Journal of Histochemistry & Cytochemistry

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Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis. A Ishida-Yamamoto, R A Eady, F M Watt, D R Roop, D Hohl and H Iizuka J Histochem Cytochem 1996 44: 167 DOI: 10.1177/44.2.8609373 The online version of this article can be found at: http://jhc.sagepub.com/content/44/2/167

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0022-1554/96/$3.30 The Journal of Histochemistry and Cytochemistry Copyright 0 1996 by The Histochemical Society, Inc

Vol. 44, No. 2, pp. 167-175, 1996 Printed i# UXA.

Original Article

I Immunoelectron Microscopic Analysis of Cornified Cell Envelope Formation in Normal and Psoriatic Epidermis' AKEMI ISHIDA-YAMAMOTO,~ROBIN A. J. EADY, FIONA M. WATT, DENNIS R. ROOP, DANIEL HOHL, and HAJIME IIZUKA Department of Dermatology, Asahikawa Medical College, AsahiRawa, Japan (AI-YHI); Department of Cell Pathology, St John i Institute of Dermatology, St ThomasIr Hospital, London, United Kingdom (AI-Y RAJE); Keratinocyte Laboratory, ICRE; London, United Kingdom (FMW); Department of Cell Biology and Dermatology, Baylor College of Medicine, Howton, Texas (DRR); and Department of Dermatology, University Hospital of Lausanne, Lausanne, Switzerland (DH). Received for publication May 15, 1995 and in revised form September 20, 1995; accepted September 25, 1995 (5A3673).

The cornified cell envelope (CE)is an insoluble, highly resistant structure formed beneath the plasma membrane of differentiating keratinocytes. It consists of various crosslinked precursor proteins, including involucrin and loricrin. However, neither the normal assembly process of CE nor its alteration in skin disorders has been fully characterized. In this study we analyzed CE formation in normal skin and in lesionalpsoriatic skin by immunoelectron microscopy. In the superficial granular cells of normal epidermis, involucrin labeling was detected along the plasma membrane, whereas loricrin staining was mostly distributed diffusely within the cells, occasionally with some additional granular aggregates within the cytoplasm and nucleus. Loricrin was also present predominantly on the desmosomal attachment plaques, co-

Introduction The cornified cell envelope (CE) is an insoluble structure formed beneath the plasma membrane during terminal differentiation of epidermal keratinocytes (for reviews, see 1-3). It is composed of several crosslinked precursor proteins, including involucrin (4-7) and loricrin (8,9). Although involucrin and loricrin are assumed to be sequentially incorporated into the CEs in this order (2,10,11), direct evidence for this is lacking. In psoriasis, altered expression of these precursor molecules, abnormal overall shapes of CEs, and altered amino acid composition have been reported (12-17). However, it is not clear whether these alterations are accompanied by a deranged assembly process of the precursor proteins during CE formation. Previous study of involucrin localization in normal and

localizing with desmoglein. In the cornified cells, involucrin labeling was reduced, whereas loriuin labeling was retained and mntinuouslydecorated the CEs,with dative sparing of the desmosomal areas. In typical psoriatic epidermis, involucrin staining was very intense but loricrin labeling was markedly reduced. The involucrin-positive CEs were formed precociously and further maturation of CE did not occur. These results suggest that formation of CE occurs sequentidy, initially involving involucrin deposition and subsequently involving loricrin inmrporation. Psoriatic epidermis demonstrates a lack of proper CE maturation. (JHsrochem Cytochem 44167-175, 19%) KEY WORDS: Involucrin; Loricrin; Skin; Keratinocytes; Psoriasis; Im-

munoelectron microscopy; Keratohyalin granules; Desmosomes.

psoriatic epidermis (18) showed that normal CEs were only temporarily involucrin-immunoreactive,whereas psoriatic CEs that were formed precociously were persistently involucrin-positive. The aim of the present study was to directly elucidate the sequential events of CE formation in normal and psoriatic epidermis. By immunoelectron microscopic assessment of involucrin, loricrin, profilaggren/filaggrin, and desmoglein deposition, we have found that the assembly of the CE occurs in a complex but very orderly fashion in normal human epidermis. In psoriasis, precocious initiation of CE formation, without loricrin, appears to prevent its maturation, resulting in the defective CEs seen in psoriatic epidermis.

Materials and Methods

' Supported in part by grants from the Ministry of Education, Science,

Sports and Culture of Japan 06454312 (HI) and 07770635 (AI-Y), by a grant from the Ministry of Health and Welfare, Japan (HI), and by a grant from the Swiss Science Foundation 3136337.92 (DH). Correspondence to: Dr. Akemi Ishida-Yamamoto, Dept. of Dermatology, Asahikawa Medical College, Nishikagura 4-5-3-11 Asahikawa, 078 Japan.

Samples of normal human skin from various body sites including scalp, face, arm, buttock, thigh, and foreskin were obtained during skin surgery or circumcision. Skin samples were also taken from chronic psoriatic plaque lesions on the forearm, shin, knee, and lumbar areas from seven patients (four men and three women; aged 57-80 years). Biopsies were taken under local anesthesia after obtaining informed consent. No patient was receiving oral retinoids. cyclosporine, or other systemic therapy.

167

ISHIDA-YAMAMOTO, EADY, WAIT. ROOP, HOHL., IIZUKA

168

Table 1. Antibodies used for immunohistochemistrv Antibody

Ig Type

Source (reference)

Reactivity

Involucrin

Biomedical Technologies, Sroughton. M A

Involucrin Involucrin Mouse loricrin C-terminal Mouse loricrin N-terminal

Involucrin” Involucrin Mouse and human loricrin Mouse and human loricrin

(43) (13)

Mouse I g G l

Partially purified human filaggrin

Mouse IgGZb

Bovine desmoglein

Human filaggrin. profilaggrin and i t s breakdown products Human, rat. and bovine desmoglein

BT60 1

Rabbit polyclonal

SY5 AF62 AF2340

Mouse Rabbit Rabbit Rabbit

BT576 DG3.10

DHI

Antigen

IgGl polyclonal polyclonal polyclonal

(8.9) DR Roop (DiSepio D et al., submitted) Biomedical Technologies Boehringer Mannheim. Mannheim. Germany

’Thc epitope of this antibody lies bctween codons 421 and 568 in human involucrin (43)

I

Figure 1. lnvolucrin and loricrin immunoelectron microscopy in normal skin. (A) lnvolucrin staining (BT601). Note diffuse intracellular labels in the granular and transitional cells and cell peripheral staining (arrowheads) in these cells and lower cornified cells. (B) Loricrin staining (AF2340). In a granular cell, labels are seen in the cytoplasm, around the keratin filaments and keratohyalingranules (k) and desmosomal attachment plaques (d). In cornifiedcells, loricrin is seen along the CEs (arrowheads). Lowicryl K l l M sections. C. cornified cells; G . granular cells; T, transitional cell. Bars = 1 pm.

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Figure 2. (A) The CEs of granular cells are immunoreactive to both involucrin (SY5, 5-nm gold) and loricrin (AF2340. 15-nm gold). (B) The labeling of involucrin (5 nm) in CE of a cornified cell is decreased, whereas that of loricrin (15 nm) is retained compared with an underlying transitional cell. Lowicryl K l l M sections. C.cornified cell; G, granular cells; T, transitional cell. Bars = 0.1 pm.

Transmission electron microscopy was performed as previously described (18). For light microscopic immunohistochemistry. 6-vm cryostat sections were fixed with cold acetone and stained either by a streptoavidin-biotin method according to the manufacturer's protocol (Histofine; Tokyo,Japan) or by an immunogold-silver staining method (18). For the latter method, the sections were preincubated in 1% bovine serum albumin (BSA). 5% normal goat serum (NGS). and 0.1% gelatin in PBS, pH 7.4. for 15 min at room temperature (RT), then incubated with one of the primary antibodies (Abs) (Table 1 ) diluted in 1% BSA. 1% NGS. and 0.1% gelatin in PBS for 1 hr at 37°C. After washing, the sections were incubated with 1-nm colloidal gold-conjugated secondary serum (Amenham International: Amersham. UK) for 1 hr at 37'C. Subsequently. the immunogold particles were enhanced using a silver staining kit (Amersham). For immunoelectron microscopy two different postembedding methods were used. The first method was carried out essentially as previously described (19). Bridly, tissue samples were cryoprotected in 15 % glycerol-PBS. then rapidly frozen in liquid propane at - 190°C. subjected to cryosubstiturion in methanol for 44-48 hr at -8O'C. and embedded in Lowicryl KllM (Chemische Werke Lowi; Waldkraiburg, Germany) at -6O'C. Polymerization was initiated under UV radiation for 48 hr at -60°C and continued for a further 48 hr at 2O'C. Alternatively. skin samples were fixed in 1% glutaraldehyde with 0.2% picric acid in PBS for 3 hr at RT. After dehydration in ethanol (50% ethanol for 10 min. three changes of 70% ethanol for 10 min each). the tissue blocks were transferred to a 2:l mixture of hard grade LR White resin (London Resin: Hampshire, UK) and 70%

ethanol and gently agitated on a rotary device for 20 min. The tissue blocks were given three changes of pure resin at 50°C in an oven for 20 min each. The blocks were placed in gelatin capsules containing a mixture of the resin and the manufacturer's accelerator in the proportion of 1.5 pI accelerator to 1 ml of the resin and were polymerized for 45 min at 50'C. The immunosraining procedures were as described elsewhere (20). Briefly. the sections were preincubated in 1% BSA. 5% NGS. and 0.1% gelatin in PBS. then incubated with one of the primary Abs (Table 1 ) . After washing, the sections were reacted with appropriate colloidal gold-conjugated secondary sera (gold particle size 5. 10. or 15 nm) (Amersham and BioCell, Cardiff, UK). For double labeling, 5- and 15-nm gold-conjugated secondary Abs were used. Negative controls included incubation in the presence of the secondary Ab alone and with primary Abs against antigens that are not expected to be present in keratinocytes [a mouse a-smooth muscle actin MAb (Dako: Glostrup, Denmark) and a rabbit polyclonal calcitonin gene-related peptide antibody (Amersham)]. To confirm the specificity of the loricrin immunoreacrion, some specimens were incubated with the loricrin antibody AF2340 which was preabsorbed with synthetic peptides corresponding to the amino-terminal 17-amino-acid residues of human loricrin and labeled with immunogold as described above. The sections were then contrasted with uranyl acetate. For quantitative analysis of involucrin and loricrin association with desmosomes, we randomly chose six prints of BT601 and AF2340 immunoelectron microscopy with IO-nm gold labels. We counted 100 gold particles for each print and determined mean percent t SD of gold particles as-

ISHIDA-YAMAMW, EADY, WAR, ROOP, HOHL, IlZUKA

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Figure 3. Loricrin (AF2340) and desmoglein (DG3.10) immunoelectron microscopy. (A) Loricrin labeling in granular cells. Many labels are associated with desmosomal attachment plaques. (E) In cornified cells, loricrin labels are associated with cell envelopes but desmosomal areas are relatively spared. (C, D) Double labeling of loricrin (15nm gold) and desmoglein (5nm gold). Loricrin is co-localized with desmoglein in a granular cell (arrows) but not in cornified cells. A, C. D. Lowicryl K l l M sections; E. LR White section. d, desmosomes; C, cornified cells; G.granular cells. Bars = 0.1 wm.

sociated with desmosome areas in the cell periphery (within 20 nm from the cell border) of superficial granular cells and lower cornified cells from normal skin.

Results ImmunoLubefing in Normal Skin Three involucrin Abs showed almost identical staining patterns. Two loricrin Abs showed almost identical staining patterns as well, and we could not tell the difference in the ultrastructural localization between C- and N-terminal epitopes recognized by the two loricrin Abs. Specimens incubated with irrelevant primary Abs or the preabsorbed loricrin antibody showed no appreciable labeling in the epidermal keratinocytes. Although two different tissue preparation methods for immunoelectron microscopy gave essentially the same staining patterns, specimens that were cryofixed and em-

bedded in Lowicryl K11M resin showed better ultrastructural preservation and greater labeling density than those that were chemically fixed and embedded in LR White resin. In normal human skin, involucrin labeling was noted diffusely in the cytoplasm from the upper spinous cells upwards. In the superficial granular cells and transitional cells, some labeling for involucrin was localized along the plasma membrane (Figure 1A). In the cornified cells, the labeling density was markedly reduced. Loricrin staining was seen in the cytoplasm and the nucleus of the granular cells. CE-associated labeling was most apparent in the cornified cells, although some labels were seen along the plasma membrane, mainly on the desmosomal attachment plaques in the granular cells (Figure 1B). Double staining with involucrin and loricrin antibodies confirmed that involucrin labels were decreased, whereas loricrin labels were retained in the cornified cells (Figures 2A and 2B).

171

CORNIFIED CELL ENVELOPE

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Figure 4. (A) Loricrin (AF2340, 15-nm gold) and filaggrin (BT576. 5-nm gold) immunoelectron microscopy. Irregularly outlined keratohyalin granules are filaggrinpositive but loricrin-negative. Loricrin immunoreactivity is seen around the keratohyalin granules and within the cytoplasm. Skin from the forehead. (6) Conventional transmission electron microscopy. Electron-dense, small round granules (arrowheads) distinct from irregularly outlined keratohyalin granules are found in the cytoplasm and nucleus of a granular cell from the upper arm. A, Lowicryl K l l M section; B, Epon 812 section. k. keratohyalin granules; n. nuclei; C.cornified cells; G.granular cell. Bars: A = 0.1 um; B = 1 pm

Of involucrin labels in the cell periphery, 18.3 t 11.7% and 29.2 t 7.0% were on the desmosomal areas in superficial granular cells and lower cornified cells, respectively. In contrast, the desmosome-associated loricrin labels were 87.3 t 3.2% and 23.7 t 7.5% of the peripheral labeling, respectively. The association of loricrin with the desmosomal attachment plaques in the granular cells (Figures 1B and 3A) was further demonstrated by double labeling of loricrin and desmoglein. These two molecules were apparently co-localized (Figure 3C). However, this association was weakened in the cornified cells. The desmosomal areas showed little loricrin labeling and co-localization of loricrin and desmoglein was no longer observed (Figures 3B-3D). Distribution patterns of loricrin in the cytoplasm and nucleoplasm of the granular cells were somewhat complicated. Although many gold particles were seen on the desmosomal areas, around

the keratohyalin granules and tonofilaments, and within the nucleus, unique structures specific for loricrin were usually not seen (Figure 1B). Double labeling of loricrin and filaggrin demonstrated that these two proteins were not co-localized and that the irregularly outlined keratohyalin granules were reactive only for filaggrin (Figure 4A). In addition to the regular keratohyalin granules, we observed small round granules, also within granular cells (Figure 4B). The latter granules were seen within the nucleus as well as in the cytoplasm. These granules were loricrin-positive but filaggrinnegative, and occurred in scalp (infrequent) and in foreskin, where loricrin immunostaining was very intense (Figure SA) (21) and the granules were frequent (Figures 5B and 5C).

Immunohbeling in Psoriatic S&in Psoriaticepidermis showed variable histological features, from typical

172

ISHIDA-YAMAMOTO, EADY, WATT, ROOP, HOHL, IIZUKA

Figure 5. Loricrin expression in the foreskin. (A) Light microscopic immunohistochemistry. Loricrin (AF62. immunogold-silver staining method) staining is very intense in granular cells. (E) lmmunoelectronmicroscopy. Loricrin (AF62) staining in foreskin epidermis. Some labels are associated with small, round intranuclearand cytoplasmic granules (arrowheads). (C) Loricrin (AF2340. 15nm gold) and filaggrin (BT576,5-nmgold) double immunoelectron microscopy. Note distinct loricrin-immunoreactivegranules (arrowhead)and filaggrin-positive granules (arrow). A, frozen section; E. C. LR White sections. d. desmosomes; n, nucleus; C.cornified cell; G . granular cell. Bars: A = 10 pm; B, C = 0.1 pm.

parakeratotic epidermis without granular cell layers to near-normal orthokeratotic epidermis with agranular cell layer (22-24). In typical psoriatic lesions, profiIaggrin/filaggrin was absent. Involucrin staining was intense (Figure 6A), whereas that of loricrin was negative or very faint (Figure 6B). Conventional and immunoelectron microscopy showed that typical psoriatic CEs were formed preco-

ciously from the midepidermis (Figure 6E) and were involucrinpositive in all layers (Figure 6F). In the nontypical areas where the granular layer was present, involucrin staining was still intense and profilaggrinlfilaggrin was focally expressed (Figure 6C). These filaggrin-positive areas were immunoreactive to loricrin as well (Figure 6D). In the nontypical areas, CEs were formed during cell con-

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Figure 6. Psoriatic epidermis. (A-D) Light microscopic immunohistochemistry. Dermal-epidermal junctions are marked with dashes. Positive involucrin (A, DH1) and negative loricrin (E, AF62) staining in a typical psoriatic lesion (immunogold-silver staining method). Filaggrin (C. ET576) and loricrin (D. AF67) are stained in serial sections of a nontypical psoriatic lesion (streptoavidin-biotin method). Note focal areas positive for both filaggrin and loricrin (arrawheads). (E,F) Typical psoriatic lesions without granular cell layer. CEs (arrowheads) are seen from the midepidermis by conventional electron microscopy (E).lnvolucrin (SY5. 5-nm gold) and loricrin (AF2340. 15-nm gold) double staining shows that CEs are positive solely for involucrin (F). (G,H) Nontypical psoriatic lesions with a granular layer. CEs (arrowheads) are seen from a cornified cell just above a granular cell by conventional electron microscopy (G). CE are positive for both involucrin (SY5.5-nm gold) and loricrin (AF2340, 15-nm gold) (H). k, keratohyalingranules. A-D. frozen sections; E.G.Epon 812 sections; F.H. Lowicryl K l l M sections. Bars: A-D = 0.1 mm; E-H = 0.1 pm.

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version from the granular to cornified cells, as in normal skin (Figure 6G), and these were labeled with both involucrin and loricrin antibodies (Figure 6H).

Discussion Several lines of evidence suggest that precursor proteins assemble sequentially during the formation of CEs. The overall shapes of isolated CEs (14) and the ultrastructure (25,26) of the CEs change as the cells differentiate. Involucrin expression precedes that of loricrin (18,21), indicating that incorporation of these CE precursors occurs in this order (2,10,11). The present results, using double labeling of involucrin and loricrin (Figures 2A and 2B), demonstrated a decrease in involucrin labeling and retention of loricrin labeling during cell conversion from granular to cornified cells, supporting the above-mentioned concept. Loss of involucrin labeling in the mature CEs may represent epitope masking. Because both involucrin and loricrin are present at the time of CE formation in the superficial granular cells, there must be a mechanism for preferential incorporation of involucrin at this stage. It might be related to the concentration of substrates or to substrate specificities of distinct transglutaminases that crosslink the CE precursor proteins (27). Although CEs are reportedly continuous with and often indistinguishable from desmosomal attachment plaques (28), various staining methods (26) and intermediate-voltage electron microscopy (29) have demonstrated noncontinuity between them. Nevertheless, there is evidence to suggest the incorporation of desmosomal components into CEs. Jessen (28) noted deposition of electron-dense materials or granules onto the desmosomes before the formation of CE. Association of loricrin with the desmosomal attachment plaques was observed in acrosyringial cells (30) and epidermal granular cells (present study). Various desmosomal components, including desmoglein, have been detected in the extracted or purified CEs immunocytochemically (6) and biochemically (31). In the present study, co-localization of loricrin with desmoglein in the granular cells was demonstrated in vivo. These observations suggest the involvement of CE components during desmosome/ corneosome maturation. There is no known mechanism for accumulation of loricrin in the desmosomal attachment plaques. However, Steinert et al. (32) suggested an association between glycine-rich loop-like structures of loricrin and those of keratins along the CEs. A similar interaction might be present in the desmosomal attachment plaques with which keratins are associated. Such glycine loop configuration is not present in involucrin. In contrast to granular cells, cornified cells did not show much loricrin labeling on the desmosomal areas. The reason for this is not clear. Subcellular localization of loricrin within the granular cells has been controversial. Steven et al. (33) found that loricrin was localized within specific granules that were distinct from filaggrinimmunoreactive granules in newborn mouse epidermis. In rodent skin and tongue epithelia, such granules have been known for many years as small electron-dense, cysteine- and sulfur-rich granules (28,3448). Yoneda et al. (39) stated that loricrin labeled filaggrin granules in human epidermis. In our previous study of normal human skin, we found loricrin accumulated within discrete granules

ISHIDA-YAMAMOTO, EADY, WATT, ROOP, HOHL, IIZUKA

in acrosyringia, but not in interappendageal epidermis, where loricrin was diffusely distributed intracellularly (30). Further examination of various body sites detected loricrin-labeled, discrete, small round granules in the human epidermis. Although such granules were extremely rare in ordinary body sites, they were relatively common in the outer leaf of foreskin. Because both acrosyrigia and foreskin are characterized by an increased content of loricrin, the formation of discrete granules might depend on the amounts of the loricrin molecules expressed. We do not have an explanation for the different results seen by others (39) in human epidermis. It is possible, however, that the latter might not have fully discriminated loricrin granules from filaggrin granules, because they did not perform double immunostaining. The biological significance of intranuclear loricrin immunoreactivity remains to be clarified. This finding appears to be a specific phenomenon, because we observed it with two different loricrin antibodies, preabsorption of the N-terminal loricrin antibody with N-terminal human loricrin peptides abolished the staining, and we observed intranuclear loricrin immunoreactivitywith three different staining procedures: an immunogold labeling method, a streptoavidin-biotin method, and an immunofluorescence method (not shown). Moreover, intranuclear loricrin granules have been observed in newborn mouse epidermis (33), which was confirmed by us (not shown). The major difference in discriminating normal and psoriatic epidermis is that loricrin is already present when CE assembly is initiated in the normal skin, whereas it is absent in typical psoriatic lesions (16). This indicates that normal maturation of CEs does not occur without appropriate synthesis of loricrin, the major constituent of normal CEs. It should be mentioned that not all psoriatic lesions were loricrin-negative.Loricrin was expressed in the areas at which a profilaggrinlfilaggrin-positivegranular layer was present (Figures 6C and 6D). In these areas, CEs were assembled when the granular cells were converted into cornified cells (Figure 6G) and became immunoreactive to both involucrin and loricrin (Figure 6H). Ultrastructurally, these CEs were close to the normal CEs. Examination of various skin diseases revealed that loricrin expression was closely linked to an orthokeratotic phenotype of epidermal keratinization (16). Parallel expression of loricrin and profilaggridfilaggrin has been observed in vitro as well (40,41). Protein kinase C, which enhances expression of loricrin and filaggrin (42), might be relevant in this context. Acknowledgments We thank Prof S. Yachiku, O r M. Morihwa, and other members of the Department of Urology, Asahikawa Medical College,forproviding us with foreskin samples. The technical assistance of Ms M. Baba and the secretarial assistance of Ms Y Maekawa are greatly appreciated

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Eady RAJ. Epidermolysis bullosa simplex (Dowling-Meara)is a genetic disease characterized by an abnormal keratin filament network involving keratins KS and K14. J Invest Dermatol 1991;97:959 21 Hohl D, Olano BR, de Viragh PA, Huber M, Detrisac CJ. Schnyder UW, Roop DR. Expression patterns of loricrin in various species and tissues. Differentiation 1993;54:25 22 Brody I. The ultrastructure of the epidermis in psoriasis vulgaris as re-

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