0022- 1 554/85/$33() The Journal of Histochemistry and Cytochemistry Vol. 33, No. 2. pp. 11 1-149. 1985 Copyright 51 1985 by The Histochemical Society. Inc. Printed in (ISA.

Original Articles

Immuno-Ultrastructural Localization of Involucrin in Squamous and Cultured Keratinocytes’

MICHAELJ. WARHOL, JURGEN ROTH, JOHN M. LUCOCQ,

GERALDINE S. PINKUS, and ROBERT H. RICE

Department of Pathology. Brigham and Women’s Hospital and Harvard Medical School (Mj.W.; G.S.P. and Dana Laboratory of Toxicologj. Harvard School ofPublic Health (R.H.R.). Boston, Massachusetts, and the Biocenter (J.R.:J.M.L.). Basel Switzerland

Received! for publication May 16, 1981 and in revised form September 19, 1984; accepted September 25, 1984 ((AOl 1 (

Involucrin immunoreactivity was localized ultrastruc- granular material and to a lesser extent with the cell pe- turally with A-gold in epidermis and cultured niphery. Upon treatment with the ionophore X537A, ker- keratinocytes embedded in Lowicryl K4M. In the skin, atm filaments were found in aggregated arrays and the immunoreactivity was found predominantly in cells of the plasma membranes became convoluted. That involucrin granular layer and inner stratum corneum. The label was immunoreactivity persisted in the cytoplasm in cultured associated primarily with amorphous cytoplasmic material cells and in vivo after cross-linking occurs could account and especially keratohyaline granules. Some labeling was for considerable isopeptide bonding detected in epidermal observed at the cell periphery, but little with fil- keratin fractions and indicates that not all the involucrin aments. Tissue samples examined without aldehyde fixa- participates in envelope formation.

tion showed relatively greater labeling in the outer KIYWORI)S: Squamous epithelium; Involucnmn; Lowicryl KIM; stratum corneum than fixed tissue. In cultured cells, the Protein A-colloidal gold. labeling was also associated primarily with cytoplasmic

cium, thus activating the cellular transglutaminase. This en- Introduction zyme forms stabilized structures by e-(y-glutamyl) lysine cross- The epidermis serves as a barrier protecting the organism from linking (4, 14 ). To help understand the fate ofinvolucrin during the environment. As squamous epithelium matures, structural this process, we have employed a low temperature embedding are synthesized that ultimately perform this protec- technique using Lowicryl K4M to precisely localize involucnin tive function. The final product ofsquamous maturation is the immunoreactivity subcellularly both in vivo and in cultured stratum conneum, which consists of a network of condensed cells. proteins and cellular debris. The keratin class of proteins is the principal component of this integument and is comple- mented by another structural element, the connified envelope Materials and Methods

(8,11,20). Normal human skin was obtained from surgical specimens: mastec- Previous biochemical studies have identified the protein tommes, amputations, and wide resectmons of tumors. Promptly after involucnin as a precursor of the highly cross-linked material surgical removal, the epidermis was separated from the dermis by referred to as envelopes ( 1 3, 1 5 ). Visualized in paraffi n-embed- crude blunt dissection and minced into 1 mm cubes. The epidermis was fixed with four different fixation protocols: 1 ) 1% glutanaldehyde ded tissue sections by immunocytochemical procedures, in- for 2 hr; 2 3% panaformaldehyde-0.P7 glutanaldehyde for 1 hn; 3) volucrin is first detected several cell layers above the basal direct immersion in 3OYc ethanol; and 1) an overnight fixation in half- layer and reaches maximal intensity at the granular layer. Plasma strength Karnovsky’s fixative. Following fixation, both the glutaral- membranes of the granular layer become permeable to cal- dehyde- and paraformaldehyde-fixed skin was washed three times in phosphate buffered saline, pH 7.4 (PBS), once in 0.5 M NH1C1 to quench unneacted aldehyde groups, and finally in PBS. The material fixed initially in half-strength Kannovsky’s fixative was postfixed for 4 ‘Supported by Public Health Service grant no. AM2 1 30 from the National Institute of Arthritis, Diabetes, Digestive and Kidney hr in osmium tetnoxide, and dehydrated and embedded in Epon for Diseases. conventional electron microscopy. Recipient of a grant from the Swiss National Science Foundation. Human epidenmal kenatinocytes were cultivated from circumcision

14!

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Figure 1

Figure 2

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Figure 1. Skin, stratum spinosum. (A) Involucnin immunoreactivity Both the aldehyde-fixeel skin specimens and the skin placed di- of granular, amc)nphous material in the cytoplasm. are nectly in 30C/( ethanol were embedded in Lowicryl KIM as previously unlabeled. (B) The granular material between the tonofibnils is labeled described I l8. The tissues were dehydrated! in I hr mncuhatmons with (small arrow), while dense tonofibnil bundles show little labeling by 30, 50, 0, 100, and 100% ethanol at 35#{176}Cand then incubated with the gold particles (large arrow). Original magnification x 1 5,000. Ban ethanol-Lowicryl mixtures in the ratio of I : 1 and 1 : 2 each for I hr = 200 nm. at - 40#{176}C.Following a 1 hr incubation with l00 Lowicryl KIM at

Figure 2. Skin, stratum granulosum and stratum conneum. (A) Ken- - 40#{176}C,the tissue was again incubated in Lowmcryl KIM overnight at atohyaline granule labeled with anti-mnvolucnin (arrows). Original mag- - 40#{176}(:.The tissue samples were then transferred to fresh Lowicryl nification X 25,000. (B) Kenatohyaline granules labeled with gold ap- in gelatin capsules and polymerization begun by exposing the capsules peaning to cc)alesce into a dense network (arrow). Original magnification tO ultraviolet (uv( light for 24 hr at - .I0C. The capsules were then x 20,000. (C) Granules labeled with anti-involucnin in the stratum brought to room temperature and polymerization was completed by corneum, visualized in unfixed skin embedded in Lowicryl (arrow). further exposure to uv light for 2 hr. Following polymerization, I Original magnification x 30,000. Bar = 100 nm. micron sections were cut and stained with toluidmne blue to select appropriate areas for thin sectioning. Thin (600-800 A) sections were picked up on nickel gniels tn immunochemical studies. All samples, from both skin and cell culture, were subjected to samples with 3T3 feeder layer support according to standard methods identical immunochemical procedures. The grids were initially mncu- ( I 2 ). Confluent cultures were examined with or without treatment with the ionophone X537A (50 jag/mI of serum-free medium) for S bateel with 0.5/ egg albumin for 5 mm at room temperature. They were then incubated with anti-mnvolucnmn at dilutions of I : 1000 or tO 8 hr at 3C as previously described ( 1 5 ). Treatedi cultures became detached from the plastic dishes as a sheet, while untreated cultures I : 1SOO for I hr at room temperature. The anti-involucnin antiserum were removed intact with Dispase (7). These specimens were fixed was that previously described ( I 5 ( and showed immunoreactivity only toward mnvolucnmn upon Western blotting ofhuman skin extracts ( 16). for 1 hr in 3% panafonmaldehyde-0. 1 % glutanaldehyde and processed in a manner identical to the epidermis samples. Aliquots were also Controls included preimmune serum and! antiserum absorbed with purified envelopes. postosmicated and! processed for conventional electron microscopy. Following incubation with antiserum, the grids were washed twice in PBS, and then incubateel with colloidal golel-Staphylococcal protein A complexes. These complexes were prepanedl as previously elescnihedi Figure 3. Anti-involucnin labels the transitional layer IT, arrowheads) ( 17), yielding gold particles Ii nm in dliameten. Following this incu- between the stratum granulosum (G) and the stratum corneum (C). hation, the grids were again washeel in PBS followed! by dmstmlleel water, Abundant label is also present in the stratum granulosum. The outer stained with uranyl acetate for 5 mm followed by leael citrate for 15 stratum conneum exhibits relatively little labeling in this aldehyde- see, and examined! in a JEOLCO JEMS 100 electron microscope. fixeel tissue. Original magnification x 5,000. Bar = 0.4 ja. Tissue postosmicateel and embedidledi in Epon was sectioned! anel stained with uranyl acetate followed! by leadl citrate.

Results

Immunochemical studies of skin sections revealed no involu- cnn immunoreactivity in the stratum basale. As visualized with colloidal gold, involucrin was detected in the stratum spi- nosum, and became most intense in the stratum granulosum and inner stratum conneum. Within the stratum spinosum, the colloidal gold predominantly labeled the amorphous, faintly electron-dense material between tonofilament bundles, with

little staining of tonofilaments or desmosomes (Figure 1 ). In the granular layer, not only was the amorphous material la- beled but also the keratohyaline granules to a striking degree (Figures 2, 3). In the transition layer of the inner stratum corneum, there was marked general staining of the cytoplasmic remnant I Figures 3, 4 ). Keratohyaline granules within this tran- sitional layer became coalescent structures that were also in- tensely labeled. Labeling of the outer layers of the stratum corneum was considerably less intense than the innermost layer (Figure 5). In these experiments, there was essentially no difference in labeling between 3C/ paraformalde- hyde-0.1% glutaraldehyde fixation and lC/r glutaralelehyde fixation. The involucrin staining pattern was also examined in on- fixed tissue placed directly in 30cA ethanol with subsequent dehydration. Involucnin immunoreactivity was retained1, and both the amorphous granular material and keratohyaline gran-

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Figure- 1. Skin, transitional Liver. The network of dense’ lihnmllar material in the transitional layer (T( is heavily Ia- hele’d with the’ antm-mnvolucnmn anti- sereim. The neutrophmlic leukocyte

within this layer ( N ) is essentially unlabeled. Original magnification

x 20,000. R,ir 0. ‘ 5 ja.

r I

ules stained (Figures 2C, 5A). Although this procedure re- Figure 5. Skin stratum dorneum. ) A ) Intense labeling of the stratum corneurn in unhxe’dl skin with some labeling at the periphery of the suitedl in P()() Preservation of ultrastructural eletails, it was cellular remnants )arrowheadls). I B) In osmicatedi and Epon-embedidiedi clear that the outer stratum corneum was relatively heavily tissue, the stratum corneum exhibits a pd’nipheral electron-diense handi labeleel compared to fixed samples. There was not only diffuse (arrowheads). This feature is not ol)servedl in unosmicatedl tissue. Onig- labeling within the cells, hut also labeling of the cell periphery inal magnification X 20,000. Bar - )() nm. (Figure 5A). Control girds incubated with 1)0th nonimmune anel at)- sorl)eel antisera revealed only backgrounel staining ofveny sparse, randomly scattered gold particles (Figure 6). Figure 6. Control samples. (A) Skin treated! with preimmune serum l’he morphology of the cultured cells with and without revealing only weak background staining within the stratum granu- monophore iS seen in Figures 7_9 Without monophore treat- losum )G). The keratohyalmne granules within this layer (arrows) are

unlabeled. Original magnification x 10,000. Bar = 100 nm. (B) Skin ment, individual intermediate filaments are arrangeel diffusely treateel with antiserum absorheel with purified! “envelopes.” Both the throughout the cells. There is little granular material in the granular layer (G( anel the transition zone IT) are unlaheledi. Remnants cytoplasm. Cell borders are relatively straight with few cell of keratohyalmne granules are seen in the transition layer (anrow(.

junctions. After treatment with ionophore, the cells have a 1)niginaI magnification X 5,000. Bar = 200 nm.

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Figures 5 and 6

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Figure 7

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Figure . (;en cultures. (A) Cultured cell fixedi in glutanaldehyde and Discussion osmium exhibiting abundiant cytoplasmic filaments. Original magnm- The dlistribution of mnvolucrin we observedi in normal skin fication X 2,000. (B) (;ultunedi cell with randomly onmentedl filaments (arrowheadls) anel a cell hordier with few intendimgitations. Original mag- closely parallels that previously described by light microscopy

nification X 1 0,000. Bar = .100 nm. (C) Ionophone-tneateei cell show- offixed sections (2). lnvolucrmn is first detectable in the stratum ing exagj.erateei mntereiigmtatmons and cytoplasmic collections of gran- spinosum and! reaches maximal levels in the stratum granu- ular material (higher magnification in E) (arrows(. Original magnification losum and stratum corneum. The staining appears cytoplasmic x 2,000. ( D lonophore-treatedi cell(s) with tonofilaments organized! into thick bundles (arrowheadis( and dlesmosome-tonofilaments com- and distinct from intermedliate filaments, consistent with bio- plexes (arrow). Original magnification x 8,000. (E) Granular material chemical observations that mnvolucrin behaves as a water-sol- in the cytoplasm of ionophore-tneated cell coalescing into lang- uble protein when purified! from cultured keratinocytes ( I 5). en granular structure (arrow). Original magnification x 5,000. Within the stratum granulosum, the anti-involucrin anti- Ban = 300 nm. serum stains not only amorphous material in the cytoplasm but also keratohyaline granules. These granules are known to contain a high molecular weight precursor of (9), a dramatically different appearance. The intermediate filaments basic protein proposed to induce keratin aggregation during become organized! into thick interwoven bundles, many of terminal differentiation (3). The pattern of filaggrin immu- which insert into diesmosomes, and granular material is visible noreactivity in human skin ( 10) is rather similar to that of between them. In some areas this granular material appears involucnin, although these proteins are biochemically andl im- to coalesce into larger granules. Cell borders show extreme munochemically distinct. This similarity in temporal expres- interdigitations, and desmosomes are more numerous. sion and localization emphasizes the profound change in char- Labeling of cultured cells with anti-involucnin reveals rel- acter of the epithelium between the stratum spinosum and atively little immunoreactivity within the cells not treated with corneum and! raises the possibility that involucrin, as a con- ionophore. The involucnin appears to be present both at the stituent of the granules, has some functional significance in periphery as well as within the cytoplasm of the cells (Figure the cytoplasm. The insertion of keratin tonofibnils into kera- 8). The ionophore-treated cells have a marked increase in tohyaline granules, pant of a elense meshwork in the transition cytoplasmic immunoreactivity. There also appears to be coa- layer between the granular and cornified layers, suggests that lescence of the involucnin into granules. In contrast to the the granules may serve as a scaffolding for the interlacing untreated cells, labeling at the cell periphery is difficult to keratin network in the stratum corneum. Formation of kera- discern. tohyaline granules appears to he an element of the maturation

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program yielding stratum corneum. Involucrin is expressed! in come incorporated into cross-linked envelopes adjacent to the normal esophageal and vaginal epmthelia, however, which lack plasma membrane, even though (in culture) it is not extractable

both strata cornea and keratohyalmne granules. after the cross-linking process takes place ( 1 S ). The data do The results with cultured keratinocytes mimic those with suggest some chemical alteration of mnvolucrin between the epielermal tissue in certain respects. The cells not treated with transition cell layer and the stratum corneum. Compared with ionophore resemble basaloid epithelmum in expression of ran- tissue placed directly in ethanol, the aldehyde-fixeel tissue cx- diomly oriented individual intermedliate filaments. After treat- hibits diminishing staining of the outer stratum corneum. In ment with monophore, the effect of altered ionic conditions is the outer cells, continued transglutaminase cross-linking could manifest in the organization of keratin into tonofibril bundles, make the reduction of mmmunoreactivity by fixation more an increase in desmosomes and tonofilament- pronounced.

complexes, and some coalescence of involucrin into granular Q uantitative evaluation of relative mnvolucrin disposition is cytoplasmic structures. Although introduced divalent ions may not possible at present in view of reduced antigenicmty in al- stimulate fibnil aggregation (5), involvement ofgranular struc- dehye!e-fixed tissue (2) and uncertain access of the antibody

tures in consolidating the keratin network is possible. In any tO highly cross-linked envelope structures in skin. The en- case, the persistence of involucnin in the cytoplasm after cross- velopes visible in mature squames of skin were not evident in linking takes place in cultured cells and in vivo could account the cultured cells after ionophore treatment, although the cell for a considerable fraction of the isopeptide bonding neporteel borders comprised detergent-resistent structures of dliameten in epidermal keratin ( 1 ), which occurs at a low level in com- comparable to the plasma membrane. Thus under the artificial parison with that in envelopes ( I 3). condition of ionophore treatment, these structures are stabi- While evidence for some localization of mnvolucnin at the lized! rapidly, whereas the much slower spontaneous cross- cell border was obtained in epidermis and cultured epidermal linking process in suspension culture can yielei the thicker cells, the observed involucrin immunoreactivity was consid- envelopes seen in skin (6). The biochemical events leading to erably more striking in the cytoplasm of maturing and even formation of these latter structures remain to he elucidated mature squames as well as the cultured cells. This finding but appear to involve several membrane-associated proteins indicates that a substantial amount of involucrin does not be- in ae!dimtion to involucrin ( I d))

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Acknowledgments 1 1. Matoltsy AG, Balsamo CA: A study of the components of con- nified epmthelmum of human skin. J Biophys Biochem Cytol I : 339, The authors thank Drs. H. Green and T.-T. Sunfor valuable advice. Ms. 1955 Christine Ridol.fi and AK. Bie/stein for technical assistance, Mr. Anthony Merolaforphotographic work, andMs. Ann Benoitforsecretarialassistance. 12. RheinwaldJG: Serial cultivation ofnonmal human epidermal ken- atinocytes. Meth Cell Biol 2 1 :229, 1980 1 3. Rice RH, Green H: The cornified envelope of terminally d!iffer- L iterature Cited entiated human epidenmal keratinocytes consists of cross-linked protein. Cell 1 1:417, I97 1. Abernathy JL, Hill RL, Goldsmith LA: #{128}-(‘y-Glutamyl) lysine cross- links in human stratum conneum. J Biol Chem 252: 1837, 1977 14. Rice RH, Green H: Relationship of protein synthesis and! trans- glutaminase activity to formation of the cross-linked envelope 2. Banks-Schlegel 5, Green H: Involucnin synthesis and tissue as- during terminal differentiation ofcultuned human epidenmal ken- sembly by kenatinocytes in natural and cultured cells. J Cell Biol atinocytes. J Cell Biol 6:705, 1978 90:732, 1981 I 5. Rice RH, Green H: Presence in human epidermal cells of a sol- 3. Dale BA, Holbrook KA, Steinert PM: Assembly of stratum con- uble protein precursor of the cross-linked envelope: activation of neum basic protein and keratin filaments in macnofibnils. Nature the cross-linking by calcium ions. Cell 18:681, 1979 276:729, 1978 16. Rice RA, Thachen SM: Involucnin: a constituent of cross-linked 4. FolkJE, FinlaysonJS: The #{128}-(‘y-glutamyl) lysine cross-link and the envelopes and marker ofsquamous maturation. In Biology of the catalytic role oftransglutaminases. Adv Protein Chem 31: 1, 1977 Integument. Edited by J Beneiter-Hahn, AG Matoltsy, KS Rich-

5. Fukuyama K, Murozuka T, Caldwell R, Epstein WL: Divalent ards. Springer-Verlag, Heidelberg, in press cation stimulation of in vitro fiber assembly from epmdenmal ken- 1 7. Roth J, Bendayan M, Onci L: Ultrastructunal localization of in- atm protein. J Cell Sci 33:255, 1978 tracellulan antigens by the use of protein A-gold complex. J His-

6. Green H: Terminal differentiation ofcultured human epidermal tochem Cytochem 26:l04, 1981 cells. Cell 11:405, 1977 18. RothJ, Bendayan M, Canlemalm E, Villiger W, Garavito M: En-

7. Green H, Kehinde 0, Thomas J: Growth of cultured epidenmal hancement of structural preservation of immunocytochemmcal cells into multiple epithelia suitable for grafting. Proc NatI Acad staining in low-temperature embedded pancreatic tissue. J His- Sci USA 76:5665, 1979 tochem Cytochem 29:663, 1981

8. Green H: The kenatinocyte as differentiated cell type. Harvey 19. Simon M, Green H: Participation of membrane-associated pro- Lect 74:101, 1979 teins in the formation of the cross-linked envelope of the kera- tinocyte. Cell 36:327, 1984 9. Handing CR, Scott KR: Histidine-nich-proteins (filaggnins): struc- tunal and functional heterogeneity during epidermal diffenentia- 20. Sun T-T, Green H: Differentiation ofthe epidermal keratmnocyte tion. J Mol Biol 170:651, 1983 in cell culture: formation of the connmfied envelope. Cell 9:11, 1976 10. Lynley AM, Dale BA: The characterization of human epidenmal filaggnin. A histidine-nich, keratin filament-aggregating protein. Biochim Biophys Acta 744:28, 1983

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