Review Skin organ culture Skin organ culture: A review M. Kataranovski and Dj. Karadaglic SUMMARY Short-term organ culture of skin explants is a useful model far research into various aspects of skin biology. The use of skin organ culture systems in defining factors which affect homeostasis in elucidating modulatory effects of biologic response modifiers, drugs and physical agents on the skin and in studying complex aspects of cutaneous biology in normal and diseased skin is reviewed. Our own data regarding organ culture of rat full-thickness skin explants have been presented in this review. Introduction Since the development of organ culture technique (1) this approach has been used for identification of various mediators in cultures of synovial, articular and other tissues and granulomas. Skin is easily organ- cultured (2,3). In this system characteristics of skin are displayed more clearly comparing with cell-culture systems, as the architecture of the tissue remains intact and cell-cell interactions are relatively undisturbed. Therefore it is suitable for investigations of various aspects of skin function and biology. Various skin organ culture systems have been described depending on the source of skin specimens (neonatal or adult skin), the size of skin explant (1-9 mm 2 or 1cm) and culture conditions (choice of liquid media, total or partial submersion, freely floating or on various supports). By using these systems it is possible to study various aspects of both normal and pathological skin biology, ex vivo or in vitro entirely. In this paper, we have reviewed the use of human and animal skin organ culture in assessing skin homeostasis and inflammatory/immune reactions, in evaluating effects of various agents on the skin and complex aspects of skin cell behavi01· involved in cel! migraton. In addition, we have presented our own data obtained by the use of organ-cultured full-thickness rat skin explants. Tissue integrity and architecture ofskin in organ culture In organ-cultured skin normal relationship between epithelial cells and between fibroblasts, epithelial cells acta dermatovenerologica A.P.A. Vol 8, 99, No 4 ---------------------- -------- 131 Skin organ culture and extracellular matrix is preserved. Thus, organ cul- tures of human skin have been utilized to study normal growth and differentiation. The tissue culturecl in a regular culture meclium with nutrient mixtures for skin cel! culture ancl growth supplements (epiclermal growth factor, insulin, hyclrocortisone, bovine pituitary extract) remains viable for a few clays, but necrosis can usually be seen by week 1 ( 4,5). The major degenerative chan- ges inclucle epidermal necrosis, destmction of the basal layer ancl separation of the epidermis from underlying basal lamina, with loss of celularity ancl a breakdown of the extracellular connective tissue stmctures in the dermis. Experiments with organ cultures established from more than 70 adult human skin specimens indi- catecl that, under appropriate conclitions (i.e. the pre- sence of serum-free, growth factor-free culture medium supplementecl with exogenous calcium ions to a fina! concentration of 1.4 nM or 3 µM of retinoic acid, human skin can be kept in organ culture histologically normal in appearance ancl biochemically active for at least 12 clays ancl up to 24 days (5). It was suggestecl that uncler these culture conditions procluction of growth factors required for the maintenance of homeostasis is favourecl. The procluction of components of extra- cellular matrix (fibronectin ancl thrombosponclin) by the organ culturecl skin in the presence of 1.4 nM Ca++ was demonstratecl also (6). As the maintenance of human skin explants in organ culture clepencls on conclitions optimized for fibroblast proliferation ancl not on those optimizecl for keratinocyte growth, a critical role for fibroblast viability ancl function was proposecl in main- taining cutaneous homeostasis (7). This organ culture model is thus suitable for stuclying mechanisms respon- sible for the maintenance of normal skin homeostasis. It has also provecl to be a useful tool for stuclying skin response to injury ancl pathophysiolgical mechanisms of alterecl clifferentiation and proliferation in psoriasis. Organ culture of psoriatic skin was recommendecl as a useful tool for deciphering pathophysiological mechanisms of aberrant keratinocyte proliferation and its possible moclulation in vitro (8). Maintenance of psoriatic lesional skin in organ culture (9) as well as mimicking the features of psoriatic skin in organ cultures of normal skin by exposing it to a growth factor-enrichecl meclium (8), provicle an experimental approach for clelineation of factors which are critical to the main- tenance of psoriatic lesion. By this approach the invol- vement of epiclermal growth factor (EG F) in maintaining the psoriatic phenotype was clemonstrated in vitro using an antibocly to the human EGF receptor (8). The effects of a vitamin A clerivative, tretinoin were followed in oi·gan cultures of psoriatic lesional skin. It was shown that keratinization of the involvecl psoriatic epiclermis is sensitively controllecl by vitamin A, with granular layers appearing in the absence of the vitamin or 132 ----E --O') o. --ro .C: o. (O 1 LL z 1- 60 50 40 30 20 10 o o 2 DNCB(%) 4 Fig 1. TNF-a levels in culture fluid of organ- cultured rat ear skin following topical application of various doses of DNCB. Values are expressed as pg TNF-cx/ml of culture fluid, determined by a commercial ELISA test. Significance at * 0.05 or ** 0.01 vs vehicle treatment. clisappearing with relatively low concentration of the vitamin (10). By using human skin organ cultures, the role of reti- noids in skin homeostasis has been extensively stucliecl. Fig 2. Tetrazolium reducing capability of skin explants cultured in the presence of DNBS. Results are expressed as absorbance of extracted formazan product. Significance at 0.05 vs control skin explants. 35 Q) * :J (/) (/) ·.;::::; 25 O') ---Q) 20 (.) C 15 ccr .o ,.._ 10 o (/) .o 5 <( o o 31 125 DNBS (µg/ml) Review 8 500 acta dermatovenerologica A.P.A. Vol 8, 99, No 4 Review It was shown that retinoids maintained viability of skin organ cultures, but the failure to maintain normal epider- mal differentiation was noted (5). An investigations into the effects of retinoic acid (RA) on epidermal homeo- stasis in a human organ culture system revealed the RA- induced expression of heparin-binding epidermal growth factor (Hb-EGF), a member of the epidermal growth factor family, which in turn might be responsible for the observed effect of the drug which promotes epidermal hyperplasia (11). Retinoic acid (RA) treatment of organ-cultured skin resulted in decreased cohe- siveness and extensive acantholysis , accompanied, in some skin specimens, with separation of the corneal from the basal and suprabasal layers (5,6). The retinoid- induced loss of epidermal cohesion could be attributed to a reduced synthesis of components of extracellular matrix (6) and/or production of proteoloytic enzymes (urokinase and tissue plasminogen activator) in the presence ofthis agent (12). Itwas suggested from these studies that proteases might influence the structural integrity of the tissue. The epidermal-dermal junction of organ cultured skin was shown to be highly suscep- tible to neutral serine proteases derived from human skin, mast cells and polymorphonuclear leukocytes (13). Thus, in vitro culture of the skin in the presence of proteolytic enzymes or agents, which induce or facilitate their activity, might provide a clue to the involvement of proteinases in skin diseases. The involvement of proteinases in the altered skin integrity was suspected in early studies of skin explants cultured with proteinases or agents, which induced proteinase activity. A loss of epidermal cohesion w as demonstrated in organ cultures of skin explants incu- bated with immunogobulin G (IgG) from pemphigus sera (14). As plasminogen enhanced the ability of pemphigus IgG to cause acantholysis in organ culture, the involvement of plasminogen activator (PA) was indicated in the process (14) . A recent study in the uPA knock-out (with targeted disruption of PA genes) neonatal mouse model of pemphigus (15) provides evi- dence that IgG from pemphigus vulgaris and pemphigus foliaceus cause the loss of celi aclhesion clirectly via binding to desmogleins, interfering thus with their fun- ction, rather than indirectly by releasing proteases. Bincling of IgG from bullous pemphigoid sera for the basement membrane zone in a skin explant culture system has been regardecl as a very sensitive methocl for the detection of bullous pemphigoid antibodies (16). The development of epidermis under the influence of the thyroid hormone, gluccocorticoids and estrogen was followecl in an explant model of fetal rat skin (17). The involvement of a variety of ligancls, activators ancl nuclear receptors in the hormone's action was revealed acta dermatovenerologica A.P.A. Vol 8, 99, No 4 Skin organ culture on the basis of expression of structural proteins (profillagrin ancl loricrin) of the stratum corneum. Organ culture as a means to study skin inflammation. Cutaneous inflammation coulcl be investigated in skin organ cul ture by monitoring the presence of vari- ous soluble biochemical ancl inflammatory/immuno- regulatory mecliators ancl expression of molecules relevant to inflammatory celi infiltrate formation. Cul ture fluids are being used to collect mediators released from skin explant for their iclentification. In this regarcl is illustrative a systematic stucly of dermal inflammation in rabbit skin following topical application of sulphur mustarcl, in which organ cultures of clevelopecl ancl healing lesions were establishecl and mediators in the culture fluicls of explants monitored (18). Organ- culturing of lesional biopsies enablecl the authors to cletermine a local turnover of serum proteins within the lesions themselves, in contrast to most studies in which leakage of serum or removal of serum proteins from lesions was reportecl (19). High levels of serum proteins were founcl in acute inflammatory lesions with rapicl t:urnover rate. It was further clemonstratecl that the serum is a major source of unbouncl extracellular protein within these lesions, serum proteinase inhibitors being a major defense against local clamage by proteinases from serum, activatecl infiltratecl leukocytes ancl skin resiclent cells (19). Lysosomal enzymes were identified among inflammatory mecliators releasecl in culture fluid of skin explants, with higher levels in the culture fluicl from healing lesions and with polymorphonuclear leukocytes ancl fibroblasts iclentified histochemically as their source (20). Byusing an in vitroapproach entirely (i.e. in vitro injrny to human skin by sulfur mustarcl) , histamine, pro- staglandin E2 and plasminogen activator were iclentified as mecliators which initiate the inflammatory response to this agent (21). Skin organ culture is suitable for stuclying soluble mecliators of cutaneous inflammation, cytokines. Both epiclermal ancl clermal elements (keratinocytes, Langer- hans cells, fibroblasts, ancl enclothelial cells) are the source of enclogenous cytokines collectecl in organ culture fluicls (22). Data obtainecl by organ-culturing the skin from various conclitions of trauma impliecl that a skin injury might evoke a local response characterizecl by inflammation-relatecl cytokine procluction. Signi- ficantly higher amounts of interelukin-6 (IL-6) were found in culture fluids of human skin specimens obtai- necl from the operative wouncls within a short tirne postoperatively, comparecl to the levels cletectecl in 133 Skin organ culture Table 1. Overview into the use of skin organ culture SKIN HOMEOSTASIS / GROWTH / DIFFERENTIATION Normal skin Maintenance in vitro Drug / biologic modifiers effects - retinoic acid - p roteinases - antibodies: p emphigus serum bullous pemphigoid serum - hormones Psoriasis les ional skin Maintenance in vitro Drug / biologic modifiers effects - EGF* - tretinoin INFLAMMATION Inciting agent - Sulphur mustard - Sulphur mustard - Operative injury - Thermal injury - Contact allergen VARIOUS AGENTS EFFECTS - Cutaneous allergens - Drugs -UV -Cold - Ionizing radiation CELL MIGRATION Human Human Human Human Human Rat (fetal) Human Human Human Rabbit Human Human Rat Rat Rat Human Human Human Human (FDE*) Human Human (vitiligo) Human Human Mouse Human Human Degenerative changes / histology ECM* Fibroblast growth / viability - epidermal hyperplasia / cohesiveness - skin integrity - acantholysys - basement membrane binding - epidermal development - histology - psoriatic phenotype - epidermal keratinization Soluble inflamatory mediators (serum proteins,lysosomal enzymes) Histamine,PGE 7 PA Cytokines -, Cytokines Cytokines Metabolic activity Cytokine mRNA Paranuclear vacuolization Adhesive molecules Cytokine-mediated aclhesive molecules Heat shock proteins Morphological, biochemical (fair follicl e) Keratinocyte proliferation Adhesion molecule Appearance Appearance Cel! aclhesion / aggregation HIV containment Phenotype Route of migration Wound healing *ECM - extracellular matrix; EGF - epidermal growth factor; FDE - fixed drug eruption fluids from uninjured skin either preoperatively or postoperatively (23) . IL-6 secretion by skin explants could be stimulated during in vitro culture by egzo- genous inflammation-related cytokines, tumour necrosis factor (TNF)-cx and IL-1 and inhibited by corticosteroids, suggesting that a local skin cytokine response might be 4,5 6 7 6, 11, 12 13 14 16 17 8,9 9 10 R e view 18,19,20,21 21 23 24 This study This study 32 36 37 38 39,40 41 42 43 44 45 46,47 48 49, 50,51 52 53, 54 134 acta dermatovenerologica A.P.A. Vol 8, 99, No 4 .Review influenced by inflammatory mediators from systemic microenvironment as well. Organ culture of rat skin was employed in our laboratory in the ex vivo evaluation of local cytokine responses in an experimental model of thermal skin injury. Increased levels of TNF-a and IL-1 in culture fluids of skin explants indicated a cytokine response in the vita! edge of the thermally injured skin early following the injury (24). We employed organ culture of rat skin in studying skin response to a cutaneous allergen, dinitrochloro- benzene (DNCB) in a previously described experimen- tally induced contact hypersensitivity reaction ( CHS) in rats (25). In this model of cutaneous inflammation/ immunity, the reaction is generated (the induction phase) and expressed (elicitation phase) in the skin. Epicutaneous application of DNCB in the elicitation phase of CHS resulted in ear swelling, reflecting a local inflammatory response to hapten. The intensity of this response could be quantitated by the "ear swelling assay" which is a measure of the intensity of local infla- mmat01y reaction in animal models of contact sensi- tivity. Recent data demonstrated the involvement of nitric oxide (NO) in the expression of CHS in mice and contribution of this inflammatory mediator to the ear swelling (26). Our preliminary data of ex vivo deter- mination of levels of NO in culture fluids of the skin from ears following topical application of DNCB in the elicitation phase of CHS, demonstrated an increase in NO (approximately 5-fold), as estimated by nitrite levels · in the Griess reaction (27). The intensity of inflammatory reaction during the elicitation of CHS in rats was evaluated by determining the levels of TNF-cx, an essential, primary cytokine involved in the expression of this experimental allergic reaction (28) in culture fluids of dorsal, cartilage free halves of ears, exposed to DNCB (Fig 1). Increased levels of TNF-a were noted with increasing doses of topically applied DNCB. The local inflammatory/immune response to skin sensitizing chemicals is preceded by the "preimmu- nological" phase of response, characterized by oxidative activities of skin cells (29). We employed rat skin organ culture to assess the potential of the skin to generate reactive oxygen species in response to DNCB. The in vitro potential of the skin to generate reactive oxygen intermediates depends in part on the activity of tetra- zolium-reducing respiratory burst oxidase (30) and could be determined via tetrazolium salts such is MTT (3-( 4 ,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) to insoluble formazan products (31). The effect of dinitrobenzene sulfonic acid (DNBS), a water- soluble DNCB analog, on tetrazolium reducing capa- bility of the skin in organ culture is presented in Fig 2. The increased reduction of MTT was shown in the presence of increasing doses of DNBS in culture. A de- Skin organ culture creased MTT reducing activity of skin specimens cul- tured with 500 µg DNBS/mL, probably reflects toxicity of DNBS, as only viable skin cells possess a MTT reducing capability. Additionally, skin organ culture en- abled an analysis of early molecular events, including expression of messenger RNAs for inflammatory cyto- kines induced in human epidermis by contact allergens or irritants (32). Cutaneous inflammation could also be evaluated by monitoring in situ (in an organ-cultured skin explant) expression of adhesion receptors on endothelial cells, leukocytes and keratinocytes, the cmcial molecules for inflammatory leukocyte extravasation, migration and their interaction with resident cells (33). Modulation of adhesion molecule expression by inflammat01y media- tors such as cytokines may be studied in organ culture. Short-term organ cul ture of neonatal foreskin was used as a model to elucidate relative effects of IL-1, TNF-cx and immune interferon (IFN-y) on the inducibile expre- ssion of activation markers of microvascular endothelial cells (34). The endothelial celi response was demon- strated immunohistochemically during in vitro culti- vation with the cytokines, localized predominantly to the postcapillary venules of the superficial vascular plexus, i.e. to those vessels most associated with infla- mmatory infiltration in vivo. The role of mast celi secre- tory activity in the initiation of cutaneous inflammation has been suggested by using a sirnilar skin organ cul ture system. A rapid induction of endothelial celi activation antigen (ELAM, CD62E), a crucial endothelial celi mole- cule for leukocyte-endothelial celi adhesion, was de- monstrated in the adjacent microvasculature following in vitro stimulation of mast cell degranulation in the perivascular space. The induction ELAM has been shown to be TNF-a- dependent, as no or only weak ELAM expression was shown in the presence of an anti- TNF antibody (35). Data obtained by this in vitro organ culture model highlighted the role of secreto1y activity of mast cells in cutaneous inflammation and established a potential link between mast cell-associated proinfla- mmatory mediators (i.e. TNF-a) and the development of a cellular inflammatory response. Evaluation oj effects oj chemical and physical agents in skin organ culture Organ culture of skin explants is widely used as an approach to assess modulato1y and/ or adverse effects of various chemical and physical agents and biologic response modifiers. These effects may be studied both ex vivo or following an in vitro exposure of the skin, by monitoring changes in skin viability, tissue structure/ acta dermatovenerologica A.P.A. Vol 8, 99, No 4 ------------------------ - - ------ 135 Skin organ culture integrity and parameters of cutaneous inflammation. Skin viability may be determined by changes in metabolic activity of skin explants measurecl by the enzymatically-mecliatecl reduction of tetrazolium salts (31) or by observing paranuclear vacuoles (termed "storage-type") associated w ith gradual cel! autolysis (36). The paranuclear vacuolization test has been recommended as a re liable test fo r evaluating clermatotoxic potential of topically applied chemicals (36) . Organ culture systems are particularly useful in tracing effects ofvarious agents both ofbiological origin or clrugs on diseased skin, where the expression of markers of inflammation was demonstrated in vivo. It was shown in organ cultures of the skin from atopic inclivicluals that ELAM (CD62E) expression on the vascular endothelium , can be incluced by in vitro exposing the skin specimens to cytokines or allergens (37). A short-term organ culture of fixecl drug eruption (FDE) lesional and nonlesional skin was employec.l to investigate the cellular and molecular events responsible for the observec.l skin inflammatory response to ingested drug (38) . The obtained results indicatec.l that the lesional keratinocytes and endothelium respondecl more rapidly and intensely than nonlesional skin to TNF-a anc.l IFN- y by expression of intercellular adhesion molecule (ICAM-1) , a major activation antigen of keratinocytes in conc.litions of inflammation ancl a general adhesion molecule on activatecl enc.lothelial cel!. ICAM-1 expre- ssion on keratinocytes and endothelium inc.lucecl by the causative drug alone was restricted to the FDE lesional skin organ cultures, which was abrogatec.l by neutra- lization ofTNF-a by anti-TNF-a antibocly. Data, which c.lemonstratecl the c.lrug-incluced ancl cytokine-depen- c.lent expression of ICAM-1 in the lesional skin spe- cimens, suggested that the incluction of expression of this activation aclhesion molecule coulc.l provicle an initial localizec.l stimulus for development of infla- mmatory response leading to activation of infilt:rating leukocytes (38). Skin response to c.lifferent environmental stimuli, including various chemical anc.l physical stressors has been assessecl in organ-cultured skin. In vitro exposure of skin explants to azetidine carboxylic acid, sodium arsenite, cadmium as well as to UV -irradiation anc.l heat resultec.l in the expression of heat shock protein HSP 72, an inclucible member of the family of stress-induced molecules required for cel! survival during and after stresses of variuos origin (39). As biologic effects ofHSP were considered primarily in association with thermo- tolerance, and therapeutic hyperthermia and UV irra- diation have been used for the treatment of various cutaneous diseases, organ-cultured skin is deemed a useful model for studying this class of cel! clefense proteins in human skin cliseases. In addition, as there is 136 an age-relatec.l decrease in the inducibility of HSP 72 in organ-cultured normal human skin ( 40), monitoring of the UV-induced expression of these molecules in organ cultures coul cl be a useful additional approach in investigation of diminished ability of agecl skin to responcl to the adverse environmental conditions and to maintain homeostasis . The exposure of organ- culturecl skin from the margins of vitiligo lesions in vitro to UV light, revealecl morphological (dendricity) ancl biochemical (catecho l oxidase ancl no raclrenaline positivity) changes as features of UV responsiveness, pointing to the hair follicle as a specialized UV receptor in human skin (41) . Organ-cultured human skin has been regardecl a useful model for evaluating the response of human skin to freezing (monitored by histology, immunolabelling ancl incorporation of raclioactive tabel by proliferating cells) cluring the clevelopment of c1yolesions as well as in eluciclating the healing process of cryosurgery wounds (42). This culture system was used in stuclying the encloth elial cel! response to ionizing radiation through the assessment of adhesion molecule (VCAM- 1, ICAM-1 and PECAM-1) expression in response to a clinically relevant dose range ( 43) . Organ culture asa modeljor studying cell migration If explants of mouse (44) ancl human (45) skin are placecl in organ culture, cells spontaneously migrate out into the culture medium surrounding the explant. The cel! emigration was observed during 3 ciays, with highest numbers of migratecl cells cluring the first 24-h of culture. Thereafter keratinocytes appear in the medium. Emi- grants from human skin specirnens comprise dendritic cells, lymphocytes and macrophages, clenclritic cells being the most abunclant among the migratecl cells . Immunophenotyping of clendritic cells, which appearecl in the meclium during 3 consecutive clays, demonstrated a high proportion ofboth epiclennal (CD l a•) ancl ciermal ( CD 1 b •) clenclritic cells ( 45). The migrato1y properties of clendritic cells interface with their capacity to initiate cutaneous immune responses through their extra- ordina1y capacity to activate T cells. Among the skin cel! emigrants, T cells could be found, preclominantly belonging to the CD4• subset (the CD4•/ CDS• subset ratio is about 2: 1), almost entirelywith a/~ T-cell recep- tor ancl with the phenotype of memo1y cells (46) . The population of cutaneous emigrant leukocytes is highly viable (more than 95%) ancl clevoicl of contaminant keratinocytes as seen using the conventional skin clisaggregation enzyme methocls, with an average yielcl of 60000 leukocytes/ cm2 (43). Thus skin organ culture systems make accessible both T lymphocytes and dendritic cells in a highly enrichecl form enabling their R eview acta dermatovenerologica A.P.A. Vol 8, 99, No 4 R eview study. Cutaneous T celi migrants were found in stable aggregates with dendritic cells which could have been formed in situ (detected ex vivo in culture fluid) and/or in vitro, from mixtures of emigratecl clenclritic ancl T cells ( 46). As denclritic cell-T celi aggregates are regardecl microenvironments in which immune activation and proliferation occur ( 47), it has been suggested that conjugation of these cells might contribute to cuta neous recall immune responses, such as the clelayecl type hy- persensitivity in which mem01y T cells are engagecl. Studying the cutaneous celi emigration ancl the corre- sponding cellular interactions may be of relevance in skin cliseases with documented immunopathology. By using organ-cultured skin, the site of cutaneous vira! HIV-1 selection was monitored and the virus was recovered from cells that had emigrated from skin ex- plants (48). Skin organ-culture systems enable studying mecha- nisms of celi emigration . A series of changes in the phenotype of dendritic cells, called "maturation", w hich include expression of molecules of the major histo- compatibility complex (MHC) class II, and an array of costimulatory and adhesion molecules was demon- strated during their migration ( 45 ,49). These phenotypic changes represent the molecular basis of acquisition of strong stimulato1y activity of dendritic cells cletected in in vitro T-cell activation assays. Denclritic cells of the epiclermis (Langerhans cells), appear to unclergo these phenotypic changes within the epidermis during skin organ culture ancl these are accompanied by their spontaneous migration within the epiclermis and then to the dermis (50). Phenotypic characteristics of DC could be modulated by cytokines of the epidermal origin (TNF-a ancl granulocyte-macrophage colony stimulating factor, GM-CSF) during organ culture, stre- ssing a microenvironmental epidermal influence on the acquired migrato1y capacity. n t? l/ r; ·u !!";' ·r- r (., r;, c .. [l. .!t.; .t · .i .. i .i.\ .l .. .1 .:. °' _; L; (") Skin organ culture Studies of the ear skin-sensitizing reaction using the mouse skin explant assay clemonstrated the involvement ofTNF-a and a /~4 integrins (a key structural component of hemidesmosomes involved in the interaction bet- ween basal epidermal cells ancl the basement mem- brane) in the regulation of clendritic celi emigration from the epidermis (51). Migration from the epiclermis through the dermis is characterized by the accumulation of dendritic cells in a characteristic string-like pattern, termed "cords", in the murine explant assay (44), and identifiecl as lymphatic vessels in human skin organ cultures (52). Migratory dendritic cells, w hich accum- ulate in the dermal lymphatic vessels, are comprised of both epidermal and clermal clendritic cells as shown by ultrastmctural and immunochemical criteria (49). Organ culture was employed in studies dealing with issues of epiclermal dendritic celi emigration from the epidermis in response to skin-sensitizing chemicals, where it was shown that these cells are stimulated to migrate speci- fi cally in response to contact sensitizing agents, but not to nonsensitizing chemicals (49). The skin organ culture model is also helpful for studying various biological aspects of wound healing in human skin including effects of growth factors (53) or transplanted keratinocytes (54), which might be of great value in better understanding the complex process that governs the healing of a human wound. In conclusion, in vitro skin organ culture systems represent suitable and useful moclels to investigate various aspects of normal and abnormal skin biology (an overview into the use of skin organ culture is presented in Table 1). These systems may be used as principal or accompanying tests in situations where a confirmation of some clinical data is needed or their functional significance is investigated. They are parti- cularly useful in studying effects of various pharmacolo- gical agents, and their possible mechanisms of action. l. Dingle J, Gordan J, editors. Cellular interactions. Invited papers presented at the symposium to celebrate the 80th birthday of the Dame Honor B. Fell. Oxford: Elsevier/North Holland Biomedical Press, 1981: 1- 289. 2. Sarkany I, Grice K, Caron G. Organ culture of adult human skin. Br J Dermatol 1965; 77: 65-76. 3. Reaven EP, Cox AJ. Organ culture of human skin. J Invest Dermatol 1965; 44: 151-6. 4. Tamni R, Jansen RT, Tam ni M. Effect of retinoic acid on human epidermis in whole skin organ culture. Arch Dermatol Res 1985; 277: 275-83. 5. Varani J, Figie EG, Schuger L, Perone P, Inman D, Griffiths CEM, Voorhees JJ. Effects of ali-trans retinoic acid and Ca++ on human skin in organ culture. AmJ Pathol 1993; 142: 1~9-98. 6. Varani], Larson BK, Parane P, Inman DR, Figiel SEG, Voorhees ]]. Ali-trans retionic acid and extracellular Ca2+ differentially influence extracellular matrix production by human skin in organ culture. AmJ Pathol 1993; 142: 1813-21. 7. Varani]. Preservation of human skin structure and function in organ culture. Histol Histopathol 1998; 13: 775-83. acta dermatovenerologica A.P.A. Vol 8, 99, No 4 - - --- --------------------------- 137 Skin organ culture 138 8. Varani]. Kang S, Stoli S, Elder JT. Human psoriatic skin in organ culture: Comparison with normal skin exposed to exogenous growth factors and effects of an antibody to the EGF receptor. Pathobiology 1998; 66: 253-9. 9. Kondo S. Maintenance of epidermal structure of psoriatic skin in organ culture. J Dermatol 1986; 13: 242-9. 10. Kondo S, Hozumi Y, Aso K. Organ culture of psoriatic lesions: Appearance of granular layers in vitamin A-free culture media. J Invest Dermatol 1992; 98: 753-7. 11. Stoli SW, Elder JT. Retinoid regulation of heparin-binding EGF-like growth factor gene expression in human keratinocytes and skin. Exp Dermatol 1998; 7: 391-7. 12. Varani J, Bumesteir B, Sitrin RG, Sholienberger SB, Inman DR, Fligiel EG, Gibbs DF, Johnson K. Expression of serine proteinases and metalloproteinases in organ-cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion. AmJ Pathol 1994; 145: 561-73. 13. Briggaman RA, Schechter NM, Fraki], Lazarus GS. Degradation of the epidermal-dermal junction by proteolitic enzymes from human skin and human polymorphonuclear leukocytes. J Exp Med 1984; 160: 1027-42. 14. Hashimoto K, Shafran KM, Webber PS, Lazarus GS, Singer KH. Anti-celi surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal celis. J Exp Med 1983; 157: 259-72. 15. Mahoney MG, Wang ZH, Stanley JR. Pemphigus vulgaris and pemphigus foliaceus antibodies are pathogenic in plasminogen activator knockout mice. J Invest Dermatol 1999; 113: 22-5. 16. Mutasim DF, Vaughan A, Supapannachart N, Farooqui J. Skin explant culture: a reliable method for detecting pemphigoid antibodies in pemphigoid sera that are negative by standard immunofluorescence and immunoblotting. J Invest Dermatol 1993; 101: 624-7. 17. Komuves LG, Hanley K, Jiang Y, Elias PM; Williams ML, Feingold KR. Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development. J Invest Dermatol 1998; lil: 429-33. 18. Harada S, Dannenberg A, Kajiki A, Higuchi K, Tanaka F, Pula P. 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Mediators, initiating the inflammatory response, released in organ culture by fuli-thickness human skin explants exposed to the irritant, sulphur mustard. J Invest Dermatol 1991; 96: 888-97. 22. Kataranovski M. Skin immune system. Cellular elements and interactions. In: Karadaglic Dj. Dermatology (in Serbian). Beograd: VIN Vojna štamparija, 1999 (in press). 23. Ueo H, Inoue H, Honda M, Uchida I, Nishimura T, Arinaga S, Nakashima H. Production of interleukin- 6 at the operative wound sites in surgical patients. J Am Coli Surg 1994; 179: 326-32. 24. Cikata B, Kataranovski M, Nikolic T, Kandolf L. Evaluation of local skin cytokine response in thermal injury by short-term organ culture offuli thickness skin explants in rats. Shock 1997; 7(Suppl):128. 25. Kandolf L, Kataranovski M, Berger S, Milosavljevic I, Karadaglic Dj. Experimentally induced contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) in rats. Arch Toxicol Kinet Xenobiot Metab 1998; 6: 267-8. 26. 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Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive ~endritic-cell T-cell conjugates. J InvestDermatol 1995; 104: 11-17. 47. Fleisher ER, Freudenthal PS, Kaplan G, Steinman RM. Antigenic-specific T lymphocytes efficiently cluster wiili dendrite cells in the primary mixed-leukocyte reaction. Celi Immunol 1988; 111: 183-95. 48. Reece JC, Handley AJ, Anstee EJ, Morrison WA, Crowe SM, Cameron PU. Hiv-1 selection by epidermal dendritic cells during transmission across human skin. J Exp Med 1998; 18: 1623-31. acta dermatovenerologica A.P.A. Vol 8, 99, No 4 - ----------------------------- 139 Skin organ culture AUTHORS' ADDRESSES 49. Weinlich G, Heine M, Strossel H, Zanella M, Stoitzner P, Ortner U, Smolle J, Koch F, Sepp NT, Schuler G, Romani N. Entry into afferent lymphatics and maturation in situ of migrating murine cutaneous dendritic cells. J InvestDermatol 1998; 110: 441-8. 50. Rambukkana A, Bos JD, Irik D, Menko WJ, Kapsenberg MI, Das PK. In situ behavior of human Langerhans cels in skin organ culture. Lab Invest 1995; 73: 521-31. 51. Price AA, Cumberbatch M, Kimber I. Alpha6 integrins are required for Langerhans celi migration from the epidermis.J Exp Med 1997; 186: 1725-35. 52. Lukas M, Strossel H, Hefel L, Imamura S, Fritsch P, Sepp NT, Schuler G, Romani N. Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model. J Invest Dermatol 1996; 106: 1293-9. 53. Kraty G. Modeling of wound healing processes in human skin using tissue culture. Microsci Res Tech 1998; 42: 345-50. 54. Moll I, Hoidek P, Schmidt H, Moll R. Characterization of epidermal wound healing in a human organ culture model: acceleration by transplanted keratinocytes. J InvestDermatol 1998; 111: 251-8. Milena Kataranovski PhD, projessor ojimmunobiology, Faculty oj Biology, University ojBelgrade; Studentska 4 and Senior research associate, Institutejor Medica! Research, Military MedicalAcademy, Crnotravska 17, 11000 Beograd, Yugos lavia Djordije Karadagli6 MD, PhD, projessor oj dermatology, Clinicjor Dermatovenereology, Military MedicalAcademy, Crnotravska 17, 11000 Beograd, Yugoslavia Review 140 - --- ------------- acta dermatovenero/ogica A.P.A. Vol 8, 99, No 4