Immunology Today, vol. 7, Nos. 7 & 8, 1986
reviewsThe skinimmunesystem Its cellular constituentsand their interactions
The term immunodermatology describes the systematic investigation of the complex mechanisms of the 'skin immune system' in health and disease. In this review Jan Bos and Martien Kapsenberg discuss the skin's vascular and lymphatic systems and the various cells which participate in the immune response. Theseindude Langerhans' cells, indeterminate cells, veiled cells, endothelial cells, mast cells, tissue macrophages and 'homing' T lymphocytes, which are all present in skin under physiological conditions. Immunodermatology is primarily directed towards an understanding of immunological mechanisms operating in skin diseases. Few investigations have been carried out on healthy skin. The skin functions as a general (physicomechanical) barrier but also synthesizes a large number of different biologically active substances. The skin is the largest organ of the human body and functions as a general defence system, while the immune system has evolved as a complementary line of defence. This intimate relationship justifies studies of the cells which exert skin immune functions under physiological conditions. It might well have been Kondo in 19221 who first described intra-epidermal lymphocytes. Fichtelius, Groth and Liden 2 suggested that the skin was a 'first level lymphoid organ'. They hypothesized that 'the lymphocytes reaching the epidermis may to a large extent be non-competent lymphoid cells which become competent during or shortly after a stay in the epidermis or close to it in the corium'. Streilein 3 coined the term 'skin-associated lymphoid tissues' (SALT) to describe: Langerhans' cells with their antigen-presenting properties; epidermotropic recirculating T-lymphocyte subpopulations (homing T lymphocytes); keratinocytes producing epidermal thymocyteactivating factor (ETAF: Refs 4, 5); and the skin draining peripheral lymph nodes, which included vascular highendothelial cells with the capacity to capture lymphocytes passing through the blood. In addition to the cells included in Streilein's original definition of SALT, one may also include a number of other immunologically relevant cells that normally populate the (epi)dermal tissues (Table 1 and Fig. 1). In this way one can describe the immune system operating in normal skin by its cellular constituents. Any claim for organ-specificity of such an immune network is thereby avoided. Other cells to be included are mast cells, tissue macrophages, granulocytes, indeterminate cells, veiled cells, vascular endothelial cells and afferent lymphatic endothelium starting in the dermis. All these cells taken together with those comprising SALT, but excluding lymph node tissue, form an intricate and complex system that we propose to call the 'skin immune system' (SIS). The number of different cell types present in normal skin and exerting immunological functions at least equals the number of cell types that until now are regarded as
Department of Dermatology and Laboratory of Histology and Cell Biology, respectively,Universityof Amsterdam,AcademischMedisch Centrum,Amsterdam,TheNetherlands 1986, Elsevier Science Publishers8.V, Amsterdam
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Jan D. Bos and Martien L. Kapsenberg having no immunological role (Table 1). Cells not involved in the immune response include eccrine and apocrine gland cells with their myoepithelial and duct cells, melanocytes, fibroblasts and fibrocytes, pericytes of the vasculature, smooth muscle and nervous tissue cells, and Merkel cells. T cells and their subsets It has been suggested that of the circulating T-cells, an as yet to be quantified number infiltrate the healthy skin and reside there for an undefined period ('homing' T cells3). This seems justified by the simple observation that normal skin always contains some scattered T lymphocytes, especially around and above the superficial venous plexus, and rarely intra-epidermally (J.D. Bos et aL, unpublished). (Epi)dermotropism of T cells becomes prominent in many dermatological disease states ranging from the rare cutaneous lymphomas to common benign inflammatory skin diseases such as eczema and psoriasis. Many questions remain to be answered about the homing T cells in skin. It has not been proved that they are 'homing'. Are they instead being recruited? Do they differ from their counterparts in other healthy connective tissues? Are there any functional or immunophenotypic characteristics by which the homing T cells of SIS differ from the scattered T cells of other connective tissues? Dendritic antigen-presenting cells As well as phagocytosing monocytes/macrophages there is another class of HLA-DR ÷, non-phagocytosing antigen-presenting cells 6'7. These include Langerhans' cells (LC), indeterminate cells, veiled cells, interdigitating cells and dendritic reticulum cells. Their subtypes, normal habitat and known markers are summarized in Table 2. The suggested migration pathways of the 'skin family' of dendritic cells present under physiological conditions in man are depicted in Fig. 2. In mouse epidermis, a new type of dendritic cell was recently described 8--11 and Romani etaL 12 have provided immunophenotypic and morphological evidence that Table I. Overviewof skin immunesystemcellsand cellsnot primarily involved in skin immune responses
Skin immunesystem(SIS)cells Keratinocytes Langerhans'cells Indeterminatecells Veiledcells Tissuemacrophages(histiocytes) Neutrophilicgranulocytes Mast cells,connectivetissuetype Mast cells, mucosaltype Vascularendothelialcells Lymphaticendothelialcells 'Homing' T cells
Non-immuneresponserelatedcells Melanocytes Merkelcells Fibroblasts/cytes Eccrinegland cells Ductcells Myoepithelial cells Apocrinesecretorycells Sebocytes Smooth musclecells Neural receptorcells Pericytes
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Immunology Today, vol. 7, Nos. 7 & 8, 1986
Fig. 1. Schematicrepresentationof type and distributionof skin immunesystem cells and their blood and lymphatic drainagesystemsdepictedin the frontal projection; cellsand structuresnot primarily involved in skin immune responses are drawn in the lateralprojection.
these Thyl + dendritic cells are related to natural killer cells. Their possible human equivalent has as yet not been discovered. Langerhans" cells
Probably the most important SIS-related cell is the Langerhans" cell (LC), an accessory, antigen-presenting cell that is strongly HLA-DR and T6 (CD1) positive 7'13. LC
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express low densities of the T4 (CD4) antigen in physiological 14 as well as pathological is conditions. They have surface receptors for C3 and the Fc fragment of IgG 16 and are bone-marrow derived 17. So-called 'Birbeck granules', which are T6 (CD1) positive 18, can be found intracellularly by electron microscopy, but the function of these structures is as yet unknown. Isolated human epidermal LC produce interleukin-1 (IL-1)19. Large numbers of LC form a regular and almost closed network of dendrites within the spinal cell layer of the epidermis (Fig. 3). A simple calculation suggests that not less than 1 x 10 9 LC are normally present in the epidermis of a human being (Table 3 and Ref. 20). This
high number underlines the antigen-presenting potential of the dendritic cells of skin. One may hypothesize that LC continuously leave the epidermis, presumably through the lymphatics, being replaced by circulating LC precursors. LC have a mean stay of approximately three weeks within mouse epidermis 17.21 , and applying this figure to man would result in a daily repopulation number of about 45 million LC for the entire skin (Table 3). Repopulation studies in patients treated by photochemotherapy (PUVA) also resulted in a mean repopulation period of three weeks 22. However, these figures represent abnormal conditions. Chernielewski, Vaigot and Prunieras 23 gave evidence for intra-epidermal cycling of LC. In-vitro analysis of the cell cycle stage of LC indicated that 1.3-3.3% and 1.0-2.5% of epidermal LC were in S and G2/M phase, respectively. These studies do not substantially differ from earlier findings in which [3H]thymidine incorporation labeling indices were found to be very low 24'2s. Nevertheless, at least part of epidermal LC may be replaced by division from an intra-epidermal LC pool.
For technical reasonswe are unableto reproducethis figure in colour. Seethe July/August issueof Immunology Today for full colour illustration.
Immunology Today, vol. 7, Nos. 7 & & 1986
reviews-
Table 2. Thefamilyof skindendriticantigen-presentingcells Dendriticantigen-presentingcell Normalhabitat Langerhans'cell Stratifiedsquamousepithelia Intermediatecell Epidermis/dermis Interdigitatingcell T-cellareasRES Veiledcell Skin-draininglymphvessels RES= reticulo-endothelial system
Fc-rec + ? ? +
C3-rec + ? ? +
HI_A-DR + + + +
T6 + + +I-
RFD1 + ?
M241 +/ + +/? ?
Indeterminate cells Breathnach 26 defined the dendritic nonkeratinocytes found in normal epidermis and lacking Birbeck granules, melanosomes or Merkel cell granules in their cytoplasm as the 'indeterminate cells'. They are HLA-DR+. Similar cells occur within the normal papillary dermis and are T6 +, as are LC (Ref. 27). Transitional forms of dermal indeterminate cells and epidermal LC have been noted 28. Monoctonal anti-M241 reacts with a minority of LC but with a substantial number of other dermal dendritic cells with morphological features of indeterminate cells29,3°. Their exact position in the migration pathways of skin dendritic cells is as yet unclear. Some of the 30ssibilities are depicted in Fig. 2.
skin (unpublished observations). In comparison with interdigitating cells, they are rare in pathological conditions. This is not surprising because B-cell accumulations are rare in dermatological disease. We have observed DRC+, HLA-DR+ dendritic reticulum cells in two cases of lymphocytoma cutis, a benign disease in which some subtypes show more or less symmetrical intradermal infiltrations resembling ectopic lymph nodes (unpublished observations). Dendritic reticulum cells are distinct from the other members of the dendritic cell family because they are mesenchymal and not bone-marrow derived 38. For this reason they have not been included in Tables 1 and 2, nor in Fig. 2.
Veiled cells Afferent lymph, as described above, contains a group of LC resembling cells known as veiled cells31. In the rabbit there is an average of 72 000 veiled cells per ml of skin-draining lymph. Veiled cells are strongly HLA-DR +, bear Fc(IgG) and C3 receptors and have no surface immunoglobulins 6. They have as yet not been demonstrated in situ in (normal) human skin lymphatics but can be presumed to be present.
Mast cells Mast cells have an important potential for regulating vasculature and possibly have an important role in T-cell trafficking (see below). Mast cells may be divided into type I (mucosal) and type II (connective tissue) cells. 'Mucosar and 'connective tissue' are misleading terms because both types occur in mucosa 39 and in the connective tissue of the dermis (T. S. Orr, unpublished). Type I mast cells are small and unlike type II cells contain no heparin. Distinction of subtypes may be obtained by an Alcian blue-Safranin staining sequence, or by electron microscopic techniques. The number of mast cells in normal human skin is quite high and has been estimated as 5120-9472 per mm 3 of dermis (mean 7225) (Ref. 40). They play an obvious role in type I immediate hypersensitivity reactions such as immunological contact urticaria.
Interdigitating cells Interdigitating cells are normally present in the T-cell areas of the reticulo-endothelial system and carry the RFD1 antigen 32 (as identified by a monoclonal antibody). They are not present in normal skin but RFD1+ dendritic cells have been detected in the inflammatory infiltrates of psoriasis 33'34, leprosy 3s and in the lesional skin of atopic dermatitis36 and pityriasis rosea37. Juxtaposition with T cells, a phenomenon also known as peripolesis, was always found and this combination may be indicative of T-cell-mediated immune reactions.
Tissue macrophages Within the papillary dermis, there are dense populations of as yet unquantified monocytes/macrophages.
Dendritic reticulum cells These cells are normally present in the B-cell areas of lymph nodes and spleen but are not present in normal
lI
-epidermis
Langerhana
cell ~
~'~-~ ~ ,ndet.~__~'~ Imitmcell---.~u~ " ~ ~Lc-p
......
q
I i
lymph-node paracorUcal
Interdigitating//~ cell - ~ / ( / jj.~,~.-~.j
afferent / ' ~ I lymph~ss~l ' ~1t ~ - - - - ~
J dOrlln~
~
\
.
Fig. 2, Schematic representation of suggested migration patterns and relationship between the varioussubsetsof dendritic accessorycellsnormallypresent in healthyskin and draining lymphsystem.
Fig. 3. Typicalexampleof T6 (CDT)positive Langerhans'cell stainingpattern in epidermisshowing darkstained dendriticcells(2-stepimmunoperoxidasetechnique; primaryantibodyanti-T6 (CD1): magnificationX 160).
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Immunology Today, vol. 7, Nos. 7 & 8, 1986
Table 3. Langerhans'cell dynamics
Ref. Skinsurface Numberof Langerhans'cellsmm 2 Total numberof Langerhans'cellsin situ within entireepidermis
2 x 106mm2 450 2 x 106 x 450 = 0.9 x 109
Repopulationof epidermiswith Langerhans'cellsafter stripping Top at days4-6 Repopulationcompletedin 14 days Maximal repopulationspeed Estimatednormal repopulationspeed
28-70mm-2 day-1 180 mm-2 day-1
These cells, also known as tissue histiocytes, are scattered, in addition, through the reticular dermis. Their role under physiological conditions may be as scavengers of debris and the degraded cell (granulocyte) substances that are continously produced. They may also have antigen-presenting functions, although such a capability has as yet not been demonstrated in situ in skin. Skin neutrophilic granulocytes41 Neutrophilis have been said to leave the blood after an average intravascular sojourn of 10 h. They do not seem to return to the blood after leaving it but their fate after entering the tissues and body cavities is not yet clear, in normal skin, neutrophils are certainly not frequent extravascularly. Their number within the postcapillary venules is high since blood flow there is decreased, and granulocytes participate in immune complex clearing under normal conditions. Skin vasculature and lymphatic drainage Blood and lymphatic vasculature connect SIS to the remainder of the body's immune system. Skin vasculature has three levels, depicted in Fig. 1 and reviewed in Ref. 42. It is around the superficial plexus and adjacent to the capillary loops that most SIS-related cells are present under physiological conditions. The postcapillary venules of the upper plexus, with a diameter of 10-20 I~m, are the sites of inflammatory cell emigration, histamineinduced vascular permeability and immune complex deposition under different pathological conditions. Endothelial cells of these postcapillary venules in skin are HI_A-DR+ and can act as antigen-presenting cells in vitro 43'44. It is evident that endothelial cells of at least part of the skin vasculature exert important functions, to be further elucidated, in the immunophysiology of the skin 4s. Skin lymphatics start with open ends within the papillary dermis, form sinuses and are initially only lined by a single layer of endothelial cells. Later on they have valves and may, in the subcutaneous layers, acquire a smooth muscle wall. Their organization is similar to that described for the vascular network with upper and lower dermal plexuses (Zhdanov, cited in Ref. 42). Lymph flow from the skin is one-directional towards the skin-related lymph nodes. Skin draining lymph nodes are especially prominent in the body folds such as the elbows, knees, and the cervical, axillary and inguinal regions.
238
SALT and SiS Several questions about the concept of SALT may be asked. Inclusion of keratinocytes in SALT may be justified
I
20
17,21 180 x 2 x 106= 360 x 106/day0.9 x 109= 42.8 x 106day-1 21 by several observations in addition to their production of the interleukin-l-like factor ETAF4,s. Keratinocytes may play a role in T-cell differentiation. Epidermal cell culture supernatant was shown to induce terminal deoxynucleotidyl transferase, an early T-cell differentiation marker, on immature human bone-marrow cells and T cells 46, and epidermal cells bound antibodies to serum thymic factor 47 and thymopoietin 48. Epidermal keratinocytes may therefore have some relationship to thymic cortical epithelial cells in T-cell differentiation but proof for intra-epidermal T-cell differentiation in vivo is still lacking. So-called epidermotropic recirculating T-lymphocyte populations are rare, in our experience, as intra-epithelial T cells are uncommon in normal skin (manuscript in preparation). Perhaps the more commonly encountered T cells in normal dermis are a pool distinct from T cells in other connective tissues. LC form part of a widely distributed family of dendritic accessory antigen-presenting cells, and the organ specificity of this cell for stratified squamous epithelial tissues does not necessarily imply a distinct and tissuespecific type of immune function. High endothelial venules (HEV) that are supposed to be necessary for the direction of organ-specific T-cell migration have not been described in healthy skin. HEV-like vasculature has, to our knowledge, only been described in a rare skin disorder called angiolymphoid hyperplasia with eosinophilia, also known as Kimura's disease 49. It is clear that other mechanisms for directing T-cell traffic must operate in skin, possibly through antigen-specific, T-cell-derived factors bound to mast cells (see below). Skin-draining lymph nodes do not receive skin lymph only, and the cells comprising SIS cannot be regarded as lymph-node tissue. It is for these reasons that we define the immune response related cells operating in skin as the SiS. SiS dynamics With SIS, lymph nodes play an important role in tolerance as well as in immunization and sensitization mechanisms. Paracortical T-cell areas which contain dendritic antigen-presenting cells and HEV which direct T-cell migration are essential in the functional cooperation between lymph nodes and SIS. LC function in vitro as stimulatory cells in epidermal cell-induced autologuos 50 and allogeneic 51 T-cell activation and in the generation of epidermal cell-induced cytotoxic T-cell responses s2. They also function as antigen-presenting cells in in-vitro T-cell activation by
ImmunologyToday,vol. 7, Nos. 7&8, 1986
protein antigens ~1. LC also play a role as stimulating cells in graft rejection in vivo s3. The role of LC in contact hypersensitivity, a classical model of delayed hypersensitivitys4'ss, is especially interesting. Sensitization starts with regular exposure of the skin to a hapten. Substances leading to allergic contact reactions generally have a molecular weight of less than 500. They first may bind to a 'carrier' before they can be immunogenic, or they may bind directly to as yet undefined molecules on antigen-presenting cell membranes. The hapten-carrier complex is called antigen or immunogen. Although the exact localization of primary sensitization is still debatable, experiments s6 have demonstrated that an animal can only be sensitized on a prepared skin flap when the connecting part contains, next to essential arteriolar and venous vessels, also the draining lymphatics. It nevertheless remains unclear whether it is the hapten, the sensitized T cell already within the skin ('peripheral sensitization'), or the LC-hapten complex that is carried to the lymph node. Recent evidence provided by Knight etal. s7 indicates that hapten-carrying dendritic cells can sensitize unprimed syngeneic mice. is thus most probably the LC-hapten complex that is involved in presentation of hapten to paracortical T cells. These become activated and are induced to proliferate and differentiate. Effector cell precursors proliferate and some of them differentiate into memory cells, whereas others become effector cells. Suppressor cell precursors are simultaneously induced to proliferate and they stop the further production of effector cells. The evidence from contact sensitization, a pathological state, may also apply to the normal situation, where immunization with a subsequent induction of a state of tolerance may take place continuously. Streileins8 has suggested that SALT represents a physiological mechanism created to deal with neoplastic events taking place continuously within the skin due to ultraviolet irradiation and the presence of potentially oncogenic viruses and carcinogenic agents. This role for LC may be significantly extended as follows. Under physiological conditions LC continuously present skin contacting antigens to the T cells in the lymph nodes. In fact, a large number of the 45 million available human LC will, every day, move through the lymphatics (presumably as veiled cells) to the regional skin-related lymph nodes. In this way, the SIS is continuously made aware of its antigenic microenvironment. Small abrasions and erosions of the physico-mechanical skin barrier that normally occur at microscopic level result in intra-epidermal injections of antigenic materials. Other water-soluble compounds will penetrate into the spinal layer and be taken up by LC. When other compartments such as neutrophils and tissue macrophages fail to clear these antigens, they are further processed by the continuously boosted T cells, without any clinical symptoms or signs. In mice, mast cells may play an important role in delayed-type h~/persensitivity (elicitation of allergic contact dermatitis ~. During sensitization, antigen-binding T-cell factors are produced and released into the circulation. These reach the (skin) mast cells which become sensitized. During elicitation, these antigen-binding Tcell factors bind the antigen and the mast cells react, releasing serotonin but without histamine release or degranulation. Serotonin induces, via receptors on postcapillary venules, vasodilatation and the formation of
reviewsinterendothelial spaces which facilitate the entry of T-cells, among them antigen-specific T cells, into the elicitation site. It is only then that the antigen-presenting, MHC class II restricted stage of delayed-type hypersensitivity develops, characterized by the development of a mononuclear cell infiltrate. Whether these antigen-binding T-cell factors play a role in normal human skin is as yet unclear. If they do, LC presentation of immunogens to these mast-cell-bound, T-cell-derived factors seems possible. The skin's micro-anatomy and immunohistology thus show a complicated and fine-tuned organization of many different immunologically active cells. Probably the most important cells within SIS are those comprising the family of skin dendritic cells. Together with recirculating and/or recruited T cells they act in the specific immune responses. Non-specific cells such as granulocytes and tissue monocytes cooperate with specific recirculating T cells in antigen clearing. T-cell migration may be directed by mast cell release of vasoactive agents through antigen binding via irnmunogen-specific T-cell factors present on mast cell membranes. As well as Fc-receptor-bearing and C3 receptor bearing dendritic cells and macrophages, the vascular endothelium of the upper dermal plexus may also take part in antigen presentation. It seems evident that the SIS form an ideal and moreover easily accessible model for the study of immunology of health and disease. R.H. Cormane, P.K. Das, W.R. Faber, R.F.H.J. Hulsmans and P.M. Lansdorp are gratefully acknowledged for their helpful discussions. Figures 1 and 2 were prepared by Mrs I.E.M. Oosterling.
References
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Nature (London) 268, 245 17 Katz, S.l., Tamaki, K. and Sachs, D. (1979) Nature (London)
282,324 18 Takahashi, S. and Hashimoto, K. (1985)J. Invest. Dermatol. 84, 469-471 19 Sauder, D.N., Dinarello, C.A. and Morhenn, V.B. (1984) J. Invest. Dermatol. 82, 605-607 20 Berman, B., Chen, V.L., France, D.S. etaL (1983) Br. J. DermatoL 109, 553-559 21 Toews, G.B., Bergstresser, P.R. and Streilein, J.W. (1980) J. Invest. DermatoL 75, 78-82 22 Friedmann, P.S., Ford, G., Ross,J. etal. (1983) Br. J. Dermatol. 109, 301-307 23 Chernietewski, J., Vaigot, P. and Prunieras, M. (1985)J. Invest. Dermatol. 84, 424-426 24 Schellander, F. and Wolff, K. (1967)Arch. Kiln. Exp. Dermatol. 230, 140-145 25 Fritsch, P. and Mantel, J. (1972)Arch. Dermatol. Forsch. 244, 44 48 26 Breathnach, A.S. (1975)J. Invest. Dermatol. 65, 2-15 27 Murphy, G.F., Bhan, A.K., Harrist, T.J. etaL (1983) Br. J. Dermatol. 108, 423-431 28 Breathnach, A.S. (1980) J. Invest. Dermatol. 75, 6-11 29 van de Rijn, M, Lerch, P.G., Bronstein, B.R. etaL (1984) Hum. Immunol. 9, 201-205 30 Murphy, G.F., Bronstein, B.R., Knowles, R.W. etal. (1985) Lab. Invest. 52,264-269 31 Drexhage, H.A., Mullink, H., de Groot, J. etal. (1979) Cell Tissue Res. 202,407-430 32 Poulter, L.W. (1983) Clin. Exp. ImmunoL 53, 513-520 33 Poulter, UW., RussellJones, R. and Hobbs, S. (1984), in Immunodermatology (MacDonald, D.M., ed.) pp. 185-188, Butterworth, Kent 34 Bos, J.D. and Krieg, S.R. (1985)Acta Dermato-Venereol. (Stockholm) 65,390-397 35 Poulter, L.W., Collings, L.A., Tung, K.S. etaL (1984) Clin. Exp. Immunol. 55, 611-617 36 Sillevis Smitt, J.H., Bos, J.D., Hulsebosch, H.J. etaL Clin. Exp. Dermatol. 11, 159-168 37 Bos, J.D., Huisman, P.M., Krieg, S.R. etaL (1985)Acta Dermato-VenereoL (Stockholm) 65, 132-137 38 Humphrey, J.H. and Sundaram, V. (1985) in Microenvironments in the Lymphoid System (Klaus, G.G.B., ed.) Adv. Exp. Med. Biol. 186, 167-170
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39 Befus, D., Lee, J., Denburg, J. eta/. (1984) ImmunoL Today 5, 218-219 40 Mikhail, G.R. and Miller-Milinska, A. (1964)J. Invest. Dermatol. 43,249-254 41 Boggs, D.R. and Wintrobe, M.M. (1974)in Harrison's Principles of Internal Medicine (Wintrobe, M.M., Thorn, G.W., Adams, R.D. et aL, eds) pp. 313-315, McG raw-Hill, New York 42 Braverman, I.M. (1983)Br. J. DermatoL 109 (suppl.), 89-98 43 Hirschberg, H., Bergh, O. J. and Thorsby, E. (1980) J. Exp. Med. 152, 249s-255s 44 Hirschberg, H., Braathen, L.R. and Thorsby, E. (1982) Immunol. Rev. 66, 57-77 45 Baldwin, WM. (1982) Immunol. Today3, 267-269 46 Rubenfeld, M.R., Silverstone, A.E., Knowles, D.A. etal. (1981 ) J. Invest. Dermatol. 77, 221-224 47 Kato, K., Ikeyama, S., Takouki, M. etal. (1981) Cell 24, 885-895 Chu, A.C., Patterson,J.A.K.,Goldstein, G. etal. (1983)], Invest Dermatol. 81, 194-I 97 49 Baum, E.W., Mitchell Sams, W. and Monheit, G.D. (1982)J. Derm. Surg. OncoL 8, 966-970 50 5tingl, G., Katz, S.I., Clement, L. etal. (1978)J. ImmunoL 121, 2005-2013 51 Braathen, L.R. and Thorsby, E. (1980) Scand. J. ImmunoL 11, 401-4O8 52 Pehamberger, H., Stingl, L.A., Pogantsch, 5. etal. (1982) Immunobiology 163, 122-123 53 Streilein, J.W., Lonsberry, UW. and Bergstresser, P.R. (1982) J. Exp. Med. 155, 863-871 54 Polak, L. (1982) Allergic Contact Dermatitis: Immunologic Considerations, in Occupational and Industrial Dermatology (Maibach, H.I. and Gellin, G.A., eds) pp. 39-64, Year Book Medical Publishers, Chicago 55 Liew, F.Y. (1984) in Advances in Inflammation Research Vol. 7 (Otterness, I., Capetola, R. and Wong, S., eds) pp. 135-147, Raven Press, New York 56 Frey, J.R and Wenk, P. (1957) Int. Arch. AilergyAppL Immunol. 11,81-100 57 Knight, S.C., Krejci, J., Malkovsky, M. etal. (1985) Cell. Immunol. 94, 427-434 58 Streilein, J.W. (1983) J. Invest. Dermatol. 80 (suppl.), 12s16s 59 Askenase, P.W. and van Loveren, H. (1983) Immunol. Today 4, 259-264
reviewsThe skinimmunesystem Its cellular constituentsand their interactions
The term immunodermatology describes the systematic investigation of the complex mechanisms of the 'skin immune system' in health and disease. In this review Jan Bos and Martien Kapsenberg discuss the skin's vascular and lymphatic systems and the various cells which participate in the immune response. Theseindude Langerhans' cells, indeterminate cells, veiled cells, endothelial cells, mast cells, tissue macrophages and 'homing' T lymphocytes, which are all present in skin under physiological conditions. Immunodermatology is primarily directed towards an understanding of immunological mechanisms operating in skin diseases. Few investigations have been carried out on healthy skin. The skin functions as a general (physicomechanical) barrier but also synthesizes a large number of different biologically active substances. The skin is the largest organ of the human body and functions as a general defence system, while the immune system has evolved as a complementary line of defence. This intimate relationship justifies studies of the cells which exert skin immune functions under physiological conditions. It might well have been Kondo in 19221 who first described intra-epidermal lymphocytes. Fichtelius, Groth and Liden 2 suggested that the skin was a 'first level lymphoid organ'. They hypothesized that 'the lymphocytes reaching the epidermis may to a large extent be non-competent lymphoid cells which become competent during or shortly after a stay in the epidermis or close to it in the corium'. Streilein 3 coined the term 'skin-associated lymphoid tissues' (SALT) to describe: Langerhans' cells with their antigen-presenting properties; epidermotropic recirculating T-lymphocyte subpopulations (homing T lymphocytes); keratinocytes producing epidermal thymocyteactivating factor (ETAF: Refs 4, 5); and the skin draining peripheral lymph nodes, which included vascular highendothelial cells with the capacity to capture lymphocytes passing through the blood. In addition to the cells included in Streilein's original definition of SALT, one may also include a number of other immunologically relevant cells that normally populate the (epi)dermal tissues (Table 1 and Fig. 1). In this way one can describe the immune system operating in normal skin by its cellular constituents. Any claim for organ-specificity of such an immune network is thereby avoided. Other cells to be included are mast cells, tissue macrophages, granulocytes, indeterminate cells, veiled cells, vascular endothelial cells and afferent lymphatic endothelium starting in the dermis. All these cells taken together with those comprising SALT, but excluding lymph node tissue, form an intricate and complex system that we propose to call the 'skin immune system' (SIS). The number of different cell types present in normal skin and exerting immunological functions at least equals the number of cell types that until now are regarded as
Department of Dermatology and Laboratory of Histology and Cell Biology, respectively,Universityof Amsterdam,AcademischMedisch Centrum,Amsterdam,TheNetherlands 1986, Elsevier Science Publishers8.V, Amsterdam
0167 - 4919/86/502.00
Jan D. Bos and Martien L. Kapsenberg having no immunological role (Table 1). Cells not involved in the immune response include eccrine and apocrine gland cells with their myoepithelial and duct cells, melanocytes, fibroblasts and fibrocytes, pericytes of the vasculature, smooth muscle and nervous tissue cells, and Merkel cells. T cells and their subsets It has been suggested that of the circulating T-cells, an as yet to be quantified number infiltrate the healthy skin and reside there for an undefined period ('homing' T cells3). This seems justified by the simple observation that normal skin always contains some scattered T lymphocytes, especially around and above the superficial venous plexus, and rarely intra-epidermally (J.D. Bos et aL, unpublished). (Epi)dermotropism of T cells becomes prominent in many dermatological disease states ranging from the rare cutaneous lymphomas to common benign inflammatory skin diseases such as eczema and psoriasis. Many questions remain to be answered about the homing T cells in skin. It has not been proved that they are 'homing'. Are they instead being recruited? Do they differ from their counterparts in other healthy connective tissues? Are there any functional or immunophenotypic characteristics by which the homing T cells of SIS differ from the scattered T cells of other connective tissues? Dendritic antigen-presenting cells As well as phagocytosing monocytes/macrophages there is another class of HLA-DR ÷, non-phagocytosing antigen-presenting cells 6'7. These include Langerhans' cells (LC), indeterminate cells, veiled cells, interdigitating cells and dendritic reticulum cells. Their subtypes, normal habitat and known markers are summarized in Table 2. The suggested migration pathways of the 'skin family' of dendritic cells present under physiological conditions in man are depicted in Fig. 2. In mouse epidermis, a new type of dendritic cell was recently described 8--11 and Romani etaL 12 have provided immunophenotypic and morphological evidence that Table I. Overviewof skin immunesystemcellsand cellsnot primarily involved in skin immune responses
Skin immunesystem(SIS)cells Keratinocytes Langerhans'cells Indeterminatecells Veiledcells Tissuemacrophages(histiocytes) Neutrophilicgranulocytes Mast cells,connectivetissuetype Mast cells, mucosaltype Vascularendothelialcells Lymphaticendothelialcells 'Homing' T cells
Non-immuneresponserelatedcells Melanocytes Merkelcells Fibroblasts/cytes Eccrinegland cells Ductcells Myoepithelial cells Apocrinesecretorycells Sebocytes Smooth musclecells Neural receptorcells Pericytes
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Immunology Today, vol. 7, Nos. 7 & 8, 1986
Fig. 1. Schematicrepresentationof type and distributionof skin immunesystem cells and their blood and lymphatic drainagesystemsdepictedin the frontal projection; cellsand structuresnot primarily involved in skin immune responses are drawn in the lateralprojection.
these Thyl + dendritic cells are related to natural killer cells. Their possible human equivalent has as yet not been discovered. Langerhans" cells
Probably the most important SIS-related cell is the Langerhans" cell (LC), an accessory, antigen-presenting cell that is strongly HLA-DR and T6 (CD1) positive 7'13. LC
236
express low densities of the T4 (CD4) antigen in physiological 14 as well as pathological is conditions. They have surface receptors for C3 and the Fc fragment of IgG 16 and are bone-marrow derived 17. So-called 'Birbeck granules', which are T6 (CD1) positive 18, can be found intracellularly by electron microscopy, but the function of these structures is as yet unknown. Isolated human epidermal LC produce interleukin-1 (IL-1)19. Large numbers of LC form a regular and almost closed network of dendrites within the spinal cell layer of the epidermis (Fig. 3). A simple calculation suggests that not less than 1 x 10 9 LC are normally present in the epidermis of a human being (Table 3 and Ref. 20). This
high number underlines the antigen-presenting potential of the dendritic cells of skin. One may hypothesize that LC continuously leave the epidermis, presumably through the lymphatics, being replaced by circulating LC precursors. LC have a mean stay of approximately three weeks within mouse epidermis 17.21 , and applying this figure to man would result in a daily repopulation number of about 45 million LC for the entire skin (Table 3). Repopulation studies in patients treated by photochemotherapy (PUVA) also resulted in a mean repopulation period of three weeks 22. However, these figures represent abnormal conditions. Chernielewski, Vaigot and Prunieras 23 gave evidence for intra-epidermal cycling of LC. In-vitro analysis of the cell cycle stage of LC indicated that 1.3-3.3% and 1.0-2.5% of epidermal LC were in S and G2/M phase, respectively. These studies do not substantially differ from earlier findings in which [3H]thymidine incorporation labeling indices were found to be very low 24'2s. Nevertheless, at least part of epidermal LC may be replaced by division from an intra-epidermal LC pool.
For technical reasonswe are unableto reproducethis figure in colour. Seethe July/August issueof Immunology Today for full colour illustration.
Immunology Today, vol. 7, Nos. 7 & & 1986
reviews-
Table 2. Thefamilyof skindendriticantigen-presentingcells Dendriticantigen-presentingcell Normalhabitat Langerhans'cell Stratifiedsquamousepithelia Intermediatecell Epidermis/dermis Interdigitatingcell T-cellareasRES Veiledcell Skin-draininglymphvessels RES= reticulo-endothelial system
Fc-rec + ? ? +
C3-rec + ? ? +
HI_A-DR + + + +
T6 + + +I-
RFD1 + ?
M241 +/ + +/? ?
Indeterminate cells Breathnach 26 defined the dendritic nonkeratinocytes found in normal epidermis and lacking Birbeck granules, melanosomes or Merkel cell granules in their cytoplasm as the 'indeterminate cells'. They are HLA-DR+. Similar cells occur within the normal papillary dermis and are T6 +, as are LC (Ref. 27). Transitional forms of dermal indeterminate cells and epidermal LC have been noted 28. Monoctonal anti-M241 reacts with a minority of LC but with a substantial number of other dermal dendritic cells with morphological features of indeterminate cells29,3°. Their exact position in the migration pathways of skin dendritic cells is as yet unclear. Some of the 30ssibilities are depicted in Fig. 2.
skin (unpublished observations). In comparison with interdigitating cells, they are rare in pathological conditions. This is not surprising because B-cell accumulations are rare in dermatological disease. We have observed DRC+, HLA-DR+ dendritic reticulum cells in two cases of lymphocytoma cutis, a benign disease in which some subtypes show more or less symmetrical intradermal infiltrations resembling ectopic lymph nodes (unpublished observations). Dendritic reticulum cells are distinct from the other members of the dendritic cell family because they are mesenchymal and not bone-marrow derived 38. For this reason they have not been included in Tables 1 and 2, nor in Fig. 2.
Veiled cells Afferent lymph, as described above, contains a group of LC resembling cells known as veiled cells31. In the rabbit there is an average of 72 000 veiled cells per ml of skin-draining lymph. Veiled cells are strongly HLA-DR +, bear Fc(IgG) and C3 receptors and have no surface immunoglobulins 6. They have as yet not been demonstrated in situ in (normal) human skin lymphatics but can be presumed to be present.
Mast cells Mast cells have an important potential for regulating vasculature and possibly have an important role in T-cell trafficking (see below). Mast cells may be divided into type I (mucosal) and type II (connective tissue) cells. 'Mucosar and 'connective tissue' are misleading terms because both types occur in mucosa 39 and in the connective tissue of the dermis (T. S. Orr, unpublished). Type I mast cells are small and unlike type II cells contain no heparin. Distinction of subtypes may be obtained by an Alcian blue-Safranin staining sequence, or by electron microscopic techniques. The number of mast cells in normal human skin is quite high and has been estimated as 5120-9472 per mm 3 of dermis (mean 7225) (Ref. 40). They play an obvious role in type I immediate hypersensitivity reactions such as immunological contact urticaria.
Interdigitating cells Interdigitating cells are normally present in the T-cell areas of the reticulo-endothelial system and carry the RFD1 antigen 32 (as identified by a monoclonal antibody). They are not present in normal skin but RFD1+ dendritic cells have been detected in the inflammatory infiltrates of psoriasis 33'34, leprosy 3s and in the lesional skin of atopic dermatitis36 and pityriasis rosea37. Juxtaposition with T cells, a phenomenon also known as peripolesis, was always found and this combination may be indicative of T-cell-mediated immune reactions.
Tissue macrophages Within the papillary dermis, there are dense populations of as yet unquantified monocytes/macrophages.
Dendritic reticulum cells These cells are normally present in the B-cell areas of lymph nodes and spleen but are not present in normal
lI
-epidermis
Langerhana
cell ~
~'~-~ ~ ,ndet.~__~'~ Imitmcell---.~u~ " ~ ~Lc-p
......
q
I i
lymph-node paracorUcal
Interdigitating//~ cell - ~ / ( / jj.~,~.-~.j
afferent / ' ~ I lymph~ss~l ' ~1t ~ - - - - ~
J dOrlln~
~
\
.
Fig. 2, Schematic representation of suggested migration patterns and relationship between the varioussubsetsof dendritic accessorycellsnormallypresent in healthyskin and draining lymphsystem.
Fig. 3. Typicalexampleof T6 (CDT)positive Langerhans'cell stainingpattern in epidermisshowing darkstained dendriticcells(2-stepimmunoperoxidasetechnique; primaryantibodyanti-T6 (CD1): magnificationX 160).
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Immunology Today, vol. 7, Nos. 7 & 8, 1986
Table 3. Langerhans'cell dynamics
Ref. Skinsurface Numberof Langerhans'cellsmm 2 Total numberof Langerhans'cellsin situ within entireepidermis
2 x 106mm2 450 2 x 106 x 450 = 0.9 x 109
Repopulationof epidermiswith Langerhans'cellsafter stripping Top at days4-6 Repopulationcompletedin 14 days Maximal repopulationspeed Estimatednormal repopulationspeed
28-70mm-2 day-1 180 mm-2 day-1
These cells, also known as tissue histiocytes, are scattered, in addition, through the reticular dermis. Their role under physiological conditions may be as scavengers of debris and the degraded cell (granulocyte) substances that are continously produced. They may also have antigen-presenting functions, although such a capability has as yet not been demonstrated in situ in skin. Skin neutrophilic granulocytes41 Neutrophilis have been said to leave the blood after an average intravascular sojourn of 10 h. They do not seem to return to the blood after leaving it but their fate after entering the tissues and body cavities is not yet clear, in normal skin, neutrophils are certainly not frequent extravascularly. Their number within the postcapillary venules is high since blood flow there is decreased, and granulocytes participate in immune complex clearing under normal conditions. Skin vasculature and lymphatic drainage Blood and lymphatic vasculature connect SIS to the remainder of the body's immune system. Skin vasculature has three levels, depicted in Fig. 1 and reviewed in Ref. 42. It is around the superficial plexus and adjacent to the capillary loops that most SIS-related cells are present under physiological conditions. The postcapillary venules of the upper plexus, with a diameter of 10-20 I~m, are the sites of inflammatory cell emigration, histamineinduced vascular permeability and immune complex deposition under different pathological conditions. Endothelial cells of these postcapillary venules in skin are HI_A-DR+ and can act as antigen-presenting cells in vitro 43'44. It is evident that endothelial cells of at least part of the skin vasculature exert important functions, to be further elucidated, in the immunophysiology of the skin 4s. Skin lymphatics start with open ends within the papillary dermis, form sinuses and are initially only lined by a single layer of endothelial cells. Later on they have valves and may, in the subcutaneous layers, acquire a smooth muscle wall. Their organization is similar to that described for the vascular network with upper and lower dermal plexuses (Zhdanov, cited in Ref. 42). Lymph flow from the skin is one-directional towards the skin-related lymph nodes. Skin draining lymph nodes are especially prominent in the body folds such as the elbows, knees, and the cervical, axillary and inguinal regions.
238
SALT and SiS Several questions about the concept of SALT may be asked. Inclusion of keratinocytes in SALT may be justified
I
20
17,21 180 x 2 x 106= 360 x 106/day0.9 x 109= 42.8 x 106day-1 21 by several observations in addition to their production of the interleukin-l-like factor ETAF4,s. Keratinocytes may play a role in T-cell differentiation. Epidermal cell culture supernatant was shown to induce terminal deoxynucleotidyl transferase, an early T-cell differentiation marker, on immature human bone-marrow cells and T cells 46, and epidermal cells bound antibodies to serum thymic factor 47 and thymopoietin 48. Epidermal keratinocytes may therefore have some relationship to thymic cortical epithelial cells in T-cell differentiation but proof for intra-epidermal T-cell differentiation in vivo is still lacking. So-called epidermotropic recirculating T-lymphocyte populations are rare, in our experience, as intra-epithelial T cells are uncommon in normal skin (manuscript in preparation). Perhaps the more commonly encountered T cells in normal dermis are a pool distinct from T cells in other connective tissues. LC form part of a widely distributed family of dendritic accessory antigen-presenting cells, and the organ specificity of this cell for stratified squamous epithelial tissues does not necessarily imply a distinct and tissuespecific type of immune function. High endothelial venules (HEV) that are supposed to be necessary for the direction of organ-specific T-cell migration have not been described in healthy skin. HEV-like vasculature has, to our knowledge, only been described in a rare skin disorder called angiolymphoid hyperplasia with eosinophilia, also known as Kimura's disease 49. It is clear that other mechanisms for directing T-cell traffic must operate in skin, possibly through antigen-specific, T-cell-derived factors bound to mast cells (see below). Skin-draining lymph nodes do not receive skin lymph only, and the cells comprising SIS cannot be regarded as lymph-node tissue. It is for these reasons that we define the immune response related cells operating in skin as the SiS. SiS dynamics With SIS, lymph nodes play an important role in tolerance as well as in immunization and sensitization mechanisms. Paracortical T-cell areas which contain dendritic antigen-presenting cells and HEV which direct T-cell migration are essential in the functional cooperation between lymph nodes and SIS. LC function in vitro as stimulatory cells in epidermal cell-induced autologuos 50 and allogeneic 51 T-cell activation and in the generation of epidermal cell-induced cytotoxic T-cell responses s2. They also function as antigen-presenting cells in in-vitro T-cell activation by
ImmunologyToday,vol. 7, Nos. 7&8, 1986
protein antigens ~1. LC also play a role as stimulating cells in graft rejection in vivo s3. The role of LC in contact hypersensitivity, a classical model of delayed hypersensitivitys4'ss, is especially interesting. Sensitization starts with regular exposure of the skin to a hapten. Substances leading to allergic contact reactions generally have a molecular weight of less than 500. They first may bind to a 'carrier' before they can be immunogenic, or they may bind directly to as yet undefined molecules on antigen-presenting cell membranes. The hapten-carrier complex is called antigen or immunogen. Although the exact localization of primary sensitization is still debatable, experiments s6 have demonstrated that an animal can only be sensitized on a prepared skin flap when the connecting part contains, next to essential arteriolar and venous vessels, also the draining lymphatics. It nevertheless remains unclear whether it is the hapten, the sensitized T cell already within the skin ('peripheral sensitization'), or the LC-hapten complex that is carried to the lymph node. Recent evidence provided by Knight etal. s7 indicates that hapten-carrying dendritic cells can sensitize unprimed syngeneic mice. is thus most probably the LC-hapten complex that is involved in presentation of hapten to paracortical T cells. These become activated and are induced to proliferate and differentiate. Effector cell precursors proliferate and some of them differentiate into memory cells, whereas others become effector cells. Suppressor cell precursors are simultaneously induced to proliferate and they stop the further production of effector cells. The evidence from contact sensitization, a pathological state, may also apply to the normal situation, where immunization with a subsequent induction of a state of tolerance may take place continuously. Streileins8 has suggested that SALT represents a physiological mechanism created to deal with neoplastic events taking place continuously within the skin due to ultraviolet irradiation and the presence of potentially oncogenic viruses and carcinogenic agents. This role for LC may be significantly extended as follows. Under physiological conditions LC continuously present skin contacting antigens to the T cells in the lymph nodes. In fact, a large number of the 45 million available human LC will, every day, move through the lymphatics (presumably as veiled cells) to the regional skin-related lymph nodes. In this way, the SIS is continuously made aware of its antigenic microenvironment. Small abrasions and erosions of the physico-mechanical skin barrier that normally occur at microscopic level result in intra-epidermal injections of antigenic materials. Other water-soluble compounds will penetrate into the spinal layer and be taken up by LC. When other compartments such as neutrophils and tissue macrophages fail to clear these antigens, they are further processed by the continuously boosted T cells, without any clinical symptoms or signs. In mice, mast cells may play an important role in delayed-type h~/persensitivity (elicitation of allergic contact dermatitis ~. During sensitization, antigen-binding T-cell factors are produced and released into the circulation. These reach the (skin) mast cells which become sensitized. During elicitation, these antigen-binding Tcell factors bind the antigen and the mast cells react, releasing serotonin but without histamine release or degranulation. Serotonin induces, via receptors on postcapillary venules, vasodilatation and the formation of
reviewsinterendothelial spaces which facilitate the entry of T-cells, among them antigen-specific T cells, into the elicitation site. It is only then that the antigen-presenting, MHC class II restricted stage of delayed-type hypersensitivity develops, characterized by the development of a mononuclear cell infiltrate. Whether these antigen-binding T-cell factors play a role in normal human skin is as yet unclear. If they do, LC presentation of immunogens to these mast-cell-bound, T-cell-derived factors seems possible. The skin's micro-anatomy and immunohistology thus show a complicated and fine-tuned organization of many different immunologically active cells. Probably the most important cells within SIS are those comprising the family of skin dendritic cells. Together with recirculating and/or recruited T cells they act in the specific immune responses. Non-specific cells such as granulocytes and tissue monocytes cooperate with specific recirculating T cells in antigen clearing. T-cell migration may be directed by mast cell release of vasoactive agents through antigen binding via irnmunogen-specific T-cell factors present on mast cell membranes. As well as Fc-receptor-bearing and C3 receptor bearing dendritic cells and macrophages, the vascular endothelium of the upper dermal plexus may also take part in antigen presentation. It seems evident that the SIS form an ideal and moreover easily accessible model for the study of immunology of health and disease. R.H. Cormane, P.K. Das, W.R. Faber, R.F.H.J. Hulsmans and P.M. Lansdorp are gratefully acknowledged for their helpful discussions. Figures 1 and 2 were prepared by Mrs I.E.M. Oosterling.
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