Use of a Serum-Free Epidermal Culture Model to Show Deleterious Effects of Epidermal Growth Factor on Morphogenesis and Differentiation

Chih-Shan J. Chen, Robert M. Lavker, Ulrich Rodeck, * Barbara Risse, * and Pamela J. J ensen Department of Dermatology, University of Pt!nnsylvania School of Medicine. and ' T he Wistar Institute, Philadelphia. Pennsylvania.

U. S.A .

The presence of serum has limited the utility of many culture models for the study of cytokine effects be­cause its complexity and variability can confound the interpretation of data. In the present study, a serum­free skin co-culture model was used to investigate the effect of exogenous epidermal growth factor (EGF) on epidermal proliferation and differentiation. Human keratinocytes cultured on collagen rafts at the air­liquid interface produced a well-differentiated epi­thelium that resembled normal epidermis. Keratin filalnents, membrane-coating granules, and kerato­hyalin granules were all observed. Epidermal differ­entiation markers keratin KlIK10, involucrin, and trans glutaminase were localized in most of the supra­basal layers, whereas profilaggrin/filaggrin was con­fined to the granular layers and stratum corneum. In the continual presence of 10-20 ng/mL EGF, the

To l11aintain normal epiderma l integrity , keratinocytes must proliferate at an appropriate rate and diffe ren­tiate according to an orderly sequence of events. The mechanisms by which keratinocyte proli feration and differentiation are regulated are largely unknown;

however, several growth f.1ctors and cytokines, acting in an auto­crine or paracrine manner, have been implicated in the control of both processes [1-3].

A central role for the epidermal growth factor receptor (EGF-R) and its ligands in the control of epidermal proliferation has been postulated on the basis of many ;/1 vitro as we lJ as ;/1 1/ ;1/0 studi es. Normal epidermis expresses the EGF-R predominantly in the prolifera tive (i.e ., basal) layer [4] . Autocrine activation of the EGF-R is suggested by evidence that keratinocytes ;/1 1/;1/0 and ;/1 1/;11'0 synthesize two of its ligands, tran sforming growth factor-a (TGF-a) [5-8] and amphireguliJl [9 -11 ]; furthermore, both TGF-a and amphiregu lin enhance keratinocyte proliferation [9 ,12,13]. EG F is also mitogenjc for keratinocytes , and although it is not produced by keratinocytes, it has been detected at sites of wound repair [14] . In hyperproUferative conditions of the epidermis, e.g., psoriasis and squamous celJ ca rcinoma , e levated levels of the

Manuscript rece ived June 17, 1994; rev ised August 9 . 1994; accepted for publication August 19. 1994.

Reprint requests to : Dr. Pallleia J. J ensen. Department of Dermatology, University of Pennsylvania, 242 C Rn, 422 C urie 13oulevard. Philadelphia. PA 19104-614 2.

epidermis was less organized, thinner, and less pro­liferative. EGF also depressed several indicators of differentiation: The number of keratohyalin granules and membrane-coating granules was greatly de­creased; antigen expression of profilaggrin/filaggrin appeared diminished by immunocytochemical stain­ing; frequent nuclear retention was noted in the relatively thickened stratum corneum-like layers. As detected by immunohistochemical staining, the ex­pression of EGF receptor in the epidermis was re­duced by exogenous EGF. These data illustrate that EGF cannot be <considered a simple mitogen. Our findings also underscore the importance of using sophisticated culture models to assess complex cyto­kine effects that may be dependent on the architec­ture of a differentiating epidermis. Key II1ol,ds: pl'Olif­el'(/tiolllkemtillocyte. ] l'I11est Demw.(oI104:107-112, 1995

EGF-R, TGF-a, and/or amphiregulin are commonly found [6-8,11,15,16].

Examination of the role of individual cytokines ;/1 1/; ,10 is com­plicated by the multitude of epithelial and m esenchym al factors that con tribute to the compl ex cutaneous microenvironment. Experi­ments usin g cell cultul'e may allow exa mination of the role of cytokine~ in a simpler and more conttoll ed environm en t. However, m any cell culture techniques fai l to reproduce fa ithfully the ;11 1/;,10

epithelial architecture and differentiation process and thus are inadequate for thorough evaluation of the role of cytokines in ke ratinocyte physiology. A considerable advan ce in cell cul ture technology has been the development of a three-dimensional co-culture model consisting of keratinocytes plated on a fibro­blast-contracted gel m aintained at the air-liquid interface [17-20). Although this model yields excellent epidermal differen­tiation and morphology, many previous formulations have included serum as an essential m cdium additive. T he presencc of such a variable. complex, and undefi ned component can confound the effects of individual cytokines on epidermal proliferation and differentiation.

To circumvent this problcm, we have used a serum-free m e­dium, which pennits growth and excellent differentiation of human ke rawl0cytes on a fibroblast-contracted collagen mattix . Use of this model to evaluate the effects of EGF on kcratinocyte prolifcr­ation and differentiation empha~izes thc potential complexity of cytokinc effec ts , somc of which m ay be observed only in the context of a tissue-like structure.

0022-202X/95/S09. 50 • SSDI0022-202X(94)00242-Y • Copyright © 1995 by The Societ)' for Investigative Dermatology. Inc.

107

108 CH EN BT At

MATEIUALS AND METHODS

Cell Culture Human nconatal fo reskin keratinocytc (second to third passage) and fibroblast (sixth to tenth passage) cultures were in itiated and propagated in, respectivcly , MCDB 153 complete medium or Du lbecco's modification ofEaglc's medium (DME) with 10% feta l bovine serum (FBS) , as dcscribed previous ly [21].

Preparation of Chemically Defined Skin Co-Culture T he proce­dure and media formu lations werc modified from prcvio us reports [19-22J. To tissuc cul ture inserts (TransweU-COL, Costar, Cambridge, MA) in six-well plates. we added 1.5 ml of cell-free co llagen solu tion consistin g of 1.25 mg/ml rat tail collagen (Collaborative Biomedical, Bcdford, MA), DME with 10'% FBS, and 50 f.Lg/m l ascorbic acid . A second collagen gel then was prepared similarly to the first, except that neonatal foreskin fi broblasts (1..5 X 10"/ ml) were incorporated and the final collagen concentration was 1.0 mg/ml. After 4-5 d of incubation at 37°C, thc medium expe lled fro m the ge l was aspirated. T he ge l was then washed, and neonatal foreskin keratin ocytes (1.5 X 105 ce lls in 50 f.L1) were seeded on top. After incubation for 1.5 h to allow attachment of the keratinocytes, more medium (5 ml) was added carefull y to cover the surface of the gel. The medium used for this submerged culture period was as follows: DME/F-1 2 mixture (3 :1 ) supplemented with a final concentration of 1.9 mM calcium chloridc (Sigma, St. Louis, MO), 7.25 mM L-glu tamine (JRH. Biosciences), 0.1 8 mM adenine (Sigma), 1 mM strontium chlo tide (Sigma), 1 mM L-scrinc (Sigma), 0.64 mM choline chloride (Sigma) , 0.1 mM ethano lamine and O-phosphoryl-ethanolamine (Sigma) , 2 f.Lg/ml linoleic acid/bovine serum albumin mixture (ratio 1:100; Sigma), 53 nM sclenious acid (Aldri ch, Milwaukee, WI), 5 f.Lg/ml insulin (Sigma), 5 f.Lg/m l trans­ferri n (GlBCO, Grand Island , NY) , 20 I'M triiodothyronine (S igma) , 0.4 f.Lg/ml hydrocortisone (Sigma), 10 nM progesterone (Sigma), 50 f.Lg/mJ gentamicin sul fa te (JRH Biosciences), 15 mM H epes (Sigma), and 10 ng/ml EGF (mouse, culture grade, Collaborative Biomedica l) . After 4 d under submerged conditions, the culture was transferred to the maintenance tray, w hich contains deeper well s (Organogenesis, Canton, MA) , and positioned at the a.ir-liquid interface with the support of a cotton filter pad (Organo­genesis). T he medium was switched to a mixture of DME/F-12 (1:1) containing the above cul ture additivcs, but not EGF or progesterone, for standard cu lturc conditions. In some experiments, we exam.ined the effects of add ition of 20 ng/ml EGF thro ugho ut the air-exposure period. In ea rly experiments, lower concentrations (1-5 ng/ml) ofEGF also were eva lua ted , but these concentrations led to rapid digestion of the co llagen gel, in agreement with a previous report [23 ], and hence made longer-term ana lysis of the effects of EGF impossible. For all experiments, the medium was changed every 2-3 d , and the cul tures were harves ted after 8-10 d of air exposure, unless otherwise noted.

T he production, purification, and characteriza tion of mouse anti-human EGF receptor IgG (MoAb 425) used in this stud y have been described [24,25J.

Light Microscopy C ultures were fixed with 4% paraf('fmaldehydc (Po lysciences, Warrington, PAl in phosphate-buffered sa l.ine overnight, dehydrated through graded ethanols, and embedded in )B-4 compound. Sections (2-3 f.Lm) were stain ed w ith hematoxylin and eos in .

E lectron Microscopy C ultures were fixed in modifi ed Karnovsky's fixative containing 2%1 paraformaldehyde and 2.5%1 g lu taraldehyde (Po ly­sciences) overnight, postfixed in 1 % osmium tetroxide (Po lysciences), dehydrated , and embedded with Epon 812 (Po lysciences) . Sections (400 A) were cut, stained w ith uranyl acetate and lead citrate, and examined with a Hitacl1i H7000 electron microscope.

Immunohistochemistry Frozen sections were sta ined immunohisto­chemica ll y [21 J using the fo llowing primary antibodies: rabbit anti-human involucrin (Biomedical Technologies, Inc., Stoughton, MA) , mouse anti­human kerati nocyte transglu taminasc type 1 (B .Cl clone; Biomedical Techno logies, Inc.), mouse anti-human profi laggrin/fi laggrin (Biomedical Techno logies, Inc.), mouse anti-keratin Kl IK1 0 (AE-20; Dr. T. T. Sun , New York University), and mouse an ti-human EGF-R [24,25J.

Proliferation Assays Cellular proliferation was qu antified by incubation with 10 f.LCi/m l [.l HJthymidine (DuPont-NEN , Boston, MA) in thymidine­free medium for 2 h to detect ce ll s that were in S phase. Sections were processed for autoradiography, and labeling indices were calculated by coun ting at least lOOO nuclei for each experimental condition. T he results were expressed as the number of labeled nuclei per 100 basa l cells. Cell s contain.ing at least 5 gra ins were conside red p ositive.

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a

Figure 1. Epidermal morphogenesis requires several days of air exposure . R ep lica te skin co-cultures were harvested at different time points and processed for jB-4 sections and hematoxylin and eosin sta ining. a: H arvested at the time of air lifting. h, c, ": Harvested after air exposure for 4, 8, and 11 days, respectively . Bar, 50 f.Lm .

RESULTS

Development of the Keratinized Epithelium Requires Sev­eral Days of Air Exposure Neonatal foreskin keratinocytes were plated onto a fibroblast-contracted co llagen matrix anchored on a collagen-coated Teflon fi lter membrane. Incubation was

--VOL. 104. NO. 1 JANUARY 1995

carried out for 4 d, at which time the culture was e levated to the air-liquid interface for a further 8-11-d incubation period. At the end of the subm erged stage, the culture was confluent, but exhibited on ly three to four layers of poorly differentiated cells;

EGF EFFECTS IN A SKIN CO-CULTURE MODEL 109

Figure 2. Ultrastructural identification of differentiation indices. Skin co-cultures were harvested after 8-10 d of air exposure and processed for electron microscopy. a: A basal keratinocyte; b: part of the stratum granulosum and stratum corneum . Note the following structures: k, keratin filaments; D, desmosomes; L, lipid droplets; KG, keratohyalin granules; Nu. nuclei; SC, stratum corneum. Al1'o",s (a) indicate basal cell-collagen matrix interf.1cc. a,b: bars, 3 /-Lm. c: Numerous membrane-coating granules with their characteristic internal lamellar structure in the upper suprabasa l layers. Bar, 1 /-Lm. d: The interf.1ce between stratum granulosum and stratum corneum. Note the exocytosis of a membrane-coating granule (astclisk) and the thickened cell membrane (al1'O"') indicating cornified envelope forma­tion. Bar, 0.25 /-Lm.

neither granular nor cornified layers were apparent (Fig 1a). By 4 d after elevation to the air-liquid interface, the epithelium was more thickened and displayed some features of differentiation, including cornified layers and sparsely distributed keratohyalin granules (Fig 1b). Upon several more days of air exposure, the epithelium exhibited the following features: small , mostly cuboidal basal cells; several stratum spinosum layers; stratum granulosum; and mostly anucleate stratum corneum-like layers (Fig 1e,d). T he terminally differentiated cells did not aPl?ear to slough off~ and hence these layers were thickened at the lo nger incubation times (Fig 1d).

Ultrastructural Feat.ures Are Consistent With a Keratinized Epithelium Ultrastructural analysis revealed that the mature cu ltured epithelium consisted of an organized basal layer (Fig 2a), 11-13 viable suprabasal layers (which included spin ous and gran­ular ce IJs), and compact, electron-dense, stratum corneum-like layers (Fig 2b). Basement membrane structure was not observed; specifically, basal lamina was absent and hemidesmosomes were not organized. Electron-opaque, round lipid droplets were common in all cell layers. Numerous desmosomes were p"esent along the cell-cell borders, in the basal as well as suprabasal layers. The basal cells were usually cuboidal with numerous cytoplasmic organelles, abundant keratin filaments, and a relatively high nucleusl cytoplasm ratio. Supra basal keratinocytes were gradually flattened and ar­ranged in an orderly pattern. The suprabasal layers corresponding to spinous cells were distinguished by the appearance of membrane­coating granules with typical lamellar internal structure (Fig 2e); in some fields of the more superficia.l layers, discharge of the mem­brane-coating granule contents was apparent (Fig 2d). The cells in the uppermost viable layers contained electron-dense keratohyalin granules reminiscent of granular layers ill l1il1o. T he stratum corne­um-like layers were composed of flattened cornified ceUs with thickened membranes (Fig 2d). Most of tlle cornified cells were anucleate, although nuclear remnants were observed in some fi elds .

These electron microscopic observations revealed that many structural and differentiation-related characteristics of human epi­dermis were reproduced in this skin co-culture model.

Localization of Proliferating Cells and Differentiation Markers Proliferative activity was analyzed using 3H thymidi.ne labeli.ng to quantify the number of cells in S phase. Proliferative keratinocytes resided almost. exclusively in the basal layer, and tlle labeling index was 11.9 ::':: 1.4% (average ::':: standard deviation. n = 5).

To examine further the differentiation pattern of tlle ill vitro epithe lium, cultures were processed for frozen sections and stained immunohistochemically with antibodies aga inst several keratillo­cyte differentiation markers, including keratin KlIK10, type 1 transglutaminase, involucrin , and profilaggrin/ filaggrin. As in nor­mal human epidermis, high-molecu lar-weight keratin Kl/Kl 0 was localized in all the suprabasal layers but was not detected in the basa l layer (Fig 3a). Human epiderma.l transglutaminase. the enzyme that m ediates envelope protein cross-linking, was localized along cell -cell borders extending from the low supra basal layers through aJJ the living layers; eBs of the basal and lowest supra basal layers were not stained (Fig 311) . Similar findings were observed for

110 C HEN E1' AL

b

Figure 3. Differentiation markers are present in suprabasal epider­mal layers. Skin co-cultu res, prepared under standard conditions (no EGF was added during the stage of air exposure; a,b,c) or in the presence of 20 ng/m l EGF throughout the 8 d of air exposure (d), were processed for frozen sections. Sections were stained with the foUowing primary antibod­ies: anti-keratin KlIKl 0 (a), anti-ep idennal transglutamin ase type 1 (b) , and anti-profi laggrin/fiJaggrin (c,d). StainiJlg for invo lucrin resembled that of transglutaminase (data not .shown). Comparison of staining intensity in c and d revea ls an attenuating effect ofEGF on profilaggrin/ filaggrin expression. Arrows indicate basal cell-collagen matrix in te rface. Bnr, 50 /Lm.

THE JOURNAL OF INVESTIGATIVE DER.MATOLOGY

involucrin, a maj or component of cornifi ed envelopes generated itt I/itro (data not shown) . Profilaggrin/filaggrin, a marker for kerato­hyalin granules, was detected in the upper supra basal layers, consisten t with the ultrastructural finding that these cells contained abundant keratohyalin granul es; profilaggrin/filaggrin also was detected in the stratum corneum-like layers (Fig 3c) .

EGF Has Deleterious Effects on Epidermal Morphogenesis and Differentiation EGF clearly has been shown to act as a keratinocyte mitogen and hence is o ne of the major growth factors used in epithelial cell cultures and m any skin equivalent m odels . In the skin co-culture model reported here, EGF was present in the submerged stage of culture to promote growth of keratinocytes on the collagen m atrix. Upon air liftiJlg, the culture m edium was switched to an EGF- free formulation and maintained as such thro ughout the second stage of culture (i.e., during air exposure) .

When the standard protocol was varied such that EGF was present throughout the keratinocyte cul ture period (i.e., during air-lifted as well as submerged periods), the epithelium revealed a number of unexpected changes. In the presence of 20 ng/ml of EGF during the en tire culture period, the labeling index was decreased to 6.8 ± 1.3%, a reduction of 43'% from control condi tions. EGF also induced altera tio ns in epidermal mo rphology: large, round basa l cells with a disorgani zed appearance alo ng a relati.vely uneven cell-matrix interface; relatively fewer supra basal layers; disorganizatio n of the suprabasa! layers, with prem aturely differentiated o r dying cells in som e fields; a marked reduction of keratohyalin granules and membrane-coating g~anules; and a thick­ened stra tum corne um with substantial nuclear retention (Fig 4a,c,d). Immunohistochemical analyses suggested an attenuatio n of profil aggrin /filaggrin express ion in the presence of exogeno us EGF (Fig 3d), consistent with the decreased number of keratohyalin granules. T he effects of EGF on epiderm al morphogenesis were abo lished by simultan eous addition of antagonist ,mti-EGF-R an­tibody, confi rming that the effects of EGF on epidermal morpho­genesis were m ediated through the EGF-R (Fig 4b). C ultures in the presence of EGF plus anti-EGF-R antibody w ere m o rph ologically comparable to control cultu res. T hese data indicate that exogenous EGF alters the normal keratinocyte differentia tion process, yielding an abnormal, disorganized epidermis.

EGF Depresses Expression of its R eceptor To begin to evalu ate localization and expression of the EGF-R in the cultured epithe lium, we stained frozen section s immunohistochemically with antibody to the EGF-R . Under standard, control cu lture conditio ns (i. e., EGF present in the submerged stage but absent du!"ing air exposure), EGF-R was detected along the cell-cell borders &om the basal layer to the middle or upper supra basal laye rs (data not shown) . When 20 ng/ml EGF was maintained throughout the stage of air exposure, the expression ofEGF-R was dramatically reduced ; only a barely detectable signal, limited to the basal cells, was observed in some fields. T hese .findings are consistent with down-regulatio n of the receptor upon ligand binding.

DISCUSSION

T he three-dimensional skin co-culture model, introduced by Bell el

al [17], has undergone many improvem ents over time, particularly with regard to m edia formulation .[19,20,26]. In the present study, we describe and characterize a skin co-culture model that permits generatio n of a well-differentiated epidermis in the absence of serum. T he fo llowing features are apparent. Proliferating cells are found almost excl usively in the basal layer , which exhibits a 3H-thymidine labeling index of 11.9%. T his proliferative index is hig her than that observed in normal human epidermis (approxi­mately 2% to 4'X,) and is in the range of that reported for pSOl:iatic les ions (approximately 12% to 24%) or other proliferative epithe lia , e.g., palm and vagiJla [27,28]. Consistent with the hyperprolifera­tive phenotype is the appearance of lipid droplets, which are also found in psoriatic lesions and during wound repair [29 ,30J. Like­wise, the slightly premature expressio n of invo lu crin and transglu­tmninase, beginning in the mid-stratum spinosum, resembles the

VOL. 104. NO . I JANUAR. Y 1995

c

pattern observed 111 psonatlc lesions or epidermis during repair [31-33], as well as in several other skin co-culture mode ls [21,34]. Other features resemble strongly the normal differentiation pattern, including expression of keratin Kl/Kl0 in aU suprabasal layers [35] .

EGF EFFECTS IN A SKIN CO-C ULTUR.E MODEL 111

Figure 4. EGF induces abnormal epidermal morphology and dif­ferentiation. n,b: Skin co-cultures were incubated with 20 ng/ml EGF (a) or 20 ng/ ml EGF plus 5 J.Lg/ ml anti-EGF receptor igG (Il) throughout the stage of air exposure (8 d) and then were processed for light microscopy. Bar, 50 J.Lm. c, d: Skin equivalents were incubated with 20 ng/ ml EGF throughout the stage of air exposure and then were processed for electron microscopy. Note retention of nuclei (Nu) in the stratuJ11 COl11cuJ11-like cells (SC) and dying or prematurely differentiated cells (mml/l) in the supra basal layers. Bnr, 5 J.L111.

Membrane-coating granules, in some fields extruding their con­tents, are common in the upperm ost spinous layers . Expression of profilaggrin /fi laggrin is limited to the granular layer, consistent with the ultrastructural localization of keratohyalin granules. Stl:a­tum corneum-like layers with cornified envelopes and only occa­sional nuclear remnants are o bserved. A notable deficiency is the lack of basem ent m embrane structure, including basal lamina and hernidesmosomes. Our data agree with those of Wilkins el nl [36], who showed excellent morphology at the light-mi croscope level in epidennis generated i/l "i/ro under similar conditions. In sum, the cu ltured epidermis has a hyperproliferative but very well-differen­tiated phenotype and thus represents a good model with which to

analyze the effects of cytokines and growth f.,cto rs on indicators of epidermal differentiation and proliferation.

Although the co-culture model that we describe here does not use serum during the epithelial growth and differentiation stages, it must be noted that the collagen gel is cast in m edium with 10% FBS, as gel contraction occurs more rapidly in the presence of serum. The total volume of FBS added per gel is 0.45 ml , and the gel is washed thoroughly after contraction and before incubation with keratinocytes in a total volume of 5 ml. Hence, the amount of FBS that can remain in the contracted gel and contaminate the medium during the keratinocyte growth phase is smalL

In the present study, we used the skin co-culture model to evaluate the effect of EGF on epidermal proliferation and differen­tiation. EGF is required during the submerged stage of culture for maximal proliferation and rapid generation of a confluent epithelial sheet (Chen, unpublished observations); .iJl contrast, the continu ed presence of EGF during the subsequent air-lifted stage of culture decreases keratinocyte proli feration. In agreement with oW' fllld­ings, Boyce 1'1 nIt showed previously in a similar model that EGF decreased the number of keratinocytes in S phase; however, Turksen el nl [23] found an enhancem ent of proliferation upon short- term addition of TGF-Cl'.

Enhancement of keratinocyte proliferation by EGF and TGF-Cl' has been ascribed to a primary efFect on cell migration [1 2], which allows physical expansion of growing colonies. Therefore. one intriguing in terpretation of our data is that EGF does not promote proliferation during the air-lifted phase of the skin-equivalent cu lture beca use the prolife rative response is not accompanied by migration during this stage . In addition, increased autocrine factor concentration or contact inhjbition, both of which may characterize a more confluent culture, also m ay influence the response to EGF. Our data suggest further that the contin ual presence of high EGF concentrations can induce aberrant differentiation, consistent with previous reports showing that EGF andl or TGF-Cl' promote main­tenance of keratinocytes in a relatively undifferentia ted state (37-39] . T hese data illustrate that EGF cannot be considered a simple mitogen; instead, under certain conditions, it inhibits proliferation and profoundly alters differentiation.

T his sl IIdy IIIns SIIp}J0I1Cd by Nn fiollnl IJlS til lltcs I!f HI'nlth grams .ROJ ·AR-I2998 (1'11), R01-El' 06769 (Rtl'[L) , nlld T J2·A R0 7465 (C·S)C), nlld by De(((srhc

F"rscllllllgsgclll eillscll~/) JU71611-1 (BR).

t Boycc ST, Hoath SB, Wickett R.Tl. Harringer MD. W illiams ML: Loss ofEGF requ ircment by cu imrcd analogues of human skin: biochemical and physiologic analyses (abstr) . J Jll llcst Denllntol 100:579. 1993.

112 C HEN ET At

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