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Pathogenesis of the Permeability Barrier Abnormality in Epidermolytic Hyperkeratosis1
Pathogenesis of the Permeability Barrier Abnormality in Epidermolytic Hyperkeratosis1 Matthias Schmuth,*² Gil Yosipovitch,³ Mary L. Williams,§¶ Florian Weber,² Helmut Hintner,** Susana Ortiz-Urda,²² Klemens Rappersberger,²² Debra Crumrine,§ Kenneth R. Feingold,* and Peter M. Elias§
§Dermatology Service and *Metabolism Section, Veterans Affairs Medical Center, §Departments of Dermatology, *Internal Medicine, and ¶Pediatrics, University of California, San Francisco, U.S.A.; ³National Skin Center, Singapore; **Department of Dermatology, General Hospital Salzburg, Austria; Departments of Dermatology, Universities of ²Innsbruck and ²²Vienna, Austria
defective organization of extracellular lamellar bilayers. The decreased lamellar material, in turn, could be attributed to incompletely secreted lamellar bodies within granular cells, demonstrable not only by several morphologic ®ndings, but also by decreased delivery of a lamellar body content marker, acid lipase, to the stratum corneum interstices. Yet, after acute barrier disruption a rapid release of preformed lamellar body contents was observed together with increased organelle contents in the extracellular spaces, accounting for the accelerated recovery kinetics in epidermolytic hyperkeratosis. Accelerated recovery, in turn, correlated with a restoration in calcium in outer stratum granulosum cells in epidermolytic hyperkeratosis after barrier disruption. Thus, the baseline permeability barrier abnormality in epidermolytic hyperkeratosis can be attributed to abnormal lamellar body secretion, rather than to corneocyte fragility or an abnormal corni®ed envelope/corni®ed-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair. Key words: corni®ed envelope/intermediate ®laments/keratin/stratum corneum/transepidermal water loss. J Invest Dermatol 117:837±847, 2001
Epidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin ®lament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of corni®cation, patients typically display an abnormality in permeability barrier function. The nature and pathogenesis of the barrier abnormality in epidermolytic hyperkeratosis are unknown, however. We assessed here, ®rst, baseline transepidermal water loss and barrier recovery kinetics in patients with epidermolytic hyperkeratosis. Whereas baseline transepidermal water loss rates were elevated by approximately 3-fold, recovery rates were faster in epidermolytic hyperkeratosis than in age-matched controls. Electron microscopy showed no defect in either the corni®ed envelope or the adjacent corni®ed-bound lipid envelope, i.e., a corneocyte scaffold abnormality does not explain the barrier abnormality. Using the water-soluble tracer, colloidal lanthanum, there was no evidence of tracer accumulation in corneocytes, despite the fragility of nucleated keratinocytes. Instead, tracer, which was excluded in normal skin, moved through the extracellular stratum corneum domains. Increasing intercellular permeability correlated with decreased quantities and
T
process of epidermal differentiation. Whereas expression of keratins 5 and 14 is restricted to the basal cell compartment, keratins 1 and 10 are produced in all suprabasal nucleated cell layers (Roop, 1995). In the nucleated cell layers, these keratins are organized into ®lament bundles that loop between desmosome plaques and the nuclear envelope providing a cytoskeleton that protects keratinocytes from mechanical injury (Fuchs and Cleveland, 1998). As the keratinocytes terminally differentiate, keratins 1 and 10 as well as several other corni®ed envelope (CE) proteins form a rigid, chemically and mechanically resistant structure that replaces the plasma membrane (Nemes and Steinert, 1999; Steinert and Marekov, 1999; Steinert, 2000). The CE, in turn, forms a scaffold upon which the lipid-enriched extracellular matrix is organized into lamellar bilayer structures that mediate the permeability barrier function of the outermost layer of the epidermis, the stratum corneum (SC) (Jackson et al, 1993; Elias et al, 1998).
he epidermis is a homeostatic, self-renewing tissue that expresses differentiation-speci®c genes sequentially as keratinocytes move outward from the basal to the granular cell layer (SG). Keratins are among the most abundant proteins produced during this vectorial
Manuscript received January 31, 2001; revised May 25, 2001; accepted for publication June 1, 2001. Reprint requests to: Dr. Matthias Schmuth, V.A. Medical Center, Metabolism Section (111F), 4150 Clement St., San Francisco, CA 94121. Email: [email protected] Abbreviations: CE, corni®ed envelope, CLE, corneocyte-bound lipid envelope; LB, lamellar body; SC, stratum corneum; SG, stratum granulosum; TEWL, transepidermal water loss. 1 We dedicate this work to Professor Peter O. Fritsch in honor of his 60th birthday. 0022-202X/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 837
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The dual-compartment, corneocyte±extracellular lamellar lipidenriched matrix of the SC serves as a protective barrier not only against external insults, but also against excess transepidermal water loss (TEWL) (Grubauer et al, 1989). Any perturbation of the permeability barrier initiates a rapid and ef®cient repair response in the underlying epidermis. Regardless of the speci®c nature of the barrier insult, i.e., mechanical (e.g., tape stripping) or chemical (e.g., solvent or detergent treatment), a metabolic response ensues in the underlying epidermis that leads to restoration of normal function (Elias, 1996). The initial response comprises an immediate exocytosis of the preformed pool of lamellar bodies from the outermost SG cell layer (Menon et al, 1992a; Elias et al, 1998). Cholesterol, fatty acid, and ceramide synthesis then increase over the next 6±9 h (Harris et al, 1997), leading to accelerated formation and further secretion of nascent lamellar bodies (Menon et al, 1992a). This response enables the re-accumulation of lipids in the SC extracellular spaces, reformation of membrane bilayers, and restoration of baseline barrier function (Grubauer et al, 1987). Whereas the importance of the lipid matrix for the maintenance of cutaneous barrier function is undisputed, the contribution of the corneocyte to the permeability barrier is less clear. A potential role for the CE scaffold in the organization of the extracellular lamellar bilayer system is suggested by lamellar ichthyosis, where transglutaminase I mutations (Huber et al, 1995; Russell et al, 1995) result not only in a defective CE formation but also abnormal permeability barrier function (Lavrijsen et al, 1993; Elias et al, 2001). The enhanced TEWL in lamellar ichthyosis has been attributed to truncation and fragmentation of extracellular lamellar arrays, which correlate with the CE defect (Elias et al, 2001), supporting a scaffold role for the CE in bilayer formation. Mutations in keratin 1 and keratin 10 have been identi®ed as the cause of the autosomal dominant form of ichthyosis, epidermolytic hyperkeratosis (EHK, also termed bullous congenital ichthyosiform erythroderma (BCIE), OMIM113800) (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992). The keratin 1/10 mutations function in a dominant-negative manner to disrupt the keratin ®lament network within the keratinocyte cytosol. Filaments retract from their attachments to desmosomal plaques and form clumps of
perinuclear shells (Anton-Lamprecht, 1983). Skin fragility to mechanical trauma, which is most pronounced in the neonatal period, is an expected consequence of this cytoskeletal defect, analogous to the fragile skin phenotype of epidermolysis bullosa, where mutations in keratins 5 or 14 produce cytoskeletal defects in the epidermal basal layer (Bonifas et al, 1991; Coulombe et al, 1991; Lane et al, 1992). The hyperkeratosis and accompanying defect in permeability barrier function are additional clinical features of EHK (Frost et al, 1968), the pathogenesis of which is less clear. Speci®cally, whether the barrier defect is due to a fragile keratinocyte, i.e., a defective corneocyte/CE scaffold, or other abnormalities is not known. To dissect the pathogenesis of the barrier abnormality in EHK, we assessed three alternative mechanisms: (i) a fragile corneocyte leading to increased transcellular permeability; (ii) an ineffective CE scaffold; (iii) aberrant lamellar body (LB) secretion. Lanthanum tracer studies revealed an intercellular rather than a transcellular route of increased water loss in EHK, ruling out a fragile corneocyte as the basis for the abnormality. Increased intercellular water movement correlated, in turn, with a decrease in the quantities and abnormal organization of the extracellular lamellar bilayer system. Finally, the decreased lamellar bilayers were associated with an accumulation of unsecreted LB in the outer SG under baseline conditions. With acute barrier disruption, however, a loss of the epidermal calcium gradient occurred that initiated a rapid release of preformed LB and appearance of organelle contents in the extracellular domains, resulting in accelerated rates of barrier recovery. MATERIALS AND METHODS Patients We investigated four patients (one female, three male, aged 12±45 y) with clinically typical EHK (Table I). The diagnosis was con®rmed by the presence of prominent hyperkeratosis, coarse keratohyalin granules, and vacuolization of the upper stratum spinosum and SG on histopathology. In some sections we additionally found acantholysis of the stratum spinosum and SG. This skin phenotype had been present in all patients since birth. Further work-up did not demonstrate any clinical or laboratory abnormalities. Mutation analysis revealed novel mutations in keratin 1 or 10 in two patients (Table I; in
Table I. Characteristics of EHK patients Patient #
Gender
Age
Clinical phenotype
Mutation
Biopsy sites
Scale
1 2 3 4
M F M M
29 12 44 17
Generalized, Generalized, Generalized, Generalized,
K10a (coil domain) ±ab ±abc K1d (tail domain)
Lower arm Abdomen Lower arm Lower leg
+ + + ±
¯exural accentuation annular ¯exural accentuation annular
a
Department of Biochemistry, University of Salzburg. Hot spot analysis of K1/10; no mutations were detected. Reduced epidermal immunolabeling of K10, Department of Dermatology, General Hospital Salzburg. d Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia. b c
Table II. Material available for pathogenesis studies Patient # 1 2 3 4 a
Material at baseline
Material after acute disruption
Assessment of LB secretiona
Acid lipase secretiona
Calcium localizationa
Lanthanum perfusiona
OsO4 post®xation
RuO4 post®xation
Pyridine treatmentb
Biopsy Scale Biopsy Scale Biopsy Scale Biopsy Scale
6h 72 h 6h ± 3h ± ± ±
+ ± + ± + ± + ±
+ ± + ± + ± + ±
+ ± ± ± + ± ± ±
+ ± + ± + ± + ±
+ ± + ± + ± + ±
+ + + + + + + +
± + ± + ± ± ± ±
Analysis possible in biopsies, but not in scale samples. Baseline only.
b
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Table III. Assessment of lamellar body secretion in EHK versus controls Type of samples (case #) EHK 1 2 3 4 Normal controls (n = 3) Other ichthyoses CIE (n = 4)a LI (n = 4)b NLSDI (n = 2)c
Lamellar bilayers
Contents of SG±SC interface
Entombed LB
Interstices-width/ corneocyte ratio
Lipase localization in SC
Decreased Decreased Decreased Decreased Replete
Decreased Decreased Decreased Decreased Replete
Present Present Present Present None
Increased Increased Increased Increased Normal
Decreased Intercellular Decreased Intercellular Decreased Intercellular Decreased Intercellular All Intercellular
Decreased Replete (dimensions
Decreased Replete abnormal) Decreased
Present None
Increased Normal
Decreased All Intercellular
Some present
Normal
Mostly Intercellular
Clefts
a
Congenital ichthyosiform erythroderma (Menon et al, 1992b). Lamellar ichthyosis (Elias et al, 2001). c Neutral lipid storage disease (Williams et al, 1985). b
Figure 1. Pathogenesis ¯ow chart for the analysis of the barrier abnormality in disorders of keratinization. First, functional assessments are used to determine TEWL levels in ichthyotic skin. If TEWL abnormalities are present, lanthanum perfusion is employed to distinguish transcellular from intercellular permeability pathways. In the case of an intercellular abnormality, a battery of ultrastructural methods is then applied to detail the morphologic basis for the intercellular defect. aElias et al, 1981; b Elias et al, 2001; cElias et al, 2000b; dpresent study.
patient 1 a mutation was detected in the coil domain 1 A of keratin 10 whereas in patient 4 the mutation was located in the tail of keratin 1). An additional patient displayed reduced immunostaining for keratin 10, but neither keratin 1 or keratin 10 mutations were detectable in this or the other remaining patient on hot spot analyis. Others have noted that a substantial proportion of patients with the EHK phenotype lack keratin 1 or 10 mutations (Porter et al, 1996; Fuchs and Cleveland, 1998). Skin from EHK patients and control subjects (three healthy male volunteers, aged 31±32 y) comprised our group of controls (Table III). None of the EHK or control subjects had employed any external medications or emollients to the studied skin areas for at least 2 wk prior to assessment of barrier function. Additional controls included historical material from normal human skin, and biopsies from a variety of individuals with other disorders of corni®cation (Table III). Functional studies To assess epidermal permeability function we measured TEWL on a 1.13 cm2 area of the volar aspect of the forearm in three EHK subjects using an evaporimeter (ServoMed, Stockholm, Sweden). TEWL values were registered in g per m2 per h after equilibration of the probe on the skin (> 60 s). All recordings were made by the same investigator (M.S.). Volunteers underwent a 15 min premeasurement rest period. Environment-related variables at the time of the study were: ambient air temperature 21.9°C±26.6°C; skin surface temperature 30.4°C±34.4°C, ambient air humidity 23%±49%; atmospheric H2O pressure 4.4±14.8 mmHg. Excess air convection was prevented by shielding the measurement zone. TEWL was measured
both under baseline conditions and following acute barrier disruption by either repeated cellophane tape stripping or acetone swabbing of the lower forearm. Barrier recovery kinetics then were assessed by measuring TEWL at 3, 6±7, 16, 24, 48, 96, 120, and 144 h after acute disruption. For calculation of the percentage change in TEWL, the following formula was used: 1 ± [(TEWL immediately after stripping ± TEWL at indicated time)/(TEWL immediately after stripping ± baseline TEWL)] 3 100%. Pathophysiology studies To assess the pathophysiologic basis for the barrier abnormality in EHK, we utilized the sequence shown in Fig 1, recently applied to Harlequin ichthyosis (Elias et al, 2000b) and lamellar ichthyosis (Elias et al, 2001). If barrier function was abnormal in an EHK individual, we next assessed whether the abnormality was due to leaky corneocytes resulting in transcellular water movement (as a putative downstream consequence of the keratinocyte fragility that is a wellknown feature of EHK), or to enhanced intercellular permeability. As the barrier abnormality was linked, instead, to enhanced intercellular permeability, we next assessed extracellular lamellar bilayer quantity and organization. If abnormal, we then sought whether the structural defect in the lamellar bilayers was due to an abnormal CE±corneocyte scaffold or to ineffective LB secretion. Biopsies from EHK skin were taken immediately before and 3 and 6 h after acute barrier disruption. Assessment of permeability pathway by lanthanum perfusion The perfusion pathway was assessed in all subjects by immersion of biopsies and/or scale in 4% lanthanum nitrate in 0.05 M Tris buffer containing 2%
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glutaraldehyde, 1% paraformaldehyde, pH 7.4, for 1 h at room temperature (Table II) (Elias et al, 1981). After lanthanum perfusion, the samples were washed and processed for electron microscopy, as described below. Assessment of lamellar bilayer system and CE-associated structures Using a combination of osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) post®xation with pyridine pretreatment, it is possible to assess the integrity of the intercellular lamellar bilayer system in relation to the CE/corneocyte-bound lipid envelope (CLE) scaffold (Elias, 1996; Elias et al, 2000b). Again, scale and biopsy samples were available for these studies (Table II). Brie¯y, all samples were pre®xed in half-strength Karnovsky's ®xative, followed by post®xation in 1% OsO4 and 0.2% RuO4, as described previously (Hou et al, 1991). Some samples were then treated for 2 h with absolute pyridine to further enhance the visualization of the CE/CLE membrane complex, as described recently (Elias et al, 2000b; 2001). After post®xation, all samples were dehydrated in a graded ethanol/propylene oxide series, and embedded in an Eponepoxy mixture. Ultrathin sections were collected and assessed either unstained or after further lead citrate contrasting in a Zeiss 10 A electron microscope operated at 60 kV. Assessment of LB secretion Several morphologic criteria have been used to assess the integrity of the LB secretory process, including (i) the amount of secreted material at the SG±SC interface (Menon et al, 1992a); (ii) the extent that the SC interstices are replete with lamellar bilayers (Menon et al, 1992b); and (iii) the presence of ``entombed'' LB
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within the corneocyte cytosol (Elias et al, 1998). We assessed all of these criteria (Table III) and, in addition, utilized the delivery of a lipid hydrolase (acid lipase), which is concentrated in LB (Menon et al, 1986), as cytochemical indicator of the extent of secretion (Menon et al, 1992b). Aldehyde-pre®xed samples were microwave-incubated with substrate containing 0.2% Tween 85 (6 inhibitor tetrahydrolipstatin, 200 mM) in a lead-capture, cytochemical method that depicts the localization of acid lipase, as described previously (Rassner et al, 1997). Prior studies on nomal controls showed abundant enzyme activity in the SC interstices, with little activity in the corneocyte cytosol (Menon et al, 1992b; Elias et al, 2001) (see Fig 8(D) also). Conversely, in some disorders of corni®cation, acid lipase is not delivered normally, and enzyme activity is retained in the corneocyte cytosol (Ghadially et al, 1992; Menon et al, 1992b) (Table III). The samples for ultrastructural cytochemistry were post®xed in OsO4 and processed as above. Ion capture cytochemistry For ultrastructural calcium localization, frozen biopsy samples (before and after acute barrier disruption, see above) were ®xed in 2% paraformaldehyde, 2% glutaraldehyde, 0.09 M potassium oxalate, containing 0.04 M sucrose. Samples were subsequently ®xed overnight at 4°C, and then post®xed in 1% OsO4 containing 2% potassium pyroantimonate, pH 7.4, for 2 h at 4°C in the dark. Tissue samples then were washed in alkalinized water (pH 10) and transferred to ethanol solutions for dehydration and embedding, as above. Statistical analyses Statistical analyses were performed using a paired or unpaired Student's t test, as appropriate. Data are expressed as mean 6 SEM.
RESULTS
Figure 2. Barrier recovery after tape stripping is accelerated in EHK epidermis. The volar forearm of EHK and normal subjects was tape-stripped, and TEWL was measured with time following barrier disruption (time 0). Accelerated barrier recovery, although evident at all early time points after disruption (i.e., 0±48 h), was signi®cant only at 48 h, because of the small number of patients (*p < 0.05 vs normal control values). At later time points, barrier recovery of EHK individuals paralleled the recovery of normal skin, and at 2±5 d, barrier recovery slowed, resulting in the higher-than-normal baseline TEWL levels characteristic of EHK.
EHK patients display abnormal baseline barrier function, but accelerated barrier recovery kinetics We ®rst assessed barrier function in the three EHK patients who where available for functional studies. As previously shown for EHK (Frost et al, 1968), baseline TEWL rates were markedly increased, with a mean of 19.7 6 1.9 g per m2 per h in patients versus 7.43 6 1.2 g per m2 per h in the three normal controls (p < 0.006, not shown). Yet, the kinetics of barrier recovery after comparable, initial insults (i.e., tape stripping to a maximal TEWL of 100 g per m2 per h) showed an accelerated repair response for the EHK patients versus the three control subjects (Fig 2). In addition, we incidently noted a remarkably fast healing of the skin biopsy sites in EHK patients. The early, more rapid recovery phase was followed by a later normalization of barrier recovery rates by 96 h (not shown). By 1 wk, TEWL levels had returned to approximately 80% of pretreatment values in both EHK and controls. Thereafter, controls and EHK patients returned to baseline levels in parallel. These results show that EHK patients, despite a prominent baseline barrier abnormality, exhibit accelerated barrier recovery rates after acute insults. The enhanced water movement in EHK occurs via intercellular domains We next assessed whether the baseline barrier abnormality in EHK could be assigned to enhanced intercellular or transcellular movement of water, utilizing divalent colloidal lanthanum as a tracer to localize the sites of enhanced water movement. Prior studies have shown that lanthanum is
Figure 3. Increased water movement occurs via intercellular pathways. Freshly obtained biopsies from EHK were exposed for 1 h to 4% colloidal lanthanum nitrate tracer in 0.05 M Tris buffer, pH 7.4, containing 2% glutaraldehyde and 2% paraformaldehyde, followed by post®xation in OsO4. (A), (B) Tracer is observed throughout the SC interstices (arrows), but was excluded from the corneocyte cytosol. OsO4 post®xation. Scale bars: 0.5 mm.
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Figure 4. Extracellular lamellar bilayer structures are abnormal in EHK. (A±C) Clefts and lacunae are present througout the SC interstices (asterisks). (C) Overall, the amount of bilayer material is reduced. (A, B, D,) Stretches of intact, normal-appearing bilayers are only focally present (arrows). RuO4 post®xation. Scale bars: 0.5 mm.
Figure 5. CE and CLE appear normal in EHK. (A) CE (arrows) in EHK appear largely normal, although there is some variation in thickness. (B) CLE (arrowheads) also appear completely normal in EHK. (A) OsO4 post®xation; (B) OsO4 post®xation after pyridine pretreatment (see Methods). Scale bars: 0.25 mm.
excluded from normal SC (Elias et al, 1977), and we con®rmed this ®nding here in the normal control samples (not shown). In contrast, lanthanum penetrated throughout the SC in EHK (Fig 3A). Moreover, in all cases and at all levels of the SC, the tracer was limited to the SC interstices (Fig 3A, B); i.e., little or no tracer was found within the cytosol of individual corneocytes. These results show that, despite evidence of keratinocyte fragility within the nucleated layers, the permeability barrier abnormality in EHK can be ascribed to enhanced movement of water, exclusively via SC extracellular domains. Although extracellular lamellar membranes are reduced and abnormal in EHK, the CE/CLE scaffold is normal We next
explored the basis for increased extracellular water movement in EHK, assessing ®rst the lamellar bilayer system. The lamellar bilayer system was markedly abnormal in all EHK samples, as assessed in RuO4 post®xed samples (Fig 4A±C). With the exception of a few foci (Fig 4A, B, insert, arrows), the amount of bilayer material generally was reduced (Fig 4C, D). Wherever bilayers were present, they appeared disrupted/interrupted by numerous clefts and lacunae that were ®lled with amorphous material (Fig 4A±C, asterisks). Both the reduction in numbers of lamellae and the interruptions of the bilayers by clefts could account for enhanced water movement through the SC interstices. Yet, the precise intercellular pathway of increased water movement could not be ascertained because lanthanum cannot be identi®ed with certainty
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Figure 6. Arrays of LB beneath plasma membrane indicate a secretory defect in EHK. Although the overall number of LB is normal in EHK, arrays of individual organelles remain restricted beneath the apical plasma membrane of outermost granular (SG) cells (A, B, arrows). (C) Lack of LB secretion results in a paucity of extracellular deposits at the SG±SC interface (asterisks). RuO4 post®xation. Scale bars: 0.5 mm.
Figure 7. LB content is entombed within corneocyte cytosol. (A, B) Entombed LB are found within the cytosol of corneocytes (A, B, short open arrows; C, solid arrow). Note again the presence of lacunae (asterisks) and areas of reduced and abnormal bilayer structures (B, C). (C) The encased LB contents focally form bilayer structures within the cytosol. RuO4 post®xation. Scale bars: 0.25 mm.
in RuO4 post®xed samples. We next assessed whether the abnormalities in lamellar membrane structure are associated with abnormalities in the CE/CLE scaffold, as occurs in lamellar
ichthyosis (Elias et al, 2001). Yet, despite the known association of keratins 1/10 with the CE (Steinert and Marekov, 1995), we noted no major abnormalities in CE structure or dimensions (Fig 5A),
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Figure 8. Further evidence of impaired secretion in EHK: acid lipase is retained within corneocytes. (A, B) The LB hydrolase, acid lipase, is largely retained within the cytosol of corneocytes in EHK (arrowheads). (C) Retained activity is colocalized with entombed LB. (D) Normal human skin demonstrates hydrolase solely in the SC interstices, indicative of effective secretion. OsO4 post®xation. Scale bars: 0.5 mm.
Figure 9. Basis for accelerated barrier recovery in EHK epidermis. (A, B) (6 h posttape stripping): No LB remain in cytosol of granular cells (SG), indicated by release of lipase activity into the intercellular spaces (B, arrows). Extent of release of contents also is shown at level of SC: no entombed organelles or cytosolic lipase activity remain in corneocyte cytosol. (C) (3 d post-tape stripping): Reappearance of additional foci of replete lamellar bilayers at time point when barrier recovery is accelerated (cf. Fig 2). (D) (6 h post-tape stripping): Lanthanum perfusion demonstrates substantial blockade of tracer movement at SG±SC interface, although foci of tracer (arrow) continue to penetrate the SC interstices. (A, B, D) OsO4 post®xation; (C) RuO4 post®xation. Scale bars: (A, B, D) 0.5 mm; (C) 0.25 mm.
and an intact CLE on the outer surface of corneocytes in all EHK samples (Fig 5B). We did not quantitate the extent of CLE biochemically, because prior studies have demonstrated normal quantities of covalently bound ceramide in EHK scale (Paige et al, 1994). These results show ®rst that an abnormality in the SC extracellular lamellar bilayer system is present in the SC of EHK under baseline conditions, which accounts for the barrier abnormality in EHK. They also show that the membrane abnormality cannot be ascribed to morphologically detectable abnormalities in the CE/CLE scaffold. LB secretion is impaired in EHK under baseline conditions We next assessed an alternative explanation for the depletion of extracellular lamellar bilayers in EHK, i.e., abnormal
production or secretion of LB. In all baseline EHK biopsies, we observed normal numbers of LB in the upper spinous and granular layers, as well as normal lamellar contents within individual organelles. Thus, LB production appears to be unimpaired in EHK. In contrast, LB secretion from outer SG cells did not occur normally. A distinctive feature was the appearance of arrays of individual organelles, localized within the peripheral cytosol, just beneath the plasma membrane (Fig 6A, B). This feature appeared primarily in cells of the outer SG layer, which also displayed retracted tono®lament bundles (not shown). Further evidence of impaired secretion in EHK included a striking paucity of secreted lamellar contents at the SG±SC interface (Fig 6A, C), and evidence of entombed LB in the form of organelle contents, encased within the cytosol of corneocytes (Fig 7A±C).
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Figure 10. Baseline Ca2+ distribution and loss of Ca2+ gradient is similar to normals in EHK. (A, B) A comparable Ca2+ gradient, with the highest Ca2+ levels in the outer SG with lower levels in subjacent layers and SC, is evident in both normal (A) and EHK (B) skin at baseline. (C, D) Loss of Ca12+ gradient after tape stripping (C) or acetone treatment (D) is accompanied by secretion of LB (SC, stratum corneum, OSG, outer stratum granulosum). OsO4 post®xation. Scale bars: 1 mm.
To further ascertain whether secretion is impaired in EHK, we next assessed the distribution of an enzyme cytochemical marker of LB contents, i.e., acid lipase (Menon et al, 1986), in EHK versus control samples. As previously reported (Menon et al, 1992b), LBderived hydrolase contents were delivered in toto to the SC interstices in normal SC (Fig 8D), leaving no residual, detectable activity within the corneocyte cytosol (Table III). In contrast, whereas EHK subjects displayed comparable labeling of cytosolic LB (Fig 8B), activity at the SG±SC interface and in the SC interstices appeared strikingly reduced (Fig 8C), further regulated by the presence of abundant, retained deposition product in the cytosol of individual corneocytes (Fig 8A, C). Moreover, in some corneocytes retained enzyme activity colocalized with residual, entombed LB contents (Fig 8C). Together, these results show that, whereas LB production is normal in EHK, impaired LB secretion accounts for the diminished extracellular lamellar bilayers that lead to the permeability barrier abnormality in EHK. Inverse calcium signaling of LB secretion accounts for accelerated barrier recovery in EHK Whereas baseline barrier function is abnormal, the kinetics of barrier recovery are faster in EHK than in normal skin (Fig 2). To assess the basis for accelerated barrier recovery in EHK, we next examined lanthanum perfusion, LB secretion, and SC membrane structures 3, 6, and 72 h after acute disruption by either tape stripping or acetone treatment. At
both 3 and 6 h, more extensive cytolysis was evident throughout the outer nucleated cell layers than in nonperturbed EHK epidermis (not shown). Yet, in contrast to baseline EHK epidermis, few LB remained in these disrupted cells; instead, organelle contents appeared in the intercellular spaces throughout the outer epidermis (Fig 9, cf. Fig 6). Further evidence for release of organelle contents also could be seen in the SC. Few corneocytes displayed entombed LB contents (Fig 9, cf. Fig 7), and lipase activity was found between, rather than within, individual corneocytes (Fig 9, cf. Fig 8). These ®ndings correlated with the presence of more widespread foci of normal-appearing membrane bilayers (Fig 9; however, the absolute quantities were still lower than in normals). Finally, after acute perturbations, resulting in LB release, lanthanum was largely (but incompletely) excluded from the SC interstices (Fig 9D). Finally, to assess whether the combination of baseline secretory defect and accelerated recovery is due to modulations in the epidermal calcium gradient, we employed ion capture cytochemistry. At baseline, we observed an accumulation of extracellular and intracellular Ca2+ in the outer SG cells with low visible levels in the SC, comparable to normal skin (Fig 10A, B). Loss of the epidermal calcium gradient with acute barrier disruption either by tape stripping or by acetone treatment occurred normally in EHK skin (Fig 10C, D). Loss of the calcium gradient was accompanied by
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secretion of preformed LB from outer SG cells in EHK (Fig 10C, D vs Fig 6; note absence of preformed LB in cytosol). These results show that the accelerated barrier recovery of EHK skin after acute perturbations can be explained by the release of preformed LB contents from cells in the outer nucleated layers of the epidermis, regulated in turn by modulations in the epidermal calcium gradient. DISCUSSION Keratin intermediate ®laments are the most prominent cytoskeletal proteins of the epidermis and its appendages, accounting for up to 85% of total cellular protein (Fuchs and Coulombe, 1992; Roop, 1995). They form copolymers consisting of equimolar amounts of at least one of two types: type I or acidic keratins (9±20) and type II or basic keratins (1±8) (Miller et al, 1993; Steinert et al, 1993). All keratins display a secondary structure with a central rod domain comprising four alpha-helices and distinctive, nonhelical, head and tail sequences. Mutations have been identi®ed in the highly conserved ends of the rod domains of keratins 1 and 10 in several forms of EHK (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992), but a signi®cant proportion of affected individuals do not have keratin 1/10 mutations (Roop, 1995; Porter et al, 1996; Fuchs and Cleveland, 1998). EHK either occurs sporadically (> 50%) or is inherited in an autosomal dominant fashion. Affected individuals are born with widespread erythema and erosions. With increased age these features diminish and are replaced by generalized epidermal thickening, indicative of epidermal hyperplasia (Irvine and McLean, 1999). The resulting hyperkeratotic plaques are prone to superinfection. The diagnosis in our patient cohort was based on the characteristic clinical picture, diagnostic histology, and the presence of a keratin 1 or keratin 10 mutation in two of the four patients (Table I). Increased expression of other CE proteins (Ishida-Yamamoto et al, 1994, 1995; Porter et al, 1996; Gerritsen et al, 1997; Reichelt et al, 1999) could compensate for the structural abnormality and further provoke the characteristic phenotype in EHK. Our results con®rm previous ®ndings of increased baseline TEWL levels in EHK patients with or without de®ned mutations (Frost et al, 1968; Lavrijsen et al, 1993). We show further that the initial rates of barrier repair after acute disruption actually occurred more rapidly in EHK subjects than in controls. The basis for the baseline permeability barrier abnormality in EHK is unknown. An earlier hypothesis held that keratinocyte fragility could lead to increased cytokine release, which in turn could drive the epidermal hyperplasia, leading to a barrier abnormality (Roop, 1995). Accordingly, we ®rst assessed whether the keratinocyte fragility in EHK leads to a more fragile, ``leaky'' corneocyte, allowing increased transcellular permeability. Indeed, an abnormality in corneocyte fragility accounts for the barrier abnormality in retinoid-treated skin, where abundant colloidal lanthanum tracer leaks into corneocytes (Elias et al, 1981). Yet, our lanthanum perfusion studies clearly showed that, despite the known fragility of keratinocytes within EHK epidermis, the increased SC permeability in EHK occurs via an intercellular rather than a transcellular pathway. Thus, a mechanism other than an incompetent corneocyte must be sought to explain the barrier defect. Instead, we found both a paucity of lamellar bilayers and distorted supramolecular architecture of those lamellar bilayers that were present in all EHK subjects. These structural changes account for the permeability barrier abnormality and the increased intercellular penetration of tracer in EHK. One mechanism whereby defective keratin 1/10 intermediate ®laments could result in abnormal extracellular lamellar bilayers could be an abnormal CE/CLE scaffold. As keratin 1 and 10 are crosslinked to the CE (Ming et al, 1994; Candi et al, 1998), they could play a hitherto unrecognized role in maintenance of the SC barrier. Accordingly, defects in the CE/CLE scaffold, even in EHK individuals with no keratin mutations, could explain the defective organization of extracellular lamellar bilayers in EHK. Indeed, an analogous defect in the CE/CLE scaffold appears to be responsible for the barrier abnormality in another keratinizing disorder with a
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defect in CE formation, i.e., lamellar ichthyosis due to transglutaminase I mutations (Lavrijsen et al, 1993; Elias et al, 2001), and in transgenic mice with a deletion of this enzyme (Matsuki et al, 1998). Barrier function also reportedly is abnormal in involucrin (another CE-constituent peptide) knockout mice,2 which otherwise display a normal phenotype (Djian et al, 2000). Finally, there is evidence for a barrier abnormality in transgenic mice expressing a mutant form of loricrin (Koch et al, 2000; Suga et al, 2000), the dominant CE peptide in epidermis (Steinert, 2000). Our ultrastructural examination did not reveal any defect of the CE/ CLE scaffold in EHK, however. As the CE/CLE scaffold cannot be invoked to explain the barrier defect, we next examined whether the reduction in lamellar bilayers in EHK extracellular domains could be attributed to decreased production (assembly, translocation) or defective secretion (exocytosis) of LB. Under baseline conditions, we observed an increased number of LB at the SG±SC interface displaying normal organelle contents. Moreover, the secretion of LB from the periphery of SG cells was impaired in EHK subjects. Thus, both assembly and translocation of LB appear to be normal, but exocytosis does not occur normally in EHK under baseline conditions. This conclusion is further supported by two additional observations, indicating impaired LB secretion in EHK: (i) the presence of abundant entombed LB contents, encased within the cytosol of corneocytes; (ii) the localization of LB-derived hydrolytic enzymes, which normally are completely secreted, within corneocytes and often colocalized with entombed LB contents. As a result of the secretory abnormality, decreased LB contents are deposited at the SG±SC interface, resulting in discontinuous lamellar bilayers, leading in turn to increased TEWL levels (Fig 4). The baseline barrier abnormality in EHK is associated with abnormal LB secretion, possibly due to an impairment of the exocytosis step. One possible explanation is that the keratin 1/10 cytoskeletal mutation could interfere with the exocytosis of LB. Prior work on lung epithelium and in adrenal secretory cells has shown that the microtubular system is crucial for exocytosis (Aunis and Bader, 1988; Zimmermann, 1989). Thus, cytoskeletal instabilities, including those caused by keratin 1/10 dominant-negative mutations in EHK, could disrupt steps that are involved in the exocytosis of LB. A precise link between the keratin 1/10 mutations and the barrier abnormality could not be established here, however, not only because only half of our patients had these mutations, but also because of limitations of experiments in humans in vivo. Animal models with de®ned defects in keratins 1/10 should allow a further dissection of the molecular pathogenesis of the LB secretory abnormality, however. Several putative mouse models of the disease have been described (Fuchs et al, 1992; Bickenbach et al, 1996; Porter et al, 1996; Reichelt et al, 1997), but available data on the permeability barrier abnormalities in these animal models do not always re¯ect the structural and functional abnormalities of EHK, presumably because the murine genotypes do not always re¯ect EHK (Reichelt et al, 1999; Elias et al, 2000a; Jensen et al, 2000). After acute barrier disruption the peripheral arrays of LB in granular cells were rapidly released (Fig 9), accounting for the accelerated recovery of permeability barrier function in EHK skin. Moreover, we found an increase in intact lamellar bilayers and exclusion of lanthanum tracer from the extracellular domains after acute barrier disruption. The basis for the release of LB after acute disruption probably does not simply re¯ect cytolysis of fragile cells with release of trapped LB. Among likely explanations could be the presence of an intact calcium gradient in EHK under baseline conditions, which, together with the mutant protein, restricts secretion. The effect of the mutant protein can be temporarily 2 Jensen JM, Djian P, Proksch E: Disturbed permeability barrier function in transgenic involucrin de®cient mice. J Invest Dermatol 112: 542, 1999 (abstr.)
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overcome by the complete calcium depletion that occurs after barrier disruption (Fig 10). Alternatively, the expression of the mutant protein could be downregulated after acute disruption, thereby allowing unrestricted LB secretion until the calcium gradient is re-established. To date, however, there is evidence that keratins 1/10 are actually upregulated after barrier disruption (Ekanayake-Mudiyanselage et al, 1998). Lastly, the pathway for LB secretion could differ under baseline versus stimulated conditions. In any case, the localization of arrays of unsecreted LB at the SG±SC interface under baseline conditions seems to re¯ect a ``geared-up'' activation state, which after an external assault is quickly transformed into a normal or supernormal repair response. A similar acceleration of the early phase of barrier repair kinetics has been reported for atopic dermatitis (Gfesser et al, 1997), but no information is available as yet for the morphologic basis of accelerated repair in atopic dermatitis. In summary, the permeability barrier abnormality in EHK can be attributed to increased intercellular water movement, despite the known fragility of keratinocytes. The barrier abnormality correlates with a reduction in extracellular lamellar membranes, which can be attributed further to impaired LB secretion. The baseline abnormality can be overcome by application of external stimuli for barrier repair. Our gratitude to the patients who have shared their time and energy by participating in this study, and to the participating nurses for their assistance. We are indebted to Dr. Sung Ku Ahn for help with ion capture cytochemistry and to Stefanie Kind for technical assistance. This work was supported by NIH grants AR 39908 (PPProjects 1 and 4) and AR 19098, and the Medical Research Service, Department of Veterans Affairs. M.S. was ®nancially supported by a grant from the Austrian Science Fund (Project J1901-MED).
REFERENCES Anton-Lamprecht I: Genetically induced abnormalities of epidermal differentiation and ultrastructure in ichthyoses and epidermolyses: pathogenesis, heterogeneity, fetal manifestation, and prenatal diagnosis. J Invest Dermatol 81:149s±156s, 1983 Aunis D, Bader MF: The cytoskeleton as a barrier to exocytosis in secretory cells. J Exp Biol 139:253±266, 1988 Bickenbach JR, Longley MA, Bundman DS, Dominey AM, Bowden PE, Rothnagel JA, Roop DR: A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis. Differentiation 61:129±139, 1996 Bonifas JM, Rothman AL, Epstein EH Jr: Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. Science 254:1202±1205, 1991 Candi E, Tarcsa E, Digiovanna JJ, Compton JG, Elias PM, Marekov LN, Steinert PM: A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate ®laments to the corni®ed cell envelope through isopeptide crosslinking by transglutaminases. Proc Natl Acad Sci USA 95:2067±2072, 1998 Cheng J, Syder AJ, Yu QC, Letai A, Paller AS, Fuchs E: The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-speci®c epidermal keratin genes. Cell 70:811±819, 1992 Chipev CC, Korge BP, Markova N, Bale SJ, DiGiovanna JJ, Compton JG, Steinert PM: A leucine-proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell 70:821±828, 1992 Coulombe PA, Hutton ME, Letai A, Hebert A, Paller AS, Fuchs E: Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66:1301±1311, 1991 Djian P, Easley K, Green H: Targeted ablation of the murine involucrin gene. J Cell Biol 151:381±388, 2000 Ekanayake-Mudiyanselage S, Aschauer H, Schmook FP, Jensen JM, Meingassner JG, Proksch E: Expression of epidermal keratins and the corni®ed envelope protein involucrin is in¯uenced by permeability barrier disruption. J Invest Dermatol 111:517±523, 1998 Elias PM: Stratum corneum architecture, metabolic activity and interactivity with subjacent cell layers. Exp Dermatol 5:191±201, 1996 Elias PM, McNutt NS, Friend DS: Membrane alterations during corni®cation of mammalian squamous epithelia: a freeze-fracture, tracer, and thin-section study. Anat Rec 189:577±594, 1977 Elias PM, Fritsch PO, Lampe M, Williams ML, Brown BE, Nemanic M, Grayson S: Retinoid effects on epidermal structure, differentiation, and permeability. Laboratory Invest 44:531±540, 1981 Elias PM, Cullander C, Mauro T, Rassner U, Komuves L, Brown BE, Menon GK: The secretory granular cell: the outermost granular cell as a specialized secretory cell. J Invest Dermatol Symp Proc 3:87±100, 1998
Elias P, Man MQ, Williams ML, Feingold KR, Magin T: Barrier function in K-10 heterozygote knockout mice. J Invest Dermatol 114:396±397, 2000a Elias PM, Fartasch M, Crumrine D, Behne M, Uchida Y, Holleran WM: Origin of the corneocyte lipid envelope (CLE): observations in harlequin ichthyosis and cultured human keratinocytes. J Invest Dermatol 115:765±769, 2000b Elias PM, Schmuth M, Uchida Y, et al: Basis for permeability barrier abnormality in lamellar ichthyosis. Exp Dermatol, in press, 2001 Frost P, Weinstein GD, Bothwell JW, Wildnauer R: Ichthyosiform dermatoses. 3. Studies of transepidermal water loss. Arch Dermatol 98:230±233, 1968 Fuchs E, Cleveland DW: A structural scaffolding of intermediate ®laments in health and disease. Science 279:514±519, 1998 Fuchs E, Coulombe PA: Of mice and men: genetic skin diseases of keratin. Cell 69:899±902, 1992 Fuchs E, Esteves RA, Coulombe PA: Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis. Proc Natl Acad Sci USA 89:6906±6910, 1992 Gerritsen MP, Kerkhof PCM, van Vlijmen-Willems IMJJ, Steijlen PM: Expression of ®laggrin, involucrin and tenascin in monogenic disorders of keratinization. Eur J Dermatol 7:164±168, 1997 Gfesser M, Abeck D, Rugemer J, Schreiner V, Stab F, Disch R, Ring J: The early phase of epidermal barrier regeneration is faster in patients with atopic eczema. Dermatology 195:332±336, 1997 Ghadially R, Williams ML, Hou SY, Elias PM: Membrane structural abnormalities in the stratum corneum of the autosomal recessive ichthyoses. J Invest Dermatol 99:755±763, 1992 Grubauer G, Feingold KR, Elias PM: Relationship of epidermal lipogenesis to cutaneous barrier function. J Lipid Res 28:746±752, 1987 Grubauer G, Elias PM, Feingold KR: Transepidermal water loss: the signal for recovery of barrier structure and function. J Lipid Res 30:323±333, 1989 Harris IR, Farrell AM, Grunfeld C, Holleran WM, Elias PM, Feingold KR: Permeability barrier disruption coordinately regulates mRNA levels for key enzymes of cholesterol, fatty acid, and ceramide synthesis in the epidermis. J Invest Dermatol 109:783±787, 1997 Hou SY, Mitra AK, White SH, Menon GK, Ghadially R, Elias PM: Membrane structures in normal and essential fatty acid-de®cient stratum corneum: characterization by ruthenium tetroxide staining and x-ray diffraction. J Invest Dermatol 96:215±223, 1991 Huber M, Rettler I, Bernasconi K, et al: Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science 267:525±528, 1995 Irvine AD, McLean WH: Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype±genotype correlation. Br J Dermatol 140:815±828, 1999 Ishida-Yamamoto A, Eady RA, Underwood RA, Dale BA, Holbrook KA: Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis). Br J Dermatol 131:767±779, 1994 Ishida-Yamamoto A, Iizuka H, Manabe M, et al: Altered distribution of keratinization markers in epidermolytic hyperkeratosis. Arch Dermatol Res 287:705±711, 1995 Jackson SM, Williams ML, Feingold KR, Elias PM: Pathobiology of the stratum corneum. West J Med 158:279±285, 1993 Jensen JM, Schutze S, Neumann C, Proksch E: Impaired cutaneous permeability barrier function, skin hydration, and sphingomyelinase activity in keratin 10 de®cient mice. J Invest Dermatol 115:708±713, 2000 Koch PJ, de Viragh PA, Scharer E, et al: Lessons from loricrin-de®cient mice. Compensatory mechanisms maintaining skin barrier function in the absence of a major corni®ed envelope protein. J Cell Biol 151:389±400, 2000 Lane EB, Rugg EL, Navsaria H, Leigh IM, Heagerty AH, Ishida-Yamamoto A, Eady RA: A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356:244±246, 1992 Lavrijsen AP, Oestmann E, Hermans J, Bodde HE, Vermeer BJ, Ponec M: Barrier function parameters in various keratinization disorders: transepidermal water loss and vascular response to hexyl nicotinate. Br J Dermatol 129:547±553, 1993 Matsuki M, Yamashita F, Ishida-Yamamoto A, et al: Defective stratum corneum and early neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte transglutaminase). Proc Natl Acad Sci USA 95:1044±1049, 1998 Menon GK, Grayson S, Elias PM: Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis. J Invest Dermatol 86:591±597, 1986 Menon GK, Feingold KR, Elias PM: Lamellar body secretory response to barrier disruption. J Invest Dermatol 98:279±289, 1992a Menon GK, Ghadially R, Williams ML, Elias PM: Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation. Br J Dermatol 126:337±345, 1992b Miller RK, Khuon S, Goldman RD: Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo. J Cell Biol 122:123±135, 1993 Ming ME, Daryanani HA, Roberts LP, Baden HP, Kvedar JC: Binding of keratin intermediate ®laments (K10) to the corni®ed envelope in mouse epidermis: implications for barrier function. J Invest Dermatol 103:780±784, 1994 Nemes Z, Steinert PM: Bricks and mortar of the epidermal barrier. Exp Mol Med 31:5±19, 1999 Paige DG, Morse-Fisher N, Harper JI: Quanti®cation of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses. 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Rassner UA, Crumrine DA, Nau P, Elias PM: Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry. Histochem J 29:387±392, 1997 Reichelt J, Bauer C, Porter R, Lane E, Magin V: Out of balance: consequences of a partial keratin 10 knockout. J Cell Sci 110:2175±2186, 1997 Reichelt J, Doering T, Schnetz E, Fartasch M, Sandhoff K, Magin AM: Normal ultrastructure, but altered stratum corneum lipid and protein composition in a mouse model for epidermolytic hyperkeratosis. J Invest Dermatol 113:329±334, 1999 Roop D: Defects in the barrier. Science 267:474±475, 1995 Rothnagel JA, Dominey AM, Dempsey LD, et al: Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis. Science 257:1128±1130, 1992 Russell LJ, DiGiovanna JJ, Rogers GR, Steinert PM, Hashem N, Compton JG, Bale SJ: Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis. Nat Genet 9:279±283, 1995 Steinert PM: The complexity and redundancy of epithelial barrier function. J Cell Biol 151:F5±F8, 2000
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Steinert PM, Marekov LN: The proteins ela®n, ®laggrin, keratin intermediate ®laments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal corni®ed cell envelope. J Biol Chem 270:17702±17711, 1995 Steinert PM, Marekov LN: Initiation of assembly of the cell envelope barrier structure of strati®ed squamous epithelia. Mol Biol Cell 10:4247±4261, 1999 Steinert PM, Marekov LN, Fraser RD, Parry DA: Keratin intermediate ®lament structure. Crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly. J Mol Biol 230:436±452, 1993 Suga Y, Jarnik M, Attar PS, et al: Transgenic mice expressing a mutant form of loricrin reveal the molecular basis of the skin diseases, vohwinkel syndrome and progressive symmetric erythrokeratoderma. J Cell Biol 151:401±412, 2000 Williams ML, Koch TK, O'Donnell JJ, Frost PH, Epstein LB, Grizzard WS, Epstein CJ: Ichthyosis and neutral lipid storage disease. Am J Med Genet 20:711±726, 1985 Zimmermann B: Secretion of lamellar bodies in type II pneumocytes in organoid culture: effects of colchicine and cytochalasin B. Exp Lung Res 15:31±47, 1989
§Dermatology Service and *Metabolism Section, Veterans Affairs Medical Center, §Departments of Dermatology, *Internal Medicine, and ¶Pediatrics, University of California, San Francisco, U.S.A.; ³National Skin Center, Singapore; **Department of Dermatology, General Hospital Salzburg, Austria; Departments of Dermatology, Universities of ²Innsbruck and ²²Vienna, Austria
defective organization of extracellular lamellar bilayers. The decreased lamellar material, in turn, could be attributed to incompletely secreted lamellar bodies within granular cells, demonstrable not only by several morphologic ®ndings, but also by decreased delivery of a lamellar body content marker, acid lipase, to the stratum corneum interstices. Yet, after acute barrier disruption a rapid release of preformed lamellar body contents was observed together with increased organelle contents in the extracellular spaces, accounting for the accelerated recovery kinetics in epidermolytic hyperkeratosis. Accelerated recovery, in turn, correlated with a restoration in calcium in outer stratum granulosum cells in epidermolytic hyperkeratosis after barrier disruption. Thus, the baseline permeability barrier abnormality in epidermolytic hyperkeratosis can be attributed to abnormal lamellar body secretion, rather than to corneocyte fragility or an abnormal corni®ed envelope/corni®ed-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair. Key words: corni®ed envelope/intermediate ®laments/keratin/stratum corneum/transepidermal water loss. J Invest Dermatol 117:837±847, 2001
Epidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin ®lament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of corni®cation, patients typically display an abnormality in permeability barrier function. The nature and pathogenesis of the barrier abnormality in epidermolytic hyperkeratosis are unknown, however. We assessed here, ®rst, baseline transepidermal water loss and barrier recovery kinetics in patients with epidermolytic hyperkeratosis. Whereas baseline transepidermal water loss rates were elevated by approximately 3-fold, recovery rates were faster in epidermolytic hyperkeratosis than in age-matched controls. Electron microscopy showed no defect in either the corni®ed envelope or the adjacent corni®ed-bound lipid envelope, i.e., a corneocyte scaffold abnormality does not explain the barrier abnormality. Using the water-soluble tracer, colloidal lanthanum, there was no evidence of tracer accumulation in corneocytes, despite the fragility of nucleated keratinocytes. Instead, tracer, which was excluded in normal skin, moved through the extracellular stratum corneum domains. Increasing intercellular permeability correlated with decreased quantities and
T
process of epidermal differentiation. Whereas expression of keratins 5 and 14 is restricted to the basal cell compartment, keratins 1 and 10 are produced in all suprabasal nucleated cell layers (Roop, 1995). In the nucleated cell layers, these keratins are organized into ®lament bundles that loop between desmosome plaques and the nuclear envelope providing a cytoskeleton that protects keratinocytes from mechanical injury (Fuchs and Cleveland, 1998). As the keratinocytes terminally differentiate, keratins 1 and 10 as well as several other corni®ed envelope (CE) proteins form a rigid, chemically and mechanically resistant structure that replaces the plasma membrane (Nemes and Steinert, 1999; Steinert and Marekov, 1999; Steinert, 2000). The CE, in turn, forms a scaffold upon which the lipid-enriched extracellular matrix is organized into lamellar bilayer structures that mediate the permeability barrier function of the outermost layer of the epidermis, the stratum corneum (SC) (Jackson et al, 1993; Elias et al, 1998).
he epidermis is a homeostatic, self-renewing tissue that expresses differentiation-speci®c genes sequentially as keratinocytes move outward from the basal to the granular cell layer (SG). Keratins are among the most abundant proteins produced during this vectorial
Manuscript received January 31, 2001; revised May 25, 2001; accepted for publication June 1, 2001. Reprint requests to: Dr. Matthias Schmuth, V.A. Medical Center, Metabolism Section (111F), 4150 Clement St., San Francisco, CA 94121. Email: [email protected] Abbreviations: CE, corni®ed envelope, CLE, corneocyte-bound lipid envelope; LB, lamellar body; SC, stratum corneum; SG, stratum granulosum; TEWL, transepidermal water loss. 1 We dedicate this work to Professor Peter O. Fritsch in honor of his 60th birthday. 0022-202X/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 837
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The dual-compartment, corneocyte±extracellular lamellar lipidenriched matrix of the SC serves as a protective barrier not only against external insults, but also against excess transepidermal water loss (TEWL) (Grubauer et al, 1989). Any perturbation of the permeability barrier initiates a rapid and ef®cient repair response in the underlying epidermis. Regardless of the speci®c nature of the barrier insult, i.e., mechanical (e.g., tape stripping) or chemical (e.g., solvent or detergent treatment), a metabolic response ensues in the underlying epidermis that leads to restoration of normal function (Elias, 1996). The initial response comprises an immediate exocytosis of the preformed pool of lamellar bodies from the outermost SG cell layer (Menon et al, 1992a; Elias et al, 1998). Cholesterol, fatty acid, and ceramide synthesis then increase over the next 6±9 h (Harris et al, 1997), leading to accelerated formation and further secretion of nascent lamellar bodies (Menon et al, 1992a). This response enables the re-accumulation of lipids in the SC extracellular spaces, reformation of membrane bilayers, and restoration of baseline barrier function (Grubauer et al, 1987). Whereas the importance of the lipid matrix for the maintenance of cutaneous barrier function is undisputed, the contribution of the corneocyte to the permeability barrier is less clear. A potential role for the CE scaffold in the organization of the extracellular lamellar bilayer system is suggested by lamellar ichthyosis, where transglutaminase I mutations (Huber et al, 1995; Russell et al, 1995) result not only in a defective CE formation but also abnormal permeability barrier function (Lavrijsen et al, 1993; Elias et al, 2001). The enhanced TEWL in lamellar ichthyosis has been attributed to truncation and fragmentation of extracellular lamellar arrays, which correlate with the CE defect (Elias et al, 2001), supporting a scaffold role for the CE in bilayer formation. Mutations in keratin 1 and keratin 10 have been identi®ed as the cause of the autosomal dominant form of ichthyosis, epidermolytic hyperkeratosis (EHK, also termed bullous congenital ichthyosiform erythroderma (BCIE), OMIM113800) (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992). The keratin 1/10 mutations function in a dominant-negative manner to disrupt the keratin ®lament network within the keratinocyte cytosol. Filaments retract from their attachments to desmosomal plaques and form clumps of
perinuclear shells (Anton-Lamprecht, 1983). Skin fragility to mechanical trauma, which is most pronounced in the neonatal period, is an expected consequence of this cytoskeletal defect, analogous to the fragile skin phenotype of epidermolysis bullosa, where mutations in keratins 5 or 14 produce cytoskeletal defects in the epidermal basal layer (Bonifas et al, 1991; Coulombe et al, 1991; Lane et al, 1992). The hyperkeratosis and accompanying defect in permeability barrier function are additional clinical features of EHK (Frost et al, 1968), the pathogenesis of which is less clear. Speci®cally, whether the barrier defect is due to a fragile keratinocyte, i.e., a defective corneocyte/CE scaffold, or other abnormalities is not known. To dissect the pathogenesis of the barrier abnormality in EHK, we assessed three alternative mechanisms: (i) a fragile corneocyte leading to increased transcellular permeability; (ii) an ineffective CE scaffold; (iii) aberrant lamellar body (LB) secretion. Lanthanum tracer studies revealed an intercellular rather than a transcellular route of increased water loss in EHK, ruling out a fragile corneocyte as the basis for the abnormality. Increased intercellular water movement correlated, in turn, with a decrease in the quantities and abnormal organization of the extracellular lamellar bilayer system. Finally, the decreased lamellar bilayers were associated with an accumulation of unsecreted LB in the outer SG under baseline conditions. With acute barrier disruption, however, a loss of the epidermal calcium gradient occurred that initiated a rapid release of preformed LB and appearance of organelle contents in the extracellular domains, resulting in accelerated rates of barrier recovery. MATERIALS AND METHODS Patients We investigated four patients (one female, three male, aged 12±45 y) with clinically typical EHK (Table I). The diagnosis was con®rmed by the presence of prominent hyperkeratosis, coarse keratohyalin granules, and vacuolization of the upper stratum spinosum and SG on histopathology. In some sections we additionally found acantholysis of the stratum spinosum and SG. This skin phenotype had been present in all patients since birth. Further work-up did not demonstrate any clinical or laboratory abnormalities. Mutation analysis revealed novel mutations in keratin 1 or 10 in two patients (Table I; in
Table I. Characteristics of EHK patients Patient #
Gender
Age
Clinical phenotype
Mutation
Biopsy sites
Scale
1 2 3 4
M F M M
29 12 44 17
Generalized, Generalized, Generalized, Generalized,
K10a (coil domain) ±ab ±abc K1d (tail domain)
Lower arm Abdomen Lower arm Lower leg
+ + + ±
¯exural accentuation annular ¯exural accentuation annular
a
Department of Biochemistry, University of Salzburg. Hot spot analysis of K1/10; no mutations were detected. Reduced epidermal immunolabeling of K10, Department of Dermatology, General Hospital Salzburg. d Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia. b c
Table II. Material available for pathogenesis studies Patient # 1 2 3 4 a
Material at baseline
Material after acute disruption
Assessment of LB secretiona
Acid lipase secretiona
Calcium localizationa
Lanthanum perfusiona
OsO4 post®xation
RuO4 post®xation
Pyridine treatmentb
Biopsy Scale Biopsy Scale Biopsy Scale Biopsy Scale
6h 72 h 6h ± 3h ± ± ±
+ ± + ± + ± + ±
+ ± + ± + ± + ±
+ ± ± ± + ± ± ±
+ ± + ± + ± + ±
+ ± + ± + ± + ±
+ + + + + + + +
± + ± + ± ± ± ±
Analysis possible in biopsies, but not in scale samples. Baseline only.
b
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Table III. Assessment of lamellar body secretion in EHK versus controls Type of samples (case #) EHK 1 2 3 4 Normal controls (n = 3) Other ichthyoses CIE (n = 4)a LI (n = 4)b NLSDI (n = 2)c
Lamellar bilayers
Contents of SG±SC interface
Entombed LB
Interstices-width/ corneocyte ratio
Lipase localization in SC
Decreased Decreased Decreased Decreased Replete
Decreased Decreased Decreased Decreased Replete
Present Present Present Present None
Increased Increased Increased Increased Normal
Decreased Intercellular Decreased Intercellular Decreased Intercellular Decreased Intercellular All Intercellular
Decreased Replete (dimensions
Decreased Replete abnormal) Decreased
Present None
Increased Normal
Decreased All Intercellular
Some present
Normal
Mostly Intercellular
Clefts
a
Congenital ichthyosiform erythroderma (Menon et al, 1992b). Lamellar ichthyosis (Elias et al, 2001). c Neutral lipid storage disease (Williams et al, 1985). b
Figure 1. Pathogenesis ¯ow chart for the analysis of the barrier abnormality in disorders of keratinization. First, functional assessments are used to determine TEWL levels in ichthyotic skin. If TEWL abnormalities are present, lanthanum perfusion is employed to distinguish transcellular from intercellular permeability pathways. In the case of an intercellular abnormality, a battery of ultrastructural methods is then applied to detail the morphologic basis for the intercellular defect. aElias et al, 1981; b Elias et al, 2001; cElias et al, 2000b; dpresent study.
patient 1 a mutation was detected in the coil domain 1 A of keratin 10 whereas in patient 4 the mutation was located in the tail of keratin 1). An additional patient displayed reduced immunostaining for keratin 10, but neither keratin 1 or keratin 10 mutations were detectable in this or the other remaining patient on hot spot analyis. Others have noted that a substantial proportion of patients with the EHK phenotype lack keratin 1 or 10 mutations (Porter et al, 1996; Fuchs and Cleveland, 1998). Skin from EHK patients and control subjects (three healthy male volunteers, aged 31±32 y) comprised our group of controls (Table III). None of the EHK or control subjects had employed any external medications or emollients to the studied skin areas for at least 2 wk prior to assessment of barrier function. Additional controls included historical material from normal human skin, and biopsies from a variety of individuals with other disorders of corni®cation (Table III). Functional studies To assess epidermal permeability function we measured TEWL on a 1.13 cm2 area of the volar aspect of the forearm in three EHK subjects using an evaporimeter (ServoMed, Stockholm, Sweden). TEWL values were registered in g per m2 per h after equilibration of the probe on the skin (> 60 s). All recordings were made by the same investigator (M.S.). Volunteers underwent a 15 min premeasurement rest period. Environment-related variables at the time of the study were: ambient air temperature 21.9°C±26.6°C; skin surface temperature 30.4°C±34.4°C, ambient air humidity 23%±49%; atmospheric H2O pressure 4.4±14.8 mmHg. Excess air convection was prevented by shielding the measurement zone. TEWL was measured
both under baseline conditions and following acute barrier disruption by either repeated cellophane tape stripping or acetone swabbing of the lower forearm. Barrier recovery kinetics then were assessed by measuring TEWL at 3, 6±7, 16, 24, 48, 96, 120, and 144 h after acute disruption. For calculation of the percentage change in TEWL, the following formula was used: 1 ± [(TEWL immediately after stripping ± TEWL at indicated time)/(TEWL immediately after stripping ± baseline TEWL)] 3 100%. Pathophysiology studies To assess the pathophysiologic basis for the barrier abnormality in EHK, we utilized the sequence shown in Fig 1, recently applied to Harlequin ichthyosis (Elias et al, 2000b) and lamellar ichthyosis (Elias et al, 2001). If barrier function was abnormal in an EHK individual, we next assessed whether the abnormality was due to leaky corneocytes resulting in transcellular water movement (as a putative downstream consequence of the keratinocyte fragility that is a wellknown feature of EHK), or to enhanced intercellular permeability. As the barrier abnormality was linked, instead, to enhanced intercellular permeability, we next assessed extracellular lamellar bilayer quantity and organization. If abnormal, we then sought whether the structural defect in the lamellar bilayers was due to an abnormal CE±corneocyte scaffold or to ineffective LB secretion. Biopsies from EHK skin were taken immediately before and 3 and 6 h after acute barrier disruption. Assessment of permeability pathway by lanthanum perfusion The perfusion pathway was assessed in all subjects by immersion of biopsies and/or scale in 4% lanthanum nitrate in 0.05 M Tris buffer containing 2%
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glutaraldehyde, 1% paraformaldehyde, pH 7.4, for 1 h at room temperature (Table II) (Elias et al, 1981). After lanthanum perfusion, the samples were washed and processed for electron microscopy, as described below. Assessment of lamellar bilayer system and CE-associated structures Using a combination of osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) post®xation with pyridine pretreatment, it is possible to assess the integrity of the intercellular lamellar bilayer system in relation to the CE/corneocyte-bound lipid envelope (CLE) scaffold (Elias, 1996; Elias et al, 2000b). Again, scale and biopsy samples were available for these studies (Table II). Brie¯y, all samples were pre®xed in half-strength Karnovsky's ®xative, followed by post®xation in 1% OsO4 and 0.2% RuO4, as described previously (Hou et al, 1991). Some samples were then treated for 2 h with absolute pyridine to further enhance the visualization of the CE/CLE membrane complex, as described recently (Elias et al, 2000b; 2001). After post®xation, all samples were dehydrated in a graded ethanol/propylene oxide series, and embedded in an Eponepoxy mixture. Ultrathin sections were collected and assessed either unstained or after further lead citrate contrasting in a Zeiss 10 A electron microscope operated at 60 kV. Assessment of LB secretion Several morphologic criteria have been used to assess the integrity of the LB secretory process, including (i) the amount of secreted material at the SG±SC interface (Menon et al, 1992a); (ii) the extent that the SC interstices are replete with lamellar bilayers (Menon et al, 1992b); and (iii) the presence of ``entombed'' LB
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within the corneocyte cytosol (Elias et al, 1998). We assessed all of these criteria (Table III) and, in addition, utilized the delivery of a lipid hydrolase (acid lipase), which is concentrated in LB (Menon et al, 1986), as cytochemical indicator of the extent of secretion (Menon et al, 1992b). Aldehyde-pre®xed samples were microwave-incubated with substrate containing 0.2% Tween 85 (6 inhibitor tetrahydrolipstatin, 200 mM) in a lead-capture, cytochemical method that depicts the localization of acid lipase, as described previously (Rassner et al, 1997). Prior studies on nomal controls showed abundant enzyme activity in the SC interstices, with little activity in the corneocyte cytosol (Menon et al, 1992b; Elias et al, 2001) (see Fig 8(D) also). Conversely, in some disorders of corni®cation, acid lipase is not delivered normally, and enzyme activity is retained in the corneocyte cytosol (Ghadially et al, 1992; Menon et al, 1992b) (Table III). The samples for ultrastructural cytochemistry were post®xed in OsO4 and processed as above. Ion capture cytochemistry For ultrastructural calcium localization, frozen biopsy samples (before and after acute barrier disruption, see above) were ®xed in 2% paraformaldehyde, 2% glutaraldehyde, 0.09 M potassium oxalate, containing 0.04 M sucrose. Samples were subsequently ®xed overnight at 4°C, and then post®xed in 1% OsO4 containing 2% potassium pyroantimonate, pH 7.4, for 2 h at 4°C in the dark. Tissue samples then were washed in alkalinized water (pH 10) and transferred to ethanol solutions for dehydration and embedding, as above. Statistical analyses Statistical analyses were performed using a paired or unpaired Student's t test, as appropriate. Data are expressed as mean 6 SEM.
RESULTS
Figure 2. Barrier recovery after tape stripping is accelerated in EHK epidermis. The volar forearm of EHK and normal subjects was tape-stripped, and TEWL was measured with time following barrier disruption (time 0). Accelerated barrier recovery, although evident at all early time points after disruption (i.e., 0±48 h), was signi®cant only at 48 h, because of the small number of patients (*p < 0.05 vs normal control values). At later time points, barrier recovery of EHK individuals paralleled the recovery of normal skin, and at 2±5 d, barrier recovery slowed, resulting in the higher-than-normal baseline TEWL levels characteristic of EHK.
EHK patients display abnormal baseline barrier function, but accelerated barrier recovery kinetics We ®rst assessed barrier function in the three EHK patients who where available for functional studies. As previously shown for EHK (Frost et al, 1968), baseline TEWL rates were markedly increased, with a mean of 19.7 6 1.9 g per m2 per h in patients versus 7.43 6 1.2 g per m2 per h in the three normal controls (p < 0.006, not shown). Yet, the kinetics of barrier recovery after comparable, initial insults (i.e., tape stripping to a maximal TEWL of 100 g per m2 per h) showed an accelerated repair response for the EHK patients versus the three control subjects (Fig 2). In addition, we incidently noted a remarkably fast healing of the skin biopsy sites in EHK patients. The early, more rapid recovery phase was followed by a later normalization of barrier recovery rates by 96 h (not shown). By 1 wk, TEWL levels had returned to approximately 80% of pretreatment values in both EHK and controls. Thereafter, controls and EHK patients returned to baseline levels in parallel. These results show that EHK patients, despite a prominent baseline barrier abnormality, exhibit accelerated barrier recovery rates after acute insults. The enhanced water movement in EHK occurs via intercellular domains We next assessed whether the baseline barrier abnormality in EHK could be assigned to enhanced intercellular or transcellular movement of water, utilizing divalent colloidal lanthanum as a tracer to localize the sites of enhanced water movement. Prior studies have shown that lanthanum is
Figure 3. Increased water movement occurs via intercellular pathways. Freshly obtained biopsies from EHK were exposed for 1 h to 4% colloidal lanthanum nitrate tracer in 0.05 M Tris buffer, pH 7.4, containing 2% glutaraldehyde and 2% paraformaldehyde, followed by post®xation in OsO4. (A), (B) Tracer is observed throughout the SC interstices (arrows), but was excluded from the corneocyte cytosol. OsO4 post®xation. Scale bars: 0.5 mm.
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Figure 4. Extracellular lamellar bilayer structures are abnormal in EHK. (A±C) Clefts and lacunae are present througout the SC interstices (asterisks). (C) Overall, the amount of bilayer material is reduced. (A, B, D,) Stretches of intact, normal-appearing bilayers are only focally present (arrows). RuO4 post®xation. Scale bars: 0.5 mm.
Figure 5. CE and CLE appear normal in EHK. (A) CE (arrows) in EHK appear largely normal, although there is some variation in thickness. (B) CLE (arrowheads) also appear completely normal in EHK. (A) OsO4 post®xation; (B) OsO4 post®xation after pyridine pretreatment (see Methods). Scale bars: 0.25 mm.
excluded from normal SC (Elias et al, 1977), and we con®rmed this ®nding here in the normal control samples (not shown). In contrast, lanthanum penetrated throughout the SC in EHK (Fig 3A). Moreover, in all cases and at all levels of the SC, the tracer was limited to the SC interstices (Fig 3A, B); i.e., little or no tracer was found within the cytosol of individual corneocytes. These results show that, despite evidence of keratinocyte fragility within the nucleated layers, the permeability barrier abnormality in EHK can be ascribed to enhanced movement of water, exclusively via SC extracellular domains. Although extracellular lamellar membranes are reduced and abnormal in EHK, the CE/CLE scaffold is normal We next
explored the basis for increased extracellular water movement in EHK, assessing ®rst the lamellar bilayer system. The lamellar bilayer system was markedly abnormal in all EHK samples, as assessed in RuO4 post®xed samples (Fig 4A±C). With the exception of a few foci (Fig 4A, B, insert, arrows), the amount of bilayer material generally was reduced (Fig 4C, D). Wherever bilayers were present, they appeared disrupted/interrupted by numerous clefts and lacunae that were ®lled with amorphous material (Fig 4A±C, asterisks). Both the reduction in numbers of lamellae and the interruptions of the bilayers by clefts could account for enhanced water movement through the SC interstices. Yet, the precise intercellular pathway of increased water movement could not be ascertained because lanthanum cannot be identi®ed with certainty
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Figure 6. Arrays of LB beneath plasma membrane indicate a secretory defect in EHK. Although the overall number of LB is normal in EHK, arrays of individual organelles remain restricted beneath the apical plasma membrane of outermost granular (SG) cells (A, B, arrows). (C) Lack of LB secretion results in a paucity of extracellular deposits at the SG±SC interface (asterisks). RuO4 post®xation. Scale bars: 0.5 mm.
Figure 7. LB content is entombed within corneocyte cytosol. (A, B) Entombed LB are found within the cytosol of corneocytes (A, B, short open arrows; C, solid arrow). Note again the presence of lacunae (asterisks) and areas of reduced and abnormal bilayer structures (B, C). (C) The encased LB contents focally form bilayer structures within the cytosol. RuO4 post®xation. Scale bars: 0.25 mm.
in RuO4 post®xed samples. We next assessed whether the abnormalities in lamellar membrane structure are associated with abnormalities in the CE/CLE scaffold, as occurs in lamellar
ichthyosis (Elias et al, 2001). Yet, despite the known association of keratins 1/10 with the CE (Steinert and Marekov, 1995), we noted no major abnormalities in CE structure or dimensions (Fig 5A),
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Figure 8. Further evidence of impaired secretion in EHK: acid lipase is retained within corneocytes. (A, B) The LB hydrolase, acid lipase, is largely retained within the cytosol of corneocytes in EHK (arrowheads). (C) Retained activity is colocalized with entombed LB. (D) Normal human skin demonstrates hydrolase solely in the SC interstices, indicative of effective secretion. OsO4 post®xation. Scale bars: 0.5 mm.
Figure 9. Basis for accelerated barrier recovery in EHK epidermis. (A, B) (6 h posttape stripping): No LB remain in cytosol of granular cells (SG), indicated by release of lipase activity into the intercellular spaces (B, arrows). Extent of release of contents also is shown at level of SC: no entombed organelles or cytosolic lipase activity remain in corneocyte cytosol. (C) (3 d post-tape stripping): Reappearance of additional foci of replete lamellar bilayers at time point when barrier recovery is accelerated (cf. Fig 2). (D) (6 h post-tape stripping): Lanthanum perfusion demonstrates substantial blockade of tracer movement at SG±SC interface, although foci of tracer (arrow) continue to penetrate the SC interstices. (A, B, D) OsO4 post®xation; (C) RuO4 post®xation. Scale bars: (A, B, D) 0.5 mm; (C) 0.25 mm.
and an intact CLE on the outer surface of corneocytes in all EHK samples (Fig 5B). We did not quantitate the extent of CLE biochemically, because prior studies have demonstrated normal quantities of covalently bound ceramide in EHK scale (Paige et al, 1994). These results show ®rst that an abnormality in the SC extracellular lamellar bilayer system is present in the SC of EHK under baseline conditions, which accounts for the barrier abnormality in EHK. They also show that the membrane abnormality cannot be ascribed to morphologically detectable abnormalities in the CE/CLE scaffold. LB secretion is impaired in EHK under baseline conditions We next assessed an alternative explanation for the depletion of extracellular lamellar bilayers in EHK, i.e., abnormal
production or secretion of LB. In all baseline EHK biopsies, we observed normal numbers of LB in the upper spinous and granular layers, as well as normal lamellar contents within individual organelles. Thus, LB production appears to be unimpaired in EHK. In contrast, LB secretion from outer SG cells did not occur normally. A distinctive feature was the appearance of arrays of individual organelles, localized within the peripheral cytosol, just beneath the plasma membrane (Fig 6A, B). This feature appeared primarily in cells of the outer SG layer, which also displayed retracted tono®lament bundles (not shown). Further evidence of impaired secretion in EHK included a striking paucity of secreted lamellar contents at the SG±SC interface (Fig 6A, C), and evidence of entombed LB in the form of organelle contents, encased within the cytosol of corneocytes (Fig 7A±C).
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Figure 10. Baseline Ca2+ distribution and loss of Ca2+ gradient is similar to normals in EHK. (A, B) A comparable Ca2+ gradient, with the highest Ca2+ levels in the outer SG with lower levels in subjacent layers and SC, is evident in both normal (A) and EHK (B) skin at baseline. (C, D) Loss of Ca12+ gradient after tape stripping (C) or acetone treatment (D) is accompanied by secretion of LB (SC, stratum corneum, OSG, outer stratum granulosum). OsO4 post®xation. Scale bars: 1 mm.
To further ascertain whether secretion is impaired in EHK, we next assessed the distribution of an enzyme cytochemical marker of LB contents, i.e., acid lipase (Menon et al, 1986), in EHK versus control samples. As previously reported (Menon et al, 1992b), LBderived hydrolase contents were delivered in toto to the SC interstices in normal SC (Fig 8D), leaving no residual, detectable activity within the corneocyte cytosol (Table III). In contrast, whereas EHK subjects displayed comparable labeling of cytosolic LB (Fig 8B), activity at the SG±SC interface and in the SC interstices appeared strikingly reduced (Fig 8C), further regulated by the presence of abundant, retained deposition product in the cytosol of individual corneocytes (Fig 8A, C). Moreover, in some corneocytes retained enzyme activity colocalized with residual, entombed LB contents (Fig 8C). Together, these results show that, whereas LB production is normal in EHK, impaired LB secretion accounts for the diminished extracellular lamellar bilayers that lead to the permeability barrier abnormality in EHK. Inverse calcium signaling of LB secretion accounts for accelerated barrier recovery in EHK Whereas baseline barrier function is abnormal, the kinetics of barrier recovery are faster in EHK than in normal skin (Fig 2). To assess the basis for accelerated barrier recovery in EHK, we next examined lanthanum perfusion, LB secretion, and SC membrane structures 3, 6, and 72 h after acute disruption by either tape stripping or acetone treatment. At
both 3 and 6 h, more extensive cytolysis was evident throughout the outer nucleated cell layers than in nonperturbed EHK epidermis (not shown). Yet, in contrast to baseline EHK epidermis, few LB remained in these disrupted cells; instead, organelle contents appeared in the intercellular spaces throughout the outer epidermis (Fig 9, cf. Fig 6). Further evidence for release of organelle contents also could be seen in the SC. Few corneocytes displayed entombed LB contents (Fig 9, cf. Fig 7), and lipase activity was found between, rather than within, individual corneocytes (Fig 9, cf. Fig 8). These ®ndings correlated with the presence of more widespread foci of normal-appearing membrane bilayers (Fig 9; however, the absolute quantities were still lower than in normals). Finally, after acute perturbations, resulting in LB release, lanthanum was largely (but incompletely) excluded from the SC interstices (Fig 9D). Finally, to assess whether the combination of baseline secretory defect and accelerated recovery is due to modulations in the epidermal calcium gradient, we employed ion capture cytochemistry. At baseline, we observed an accumulation of extracellular and intracellular Ca2+ in the outer SG cells with low visible levels in the SC, comparable to normal skin (Fig 10A, B). Loss of the epidermal calcium gradient with acute barrier disruption either by tape stripping or by acetone treatment occurred normally in EHK skin (Fig 10C, D). Loss of the calcium gradient was accompanied by
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secretion of preformed LB from outer SG cells in EHK (Fig 10C, D vs Fig 6; note absence of preformed LB in cytosol). These results show that the accelerated barrier recovery of EHK skin after acute perturbations can be explained by the release of preformed LB contents from cells in the outer nucleated layers of the epidermis, regulated in turn by modulations in the epidermal calcium gradient. DISCUSSION Keratin intermediate ®laments are the most prominent cytoskeletal proteins of the epidermis and its appendages, accounting for up to 85% of total cellular protein (Fuchs and Coulombe, 1992; Roop, 1995). They form copolymers consisting of equimolar amounts of at least one of two types: type I or acidic keratins (9±20) and type II or basic keratins (1±8) (Miller et al, 1993; Steinert et al, 1993). All keratins display a secondary structure with a central rod domain comprising four alpha-helices and distinctive, nonhelical, head and tail sequences. Mutations have been identi®ed in the highly conserved ends of the rod domains of keratins 1 and 10 in several forms of EHK (Cheng et al, 1992; Chipev et al, 1992; Rothnagel et al, 1992), but a signi®cant proportion of affected individuals do not have keratin 1/10 mutations (Roop, 1995; Porter et al, 1996; Fuchs and Cleveland, 1998). EHK either occurs sporadically (> 50%) or is inherited in an autosomal dominant fashion. Affected individuals are born with widespread erythema and erosions. With increased age these features diminish and are replaced by generalized epidermal thickening, indicative of epidermal hyperplasia (Irvine and McLean, 1999). The resulting hyperkeratotic plaques are prone to superinfection. The diagnosis in our patient cohort was based on the characteristic clinical picture, diagnostic histology, and the presence of a keratin 1 or keratin 10 mutation in two of the four patients (Table I). Increased expression of other CE proteins (Ishida-Yamamoto et al, 1994, 1995; Porter et al, 1996; Gerritsen et al, 1997; Reichelt et al, 1999) could compensate for the structural abnormality and further provoke the characteristic phenotype in EHK. Our results con®rm previous ®ndings of increased baseline TEWL levels in EHK patients with or without de®ned mutations (Frost et al, 1968; Lavrijsen et al, 1993). We show further that the initial rates of barrier repair after acute disruption actually occurred more rapidly in EHK subjects than in controls. The basis for the baseline permeability barrier abnormality in EHK is unknown. An earlier hypothesis held that keratinocyte fragility could lead to increased cytokine release, which in turn could drive the epidermal hyperplasia, leading to a barrier abnormality (Roop, 1995). Accordingly, we ®rst assessed whether the keratinocyte fragility in EHK leads to a more fragile, ``leaky'' corneocyte, allowing increased transcellular permeability. Indeed, an abnormality in corneocyte fragility accounts for the barrier abnormality in retinoid-treated skin, where abundant colloidal lanthanum tracer leaks into corneocytes (Elias et al, 1981). Yet, our lanthanum perfusion studies clearly showed that, despite the known fragility of keratinocytes within EHK epidermis, the increased SC permeability in EHK occurs via an intercellular rather than a transcellular pathway. Thus, a mechanism other than an incompetent corneocyte must be sought to explain the barrier defect. Instead, we found both a paucity of lamellar bilayers and distorted supramolecular architecture of those lamellar bilayers that were present in all EHK subjects. These structural changes account for the permeability barrier abnormality and the increased intercellular penetration of tracer in EHK. One mechanism whereby defective keratin 1/10 intermediate ®laments could result in abnormal extracellular lamellar bilayers could be an abnormal CE/CLE scaffold. As keratin 1 and 10 are crosslinked to the CE (Ming et al, 1994; Candi et al, 1998), they could play a hitherto unrecognized role in maintenance of the SC barrier. Accordingly, defects in the CE/CLE scaffold, even in EHK individuals with no keratin mutations, could explain the defective organization of extracellular lamellar bilayers in EHK. Indeed, an analogous defect in the CE/CLE scaffold appears to be responsible for the barrier abnormality in another keratinizing disorder with a
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defect in CE formation, i.e., lamellar ichthyosis due to transglutaminase I mutations (Lavrijsen et al, 1993; Elias et al, 2001), and in transgenic mice with a deletion of this enzyme (Matsuki et al, 1998). Barrier function also reportedly is abnormal in involucrin (another CE-constituent peptide) knockout mice,2 which otherwise display a normal phenotype (Djian et al, 2000). Finally, there is evidence for a barrier abnormality in transgenic mice expressing a mutant form of loricrin (Koch et al, 2000; Suga et al, 2000), the dominant CE peptide in epidermis (Steinert, 2000). Our ultrastructural examination did not reveal any defect of the CE/ CLE scaffold in EHK, however. As the CE/CLE scaffold cannot be invoked to explain the barrier defect, we next examined whether the reduction in lamellar bilayers in EHK extracellular domains could be attributed to decreased production (assembly, translocation) or defective secretion (exocytosis) of LB. Under baseline conditions, we observed an increased number of LB at the SG±SC interface displaying normal organelle contents. Moreover, the secretion of LB from the periphery of SG cells was impaired in EHK subjects. Thus, both assembly and translocation of LB appear to be normal, but exocytosis does not occur normally in EHK under baseline conditions. This conclusion is further supported by two additional observations, indicating impaired LB secretion in EHK: (i) the presence of abundant entombed LB contents, encased within the cytosol of corneocytes; (ii) the localization of LB-derived hydrolytic enzymes, which normally are completely secreted, within corneocytes and often colocalized with entombed LB contents. As a result of the secretory abnormality, decreased LB contents are deposited at the SG±SC interface, resulting in discontinuous lamellar bilayers, leading in turn to increased TEWL levels (Fig 4). The baseline barrier abnormality in EHK is associated with abnormal LB secretion, possibly due to an impairment of the exocytosis step. One possible explanation is that the keratin 1/10 cytoskeletal mutation could interfere with the exocytosis of LB. Prior work on lung epithelium and in adrenal secretory cells has shown that the microtubular system is crucial for exocytosis (Aunis and Bader, 1988; Zimmermann, 1989). Thus, cytoskeletal instabilities, including those caused by keratin 1/10 dominant-negative mutations in EHK, could disrupt steps that are involved in the exocytosis of LB. A precise link between the keratin 1/10 mutations and the barrier abnormality could not be established here, however, not only because only half of our patients had these mutations, but also because of limitations of experiments in humans in vivo. Animal models with de®ned defects in keratins 1/10 should allow a further dissection of the molecular pathogenesis of the LB secretory abnormality, however. Several putative mouse models of the disease have been described (Fuchs et al, 1992; Bickenbach et al, 1996; Porter et al, 1996; Reichelt et al, 1997), but available data on the permeability barrier abnormalities in these animal models do not always re¯ect the structural and functional abnormalities of EHK, presumably because the murine genotypes do not always re¯ect EHK (Reichelt et al, 1999; Elias et al, 2000a; Jensen et al, 2000). After acute barrier disruption the peripheral arrays of LB in granular cells were rapidly released (Fig 9), accounting for the accelerated recovery of permeability barrier function in EHK skin. Moreover, we found an increase in intact lamellar bilayers and exclusion of lanthanum tracer from the extracellular domains after acute barrier disruption. The basis for the release of LB after acute disruption probably does not simply re¯ect cytolysis of fragile cells with release of trapped LB. Among likely explanations could be the presence of an intact calcium gradient in EHK under baseline conditions, which, together with the mutant protein, restricts secretion. The effect of the mutant protein can be temporarily 2 Jensen JM, Djian P, Proksch E: Disturbed permeability barrier function in transgenic involucrin de®cient mice. J Invest Dermatol 112: 542, 1999 (abstr.)
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overcome by the complete calcium depletion that occurs after barrier disruption (Fig 10). Alternatively, the expression of the mutant protein could be downregulated after acute disruption, thereby allowing unrestricted LB secretion until the calcium gradient is re-established. To date, however, there is evidence that keratins 1/10 are actually upregulated after barrier disruption (Ekanayake-Mudiyanselage et al, 1998). Lastly, the pathway for LB secretion could differ under baseline versus stimulated conditions. In any case, the localization of arrays of unsecreted LB at the SG±SC interface under baseline conditions seems to re¯ect a ``geared-up'' activation state, which after an external assault is quickly transformed into a normal or supernormal repair response. A similar acceleration of the early phase of barrier repair kinetics has been reported for atopic dermatitis (Gfesser et al, 1997), but no information is available as yet for the morphologic basis of accelerated repair in atopic dermatitis. In summary, the permeability barrier abnormality in EHK can be attributed to increased intercellular water movement, despite the known fragility of keratinocytes. The barrier abnormality correlates with a reduction in extracellular lamellar membranes, which can be attributed further to impaired LB secretion. The baseline abnormality can be overcome by application of external stimuli for barrier repair. Our gratitude to the patients who have shared their time and energy by participating in this study, and to the participating nurses for their assistance. We are indebted to Dr. Sung Ku Ahn for help with ion capture cytochemistry and to Stefanie Kind for technical assistance. This work was supported by NIH grants AR 39908 (PPProjects 1 and 4) and AR 19098, and the Medical Research Service, Department of Veterans Affairs. M.S. was ®nancially supported by a grant from the Austrian Science Fund (Project J1901-MED).
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