Evidence That the Corneocyte Has a Chemically Bound Lipid Envelope

Donald C. Swartzendruber, Ph.D. , Phili p W. Wertz, Ph.D., Kathi C. Madison, M.D ., and Donald T. Downin g, Ph.D. Marsh a ll r~csca r c h Laborato ri es. Department of I ermatology, University of Iowa Coll ege of Medi cin e, Iowa C ity. Iowa. U .S.A.

Th e stratum corneum of mammali an epidermis contain s a leaves the lu cent band that has been termed the corneocyte mix ture of ceramides, free fatty acids, choles terol, and cho­ plasma membrane. T he subsequent alkaline hydrolysis and les teryl sul fate, amounting to 14% of the dry weight of lipid extraction remove the lucent ba nd, which must there­ the tissue, that can be removed by exhaustive extracti on fore contain the w-hydroxyacylsphingosines. From the re­ with c hl oroform/ methanol. Subsequent mild alkaline hy­ sul ts of in situ derivati za tion of these lipids and the con­ drolysis liberates additional lipid, consisting almost exclu­ struction of molecul ar models, it is inferred that the bound sivel y of C 30-C 34 w-h ydroxyacids in amide li nkage with lipids exist in es ter lin kage with protein on the surface of sphingosin e, equal to 2% of the ti ss ue mass. In the present the corneocyte envelope. T he tightl y packed hydroxy­ study, transmiss ion electron microscopy was used to dem­ acylsphingosin e molecul es thus form a lipid envelope for onstra te th at the initial extra ction removes the intercellular each co rneocyte. J 11111 esr Dennarol 88:709-713, '/ 987 la mellae that constitute the epidermal water barrier but

he horn y layer of mammalian epidermis contains about trypsin (type III , Sigma C hemica l Co, St Lo uis, Missouri) for I 14% by weight of lipid that is extrac table with polar h. T he un cornified cells were scraped off with a spatul a and the solvellts. T hi s lipid consists of ce ramides (40-50%), trypsin trea tment and scraping were repea ted . Histology showed free fatty acids (15-25%), choles terol (20- 25 %), and onl y occas ional uncornified ce ll s remaining on the sheets of stra­ chobteryl sulfate (5-10%) 11 ,21. T he lipid is ar­ tum co rneUIll , w hich were dried and weighed. Tranged in broad lamell ar sheets between the corn eocytes, w here Extraction of Unbound Lipids T he dried stratum corn cum it appears to co nstitute the epidermal permea bility ba rrier [3,41. It has b een pos tulated that the shee ts arc formed by the edge- to­ was pl aced in a succession of chl orofor m:methanol mixtures (2: 1, 1:1 , and 1 :2) for 2 h cach to extract the free lipids. T he same edge fu sion ofAattened li pid vesicles extruded fro m the outermost seq uence of extracti ons was thell repeated for 1 h each. T he sol­ ceUs of the via ble epidermis [41. Each sheet therefore co nsists o f ve nt extracts were co mbined and evaporated to recover the ex­ two Ii p id bi layers, and there may be one, two, or more, such tracted li pids. sheets in each intercellular space . Transmiss ion electron micrographs of epid ermis show an elec­ Extraction of Esterified Lipids T he sheets of solvent-ex­ tron-lu cent region, usuall y referred to as the horn y ce ll plas ma tracted stratum corneum were placed in 1 M N aOH in 90% meth­ me m brane, between the intercellular lamell ae and the proteina­ anol at 60°C fo r 1 h. The suspension was then filtered and the ceo u s corn eocyte envelope [5-81. Evidence obtain ed in th e present recovered stratum co rneum sheets were extracted with chl oro­ stu d y indica tes that this lu cent band consists o f a mono molecul ar form:methanol as described above. T he methanoli c NaOH filtrate layer ofN-(w-hydroxyacyl) sphingosin es that arc chemi ca ll y bound was neutrali zed with aqueous HC I and extracted with chl oro­ to the h o rn y cel l enve lope. T hus, in addition to the rough, chem­ form, w hich was separated and combined with the chl oro­ icall y I"csistant envelo pe of cross-linked protein , each co rneocyte form / methanol extracts of the recovered tissue. T he solvent was appears to possess a lipid enve lope that may function both in then evaporated to recover the lipids liberated by the alka li trea t­ corn eocyte cohes ion and in the barrier properties of the epic! ermis. ment. MATERIALS AND METHODS Lipid Analysis T he recovered li pids were analyzed by quan­ titative thi n-layer chromatography, for w hich aliquots of solu­ Isolat:ion of Stratum Corneum Pi eces of fresh pig skin were tions in chl oroform/ methanol, containing 10-20 JLg of total lipid, placed in 60°C water for 1 min and the epidermis was then scraped were applied to 6-mm lanes on glass plates coated with 0.25 mm­ off in s heets with a spatula. T he sheets were in cubated with 1 % thi ck layers of sili ca gel H . T he chroma tograms were developed with hex3n e:ether: aceti c acid (70:30:1 ) to resolve nonpolar lipids WI; with chl oroform:methanol:ace ti c acid (1 90:9: I) to resolve lip­ ids of intermediate polarity such as ce ramides [1 01; or with chlo­ Man u script received N ovember 12, 1986; accepted fo r publi cation Jan­ ro form:methanol:water (40: 10: 1) to resolve the more polar li pids, uary 1 2. 1987. in cl uding glucosylceramides and choles teryl sul fate 111 ,12]. After T his study was supported in part by grants from the U.S. Pu bli c Hea lth Se rvice (AM32374 and AM0 16 10) and by r~i c h ardso n - Vi c k s. In c., Wil ton, devel opment, the chromatograms were sprayed with 50% H2S04 Connec t icut. and hea ted to 220°C to char the lipids. After cooling, the chro­ Repri nt reques ts to: Donald T . Downin g, Ph. D. , 27U Medical Labo­ matograms were sca nned with a photodensitometer (S himadzu rato rics. Uni ve rsity oflowa College of Medi ci ne. Iowa C ity. Iowa 52242. CS-930) to quantify the lipid mi xtures 191.

0022-202X/ 87/S03.50 Copyright © 1987 by T he Society for Inves tiga ti ve Dermatology. Inc.

709 710 SWA lnZENDRUUER ET AL T H E JOURNAL O F IN VESTIGATIVE DERMATOLOGY

Figure l.. Transmi ss ion elcctron mi cro­ graph or iso lated, uncxtracted stratum co rneull1 . Thc intcrcellul ar spa ces arc fi lled with multiple alternating den se Jnd less dcnse lines rcpresenting the sheets or lipid lame ll ac. Bar = 100 n111 .

Electron Microscopy Small pieces of the isolated stratum cor­ Ultrastructure of Completely Delipidized Stratum Cor­ neum obtain ed prio r to extracti on, after extra ction w ith chl o­ neunl Electro n llIi crographs of the tissue recovered after re­ roform / methano l mixtures, and after hydrolys is and extracti o n moval of the chemicall y bo und lipid (Fig 4) show that the lucent of the bound lipids, were exa mined. The ti ssues were fixed in bands adj acent to the cell envelopes had completel y disappeared. 2.5% g lutaraldehyde/O. l M cacodylate buffer at pH 7.2 and post­ In addition, the envelo pes partiall y separated from the contents fixed in 0.2% ruthenium tetroxide in cacodylate buffer. The tis­ o f the ce ll s and beca me grea tl y convolu ted, as tho ugh they had sues were then dried b y extraction w ith g raded acetone solutions been much in creased in area. and embedded in Spurr's low- viscosity epoxy resin. Ultrathin D ISCU SS IO N sections were cut and stained w ith uranyl acetate and le ad citrate before examination in a Hitachi M odel H-600 electron micro­ The present study has dem o nstrated that exhausti ve extraction of scope. stratum corneum w ith polar lipid solvent, w hile removin g the intercellular lamellae, did no t eliminate the lu cent bands adjacent RESULTS to the corneocyte envelo pe. That the lu cent bands result fro m Ultrastructure and Lipid Content of Isolated Stratum Cor­ chemica ll y bound lipid is suppo rted by the dis:lppearance of these neum Figure 1 illustrates an electro n micrograph of isolated , bands fo ll owin g alka li treatment and extr:l cti on of the liberated unextracted stratum corneum. The interce llul ar space contain s li pid. Analysis of the li pids extractcd fro m isolated stratum cor­ the Illultiple, alternating dense and lu cent bands that represent a neum prio r to alka li treatment confirmed the accepted view thar cross secti o n of the sheets of li pid lam ell ae. The broad, dense the intercellular lamcllae arc composed of ce ramides, free fatty bands on either side o f the intercellular space arc the cross- linked acids, free cholesterol, and cbo lesteryl sul fa te /1 ,21. The bound protein en ve lopes of adjacent corneocytes [1 3, 14 /. li pids released from solvent-extracted stratum corneum by mild The lipid obtained fro m isolated stratum corneum by ex haus­ alka line hydrolysis consisted almost exclusively o f the ceramides tive extraction with chlo roform/ m ethanol has bccn shown pre­ ofC.' IJ - C.,~ w-h ydroxyacids. Since the hydrolysis completely elim­ viously to consist of ceramides, free fatty acids, cholesterol, and in ated the lu cent bands adjacent to the cell envclo pes, it can be cho lcsteryl su lfate [1, 2/. In the present study, extraction of the concluded that t be m ateri al responsible for the lu cent bands is the stratum corn eum sa mples yielded a similar mixture of lipids, hydroxyceramide mixture that was recovered after hydro lysis. amounting to 14% of the dry weight of recovered ti ssue, that The onl y fun cti o nal gro ups in these m o lecules cap:lblc of an alkali­ was analyzed by quantitative thin-layer chro matography, the re­ labile chemica l lin kage arc the h ydroxyl g ro ups, so the binding sults of w hi ch are shown in Table I. presumably in volves one or m o re ofthc hydroxyl g roups of each Ultrastructure and Lipid Content of Solvent-Extracted StratUln Corneum Figure 2 shows that after chl oro­ forlll / m ethano l extracti on the intercellular lam ell ae were absent, Table I. Composition (Wt %) of Lipids E xtracted Fro m but a lucent band rem ain ed adj acent to each ce ll envel ope. Where Stratum Corneum Using C hlo roform/ Methanol the lu cent bands o f adjacent cell s were in contact, their o uter (Mea ns of 3 Analyses) surfaces united to fo rm a single dense lin e, as no ted by previous in vesti gators /5,7J. After treatm ent w ith m ethano li c sodium hy­ Cholcsteryl cs tns 1.7 :t 1.1 droxide, the solvent-extracted stratum corn eul11 yielded addi­ Triglycc rid es 2.8 :±: 0.7 tional chl oroform/ m ethano l extractable li pid am ounting to 2% Free ratty acid s 13. 1 :t 1.6 by weight of the tissue. T hi s lipid ha s been shown /'151 to consist Free cholesterol 26.0 :±: 0.6 alm ost entirely o f N-(w-h yd roxyacyl) sphingosines, a represen­ Ce ralllidcs 45.8 :±: 2. 1 Glu cosy lccramides 1.0 :t 0.6 tative structure o f w hi ch is shown in Fi g 3. The detailed com­ Cholcs teryl sulfate 3. 9 :±: 1.4 position of this m aterial isolated in the present study is shown in UnidentifIed 5.7 :±: 0.9 Table II. VOL. l:l~. NO. (\ JUN E 1987 T H E O RN EOCYTE LIPID EN VELO PE 711

Figure 2. Transmiss ion electron micro­ graph of isolated, chi oroform/ lll ethano l­ extracted stratum corneum. T he intercel­ lular lamellae arc absent, but th e lu cen t bands adjacent to the.ho rn y cdl envelopes. representi ng the lipid envel ope. remain. Bnr = 100 nm.

m o lec ule in ester lin kage w ith corneocyte protein . The specifi c faces of the cell ). As the specific gravity of lipid is approximatel y hyd r oxyl groups in volved in this linkage were indicJted in a 0.9, and that of ho rn is 1. 3 (determined by us fo r cow ho rn), the previ o us study by in situ deri va ti za ti on of the ceramide 1,3-hy­ lipid of the lucent band should amoullt to about 1. 9% of the droxyl g ro ups w ith acetone 11 51. Half o f the ceramides fo rmed wcig ht of the corneocyte. This is in good agreement w ith th.: a 1 ,3-isopro pyli dene deri va ti ve, showing that their polar head 2% actuall y recovered fro m chemica l attachment to stratum cor­ g ro ups were uncombined in the native state, so that chemical ncum. This ca lculation ca n al so be used to argue that virtuall y att ach m ellt must have been via the w-hydroxyl g roup. For the all o f the chemica ll y bound lipid in the corneocyte is to be fo und remai ning ceramides, fa ilure to fo rm an acetonide pro bably m ea ns in the lu cent band. that one of the sphingosine hydroxyls was derivati zed in the nati ve Third, cytochemical reaction w ith fi lipin indica tes that the cor­ state. While this may have constituted th e linkage to the corn eo­ neocyte m embrane does no t contain cholesterol 11 7]. Present evi­ cyte, the ce ramides that fa iled to fo rm an acetonide might also dence does not eliminate the possibility that unbound ceramides have b een bound to the corneocyte by their w-h yd roxyl fun cti on and free fatty rlcids take part in the corneocyte m embrane and are as VIIe ll as having a sphingosine hydroxyl bl ocked b y some other eliminated in the initial solvent extraction. If this were the case, moiety. Althoug h the results of the acetonide experiment alonc however, there would seem to be no basis for the apparent ex­ cann o t be conclusive, additio nal evidence ca n be assem bled to clusion o f cho lesterol from the membrane. suppo rt the hypo thesis that the w-hydroxyceramides arc attached Fina ll y. in considering the nature o f the substrate to which the to the corneocyte envelo pe, lulf via their w-h ydroxyl gro ups and corncocyte hydroxyceramides might be attached, it is apparent hal f v ia their polar head g ro ups. tlut a ri ch source of otherwise uninvolved ca rboxyl g ro ups is First, multiple measurem ents on electron micrographs con­ required. One is immediately struck by the observation that in­ fi rmed the repo rt by Lavker 161 that the lu cent la yer attached to volu crin , the principal pro tein that becomes bo und into the cor- th e corneocyte envelo pe is about 4 nm across. This is similar to th e thickness of the no npo lar region of a lipid bilayer, and is thc sam e as the length of the hydroxyceramide m olecules mcasured Table II. Detailed Compositio n of from the w-hydroxyl fun ction to the ceramide hea d g roup. This w-Hydroxyacylsphingosin es Released From Isolated, Extracted su ggests tlut the ceramide m olecules arc assembled in cl ose-packed Stratum Corneum by Mild Alkaline H ydrolysis. arra y with the sphingosine chains folded in to the sa me lipid regio n Sphin gosincs as the hydroxyacid c1uins, as shown in Fi gs 5 and 6. Such an

Figure 4. T ransmission el ectron micro­ graph of isolatcd stratum corneum after extrac ti on w ith chl o roform/ methano l fol­ lowed by hydro lys is and extrac tio n of the ester-linked w-h ydroxyacylsphingosines. T he lu cent band normall y seen adj acent to thc protein en vel o pe has been re moved and the protei n envelope has become highl y convoluted and largely torn frce fro m the ce ll contents. Bar = 100 nm.

neocyte envelo pe, contain s 20 glutamate moieties per 100 amino acid s, in addition to the 25 glu ta mine residues that are avail able fo r the E- (y-glutam yl) lys ine isopeptide cross li nks that immo­ bilize the protein in th e corn eocyte envelo pe [1 4, 18, 19]. Thus, it is easy to imagin e regio ns of the in volucrin chain in w hich free ca rboxyl g ro ups are suffi ciently abundant to bind all of the pri­ mary hydroxyl g roups on o ne surface o f a cl osely packed array of hydroxyceramides . Constructi o n of a mo lecular model (Fi g 6) shows that a peptide regio n in which each alternate residue is glutamate, and w hich is in the ,B- pleated sheet confo rmati on, w ill cl osely fi t the positio ns of all of the primary hydroxyl fun ctions o f an alternating array of hydroxyceramides. While it is known that pro tein s having a hi g h pro po rtio n of ioni zed groups are inca pable o f assuming the ,B-pleated sheet con­ fo rmati on, this constraint woul d not apply to the involu crin chains in th e corn eocyte envelo pe if cllJrges o n the glutamate ca rboxyls were neutrali zed by es terifi catio n. Perhaps the expansion of the envelo pe fo ll o wing release o f the es terified lipids res ults fro l11 mutual repul sion o f the liberated y-carboxyl g ro ups of the glu­ Figure 5. T he postulated arrangem ent of w-hydroxyacylsphingosin es es­ terified w ith altern atin g y-glutam yl g roups in a tJ- pleated po lypeptide tamate res idues . chain .

Figure 6. A m olecul ar model showing the favorable spacin g o btain ed w hen an alternatin g array of w-hyd roxyacylsphin­ gosin cs is esteri fie d w ith altern ating g lu­ tam yl rcsid ucs in a tJ-plea ted chain .

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Nanomoters VOL. 88. N O.6 JUN E 1987 T H E CORNEOC YTE LIPI D ENVELOPE 713

In transmission electron micrographs, the lu cent region at­ m odifica tions in keratinizing epithelia. Am J Anat 130:93-120, tached to the corneocyte envelope has the appea ran ce of a normal 197 1 cell m e mbrane. There is little change in this appearance as ce ll s 6. Lavker RM: M embrane coating g ranules: the f.1 tc of thc dischargcd pass fro m the granular layer into the horny layer, other than the lamell ae. J Ultrastruct Res 55:79- 86, 1976 ap pare nt mergin g of the inner dense band o f the membrane with 7. Eli as PM, Grayson S, Lampe MA, Williams ML, Brown BE: The the protein envelo pe. It is natural, therefore, that inves ti ga tors intcrcorneoctye space, in Stratum Corncum. Edited by R Marks, should have referred to this lucent region as the pl as ma membrane G Plewig. Heidelberg, Sprin ger-Verlag, 1983, pp 53-67 of the corneocyte. However, this fea ture is now known to have 8. Hayward AF: Ultrastructural changes in contents of membranc­ little in common with true ce ll membranes, sin ce it contains no coatin g g ranules aftcr extrusion from epitheli al cell s of ham ster phospholi pids [1 ,21. Al so, the li pids of which it is composed, check po uch. Cell Tissue Res 187:323-33 1, 1978 bein g largely saturated, long-chain compounds, are ce rtainly in 9. Downing DT: Phorcdcnsito merr y in the thin layer chrom atographic the gel phase rather than the fluid , liquid crystalline phase of analysis of neutral lipids. J C hroll1Jtog r 38:91-99, 1968 biolog ic membranes, and as a res ult will be highly impermeable 10. Wertz PW, Downing DT: Ceramides of pig epidermis: structure to v.rater. It seems probable that the corneocyte membrane pos­ detcrmination. J Lipid Res 24:759-765, 1983 sesses pass ive physica l properties that arc benefici al, including the II. Wertz PW, Downing DT: Glu cosylccramides of pi g epidermis: provis i o n of cohesion between the protein envelope and th e in­ structure determination. J Lipid Res 24: 11 35-11 39, 1983 tercell ular lamell ae and provision of an efficient barrier to passage 12. Wertz PW, Downin g DT, Freinkel RK, Trazyk TN: Sphingolipids ofv.ratcr into or out of the corneocyte. Beca use of these properties, of the stratum corneum and lamellar granules of fe tal rat epider­ and b ecause of the marked divergence from the properties and mis. J In vest Dermatol 83:193-195, 1984 functio ns of a true pl as ma membrane, it would seem reasonable 13. Mato ltsy AG, Matoltsy MN: The membrane pro tein of horny cell s. to discontinue that designati on. It might perhaps be useful to refer J In vest DermatoI 46: 127-129, 1966 to the bound lipids as the co rneocyte lipid envelope. 14. Simon M , G rcen H : Enzymati c cross-linking ofin volucrin and other protein s by keratinocyte particulates in vi tro. C ell 40:677-683, 1985 15. Wertz PW, Downing DT: ovalentiy bound w-h ydroxyacylsphin­ ](EFERENCES gosin c in the stratum corncum. Biochim Biophys Acta 917: 10S­ III , 1987 1. Gray G M , White RJ , Willi am s RH, Yardley HJ : Lipid composi tio n 16. Plew ig G, Scheuber E, Reuter B, Waidelich W: Thickness of cor­ of the superfi cial stratum corneum cell s of the epid ermis. Br J neocytes, in Stratum Corncum. Edited by R Mark s, G Plcwig. Dermarcl 106:59-63, 1982 H cid elberg, Springer- Verla g, 1983, pp 17 1- 174 2. Long SA, Wertz PW, Strauss JS, Downing DT: Human straml11 17. Kitajima Y, Sekiya T, Mori S, Nozawa Y, Yaoita H: Freeze-fra cture corneul11 polar lipids and desquamation. Arch Dermarcl Res 277: cytochcmical study of membrane systcms in human epidcrmis 284-287, 1985 using filipin as a probc for cholestero l. J In vcst Dermatol 84: 3. E lias PM, Friend DS: The permcability barrier in mammalian epi­ 49-153, 1985 dermis. J Cell Bioi 65: 180-19 1, 1975 18. Peterson LL, Zcttcrgren JG, Wuepper KD: Biochemistry of trans­ 4. Landmann L: Epidermal perm ea bility barrier: transformation of la­ g lutaminascs and cross-linking in the skin. J In vest Dcrlllarol 81 mellar granule-disks into intercellular sheets by a membrane fusion (s uppl):95s-100s, 1983 process, a freeze-fracmre study. J In vest Derlllarci 87:202-209, 19. Simon M, G reen H : Participati on o f m embranc-associated proteins 1986 in the fo rmation of the cross-linked envelo pc of thc keratin ocyte. 5. M a rtinez m., Peters A: Membrane-coatin g g ranules and membrane Cell 36:827-834, 1984