Cohesion and desquamation of epidermal stratum corneum.

Cohesion and Desquamation of Epidermal Stratum Corneum

MIRIAM M. BRYSK . SRINIVASAN RA]ARAMAN

With 27 Figures and 4 Tables

)! SEMPER

~

GUSTAV FISCHER VERLAG· STUTTGART· NEW YORK· 1992

MIRIAM M. BRYSK, Ph.D.

Department of Dermatology SRINIVASAN RAJARAMAN, M.D.

Department of Pathology University of Texas Medical Branch Galveston, TEX 77550 (USA)

Acknowledgement Supported by Grant Mo. DE 08477 from the National Institute of Dental Research.

Die Deutsche Bibliothek - CIP-Einheitsaufnahme Brysk, Miriam M.: Cohesion and desquamation of epidermal stratum corneum / Miriam M. Brysk ; Srinivasan Rajaraman. - Stuttgart; Jena ; New York: G. Fischer, 1992 (Progress in histochemistry and cytochemistry; Vol. 25, No.1) ISBN 3-437-11459-X (Stuttgart ...) ISBN 1-56081-355-5 (New York) NE: Rajaraman, Srinivasan:; GT Library of Congress Card-No. 88-20469 Published jointly by: Gustav Fischer VerlagNCH Publishers 220 East 23rd Street, Suite 909, New York, New York 10010 Gustav Fischer Verlag Wollgrasweg 49, D-7000 Stuttgart 70 (Hohenheim) FRG © Gustav Fischer Verlag· Stuttgart· Jena· New York· 1992 AIle Rechte vorbehalten Gesamtherstellung: Laupp & Gobel, NehrenlTiibingen Printed in Germany

Contents 1 2 3 3.1

3.2 3.3 3.4 3.5

3.6 4

5 6 6.1

6.2 6.3 7 7.1

7.2 7.3 7.4 8 9 10 11

Introduction to epidermal differentiation Differentiation in cultured epidermal cells Molecules in epidermal differentiation . Cytokeratins Filaggrin . Involucrin . . Loricrin . . . Glycoproteins . Lipids . . . . . Biogenesis of the stratum corneum . Characteristics of the stratum corneum Molecules in cohesion and desquamation Lipids . Glycoproteins . Proteinases . Model systems for the study of desquamation . In vitro aggregation of squames Diseases . Tissue culture models . . . . . . . Modulation by exogenous factors Perspective Summary .. References . . Subject index

1

3 4 4

4 5 5 7 12 12 13 16 17 18

23 25 25 31 34

36 42 43 45 55

1 Introduction to epidermal differentiation The skin is one of the largest organs of the body; in the average adult its surface exceeds 2 m2 • The skin constitutes a physical barrier between the organism and its environment. It is composed of two distinct entities. The dermis contains all the vascular and nerve networks, as well as specialized excretory and secretory glands; its principal cell type is the fibroblast. The epidermis is a stratified epithelium; its principal cell type is the keratinocyte. The epidermis is subdivided into several distinct cell strata: basal, spinous, granular, and stratum corneum. The layers differ in the extent of their differentiation. The basal cells have the highest nuclear to cytoplasmic ratio of all the keratinocytes. Mitosis occurs only in the basal cells. They are attached by hemidesmosomes to the basement membrane above the dermis (BRIGGAMAN and WHEELER 1975). The spinous cells are flatter, contain more abundant desmosomes, and have smaller nuclei. Lamellar granules become evident in the upper spinous cells (MATOLTSY and PARAKKAL 1965; LAVKER 1976). Cells in the granular layer are flat and polygonal; they contain the distinctive cytoplasmic inclusions, the keratohyalin granules. This cell layer is the transition zone between the viable epidermis and the overlying anucleated stratum corneum. The stratum corneum has lost all the organelles and consists of stacked lamellae. A schematic showing the structure of the epidermis is illustrated in Fig. 1.

Epidermis --- --- -- -stra tu m

corneum

layer c:~;F.;:~~;~~~~E;;~----·--··-···--·-granular -keratohyalin granules

.....:::::---c:::-- -----------------------s pin 0 u s

······nucleus - --- - ---- --- - -. - - -. basa I

layer

membrane

Dermis ,Fig. 1. Schematic of the epidermis.

layers

2 . M. M. Brysk . S. Rajaraman

Stratum corn urn

=

Granular

Spinous

= Basal

.. 8M

D rmi

Fig. 2 shows the ultrastructure of the epidermis; included are all the cell layers as well as the major events in the differentiative process. The entire population of keratinocytes in the epidermis is in a continual process of cell replacement. The loss of the outer squames from the stratum corneum is balanced by mitosis of the basal cells. In a sense, epidermal differentiation is a «programmed suicide» dedicated to the constant renewal of the outer barrier. It is a homeostatic process in which the thickness and integrity of the epidermis is continually maintained. During stratification, the cells change in size,

Cohesion and desquamation of epidermal stratum corneum . 3

shape, and function. The time of transit of a keratinocyte from the basal through the granular cell layers is about 6-8 weeks, while the stratum corneum takes an additional two weeks (HALPRIN 1972; BERGSTRESSER and TAYLOR 1977). The regulation of proliferation is under the control of a variety of endogenous and exogenous factors, including extracellular substrata, divalent cations, growth factors, chalones, cytokines, hormones, and cyclic nucleotides. Some of these factors also regulate terminal differentiation and desquamation.

2 Differentiation in cultured epidermal cells The oldest in vitro system is organ culture, in which the structural integrity of the skin is maintained intact. A related technique is explant culture; in this system both fibroblasts and epidermal cells migrate out of the skin tissue. In epidermal cell culture systems, the epidermis is separated from the dermis by incubation with enzymes such as trypsin. The dissociated keratinocytes are then propagated on different substrata and in media of different composition. The differentiation of keratinocytes in vitro is influenced by their environment; they display a variety of marker proteins that may qualitatively differ from those in the native epidermis. In most earlier epidermal culture systems, dissociated keratinocytes were seeded directly onto plastic and grown in media which contained fetal calf serum. The cells in such cultures became multilayered and stratified but lacked the granular layer and a true stratum corneum (see review by HOLBROOK and HENNINGS 1983). Subculturing became feasible when keratinoeytes were cultured on feeder layers consisting of lethally-irradiated 3T3 fibroblasts (RHEINWALD and GREEN 1975). HENNINGS et al. (1980) developed a culture system that permits the selective growth of mitotic keratinocytes in monolayers. Keratinocytes are maintained in media of low extracellular Caz+ concentrations « 0.1 mM, as against 1-2 mM in normal media). In such media, desmosome formation and stratification do not occur and the cells can be passaged many times. When Caz+ is added, the keratinocytes differentiate and stratify. A serum-free medium (at 0.1 mM Caz+) has been developed by BOYCE and HAM (1983). It includes bovine pituitary extract, growth factors and other supplements. The advantage of the low Caz+ systems is that the differentiation processes can be easily modulated with Caz+, serum, and other growth regulators, without the need for fibroblast feeder layers. Keratinocytes cultured in all the above mentioned systems fail to produce a complete multilayered structure comparable to epidermis in vivo. On the other hand, keratinocytes cultured at the air-liquid interface closely mimic the physiological conditions of the epidermis in vivo (LILLIE et al. 1980; PRUNIERAS et al. 1983). BERNSTAM et al. (1986) showed that epidermal cells grown on collagen rafts at the air-liquid interface develop and maintain multilayered strata that are similar to epidermis in vivo. They demonstrated the formation of a granular layer containing keratohyalin granules, lamellar


granules, cross-linked envelopes, and up to 20 non-nucleated cell layers resembling a true stratum corneum. Even greater stratification is observed when keratinocytes in lifted cultures are grown on dead dermis (WILLIAMS et al. 1988; MADISON et al. 1988).

3 Molecules in epidermal differentiation 3.1 Cytokeratins Keratinocytes synthesize and express numerous different structural proteins, cell adhesion glycoproteins, and lipids during their maturation. Keratin intermediate filaments comprise a large fraction of the cytoskeleton of epidermal cells (for a review see SUN et al. 1983). They are water- insoluble and desmosome-associated. The keratin family includes approximately 20 different proteins. Two distinct classes of keratins can be identified according to their size and their amino acid sequences: Type I subunits are acidic (pKj 4.5-5.5) and small (molecular weights 40-56.5 kD); type II subunits are basic (pKj 6.5-7.5) and larger (molecular weights 53-67 kD) (FUCHS et al. 1981; KIM et al. 1983; MAGIN et al. 1986). There are about ten different members of each type and that differ in immunoreactivity (SCHEFFER et al. 1982) and ability to pair (EICHNER at al. 1986) Each member of the family is encoded by a single mRNA (ECKERT and GREEN 1984; FUCHS et al. 1981; KIM et al. 1983). One member of each class is required for filament formation (CREWTHER et al. 1983; EICHNER et al. 1986; FRANKE et al. 1986). The filaments are stabilized by disulfide bonds (SUN and GREEN 1978). Keratin synthesis is regulated in a differentiation-dependent fashion, with the higher molecular weight keratins synthesized in the more stratified cell layers (FUCHS and GREEN 1980; MOLL et al. 1982). Differences in the end domains of different keratin pairs may be important in determining the properties of the resulting filaments, as well as filament interactions with other proteins and organelles (GIUDICE and FUCHS 1987). By their interaction with filaggrin, the keratins playa major role in the formation of the insoluble filamentous structures that confer rigidity to the terminally differentiated squames in the lower stratum corneum.

3.2 Filaggrin Another protein that contributes to the structure of the corneocyte is filaggrin (for a review see DALE et al. 1990). It is so named because of its ability to aggregate intermediate filaments in vitro into parallel-oriented macrofibrils (DALE et al. 1978). It has been proposed that it functions in the lower stratum corneum to stabilize the keratin filaments in closely-packed structures (STEINERT et al. 1981). Filaggrin is synthesized in the keratohyalin granules of the granular cells from a very large phosphoprotein called


profilaggrin (UGEL 1975; BALL et al. 1978; LONSDALE-EcCLES et al. 1982). During the transition from the granular layer, the profilaggrin is dephosphorylated and proteolytically cleaved to filaggrin. The synthesis of profilaggrin and its conversion to filaggrin occur only in those tissues that synthesize high molecular weight keratins. Filaggrins can be separated by 2-dimensional gel electrophoresis into a family of proteins that are species specific (HARDING and SCOIT 1983). The intracorneocyte filaggrins are shortlived in the lower stratum corneum; they become further degraded to generate a pool of free amino acids in the outer corneum (SCOIT et al.b. 1982 b). RICHARDS et al. (1988) report that filaggrin also serves as a substrate for transglutaminase and that it is incorporated into the cornified envelope; it accounts for approximately 10% of the cell envelope proteins.

3.3 Involucrin In addition to the keratins, keratinocytes express a number of precursor proteins required for the biogenesis of the cornified envelopes. The best characterized of these proteins is involucrin (SUN and GREEN 1976), which has a molecular weight of 80-90 kD, is acidic, and contains 46% glutamate residues. The keratinocytes initiate involucrin synthesis in the suprabasal cell layers (BANKS-SCHLEGEL and GREEN 1981; WAIT and GREEN 1982; WAIT 1984). Ca2 + ions activate an epidermal transglutaminase that catalyzes the formation of E-(t-glutamyl) lysine bonds between the glutamyl groups of involucrin and the primary amino group of lysine of other proteins. In this fashion, involucrin becomes cross-linked into the cornified envelope. Immunofluorescent studies indicate that after cross-linking, involucrin is present in a peripheral, sub-membrane location of the envelope. The cornified envelope confers rigidity and insolubility to the cell peripheries of the corneocytes. In addition to involucrin, other proteins have been shown to become cross-linked by transglutaminase and transported into the cornified envelopes; they include keratolinin (ZEITERGREN et al. 1984) and other membrane proteins (which have not been named) that are rendered insoluble when cultured keratinocytes are stimulated to form envelope proteins with Ca2 + ionophores (SIMON and GREEN 1984). Other proteins that are potential envelope precursors have also been reported (HANIGAN and GOLDSMITH 1978; KUBILUS and BADEN 1982).

3.4 Loricrin Loricrins are a family of cell envelope proteins with highly variable sequences (HOHL et al. 1991) which are expressed in all mammalian stratified epithelia (HOHL 1990 a, b). They are expressed late in epidermal differentiation (HOHL et al. 1991; MEHREL et al.


Table 1. Fluorochrome-conjugated lectins binding to human epidermis. Cell layer Lectin

Sugar specificity

Basal

Suprabasal

Canavalia ensiformis

a-D-man a-D-glc

+

+

Pisum sativum (PSA)

a-D-man a-D-glc

+

+

Lens culinaris (LCA)

a-D-man a-D-glc

+

+

Ricinus communis

~-D-gal

+

+

(Con A)

(RCA-I)

Bandeiraea simplicifolia

a-D-gal

+

a-L-fuc

+ + +

(BSA-I)

Ulex europaeus (UEA-l)

+

+

~-GlcNAc

+ +/-

+ +

Glycine maximum (SBA)

~-GlcNAc

+

+

Sophora japonica

galNAc

+

Dolichos biflorus Helix pomatia (HPA)

a-D-galNAc a-D-galNAc

+

Triticum vulgare (WGA)

~-glcNAc

NeuNAc

Bandeiraea simplicifolia (BSA-II)

Arachis hypogaea (PNA)

Gal~I-3gaINAc Gal~ 1-4galNAc

Galactose

Limulus polyphemus

NeuNAc

-

+

Stratum corneum

Reference

NEMANIC et ~l. (1980) REANO et al. (1982) BELL & SKERROW (1984) VIRTANEN et al. (1986) NEMANIC et al. (1980) BELL & SKERROW (1984) VIRTANEN et al. (1986) NEMANIC et al. (1980) BELL & SKERROW (1984) VIRTANEN et al. (1986) NEMANIC et al. (1980) REANO et al. (1982) VIRTANEN et al. (1986) NEMANIC et al. (1980) REANO et al. (1982) BELL & SKERROW (1984) NEMANIC et al. (1980) VIRTANEN et al. (1986) REANO et al. (1982) NEMANIC et al. (1980) REANO et al. (1982) BELL & SKERROW (1984) NEMANIC et al. (1980) NEMANIC et al. (1980) REANO et al. (1982) BELL & SKERROW (1984) NEMANIC et al. (1980) REANO et al. (1982) REANO et al. (1982) REANO et al. (1982) BELL & SKERROW (1984) MANSBRIDGE et al. (1984) NEMANIC et al. (1980) REANO et al. (1982) VIRTANEN et al. (1986) WATT (1984) REANO et al. (1982) BELL & SKERROW (1984)


1990). Human loricrin is a 26 kD cationic protein that is rich in cysteine, serine, and glycine; it is extremely insoluble due to cross linking by disulfide bonds. Loricrin initially accumulates in certain sites of the keratohyalin granules, termed L-granules (STEVEN et al. 1990), which are likely to be the sulfur-rich and dense homogeneous deposits reported by FUKUYAMA and EpSTEIN (1969). They are distinct from the large irregularly shaped F-granules that are labeled with the filaggrin antibody (STEVEN et al. 1990). Loricrin subsequently becomes incorporated as a major component of the cornified envelope; it is cross-linked by the N-e-(t-glutamyl) lysine isodipeptide bond to the inner cytoplasmic surface of the cornified cell envelope. Loricrin is believed to impart a flexible character important to the resilience of the stratum corneum.

3.5 Glycoproteins Cell surface glycoproteins playa major role in many diverse cellular functions. They are often associated with stages in cellular differentiation. Epidermal cell surface glycoproteins have been studied on whole skin sections in which the sugar residues are identified by a variety of techniques. Fluorescein-conjugated lectins have been used on frozen skin sections to distinguish the binding of lectins with different sugar specificities to the various layers of the epidermis (BRABEC et al. 1980; REANO et al. 1982; NEMANIC et al. 1983; VIRTANEN et al. 1986). A compilation of the various studies is shown in Table 1. Some lectins bind to all the viable epidermal layers while others are layerspecific and bind to only the basal cells or spinous or granular cell layers; none of the lectins bind to the stratum corneum, in this technique. In order to identify individual glycoproteins binding to different lectins, we have used a complementary procedure. Epidermal glycoproteins are extracted with detergents; they are then separated by gel electrophoresis. Individual molecules are identified by overlaying the gels with radiolabeled lectins (BRYSK and SNIDER 1982 a, b; BRYSK et al. 1984, 1986; BRYSK and MILLER 1984). Using this approach we examined enzymedissociated native epidermis in which the stratum corneum was enzymatically separated from the viable cell layers and the viable cells were further fractionated on gradients of PERCOLL (BRYSK et al. 1981). Fig. 3 displays the fractionated cells; the most dense small basal cells sediment to the bottom of the gradient, while the suprabasal cells fractionate in the top layers of the gradient. Fig. 4 displays the glycoprotein profiles of the basal, suprabasal, and cornified cells after gel electrophoresis and reaction with 125I_Con A. Using this approach we have identified over 20 glycoproteins of different molecular weights in the intact epidermis. The basal cell profile resembles that of the suprabasal cells but is strikingly different from the stratum corneum. Several bands appear unique to the stratum corneum, mostly below 50 kD, with the most prominent at 40 kD and 30 kD. The bands for the viable cell layers are displayed mostly at > 50 kD, extending to the very top of the gel (> 300 kD) (BRYSK and MILLER 1984).


Fig. 3. Epidermal cells separated on discontinuous isokinetic gradients of PERCOLL. a: mixture of dissociated cells; b: cell populations separated at a density of 1.023 gm/ml; c: at 1.036 gm/ml; d: at 1.060 gm/ml; e: at 1.090 gm/ml. Bar = 25 I-lm. - From BRYSK et al. (1981), reproduced with permission.

Cohesion and desquamation of epidermal stratum corneum· 9

200-

92.568~

46-

> 30-

a b

c

d

Fig. 4. Glycoproteins of different populations of epidermal cells obtained from fresh epidermis which have been fractionated on a PERCOLL gradient. Autoradiogram of an SDS polyacrylamide gel after overlay with 125I_Con A. a: whole epidermis; b: basal cells; c: suprabasal cells; d: stratum corneum. The numbers identify molecular weight standards; the arrows point to the bands at 40 kD and 80 kD. - From BRYSK and MILLER (1984), reproduced with permission.

Using the system developed by HENNINGS et al. (1980), we removed the stratum corneum, and cultured the keratinocytes in media of different Ca2 + concentrations (BRYSK et al. 1982 b). Fig. 5 displays the autoradiograms of the molecules that were radiolabeled. Again we observed a large number of Con A binding glycoproteins. Densitometer scans of the gels (Fig. 6), show that glycoproteins were labeled at similar molecular weights in each case. However, the glycoproteins of the undifferentiated cells (grown at low Ca2 +) were markedly different in the intensities of the labeled bands, from those cultured at high Ca2 +. The most prominent bands of the undifferentiated cells were of molecular weights above 80 kD. The differentiated cells displayed the most prominent bands in a doublet at 75 kD and 80 kD. We also examined the floating cornified envelopes shed into the culture medium after the Ca2 + switch. Their glycoprotein profile was very similar to that of the differentiated cells that were still attached. The differences in the glycoprotein profiles between cells in vivo and in vitro are most likely to be due to differences in the extent of differentiation in vitro. In the fresh


a

b

c

d

e

f

9

Fig. S. Con A glycoproteins from different populations of keratinocytes in vitro. Photograph of an SDS polyacrylamide gel. a-e: stained with Coomassie blue. Remaining lanes are autoradiograms after reaction with 1251_ Con A. Tracks a, d, f are for cells grown at 1.S mM Ca2+ (differentiated); tracks b, e, g are for cells grown at 0.07 mM Ca2+ (undiffererentiated). Track c displays the standards. Tracks f and g are for controls in which lectin binding is inhibited by a-methyl-D-mannoside. - From BRYSK and SNIDER (1982 a), reproduced with permission.

epidermis, we had removed the stratum corneum prior to further cell fractionation. Cells in culture, while they express fewer cell layers, do form cornified envelopes. We can also distinguish between different diseases of altered epidermal differentiation on the basis of their Con A binding glycoprotein profiles (BRYSK et al. 1984). The profiles for normal callus and for diseases with an overaccumulation of the stratum corneum resemble those of the normal stratum corneum, particularly in the overexpression of the 40 kD glycoprotein. In contrast to our results in culture with Con A, we found only 3 major glycoproteins labeled with iodinated wheat germ and Ricinus communis agglutinins, mostly at higher molecular weights (BRYSK and SNIDER 1982 a). The Con A glycoproteins at 30 and 40 kD are unique to the stratum corneum and do not bind to these other lectins. Other investigators have used related methods to study glycoprotein changes during epidermal differentiation. Epidermal cell slices in organ culture or keratinocytes in primary culture were incubated with different radiolabeled monosaccharide precursors to show metabolic labeling into different glycoproteins. After gel electrophoresis of extracted fractions, about 20 glycoproteins in the range of 70 kD to > 200 kD were


>~

> ~

u

~

o

Cl

~

a:

w

>

~

w

a:

200 125 92.5 68

46

30

MOLECULAR WEIGHT

X

10-3

Fig. 6. Densitometer scans of autoradiograms of Con A binding glycoproteins expressed by keratinocytes in vitro. Curve (a) is for cells grown at 0.07 mM Ca2 + (undifferentiated); curve (b) is for cells grown at 1.5 mM Ca2 + (differentiated); curve (c) is for cells shed into the culture medium when the Ca2+ concentration was switched from 1.5 mM to 0.07 mM. - From BRYSK and SNIDER (1982 a), reproduced with permission.

identified in organ culture which incorporated labeled glucosamine (KING et al. 1980; KING and TABIOWO 1982; ROBERTS 1987). Many of the major glycoproteins were similar in molecular weight to previously identified components of desmosomes (GORBSKY and STEINBERG 1981). KING et al. (1987) observed that the major glycoproteins that bind to Con A (between 44 kD and 78 kD) arise from the degradation of desmosomal molecules during terminal differentiation. These glycoproteins are recognized by antisera raised against desmoglein II and desmocollin glycopeptides. We also found an 80 kD doublet band on our electrophoretic gels that was present in the viable cell layers as well as in the stratum corneum (BRYSK and MILLER 1984); it is likely to be of desmosomal origin. A number of glycoprotein changes in cultured cells has also been noted by the selective incorporation of radiolabeled fucose. Fucose is incorporated preferentially by the stratified epidermis, especially the granular cells (ROBERTS 1987;


BELL and SKERROW 1984; ZIESKE and BERNSTEIN 1982). In contrast, Bandieraea simplicifolia lectin selectively binds to basal cell glycoproteins. During the transition out of the basal layer, the a-galactose residues in the basal cells become fucosylated with differentiation (ZIESKE and BERNSTEIN 1984). WATT (1984) also observed that, in keratinocytes grown at low Ca2+ concentrations, there is a selective migration of differentiating cells from the basal layer which are recognized by the peanut lectin (~ galactose-specific). The differentiating cells showed a pattern of labeling similar to that observed with the antibody to involucrin. Using radiolabeled galactose, two peanut lectin binding molecules of 11 0 kD and 250 kD were synthesized by keratinocytes in response to Ca2 + elevation of the culture medium (MORRISON et al. 1988). DABELSTEEN et al. (1984 a, b), using monoclonal antibodies that recognize blood group-specific saccharide moieties, found that human epidermal cells express sugar residues in a bloodgroup independent way that is, however, differentiation dependent.

3.6 Lipids The distribution of lipid profiles also changes with epidermal differentiation. LAMPE et al. (1983) quantified the lipids in the basal, suprabasal, and cornified layers of human epidermis. There is a progressive depletion of phospholipids during differentiation coupled with repletion of sterols and sphingolipids. The sphingolipids, barely present in the lower epidermis, account for about 20% of the lipids in the stratum corneum. The most dramatic changes in lipid profiles occur during the transition from the granular layer to the stratum corneum (LAMPE et al. 1983; WERTZ et al. 1986). Cultured keratinocytes differ from the intact epidermis in their lipid composition. In cultured keratinocytes, the nonpolar lipid content is similar to that of basal cells in vivo, the phosphilipid content is reduced, and the ceramide content approaches that of the epidermis (MADISON et al. 1986).

4 Biogenesis of the stratum corneum There is a definite transition zone between the granular layer and the stratum corneum. This zone is the site of many degradative events, resulting in a variety of structural and functional changes. It is within this region that most cytoplasmic organelles are destroyed, presumably by the action of a variety of degradative enzymes (proteinases, hydrolases, glycosidases, DNase and RNase). The nuclei disappear, as do the mitochondria, keratohyalin granules and membrane-coating (or lamellar) granules. Many lipids disappear, and new lipids are released from the extrusion of the lamellar granules. The transition is accompanied by distinct changes in lipid composition, including a depletion of phospholipids and glycolipids (GRAY et al. 1978; YARDLEY and SUMMERLY 1981;


LAMPE et al. 1983), retention and enrichment of neutral lipids (ELIAS et al. 1972; GRAYSON and ELIAS 1982), and the generation of large amounts of sphingolipids (SWARTZENDRUBER et al. 1987; SWARTZENDRUBER et al. 1988). The keratinocytes lose approximately 70% of their dry weight as they enter the stratum corneum (MEYER et al. 1970); they also become flattened into polyhedral structures. Keratins account for 80% of the stratum corneum proteins. They may be responsible for the insoluble packed skeletal patterns observed in the lower stratum corneum (STEINERT et al. 1981; DALE et al. 1978). Involucrin becomes incorporated into the cornified envelope (RICE and GREEN 1978; RICE and GREEN 1979). The envelope proteins become covalently crosslinked; they are resistant to treatment with alkali (MATOLTSY and MATOLTSY 1966), detergents and reducing agents (SUN and GREEN 1976; GREEN 1977). The cornified envelope accounts for the insolubility and rigidity of the individual corneocytes. Profilaggrin is also enzymatically degraded to filaggrin during the transition; it then presumably forms macrofibrils with the basic keratins in the lower stratum corneum.

5 Characteristics of the stratum corneum The stratum corneum functions as the body's main defense to the environment. It acts as a shield against dehydration and damage from physical, chemical, and biological agents. In doing so, it acts as a barrier to the inward and outward transport of water and of a variety of pharmacological agents. Because it is a biologically active degradative cell layer, many macromolecules are enzymatically altered in it prior to desquamation. . The stratum corneum contains about 15-20 layers of flat thin squames which are approximately 0.5 !-lm thick and 40 !-lm wide (PLEWIG and MARPLES 1970). The geometrical pattern of the corneocytes is revealed by silver staining of separated epidermis (Fig. 7). In sections of the epidermis after hydration in buffered-alkali solutions, it is possible to observe 10-20 layers of squames stacked in an orderly fashion; with cells in contact with the squames directly above and below (Figs. 8 and 9) (MACKENZIE 1969; MENTON and EISEN 1971). The lamellar stacked sheets also show regular interdigitations of adjacent stacks (MACKENZIE and LINDEN 1973). Cell flattening occurs 3-5 cell layers beneath the lowest layers of the stratum corneum. Remnants of desmosomes are evident throughout the stratum corneum in ultrastructural photomicrographs. Desquamation is the final event in epidermal differentiation. The cohesive forces holding corneocytes together decrease in the outer lamellae, permitting an orderly shedding process. The molecules localized within the intercorneal matrix modulate cohesion and desquamation. The molecular events are difficult to study because the squames are dead cells, tightly stacked and extremely insoluble. The stratum corneum evolves in two major structural and functional stages (KING et al. 1979). The innermost layer is so tightly bound that it resists disruption by most methods (BOWSER and WHITE 1985). In the middle region, the intercorneal cohesive


Fig. 7. Structure of mouse stratum corneum that was silver stained after removal of the epidermis with EDTA; bar = 30 IJ,m. - Courtesy of Dr. Ian C. Mackenzie.

Cohesion and desquamation of epidennal stratum corneum· 15

b

Fig. 8. Mammalian ear epidermis. Frozen section stained with methylene blue and expanded in Sorenson-Walburn buffer (pH 9.4) a: hamster ear; b: human; bar = 30 fim. - Courtesy of Dr. Ian C. Mackenzie.


Fig. 9. Rhesus monkey epidermis prepared as in Fig. 8. Note the interdigitations of the stratum corneum squames depicted in Figs. 8 and 9. - Courtesy of Dr. Ian C. Mackenzie.

forces loosen and many macromolecules within the squames disappear, among them filaggrin (SCOTI et al. 1982 a). Filaggrin is degraded by proteinases into a pool of free amino acids in the upper stratum corneum (SCOTI et al. 1982 b). Some of these amino acids are further metabolized to produce other compounds such as urocanic acid (SCOTI et al. 1981), which acts as an ultraviolet light blocker (ZENISEK et al. 1955), and pyrrolidone carboxylic acid, which functions in moisture retention at low humidities (LADEN and SPITZER 1967). In the outer stratum corneum, the remaining desmosomal structures are somehow ruptured and the corneocytes desquamate.

6 Molecules in cohesion and desquamation Many molecules in the stratum corneum have been shown to playa role in cohesion and desquamation. Among them are lipids (ELIAS 1983; LONG et al. 1985; WILLIAMS and ELIAS 1987; WILLIAMS 1984; SWARTZENDRUBER et al. 1989; RANASINGHE et al.


1986), proteins (BISSETT et al. 1987; WHITE et al. 1988), glycoproteins (BRYSK et al. 1986, 1988, 1989), and enzymes (EpSTEIN et al. 1981; NEMANIC et al. 1983; LUNDSTROM and EGELRUD 1988).

6.1 Lipids The stratum corneum has been depicted as a two-compartment system consisting of corneocytes bounded by lipid-enriched intercellular domains (ELIAS 1983; ELIAS and FRIEND 1975). The squames (or bricks) are surrounded by a rigid cell envelope. The intercellular space (or mortar) is composed largely of neutral lipids. The mortar accounts for 10-30% of the volume of the stratum corneum (ELIAS and LEVENTHAL 1979). The lipids within this matrix are considered to be responsible for the permeability and barrier function of the stratum corneum; after lipid extraction, the barrier properties are lost (SWEENEY and DOWNING 1970). Thus, the selective permeability function appears to be controlled by the macromolecules in the intercorneal spaces (SQUIER 1973; ELIAS and FRIEND 1975). Many of the lipid components in the stratum corneum are derived from lamellar granules. These granules first appear in the upper spinous layers of the differentiating epidermis (MATOLTSY and PARAKKAL 1965; LAVKER 1976). Their number is greatly increased in the granular layer. As the lamellar granules migrate into the stratum corneum (ODLAND and HOLBROOK 1981), they extrude their lipids which then assume the characteristic lamellar structure of the horny layer (LAVKER 1976; HAYWARD 1978; MADISON et al. 1988). The granules are rich in lysosomal enzymes: phosphatases, hydrolytic enzymes such as aryl sulfatase and others (WOLFF and SCHREINER 1968; TAKAKI 1974; SQUIER and WATERHOUSE 1970; GONZALES et al. 1976). Many of these enzymes contribute to the degradative events that occur in the granular cell layer. The lamellar granules are devoid of phospholipids (ELIAS et al. 1977). They do contain glycoconjugates, as observed by ultrastructural histochemistry (ASHRAF! et al. 1977). The granules increase in number in hyperplastic lesions, such as psoriasis (MOTTAZ and ZELICKSON 1975; their number is decreased in diseases of hyperkeratinization, such as X-linked ichthyosis. Lipid lamellae, presumably derived from lamellar granules, have recently been reported in the upper layers of normal stratum corneum (MADISON et al. 1987). Many changes in lipid composition become evident in the transition zone between the granular layer and the stratum corneum. There is a depletion of phospholipids and glycolipids (GRAY and WHITE 1978; YARDLEY and SUMMERLY 1981; LAMPE et al. 1983), a retention and enrichment of neutral lipids (ELIAS et al. 1972; GRAYSON and ELIAS 1982), and the generation of large amounts of sphingolipids (SWARTZENDRUBER et al. 1987; SWARTZENDRUBER et al. 1988). About 10% of the dry mass of the stratum corneum consists of lipids (FRIBERG et al. 1990), of which 25% are sphingolipids. Other


major lipid components include cholesterol (20-25%), free fatty acids (15-20%) and cholesterol sulfate (5-10%) (GRAY et al. 1982; LONG et al. 1985). The stratum corneum contains no phospholipids or glycolipids (LAMPE et al. 1983). Sphingolipids, particularly ceramides, are considered critical constituents responsible for the epidermal permeability barrier (see reviews by ELIAS et al. 1983; WERTZ and DOWNING 1982). Polar organic solvents that remove sphingolipids and neutral lipids from the stratum corneum are capable of abrogating the permeability barrier (GRUBAUER et al. 1989). In addition, topical application of sphingolipids restores the water-retaining properties of detergentextracted stratum corneum (IMOKAWA et al. 1986). ELIAS (1981) proposed a layered structure for the lipids of the stratum corneum that would function as the water barrier. FRIBERG et al. (1990), however, extracted the ceramides from the stratum corneum and found that they would not form a layered structure; a layered structure formed spontaneously from the extracted free fatty acids in the pH range of the skin, 4.5-6.0. They conclude that the water barrier in the stratum corneum depends on the formation of a layered structure and is independent of the chemical nature of the individual lipid components. It remains to be resolved how the layered structure forms, and how it functions as a barrier. In addition to the lipids involved in the water barrier, the corneal matrix also contains a lipid envelope surrounding the rigid cornified envelope. The lipid envelope contains omega-hydroxylacyl sphingosine (WERTZ and DOWNING 1987) in ester linkage to a component of the cornified envelope which may be involucrin. SWARTZENDRUBER et al. (1987) proposed that this acyl ceramide may be covalently attached to the glutamate residues of involucrin, and that it functions in corneocyte cohesion. It is not clear whether it also affects desquamation.

6.2 Glycoproteins We were the first to show the presence of glycoproteins in the stratum corneum (BRYSK and MILLER 1984; BRYSK et al. 1984, 1986; CHEN et al. 1986). Because the stratum corneum did not react by immunofluorescence with some 14 different lectins, it was believed to be devoid of glycoconjugates (NEILAND 1973; VAN LIS and KALSBEEK 1975; HOLT et al. 1979; NEMANIC et al. 1983; REANO et al. 1982; BRABEC et al. 1980). Figure 10 illustrates the lack of binding of fluorescein-conjugated Con A to the stratum corneum. Using SDS-polyacrylamide gels of detergent-solubilized proteins from purified stratum corneum, we detected glycoproteins that bound iodinated Con A (BRYSK and SNIDER 1982 b; BRYSK et al. 1984). Several of these molecules are unique to the stratum corneum (Fig. 4) (BRYSK et al. 1984; BRYSK and MILLER 1984; CHEN et al. 1986). Among them are molecules with molecular weights of 30 kD, 40 kD and 80 kD. Apparently, detergent soltibilization followed by gel electrophoresis exposes sugar residues that are not accessible to in situ binding techniques, presumably because of masking by surrounding lipids. We have studied the 30 kD and 40 kD glycoproteins

Fig. 10. Localization of fluorescein-conjugated Con A to frozen sections of (a) heat-separated epidermis, (b) trypsin-separated epidermis, (c) purified stratum corneum. (x200). - From BRYSK and MILLER (1984), reproduced with permission.


Table 2. Effects of saccharides on agglutination by desquamin. Saccharide N ,N-Diacetylchitobiose N -Acetylglucosarnine N-Acetylneuraminic acid N -Acetylgalactosamine D-Galactosamine D-Glucosamine D-Glucose a-Methyl-D-mannoside D-Galactose Lactose L-Fucose Methyl-a-D-galactopyranoside Methyl-p-D-galactopyranoside

Concentration inhibiting hemagglutination (mM) 10-5 10-5 10-5 10-5 10-4 10-3 1

>50 >50 >50 >50 >50 >50

(CHEN et al. 1986; BRYSK at al. 1986). A 78 kD molecule has been identified as a desmosomal component in the stratum corneum (KING et al. 1987). We have isolated and partially characterized the 40 kD glycoprotein form the stratum corneum (BRYSK et al. 1986). One of its unique properties is that it hemagglutinates trypsinized rabbit erythrocytes and is thus an endogenous lectin. It has affinity for amino sugars; other saccharides do not inhibit hemagglutination even at concentrations of over 50 mM (Table 2). For most endogenous lectins, the inhibition of hemagglutination by specific saccharides occcurs in the mM concentration range. In contrast, the inhibition of the 40 kD glycoprotein occurs in the I-lM range. The high avidity of binding suggests that this glycoprotein is a major carbohydrate-binding and crosslinking molecule that holds adjacent molecules together in the stratum corneum. It should logically, therefore, play an important role in desquamation. Accordingly, we have named it desquamin. We have prepared polyclonal and monoclonal antibodies to desquamin. Both types of antibodies localize, by immunofluorescence, to the stratum corneum (BRYSK et al. 1991) (Fig. 11). By ultrastructural immunolocalization, the molecule is localized to the lipid envelope surrounding the cornified cells (BRYSK et al. 1986) (Fig. 12). The stratum corneum is devoid of enzymes required for protein synthesis or glycosylation but contains many degradative enzymes. Therefore, it is not possible for desquamin to be synthesized in the stratum corneum. It must be formed by degradation of a glycosylated precursor already synthesized in the viable cell layers. On Western blots of epidermal cell extracts, the antibody to desquamin recognizes a large glycoprotein (about 600 kD). We believe that the 600 kD molecule is the precursor, and we have named it predesquamin. Monoclonal antibodies to predesquamin localize to the upper granular layer and to the stratum corneum. By ultrastructural immunolocali-

Cohesion and desquamation of epidermal stratum corneum' 21

Fig. 11. Indirect immunofluorescent localization of antibodies to desquamin. a:" polyclonal antibody; b: monoclonal antibody (x250).

zation, this antibody labels the surfaces of the squames diffusely (Fig. 13). It is likely that predesquamin is degraded within the transition zone between the granular layer and the stratum corneum, and that desquamin is transported to the lipid envelope, perhaps by binding to the amino sugars of glycoproteins located in the lipid envelopes of the squames.


~~_lL.o;:-_..;.1..._m

_

Fig. 12. Ultrastructural immunolocalization of polyclonal antibody to desquamin to the stratum corneum. The antibody localizes to the lipid envelopes. a: Squames in the outer stratum corneum; b: squames in the inner stratum corneum. Arrows point to the reaction product. - From BRYSK et al. (1986), reproduced with permission.


Fig. 13. Ultrastructural immunolocalization of a polyclonal antibody to predesquamin to the intracellular spaces of the lamellae of the lower stratum corneum. Immunogold methodology. (x30,OOO).

6.3 Proteinases Corneocytes lack a distinct cell membrane; nevertheless, desmosomal junctions persist throughout the stratum corneum. For desquamation to take place, these junctions


must be severed. The chemical composition of desmosomes becomes altered during terminal differentiation (KONOHANA et al. 1987; KING et al. 1987). Desmosomal degradation begins in the granular layer. Unlike the desmosomes in the inner corneal layers which are masked by lipids, those in the superficial layers become more susceptible to the action of proteinases (BOWSER and WHITE 1985; KING et al. 1987). EGELRUD et al. (1988) developed an in vitro desquamation system in which agitation of pieces of plantar stratum corneum, when exogenously incubated with trypsin, led to squame dissociation. The only observable ultrastructural change was an apparent degradation of desmosomal plates. The cell shedding was unipolar from the outer side of the corneum. BISSETT et al. (1987) reported that stratum corneum could be dissociated into free squames by detergents; exogenous trypsin and calcium enhanced the dissociation. Extended studies by LUNDSTROM and EGELRUD (1990) implicate endogenous proteolysis, by a chymotrypsin-like proteinase of the desmosomal protein desmoglein I, as being responsible for squame shedding; the process is inhibited by the proteinase inhibitors aprotinin and chymostatin (LUNDSTROM and EGELRUD 1990; EGELRUD and LUNDSTROM 1991). The inference from plantar callus to stratum corneum is hampered by the facts that callus, by definition, does not ordinarily desquamate and that the in vitro process requires tissue hydration. The stratum corneum is both a desquamative and a degradative tissue layer. A plethora of proteolytic and glycolytic enzymes destroy squame contacts and desmosomes during desquamation. By selective use of chromogenic peptides many different proteinases have been shown to be present in the stratum corneum. Trypsin-like and chymotrypsin-like enzymes promote desquamation by selectively degrading desmosomal proteins in the outer corneum (LUNDSTROM and EGELRUD 1988; EGELRUD and LUNDSTROM 1989; EGELRUD et al. 1988). A number of specific glycosidases have also been identified by use of substrates such as nitrophenol-conjugated sugars (NEMANIC et al. 1983). In addition, microorganisms often inhabit the outer surface of the stratum corneum and secrete active enzymes; for example, Staphylococcus aureus produces endoproteinase Glu-C. If desquamin is to playa major role in the cohesion and dehiscence of the stratum corneum, it must be able to survive the hostile degradative environment in which other macromolecules are destroyed by proteolysis. Desquamin is resistant to most proteinases, including trypsin, chymotrypsin, pepsin, papain, and endoproteinase Glu-C;it is degraded by proteinase K and by pronase (BRYSK et al. 1991). Trypsin and chymotrypsin-like enzymes and endoproteinase Glu-C have been reported to be present in the stratum corneum; proteinase K has not, nor has pronase. In the native tissue, desquamin is embedded in a lipid envelope, whose shielding effect would tend to render desquamin even less susceptible to endogenous proteolysis. Glycoproteins are more resistant to proteolytic degradation than are non-glycosylated proteins. Glycosylation has been suggested as a mechanism for protecting proteins from proteolysis (OVERTON 1982). When we pretreated desquamin with glycosidases prior to using proteolytic


enzymes, however, we did not alter the resistance of desquamin to enzymatic degradation, suggesting that desquamin survives intact during desquamation.

7 Model systems for the study of desquamation Much interest has focused on those molecules thought to be responsible for the cohesion of the stratum corneum. In trying to understand the processes responsible for the cohesion of the stratum corneum, a variety of solvents, detergents and enzymes have been used to loosen intercorneal bonds (BOWSER and WHITE 1985; ELIAS 1983; SMITH et al. 1982; KOCK et al. 1988; BRYSK et al. 1989). Both the solubilized molecules and the structures remaining after extraction have been studied as to their roles in desquamation.

7.1 In vitro reaggregation of squames

In order to demonstrate that desquamin functions as a lectin in the stratum corneum, we developed a model system for dispersing and reaggregating corneocytes (BRYSK et al.

Fig. 14. Appearance of isolated stratum corneum and dissociated squames by light microscopy. a: Cross-section of stratum corneum; b: dissociated single squames (x160).


1988, 1989). We were able to obtain stratum corneum from fresh epidermis by incubation of the tissue with trypsin (Fig. 14 a). The corneum could then be dispersed into single squames by three different techniques: mechanically, with ether, and with detergent (Fig. 14 b). We were able to recombine the corneocytes prepared by mechanical dispersion into a bilayered lamellar structure that resembled the normal stratum corneum (Fig. 15). This reconstituted stratum corneum mimicked the original (predissociation) structure (Fig. 16). In particular, the intercellular spaces between adjacent cells were similar to those in the intact tissue. It appears that this procedure had not removed any factors essential to the structural integrity of the stratum corneum. The squames obtained by dispersion of tissue with ether could also be reaggregated. The resulting multilayered structure differed markedly from the undissociated tissue, in that it lacked intercellular spaces, convolutions or inter-digitations; only a lucent band and some osmiophilic deposits were evident (Fig. 17). The reaggregated structure could not be redispersed with ether. The original ether treatment probably extracts most of the interlamellar lipids. This suggests that the neutral lipids are necessary for desquama-

Fig. 15. Reaggregated structure from single squames obtained after mechanical dispersion of the stratum corneum. Arrows point to desmosomal contacts. (x29,OOO). (Brysk et al. 1989, reproduced with permission).


Fig. 16. Ultrastructural view of purified undissociated stratum corneum. Arrows point to desmosomal contacts. (x29,OOO). - From BRYSK et al. (1989), reproduced with permission.

tion. Ether extraction still leaves the glycoproteins and an ester-bound lipid, omegahydroxyacylsphingosine, intact (SWARTZENDRUBER et al. 1987). After removal of the neutral lipids, the glycoproteins and their ligands assume a more uniform compact orientation with more tightly bound squames. A similar structure had been reported after leaching out the lipids from intact stratum corneum with other organic solvents such as chloroform-methanol (SWARTZENDRUBER et al. 1987). The reaggregated stratum corneum after organic solvent extraction does not exhibit desmosomal attachment structures. Single squames obtained by dispersion in detergents could not be aggregated into a multilayered structure. The detergent solubilizes both the lipids and the glycoproteins. SMITH et al. (1982) reported that ether-dispersed squames lost their ability to reaggregate if they were pretreated with trypsin or glycolytic enzymes. Both types of enzymes cleave cell surface proteins, including glycoproteins. Thus, the combined damage from the ether and the enzyme is equivalent to that from detergent extraction. The key factor in the loss of corneocyte adhesion is the removal of the glycoproteins. Clearly, glycoproteins are crucial to the cohesion of the stratum corneum whether lipids are present or


Fig. 17. Reaggregated structure from single squames obtained after ether dispersion of the stratum corneum. (x29,OOO). - From BRYSK et al. (1989), reproduced with permission.

not. Lipids may help provide the appropriate stereochemical orientation in such a way that the requisite domains of the glycoproteins are exposed for interaction. Unlike the intact tissue, the reconstituted stratum corneum after ether extraction expressed lectin binding to Con A, WGA ~-glcNAc, NeuNAc) and SBA (~-glcNAc), though not to UEA-1 (a-L-fucose) or BSA-B 4 (a-D-gal) (BRYSK et al. 1988). It is likely that, in the intact tissue, the lipids mask the saccharide moieties of the glycoproteins, which are exposed after delipidation. We have developed an aggregation model for testing the effect of a variety of agents on corneocyte adhesion. Ether-dispersed squames are processed through an acetone transition phase from which the squames can reaggregate when transferred to an aqueous phase (BRYSK et al. 1988). This system has allowed us to modulate the aggregation process with lectins, sugars and the antibody to desquamin. Reaggregation is inhibited with amino sugars, with the lectins WGA (~-glcNAc, NeuNAc) and SBA (~-glcNAc) specific for amino sugars, as well with the antibody to desquamin. We have used this system to corroborate our hypothesis that glycoproteins, and desquamin in particular, play a major role in the adhesion of the stratum corneum. Lectins have been used to induce agglutination of many diverse cell types. Because of their multivalent structure,


lectins can form cross-links between appropriate receptors on adjacent cells. Receptor mobility has been shown to be crucial to the aggregation process (HARDING and GALLAGHER 1982; GLENNEY et al. 1979). It has also been reported that cells which have lost their lectin receptor mobilities can still bind to lectins, but are not agglutinated by them (WANG et al. 1982). Among the epidermal cells, the differentiated cells (past the upper spinous layers) lose the mobility of their Con A receptors (TAGIKAWA et al. 1983). Particularly for the stratum corneum, which consists of dead squames without a cell membrane, the process of agglutination cannot take place through cross-linkage by lectins. The mode of action that we postulate for desquamin in our aggregation system is that, as the flat squames slide past each other, desquamin on one corneocyte attaches to an amino sugar site on an adjacent cell. We have shown that this process can be disrupted by introducing an excess of amino sugars into the system; the free sugars bind to the endogenous lectin, preventing it from reacting with its receptor on another cell. Indeed, we observe inhibition of aggregation with amino sugars but not with other sugars. The process can also be blocked with exogenously added lectins which bind to the amino sugar sites, making them unavailable for binding to ,desquamin. We observe inhibition with WGA (~-glcNAc, NeuNAc) and SBA (~-glcNAc), but not with UEA-1 (a-L-fucose) or BSA-B 4 (a-D-gal) (Tables 3 and 4). Table 3. Effects of different lectins on squame aggregation. Lectin

Sugar specificity

WGA

N -Acetylglucosamine N-Acetylneuraminic acid N -Acetylgalactosamine D-mannose L-Fucose a-D-galactose

SBA Con A UEA-1 BSA-B 4

Concentration inhibiting aggregation (l-tg/assay) 10 200 100

Table 4. Effects of saccharides on corneocyte aggregation. Saccharide

Concentration inhibiting aggregation (M)

N-acetylneuraminic acid N -acetylglucosamine N -acetylgalactosamine a-methyl-D-mannoside a-D-galactose L-fucose

0.1 0.2 0.2 0.3


200

92.5 68

46

30

a b

>t-

C

normal epidermis normal callus

keratoderma

> t-

U

«0

d

pachyonychia congenita

£:)

«a:: w

e

> t-

«.....J

psoriasis

w

a::

epidermolytic hyperkeratosis

9 basal cell '-'-------; carci noma 200

92.568

46

30

MOLECULAR WEIGHT

X

Fig. 18. Densitometer scans of gels from Fig. 19. - From permlsslOn.

10-3 BRYSK

et al. (1984), reproduced with


7.2 Diseases Insight into stratum corneum cohesion and desquamation has come from studies of scaling skin disorders. We examined the distribution of Con A binding glycoproteins, and of desquamin in particular, in normal skin (epidermis and callus) and in epidermis from several diseases of aberrant differentiation (BRYSK et al. 1984; THOMAS et al. 1984). Fig. 18 displays the glycoprotein profiles. Desquamin, the 40 kD glycoprotein, is the most prominent molecule both in normal skin and in callus (which consists primarily of accumulated stratum corneum). Keratoderma is a hyperkeratotic disease that clinically resembles normal callus; it shows a prominent peak at 40 kD. Pachyonychia congenita exhibits acanthosis and is also characterized by the absence of a granular layer and an overaccumulation of stratum corneum; it displays an even greater desquamin expression. We also examined the epidermis in diseases with a truncated pattern of terminal differentiation. Psoriasis involves abnormally high cell proliferation and turnover, with a thickened spinous layer, attenuated granular layer, parakeratosis, and corneal cells which shed as clumps (not singly, as in normal epidermis); desquamin is hardly expressed, if at all, in this disease. Epidermolytic hyperkeratosis is a hyperproliferative skin disease characterized by thickened spinous and corneal layers, but with the granular layer vacuolated and degenerated as a result of overproduction of proteinases; the expression of desquamin is also reduced. Both psoriasis and epidermolytic hyperkeratosis have been classed as in the family of hyperproliferative ichthyoses (WILLIAMS

normal epidermis

Fig. 19. Autoradiographs of SDS polyacrylamide gels of extracts from lesional disease tissue reacted with 125I_Con A. Each lane corresponds to a biopsy specimen from a different patient. The same amount of protein was used for each lane; scans are from the same slab gel. - From BRYSK et al. (1984), reproduced with permission.


and ELIAS 1985) because of their abnormal retention of the stratum corneum and their aberrant desquamation. In basal cell carcinoma, keratinocytes do not differentiate at all, and desquamin is absent. In addition to the expression of desquamin, we observed that the overall glycoprotein profiles in biopsy samples were different for each disease and were distinguishable from normal epidermis and callus (Fig. 19). The pattern recognition can be expressed in terms of the glycoproteins at 80 kD, 50 kD and 40 kD. All the disease samples had an intense band at 50 kD whose counterpart was barely visible for normal skin. We do not yet know the functional role of this glycoprotein (Fig. 18). We have also isolated a 30 kD glycoprotein from human skin (CHEN et al. 1986). A monospecific polyclonal antibody to this glycoprotein immunolocalizes only to the lipid envelopes of the stratum corneum. Functional analyses reveal the 30 kD glycoprotein to be a potent chemokinetic molecule; it activates the migration of polymorphonuclear neutrophils in collagene gels (RA]ARAMAN et al. 1987). Unlike desquamin, which is not expressed in psoriasis, the 30 kD molecule is expressed in all the viable epidermal layers in lesional and nonlesional psoriatic skin (Fig.20). It thus may be responsible for some of the pathophysiology of psoriasis. Other glycoproteins are also aberrantly expressed in psoriatic epidermis (MANSBRIDGE and KNAPP 1984). Abnormal lipid profiles in the stratum corneum have also been reported for psoriasis and for other scaling disease disorders in which desquamation is impaired. The ichthyoses constitute a group of acquired and genetic disorders in which scales accumulate on the skin surface. Normally, lipids of shed corneocytes are poor in cholesterol sulfate (RANASINGHE et al. 1986; LONG et al. 1985). However, both psoriatic and lamellar ichthyotic scales display increased levels of free cholesterol and decreased levels of esterified cholesterol when compared with controls (SCHMIDT et al. 1977). Higher levels of cholesterol sulfate also accumulate in recessive X-linked ichthyosis (EpSTEIN et al. 1981). Patients with this disease lack the enzyme steroid sulfatase (SHAPIRO et al. 1978). In the absence of this enzyme, patients accumulate high levels of the substrate, cholesterol sulfate, in the stratum corneum (EpSTEIN et al. 1981; WILLIAMS and ELIAS 1981). These results suggest that desulfation of the substrate is necessary for normal desquamation (EpSTEIN et al. 1988). Chemical irritants can also elicit abnormal desquamation. The topical application of the surfactant sodium dodecyl sulfate (SDS) induces scaly and dry skin (PROTTEY and FERGUSON 1975; IMOKAWA 1980). We have found that SDS extracts most of the glycoproteins from the stratum corneum (BRYSK et al. 1984). SDS extraction also changes the free-cholesterol to cholesterol-ester ratios (FULMER and KRAMER 1986). We believe that this detergent induces abnormal scaling by extracting both classes of macromolecules; and that both glycoproteins and lipids are necessary for the normal desquamation process. In addition to glycoproteins and lipids, the cytoskeletal proteins are also aberrantly expressed in diseases of altered epidermal differentiation. The 65-67 kD basic keratins are present only if the lesions retain their keratinized morphology (WEISS et al. 1983,


Fig. 20. Immunofluorescent localization of the antibody to the 30 kD glycoprotein on frozen sections. Panel (a) normal epidermis; (b) non-lesional psoriatic skin; (c) lesional psoriatic skin. (x160). - From RAJARAMAN et al. (1987).


1984). These keratins are reduced in psoriasis. The acidic small keratins characteristic of hyperproliferative epidermis are absent in ichthyosis vulgaris. The expression of filaggrin is reduced or attenuated in several differentiative disorders, among them psoriasis, ichthyoses vulgaris and X-linked ichthyosis (SYBERT et al. 1985; FLECKMAN et al. 1987; KANITAKIS et al. 1988; THIVOLET 1988).

7.3 Tissue culture models Numerous culture systems have been developed for the study of the proliferation and differentiation of epidermal cells (see review by HOLBROOK and HENNINGS 1983). MACKENZIE et al. (1985) used organ cultures of mouse ear skin to show that, in media supplemented with cortisone, there was a linear accumulation of stratum corneum cell layers that mimicked that observed in vivo. The accumulated cells were loosely bound and could be removed by treatment with detergent. The main documented change in the accumulated cells was a reduction in cholesterol sulfate. From these results, one might suspect the presence of a catalytically active sterol sulfatase within the stratum corneum; however, no such activity has been demonstrated (RANASINGHE et a. 1986). While the organ cultures showed a pile-up of outer squames, no actual desquamation was evident. Keratinocytes cultured at the air-liquid interface most closely resemble the epidermis in vivo; they show a nearly complete pattern of terminal differentiation (MACKENZIE and FUSENIG 1983; PRUNIERAS et al. 1983; LILLIE et al. 1980; BERNSTAM et al. 1986). These cultures have a distinct granular layer and a normal multilayered stratum corneum. They also exhibit lamellar bodies and lipid profiles which resemble the native tissue (WILLIAMS et al. 1988; MADISON et al. 1988; ROSDY and CLAUS 1990). We have also found that such cultures express desquamin in the corneal layers (BRYSK, unpublished observations). While air-liquid interface cultures resemble the normal epidermis, the stratum corneum that is formed does not desquamate. This suggests that stratum corneum cohesion is regulated by a different mechanism than is dehiscence or desquamation. It is likely that regulatory mechanisms which are operative in the normal epidermis are absent in culture and, therefore, the cultures fail to desquamate. Possible requisite agents include cytokines and growth factors. Numerous submerged culture systems have also been described (HOLBROOK and HENNINGS 1983). In some systems, cells are grown on plastic; in others, a variety of dermal feeder layers have been used. The best feeder layers have been lethally-irradiated 3T3 fibroblasts (RHEINWALD and GREEN 1975). As a rule, submerged cultures exhibit a truncated pattern of differentiation and stratification, with the absence of a distinct granular layer, stratum corneum or lamellar bodies. Stratification is dependent on the Ca2 + concentration of the culture medium (HENNINGS et al. 1980) and the presence of fetal calf serum. Serum-free culture systems have also been developed for the growth of keratinocytes (BOYCE and HAM 1983). Human keratinocytes stratify when the Ca2 +


concentration of the culture medium exceeds that of 0.1 mM. The extent of differentiation increases when serum is also added exogenously. In submerged cultures, keratinocytes shed some cornified envelopes, but as a rule do not desquamate.

•

~a

•

All

Fig. 21. Phase contrast micrographs of human keratinocytes in primary culture. Panels (A) and (B) are for cells grown in KGM medium (0.1 mM Ca2 +); (C) and (D) are for cells grown in KGM medium with added Ca2+ (1.5 mM); (E) and (F) are for cells grown in DMEM containing 10% fetal calf serum. Panels A, C, and E are of cells with no lFN-y; B, D, and E are of cells grown in medium supplemented with 100 D/ml of lFN-y. (xl000). - From Brysk et al. (1991), reproduced with permission.


7.4 Modulation by exogenous factors NICKOLOFF et at. (1984) observed increased cell shedding in keratinocytes cultured with interferon-yo This cytokine has a multiplicity of effects on cultured keratinocytes. IFN-y alters epidermal differentiation (BASHAM et at. 1984; BASHAM et at. 1985) and, in particular, augments the expression of cell surface molecules related to adhesion (DusTIN et at. 1986; GRIFFITHS et at. 1989; REANO et at. 1990). We have found that IFN-y modulates desquamation, as well as the expression of the desquamins (BRYSK et at. 1991). We cultured human keratinocytes in low Ca2 + (0.1 mM), in high Ca2 + (1.5 mM), and in high Ca2+ with serum. In all three systems, IFN-y induced cell shedding and an altered phenotype. In the serum-containing medium supplemented with IFN-y, the outer squames were six times larger than normal; they resembled, in size and shape, the outer squames from intact epidermis (Fig. 21). Ultrastructural features of the keratinocytes at low Ca 2+ show cell piling and a lack of desmosomes (Fig. 22). In the serum-containing medium, the cells grown without IFN-y show a normal organized stratification pattern (Fig. 23). On the other hand, when IFN-y was added to this medium, there resulted a disorderly pattern of stratification with larger outer squames, and fewer celle nuclei (Fig. 24). We also showed, by in situ immunolocalization with antibodies (Fig. 25) and by Western blotting (Fig. 26), that IFN-y in the presence of serum induces the expression of predesquamin and desquamin. Neither desquamin is formed in submerged cultures, except when the medium is supplemented with both serum and IFN-y. This cytokine has been shown to be a normal constituent of epidermis; it may thus regulate desquamation in vivo, in concert with other growth factors. In submerged cultures, keratinocytes do not form a granular layer nor a multilayered stratum corneum. Nonetheless, a number of molecules associated with terminal differentiation in vivo are expressed, among them the higher molecular weight keratins and filaggrin. We find that the expression of keratins and filaggrin is unaffected by the presence of IFN-y in the culture medium (Fig. 27). On the other hand, the desquamins and desquamation are induced in submerged cultures in response to serum and IFN-y (BRYSK et at. 1991). Our results suggest that IFN-y may be inducing a pattern of differentiation with some abnormal characteristics (most notably: fewer cell layers, fewer nuclei, and larger outer squames) in a truncated pattern of terminal differentiation which also promotes desquamation. Keratinocytes grown at the air-liquid interface express the desquamins, and form lipids in a profile that resembles normal epidermis (WILLIAMS et at. 1988). Nonetheless, the squames in such cultures do not desquamate. These cumulative findings suggest that normal desquamation in vivo requires not only a complete pattern of terminal differentiation but, in addition, regulation by exogenous agents produced in vivo but which are absent in vitro. A variety of cytokines, hormones, and growth factors modulate epidermal differentiation in vivo. Some of these may be present in serum, others may be formed within the tissue. BIKLE et at. (1989) showed that IFN-y could regulate the production of 1,25-(OHhD3 by cultured keratinocytes.


Fig. 22. Ultrastructure of keratinocytes grown at low Ca2+. A: without IFN-y; B: with IFN-y (A: x7,OOO; B: x2S,OOO). - From BRYSK et al. (1991), reproduced with permission.


Fig. 23. Ultrastructure of keratinocytes grown in DMEM with fetal calf serum without IFN-y (A: x7,OOOj B: x28,OOO). - From BRYSK et al. (1991), reproduced with permission.


Fig. 24. Ultrastructure of keratinocytes grown in DMEM with fetal calf serum with IFN-y (A: x7,OOO; B: x28,OOO). - From BRYSK et al. (1991), reproduced with permission.


Fig. 25. Immunofluorescent localization of predesquamin and desquamin monoclonal antibodies. A and C: fresh tissue; Band D: keratinocytes grown in serum and with IFN-y; predesquamin (A, B) and desquamin (C, D). (x500). - From BRYSK et al. (1991), reproduced with permISSIOn.

Such a regulation was modulated by a variety of factors, among them the Ca2 + concentration and the presence of serum. Factors in serum that could also participate in this process include retinoids (EICHNER 1986), glucocorticoids (MARCELO and TOMICH 1983), tumor necrosis factor (MURPHY et al. 1988; TAKUMA et al. 1987), transforming growth factors (COFFEY et al. 1987; SHIPLEY et al. 1986), colony stimulating factors (DANBURG and SAUDER 1986), and interleukin-1 (LE and VILCEK 1987; TSAI and GAFFNEY 1987). Interleukin-1 levels are elevated in the stratum corneum (GAHRING et al.


(

.., DOWNING, D. T.: Lipids of epidermis and keratinized and non-keratinized oral epithelia. - Compo Biochem. Physiol. 83B, 529-531 (1986). WERTZ, P. W., DOWNING, D. T.: Glycolipids in mammalian epidermis: structure and function of the water barrier. - Science 217, 1261-1262 (1982). -: : Covalently bound omegahydroxyacylsphingosine in the stratum corneum. - Biochim. biophys. Acta (Arnst.) 917,108-111 (1987). WHITE, S. H., MIREJOVSKY, D., KING, G. I.: Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study. - Biochemistry 27, 3725-3732 (1988). WILLIAMS, M. L., BROWN, B. E., MONGER, D. J., GRAYSON, S., ELIAS, P. M.: Lipid content and metabolism of human keratinocyte cultures grown at the air-liquid interface. - J. cell. Physiol. 136, 103-110 (1988). WILLIAMS, M. L., ELIAS, P. M.: Stratum corneum lipids in disorders of cornification. I. Increased cholesterol sulfate content in recessive X-linked ichthyosis. - J. clin. Invest. 68, 1404 -1410 (1981). -: Elevated n-alkanes in congenital ichthyosiform erythroderma. Phenotypic differentiation of two types of autosomal recessive ichthyosis. - J. clin. Invest. 74, 296-300 (1984). -: The ichthyoses. - In: Pathogenesis of Skin Disease (eds. Thiers, B. H., Dobs, M. R. L.), 519-551. - Churchill-Livingstone, New York 1985. -: The extracellular matrix of stratum corneum: role of lipids in normal and pathological function. - (CRC Crit. Rev.) Therap. Drug Carrier Syst. 3,95-122 (1987). WOLFF, K., SCHREINER, E.: An electron microscopic study on the coat of keratinocytes and intercellular space of epidermis. - J. invest. Dermatol. 51, 418-430 (1968). YARDLEY, H. J., SUMMERLY, R.: Lipid composition and metabolism in normal and diseased epidermis. - Pharmacol. Ther. 13, 357-383 (1981). ZENISEK, A., KRAL, J. A., HAIS, I. M.: Sunscreening effect of urocanic acid. - Biochim. biophys. Acta (Arnst.) 18,589-591 (1955). ZETTERGREN, J. G., PETERSON, L. L., WUEPPER, K. D.: Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue. - Proc. natl. Acad. Sci. USA 81, 238-242 (1984). ZIESKE, J. D., BERNSTEIN, I. A.: Modification of cell surface glycoprotein: addition of fucosyl residues during epidermal differentiation. - J. Cell BioI. 95, 626-631 (1982). -: Epidermal fucosylation of a cell surface glycoprotein. - Biochem. Biophys. Res. Commun. 119, 1028-1033 (1984).

Electrical properties of the epidermal stratum corneum.

Comparison of lipids from epidermal and palatal stratum corneum.

TMPRSS13 deficiency impairs stratum corneum formation and epidermal barrier acquisition.

Epidermal thermal conductivity and stratum corneum hydration in cat footpad.

Development of the stratum corneum.

Stratum corneum lipid function.

Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Skin diseases associated with the depletion of stratum corneum lipids and stratum corneum lipid substitution therapy.

Hand eczema and stratum corneum ceramides.

The fibrous proteins of stratum corneum.

The physics of stratum corneum lipid membranes.

Thermal analysis of human stratum corneum.

Facial skin pigmentation is not related to stratum corneum cohesion, basal transepidermal water loss, barrier integrity and barrier repair.

Stratum corneum lipid abnormalities in atopic dermatitis.

Atopic dermatitis and the stratum corneum: part 1: the role of filaggrin in the stratum corneum barrier and atopic skin.

Sampling the stratum corneum for toxic chemicals.

The stratum corneum lipid thermotropic phase behavior.

The stratum corneum: a moving subject.

Effects of petrolatum on stratum corneum structure and function.

The biochemistry and function of stratum corneum lipids.

Molecular basis for stratum corneum maturation and moisturization.

Interaction between corneocytes and stratum corneum lipid liposomes in vitro.

Artifactual Stratum Corneum Calcification of the Beagle Dog Tongue.

The physical chemistry of the stratum corneum lipids.