Pathogenesis of the Permeability Barrier Abnormality inEpidermolytic 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,* andPeter 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

Epidermolytic hyperkeratosis is a dominantly in-herited ichthyosis, frequently associated with muta-tions in keratin 1 or 10 that result in disruption ofthe keratin ®lament cytoskeleton leading to keratino-cyte fragility. In addition to blistering and a severedisorder of corni®cation, patients typically display anabnormality in permeability barrier function. Thenature and pathogenesis of the barrier abnormalityin epidermolytic hyperkeratosis are unknown, how-ever. We assessed here, ®rst, baseline transepidermalwater loss and barrier recovery kinetics in patientswith epidermolytic hyperkeratosis. Whereas baselinetransepidermal water loss rates were elevated byapproximately 3-fold, recovery rates were faster inepidermolytic hyperkeratosis than in age-matchedcontrols. Electron microscopy showed no defect ineither the corni®ed envelope or the adjacent corni-®ed-bound lipid envelope, i.e., a corneocyte scaffoldabnormality does not explain the barrier abnormal-ity. Using the water-soluble tracer, colloidal lantha-num, there was no evidence of tracer accumulationin corneocytes, despite the fragility of nucleatedkeratinocytes. Instead, tracer, which was excluded innormal skin, moved through the extracellularstratum corneum domains. Increasing intercellularpermeability correlated with decreased quantities and

defective organization of extracellular lamellarbilayers. The decreased lamellar material, in turn,could be attributed to incompletely secreted lamellarbodies within granular cells, demonstrable not onlyby several morphologic ®ndings, but also bydecreased delivery of a lamellar body contentmarker, acid lipase, to the stratum corneum inter-stices. Yet, after acute barrier disruption a rapidrelease of preformed lamellar body contents wasobserved together with increased organelle contentsin the extracellular spaces, accounting for the accel-erated recovery kinetics in epidermolytic hyperkera-tosis. Accelerated recovery, in turn, correlated with arestoration in calcium in outer stratum granulosumcells in epidermolytic hyperkeratosis after barrierdisruption. Thus, the baseline permeability barrierabnormality in epidermolytic hyperkeratosis can beattributed to abnormal lamellar body secretion,rather than to corneocyte fragility or an abnormalcorni®ed envelope/corni®ed-bound lipid envelopescaffold, a defect that can be overcome by externalapplications of stimuli for barrier repair. Key words:corni®ed envelope/intermediate ®laments/keratin/stratumcorneum/transepidermal water loss. J Invest Dermatol117:837±847, 2001

The epidermis is a homeostatic, self-renewing tissue thatexpresses differentiation-speci®c genes sequentially askeratinocytes move outward from the basal to thegranular cell layer (SG). Keratins are among the mostabundant proteins produced during this vectorial

process of epidermal differentiation. Whereas expression of keratins5 and 14 is restricted to the basal cell compartment, keratins 1 and10 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 thenuclear envelope providing a cytoskeleton that protects keratino-cytes from mechanical injury (Fuchs and Cleveland, 1998). As thekeratinocytes terminally differentiate, keratins 1 and 10 as well asseveral other corni®ed envelope (CE) proteins form a rigid,chemically and mechanically resistant structure that replaces theplasma membrane (Nemes and Steinert, 1999; Steinert andMarekov, 1999; Steinert, 2000). The CE, in turn, forms a scaffoldupon which the lipid-enriched extracellular matrix is organizedinto lamellar bilayer structures that mediate the permeability barrierfunction of the outermost layer of the epidermis, the stratumcorneum (SC) (Jackson et al, 1993; Elias et al, 1998).

Manuscript received January 31, 2001; revised May 25, 2001; acceptedfor 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: matthias.schmuth@uibk.ac.at

Abbreviations: CE, corni®ed envelope, CLE, corneocyte-bound lipidenvelope; LB, lamellar body; SC, stratum corneum; SG, stratumgranulosum; TEWL, transepidermal water loss.

1We dedicate this work to Professor Peter O. Fritsch in honor of his 60thbirthday.

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

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The dual-compartment, corneocyte±extracellular lamellar lipid-enriched matrix of the SC serves as a protective barrier not onlyagainst external insults, but also against excess transepidermal waterloss (TEWL) (Grubauer et al, 1989). Any perturbation of thepermeability barrier initiates a rapid and ef®cient repair response inthe underlying epidermis. Regardless of the speci®c nature of thebarrier insult, i.e., mechanical (e.g., tape stripping) or chemical(e.g., solvent or detergent treatment), a metabolic response ensuesin the underlying epidermis that leads to restoration of normalfunction (Elias, 1996). The initial response comprises an immediateexocytosis of the preformed pool of lamellar bodies from theoutermost SG cell layer (Menon et al, 1992a; Elias et al, 1998).Cholesterol, fatty acid, and ceramide synthesis then increase overthe next 6±9 h (Harris et al, 1997), leading to accelerated formationand further secretion of nascent lamellar bodies (Menon et al,1992a). This response enables the re-accumulation of lipids in theSC extracellular spaces, reformation of membrane bilayers, andrestoration of baseline barrier function (Grubauer et al, 1987).

Whereas the importance of the lipid matrix for the maintenanceof cutaneous barrier function is undisputed, the contribution of thecorneocyte to the permeability barrier is less clear. A potential rolefor the CE scaffold in the organization of the extracellular lamellarbilayer system is suggested by lamellar ichthyosis, where transglu-taminase I mutations (Huber et al, 1995; Russell et al, 1995) resultnot only in a defective CE formation but also abnormal permea-bility barrier function (Lavrijsen et al, 1993; Elias et al, 2001). Theenhanced TEWL in lamellar ichthyosis has been attributed totruncation and fragmentation of extracellular lamellar arrays, whichcorrelate with the CE defect (Elias et al, 2001), supporting a scaffoldrole for the CE in bilayer formation.

Mutations in keratin 1 and keratin 10 have been identi®ed as thecause of the autosomal dominant form of ichthyosis, epidermolytichyperkeratosis (EHK, also termed bullous congenital ichthyosiformerythroderma (BCIE), OMIM113800) (Cheng et al, 1992; Chipevet al, 1992; Rothnagel et al, 1992). The keratin 1/10 mutationsfunction in a dominant-negative manner to disrupt the keratin®lament network within the keratinocyte cytosol. Filaments retractfrom their attachments to desmosomal plaques and form clumps of

perinuclear shells (Anton-Lamprecht, 1983). Skin fragility tomechanical trauma, which is most pronounced in the neonatalperiod, 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 inthe epidermal basal layer (Bonifas et al, 1991; Coulombe et al, 1991;Lane et al, 1992). The hyperkeratosis and accompanying defect inpermeability 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 fragilekeratinocyte, i.e., a defective corneocyte/CE scaffold, or otherabnormalities is not known. To dissect the pathogenesis of thebarrier abnormality in EHK, we assessed three alternative mech-anisms: (i) a fragile corneocyte leading to increased transcellularpermeability; (ii) an ineffective CE scaffold; (iii) aberrant lamellarbody (LB) secretion. Lanthanum tracer studies revealed anintercellular rather than a transcellular route of increased waterloss in EHK, ruling out a fragile corneocyte as the basis for theabnormality. Increased intercellular water movement correlated, inturn, with a decrease in the quantities and abnormal organization ofthe extracellular lamellar bilayer system. Finally, the decreasedlamellar bilayers were associated with an accumulation ofunsecreted LB in the outer SG under baseline conditions. Withacute barrier disruption, however, a loss of the epidermal calciumgradient occurred that initiated a rapid release of preformed LB andappearance 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, aged12±45 y) with clinically typical EHK (Table I). The diagnosis wascon®rmed by the presence of prominent hyperkeratosis, coarsekeratohyalin granules, and vacuolization of the upper stratum spinosumand SG on histopathology. In some sections we additionally foundacantholysis of the stratum spinosum and SG. This skin phenotype hadbeen present in all patients since birth. Further work-up did notdemonstrate any clinical or laboratory abnormalities. Mutation analysisrevealed 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 M 29 Generalized, ¯exural accentuation K10a (coil domain) Lower arm +2 F 12 Generalized, annular ±ab Abdomen +3 M 44 Generalized, ¯exural accentuation ±abc Lower arm +4 M 17 Generalized, annular K1d (tail domain) Lower leg ±

aDepartment of Biochemistry, University of Salzburg.bHot spot analysis of K1/10; no mutations were detected.cReduced epidermal immunolabeling of K10, Department of Dermatology, General Hospital Salzburg.dJefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia.

Table II. Material available for pathogenesis studies

Patient #Material atbaseline

Material afteracute disruption

Assessment ofLB secretiona

Acid lipasesecretiona

Calciumlocalizationa

Lanthanumperfusiona

OsO4 post-®xation

RuO4 post-®xation

Pyridinetreatmentb

1 BiopsyScale

6 h72 h

+±

+±

+±

+±

+±

++

±+

2 BiopsyScale

6 h±

+±

+±

±±

+±

+±

++

±+

3 BiopsyScale

3 h±

+±

+±

+±

+±

+±

++

±±

4 BiopsyScale

±±

+±

+±

±±

+±

+±

++

±±

aAnalysis possible in biopsies, but not in scale samples.bBaseline only.

838 SCHMUTH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

patient 1 a mutation was detected in the coil domain 1 A of keratin 10whereas 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 orthe other remaining patient on hot spot analyis. Others have noted that asubstantial proportion of patients with the EHK phenotype lack keratin 1or 10 mutations (Porter et al, 1996; Fuchs and Cleveland, 1998). Skinfrom EHK patients and control subjects (three healthy male volunteers,aged 31±32 y) comprised our group of controls (Table III). None of theEHK or control subjects had employed any external medications oremollients to the studied skin areas for at least 2 wk prior to assessmentof barrier function. Additional controls included historical material fromnormal human skin, and biopsies from a variety of individuals with otherdisorders of corni®cation (Table III).

Functional studies To assess epidermal permeability function wemeasured TEWL on a 1.13 cm2 area of the volar aspect of the forearmin three EHK subjects using an evaporimeter (ServoMed, Stockholm,Sweden). TEWL values were registered in g per m2 per h afterequilibration of the probe on the skin (> 60 s). All recordings weremade by the same investigator (M.S.). Volunteers underwent a 15 minpremeasurement rest period. Environment-related variables at the time ofthe study were: ambient air temperature 21.9°C±26.6°C; skin surfacetemperature 30.4°C±34.4°C, ambient air humidity 23%±49%;atmospheric H2O pressure 4.4±14.8 mmHg. Excess air convection wasprevented by shielding the measurement zone. TEWL was measured

both under baseline conditions and following acute barrier disruption byeither repeated cellophane tape stripping or acetone swabbing of thelower forearm. Barrier recovery kinetics then were assessed by measuringTEWL at 3, 6±7, 16, 24, 48, 96, 120, and 144 h after acute disruption.For calculation of the percentage change in TEWL, the followingformula was used: 1 ± [(TEWL immediately after stripping ± TEWL atindicated time)/(TEWL immediately after stripping ± baselineTEWL)] 3 100%.

Pathophysiology studies To assess the pathophysiologic basis for thebarrier abnormality in EHK, we utilized the sequence shown in Fig 1,recently applied to Harlequin ichthyosis (Elias et al, 2000b) and lamellarichthyosis (Elias et al, 2001). If barrier function was abnormal in an EHKindividual, we next assessed whether the abnormality was due to leakycorneocytes resulting in transcellular water movement (as a putativedownstream consequence of the keratinocyte fragility that is a well-known feature of EHK), or to enhanced intercellular permeability. Asthe barrier abnormality was linked, instead, to enhanced intercellularpermeability, we next assessed extracellular lamellar bilayer quantity andorganization. If abnormal, we then sought whether the structural defectin the lamellar bilayers was due to an abnormal CE±corneocyte scaffoldor to ineffective LB secretion. Biopsies from EHK skin were takenimmediately before and 3 and 6 h after acute barrier disruption.

Assessment of permeability pathway by lanthanum perfusion The perfusionpathway was assessed in all subjects by immersion of biopsies and/orscale in 4% lanthanum nitrate in 0.05 M Tris buffer containing 2%

Table III. Assessment of lamellar body secretion in EHK versus controls

Type of samples(case #) Lamellar bilayers

Contents of SG±SCinterface Entombed LB

Interstices-width/corneocyte ratio Lipase localization in SC

EHK1 Decreased Decreased Present Increased Decreased Intercellular2 Decreased Decreased Present Increased Decreased Intercellular3 Decreased Decreased Present Increased Decreased Intercellular4 Decreased Decreased Present Increased Decreased Intercellular

Normalcontrols (n = 3)

Replete Replete None Normal All Intercellular

Other ichthyosesCIE (n = 4)a Decreased Decreased Present Increased DecreasedLI (n = 4)b Replete (dimensions Replete

abnormal)None Normal All Intercellular

NLSDI (n = 2)c Clefts Decreased Some present Normal Mostly Intercellular

aCongenital ichthyosiform erythroderma (Menon et al, 1992b).bLamellar ichthyosis (Elias et al, 2001).cNeutral lipid storage disease (Williams et al, 1985).

Figure 1. Pathogenesis ¯ow chart for theanalysis of the barrier abnormality indisorders of keratinization. First, functionalassessments are used to determine TEWL levels inichthyotic skin. If TEWL abnormalities arepresent, lanthanum perfusion is employed todistinguish transcellular from intercellularpermeability pathways. In the case of an inter-cellular abnormality, a battery of ultrastructuralmethods is then applied to detail the morphologicbasis for the intercellular defect. aElias et al, 1981;bElias et al, 2001; cElias et al, 2000b; dpresentstudy.

VOL. 117, NO. 4 OCTOBER 2001 BARRIER DEFECT IN EPIDERMOLYTIC HYPERKERATOSIS 839

glutaraldehyde, 1% paraformaldehyde, pH 7.4, for 1 h at roomtemperature (Table II) (Elias et al, 1981). After lanthanum perfusion, thesamples were washed and processed for electron microscopy, as describedbelow.

Assessment of lamellar bilayer system and CE-associated structures Using acombination of osmium tetroxide (OsO4) and ruthenium tetroxide(RuO4) post®xation with pyridine pretreatment, it is possible to assessthe integrity of the intercellular lamellar bilayer system in relation to theCE/corneocyte-bound lipid envelope (CLE) scaffold (Elias, 1996; Eliaset al, 2000b). Again, scale and biopsy samples were available for thesestudies (Table II). Brie¯y, all samples were pre®xed in half-strengthKarnovsky's ®xative, followed by post®xation in 1% OsO4 and 0.2%RuO4, as described previously (Hou et al, 1991). Some samples werethen treated for 2 h with absolute pyridine to further enhance thevisualization of the CE/CLE membrane complex, as described recently(Elias et al, 2000b; 2001). After post®xation, all samples were dehydratedin a graded ethanol/propylene oxide series, and embedded in an Epon-epoxy mixture. Ultrathin sections were collected and assessed eitherunstained or after further lead citrate contrasting in a Zeiss 10 A electronmicroscope operated at 60 kV.

Assessment of LB secretion Several morphologic criteria have been usedto assess the integrity of the LB secretory process, including (i) theamount of secreted material at the SG±SC interface (Menon et al,1992a); (ii) the extent that the SC interstices are replete with lamellarbilayers (Menon et al, 1992b); and (iii) the presence of ``entombed'' LB

within the corneocyte cytosol (Elias et al, 1998). We assessed all of thesecriteria (Table III) and, in addition, utilized the delivery of a lipidhydrolase (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 withsubstrate containing 0.2% Tween 85 (6 inhibitor tetrahydrolipstatin,200 mM) in a lead-capture, cytochemical method that depicts thelocalization of acid lipase, as described previously (Rassner et al, 1997).Prior studies on nomal controls showed abundant enzyme activity in theSC interstices, with little activity in the corneocyte cytosol (Menon et al,1992b; Elias et al, 2001) (see Fig 8(D) also). Conversely, in somedisorders of corni®cation, acid lipase is not delivered normally, andenzyme activity is retained in the corneocyte cytosol (Ghadially et al,1992; Menon et al, 1992b) (Table III). The samples for ultrastructuralcytochemistry were post®xed in OsO4 and processed as above.

Ion capture cytochemistry For ultrastructural calcium localization, frozenbiopsy samples (before and after acute barrier disruption, see above) were®xed in 2% paraformaldehyde, 2% glutaraldehyde, 0.09 M potassiumoxalate, containing 0.04 M sucrose. Samples were subsequently ®xedovernight 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. Tissuesamples then were washed in alkalinized water (pH 10) and transferredto ethanol solutions for dehydration and embedding, as above.

Statistical analyses Statistical analyses were performed using a pairedor unpaired Student's t test, as appropriate. Data are expressed as mean 6SEM.

RESULTS

EHK patients display abnormal baseline barrier function,but accelerated barrier recovery kinetics We ®rst assessedbarrier function in the three EHK patients who where available forfunctional studies. As previously shown for EHK (Frost et al, 1968),baseline TEWL rates were markedly increased, with a mean of19.7 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, thekinetics of barrier recovery after comparable, initial insults (i.e., tapestripping to a maximal TEWL of 100 g per m2 per h) showed anaccelerated repair response for the EHK patients versus the threecontrol subjects (Fig 2). In addition, we incidently noted aremarkably fast healing of the skin biopsy sites in EHK patients.The early, more rapid recovery phase was followed by a laternormalization of barrier recovery rates by 96 h (not shown). By 1wk, TEWL levels had returned to approximately 80% ofpretreatment 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 baselinebarrier abnormality, exhibit accelerated barrier recovery rates afteracute insults.

The enhanced water movement in EHK occurs viaintercellular domains We next assessed whether the baselinebarrier abnormality in EHK could be assigned to enhancedintercellular or transcellular movement of water, utilizing divalentcolloidal lanthanum as a tracer to localize the sites of enhancedwater movement. Prior studies have shown that lanthanum is

Figure 2. Barrier recovery after tape stripping is accelerated inEHK epidermis. The volar forearm of EHK and normal subjects wastape-stripped, and TEWL was measured with time following barrierdisruption (time 0). Accelerated barrier recovery, although evident at allearly time points after disruption (i.e., 0±48 h), was signi®cant only at48 h, because of the small number of patients (*p < 0.05 vs normalcontrol values). At later time points, barrier recovery of EHK individualsparalleled the recovery of normal skin, and at 2±5 d, barrier recoveryslowed, resulting in the higher-than-normal baseline TEWL levelscharacteristic of EHK.

Figure 3. Increased water movement occursvia intercellular pathways. Freshly obtainedbiopsies from EHK were exposed for 1 h to 4%colloidal lanthanum nitrate tracer in 0.05 M Trisbuffer, pH 7.4, containing 2% glutaraldehyde and2% paraformaldehyde, followed by post®xation inOsO4. (A), (B) Tracer is observed throughout theSC interstices (arrows), but was excluded from thecorneocyte cytosol. OsO4 post®xation. Scale bars:0.5 mm.

840 SCHMUTH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

excluded from normal SC (Elias et al, 1977), and we con®rmed this®nding here in the normal control samples (not shown). Incontrast, lanthanum penetrated throughout the SC in EHK(Fig 3A). Moreover, in all cases and at all levels of the SC, thetracer was limited to the SC interstices (Fig 3A, B); i.e., little or notracer was found within the cytosol of individual corneocytes.These results show that, despite evidence of keratinocyte fragilitywithin the nucleated layers, the permeability barrier abnormality inEHK can be ascribed to enhanced movement of water, exclusivelyvia SC extracellular domains.

Although extracellular lamellar membranes are reduced andabnormal in EHK, the CE/CLE scaffold is normal We next

explored the basis for increased extracellular water movement inEHK, assessing ®rst the lamellar bilayer system. The lamellar bilayersystem was markedly abnormal in all EHK samples, as assessed inRuO4 post®xed samples (Fig 4A±C). With the exception of a fewfoci (Fig 4A, B, insert, arrows), the amount of bilayer materialgenerally was reduced (Fig 4C, D). Wherever bilayers werepresent, they appeared disrupted/interrupted by numerous cleftsand lacunae that were ®lled with amorphous material (Fig 4A±C,asterisks). Both the reduction in numbers of lamellae and theinterruptions of the bilayers by clefts could account for enhancedwater movement through the SC interstices. Yet, the preciseintercellular pathway of increased water movement could not beascertained because lanthanum cannot be identi®ed with certainty

Figure 4. Extracellular lamellar bilayerstructures are abnormal in EHK. (A±C) Cleftsand lacunae are present througout the SCinterstices (asterisks). (C) Overall, the amount ofbilayer material is reduced. (A, B, D,) Stretches ofintact, normal-appearing bilayers are only focallypresent (arrows). RuO4 post®xation. Scale bars:0.5 mm.

Figure 5. CE and CLE appear normal inEHK. (A) CE (arrows) in EHK appear largelynormal, although there is some variation inthickness. (B) CLE (arrowheads) also appearcompletely normal in EHK. (A) OsO4

post®xation; (B) OsO4 post®xation after pyridinepretreatment (see Methods). Scale bars: 0.25 mm.

VOL. 117, NO. 4 OCTOBER 2001 BARRIER DEFECT IN EPIDERMOLYTIC HYPERKERATOSIS 841

in RuO4 post®xed samples. We next assessed whether theabnormalities in lamellar membrane structure are associated withabnormalities in the CE/CLE scaffold, as occurs in lamellar

ichthyosis (Elias et al, 2001). Yet, despite the known association ofkeratins 1/10 with the CE (Steinert and Marekov, 1995), we notedno major abnormalities in CE structure or dimensions (Fig 5A),

Figure 6. Arrays of LB beneath plasmamembrane indicate a secretory defect inEHK. Although the overall number of LB isnormal in EHK, arrays of individual organellesremain restricted beneath the apical plasmamembrane of outermost granular (SG) cells (A, B,arrows). (C) Lack of LB secretion results in apaucity of extracellular deposits at the SG±SCinterface (asterisks). RuO4 post®xation. Scale bars:0.5 mm.

Figure 7. LB content is entombed withincorneocyte cytosol. (A, B) Entombed LB arefound within the cytosol of corneocytes (A, B,short open arrows; C, solid arrow). Note again thepresence of lacunae (asterisks) and areas of reducedand abnormal bilayer structures (B, C). (C) Theencased LB contents focally form bilayer structureswithin the cytosol. RuO4 post®xation. Scale bars:0.25 mm.

842 SCHMUTH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

and an intact CLE on the outer surface of corneocytes in all EHKsamples (Fig 5B). We did not quantitate the extent of CLEbiochemically, because prior studies have demonstrated normalquantities of covalently bound ceramide in EHK scale (Paige et al,1994). These results show ®rst that an abnormality in the SCextracellular lamellar bilayer system is present in the SC of EHKunder baseline conditions, which accounts for the barrierabnormality in EHK. They also show that the membraneabnormality cannot be ascribed to morphologically detectableabnormalities in the CE/CLE scaffold.

LB secretion is impaired in EHK under baselineconditions We next assessed an alternative explanation for thedepletion of extracellular lamellar bilayers in EHK, i.e., abnormal

production or secretion of LB. In all baseline EHK biopsies, weobserved normal numbers of LB in the upper spinous and granularlayers, as well as normal lamellar contents within individualorganelles. Thus, LB production appears to be unimpaired in EHK.In contrast, LB secretion from outer SG cells did not occurnormally. A distinctive feature was the appearance of arrays ofindividual organelles, localized within the peripheral cytosol, justbeneath the plasma membrane (Fig 6A, B). This feature appearedprimarily in cells of the outer SG layer, which also displayedretracted tono®lament bundles (not shown). Further evidence ofimpaired secretion in EHK included a striking paucity of secretedlamellar contents at the SG±SC interface (Fig 6A, C), and evidenceof entombed LB in the form of organelle contents, encased withinthe cytosol of corneocytes (Fig 7A±C).

Figure 8. Further evidence of impairedsecretion in EHK: acid lipase is retainedwithin corneocytes. (A, B) The LB hydrolase,acid lipase, is largely retained within the cytosol ofcorneocytes in EHK (arrowheads). (C) Retainedactivity is colocalized with entombed LB. (D)Normal human skin demonstrates hydrolase solelyin the SC interstices, indicative of effectivesecretion. OsO4 post®xation. Scale bars: 0.5 mm.

Figure 9. Basis for accelerated barrierrecovery in EHK epidermis. (A, B) (6 h post-tape stripping): No LB remain in cytosol ofgranular cells (SG), indicated by release of lipaseactivity into the intercellular spaces (B, arrows).Extent of release of contents also is shown at levelof SC: no entombed organelles or cytosolic lipaseactivity remain in corneocyte cytosol. (C) (3 dpost-tape stripping): Reappearance of additionalfoci of replete lamellar bilayers at time point whenbarrier recovery is accelerated (cf. Fig 2). (D) (6 hpost-tape stripping): Lanthanum perfusiondemonstrates substantial blockade of tracermovement at SG±SC interface, although foci oftracer (arrow) continue to penetrate the SCinterstices. (A, B, D) OsO4 post®xation; (C)RuO4 post®xation. Scale bars: (A, B, D) 0.5 mm;(C) 0.25 mm.

VOL. 117, NO. 4 OCTOBER 2001 BARRIER DEFECT IN EPIDERMOLYTIC HYPERKERATOSIS 843

To further ascertain whether secretion is impaired in EHK, wenext assessed the distribution of an enzyme cytochemical marker ofLB contents, i.e., acid lipase (Menon et al, 1986), in EHK versuscontrol samples. As previously reported (Menon et al, 1992b), LB-derived hydrolase contents were delivered in toto to the SCinterstices in normal SC (Fig 8D), leaving no residual, detectableactivity within the corneocyte cytosol (Table III). In contrast,whereas EHK subjects displayed comparable labeling of cytosolicLB (Fig 8B), activity at the SG±SC interface and in the SCinterstices appeared strikingly reduced (Fig 8C), further regulatedby the presence of abundant, retained deposition product in thecytosol of individual corneocytes (Fig 8A, C). Moreover, in somecorneocytes 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 secretionaccounts for the diminished extracellular lamellar bilayers that leadto the permeability barrier abnormality in EHK.

Inverse calcium signaling of LB secretion accounts foraccelerated barrier recovery in EHK Whereas baseline barrierfunction is abnormal, the kinetics of barrier recovery are faster inEHK than in normal skin (Fig 2). To assess the basis for acceleratedbarrier recovery in EHK, we next examined lanthanum perfusion,LB secretion, and SC membrane structures 3, 6, and 72 h afteracute disruption by either tape stripping or acetone treatment. At

both 3 and 6 h, more extensive cytolysis was evident throughoutthe outer nucleated cell layers than in nonperturbed EHKepidermis (not shown). Yet, in contrast to baseline EHKepidermis, few LB remained in these disrupted cells; instead,organelle contents appeared in the intercellular spaces throughoutthe outer epidermis (Fig 9, cf. Fig 6). Further evidence for releaseof organelle contents also could be seen in the SC. Few corneocytesdisplayed entombed LB contents (Fig 9, cf. Fig 7), and lipaseactivity was found between, rather than within, individualcorneocytes (Fig 9, cf. Fig 8). These ®ndings correlated with thepresence of more widespread foci of normal-appearing membranebilayers (Fig 9; however, the absolute quantities were still lowerthan in normals). Finally, after acute perturbations, resulting in LBrelease, lanthanum was largely (but incompletely) excluded fromthe SC interstices (Fig 9D).

Finally, to assess whether the combination of baseline secretorydefect and accelerated recovery is due to modulations in theepidermal calcium gradient, we employed ion capture cytochem-istry. At baseline, we observed an accumulation of extracellular andintracellular Ca2+ in the outer SG cells with low visible levels in theSC, comparable to normal skin (Fig 10A, B). Loss of the epidermalcalcium gradient with acute barrier disruption either by tapestripping or by acetone treatment occurred normally in EHK skin(Fig 10C, D). Loss of the calcium gradient was accompanied by

Figure 10. Baseline Ca2+ distribution andloss of Ca2+ gradient is similar to normals inEHK. (A, B) A comparable Ca2+ gradient, withthe highest Ca2+ levels in the outer SG withlower levels in subjacent layers and SC, is evidentin 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 bysecretion of LB (SC, stratum corneum, OSG,outer stratum granulosum). OsO4 post®xation.Scale bars: 1 mm.

844 SCHMUTH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

secretion of preformed LB from outer SG cells in EHK (Fig 10C,D vs Fig 6; note absence of preformed LB in cytosol). These resultsshow that the accelerated barrier recovery of EHK skin after acuteperturbations can be explained by the release of preformed LBcontents 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 cytoskeletalproteins of the epidermis and its appendages, accounting for up to85% of total cellular protein (Fuchs and Coulombe, 1992; Roop,1995). They form copolymers consisting of equimolar amounts ofat least one of two types: type I or acidic keratins (9±20) and type IIor basic keratins (1±8) (Miller et al, 1993; Steinert et al, 1993). Allkeratins display a secondary structure with a central rod domaincomprising four alpha-helices and distinctive, nonhelical, head andtail sequences. Mutations have been identi®ed in the highlyconserved ends of the rod domains of keratins 1 and 10 in severalforms of EHK (Cheng et al, 1992; Chipev et al, 1992; Rothnagelet al, 1992), but a signi®cant proportion of affected individuals donot 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. Affectedindividuals are born with widespread erythema and erosions. Withincreased age these features diminish and are replaced by general-ized epidermal thickening, indicative of epidermal hyperplasia(Irvine and McLean, 1999). The resulting hyperkeratotic plaquesare prone to superinfection. The diagnosis in our patient cohortwas based on the characteristic clinical picture, diagnostic histology,and the presence of a keratin 1 or keratin 10 mutation in two of thefour patients (Table I). Increased expression of other CE proteins(Ishida-Yamamoto et al, 1994, 1995; Porter et al, 1996; Gerritsenet al, 1997; Reichelt et al, 1999) could compensate for the structuralabnormality and further provoke the characteristic phenotype inEHK. Our results con®rm previous ®ndings of increased baselineTEWL levels in EHK patients with or without de®ned mutations(Frost et al, 1968; Lavrijsen et al, 1993). We show further that theinitial rates of barrier repair after acute disruption actually occurredmore rapidly in EHK subjects than in controls. The basis for thebaseline permeability barrier abnormality in EHK is unknown. Anearlier hypothesis held that keratinocyte fragility could lead toincreased cytokine release, which in turn could drive the epidermalhyperplasia, leading to a barrier abnormality (Roop, 1995).Accordingly, we ®rst assessed whether the keratinocyte fragilityin EHK leads to a more fragile, ``leaky'' corneocyte, allowingincreased transcellular permeability. Indeed, an abnormality incorneocyte fragility accounts for the barrier abnormality inretinoid-treated skin, where abundant colloidal lanthanum tracerleaks into corneocytes (Elias et al, 1981). Yet, our lanthanumperfusion studies clearly showed that, despite the known fragility ofkeratinocytes within EHK epidermis, the increased SC permeabil-ity in EHK occurs via an intercellular rather than a transcellularpathway. Thus, a mechanism other than an incompetentcorneocyte must be sought to explain the barrier defect. Instead,we found both a paucity of lamellar bilayers and distortedsupramolecular architecture of those lamellar bilayers that werepresent in all EHK subjects. These structural changes account forthe permeability barrier abnormality and the increased intercellularpenetration of tracer in EHK.

One mechanism whereby defective keratin 1/10 intermediate®laments could result in abnormal extracellular lamellar bilayerscould be an abnormal CE/CLE scaffold. As keratin 1 and 10 arecrosslinked to the CE (Ming et al, 1994; Candi et al, 1998), theycould play a hitherto unrecognized role in maintenance of the SCbarrier. Accordingly, defects in the CE/CLE scaffold, even in EHKindividuals with no keratin mutations, could explain the defectiveorganization of extracellular lamellar bilayers in EHK. Indeed, ananalogous defect in the CE/CLE scaffold appears to be responsiblefor the barrier abnormality in another keratinizing disorder with a

defect in CE formation, i.e., lamellar ichthyosis due to transglu-taminase I mutations (Lavrijsen et al, 1993; Elias et al, 2001), and intransgenic 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 other-wise display a normal phenotype (Djian et al, 2000). Finally, there isevidence for a barrier abnormality in transgenic mice expressing amutant form of loricrin (Koch et al, 2000; Suga et al, 2000), thedominant CE peptide in epidermis (Steinert, 2000). Ourultrastructural 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 barrierdefect, we next examined whether the reduction in lamellarbilayers in EHK extracellular domains could be attributed todecreased production (assembly, translocation) or defective secre-tion (exocytosis) of LB. Under baseline conditions, we observed anincreased number of LB at the SG±SC interface displaying normalorganelle contents. Moreover, the secretion of LB from theperiphery of SG cells was impaired in EHK subjects. Thus, bothassembly and translocation of LB appear to be normal, butexocytosis does not occur normally in EHK under baselineconditions. This conclusion is further supported by two additionalobservations, indicating impaired LB secretion in EHK: (i) thepresence of abundant entombed LB contents, encased within thecytosol of corneocytes; (ii) the localization of LB-derivedhydrolytic enzymes, which normally are completely secreted,within corneocytes and often colocalized with entombed LBcontents. As a result of the secretory abnormality, decreased LBcontents are deposited at the SG±SC interface, resulting indiscontinuous lamellar bilayers, leading in turn to increasedTEWL levels (Fig 4).

The baseline barrier abnormality in EHK is associated withabnormal LB secretion, possibly due to an impairment of theexocytosis step. One possible explanation is that the keratin 1/10cytoskeletal mutation could interfere with the exocytosis of LB.Prior work on lung epithelium and in adrenal secretory cells hasshown that the microtubular system is crucial for exocytosis (Aunisand Bader, 1988; Zimmermann, 1989). Thus, cytoskeletal instabil-ities, including those caused by keratin 1/10 dominant-negativemutations in EHK, could disrupt steps that are involved in theexocytosis of LB. A precise link between the keratin 1/10mutations and the barrier abnormality could not be establishedhere, however, not only because only half of our patients had thesemutations, but also because of limitations of experiments in humansin vivo. Animal models with de®ned defects in keratins 1/10 shouldallow a further dissection of the molecular pathogenesis of the LBsecretory abnormality, however. Several putative mouse models ofthe disease have been described (Fuchs et al, 1992; Bickenbach et al,1996; Porter et al, 1996; Reichelt et al, 1997), but available data onthe permeability barrier abnormalities in these animal models donot always re¯ect the structural and functional abnormalities ofEHK, presumably because the murine genotypes do not alwaysre¯ect EHK (Reichelt et al, 1999; Elias et al, 2000a; Jensen et al,2000).

After acute barrier disruption the peripheral arrays of LB ingranular cells were rapidly released (Fig 9), accounting for theaccelerated recovery of permeability barrier function in EHK skin.Moreover, we found an increase in intact lamellar bilayers andexclusion of lanthanum tracer from the extracellular domains afteracute barrier disruption. The basis for the release of LB after acutedisruption probably does not simply re¯ect cytolysis of fragile cellswith release of trapped LB. Among likely explanations could be thepresence of an intact calcium gradient in EHK under baselineconditions, which, together with the mutant protein, restrictssecretion. The effect of the mutant protein can be temporarily

2Jensen JM, Djian P, Proksch E: Disturbed permeability barrier functionin transgenic involucrin de®cient mice. J Invest Dermatol 112: 542, 1999(abstr.)

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overcome by the complete calcium depletion that occurs afterbarrier disruption (Fig 10). Alternatively, the expression of themutant protein could be downregulated after acute disruption,thereby allowing unrestricted LB secretion until the calciumgradient is re-established. To date, however, there is evidence thatkeratins 1/10 are actually upregulated after barrier disruption(Ekanayake-Mudiyanselage et al, 1998). Lastly, the pathway for LBsecretion could differ under baseline versus stimulated conditions. Inany case, the localization of arrays of unsecreted LB at the SG±SCinterface under baseline conditions seems to re¯ect a ``geared-up''activation state, which after an external assault is quickly trans-formed into a normal or supernormal repair response. A similaracceleration of the early phase of barrier repair kinetics has beenreported for atopic dermatitis (Gfesser et al, 1997), but noinformation is available as yet for the morphologic basis ofaccelerated repair in atopic dermatitis.

In summary, the permeability barrier abnormality in EHK can beattributed to increased intercellular water movement, despite theknown fragility of keratinocytes. The barrier abnormality correlateswith a reduction in extracellular lamellar membranes, which can beattributed further to impaired LB secretion. The baseline abnor-mality can be overcome by application of external stimuli forbarrier 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 (PP-

Projects 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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