. 1995; 104: 391-395Crossref PubMed Scopus (62) Google Scholar). Therefore, it seems that CE formation occurs so rapidly that not all lamellar granules reach the apical cell surface and release their contents. Increased intracellular calcium is a common trigger for exocytosis of Golgi-derived secretory granules in many cell types (Madison and Howard, 1996Madison K.C. Howard E.J. Ceramides are transported through the Golgi apparatus in human keratinocytes in vitro.J Invest Dermatol. 1996; 106: 1030-1035Crossref PubMed Scopus (34) Google Scholar;Madison et al., 1998Madison K.C. Sando G.N. Howard E.J. True C.A. Gilbert D. Swartzendruber D.C. Wertz P.W. Lamellar granule biogenesis. a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi.J Invest Dermatol Symp Proc. 1998; 3: 80-86Abstract Full Text PDF PubMed Scopus (79) Google Scholar), and also for protein cross-linking by transglutaminases. It has been shown that intracellular calcium levels are elevated in the terminal differentiation of keratinocytes (Menon 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) and A23187, a calcium ionophore, induces CE formation, which is accompanied with membrane-associated transglutaminase 1 activation in normal human keratinocytes (Takahashi et al., 2000Takahashi H. Aoki N. Nakamura S. Asano K. Ishida-Yamamoto A. Iizuka H. Cornified cell envelope formation is distinct from apoptosis in epidermal keratinocytes.J Dermatol Sci. 2000; 23: 161-169Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Therefore, it seems that upon the elevation of intracellular calcium, elafin stored within granules are released extracellularly, CE formation is initiated, and ultimately secretory granules are unable to fuse with the plasma membrane and disintegrate (Figure 7). Elafin molecules are then dispersed into the cytoplasm and cross-linked on to the CE from inside the cells. Our data also support the "precursor availability hypothesis", whereby transglutaminases utilize various substrates that are available at the time of CE assembly (Robinson et al., 1997Robinson N.A. Lapic S. Welter J.F. Eckert R.L. S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.J Biol Chem. 1997; 272: 12035-12046Crossref PubMed Scopus (198) Google Scholar). Although elafin is not usually detected in normal human epidermal CE, it can be a major component in psoriatic epidermis where elafin expression is greatly increased. Further studies concerning protease inhibitory activities of cross-linked elafin will provide more insights into the biologic roles of elafin as a component of CE. This study was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to AI-Y (14570794) and to HT (136708607) and a grant from the Ministry of Health and Welfare, Japan to HI. Electron microscopy was performed in the Electron Microscopy Unit, Central Laboratory for Research and Education, Asahikawa Medical College.