0022-202X/ 80/ 7501-0006$02.00/0 THE JOURNAL OF INVESTIGATIVE D ERMATOLOGY, 75:6-11, 1980 Vol. 75, No.1 Copyrighl © 1980 by The Williams & Wilkins Co. Printed in U.S.A. Branched Cells in the Epidermis: An Overview

A. S. BREATHNACH, M.Sc., M.D. Department of Anatomy, St. Mary's Hospital Medical School (University of London), London, W.2, UK

Current knowledge concerning the nature, lineage, due, no doubt, to overconcentration upon their own pet clear and function of the Langerhans cell, Merkel cell, and, to cells, to the fact that no specific stains for lymphocytes had a lesser extent, the melanocyte, are reviewed under been evolved, to the unacceptability of Andrew's conclusions headings that emphasize the confederate constitution of [ 4] that lymphocytes were capable of differentiating into prac­ the epidermis as a compound tissue composed of a vari­ tically every other variety of epidermal cell, and finally to the ety of cellular elements; the role of the lymphocyte as a lack of an obvious reason for their presence there at all under component of normal epidermis is also considered. It normal circumstances. appears that the function of the Langerhans cell has Today; a further 20 yr along the way, this question of intrae­ finally been established, i.e., it serves as a front-line pidermallymphocytes has become an issue of vital importance, element in immune reactions of the skin. Develop­ not only in relation to individual immunopathologic situations, mentally, it is of mesenchymal origin. The Merkel cell but also from the wider points of view put forward by Fichthel­ still presents a number of problems centering around ius, Groth, and Liden [5] and Streilein [6] in connection with questions of its lineage, the nature of its characteristic the skin as an immunologic inductive microenvironment, and granules, and the "synaptic" relationship between it and the possible existence of a subpopulation of lymphocytes sub­ the associated neurite. The melanocyte continues to hold serving a skin-associated lymphoid tissue (SALT). These and the attention of investigators, mainly from the point of similar concepts will, no doubt, be further aired at this Sym­ view of the chemistry of melanin mid the rational treat­ posium, and were Warren Andrew with us today he would ment of pigmentary disorders based upon findings de­ sw·ely feel fully vindicated. Certainly, most workers now agree rived from fundamental research into all aspects of its with his view that the lymphocyte is a component cell of normal biology. epidermis and that there is a continuous traffic, in both direc­ tions, of lymphocytes across the epidermal-dermal junction. Most electron microscopists would probably claim to be able Billingham [1] 31 yr ago emphasized that the epidermis is a to recognize a lymphocyte on the grounds of general ultrastruc­ "compound tissue composed of cells of at least two distinct ture, but their absolute, positive identification and distinction races." Nowadays, most of us would probably extend his 2 races from other intraepidermal mononuclear and "indeterminate" to 5, and a few would even suggest 6. Although all perform their cells (see below) will continue to present a problem until some functions at the body surface, some of these cells are more immunologic electron microscopic or other specific marker specifically related to this environment than others, and, in­ technique can be devised for their demonstration in situ in deed, may have little in common with their neighbors apart normal epidermis. The cells in question are presumably T from their location. Nevertheless, it would be a mistake to lymphocytes, and in mice, therefore, might be expected to react consider them isolated elements and the headings under which ~o la~eled anti-8 antibody, but I am not aware that anyone has I propose to deal with them serve mainly to emphasize their Identified them by this technique in normal murine epidermis. confederacy rather than their interdependence. They are very McDonald and his colleagues [7] have developed a specific anti­ broad collective terms, any of which might have served as an human-T~~ell antiserum, which-labeled by peroxidase-conju­ alternative title for this Symposium. gated antummunoglobulin antiserum-they have used for the demonsb'ation of epidermal T cells in contact dermatitis, lichen CLEAR CELLS IN THE EPIDERMIS planus, and mycosis fungoides. By June 1979 they had not In routine hematoxylin-and-eosin-stained sections, "clear attempted this technique on normal human epidermis, but cells" [2], i.e., cells with nuclei surrounded by relatively un­ agreed to do so at my suggestion, and right now I am awaiting stain ed or "clear" cytoplasm, are a regular feature of both basal the results. Still no luck (May 1980) . and suprabasal layers of the epidermis. These include the It need hardly be stressed that attempts to estimate, even branched cells (melanocytes, Langerhans cells, and, in particu­ roughly? population densities of any of the above cell types on lar regions, Merkel cells) , each of which can now individually ~he _basis of numbe~s of epidermal "clear cells" are no longer be distinguished by special light microscopic techniques, and J';!Stified_, bu~ atte~twn should be drawn to 2 minor misconcep­ more certainly, by electron microscopy, on the basis of specific tions, still farrly wtdespread, i.e., that all of the basal layer clear marker organelles. Andrew and Andrew [3] 30 yr ago concluded cells are melanocytes and that Langerhans cells occupy only that lymphocytes also formed a proportion of the normal epi­ the suprabasal layers. They do not; Langerhans cells can be dermal population of "clear cells," but this view gained little observed quite commonly in the basal layer by both light and acceptance and 20 yr later, Andrew [ 4], reviewing some publi­ electron microscopy. Keratinocytes may also appear as "clear cations, somewhat plaintively remarked: "it has seemed rather cells," especially in embryonic and fetal epidermis as a result of surprising to us that . . . there has not been a return to the a high content of cytoplasmic glycogen. This feature and the question as to whether or not lymphocytes do occur in the fact that technical developments now permit certain identifi­ normal epidermis of man and other mammals, and if so, what cation of the individual cells to which it once gave blanket their role and destiny may be in such a situation." This general coverage lead me to suggest that the redundant and confusing lack of interest on the part of other workers at the time was term "clear cell" be dropped from usage. Ultrastructurally, in well-fixed material melanocytes, Lan­ gerhans cells, and Merkel cells do not exhibit cytoplasmic Heprint requests to: A. S. Breathnach, M.Sc., M.D., Department of Anatomy, St. Mary's Hospital Medical School, London, W.2, United sluinkage, one of the reasons they appear as "clear cells" in Kingdom. light microscopic preparations. But there is a certain ultrastruc­ Abbreviations: tural feature of melanocyte$ upon which I want to comment. In EPU: epidermal proliferative unit clinically perfectly normal skin, one frequently sees individual SALT: skin-associated lymphoid tissue melanocytes with mitochondria exhibiting swelling, vacuola- 6 July 1980 BRANCHED CELLS IN EPIDERMIS 7 tion, and loss of cristae and internal substance, even though been elaborated upon by English (14-16], who has described mitochondria of flanking keratinocytes, adjacent melanocytes, the occunence of "transitional cells" with features allegedly and a variety of dermal cells appear perfectly sound. The same intermediate between those of Merkel cells and keratinocytes phenomenon may be observed in melanocytes in a variety of in Haarscheiben of skin of cat, newborn rat, and pig's snout. pigmentary disorders, and the question of whether or not it has The facts that "transitional cells" are prominent in maturing pathological significance arises in this latter situation. One has Haarscheiben of newborn rat skin and that both they and the impression that this phenomenon has only become promi­ Merkel cells are reduced in number after denervation of Haar­ nent since the introduction of glutaraldehyde, appropriately schebein have also been advanced in support of the modified­ buffered, as a routine fixative. It seems to have been less keratinocyte hypothesis. It is true that some of the cells pictured common with the old Palade fixative, an indication that it may by English [15,16] seem to fit the morphological bill of her be due to a defect of primary processing of the tissue ("poor argument, but nevertheless the evidence remains circumstantial fixation," the nightmare of electron microscopists), though it is and must be weighed against other evidence-referred to below difficult to imagine that quality of processing could vary to such and admittedly equally circumstantial-that the Merkel cell is a degree over a distance little greater than the width of the an immigrant element that enters the epidermis during fetal intercellular space. In my experience it rar ely occurs with fetal life [17-19). It is my opinion that to suggest that Langerhans melanocytes or with inactive or moderately active cells, such as cells and Merkel cells are modified keratinocytes goes against albino melanocytes, or those of pale areas of freckled skin. common sense. The epidermal keratinocyte is itself a highly These overall observations lead me to muse as follows. specialized cell, programmed toward differentiation in a partic­ It may be that mitochondria of melanocytes differ generally ular direction. That selected individual cells of this type should in some way from those of immediately adjacent cells in being dedifferentiate and redifferentiate into another cell type of particularly difficult to preserve, or in being particularly sus­ totally different structme and function I find very difficult to ceptible to autolytic change, and rapidly so, so that they start accept. to swell, etc., even before the fixative reaches them; they may Whatever their derivation, none of the epidermal nonkerati­ be similar in this respect to certain vesicular structmes of nocytes seem to be desquamated under normal cn·cumstances. peripheral nerve terminals. There may, on the other hand, be Melanocytes are normally strictly confined to the basal layer, some featme of the plasma membrane of the melanocyte, such as are Merkel cells, and Langerhans cells are ra!'ely, if ever, as permeability, that prevents the fixative from getting into the seen beyond the upper spinous level; no trace of any can be cell as rapidly as it does into the adjacent keratinocytes; gap found in the stratum corneum. This raises the question of how junctions, or even desmosomes might aid entry of fixatives into these cells maintain then· position, or station, against the stream the adjacent keratinocytes; this mitochondrial phenomenon is of keratinocytes moving toward the swface, either singly or in not exhibited by Langerhans cells, which likewise Jack desmo­ columns. The situation is essentially similar to certain morpho­ somes, whereas it is manifested to some extent by Merkel cells, genetic tissue rearrangements occurring in embryogenesis in­ which have them. Finally, it could be that, at any given time, volving "sorting out" of mixed-cell populations. Such rearrange­ there exist "phase" or other differences between individual ments are thought to be driven and guided by variations in members of the melanocyte population that account for the mutual cellular adhesiveness as well as by locomotor activity fact that frequently, of 2 closely positioned cells, only 1 exhibits that may be classed as "active" or "passive" [20). Intercellular mitochondrial swelling, etc. If, as supposed, there is a normal adhesion is now generally thought of as involving interactions turnover of melanocytes of the basal layer at any particular of not only extracellular structmes such as exposed portions of time, there must be young, middle-aged, and venerable cell membrane proteins and smface coats, or glycocalyces (in­ melanocytes present. The phenomenon might be related to this, tercellular "cement"), but also intracellular elements that can and, perhaps, to associated variations in melanogenic activity. control these surface components as well as influence the overall Whatever may be the case, it is necessary to establish the shape and surface topography of the cell. The terms "stick" and normal range of variability of this feature of swelling and "grip," respectively, have been introduced to give expression to vacuolation of mitochondria of melanocytes since it appears these 2 factors, and it seems that intracellular cytoskeletal that the mitochondrion may be a sensitive morphological indi­ elements (e.g., filaments) are of importance in consolidating or cator of melanocyte pathophysiology. relaxing the "grip" [21]. Changes in the natme of adhesion between keratinocytes dming mitosis, and during lateral and NONKERATINOCYTES IN THE EPIDERMIS vertical migration, have received increasing attention in recent The term "nonkeratinocyte" specifies an epidermal cell that years [9,22,23-25], and these must also be considered in any is not of prime ectodermal lineage and that does not undergo analysis of "station-keeping" by melanocytes and Langerhans keratinization or desquamation. Clearly, the melanocyte and cells since the keratinocytes serve as a moving substratum for the lymphocyte fall into this category, and although most the other 2 classes of cells. Without being able to present investigators maintain that the same applies to the Langerhans defmite evidence, I think that local variations in "grip" rather cell and the Merkel cell, others still have lingering doubts. than in " tick" are likely to be of major significance in enabling Reams and Tompkins [8) produced what at the time seemed melanocytes and Langerhans cells to maintain then· positions, to be incontrovertible experimental evidence that in the mouse and that the dendritic shape of both cells may be in large part Langerhans cells arise as division products of basal keratino­ determined by this requn·ement and may even facilitate it. The cytes, but this proposition has received little support, and their phenomenon of attachment of Langerhans granules to the experimental procedmes have been criticized [9]. It seems to plasma membrane [25] might be considered in this general me that the argument is about to be overtaken by events context. It could be a manifestation of addition to or subtraction recently reported by Katz et al [10,11], who claim to have from the plasma membrane, associated with movement and equally weighty experimental evidence of the origin of Langer­ overall changes in shape. It is significant that this feature is hans cells from cells originating in bone marrow. I shall need to much more frequently observed with cells of histiocytosis X refer to this evidence again in the next section on epidermal [26], and the suggestion is consis.tent with both the "secretion" immigrants, so shall leave it for the moment, and, no doubt, we and the "endocytosis" theories [27] of the origin and mode of shall hear more of it tomorrow when Dr. Katz deals in detail formation of the characteristic Langerhans granule. The signif­ with the ontogeny of the Langerhans cell. icance of the granule is a major problem, and it will be of The suggestion that the Merkel cell is a specialized, or interest to see if eventually the granule can be specifically modified, epidermal keratinocyte originally a1·ose from ultra­ related to the presumed immunologic function of the cell or to structural studies demonstrating cytoplasmic filaments and des­ some other requn·ement. This matter should receive attention mosomal contacts with neighboring cells [12,13]. This view has dming our discussions; should no solution to the problem 8 BREATHNACH Vol. 75, No. 1 emerge, I hope that realistic suggestions for its further investi­ gerhans cell is of mesenchymal lineage has been knocking gation will be advanced. around for quite a while now [17,27], greatly reinforced by recent immunologic evidence [37,38), and it will be of interest EPIDERMAL IMMIGRANTS to see how this new experimental supportive evidence of Katz Notable among critical experiments related to mammalian et al [10,11) will stand up to scrutiny, particularly from the skin is that of Mary Rawles [28] demonstrating conclusively point of view of procedures. that melanocytes are derived from the neural crest and that This question of the developmental origin of the Langerhans they can therefore be classed as immigrant elements within the cell is now ripe for close examination and discussion, and I hope epidermis. At the time (1948), and for a considerable period a consensus view on the matter will be an outcome of this thereafter, the Schwann cell and "effete melanocyte" hy­ Symposium. Intimately tied up with it is the problem of the so­ potheses of the nature of the Langerhans cell held sway; each called indeterminate cell, which has befogged many a mind over was supported by a formidable body of circumstantial evidence the years. The recent demonstration by Rowden, Phillips, and and some seductive arguments [29-31]. These did not greatly Lewis [39] that the bulk of "indeterminate cells" express surface impress Silvers [32], who decided to put the matter to the test antigen associated with Langerhans cells conclusively confirms by duplicating Rawles' [28] experimental production of neural­ their relationship and goes a long way toward making seem crest-free (and therefore melanocyte-free and Schwann-cell­ reasonable the suggestion that they and morphologically similar free) mouse skin and by examining it for the presence of cells present in both fetal and postnatal dermis are precursor, Langerhans cells. Silvers [32] found aureophilic dendritic cells or immature, Langer hans cells [ 40]. On a previous occasion and concluded that Langerhans cells were neither of neural (9], I made a plea for outlawing the terms "indeterminate cell," crest origin nor related to melanocytes. "a-dendritic cell," etc. I do so again, now with even greater This apparently equally critical experiment was criticized on justification. the grounds (a) that the absence of melanocytes had not been The suggestion that mammalian Merkel cells are also epider­ absolutely proven, (b) that although aureophilic dendritic cells mal immigrants arose from their presence in the dermis of had been present in the dermis, their presence in epidermis had human fetal skin, and their close association with developing not been conclusively established by the illustrations, and (c) nerve terminals in this situation indicated a relationship to that the gold impregnation techniques in general were highly Schwann cells and possibly derivation from the neural crest capricious and nonspecific in nature (33]. It was suggested that [17-19,41]. A close morphological resemblance to a variety of the experiment be repeated with electron microscopic tech­ neurosecretory cells, some of which certainly are derived from niques since these would provide more certain criteria for the neural crest, also argues in favor of a neural lineage [ 42, identification of both melanocytes and Langerhans cells. The 43], but the evidence is entirely circumstantial and must be results proved to be reproducible, and the previous conclusions balanced against evidence, already referred to, that they are were apparently fully confirmed (34]. However, Reams, Scog­ modified keratinocytes (14-16]. As with the issues of the origin gins, and Zelickson [35] at the same time were embarking on an of the melanocyte and the Langerhans cell, the issue of the essentially similar critical experiment, and they reported exactly origin of the Merkel cell can only be decided by a critical opposite results, i.e., an absence of Langerhans cells in neural­ embryologic marking or ablation experiment; perhaps the hold­ crest-free skin, an indication that they were of neural crest ing of this Symposium will encourage someone to institute it origin and therefore· immigrants. Impasse. In the meantime, for some mammal. Tweedle [44) has reported such an experi­ continuing ultrastructural and other studies were gradually ment in amphibian larvae. He produced "aneurogenic" larvae undermining the view that Langerhans cells and melanocytes of Ambystoma maculatum by removing the entire nervous were related [27 ,36], and this situation led Reams to repeat his system, including the neural crest, and subsequently found critical experiment. He then reported (8] that the results af­ Merkel cells in the epidermis. He has certainly proved the firmed "the conclusion of Breathnach et al [1968] that epider­ nonneural origin of Merkel cells in this species, but whether or mal Langerhans cells are not of neural crest origin, and thus not the results can be extrapolated to other species, including cannot be related to melanocytes." mammals, remains to be seen. Even if the nervous system has My colleagues and I [34) were suitably gratified with this been eliminated as a source, the question of their actual origin volte-face, but our joy was somewhat tempered by Reams and remains. Tompkins' [8) suggestion that there was a flaw inherent in In this general context, the question of dependence of Merkel Silvers' original experiment, and in its repetition. Silvers [32,34] cells on innervation may be mentioned. It is clear that in both had grafted his neural-crest-free limb buds onto the spleens of amphibia [ 45] and mammals [ 46] they can develop and differ­ histocompatible adult mice, and had not considered the possi­ entiate fully in ontogeny independently of contact with nerve bility that Langerhans cells might have invaded the grafts from terminals, but although in the former group they appear to be the splenic beds. At the time, there was no hard and fast unaffected by subsequent denervation [45], in some species of evidence for the occurrence of Langerhans cells in spleen, a the latter group denervation is followed by degenerative situation that has since changed. The experiment, therefore, changes and loss in numbers [15,16,47,48). Diamond [ 49] has was not as critical as it appeared, even though the conclusions suggested that in the salamander, Merkel cells are involved in reached were correct. However, Reams and Tompkins' experi­ regulating terminal nerve fields by acting as target elements for mental procedures [8] were above criticism in this respect mechanosensory axons in both development and regeneration. because they had used chick embryos as a cultivation site for None of these observations necessarily indicate that Merkel the neural-crest-free mouse skin grafts, and Langerhans cells cells are modified keratinocytes. were apparently absent from the chicks (or were they?). I draw The establishment early in development of an epidermal­ attention to these matters for 2 reasons. First, the collaborative dermal interface and basal lamina has led to the concept of a study conducted by Silvers, others, and me [34] has been widely highly selective barrier, or dividing region, that is rarely if ever quoted as THE critical experiment proving that Langerhans cells breached by whole cells under normal postnatal circumstances. are not of neural crest origin, and insufficient credit has been This concept now seems•too rigid in the light of probable 2-way allotted to the work of Reams and Tompkins (8]. Second, new traffic across the junction of both lymphocytes and Langerhans and apparently even more critical experiments related to the cells, subserving a continuous monitoring role with respect to origin and lineage of Langerhans cells have recently been re­ external antigen. Immigration and emigration across the fron­ ported. These are the transplantation and chimera studies by tier is freer than previously thought; however, in accepting this Katz et al [10,11], which, it has been claimed, indicate that fact, one may well ask why one so rarely encounters cells epidermal Langerhans cells are derived from precursor cells actually en passage. Maybe, as does mitosis, movement occw·s originating in bone marrow. Certainly, the view that the Lan- in the depth of night. July 1980 BRANCHED CELLS IN EPIDERMIS 9 EPIDERMAL SYMBIONTS T he close relationship between epidermal Langerhans cells and t heir combined role as a peripheral out­ This concept expresses a close anatomical and functional and lymphocytes of the immunologic system might be cited as further association, a nd biological balance, between epidermal cells of post idermal symbiosis, b ut this extension may be different developmental lineages. T here is some doubt about examples of ep concept a bit too far. And what of Merkel cells? who should be given credit for first introducing it, but according stretching t he all the other cells u nder consideration, are con­ to Niebauer [50], the honor belongs to Caudiere [51]. Rappaport T hey, unlike inocytes by desmosomes, and might [52] w as apparently t he fust to relate it directly to the mela­ nected to adjacent kerat having the most intimate functional nocyte when stating t hat transference and storage of pigment therefore be t hought of as wever, the Merkel cell is also directly within keratocytes might act as a regulating mechanism for the relationship with them. Ho d n erve terminals, and it is in this rate of primru-y melanogenesis. The idea was further d eveloped related to its associate function is usually though t to reside. by Pinkus et al [53] and fi nally elaborated b y F itzpatrick and relationship t hat its main ationship lue ill understood, some are me [54] into the concept of an "epidermal melanin unit." The Some aspects of this rel rsy; in fact, the Merkel cell is now in th eory stresses that the organization of epidermal pigmentation even m atters o f controve il recently by t he Langerhans cell, i.e., in all its aspects is a cooperative exercise involving a close the position occupied u nt about its structw·e and relationships, interaction b etween m elanocytes and keratinocytes, and has although we know much lineage (see above) and fu nction is proved u seful in giving expression to such featw·es as regional, evidence concerning its stantial and is open to conflicting interpretations. racial, a nd species diffe rences in pigmentation, and in the mainly circum his issue deals with the Merkel cell I analysis of genetic and other control mechanisms [ 4], as well as Since only 1 p aper in t ing, to deal with some of the pr;b­ of certain pigmentary disorders [55,56). No doubt Dr. Quevedo should like, before conclud nts in somewhat greater depth. will refer to the melanin unit in these and other contexts dming lems it still prese upon i t except to remark his t alk, so I shall not dwell further THE MERKEL CELL that the key process involved in the relationship between the 2 cells, the t ransfer of melanosomes in intact epidermis in vivo, The mammalian Merkel cell-nemite complex constitutes a still requires full elucidation. I am n ot entirely convinced b y slowly a dapting mechanoreceptor [70), but t he exact mecha­ the "penetration and nipping off of the dendr ites" explanation nism of stinmlus t ransduction r emains to be established. T he of the mechanism since I have never seen within a keratinocyte most commonly held v iew is t hat the Merkel cell is the prime a cl uster of melanosomes invested by 2 distinct membranes transducer element. It is t hought to detect vertical, shearing, or representing t he ingested segments of plasma membranes of other deformities of the e pidermis via its villous processes melanocyte and keratinocyte. Has anybody? As somebody who (spines) and desmosomal attachments to keratinocytes, and to called me up recently on this point remarked, such a method of thereby give rise to a receptor potential that activates the axon transfer of a membrane- bound granule from cell to cell seems by a synaptic mechanism involving release of a transmitter unique in biology. Recent o bservations t hrough high-voltage substance fro m the dense-core cytoplasmic gxanules. However electron microscopy on guinea pig melanocytes in tissue cuJ tm e despite efforts of a number of workers over the year~ [57] indicate that dendritic processes of melanocytes neither [ 42,48, 71 ,72 ], no histochemical, pharmacologic, or uitracyto­ penetrate into nor axe engulfe d b y keratinocytes, and that chemical technique has yet provided unequivocal evidence that mela nosomes ru-e transferred to keratinocytes as individual the g ranules contain a neurotransmitter substance, as their gxanules. These matters require fm ther study. appearance strongly suggests. T he neru-est approach to success On the basis of quantitative studies, Wolff and Winklemann in this connection i s Crowe and Whitear's [73] demonstration [58] in analogy to the concept o f an epidermal/ melanin unit of quinacrine flu orescence of Merkel cells in Xenopus laevis postulated the existence of an "epidermal Langerhans cell unit" but t hey were unable to localize it to the dense-core granules: of unknown function, a nd Allen and Patten [59,60) identified Continuing failure to demonstrate a transmitter su bstance this in m ouse skin as an "epidermal proliferative unit" (EP U) together with the fact that the number of granules is much consisting of 9 or 10 basal keratinocytes and 1 Langerhans cell greater than that required to provide adequate activation of the functioning together to provide controlled r eplacement of the alleged "synapses," has led to speculation that they may have epidermal comified cells. This idea fi tted in very well wi th the other functions. Fox and Whitear [74] suggest that in amphib­ vertical organization of epidermal keratinocytes in columns ians t he g r anules may release theil: contents into the general with a Langerhans cell centrally situated b eneath each column, cytoplasm, from which they could be conveyed along the villous "ideally placed to provide a controlling role in the EP U" [60]. processes into the intercellular space, where t hey could affect It seemed as if t he f unction of the Langerhans cell was finally naked nerve terminals or even the general epidermal cells. Even established as being directly related to keratinocyte kinetics, if t his hypothesis is valid, t he p roblem of the nature of the and that this "unlaid ghost of the epidermis" [61), and those of granule contents remains. us whom it had h aunted over the years, could rest in peace at Failme to demonstrate a transmitter substance within Mer­ last. H owever, it was soon p ointed out t hat the relationship kel cell granules is also leading some workers to question the between L angerhans cells a nd cell columns found in mouse functional reality of "synapses" between Merkel cell and asso­ epidermis is not typical [ 62], a nd in the meantime t he initial ciated nemite. These "synapses" are sparsely distributed local­ immunologic studies by S ilberberg a nd her colleagues [63,64) ized zones of specialization of apposed m embranes associated were being reported. These and subsequent studies by the same with accumulation of vesicles in t he neurite and sometimes gxoup [37,38), and by others present here t oday (65- 68], as we though exceptionally, of dense-core granules in the Merkel cell: all know, soon put a brake upon the speculations of the kineti­ It is not clear on morphological gr ounds w hat should be re­ cists, and this a lmost perfe ct example o f symbiosis has been gru·ded as "presynaptic" and "postsynaptic," a s ituation that surrendered to immunology. But t here remains at least 1 voice has led to the idea that a form of reciprocal synaptic function crying in the w ilderness of cunent fashionable o pinion. It is may exist at the s ite of Merkel cell-neurite apposition [75]. that of Schweitzer of Heidelberg (69], who, on the basis o f Gottschaldt* has pointed out t hat "synapses" are not sufficient observations o f changing patterns of keratinization in embry­ in number or extent to provide t he ru·ea of depolru·ized nemite onic, neonatal, and postnatal mouse tail, claims t hat Langer­ membrane required to give rise to an action potential of 1,000 hans cells do exer t some control over certain aspects of keratin­ impulses/ sec. T his ru·gument and the fact that in some animals ization. E ven if their prime function is immunologic, additional such as birds a nd amphibians (76] Merkel cell-new·ite com­ fu nctions ru-e conceivable; after all, s ubsidiary functions fo r plexes ru·e rapidly adapting mechanoreceptors, rather than melanocytes ru-e constantly being s peculated upon, mainly be­ slowly adapting ones (as in mammals), has led Gottschaldt to cause of their occurrence in tissues to which solar r adiation can have no access. • Gottschaldt KM , personal communication. 10 BREATHNACH Vol. 75, No. 1

q uestion the role of the Merkel cell as the prime transducer 6. Streil ein JW: Lymphocyte traffic, T-cell malignancies and the skin. element, and even its functional identity in all vertebrate J Invest Dermatol 71:167-171, 1978 7. McDonald DM, Schmitt D, Germain D, Thivolet J : Ultrastructural classes. He believes it may be more an auxiliary cell than an demonstration ofT cells in cutaneous t issue sections using spe­ effector cell, an idea in line with M unger's suggestion [75] of a cific anti-human T cell antiserum. Br J Dermatol 99:641-646, "trophic" relationship between it and the neurite, which in a 1978 limited sense can be said to exist during development in the 8. Reams WM, Tompkins SP: A developmental study of murine epidermal Langerhans cells. Dev Bioi 31:114-123, 1973 salamander [ 45,49]. 9. Breathnach AS: Aspects of epidermal ultrastructure. J Invest Der­ The very close association between Merkel cell and neurite matol 65:2-15, 1975 makes it difficult to formulate an experimental approach to 10. Katz SI, Tamaki K, Sachs D: Epidermal Langerhans cells a re investigation of their individual roles in stim ulus transduction. derived from and are repopulated by mobile precursor cells which originate in bone marrow. Clinical Research 27:446A, 1979 Denervation experiments have not been rewarding. However, 11. Katz SI, Kunihiko T, Sachs DH: Epidermal Langerhans cells are an observation by Budtz [77] raises hopes of a more promising derived from cells which originate in bone marrow. Nature, in avenue. He has reported that Merkel cells degenerated and press disappeared 14 days after removal of the pars distalis of the 12. Munger BR: The intraepidermal innervation of the s nout skin of pituitary the opossum. A light and electron microscope study with obser­ gland in the toad. Apart from the intrinsic interest of vations on the nature of Merkel's Tastzelle. J Cell Bioi 26:79-97, this demonstration of Merkel cell dependence upon, presum­ 1965 ably, some pituitary hormone, it would appear to offer the 13. Lyne AG, Hollis DE: Merkel cells in the s heep epidermis during opportunity for electroph ysiological and other investigations of fetal development. J Ultrastruct Res 34:464-472, 1971 the neurite divorced from its assoc 14. English KB: Cell types in cutaneous type I m echanoreceptors iated Merkel cell. It will be (Haarscheiben) and their alteration with injury. Am J Anat 141: of interest to see if partial hypophysectomy produces the sam e 105-126, 1974 result in oth er classes and species of animals, and if the terminal 15. English KB: Morphogenesis of Haarscheiben in rats. J Invest neurite is affected in any way by th e procedure. There must be Dermatol 69:58-67, 1977 thousands of surgically or chemically h ypophysecto 16. English KB: The ultrastructure of cutaneous type I mechanorecep­ mized tors (Haarscheiben) in cats following denervation. J Comp Neurol beasts in laboratories throughout the world that could provide 172:137- 164, 1971 an answer to this question after simple biopsy. 17. Breathnach AS: An Atlas of the Ultrastructure of Human Skin. London, Churchill, 1971 CONCLUSION 18. Breathnach AS: Embryology of human skin: A review of ultrastruc­ tural studies. J Invest Dermatol 57:133-143, 1971 Of the 3 types of branched cell discussed in this Symposium, 19. Winklemann RK, Breathnach AS: The Merkel cell. J Invest Der­ the melanocyte is by far the most studied; it has already been matol 60:2-15, 1973 the subject of 10 International P igment Cell Conferences (the 20. Steinberg MS, Wiseman LL: Do morphogenetic t issue re-arrange­ ments require active cell movements? J Cell Bioi 55:606-615, 11th is scheduled to take place in Japan in 1980) , and was th e 1972 subject of the 16th Symposium on the Biology of Skin held at 21. Rees DA, Lloyd CW, Thorn D: Control of grip and stick in cell the Oregon Regional Primate Research Center in 1966. At that adhesion through lateral r elationships of membrane glycopro­ Symposium, 3 papers dealt with th e Langerhans cell, and t he teins. Nature 267:1 24-128, 1977 22. Campbell RD, Campbell JH: Origin and continuity of desmosomes, editors of the published proceedings [78) remarked in their Origin and Continuity of Cell Organelles. Edited by J Reinert, H preface that "the particular identity, origin, a nd possible func­ Ursprung. Berlin, Springer-Verlag, 1971, pp 261-298 tion of this cell still escapes us." Undoubtedly t he Langerhans 23. Allen TD, Potten CS: Desmosomal form, fate and function in cell owed its inclusion to the lingering suspicion that it belonged mammalian epidermis. J Ultrastruct Res 51:94-105, 1975 24 . Skerrow CJ: Intercellular adhesion and its role in epidermal differ­ to the melanocyte lineage of cells, but it was at t hat Symposium entiation. Investigative and Cell Pathology 1:23-37, 1978 that this relationship was critically questioned on the basis of 25. Breathnach AS: Observations on cytoplasmic organelles in Langer­ ultrastructural observations, including reports of Basset's find­ hans cells of human epidermis. J Anat 98:265-270, 1964 ings in histiocytosis X [79), and Wolff a nd Winklemann's [80] 26. Basset F, Soler P, Wyllie L, Mazin F, Turiaf J : Langerhans cells and lung interstitium. Ann NY Acad Sci 278:599- 611, 1976 enzyme histochemical studies. Certainly, that 1966 Symposium 27. Wolff K: The Langerhans cell. Curr Probl Dermatol 4:79-145, 1972 represents a milestone in the lengthy story of the Langerhans 28. Rawles ME: Origin of pigment cells from the neural crest in the cell in that it liberated it from the restrictions of fixed ideas mouse embryo. Physiological Zoology 20:248-266, 1948 then current. It is entirely appropriate therefore that we should 29. Ferreira-Marques J : Systema sensitivum in traepidermicum. Die Langerhannschen zellen als receptoren d es hellen schmerzes. discuss once again here in Oregon the exciting developments Doloriceptors. Archiv fur Dermatologie und Syphilis 193:191- that have resulted from major rethinking over the past decade. 250, 1951 These developments lie mainly in the field of immunology, and 30. Masson P: My conception of cellular nevi. Cancer 4:9-38 1951 in this overview I h ave made only passing reference to th em as 31. Billingham RE, Medawar PB: A study of the branched c~ ll s of the mammalian epidermis with special reference to the face of their they will be extensively reviewed in presentations to follow. I division products. Philos Trans R Soc Lond [Bioi] 237:151-171 am glad to see the inclusion of 3 papers on melanocytes, not 1953 ' only for old times' sake, but to emph asize the fact t hat t hough 32. Silvers WK: A histological and experimental approach to determine they no longer claim a family relationship with the· Langer hans the relationship between gold-impregnated dendritic cells and melanocytes. Am J Anat 100:225-240 cell, the 2 may still interact in a significant 1957 manner within the 33. Breathnach AS: The cell of La n gerha~s. Int Rev Cytol 18:1-28 epidermis [81]. The Merkel cell, with just 1 paper, still presents 1965 ' a number of interesting problems. It is interesting to speculate 34. Breathnach AS, Silvers WK, Smith J, Heyner S: Langerhans ce lls that, had he lived longer, Paul Langerhans might have become in mouse skin experimentally deprived of its neural crest com­ interested in ·the Merkel cell. He was close enough to it on ponent. J Invest Dermatol 50: 147-160, 1968 35. Reams WM, Scoggins RB, Ze lickson AS: An experimental and EM another occasion [82]. analysis of the origin of Langerhans ce lls. Association of Southern Biologists Bulletin 14:38, 1967 REFERENCES 36. Breathnach AS, Wyllie LM-A: The problem of the Langerhans cells, Advances in Biology of Skin, vo l VIII, The Pigmentary 1. Billingham RE: Dendritic ce lls. J Anat 82:93-109, 1948 · System. Ed1ted by W Montagna, F Hu. Oxford, Pergamon, 1967, 2. Masson P: Les naevi pigmentai..res, tumeurs nerveuses. Annales pp 97-113 d'Anatomie Pathologique et d'Anatomie Normale Medico-Chi­ 37. Si!berberg-Sinakin I, Baer RL, Thorbecke GJ: Langerhans ce lls. A rurgicale 3:4 17-453, 657-696, 1926 1:eview of their nature with emphasis on their immunologic func­ 3. Andrew W, Andrew NV: Lymphocytes in the normal epidermis of tions. Prog Allergy 24:268-294, 1978 the rat and of man. Anat Rec 104:217-232, 1948 38. Silberberg-Sinakin I, Thorbecke GJ: The Langerhans cell , The 4. Andrew W: Differentiation of migrant cells in the human epidermis Reticuloendothelial System, vo l I. Edited by I Carr, WT Deans. and the non-specificity of the germ-layers. Arch Histo Jpn 29: New York, Plenum Press, in press 2ll-235, 1968 39. Rowden G, Phillips TM, Lewis MG: la antigens on indeterminate 5. Fichthelius KE, Groth 0 , Liden S: The skin, a f:trst level lymphoid cells of the epidermis: Immunoelectronmicroscopic studies of organ'? Int Arch Allergy Appl Immunol 37:607-620, 1970 surface antigens. Br J D ermatol 100:531-542, 1979 July 1980 BRANCHED CELLS IN EPIDERMIS 11

40. Breathnach AS: Electron micrographs from a collection. Clin Exp Lyne, BF Short. Sidney, Angus and Robertson, 1965, pp 1-24 Dermatol 2:1-16, 1977 62. Mackenzie IC: Ordered structme of the epidermiP>. J Invest Der­ ~1. Hashimoto K: The ultrastructme of human embryos. X, Me1·kel matol 65:45-51, 1975 tactile cells in the finger and nail. J Anat 111:99-120, 1972 63. Silberberg I, Baer RL, Rosenthal SA: Circulating Langerhans cells 42. Winklemann RK: The Me1·kel cell system and a compaTison be­ in a dermal vessel. Acta Dermato Venereol (Stockh) 54:81-86 tween it and the new-osecretory or APUD cell system. J Invest 1974 ' DeTmatol 69:41-46, 1977 64. Silberberg-Sinakin I, Thorbecke GJ, Baer RL, Rosenthal SA Ber­ 43. Fujita T: Concept of pamneuTons. Arch Histol Jpn 40 [suppl]:l-12, ezowsky V: Antigen-bearing Langerhans cells in skin, d~rmal 1977 lymphatics and in lymph nodes. Cell Immunol25:137-l51 1976 44. Tweedle CD: Ultrastructme of Merkel cell development in aneu­ 65. Shelley WB, Juhlin L: Selective uptake of contact allerg~ns by rogenic and control amphibian larvae (Ambystoma). Nemosci­ Langerhans cells. Arch Dermatol 113:187-192, 1977 ence 3:481-486, 1978 66. Sting! G, Katz SI, Clement L, Green I, Shervach EM: Immunologic 45. D iamond J, Cooper E, Tmner C, Macintyre L: Trophic regulation functions of Ia-beru·ing epidermal Langerhans cells. J Immunol of nerve sprouting. Science 193:371-377, 1976 121:2005-2013, 1978 46. Suzuki H, Ochiai T , Shunichi B: Merkel cells in the newborn rats 67. Sting! G, Katz SI, Shervach EM, Rosenthal AS, Green 1: Analogous and the denervated animals. J Invest Dermatol 72:271, 1979 functiOns of macrophages and Langerhans cells 111 the initiation 47 . Kurosumi K, Suzuki H: Fine structure of the dendl·itic cells and of the immune response. J Invest Derma to! 71:59-64, 1978 Merkel cells in the epidermis of vaTious mammals. Japanese 68. Rowden G: Immuno-electron microscopic studies of surface recep­ Journal of Dermatology; Series B (Tokyo) 81:343-351, 1971 tors and antigens of human Langerhans cells. Br J Dermatol 97: 48. K urosumi K, Kmosumi U, !none K: Morphological and morpho­ 593-608, 1977 metric studies with the electron microscope on the Merkel cells 69. Schweizer J: Langerhans cells, epidermal growth control mecha­ and associated nerve terminals of normal and denervated skin. nisms and type of keratinization, The epidermis in Disease. ATch Histol Jpn 42:243-261, 1979 Edited by R Mru·ks. Basel, Kru·ger, in press 49. Diamond J: The regulation of nerve sprouting by extrinsic influ­ 70. Iggo A, Muir AR: 1~he structure and function of slowly adapting ences, The Nemosciences: Fomth Study Program. Edited by FO touch corpuscles 111 harry skm. J Physwl (PaTis) 200:763-796 Schmitt, FC WaTden. Boston, Massachusetts Institute of Tech­ 1969 ' nology Press, 1979, pp 937-955 71. Kmosumi K: Pru·aneuronic and related cells in the mammalian 50. Niebauer G: Dendl·itic cells of human skin, Experimental Biology skin, Chromaffin, Enterochromaffin and Related Cells. Edited by and Medicine, vol 2. Edited byE Hagen, W Wechsler, F Zilliken. RE Coupland, T Fujita. Amsterdam, Elsevier, 1976, pp 187-189 Basel, KaTger, 1968, pp 1-44 72. Tachibana T: The Merkel cell in the labial ridge of the anman 51. Caudiere M: Recherches sur !'evolution des cellules pigmentaires tadpole. Anat Rec 191:487-502, 1978 das certains epitheliomes envahissant l'epiderme. Ann Anat Pa­ 73. Crowe R, Whitear M: Quinacrine fluorescence of Merkel cells in thol (PaTis) 3:119-145, 1926 Xenopus laevis. Cell Tissue Res 190:273-283, 1978 52. Rappaport BZ: Studies on atopic dermatitis. Arch Pathol 61:318- 74. Fox H, Whitear M: Observations on Merkel cells ·in amphibians. 321, 1956 Biologie Cellulaire 32:223-232, 1978 53. Pinkus H, StaTicco RJ, Kropp PJ, Fan J: The symbiosis of mela­ 75. Munger BL: Neural-epithelial interactions in sensory receptors. J nocytes and human epidermis under normal and abnormal con­ Invest Dermatol 69:27-40; 1977 ditions, Pigment Cell Biology. Edited by W Gordon. New York, 76. Parducz A, Leslie RA, Cooper E, Turner CJ, Diamond J: The Academic Press, 1959, pp 127-138 Merkel cells and the rapidly adapting mechanoreceptors of the 54. Fitzpatrick TB, Breathnach AS: Das epidermale Melanin-Einheit­ salamander skin. Neuroscience 2:511-521, 1977 System. Dermatologische Wochenschrift 147:481-489, 1963 77. Budtz PE: Epidermal structure a~d dynamics of the toad (Bufo 55. Quevedo WC Jr: The control of color in mammals. American bufo), depnved of the pru·s d1stahs of the pitUi tru·y gland. Journal Zoologist 9:531-540, 1969 of Zoology, 189:57-92, 1979 56. Quevedo WC Jr: Epidermal melanin units: Melanocyte-keratino­ 78. Montagna W, F Hu (eds): Advances in Biology of Skin vol VIII cyte interactions. American Zoologist 12:35-41, 1972 The Pigmentru·y System. Oxford, Pergamon, 1967 ' ' 57. Merriman JA, Nieland ML, Wedmore RJ: Guinea-pig keratinocytes 79. Breathnach AS, ':Vyllie LMA: The problem of the Langerhans cells, and melanocytes in tissue culture: Scanning transmission, and Advances m Bwlogy of Skm, vol VIII, The Pigmentru·y System. high voltage electron microscope observations. J Microsc 116: Edited by W Montagna, F Hu. Oxford, Pergamon, 1967, pp 97- 243-253, 1979 113 58. Wolff K, Winklemann RK: Quantitative studies on the Langerhans 80. Wolff K, Winklemann RK: Non-pigmentary enzymes of the cell population of guinea pig epidermis. J Invest Dermatol 48: melanocyte-Langerhans cell system, Advances in Biology of Skin 504-513, 1967 vol VIII, T he P igmentru·y System. Edited by W Montagna F Hu' 59. Allen TD, Potten CS: Fine-structural identification and organiza­ Oxford, Pergamon, 1967, pp 135-167 ' · tion of the epidermal proliferative unit. J Cell Sci 15:291-319, 81. Griscelli C, DuJ"andy A, Guy-Grat~d D , Daguillru·d. F, Herzog C, 1974 Prumeras M: A syndl"ome assoc1atmg partial albm1sm and im­ 60. Pot ten CS, Allen TD: The fin e structure and cell kinetics of mouse munodeficiency. Am J Med 65:691-702, 1978 epidermis after wounding. J Cell Sci 17:413-447, 1975 82. Langerhans P: Zur pathologishen Anatomie der Tastkorper. Archiv 61. Billingham RE, Silvers WK: Some unsolved problems in the biology fur Pathologische Anatomie und Physiologie und fur Klin Med of skin, Biology of the Skin and Hair Growth. Edited by AG 45:413-417, 1869