Pathogenesis of the Permeability Barrier Abnormality in Epidermolytic Hyperkeratosis11We dedicate this work to Professor Peter O. Fritsch in honor of his 60th birthday.
2001; Elsevier BV; Volume: 117; Issue: 4 Linguagem: Inglês
10.1046/j.0022-202x.2001.01471.x
ISSN1523-1747
AutoresMatthias Schmuth, Gil Yosipovitch, Mary L. Williams, Florian Weber, Helmut Hintner, Susana Ortiz‐Urda, Klemens Rappersberger, Debra Crumrine, Kenneth R. Feingold, Peter M. Elias,
Tópico(s)Hair Growth and Disorders
ResumoEpidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin filament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of cornification, 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, first, 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 cornified envelope or the adjacent cornified-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 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 findings, 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 cornified envelope/cornified-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair. Epidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin filament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of cornification, 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, first, 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 cornified envelope or the adjacent cornified-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 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 findings, 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 cornified envelope/cornified-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair. cornified envelope, CLE, corneocyte-bound lipid envelope lamellar body stratum corneum stratum granulosum transepidermal water loss The epidermis is a homeostatic, self-renewing tissue that expresses differentiation-specific 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 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, 1995Roop D. Defects in the barrier.Science. 1995; 267: 474-475Crossref PubMed Scopus (166) Google Scholar). In the nucleated cell layers, these keratins are organized into filament bundles that loop between desmosome plaques and the nuclear envelope providing a cytoskeleton that protects keratinocytes from mechanical injury (Fuchs and Cleveland, 1998Fuchs E. Cleveland D.W. A structural scaffolding of intermediate filaments in health and disease.Science. 1998; 279: 514-519Crossref PubMed Scopus (793) Google Scholar). As the keratinocytes terminally differentiate, keratins 1 and 10 as well as several other cornified envelope (CE) proteins form a rigid, chemically and mechanically resistant structure that replaces the plasma membrane (Nemes and Steinert, 1999Nemes Z. Steinert P.M. Bricks and mortar of the epidermal barrier.Exp Mol Med. 1999; 31: 5-19Crossref PubMed Scopus (414) Google Scholar;Steinert and Marekov, 1999Steinert P.M. Marekov L.N. Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia.Mol Biol Cell. 1999; 10: 4247-4261Crossref PubMed Scopus (121) Google Scholar;Steinert, 2000Steinert P.M. The complexity and redundancy of epithelial barrier function.J Cell Biol. 2000; 151: F5-F8Crossref PubMed Google Scholar). 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., 1993Jackson S.M. Williams M.L. Feingold K.R. Elias P.M. Pathobiology of the stratum corneum.West J Med. 1993; 158: 279-285PubMed Google Scholar;Elias et al., 1998Elias P.M. Cullander C. Mauro T. Rassner U. Komuves L. Brown B.E. Menon G.K. The secretory granular cell: the outermost granular cell as a specialized secretory cell.J Invest Dermatol Symp Proc. 1998; 3: 87-100Abstract Full Text PDF PubMed Scopus (110) Google Scholar). The dual-compartment, corneocyte-extracellular lamellar lipid-enriched 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., 1989Grubauer G. Elias P.M. Feingold K.R. Transepidermal water loss: the signal for recovery of barrier structure and function.J Lipid Res. 1989; 30: 323-333Abstract Full Text PDF PubMed Google Scholar). Any perturbation of the permeability barrier initiates a rapid and efficient repair response in the underlying epidermis. Regardless of the specific 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, 1996Elias P.M. Stratum corneum architecture, metabolic activity and interactivity with subjacent cell layers.Exp Dermatol. 1996; 5: 191-201Crossref PubMed Scopus (105) Google Scholar). The initial response comprises an immediate exocytosis of the preformed pool of lamellar bodies from the outermost SG cell layer (Menon et al., 1992aMenon G.K. Feingold K.R. Elias P.M. Lamellar body secretory response to barrier disruption.J Invest Dermatol. 1992; 98: 279-289Crossref PubMed Scopus (234) Google Scholar;Elias et al., 1998Elias P.M. Cullander C. Mauro T. Rassner U. Komuves L. Brown B.E. Menon G.K. The secretory granular cell: the outermost granular cell as a specialized secretory cell.J Invest Dermatol Symp Proc. 1998; 3: 87-100Abstract Full Text PDF PubMed Scopus (110) Google Scholar). Cholesterol, fatty acid, and ceramide synthesis then increase over the next 6-9 h (Harris et al., 1997Harris I.R. Farrell A.M. Grunfeld C. Holleran W.M. Elias P.M. Feingold K.R. Permeability barrier disruption coordinately regulates mRNA levels for key enzymes of cholesterol, fatty acid, and ceramide synthesis in the epidermis.J Invest Dermatol. 1997; 109: 783-787Crossref PubMed Scopus (98) Google Scholar), leading to accelerated formation and further secretion of nascent lamellar bodies (Menon et al., 1992aMenon G.K. Feingold K.R. Elias P.M. Lamellar body secretory response to barrier disruption.J Invest Dermatol. 1992; 98: 279-289Crossref PubMed Scopus (234) Google Scholar). 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., 1987Grubauer G. Feingold K.R. Elias P.M. Relationship of epidermal lipogenesis to cutaneous barrier function.J Lipid Res. 1987; 28: 746-752Abstract Full Text PDF PubMed Google Scholar). 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., 1995Huber M. Rettler I. Bernasconi K. et al.Mutations of keratinocyte transglutaminase in lamellar ichthyosis.Science. 1995; 267: 525-528Crossref PubMed Scopus (406) Google Scholar;Russell et al., 1995Russell L.J. DiGiovanna J.J. Rogers G.R. Steinert P.M. Hashem N. Compton J.G. Bale S.J. Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis.Nat Genet. 1995; 9: 279-283Crossref PubMed Scopus (308) Google Scholar) result not only in a defective CE formation but also abnormal permeability barrier function (Lavrijsen et al., 1993Lavrijsen A.P. Oestmann E. Hermans J. Boddé H.E. Vermeer B.J. Ponec M. Barrier function parameters in various keratinization disorders: transepidermal water loss and vascular response to hexyl nicotinate.Br J Dermatol. 1993; 129: 547-553Crossref PubMed Scopus (99) Google Scholar;Elias et al., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar). 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., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar), supporting a scaffold role for the CE in bilayer formation. Mutations in keratin 1 and keratin 10 have been identified as the cause of the autosomal dominant form of ichthyosis, epidermolytic hyperkeratosis (EHK, also termed bullous congenital ichthyosiform erythroderma (BCIE), OMIM113800) (Cheng et al., 1992Cheng J. Syder A.J. Yu Q.C. Letai A. Paller A.S. Fuchs E. The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes.Cell. 1992; 70: 811-819Abstract Full Text PDF PubMed Scopus (274) Google Scholar;Chipev et al., 1992Chipev C.C. Korge B.P. Markova N. Bale S.J. DiGiovanna J.J. Compton J.G. Steinert P.M. A leucine-proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis.Cell. 1992; 70: 821-828Abstract Full Text PDF PubMed Scopus (244) Google Scholar;Rothnagel et al., 1992Rothnagel J.A. Dominey A.M. Dempsey L.D. et al.Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis.Science. 1992; 257: 1128-1130Crossref PubMed Scopus (306) Google Scholar). The keratin 1/10 mutations function in a dominant-negative manner to disrupt the keratin filament network within the keratinocyte cytosol. Filaments retract from their attachments to desmosomal plaques and form clumps of perinuclear shells (Anton-Lamprecht, 1983Anton-Lamprecht I. Genetically induced abnormalities of epidermal differentiation and ultrastructure in ichthyoses and epidermolyses: pathogenesis, heterogeneity, fetal manifestation, and prenatal diagnosis.J Invest Dermatol. 1983; 81: 149s-156sCrossref PubMed Scopus (81) Google Scholar). 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., 1991Bonifas J.M. Rothman A.L. Epstein Jr., E.H. Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities.Science. 1991; 254: 1202-1205Crossref PubMed Scopus (330) Google Scholar;Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (507) Google Scholar;Lane et al., 1992Lane E.B. Rugg E.L. Navsaria H. Leigh I.M. Heagerty A.H. Ishida-Yamamoto A. Eady R.A. A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (322) Google Scholar). The hyperkeratosis and accompanying defect in permeability barrier function are additional clinical features of EHK (Frost et al., 1968Frost P. Weinstein G.D. Bothwell J.W. Wildnauer R. Ichthyosiform dermatoses. 3. Studies of transepidermal water loss.Arch Dermatol. 1968; 98: 230-233Crossref PubMed Scopus (45) Google Scholar), the pathogenesis of which is less clear. Specifically, 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. We investigated four patients (one female, three male, aged 12–45 y) with clinically typical EHK Table I. The diagnosis was confirmed 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 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., 1996Porter R.M. Leitgeb S. Melton D.W. Swensson O. Eady R.A. Magin T.M. Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.J Cell Biol. 1996; 132: 925-936Crossref PubMed Scopus (84) Google Scholar;Fuchs and Cleveland, 1998Fuchs E. Cleveland D.W. A structural scaffolding of intermediate filaments in health and disease.Science. 1998; 279: 514-519Crossref PubMed Scopus (793) Google Scholar). 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 cornification Table III.Table ICharacteristics of EHK patientsPatient #GenderAgeClinical phenotypeMutationBiopsy sitesScale1M29Generalized, flexural accentuationK10aDepartment of Biochemistry, University of Salzburg. (coil domain)Lower arm+2F12Generalized, annular–aDepartment of Biochemistry, University of Salzburg.bHot spot analysis of K1/10; no mutations were detected.Abdomen+3M44Generalized, flexural accentuation–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.Lower arm+4M17Generalized, annularK1dJefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia. (tail domain)Lower leg–a Department of Biochemistry, University of Salzburg.b Hot spot analysis of K1/10; no mutations were detected.c Reduced epidermal immunolabeling of K10, Department of Dermatology, General Hospital Salzburg.d Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia. Open table in a new tab Table IIMaterial available for pathogenesis studiesPatient #Material at baselineMaterial after acute disruptionAssessment of LB secretionaAnalysis possible in biopsies, but not in scale samples.Acid lipase secretionaAnalysis possible in biopsies, but not in scale samples.Calcium localizationaAnalysis possible in biopsies, but not in scale samples.Lanthanum perfusionaAnalysis possible in biopsies, but not in scale samples.OsO4 post-fixationRuO4 post-fixationPyridine treatmentbBaseline only.1Biopsy6 h++++++–Scale72 h–––––++2Biopsy6 h++–++++Scale––––––++3Biopsy3 h++++++–Scale––––––+–4Biopsy–++–+++–Scale––––––+–a Analysis possible in biopsies, but not in scale samples.b Baseline only. Open table in a new tab Table IIIAssessment of lamellar body secretion in EHK versus controlsType of samples (case #)Lamellar bilayersContents of SG–SC interfaceEntombed LBInterstices-width/ corneocyte ratioLipase localization in SCEHK 1DecreasedDecreasedPresentIncreasedDecreased Intercellular 2DecreasedDecreasedPresentIncreasedDecreased Intercellular 3DecreasedDecreasedPresentIncreasedDecreased Intercellular 4DecreasedDecreasedPresentIncreasedDecreased IntercellularNormal controls (n = 3)RepleteRepleteNoneNormalAll IntercellularOther ichthyoses CIE (n = 4)aCongenital ichthyosiform erythroderma (Menon et al, 1992b).DecreasedDecreasedPresentIncreasedDecreased LI (n = 4)bLamellar ichthyosis (Elias et al, 2001).Replete (dimensionsReplete abnormal)NoneNormalAll Intercellular NLSDI (n = 2)cNeutral lipid storage disease (Williams et al, 1985).CleftsDecreasedSome presentNormalMostly Intercellulara Congenital ichthyosiform erythroderma (Menon et al., 1992bMenon G.K. Ghadially R. Williams M.L. Elias P.M. Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation.Br J Dermatol. 1992; 126: 337-345Crossref PubMed Scopus (109) Google Scholar).b Lamellar ichthyosis (Elias et al., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar).c Neutral lipid storage disease (Williams et al., 1985Williams M.L. Koch T.K. O'Donnell J.J. Frost P.H. Epstein L.B. Grizzard W.S. Epstein C.J. Ichthyosis and neutral lipid storage disease.Am J Med Genet. 1985; 20: 711-726Crossref PubMed Scopus (65) Google Scholar). Open table in a new tab 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)] × 100%. To assess the pathophysiologic basis for the barrier abnormality in EHK, we utilized the sequence shown in Figure 1, recently applied to Harlequin ichthyosis (Elias et al., 2000bElias P.M. Fartasch M. Crumrine D. Behne M. Uchida Y. Holleran W.M. Origin of the corneocyte lipid envelope (CLE): observations in harlequin ichthyosis and cultured human keratinocytes.J Invest Dermatol. 2000; 115: 765-769Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and lamellar ichthyosis (Elias et al., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar). 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 well-known 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. 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% glutaraldehyde, 1% paraformaldehyde, pH 7.4, for 1 h at room temperature Table II (Elias et al., 1981Elias P.M. Fritsch P.O. Lampe M. Williams M.L. Brown B.E. Nemanic M. Grayson S. Retinoid effects on epidermal structure, differentiation, and permeability.Laboratory Invest. 1981; 44: 531-540PubMed Google Scholar). After lanthanum perfusion, the samples were washed and processed for electron microscopy, as described below. Using a combination of osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) postfixation 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, 1996Elias P.M. Stratum corneum architecture, metabolic activity and interactivity with subjacent cell layers.Exp Dermatol. 1996; 5: 191-201Crossref PubMed Scopus (105) Google Scholar;Elias et al., 2000bElias P.M. Fartasch M. Crumrine D. Behne M. Uchida Y. Holleran W.M. Origin of the corneocyte lipid envelope (CLE): observations in harlequin ichthyosis and cultured human keratinocytes.J Invest Dermatol. 2000; 115: 765-769Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Again, scale and biopsy samples were available for these studies Table II. Briefly, all samples were prefixed in half-strength Karnovsky's fixative, followed by postfixation in 1% OsO4 and 0.2% RuO4, as described previously (Hou et al., 1991Hou S.Y. Mitra A.K. White S.H. Menon G.K. Ghadially R. Elias P.M. Membrane structures in normal and essential fatty acid-deficient stratum corneum: characterization by ruthenium tetroxide staining and x-ray diffraction.J Invest Dermatol. 1991; 96: 215-223Abstract Full Text PDF PubMed Google Scholar). 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., 2000bElias P.M. Fartasch M. Crumrine D. Behne M. Uchida Y. Holleran W.M. Origin of the corneocyte lipid envelope (CLE): observations in harlequin ichthyosis and cultured human keratinocytes.J Invest Dermatol. 2000; 115: 765-769Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar;Elias et al., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar). After postfixation, all samples were dehydrated in a graded ethanol/propylene oxide series, and embedded in an Epon-epoxy 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. 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., 1992aMenon G.K. Feingold K.R. Elias P.M. Lamellar body secretory response to barrier disruption.J Invest Dermatol. 1992; 98: 279-289Crossref PubMed Scopus (234) Google Scholar); (ii) the extent that the SC interstices are replete with lamellar bilayers (Menon et al., 1992bMenon G.K. Ghadially R. Williams M.L. Elias P.M. Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation.Br J Dermatol. 1992; 126: 337-345Crossref PubMed Scopus (109) Google Scholar); and (iii) the presence of “entombed” LB within the corneocyte cytosol (Elias et al., 1998Elias P.M. Cullander C. Mauro T. Rassner U. Komuves L. Brown B.E. Menon G.K. The secretory granular cell: the outermost granular cell as a specialized secretory cell.J Invest Dermatol Symp Proc. 1998; 3: 87-100Abstract Full Text PDF PubMed Scopus (110) Google Scholar). 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., 1986Menon G.K. Grayson S. Elias P.M. Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis.J Invest Dermatol. 1986; 86: 591-597Crossref PubMed Scopus (97) Google Scholar), as cytochemical indicator of the extent of secretion (Menon et al., 1992bMenon G.K. Ghadially R. Williams M.L. Elias P.M. Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation.Br J Dermatol. 1992; 126: 337-345Crossref PubMed Scopus (109) Google Scholar). Aldehyde-prefixed samples were microwave-incubated with substrate containing 0.2% Tween 85 (± inhibitor tetrahydrolipstatin, 200 µM) in a lead-capture, cytochemical method that depicts the localization of acid lipase, as described previously (Rassner et al., 1997Rassner U.A. Crumrine D.A. Nau P. Elias P.M. Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry.Histochem J. 1997; 29: 387-392Crossref PubMed Scopus (32) Google Scholar). Prior studies on nomal controls showed abundant enzyme activity in the SC interstices, with little activity in the corneocyte cytosol (Menon et al., 1992bMenon G.K. Ghadially R. Williams M.L. Elias P.M. Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal and abnormal desquamation.Br J Dermatol. 1992; 126: 337-345Crossref PubMed Scopus (109) Google Scholar;Elias et al., 2001Elias P.M. Schmuth M. Uchida Y. et al.Basis for permeability barrier abnormality in lamellar ichthyosis.Exp Dermatol. 2001Google Scholar) (see Figure 8(d)also). Conversely, in some disorders of cornification, acid lipase is not delivered normally, and enzyme activity is retained in the corneocyte cytosol (Ghadially et al., 1992Ghadially R
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