fifty years of cell biology in the journal of

Fifty Years of Cell Biology in The Journal ofInvestigative DermatologyEdward J. O’Keefe, M.D.

CELL BIOLOGYAlthough the culture of skin in explant culture and the culture ofepidermal keratinocytes comprise the dominant theme in cell biologydescribed in The Journal of Investigative Dermatology in its 50 years ofpublication, only in the last half of that time, since about 1960, havethese studies been numerous. In the last 10 years or less, the utility of thenow established culture methods has enabled the beginning of moreincisive mechanistic studies, which are too varied to describe in a shortessay except in a very limited way. Therefore, I have chosen to trace thedevelopment of methods of culturing epithelium and to select points ofinvestigations made possible by these methods, and then to note somestudies in cell biology not dependent on culture methods. Many areas ofinvestigation, such as pigment cell culture, Langerhans cells, immuno-logic studies, ultrastructure, extracellular matrix, and epidermal kinetics,have been omitted or are discussed elsewhere in this issue.

Organ Culture of Skin Attempts at cell culture predate organ culture instudies published in the JID, but organ culture was probably morewidespread in the earlier years. In 1966, Karasek [1] studied epithelialoutgrowth from epidermis in vitro and showed that the process involvedDNA synthesis, not just spreading or migration of the cells. Rothberg [2]was able to cultivate embryonic chicken skin in a chemically definedmedium and studied the response of the epidermis to vitamin A, whichreduced stratification and synthesis of mucopolysaccharide. Chopra andFlaxman [3] found that retinoic acid increased labeling index andgrowth, but particularly increased the area covered by keratinocyteexplants after an extended period of culture (5 wk).

In 1967, Flaxman et al [4] studied maturation of cells and tissueorganization of epithelial outgrowths from skin explants and noted thatcells attached to plastic were the least differentiated, indicating that thelocation of nutrients did not dictate differentiation, as the maturationgradient was opposite to that predicted on the basis of availability ofnutrients. These authors felt that attachment was important in determin-ing proliferative potential of epithelial cells. Reaven and Cox [5] culturedadult human skin in defined medium and studied the effects of tapestripping on subsequent culture as well as the effects of temperature,oxygen tension, pH, and serum. Tape stripping was found to produceincreased in vitro labeling after 3–4 d of culture. Bishop and Cox [6]found that a 10-fold increase in labeled mitoses after the fourth day ofculture followed tape stripping of human skin subsequently studiedin vitro. This remarkable finding, which indicates that skin stimulated bytape stripping ‘‘remembers’’ the event even after transfer to in vitroconditions, requires further study. Halprin et al [7] cultured mouse skinexplants in vitro and demonstrated growth and differentiation.

In 1970, Singh and Hardy [8] used a novel method to cultureembryonic rat skin. These authors implanted skin explants in intraper-itoneal chambers in rats and found that the tissue was still growing after25 d. Kreider et al [9] transplanted human skin to the hamster cheekpouch, which had in turn been grafted on the thoracic wall, because theybelieved that the cheek pouch was ‘‘immunologically privileged’’ and,therefore, could tolerate a homograft. Survival of the graft wasdemonstrated for 6–8 wk and was more prolonged if the hamster hadbeen treated with cortisone. Frater and Whitmore [10] were able toculture newborn mouse follicles in a collagen gel only if a tryptic digestof mouse embryo extract was present in the medium, even though theepithelium would grow without such extract. It was of interest that theembryo extract had to be digested with trypsin to be effective in hairfollicle growth. Frater [11] subsequently demonstrated differentiation ofmurine follicular cells in vitro and showed that this occurred only in thepresence of the embryo extract. Flaxman and Harper [12] were able toculture organ explants of skin for 7 d in defined medium, but DNAsynthesis was observed for only 5 d without serum. Stenn and Stenn [13]also cultured epithelial explants in defined medium using adult mouseesophagus, but found that the tissue was viable for only 3 d.

The putative epidermal ‘‘chalone,’’ a concept championed by Elgjo[14], was studied by Chopra et al [15], among others. Usingautoradiography to assess growth, these authors found that human skinextracts were inhibitory to the growth of human epidermis in organculture even in the absence of epinephrine, which had been thought tobe necessary to demonstrate the effects of the chalone. The chalonequestion was also addressed by Rothberg and Arp [16], who demon-strated inhibition of DNA synthesis by epidermal extracts and no effect ofepinephrine. This complex problem was reviewed by Duell et al [17].More recently, Elgjo et al [18] have described a purified pentapeptidewith characteristics of the chalone.

The role of cyclic AMP in growth of epithelium in organ culture hasbeen described in some detail in the Journal. Harper et al [19] soughtdifferences between psoriatic and normal skin in outgrowth cultures byperturbing growth with dibutyryl cyclic AMP and other agents affectingcyclic AMP (epinephrine, isoproterenol, histamine, sodium fluoride, andtheophylline). Agents elevating cyclic AMP inhibited growth but therewas no difference in the results using psoriatic tissue. Kariniemi [20]cultured psoriatic and normal human skin in diffusion chambers inintraperitoneal chambers in mice and found no difference in autoradio-graphic labeling of explants. These findings are in agreement with studiesof cultured cells. Taylor et al [21], in further studies of skin explants,showed that theophylline and other agents, which inhibit DNA synthesisat high concentrations, may do so not by stimulating cyclic AMP but byanother, unknown mechanism, as concentrations that were shown toraise cyclic AMP were not necessarily inhibitory. The action of agentsstimulating cyclic AMP may be complex, because stimulation of cyclicAMP concentrations is associated with stimulation of proliferation, notinhibition of growth, in keratinocyte cell culture (see below).

Nieland et al [22] used organ culture to try to investigate theproduction in the epithelium of glycogen, which they noted accom-panied inflammation or injury. Glycogen accumulation in vitro wasstimulated by galactosamine but not by other simple sugars. In 1975,

0022-202X/89/$03.50 Copyright & 1989 by The Society for Investigative Dermatology, Inc.

105S

REVIEW ARTICLE

This work was supported by National Institutes of Health grants AR25871 andAR36497.

Department of Dermatology, University of North Carolina School ofMedicine, Chapel Hill, North Carolina, U.S.A.

Abbreviations: Con-A, concanavalin A; EGF, epidermal growth factor; ETAF,epidermal thymocyte activating factor; IL-1, interleukin 1

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by Elsevier - Publisher Connector

https://core.ac.uk/display/82555264?utm_source=pdf&utm_medium=banner&utm_campaign=pdf-decoration-v1

Flaxman and Harper [23] studied the in vitro control of keratinocyteproliferation in explants by various physiologic and pharmacologicagents, further defining the in vitro behavior of skin in organ culture.Growth was inhibited by histamine and the inhibitory effect reversed byantihistamines [24]. Later, Aoyagi et al [25] also found that histamineinhibited explant growth from pig epidermis, an effect that was preventedby the more selective antihistamine, cimetidine. Flaxman et al [26] foundthat methotrexate was inhibitory to keratinocyte proliferation in vitro, incontrast with the findings of Liu and Karasek [27] in relatively quiescenthigh-density cell cultures, and suggested that de novo synthesis ofnucleotides was probably of significance in the outgrowth cultures. Holt[28] found that skin organ culture showed enhanced growth in responseto thyroid hormone, triiodothyronine, and thought this probablyrepresented a direct effect on the epithelium not mediated through thedermal component. The expression of basement membrane antigens byorgan explants was examined by Didierjean et al [29], who found thatthe epithelium as able to elaborate its characteristic antigens even ondead pig dermis.

Cell Culture of Epidermis In 1957, Wheeler et al [30] described thedevelopment in culture of an apparently transformed human epithelialcell derived from a biopsy of normal skin. The mechanism of thisphenomenon was not clear. In an early paper on methods of culturingisolated epidermal cells published in the JID Cruickshank et al [31], in1960, described the separation of dermis from epidermis in a trypsinsolution, production of an epidermal cell suspension, and plating of thesein high density on a glass surface. This methodology was developedfurther by Briggaman et al [32] in a study in which postembryonic humancells were cultured successfully for longer periods. In 1970, Laerum andBoyum [33] developed a method for separation of basal cells from moredifferentiated keratinocytes and cultured basal cells from hairless mouseepidermis intraperitoneally in plastic chambers, in which the cells wereisolated from the environment by Millipore filters. In 1970, Yuspa et al[34] reported the growth of fetal mouse keratinocytes in culture for 4–5 dand subsequent replating as a single cell preparation in chambers on thebacks of mice. These grafted cells contained both fibroblasts andkeratinocytes, and the grafts produced follicles and hair and alsopigment. The production of follicles in the skin presumably dependedon the fetal nature of the transplanted cells.

Karasek and Charlton [35] subsequently reported the growth ofpostembryonic skin epithelial cells using collagen gels. Growth wasimproved by the collagen and also by conditioned medium. Moore andKarasek [36] used the method of Laerum and Boyum [33] to separatebasal and differentiated cells and successfully cultivated postembryonicepithelium from mouse, rabbit, and human. They found that cells thatattach to collagen were the germinative population; that is, were capableof synthesizing DNA and protein. These cells could be discriminated bytheir characteristically high ratio of nucleus to cytoplasm. Vaughan andBernstein [37] also believed that trypsinized rat skin released basal cellsfirst when epidermis was agitated in solution, as these were the cells thatincorporated thymidine in culture. Fusenig and Worst [38] describedprolonged culture of adult mouse epidermal cells for the first time andshowed minimal contamination by fibroblasts.

The introduction of specific growth factors enhanced the success ofculture and permitted mechanistic studies. In 1963, Cohen and Elliott[39] described the action of a purified protein from the sub-maxillarygland of the mouse, which was capable of eliciting precocious separationof the eyelids, a phenomenon that depended on the keratinization of thetwo opposing lids (Fig 1), and also the premature eruption of teeth innewborn mice. A subsequent review noted that because the factor,known as epidermal growth factor (EGF), could stimulate the growth ofisolated embryonic epithelium and corneal epithelium, it was clearlyacting not through the dermis but on epidermal cells [40]. Another agent,retinoic acid, was found by Christophers [41] to be useful in the cultureof guinea pig epidermal cells. In cells grown with McCoy’s medium and10% fetal calf serum, retinoic acid appeared to increase attachment ofcells and enhanced DNA synthesis, apparently in agreement with the

previously noted in vivo stimulation associated with topical applicationor oral administration in vitamin A-deficient animals as described bySherman [42]. Stimulation of growth in vitro by retinoids has previouslybeen described in skin organ culture [3], but was recently shown by Tonget al [43] to occur in cell culture if the appropriate limiting conditions canbe found.

Inclusion of proceedings of the annual symposia on the biology ofskin has provided important reviews of various areas of skin biology inthe Journal. In the July 1975 issue, entitled ‘‘The Epidermis,’’ Karasekreviewed the growth and maturation of epithelial cells in vitro frompostembryonic skin. Liu and Karasek [27] later reported improvements inepithelial cell culture in an important study. They plated rabbitkeratinocytes at high density and found that negligible DNA synthesisoccurred in the primary culture; they were then able to passage the cells,at which time they demonstrated an increase in cell number andthymidine incorporation and associated sensitivity to methotrexate notdemonstrable in the primary culture. These investigators also reportedthat both collagen gels and L-serine enhanced proliferative growth. Asubsequent paper [44] further described the use of collagen gels,L-serine, and corticosteroids in cultures. L-serine (0.4 mM) anddexamethasone (1 mg/ml) were stimulatory to growth in these culturesof human epidermal keratinocytes. Also in 1978, Carpenter describedwhat had been learned about EGF in the 15 years since its discovery in animportant review. Elsewhere, Rheinwald and Green described the clonalgrowth of keratinocytes on 3T3 feeder layers [45] and also showed thatthe keratinocytes proliferated in response to EGF [46] (Fig 2). In avariation of the method of Rheinwald and Green, Kondo et al [47]described the growth of human epidermal cells on 3T3 feeder layersusing Millipore filters to facilitate examination of epithelial growthin vitro.

In attempts to examine the biology of cultured keratinocytes,Gilchrest [48] studied actinically damaged or protected skin and foundthat keratinocytes from protected skin produced more generations in vitroand that a reduction in generations was proportional to the severity of theactinic damage and the age of the donor. It was found that platingefficiency was substantially higher for the damaged skin. This wasthought possibly to reflect the promoting activity of the actinic damage,which may have increased the proliferative activity of the basal cellpopulation, while at the same time shortening the apparent longevity ofthe keratinocytes because of their advanced apparent senescence. In1979, Prunieras [49] reviewed the field of cell culture with particularemphasis on the problem of the study of morphogenesis using tissueculture and commented on the eventual possibility of exploring theinteraction of keratinocytes with other cultured cells such as Langerhanscells or melanocytes.

In 1980, Stanley et al [50] extended the experiments of Moore andKarasek [36]. Studying the cells that attach to collagen, which had earlier

Figure 1. Cross sections of the eyelid area from control (a) and experimental

(b) 8-d-old rats. The experimental animal had received daily injections (1 mg/g

body weight) of the active protein. (�100.) From Cohen and Elliott [39].

106S O’KEEFE THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

been found to be the germinative population, these investigators foundthat such cells could also be identified by their content of pemphigoidantigen and the absence of an upper cytoplasmic antigen, confirming thatthe cells that adhere to collagen were basal cells.

In 1980, Hawley-Nelson et al [51] described a method for the cultureof human epidermal cells that permitted subculture 2–3 times and usedreduced calcium concentrations, which these investigators had pre-viously found to prevent stratification of mouse keratinocytes. A collagensubstrate was also found to enhance growth in this culture system.Because calcium stimulates differentiation, a calcium signal could alsobe used to detect malignant or possibly premalignant cells altered bycarcinogens, as these cells responded poorly to calcium and did notterminally differentiate and cease to proliferate (Fig 3); this finding hasprovided an important in vitro correlate of carcinogenesis [52].

In another example of the utility epidermal cell culture in elucidatingmechanisms, Yaar et al [53] found that retinoic acid decreased theterminal differentiation of keratinocytes, as assessed by measurement ofthe production of envelopes and cross-linked keratin,with maximaleffects at 0.5–5 mg/ml. Milstone et al [54] found that retinoic acid causedpremature desquamation of cells from confluent cultures of keratino-cytes. Furthermore, the desquamated cells had a reduced ratio of proteinto DNA, indicating that they were less fully differentiated in the presenceof retinoic acid. Differences were found between calf esophagus andhuman foreskin keratinocytes. Retinoic acid decreased proliferation ofthe calf cells. In the case of human foreskin cells, which showed areduced proliferation after weeks in culture in control cultures, thedecrease was reduced in the presence of retinoic acid (3.3mM). Thethickness of the culture and the numbers of cell layers were reduced byretinoic acid in both types of cultures.

The action of vitamin D on cultured keratinocytes has been reportedin the Journal. Smith et al [55] grew keratinocytes in serum-freeconditions and demonstrated a dose-dependent decrease in basal cells,an increase in desquamating cells, and inhibition of DNA synthesis bythe active form of vitamin D, 1,25-dihydroxy vitamin D3, and suggestedthat this hormone may play a role in differentiation and development.These investigators also showed that the action of vitamin D was relatedto the concentration of the receptor in the skin [56].

Immunologic studies began to be directed at keratinocyte cultures. In1982, Morhenn et al [57] showed that cultured human epidermal cellsdid not have characteristics of Langerhans cells by rosetting oferythrocytes or electron microscopy, suggesting that the Langerhanscells were lost during culture. They also proposed a method for theculture of keratinocytes on a gelatin membrane without plastic. An

immunologic function was ascribed to epidermal cells after Sauder et al[58] demonstrated the production of epidermal thymocyte activatingfactor (ETAF) in keratinocyte cultures. Luger et al [59] also found that aninterleukin l(IL-l)-like factor was produced by cultured keratinocytes. Thematerial in their supernatants increased IL-2 production by humanperipheral blood lymphocytes in the presence of concanavalin A(Con-A), was mitogenic for fibroblasts, was chemotactic for neutrophils,and increased serum amyloid A production by murine hepatocytes. TheETAF activity was increased by sublethal doses of ultraviolet light [60]both in vitro and in vivo, a finding with major implications for cutaneousbiology (Fig 4). Kupper and McGuire [61] found that micromolarconcentrations of hydrocortisone suppressed production of ETAF. Goslenet al [62] found that extracts of basal cell carcinomas could stimulatecollagenase production from fibroblasts and suggested that these extractsmight contain ETAF or other IL-l-like material. Interleukin-1 had beenshown elsewhere to enhance collagenase production by fibroblasts.Hernandez et al [63] established basal cell carcinoma cells in culture andfound that supernatants from these cultures increased collagenaseproduction. The supernatants also increased thymocyte proliferation,which indicated that they contained an IL-1-like material. These authorssuggested that the basal cells demonstrated aberrant release of themediator of this kind of activity. Evidence was also presented byWoodley et al [64] that keratinocytes were able to produce collageno-lytic activity against collagen types I and IV, a finding that led to thedemonstration by Petersen et al [65] of the production of collagenase bykeratinocyte cultures. Interleukin-1 did not stimulate collagenase inkeratinocytes.

A novel phenomenon in keratinocyte cultures reported by Ristow [66]was the partial arrest of growth of mouse epidermal keratinocytes bytreatment with serum-free medium containing 0.06 mM calcium for 4 d.Peripheral blood leukocytes were shown to stimulate such cultures toincorporate tritiated thymidine and label a substantial proportion of theirnuclei. More recently, Ristow [67] has reported elsewhere the stimulationof growth of keratinocytes byIL-1.

At a time of major interest in the role of cyclic AMP in cellulargrowth, several laboratories published studies of the possible role of

Figure 2. Effect of EGF on growth of keratinocytes. Numbers indicate ng/ml of

EGF. From Rheinwald and Green [46].Figure 3. Effect of calcium switch on DNA synthesis. Primary keratinocytes or

cells from lines 308, D, and F were each grown to confluence in medium with

0.05 mM calcium, then switched to medium with 1.2 mM calcium. [3H]

Thymidine (1 mCi/ml) was added 23 h after the calcium switch, and cells were

harvested at 24 h. The specific activity of DNA (cpm/mg DNA) was

determined as a measure of the rate of DNA synthesis. Values in low-calcium

medium are indicated by the open bars; values in high-calcium are shown by

the solid bars. From Hawley-Nelson et al [51].

VOL. 92, NO. 4, SUPPLEMENT, APRIL 1989 FIFTY YEARS OF CELL BIOLOGY 107S

cyclic nucleotides in keratinocyte growth in the JID. Wilkinson et al [68]found that cyclic AMP concentrations varied with the cell cycle inkeratinocytes; concentrations were low in G1 and high in cells with highDNA content (G2) as shown by flow cytometry. S-phase could not beassessed specificially. Effects of cyclic AMP were also studied by Okadaet al [69], who found that cholera toxin produced 100-fold increases incyclic AMP but was effective in increasing growth of keratinocytes, ashas been demonstrated elsewhere by Green [70], only in subconfluentcells in cultures. The cultures of Okada et al were plated in high calciumin the presence of serum. In other studies related to cyclic AMP,Orenberg et al [71] reviewed findings for adrenergic receptors in culturedepidermal keratinocytes, which are sensitive to catecholamines, hista-mine, adenosine, and PGE1 and PGE2 and found that the keratinocytereceptor behaved like a beta2 receptor. This conclusion was based onintracellular cyclic AMP concentrations after incubation for 2 h in thepresence of a phosphodiesterase inhibitor.

Other types of receptors have also been studied in keratinocytes inculture. O’Keefe et al [72] demonstrated the presence of EGF receptors incultured keratinocytes and showed that the binding of EGF wasdramatically increased by reduction of calcium ion, a condition thatfavored keratinocyte growth [73]. Fleckman et al [74] found that EGFstimulated ornithine decarboxylase activity in keratinocyte cultures.Baden and Kubilus [75] reported production of rat keratinocyte lines fromcultures of newborn rat keratinocytes grown in the absence of EGF. Thesecells were able to grow without 3T3 cells and were polyploid but wouldnot grow in soft agar and had desmosomes, cell envelopes, andkeratohyalin granules. Misra et al [76] demonstrated the binding ofinsulinlike growth factor I/somatomedin-C in human keratinocytes inculture.

Isseroff et al [77] demonstrated the presence of plasminogen activatorin differentiating mouse keratinocytes and found that activity was highestin shed cells and increased at confluence. The plasminogen activatoractivity peaked as DNA synthesis declined. Subsequently, these authors[78] found that plasminogen was demonstrable in the basal layer of theepidermis when antiplasminogen antibody was used to stain theepidermis. An instructive point in this paper was the requirement forthe demonstration of plasminogen for affinity-purified antibody to reduce

background. The authors proposed that this demonstration providedevidence that plasmin, generated by epidermal plasminogen activatorfrom plasminogen, may have a role in epidermal differentiation.Furthermore, the location of plasminogen in the basal layer suggestedthat it may be consumed during the course of differentiation. Hashimotoet al [79] also demonstrated plasminogen activator in culturedkeratinocytes and showed that conditioned medium could cleave fibrin.The activity was stimulated by cholera toxin or EGF. Morioka et al [80]found that migrating keratinocytes express urokinase-type plasminogenactivator at the migrating edge of a wound. They proposed that theenzyme may cleave fibronectin and that such cleavage may be necessaryfor migration of cells.

Although there was great hope that the use of cell culture wouldpermit the identification of in vitro correlates for the psoriatic phenotype,Baden et al [81] found no differences in growth characteristics of normaland psoriatic keratinocytes alone or of keratinocytes grown with psoriaticor normal fibroblasts as a feeder layer. Liu and Parsons [82] found nodifference in growth rate, cell detachment from culture, metaboliclabeling with leucine or thymidine, or mitotic cells between psoriatic andnormal keratinocytes in an extensive in vitro study.

Keratinocyte cultures have also been used to study the effects ofdrugs. A provocative effect demonstrated by Baden and Kubilus [83] wasthe ability of minoxidil at 70mg/ml to increase the apparent survival ofcultures of keratinocytes, appearing to slow their senescence, acharacteristic in common with EGF. The time after which cells couldbe passaged after confluence was increased by either minoxidil or EGF.Cohen et al [84] also found a possible stimulation of growth of mousekeratinocytes by minoxidil at concentrations of 5–20mg/ml. CyclosporinA, generally thought to improve psoriasis through its action onlymphocytes, was shown to inhibit keratinocyte growth in vitro atclinically relevant concentrations [85,86].

An important enhancement in the ability to culture keratinocytes wasdevised by Boyce and Ham [87], who developed a chemically definedmedium capable of supporting clonal culture of keratinocytes. Althoughtechnically more difficult to perform than culture as described byRheinwald and Green with a 3T3 feeder layer, this unique methodpermitted culture in the absence of serum. This and other papersappeared in a 1983 supplement to the JID entitled ‘‘The Biology of theKeratinocyte In Vitro,’’ which provided useful reviews of many aspects ofkeratinocyte biology including involucrin, filaggrin, keratin classes,envelope production, action of retinoids, and the question of epidermalstem cells.

In the last several years, many different mechanisms have beenstudied in keratinocyte cultures as the utility of these cultures hasincreased and more investigators have studied keratinocyte biologyin vitro. Albers and Taichman [88] devised an ingenious method todetermine what proportion of keratinocytes in culture continued todivide and presented evidence that only about 77% of dividing cellscontinue to divide in a subsequent cell cycle, thus providing a directmeasurement of the rate of loss of cells from the proliferativecompartment in vitro. The ability of retinoic acid to affect pemphigusand pemphigoid antigens was studied by Thivolet et al [89], who foundthat retinoic acid affected differentiation but did not always affect specificantigens in the expected way. For example, although retinoic acid coulddecrease certain differentiation markers, such as keratohyalin granules orthe 67-kD keratin, it increased apparent amounts of pemphigus antigen,as demonstrated by immunofluorescence. On the other hand, if calciumwas raised, increasing these differentiated functions, pemphigus antigenwas also increased in that setting. In vitamin A-deficient serum, whencultures were well differentiated pemphigus antigen was depleted. Theauthors concluded that because of these complex findings pemphigusantigen is not a good marker of differentiation of keratinocyte cultures.

Milstone and LaVigne [90] demonstrated for the first time that TCDD,which causes epidermal hyperplasia in animals and chloracne inhumans, produced hyperplasia in pure cultures of keratinocytes,increasing cells, protein, and thymidine incorporation. The concern has

Figure 4. Effect of in vitro UV on the intracellular (J—J) and extracellular

(K—K) production of ETAF activity by the squamous cell carcinoma line

(SSC). From Ansel et al [60].


arisen that studies of thymidine incorporation in epidermal cell culturemay give rise to artifacts. Milstone and LaVigne [91] investigated thereason for nonrandom distribution of labeled nuclei when keratinocyteswere incubated with tritiated thymidine. This had been thought to be atechnical artifact, possibly based on the inability of thymidine to diffusethrough stratified layers of cornified cells. The results indicated clearlythat such labeling was not artifactual but was a characteristic of thecultures.

The production of fibronectin by keratinocytes [92,93], the attach-ment [94], spreading [95], and migration [96] of keratinocytes onfibronectin, and the expression of fibronectin receptor function [95] haveprovided a foundation for the idea, described in some detail in JIDarticles, that fibronectin may provide one of the earliest adhesion factorsfor keratinocytes migrating over a wound, before the production of othermatrix factors and the subsequent organization of the basementmembrane. Fibronectin in the region of the basement membrane canindeed be contributed by the epithelium [97] or by basal cell carcinomacells [98]. Fibronectin messenger-RNA was recently demonstrated inkeratinocytes [99] and enhanced production of keratinocyte fibronectinwas demonstrated in response to transforming growth factor-beta [100].A block in the ability of keratinocytes to spread occurs in vitro when thecells reach confluence and is removed after several days of subculture atsub-confluent concentrations [101]. Similar failure to spread occursin vivo, as newly plated keratinocytes from skin spread poorly, and thissimilarly improves after culture for several days, at a time when the cellsbegin to recognize fibronectin [102]. Because cells in healing epitheliumare required to spread and migrate, fibronectin receptor function isemerging as a critical feature of wound healing. The actions of a plasma-derived spreading factor, epibolin, have been described by Stenn et al[103], who presented evidence that protein kinase C may be involved inepibolin-mediated spreading. Negi et al [104] described cell detachment,more marked in the presence of complement, produced by a monoclonalantibody to a 35-kD cell surface protein.

In the last decade cell culture has been used for a large number ofdifferent types of studies, some of which are described in other sectionsof this volume. Pigment cell studies, the culture of melanocytes, and theproduction of extracellular matrix proteins have been described.

Other Studies in Cell Biology Other studies in cell biology are toovaried to group either by subject or by methodology in this shortsummary but have used subcellular fractions from skm, intact skin,fibroblasts or other types of cells, or intact animals.

Eye-derived growth factor was shown to reduce epithelial resurfacingtime in skin wounds [105]. Basic fibroblast growth factor, which is, likeeye-derived growth factor, a member of the family of heparin-bindinggrowth factors, was shown to stimulate growth of keratinocytes [106].Epidermal growth factor was shown to slow hair growth and moreremarkably, rapidly to induce catagen’ in sheep, facilitating a ‘‘chemi-cal’’ shearing [107,108]. Lord and Ziboh [109] demonstrated binding ofprostaglandin E2 to membranes from human skin and inhibition of thisbinding by ultraviolet B irradiation. The action of corticosteroids wasexamined by Runikis et al [110], who noted an increased growth ofcultured human fibroblasts by glucocorticoids, a different finding fromthat in mouse cells, which were inhibited. Ponec et al [111]demonstrated specific binding of corticosteroids to human skin fibroblastreceptors and related their findings to potency of growth inhibition. Theglucocorticoid receptor in human skin cytosol was characterized in somedetail by Leiferman et al [112]. Ponec et al [113] correlated binding ofcorticosteroids with clinical efficacy. Sarnstrand et al [114] found areduction of glycosaminoglycan metabolism in fibroblasts in response tocorticosteroids, which was thought possibly to reflect atrophogenicchanges.

Epidermal growth factor receptors were visualized in epidermis byNanney et al [115] and in epidermal appendages as well. The bindingwas substantially altered in psoriasis [116]; binding was detected notonly in the basal cell layer but also in upper layers of the epidermis.Green and Couchman [117] also found EGF receptors in epithelium

using similar autoradiographic and antireceptor antibody stainingtechniques. Green and Couchman [118] subsequently confirmed thefinding of Nanney et al [115] that binding detected by 125I-labeled EGFwas less extensive than demonstration of receptor by antireceptorantibody, indicating that receptor was present that could not bind EGF.The significance of such occult receptors is uncertain.

Bullous pemphigoid antigen, long thought to be a base-ment membrane antigen, may be primarily intracellular. Severalstudies indicate that the antigen is better demonstrated in permeabilizedcells and may be associated with hemidesmosomes (Fig 5) [119–121].In other recent studies, chemotaxis of keratinocytes was demon-strated, and chemoattraction was marked in response to IL-1 orconditioned medium from cultured smooth muscle cells [122]. Also,Madison et al [123] developed conditions for expression of lamellargranules in vitro.

This review demonstrates a remarkable change in the quality andbreadth of studies in cell biology published in the JID. These studies andothers described in this issue show the continual development of the JIDwith contributions of more scientifically rigorous work and broader scopeinto a major repository of studies in cutaneous biology. The incorporationand reporting of developments in culture methods has been the majorarea of this change, as this has been a requisite for many studies of cyclicAMP and growth, growth factors, receptors, wound healing, mechanismof action of drugs at a cellular level, and others. Although culturemethods as such and the effects of various agents on culture itself wereemphasized initially, current studies more often involve biologic orbiochemical mechanisms elucidated in culture.

The increasing scope and sophistication of these studies bodes wellfor future work if the momentum can be maintained. The JID is theappropriate vehicle for representation in cutaneous biology of theongoing revolution in biologic science. We can, therefore, expect to seemore molecular and biochemical studies in the near future and thecontinuing emergence of the JID as a major means of communication,and not only publication, for investigators in cutaneous biology.

Figure 5. Binding of bullous pemphigoid antibodies by trypsin-dissociated

epidermal basal cells. This panel shows basal cells in suspension pretreated

with saponin before incubation with BP serum and fluorescent conjugate.

Under these conditions all basal cells bound BP antibody, producing a diffuse

granular pattern of fluorescence. From Mutasim et al [120].


REFERENCES

1. Karasek MA: In vitro culture of human skin epithelial cells. J InvestDermatol 47:533–540, 1966

2. Rothberg S: The cultivation of embryonic chicken skin in a chemicallydefined medium, and the response of the epidermis to excess of vitaminA. J Invest Dermatol 49:35–38, 1967

3. Chopra DP, Flaxman BA: The effect of vitamin A on growth anddifferentiation of human keratinocytes in vitro. J Invest Dermatol64:19–22, 1975

4. Flaxman BA, Lutzner MA, Van Scott EJ: Cell maturation and tissueorganization in epithelial outgrowths from skin and buccal mucosain vitro. J Invest Dermatol 49:322–332, 1967

5. Reaven EP, Cox AJ: Behavior of adult human skin in organ culture. II.Effects of cellophane tape stripping, temperature, oxygen tension, pH,and serum. J Invest Dermatol 50:118–128, 1968

6. Bishop SC, Cox AJ: Autoradiographic study of the DNA synthetic activityin human skin cultured in vitro: effects of stripping and the inhibition byglucosamine. J Invest Dermatol 62:74–79, 1974

7. Halprin KM, Lueder M, Fusenig NE: Growth and differentiation ofpostembryonic mouse epidermal cells in explant cultures. J InvestDermatol 72:88–98, 1979

8. Singh A, Hardy MH: Skin and isolated epidermis in intraperitonealdiffusion chambers. J Invest Dermatol 55:57–64, 1970

9. Kreider JW, Haft HM, Roode PB: Growth of human skin on the hamster.J Invest Dermatol 57:66–71, 1971

10. Frater R, Whitmore PG: In vitro growth of postembryonic hair. J InvestDermatol 61:72–81, 1973

11. Frater R: In vitro differentiation of mouse hair follicle cells. J InvestDermatol 64:235–239, 1975

12. Flaxman BA, Harper RA: Organ culture of human skin in chemicallydenned medium. J Invest Dermatol 64:96–99, 1975

13. Stenn K, Stenn J: Organ culture of adult mouse esophageal mucosa in adefined medium. J Invest Dermatol 66:302–305, 1976

14. ElgjoK: Chalone inhibition of cellular proliferation. J Invest Dermatol59:81–83, 1972

15. Chopra DP, Yu RJ, Flaxman BA: Demonstration of a tissue specificinhibitor of mitosis of human epidermal cells in vitro. J Invest Dermatol59:207–210, 1972

16. Rothberg S. Arp BC: Inhibition of epidermal and dermal DNA synthesisby mouse and cow-snout epidermal extracts. J Invest Dermatol64:245–249, 1975

17. Duell EA, Kelsey WH, Voorhees JJ: Epidermal chalone, past to presentconcept. J Invest Dermatol 65:67–70, 1975

18. Elgjo K, Reichelt KL, Hennings H, Michael D, Yuspa SH: Purifiedepidermal pentapeptide inhibits proliferation and enhances terminaldifferentiation of cultured mouse epidermal cells. J Invest Dermatol87:555–558, 1986

19. Harper RA, Flaxman BA, Chopra DP: Mitotic response of normal andpsoriatic keratinocytes in vitro to compounds known to affectintracellular cyclic AMP. J Invest Dermatol 62:384–387, 1974

20. Kariniemi A-L: Culture of psoriatic and uninvolved human skin indiffusion chambers in mice. J Invest Dermatol 63:388–391, 1974

21. Taylor JR, Halprin KM, Levine V: Inhibitors of epidermal cell DNAsynthesis in surviving pig skin in vitro. J Invest Dermatol 74:125–130,1980

22. Nieland ML, Wanger NS, Hambrick GW: Keratinocyte glycogen: anultrastructural study of the epidermal response to galactosamine andglucose in organ culture. J Invest Dermatol 56:113–126,1971

23. Flaxman BA, Harper RA: In vitro analysis of the control of keratinocyteproliferation in human epidermis by physiologic and pharmacologicagents. J Invest Dermatol 65:52–59, 1975

24. Harper RA, Flaxman BA: Effect of pharmacologic agents on humankeratinocyte mitosis in vitro. J Invest Dermatol 65:400–403,1975

25. Aoyagi T, Adachi K, Halprin KM, Levine V, Woodyard CW: The effect ofhistamine on epidermal outgrowth: its possible dual role as an inhibitorand stimulator. J Invest Dermatol 76:24–27, 1981

26. Flaxman BA, Harper RA, Chiarello S, Feldman AM: Effects ofmethotrexate on proliferation of human keratinocytes in vitro. J InvestDermatol 68:66–69, 1977

27. Liu S-C, Karasek M: Isolation and serial cultivation of rabbit skinepithelial cells. J Invest Dermatol 70:288–293, 1978

28. Holt PAJ: In vitro responses of the epidermis to triiodothyronine. J InvestDermatol 71:202–204, 1978

29. Didierjean L, Woodley D, Regnier M, Prunieras M, Saurat JH: Skinexplant cultures: expression of cytoplasmic differentiation antigens inoutgrowth cells. J Invest Dermatol 76:38–41, 1981

30. Wheeler CE, Canby CM, Cawley EP: Long-term tissue culture ofepithelial-like cells from human skin. J Invest Dermatol 29:383–392,1957

31. Cruickshank CND, Cooper JR, Hooper C: The cultivation of cells fromadult epidermis. J Invest Dermatol 34:339–342, 1960

32. Briggaman RA, Abele DC, Harris SR, Wheeler CE Jr: Preparation andcharacterization of a viable suspension of postembryonic humanepidermal cells. J Invest Dermatol 48:159–168, 1967

33. Laerum OD, Boyum A: The separation and cultivation of basaldifferentiating cells from hairless mouse epidermis. J Invest Dermatol54:279–287, 1970

34. Yuspa SH, Morgan DL, Walker RJ, Bates RR: The growth of fetal mouseskin in cell culture and transplantation to F1 mice. J Invest Dermatol55:379–389, 1970

35. Karasek MA, Charlton ME: Growth of postembryonic skin epithelial cellson collagen gels. J Invest Dermatol 56:205–210, 1971

36. Moore JT, Karasek MA: Isolation and properties ofagerminative and non-germinative cell population from postembryonic mouse, rabbit, andhuman epidermis. J Invest Dermatol 56:318–324, 1971

37. Vaughan FL, Bernstein IA: Studies of proliferative capabilities in isolatedepidermal basal and differentiated cells. J Invest Dermatol 56:454–466,1971

38. Fusenig NE, Worst PKM: Mouse epidermal cell cultures. J InvestDermatol 63:187–193, 1974

39. Cohen S, Elliott GA: The stimulation of epidermal keratinization by aprotein isolated from the submaxillary gland of the mouse. J InvestDermatol 40:1–6, 1963

40. Cohen S: Epidermal growth factor. J Invest Dermatol 59:13–16,1972

41. Christophers E: Growth stimulation of cultured postembryonic epidermalcells by vitamin A acid. J Invest Dermatol 63:450–455, 1974

42. Sherman BS: The effect of vitamin A on epithelial mitosis in vitro andin vivo. J Invest Dermatol 37:469–480, 1961

43. Tong PS, Mayes DM, Wheeler LA: Differential effects of retinoids onDNA synthesis in calcium-regulated murine epidermal keratinocytecultures. J Invest Dermatol 90:861–868, 1988

44. Liu S-C, Karasek M: Isolation and growth of adult human epidermalkeratinocytes in cell culture. J Invest Dermatol 71:157–163, 1978

45. Rheinwald JG, Green H: Serial cultivation of strains of humankeratinocytes: the formation of keratinizing colonies from single cells.Cell 6:331–334, 1975

46. Rheinwald JG, Green H: Epidermal growth factor and the multiplicationof cultured human epidermal keratinocytes. Nature 265:421–424, 1977

47. Kondo S, Aso K, Namba N: Culture of normal human epidermal cellswith 3T3 feeders on Millipore filters. J Invest Dermatol 72:85–87, 1979

48. Gilchrest BA: Relationship between actinic damage and chronologicaging in keratinocyte cultures of human skin. J Invest Dermatol72:219–223, 1979

49. Prunieras M: Epidermal cell cultures as models for living epidermis.J Invest Dermatol 73:135–137, 1979

50. Stanley JR, Foidart J-M, Murray JC, Martin GR, Katz SI: The epidermalcell which selectively adheres to a collagen substrate is the basal cell.J Invest Dermatol 74:554–558, 1980


51. Hawley-Nelson P, Sullivan JE, Kung M, Hennings H, Yuspa SH:Optimized conditions for the growth of human epidermal cells inculture. J Invest Dermatol 75:176–182, 1980

52. Hennings H, Michael D, Lichti U, Yuspa SH: Response of carcinogen-altered mouse epidermal cells to phorbolester tumor promoters andcalcium. J Invest Dermatol 88:60–65,1987

53. Yaar M, Stanley JR, Katz SI: Retinoic acid delays the terminaldifferentiation of keratinocytes in suspension culture. J Invest Dermatol76:363–366, 1981

54. Milstone LM, McGuire J, LaVigne JF: Retinoic acid causes prematuredesquamation of cells from confluent cultures of stratified squamousepithelia. J Invest Dermatol 79:253–260, 1982

55. Smith EL, Walworth NC, Holick MF: Effect of 1-alpha, 25-dihydroxy-vitamin D3 on the morphologic and biochemical differentiation ofcultured human epidermal keratinocytes grown in serum-free condi-tions. J Invest Dermatol 86:709–714, 1986

56. Horiuchi N, Clemens TL, Schiller AL, Holick MF: Detection anddevelopmental changes of the 1,25-(OH) 2-D3 receptor concentrationin mouse skin and intestine. J Invest Dermatol 84:461–464, 1985

57. Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engleman EG: Culturedhuman epidermal cells do not synthesize HLA-DR. J Invest Dermatol78:32–37, 1982

58. Sauder DN, Carter CS, Katz SI, Oppenhein JJ: Epidermal cell productionof thymocyte activating factor (ETAF). J Invest Dermatol 79:34–39, 1982

59. Luger TA, Stadler BM, Luger BM, Sztein MB, Schmidt JA, Hawley-NelsonP, Grabner G, Oppenhein JJ: Characteristics of an epidermal cellthymocyte-activating factor (ETAF) produced by human epidermal cellsand a human squamous cell carcinoma cell line. J Invest Dermatol81:187–193, 1983

60. Ansel JC, Luger TA, Green I: The effect of in vitro and in vivo UVirradiation on the production of ETAF activity by human and murinekeratinocytes. J Invest Dermatol 81:519–523, 1983

61. Kupper TS, McGuire J: Hydrocortisone reduces both constitutive andUV-elicited release of epidermal thymocyte activating factor (ETAF) bycultured keratinocytes. J Invest Dermatol 87:570–573, 1986

62. Goslen JB, Eisen AJ, Bauer EA: Stimulation of skin fibroblast collagenaseproduction by a cytokine derived from basal cell carcinomas. J InvestDermatol 85:161–164, 1985

63. Hernandez AD, Hibbs MS, Postlethwaite AE: Establishment of basal cellcarcinoma in culture: evidence for a basal cell carcinoma-derivedfactor(s) which stimulates fibroblasts to proliferate and release collage-nase. J Invest Dermatol 85:470–475, 1985

64. Woodley DT, Kalebec T, Banes AJ, Link W, Prunieras M, Liotta L: Adulthuman keratinocytes migrating over nonviable dermal collagen producecollagenolytic enzymes that degrade type I and type IV collagen. J InvestDermatol 86:418–423, 1986

65. Petersen MJ, Woodley DT, Stricklin GP, O’Keefe EJ: Constitutiveproduction of procollagenase and tissue inhibitor of metalloproteinasesby human keratinocytes in culture. J Invest Dermatol 92:156–159,1989

66. Ristow H-J: Stimulation of DNA synthesis in cultured mouse epidermalcells by human peripheral leukocytes. J Invest Dermatol 79:408–411,1982

67. Ristow, H-J: A major factor contributing to epidermal proliferation ininflammatory skin diseases appears to be interleukin-1 or a relatedprotein. Proc Nad Acad Sci USA 84:1940–1944, 1987

68. Wilkinson DI, Liu S-C, Orenberg EK: Cyclic nucleotide content ofpassaged keratinocytes in culture during various growth stages. J InvestDermatol 77:385–388, 1981

69. Okada N, Kitano Y, Ichihara K: Effects of cholera toxin on proliferation ofcultured human keratinocytes in relation to intracellular cyclic AMPlevels. J Invest Dermatol 79:42–47, 1982

70. Green H: Cyclic AMP in relation to proliferation of the epidermal cell: anew view. Cell 15:801–811, 1978

71. Orenberg EK, Pfendt EA, Wilkinson DI: Characterization of alpha-andbeta-adrenergic agonist stimulation of adenylate cyclase activity inhuman epidermal keratinocytes in vitro. J Invest Dermatol 80:503–507,1983

72. O’Keefe E, Battin T, Payne R: Epidermal growth factor receptor in human

epidermal cells: direct demonstration in cultured cells. J Invest Dermatol

78:482–487, 1982

73. O’Keefe EJ, Payne RE Jr: Modulation of the epidermal growth factor

receptor of human keratinocytes by calcium ion. J Invest Dermatol

81:231–235,1983

74. Fleckman P, Langdon R, McGuire J: Epidermal growth factor stimulates

ornithine decarboxylase activity in cultured mammalian keratinocytes.

J Invest Dermatol 82:85–89, 1984

75. Baden HP, Kubilus J: The growth and differentiation of cultured newborn

rat keratinocytes. J Invest Dermatol 80:124–130, 1983

76. Misra P, Nickoloff BJ, Morhenn VB, Hintz RL, Rosenfeld RG:

Characterization of insulin-like growth factor-I/somatomedin-C receptors

on human keratinocyte monolayers. J Invest Dermatol 87:264–267, 1986

77. Isseroff RR, Fusenig, NE, Rifkin DB: Plasminogen activator in differ-

entiating mouse keratinocytes. J Invest Dermatol 80:217–222, 1983

78. Isseroff RR, Rifkin DB: Plasminogen is present in the basal layer of the

epidermis. J Invest Dermatol 80:297–299, 1983

79. Hashimoto K, Singer KH, Lide WB, Shafran K, Webber P, Morioka S,

Lazarus GS: Plasminogen activator in cultured human epidermal cells.


80. Morioka S, Lazarus GS, Baird JL, Jensen PJ: Migrating keratinocytes

express urokinase-type plasminogen activator. J Invest Dermatol

88:418–423, 1987

81. Baden HP, Kubilus J, Macdonald MJ: Normal and psoriatic keratinocytes

and fibroblasts compared in culture. J Invest Dermatol 76:53–55, 1981

82. Liu S-C, Parsons CS: Serial cultivation of epidermal keratinocytes from

psoriatic plaques. J Invest Dermatol 81:54–61, 1983

83. Baden HP, Kubilus J: Effect of minoxidil on cultured keratinocytes.


84. Cohen RL, Alves ME, Weiss VC, West DP, Chambers DA: Direct effects

of minoxidil on epidermal cells in culture. J Invest Dermatol 82:90–93,

1984

85. Furue M, Gaspari AA, Katz SI: The effect of cyclosporin A on epidermal

cells. II. Cyclosporin A inhibits proliferation of normal and transformed

keratinocytes. J Invest Dermatol 90:796–800, 1988

86. Fisher GJ, Duell EA, Nickoloff BJ, Annesley TM, Kowalke JK, Ellis CN,

Voorhees JJ: Levels of cyclosporin in epidermis of treated psoriasis

patients differentially inhibit growth of keratinocytes cultured in serum

free versus serum containing media. J Invest Dermatol 91:142–146, 1988

87. Boyce ST, Ham RG: Calcium-regulated differentiation of normal human

epidermal keratinocytes in chemically defined clonal culture and serum-

free serial culture. J Invest Dermatol 81:33s–40s, 1983

88. Albers KM, Taichman LB: Kinetics of withdrawal from the cell cycle in

cultured human epidermal keratinocyes. J Invest Dermatol

82:161–164,1984

89. Thivolet CH, Hintner HH, Stanley JR: The effect of retinoic acid on the

expression of pemphigus and pemphigoid antigens in cultured human

keratinocytes. J Invest Dermatol 82:329–334, 1984

90. Milstone LM, LaVigne JF: 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces

hyperplasia in confluent cultures of human keratinocytes. J Invest

Dermatol 82:532–534, 1984

91. Milstone LM, LaVigne JF: Heterogeneity of basal keratinocytes: nonran-

dom distribution of thymidine-labelled basal cells in confluent cultures is

not a technical artifact. J Invest Dermatol 84:504–507, 1985

92. O’Keefe EJ, Woodley D, Castillo G, Russell N, Payne RE, Jr: Production

of soluble and cell associated fibronectin by cultured keratinocytes.


93. Kubo M, Norris DA, Howell SE, Ryan SR, Clark RA: Human

keratinocytes synthesize and secrete, and deposit fibronectin in the

pericellular matrix. J Invest Dermatol 82:580–586, 1984

94. Clark RA, Folkvord JM, Wertz RL: Fibronectin, as well as other

extracellular matrix proteins, mediate human keratinocyte adherence.



95. Toda K, Grinnell F: Activation of human keratinocyte fibronectinreceptor function in relation to other ligand-receptor interactions.J Invest Dermatol 88:412–417, 1987

96. O’Keefe EJ, Payne RE Jr, Russell N, Woodley DT: Spreading andenhanced motility of human keratinocytes on fibronectin. J InvestDermatol 85:125–130, 1985

97. O’Keefe, EJ, Woodley DT, Falk RJ, Gammon WR, Briggaman RA:Production of fibronectin by epithelium in a skin equivalent. J InvestDermatol 88:634–639, 1987

98. Grimwood RE, Ferris CF, Nielsen LD, HuffJC, Clark RA: Basal cellcarcinomas grown in nude mice produce and deposit fibronectin in theextracellular matrix. J Invest Dermatol 87:42–46, 1986

99. Peltonen J, Jaakkola S, Lask G, Virtanen I, Uitto J: Fibronectin geneexpression by epithelial tumor cells in basal cell carcinoma: animmunocytochemical and in situ hybridization study. J Invest Dermatol91:289–293, 1988

100. Wikner NE, Persichitte KA, Baskin JA, Nielsen LD, Clark RA:Transforming growth factor-beta stimulates the expression of fibronectinby human keratinocytes. J Invest Dermatol 91:207–212, 1988

101. Stenn KS, Milstone LM: Epidermal cell confluence and implications for atwo-step mechanism of wound closure. J Invest Dermatol 83:445–447,1984

102. Takashima A, Grinnell F: Fibronectin-mediated keratinocyte migrationand initiation of fibronectin receptor function in vitro. J Invest Dermatol85:304–308, 1985

103. Stenn KS, Core NG, Halaban R: Phorbol ester serves as a coepibolin inthe spreading of primary guinea pig epidermal cells. J Invest Dermatol87:754–757, 1986

104. Negi M, Lane A, McCoon PE, Fairley JA, Goldsmith LA: Monoclonalantibody to a 35-kD epidermal protein induces cell detachment. J InvestDermatol 86:634–637, 1986

105. Fourtanier AY, Courty J, Muller E, Courtois Y, Prunieras M, Barritault D:Eye-derived growth factor isolated from bovine retina and used forepidermal wound healing in vivo. J Invest Dermatol 87:76–80, 1986

106. O’Keefe EJ, Chiu ML, Payne RE: Stimulation of growth of keratinocytesby basic fibroblast growth factor. J Invest Dermatol 90:767–769,1988

107. Moore GPM, Panaretto BA, Carter NB: Epidermal hyperplasia and woolfollicle regression in sheep infused with epidermal growth factor. J InvestDermatol 84:172–175, 1985

108. Hollis DE, Chapman RJE: Apoptosis in wool follicles during mouseepidermal growth factor (mEGF)-induced catagen regression. J InvestDermatol 88:455–458, 1987

109. Lord JT, Ziboh VA: Specific binding of prostaglandin E2 to membranepreparations from human skin: receptor modulation by UVB-irradiationand chemical agents. J Invest Dermatol 73:373–377, 1979

110. Runikis JO, Mclean DI, Stewart WD: Growth rate of cultured humanfibroblasts increased by glucocorticoids. J Invest Dermatol 70:348–351,1978

111. Ponec M, De Kloet ER, Kempenaar JA: Corticoids and human skinfibroblasts: intracellular specific binding in relation to growth inhibition.J Invest Dermatol 75:293–296, 1980

112. Leiferman KM, Schroeter A, Kirschner MK, Spelsberg TC: Characteriza-tion of the glucocorticoid receptor in human skin. J Invest Dermatol81:355–360, 1983

113. Ponec M, Kempenaar JA, DeKloet ER: Corticoids and cultured humanepidermal keratinocytes: specific intracellular binding and clinicalefficacy. J Invest Dermatol 76:211–214, 1981

114. Sarnstrand B, Brattsand R, Malmstrom A: Effect of glucocorticoids onglycosaminoglycan metabolism of cultured human skin fibroblasts.J Invest Dermatol 79:412–417, 1982

115. Nanney LB, McKanna JA, Stoscheck CM, Carpenter G, King LE:Visualization of epidermal growth factor receptors in human epidermis.J Invest Dermatol 82:165–169, 1984

116. Nanney LB, Stoscheck CM, Magid M, King LE: Altered [125I] epidermalgrowth factor binding and receptor distribution in psoriasis. J InvestDermatol 86:260–265, 1986

117. Green MR, Couchman JR: Distribution of epidermal growth factorreceptors in rat tissues during embryonic skin development, hairformation, and the adult hair growth cycle. J Invest Dermatol83:118–123, 1984

118. Green MR, Couchman JR: Differences in human skin between theepidermal growth factor receptor distribution detected by EGF bindingand monoclonal antibody recognition. J Invest Dermatol 85:239–245,1985

119. Westgate GE, Weaver AC, Couchman JR: Bullous pemphigoid antigenlocalization suggests an intracellular association with hemidesmosomes.J Invest Dermatol 84:218–224, 1985

120. Mutasim DF, Takahashi Y, Labib RS, Anhalt GJ, Patel HP, Diaz LA: Apool of pemphigoid antigen(s) is intracellular and associated with thebasal cell cytoskeleton-hemidesmosome complex. J Invest Dermatol84:47–53, 1985

121. Regnier M, Vaigot P, Michel S, Prunieras M: Localization of bullouspemphigoid antigen (BPA) in isolated human keratinocytes. J InvestDermatol 85:187–190, 1985

122. Martinet N, Harne LA, Grotendorst GR: Identification and characteriza-tion of chemoattractants for epidermal cells. J Invest Dermatol90:122–126, 1988

123. Madison KC, Swartzendruber DC, Wertz PW, Downing DT: Lamellargranule extrusion and stratum corneum intercellular lamellae in murinekeratinocyte cultures. J Invest Dermatol 90:110–116, 1988


fifty years of cell biology in the journal of

Documents