Langerhans Cells: Antigen Presenting Cells of the Epidermis

Immunobiol., vol. 168, pp. 285-300 (1984)

1 Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida, U.S.A. 2 Department of Dermatology, Southwestern Medical School, Dallas, Texas, U.S.A.

Langerhans Cells: Antigen Presenting Cells of the Epidermis

J. W. STREILEIN1 and P. R. BERGSTRESSER2

Abstract While epidermis in the skin provides an excellent barrier to the environment, it is an incomplete one. Some antigenic material can penetrate through the stratum corneum (or be introduced pathologically) where strategically placed epidermal Langerhans cells reside. In this review, we have assembled relevant data concerning the antigen presenting potential of epidermal Langerhans cells. Strong circumstantial evidence derived from in vitro studies of epidermal cell suspensions enriched for Langerhans cells indicates that Langerhans cells possess this capability. In vivo studies with intact skin indicate that critical numbers of functioning Langerhans cells are essential for successful induction of contact hypersensitivity by epicutaneously applied haptens. And within the past several months, experiments with purified preparations of epidermal Langerhans cells have proven that these cells, and perhaps they alone among epidermal cells, possess the capacity of processing and presenting haptenic determinants to the immune system. The challenge for the future is to determine the extent to which this unique property of Langerhans cells affords physiologic protection to the skin and under what pathologic circumstances altered Langerhans cell function leads to disease.

Introduction

The enormous amount of information that has been generated recently concerning the role of Langerhans cells in cutaneous immune reactivities makes it difficult to believe that the proposal that Langerhans cells function as antigen presenting cells in skin is only a decade old. The signal discovery responsible for this burst of experimental interest was the observation of SILBERBERG and her colleagues in 1973 (1, 2) that lymphocytes cluster around epidermal Langerhans cells in contact hypersensitivity reactions. During this past decade, numerous in vitro experiments using epidermal cells partially enriched for Langerhans cells have been conducted, chiefly by G. STINGL and his various collaborators (3, 4), to document the role of Langerhans cells in antigen presentation. A parallel series of in vivo studies emanating predominantly from the laboratories of BERGSTRESSER and STREILEIN (5) have examined the role of epidermal Langerhans cells in vivo Abbreviations: SALT = Skin Associated Lymphoid Tissues; UVR MHC = Major Histocompatibility Complex.

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in the induction of contact hypersensitivity in mice. Implicit in the assignment of a putative role for Langerhans cells in antigen presentation is the likelihood that these cells play an important role in protective host defense mechanisms that are operative in cutaneous malignancies and in viral infections.

Historical development of knowledge of Langerhans cells More than a 100 years ago PAUL LANGERHANS, working with a gold impregnation stain on tissue sections of human skin, discovered a population of cells with a dendritic configuration within epidermis (6). The staining reaction with gold suggested to this investigator that these cells were cutaneous nerve endings and served as remote outposts of the nervous system. Subsequently, a second population of melanin-producing dendritic cells (melanocytes) was also identified to exist within the epidermis, and because of their similar morphologic shapes, it was proposed that Langerhans cells were related in some way to melanocytes. This view was strengthened in the early 1950s by BILLINGHAM and MEDAWAR (7), who conducted a careful series of experiments in melanotic and amelanotic guinea pig skin, from which they concluded, almost exclusively on circumstantial evidence, that Langerhans cells were effete melanocytes. The next important observation concerning this issue was made in 1961 by BIRBECK (8) who noted that Langerhans cells contained within their cytoplasm a unique organelle which now carries the name of Birbeck granule. He observed that these granules were never present within melanocytes or keratinocytes, and suggested that Langerhans cells might be an independent and unique population of epidermal cells. The idea that there was a unitary relationship between Langerhans cells and melanocytes through a common origin from the neurocrest during ontogeny was finally laid to rest by an elegant study conducted by BREATHNACH and his colleagues (9). These workers demonstrated that mouse skin that was experimentally deprived of infiltrating neurocrest elements would acquire Langerhans cells, but not melanocytes. Thus, it was formally established that Langerhans cells represent a unique epidermal cell population, albeit of unknown origin and function. Because of the regular spacing of Langerhans cells in a grid-like pattern through mammalian epidermis, an idea emerged that perhaps Langerhans cells in some way were related to the orderly alignment of epidermal cells from the basal cell layer into a keratinized superficial stratum corneum. Several investigators proposed: (a) that there might exist an epidermal proliferative unit in which a finite number of keratinocytes constellated near an eccentrically placed Langerhans cell, and (b) that keratinocytes from this unit emerged from the basalar layer and underwent definitive

Langerhans Cells Present Antigen in Skin' 287 differentiation into fully keratinized squamous cells of the stratum corneum. The implication was made that this orderly process was supervised by the attendant Langerhans cell (10, 11, 12). This brief survey of the historical progression of ideas concerning Langerhans cells is more completely summarized in the definitive review by WOLFF in 1972 (13). SILBERBERG's observations of a histologic relationship between Langerhans cells and infiltrating lymphocytes in contact sensitivity reactions was made at about that time and published in 1973. This link forged between Langerhans cells and the immune system has progressed vigorously until today when virtually all studies of epidermal Langerhans cells concern their role in immunology and ignore other possible types of biologic activity.

Cell biology of Langerhans cells PAUL LANGERHANS' original description of these intraepidermal cells relied upon a histologic technique of gold impregnation for their identification. It was subsequently learned that among epidermal cells, Langerhans cells uniquely expressed high amounts of the cell surface enzyme ATPase (14). Histochemical techniques were devised to identify Langerhans cells by virtue of this surface marker. ATPase was useful in distinguishing Langerhans cells from the other cells that resemble them, that is, epidermal dendritic cells such as melanocytes, Merkel cells, and indeterminate cells, as well as from typical keratinocytes. In truth, all nucleated cells have cell surface ATPase, however, Langerhans cells stand alone among epidermal cells in their enormous quantitative expression of this enzyme. Unequivocal documentation at the microscopic level of Langerhans cells was achieved with a description of the unique cytoplasmic granule by BIRBECK. Since 1961, this cytoplasmic organelle has been used as the final arbiter of whether or not a cell is a Langerhans cell. In fact, it is the presence of these granules within the cells of the disease histocytosis X which indicated that Langerhans cells could be identified outside the epidermis. Beginning 10 years ago, as awareness of a potential role for Langerhans cells in immune reactions developed, a variety of cell surface molecules were searched for and identified on Langerhans cells. Notable among these are the Fc receptor for IgG, and the receptor for the C3b component of complement (15). It was also discovered that Langerhans cells express class II major histocompatibility complex (MHC) molecules, designated as Ia in the mouse, and HLA D/DR in man (3, 16, 17, 18, 19,20). It has gradually become apparent that while Ia expression is an important feature of Langerhans cells, it is not a constant feature. In fact, a subpopulation of Langerhans cells exists which is Ia negative. Proof of this comes from work with monoclonal antibodies in human epidermis where the reagent OKT6

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routinely identifies more Langerhans cells than is appreciated when an antiHLA D/DR reagent is employed (21). This remains a unique observation in man since no comparable universal marker, such as that identified by OKT6, is available in rodent species. As will be appreciated later, the ability to identify all Langerhans cells and the realization that there may be subpopulations of Langerhans cells may have important implications for the range and possible discrepancies in immunobiologic reactivities reported for these cells. At the very least, the evidence using OKT6 would suggest that the number of Langerhans cells in various cutaneous sites in mice are likely to be underestimated when the fluorescent antibody technique using an anti-Ia reagent is used to identify them. The c~ll-surface molecules just described can be used to identify Langerhans cells as distinct from other epidermal cells. It is important to point out that, like all nucleated cells, Langerhans cells express the glycoproteins encoded by class I major histocompatibility complex molecules. In addition, they express on their surfaces molecules which in mice (L y5 antigen) and man (T200) mark them as descendants of precursor cells residing within the bone marrow. Langerhans cells were first described within the epidermis of body wall skin. Within this typical site, Langerhans cells assume a superbasalar position and a dendritic configuration. They are spaced relatively regularly with a density of approximately 800 to 1200 per square mm (22, 23). This description is typical of Langerhans cells in the skin of virtually every mammalian species that has been examined. Among genetically disparate strains of mice there are minor variations in the density of Langerhans cells within body wall skin, but all fit into this same general pattern (24). As skin is modified for particular functions at various regions, such as tail skin of the mouse and the skin on the sole of the foot, the density of Langerhans cells falls to a third to half the densitiy found in normal body wall skin (25, 26). Langerhans cells have been amply demonstrated intraepithelially within the oral mucous membrane (27), the membranes of the vagina and ectocervix (28), and even in the rumen of sheep (29). Hamster cheek pouch epithelium contains only 10-20 % the number of Langerhans cells found in normal hamster body wall skin (22). In mouse tail skin, Langerhans cells are distributed unevenly in the following pattern: the scales of the mouse tail skin, which are parakeratotic, are devoid of Langerhans cells. The surrounding interscalar regions are orthokeratotic, and Langerhans cells are amply present there. Experimental advantage of this unusual distribution has been taken to test the immune properties of mouse skin that is relatively depleted of Langerhans cells. Normal corneas of every species yet examined are devoid of Langerhans cells (22, 30, 31, 32). At the circumferential margins of the limbic attachments of the cornea to the sclera, Langerhans cells are arranged in a circular fashion with only a rare cell penetrating into the cornea epithelium proper.

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While Langerhans cells are normally absent from the columnar epithelium of the trachea, in smokers, in whom metaplastic changes occur in the tracheal epithelium (33), and in Vitamin A deficiency (34), Langerhans cells appear when the metaplastic change converts the native epithelium into a stratified squamous form. However, observations in humans with fibrotic lung disorders suggest that squamous metaplasia may not be the only reactive process which leads to such infiltration (33). Langerhans cells have also been identified within the thymus, especially among the epithelial cells of the medulla (35). From this long list of cutaneous and epithelial sites containing Langerhans cells, the idea has emerged that Langerhans cells occur in those epithelial tissues in which stratified squamous epithelium undergoes keratinization. Exceptions to this rule exist, however. Cells with Birbeck granules have been identified in peripheral lymph nodes (36) and in two pathologic circumstances, histocytosis X and eosinophilic granuloma (37, 38). In these tissues, typical Langerhans cells exist with no direct histologic interaction with keratinizing epithelial structures. The numbers of identifiable Langerhans cells within skin can be changed dramatically by several forms of local treatment. The best studied example is exposure of skin to ultraviolet radiation (UVR) which has the effect of dramatically reducing the number of ATPase positive and la positive cells within epidermis. This effect is achieved in human, mouse, and hamster skin and is presumably a general biological phenomenon (22, 39, 40, 41, 42). Part of this effect has to do with the capacity of UVR, especially at lower doses, to cause resident Langerhans cells to modulate some molecules off their surfaces, rendering these markers no longer detectable. This is the case with ATPase and la. However, with higher doses of UV radiation in humans, there is actual loss of exposed Langerhans cells. Treatment of skin with X or gamma irradiation has little if any effect on resident Langerhans cells as detected by these conventional assays (24). By contrast, local treatment with corticosteroids applied epicutaneously produces a dramatic decrease in the number of identifiable la positive cells (40,43,44). However, use in man of the OKT6 antibody indicates that virtually all of the original density of Langerhans cells is preserved following corticosteroid therapy since the number of OKT6 positive cells remains virtually unchanged, or is only slightly reduced (44). Thus, the effect of cortisone is to cause the cells to erase la expression from their surface without causing them to die or to leave the epidermis in appreciable numbers. Another method for reducing Langerhans cells within epidermis is to strip the stratum corneum repeatedly with cellophane tape (45, 46). By tape stripping it is possible to obliterate virtually all Langerhans cells in the stripped areas. For an interval of several days thereafter, stripped regions of epidermis become devoid of Langerhans cells. Since evidence suggests that Langerhans cells are only poorly able to enter into mitosis (23, 47, 48) in

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situ, their replenishment in tape-stripped areas is thought to result from their migration into skin from an extracutaneous source. This matter is discussed in more detail below. The fact that mur~ne Langerhans cells express the L y5 marker indicates that they must be of bone marrow origin. This has been formally proven by KATZ et al. (49), and by FRELINGER et al. (50), who studied radiation chimeras. The former group observed over the course of several weeks after irradiation and reconstitution of mice with allogeneic hematopoietic cells that dendritic cells of donor origin appeared in recipient epidermis as early as 14 days, and in significant numbers by 30 days. FRELINGER et aI., demonstrated that Ia molecules made by epidermal cells of these chimeras were of the donor genetic type. Thus, epidermal Langerhans cells are the descendants of a precursor which resides among hematopoietic cells. Langerhans cells appear to be able to migrate into the epidermis from a hematogenous source throughout life. They are first identified in fetal skin in mice on the 16th day of gestation (26). The studies of KATZ et al. (49), indicate that in adult radiation chimeras donor Langerhans cells gradually come to repopulate the unperturbed epidermis during the course of several weeks. We have conducted tape stripping experiments in conjunction with skin grafts as well as in radiation chimeras to document that when epidermis is perturbed severely by tape-stripping, Langerhans cells from a hematogenous source can infiltrate skin extremely rapidly (46). As early as 4 days after tape-stripping, Langerhans cells can be seen to enter into the epidermis and by seven days their numbers are virtually normal. In an ingenious series of studies, KREUGER and his collaborators (51) have used xenogeneic and allogeneic orthotopic skin grafts applied to nude (athymic) mice to make the following points about Langerhans cells: (a) they are very long-lived cells, since grafts on nude mice continue to retain Langerhans cells of original donor type for weeks and months; (b) MHC incompatibility plays no role in guiding or modifying Langerhans cell entry into epidermis; (c) species determinants do appear to limit Langerhans cell migration since guinea pig or human skin placed on nude mice fails to permit mouse Langerhans cells from penetrating into the epidermis. In summary, Langerhans cells can be readily identified and separated from other epidermal cells on the basis of unique surface markers and cytologic structures. Their regular distribution in body wall skin cranges as skin is modified for various site-specific properties. Moreover, Langerhans cells can be found in most sites of keratinizing stratified squamous epithelium, as well as in selected sites of the lymphoreticular apparatus. Langerhans cells can be functionally as well as physically depleted from body wall skin when a variety of perturbants are applied. Langerhans cells can replenish skin from a hematogeneous source with considerable rapidity, indicating that they are a mobile rather than fixed inhabitant of the epidermal compartment. A more definitive detailed review of this material has recently appeared (52).

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Immunobiology of Langerhans cells For years the macrophage has assumed a central role in our understanding of antigen processing and presentation for induction of immune responses (53, 54, 55). Over the last five years, another population of bone marrow derived cells has been described by STEINMAN and his colleagues (56), the dendritic cell, which is now also recognized as an extremely important cell that is capable of antigen processing and presentation (57). While macrophages, Langerhans cells and dendritic cells share a common origin among the bone marrow stem cell pool, it is not clear that they are of the same cell lineage. Moreover, Langerhans cells share some but not other properties with macrophages or with dendritic cells (58). Macrophages, dendritic cells, and Langerhans cells are each capable of migrating from its site of origin to somatic tissues. All three cell types display the property of adherence to glass and plastic surfaces. However, dendritic cells differ from macrophages in this property; following their initial adherence in vitro to a surface, dendritic cells release from that surface beyond two and less than twenty four hours. By contrast, the strength of macrophage adherence only increases during this interval of time. Historically and functionally, the dominant feature of macrophages is their capacity to phagocytize particulate matter from the extracellular environment. While all three cell types (macrophages, dendritic cells, and Langerhans cells) share the property of endocytosis, neither Langerhans cells nor dendritic cells display appreciable phagocytic function. This realization strongly supports the contention that phagocytosis is not an essential prerequisite to processing and presentation of antigens by antigen presenting cells. Surface expression of class II MHC molecules is thought to be an essential feature of antigen presenting cells. It would appear that surface expression of class II molecules is constitutive in dendritic cells and Langerhans cells, but is inducible in macrophages (55). However, at the time of antigen presentation, all three types bear class II molecules. Macrophages and Langerhans cells share the properties of surface Fc and C3b receptors, but dendritic cells apparently do not (56). All three cell types have been demonstrated to secrete interleukin 1. Thus, it can be seen that Langerhans cells possess an array of features which they share partially with antigen presenting macrophages and dendritic cells, and which makes them excellent candidates for antigen presenting functions. Evidence in favor of a role for Langerhans cells in antigen presentation has been generated from in vitro as well as in vivo studies. In the former, it is now known that allogeneic mixed epidermal cell-lymphocyte cultures result in a proliferative response on the part of the latter cell type (59, 60). Using rosetting techniques that allow for the enrichment of Langerhans cells within an epidermal cell suspension prepared by trypsinization, STINGL et a1. (61), have demonstrated in the guinea pig that the Langerhans cell is responsible for this allogeneic stimulation. In addition, these inves-

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tigators have used similar Langerhans cell enriched suspensions to demonstrate that, following antigen pulsing, these cells are able to present antigen effectively to specifically primed T lymphocytes (which then undergo proliferation). It has also been demonstrated that cytotoxic T cells can be generated in cultures in which the source of target alloantigens is provided by an epidermal cell preparation enriched for Langerhans cells (62). Several groups of investigators documented that UVR exposure robs epidermal cell suspensions of their putative antigen presenting and mixed lymphocyte inducing activity (63, 64, 65). To the present, evidence such as that just enumerated makes a strong circumstantial case for the capacity of Langerhans cells to function as antigen presenting cells in vitro. Experimental efforts to produce purified populations of Langerhans cells have only reached success within the past months. As a consequence, experimental details concerning the antigen presenting potential of purified Langerhans cells in comparison to purified keratinocytes or other epidermal cell populations must yet be accomplished. Our laboratory, as well as several others, have attempted to define the antigen presenting function of Langerhans cells in vivo. Chiefly, the strategy has been to take advantage of skin in which Langerhans cells are either naturally or artifically depleted and compare it with normal skin for its capacity to support the induction of contact hypersensitivity to epicutaneously applied haptens. We have employed mouse corneas (which are devoid of Langerhans cells) to demonstrate that cornea grafts lack the capacity to immunize recipient mice to Ia alloantigens (66). We have altered corneas by treating them with an irritant which brings Langerhans cells into the central epithelium and demonstrated that cornea grafts containing Langerhans cells display Ia immunogenicity (67). More importantly, we and others have applied hapten via mouse tail skin, hamster cheek pouch epithelium, through tape-stripped as well as ultraviolet radiation treated body wall skin sites (68, 69, 70, 71). In each instance, we have documented that these cutaneous sites which are deficient in normal Langerhans cells fail to support the induction of contact hypersensitivity. In fact, cutaneous surfaces that are depleted of normal Langerhans cells permit epicutaneously applied hapten to induce a systemic state of specific unresponsiveness. By using low dose UVR to abdominal wall skin in mice, and by examining several different genetically disparate strains of inbred mice, we have determined that some but not all genetic stocks of mice are resistant to unresponsiveness induced by UVR plus hapten. By using appropriate segregant genetic populations as well as H-2 con genic recombinant strains of mice we have determined that two and probably three independent genetic loci govern this process. One locus resides within the major histocompatibility complex. By using these experiments in conjunction with tape-stripping followed by hapten painting at the stripped site we have produced evidence which suggests that two different pathways of antigen

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presentation may participate following cutaneous application of hapten. One pathway, which appears to be present in the UVR suppressible mice, requires epidermal Langerhans cells for effective antigen presentation to take place. The other pathway, which apparently is not present in UVR suppressible mice, allows hapten to interact with antigen presenting cells other than Langerhans cells and leads to induction of typical contact hypersensitivity. Thus, it would appear that epicutaneously applied hapten can be effectively processed on the one hand by epidermal Langerhans cells, and on the other, by antigen presenting cells beyond the epidermis. We have come to the same conclusions about a critical in vivo role in antigen presentation for epidermal Langerhans cells from a different line of investigation. Skin grafts have been fashioned from body wall skin of mice one hour after epicutaneous painting with immunizing doses of hapten (72). When these grafts are placed orthotopically on syngeneic recipient mice, the latter develop typical contact hypersensitivity as revealed by ear swelling in response to local challenge. When similar grafts are placed on H-2 incompatible recipient mice, contact hypersensitivity is not induced. Since hapten-derivatized skin grafts are able to induce contact hypersensitivity in semiallogeneic recipients, in which one parent is syngeneic with the graft donor, we have concluded that all of the essential antigen presenting elements required for induction of contact hypersensitivity must reside within the haptenated skin grafts and that the effectors of contact hypersensitivity are restricted by graft-specific antigens encoded by the MHC. By judicious use of tape-stripping (to remove Langerhans cells) and exposure to 60°C of one hour (to eliminate all viable cells), we have been able to demonstrate that the antigen presenting potential of these skin grafts resides among a viable cell population that is eliminated by tape-stripping, i.e. Langerhans cells. Thus, a considerable body of circumstantial evidence can be mobilized to support the hypothesis that epidermal Langerhans cell are essential antigen presenting cells within the skin. Definitive proof of antigen presenting function of Langerhans cells in vivo has had to await the technological advances necessary to produce purified populations of these cells from normal epidermis. Partially purified populations of murine epidermal cells that are hapten derivatized and used to induce sensitization have given conflicting answers. PTAK et al. (73), have used Fc receptor-bearing cellenriched epidermal suspensions prepared from mouse tail skin. When hapten derivatized and inoculated intravenously into syngeneic mice, they claim that these cells produce contact hypersensitivity. TAMAKI et al. (74), by contrast have claimed that similar preparations inoculated intravenously induce unresponsiveness. Dendritic cells and induced peritoneal macrophages represent the only known populations of hapten derivatized lymphoreticular cells that are capable of inducing contact and delayed hypersensitivity following intravenous inoculation in mice (75). Alternatively, hapten-derivatized spleen cells, thymocytes, lymph node cells, and

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uninduced peritoneal macrophages induce unresponsiveness when inoculated by the intravenous route. In fact, UVR treated hapten-conjugated epidermal cells also induce unresponsiveness when inoculated intravenously (76). Therefore, a definitive test of the antigen presenting capabilities of Langerhans cells would be to take purified preparations and determine whether following haptenization they induce sensitization or unresponsiveness by the intravenous route. Technological success in this area has recently been achieved in our laboratory by SULLIVAN et al. (77), using monoclonal antibodies against murine Ia antigen and the fluorescent activated cell sorter. With epidermal cell preparations that are greater than 98 % Ia positive, SULLIVAN has demonstrated that as few as 6,000 haptenderivatized cells inoculated intravenously into syngeneic mice produces intense systemic contact hypersensitivity. Similar (as well as larger) numbers of haptenated, purified keratinocytes or hap ten ated spleen cells produce no such effect. On the basis of this and supporting evidence, we feel confident in stating that epidermal Langerhans cells are able to process and present antigen. Moreover, they appear to be capable of delivering to the immune system only an unambiguous «up» signal, leading obligatorily to the induction of contact hypersensitivity. Since we are unable to show that intravenously delivered haptenated Langerhans cells induce any component of down regulation, we propose that Langerhans cells stand alone among epidermal cells in their capacity to deliver an unambiguous inductive signal following antigen exposure.

Future directions in Langerhans cell immunobiology

Numerous unresolved questions remain concerning the immunobiologic function of Langerhans cells. Direct and indirect evidence suggests that Langerhans cells may not be a unitary population of equally differentiated cells within the skin. Clearly, there are cells within the epidermis with cytoplasmic Birbeck granules, but without surface expression of class II MHC molecules. When and if these cells can be separated away from the majority Ia positive Langerhans cells, it should be possible to determine whether the Ia negative cells are able to conduct antigen processing and presentation. Recently it has been reported that mouse skin contains within it (probably within the epidermis) a source of a down regulating signal which in the presence of epicutaneously applied hapten leads to the induction of unresponsiveness (78). It is possible that Ia negative Langerhans cells are the source of this signal. Whether Langerhans cells express I-J determinants on their surfaces, a feature which seems to be characteristic of the epidermal source of the down regulating signal, is at present unknown. The epidermis also contains within it a newly described population of dendritic cells which are neither Langerhans cells nor atypical keratino-

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cytes. These cells, which are Thy-1 positive in the mouse, are Ia negative, and Ly5 positive (79, 80). They are of bone marrow origin (81) and are capable of migrating into the epidermis from a hematogenous source during adult life. However, the kinetics of their migration are distinctly different from epidermal Langerhans cells. Their biologic function is unknown, although it would appear that they are not conventional T lymphocytes nor are they natural killer cells of the classically described type. They also represent candidates for the source of down regulating signals that emanate from the epidermis. Experiments examining this possibility using purified populations of Thy-1 positive cells from murine epidermis are underway. The original studies which linked Langerhans cells to the immune system were based on histologic examination of contact hypersensitivity reactions. One might have presumed that it would have been easy to demonstrate a role for Langerhans cells in the expression of cutaneous immunity. Far from the case, to the present, it has been impossible to demonstrate that there is a critical role for Langerhans cells at sites of epicutaneous challenge for contact hypersensitivity. However, there is evidence to suggest that Langerhans cells participate in some manner in resistance to ticks in guinea pigs (82, 83) and much remains to be done to investigate the role of these cells in other infectious diseases of the skin.

Relationship of Langerhans cells to skin associated lymphoid tissues (SALT) Over the past decade we have proposed that an integrated, organized set of lymphoid structures exists which constellate around skin (84, 84). We have suggested that these skin associated lymphoid tissues (SALT) are comprised of (1) a specialized set of antigen presenting cells within the epidermis, Langerhans cells; (2) distinctive populations of recirculating T lymphocytes that preferentially infiltrate the skin, especially the epidermis; (3) keratinocytes that provide an anatomically distinct environment for these lymphoreticular cells and secrete into that environment immunoregulatory molecules that can profoundly affect the consequences of immune recognition and differentiation; and (4) a set of draining peripheral lymph nodes, integrating this multicellular system that contains, along with the dermis, blood vessels with endothelial cells whose surfaces capture lymphocytes passing through the blood. In an anatomic sense, skin is uniquely situated, and as a consequence, it faces a unique spectrum of antigenic demands. The skin is bombarded daily with the potentially damaging effects of ultraviolet radiation, raising the risk of neoplastic transformation of cells, especially within the epidermis. Neoplastic transformation, especially that induced by ultraviolet light, is associated with expression of unique neoantigens on the surfaces of the

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neoplastic cells (86). It is within this context that a formulation of SALT addresses a physiologic mechanism created to deal with this special demand placed on skin. The scenario by which SALT operates might be stated as follows: Neoplastic transformation of (for example) keratinocytes results in the expression of surface neoantigen(s). When transferred to the ubiquitous Langerhans cells, a neoantigen is processed and presented in immunogenic form. One of two subsequent events may then transpire; either (1) peripatetic T lymphocytes with predetermined affinity for the epidermis and with immunologic specificity for the neoantigen migrate into the epidermis, recognize the neoantigen presented on Langerhans cells, transform in situ into effector cells, and directly destroy the adjacent malignant cells, or (2) neoantigen-bearing Langerhans cells «drop down» into the dermis, flow with the draining lymph into the regional lymph node, settle into the cortex, and form a nidus for the attraction of antigen-specific lymphocytes that are activated within the node, proliferate, and then disseminate systemically to return predominantly to the skin in order to effect destruction of the neoplastic keratinocytes. We are beginning to collect relevant data supporting the second of these options. However, to the present, essentially no information exists that indicates that T cells can be activated directly in situ, can destroy the offending cells, and yet never send this signal to be amplified and disseminated systematically. It is conceivable that this may be the fundamental mechanism by which SALT works. Ultraviolet radiation, as an oncogenic agent, oncogenic viruses, and other carcinogenic agents may act to induce cutaneous neoplasia. Ultraviolet radiation, however, has other effects on the skin that make it particularly appropriate in this consideration. It induces melanization of skin and alters remarkably the functional properties of Langerhans cells with regard to antigen presentation. We would presume that under normal circumstances the immediate effects of UVR (that impair antigen presentation by Langerhans cells) prove to be transient. This is so in humans because activation of melanogenesis by UVR rapidly provides the epidermis with some photoprotection allowing Langerhans cells to restore their immune functional properties even in the face of repeated exposure to the radiation. Failure of SALT could occur through numerous pathways. Inadequate melanization would permit cutaneous cells, including Langerhans cells, to be exposed chronically to the deleterious effects of UVR; in the case of Langerhans cells, ultraviolet-induced inability to process neoantigens effectively would lead to the establishment of specific unresponsiveness rather than specific sensitization. In this manner, chronic impairment of the presentation of neoantigens by Langerhans cells would promote, rather than prevent, the escape of neoplastic cells from detection by the immune system. Reduction in the numbers and varieties of antigen-specific recirculating lymphocytes, as might be achieved by chronic immunosuppression, would similarly disrupt SALT, robbing the organism of the antigenspecific and tissue-destructive components of the surveillance mechanism.

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