Biology of Langerhans cells: analysis by experiments to deplete Langerhans cells from human skin

In vivo studies have demonstrated that various treatments of skin, e.g., UV irradiation, topical corticosteroids, and tape-stripping, will temporarily deplete the epidermis of Langerhans cells (LC). Whether this loss represents simply a loss of cell surface markers unique to LC, or actual depletion of cells, is unknown. By design, normal human skin transplanted to the congenitally athymic (nude) mouse is a system devoid of circulating precursors for human LC. Because LC have been shown to be of bone marrow origin, any depletion of these cells in this system should be permanent. Treatments to deplete LC from human skin grafts on nude mice after grafting included: (a) large doses of UV radiation (400 mJ/cm2 every 48 h, X 3), (b) potent high-dose topical corticosteroids (2.5 mg betamethasone valerate/cm2 every day, X 5), (c) tape-stripping (X 20). Treatments before grafting included: (a) treating donor skin with 900 R of gamma irradiation, (b) complement fixing monoclonal antibody to Ia-like antigens of LC, followed by fresh complement, (c) monoclonal antibody conjugated to toxins. Quantitation of the number of LC was analyzed on control and treated epidermal sheets using immunodiagnostic reagents, anti-HLA-DR, and surface ectoenzymes , ATPase. Results show that both UV irradiation and topical corticosteroids reduce the number of LC by these analyses. However, within 3 weeks, recovery to pretreatment levels has occurred. X-irradiation and tape-stripping were without effect. Despite evidence that the monoclonal antibody, complement, and toxic systems were delivered to the LC within the epidermis, there is no evidence that these treatments resulted in a decrease in LC. It appears that LC are currently either long-lived or replaced locally from a proliferative pool and that certain cell membrane determinants of human LC are somewhat differentially sensitive to UV radiation and corticosteroids.     

Further evidence for the self-reproducing capacity of Langerhans cells in human skin

The limited number of Langerhans cells (LC) in the epidermis is one of the main reasons for the technical difficulties in resolving the question of LC kinetics. In the present paper, we describe a method to evaluate the LC replication potential in epidermis. The procedure is based on the specific incorporation of bromodeoxyuridine (BrdU), a thymidine analogue, into the DNA during the S-phase of the cell cycle. Mice, bearing human skin grafts, were injected s.c. every 6 h for up to 17 days with BrdU. At different times, the incorporated BrdU as well as the human epidermal LC were revealed on skin sections using anti-BrdU and OKT-6 monoclonal antibodies, respectively. After 6 h, 4.9% of the LC were labeled with BrdU. Then, the number of OKT-6(+) BrdU(+) cells increased in a linear manner and achieved 34% at 120 h, 67% at 240 h, and 94% at 400 h during the course of continuous labeling procedures. Based on this result we calculated a total cell cycle time of 392 h (16.3 days) and 12 h for the S-phase for human epidermal LC. Applying this technique, we were able to show also that 48 h after local treatment with 12-O-tetradecanoylphorbol-13-acetate or after stripping, the number of BrdU-labeled LC was considerably increased. Furthermore, after i.p. injection of colchicine in the nude mouse, human epidermal LC undergoing mitosis were evidenced by electron microscopy in the graft. From these results we conclude that the LC are actively cycling--therewith a self-reproducing cell population in human epidermis.     

Improvement of epidermal differentiation and barrier function in reconstructed human skin after grafting onto athymic nude mice

To determine whether epidermis reconstructed in vitro at the air-liquid interface on de-epidermized dermis has the capacity to normalize the expression of differentiation-specific markers, its lipid composition and stratum corneum barrier properties, human skin equivalents were transplanted onto athymic nude mice and investigated at different stages ranging from 1 to 4 months after grafting. Indirect immunofluorescence with species- or non-species-specific antibodies revealed that as early as 1 month after transplantation keratinization, and involucrin, loricrin and transglutaminase patterns were normalized. Human melanocytes were observed in the basal layer of the pigmented graft. As revealed by high-performance thin-layer chromatography and transmission electron microscopy after ruthenium tetroxide fixation, the lipid profile and the intracellular lamellar organization were similar to those found in natural epidermis. Transepidermal water loss measurements and penetration studies showed that the barrier properties of the reconstructed epidermis after transplantation were comparable to those of normal human skin.     

Characterization of the chick chorioallantoic membrane model as a short-term in vivo system for human skin

Abstract We report on the cultivation and characterization of human skin on the chorioallantoic membrane of chicken eggs with the aim of replacing animals in short-term investigations in dermatology. Adult human split-thickness skin was grafted onto the chorioallantoic membrane of 5-day chick embryos. Grafts and surrounding host tissue were examined daily by in vivo stereomicroscopy and in histological sections and were characterized using a panel of monoclonal antibodies. The skin grafts were completely incorporated into the chorioallantoic membrane 2 days after transplantation. A remarkable angiogenesis occurred towards the grafts. Skin tissues revascularized within 2 or 3 days by reperfusion of the existing graft vasculature. Anastomosis of host and graft blood vessels occurred and the transplanted skin was nourished by the host blood supply as indicated by nucleated chick erythrocytes in the skin vessels. The skin grafts on the chorioallantoic membrane preserved an almost entire human phenotype. Besides a fully differentiated human epidermis and dermis containing all the cellular and extracellular constituents such as skin immune cells, capillary vessels composed of human endothelial cells were enclosed by a basement membrane of human origin. The integrin expression pattern formed in human skin transplants 5 days after grafting was identical to that of human skin controls before grafting. Received: 3 September 1998 / Received after revision: 12 November 1998 / Accepted: 13 November 1998     

A new dermal equivalent: the use of dermal fibroblast culture alone without exogenous materials

BACKGROUND: During the past decade, several kinds of skin equivalents have been developed. However, the dermal equivalents have all contained exogenous materials, which can be difficult to obtain and a source of infections. OBJECTIVES: The aim of this study was to develop a new dermal equivalent by culturing dermal fibroblasts alone without exogenous materials and to evaluate its applicability in vitro and in vivo. METHODS: The postconfulent cultures of dermal fibroblasts in serum containing medium, that was supplemented with epidermal growth factor, insulin, hydrocortisone, transferrin and triiodothyronine for 3 weeks, produced a fibrous sheet that was visible macroscopically. To construct a skin equivalent, epidermal keratinocytes were cultured on the top of the fibrous sheet at the air-liquid interface. To evaluate its fate in vivo, the fibrous sheet was grafted into a nude mouse. RESULTS: Histologically, the fibrous sheet showed dermis-like tissue that consisted of an extracellular matrix around dermal fibroblasts, and revealed collagen fibers by Masson-trichrome staining. The components of dermal matrix such as type I collagen, type III collagen, elastin, fibrillin-1 and fibronectin were diffusely expressed. Some collagen fibrils were found by electron microscopy. In the skin equivalent, a multilayered epidermis with a horny layer was formed. Some differentiation markers (keratin 1 and 10, and involucrin) and the components of basement membrane (beta4 integrin chain, type IV and VII collagens) were expressed in a similar fashion to those in normal skin in vivo. Ultrastructurally, basement membrane zone such as hemidesmosomes, lamina lucida and lamina densa was found, although it was still incomplete. When the fibrous sheet was grafted in vivo, it revealed blood vessels that were derived from the nude mouse, and persisted for 4 weeks. CONCLUSION: These findings demonstrated that a new dermal equivalent, closely resembling a dermis in vivo, could be constructed by culturing dermal fibroblasts alone in a special culture medium. In addition, the dermal equivalent may be useful for experimental and clinical purposes, such as the reconstruction of a skin equivalent in vitro and grafting in vivo.     

Growth and differentiation of human epidermal cultures used as auto- and allografts in humans

Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo-epidermal basement membrane was newly synthesized during the first few days following transplantation onto de-epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.     

Human skin grafts on nude athymic mice: A light microscopic stereological study

Summary A morphometric procedure is presented, which allows quantitative information to be obtained from the epidermis at the light microscope level. The application of this procedure to human skin grafted to the nude mouse revealed acanthosis of the grafted epidermis compared to the original donor skin. All epidermal layers were thicker, but the increase in the granular layer was especially marked. The ratio of the basement membrane surface to the epithelial surface showed no significant change. A possible explanation for the acanthosis of the graft might be the higher mechanical stress on the nude mouse compared to the original site on the abdomen. This adaptation of the grafted epidermis does not limit the usefulness of this animal model for dermatological research, when it is assessed by objective methods, allowing statistical comparison as described here.     

银屑病皮损中郎格罕氏细胞的初步观察

本文应用ATP酶法和抗T6单克隆抗体OKT6直接免痊荧光法,对6例寻常型银屑病皮损及其邻近正常皮中的LC进行了初步对比观察.结果两法在正常皮肤部位显示表皮内的LC带有树枝状突起,分布均匀,主要位于中层;且OKTs法尚可显示真皮内少最散在的LC.而皮损部位则表皮内LCJL平消失,偶见1~2七且其突起消失.呈小圆圈状;真皮内未见可辨认的LC.以上所见提示皮肤中的LC可能与银屑病的发病机理有关.     

Melanocyte autologous grafting for treatment of leukoderma

Patients with three types of leukoderma--vitiligo, idiopathic guttate hypomelanosis, and postinflammatory leukoderma--had successful repigmentation after transplantation of autologous melanocytes. The procedure was performed easily by producing blisters on normal skin and on depigmented lesions. Blisters were produced by suction or by freezing with liquid nitrogen. The roof of the blister from donor skin was grafted to the raw surface of the recipient site. Repigmentation was visible within 7 to 14 days. Direct immunofluorescence staining with bullous pemphigoid antibodies suggested that the separation of the epidermis from the dermis occurs within the lamina lucida. Histochemical studies confirmed the absence of dopa-positive cells in the areas of leukoderma prior to grafting. Melanocytes were present in the successful grafts.     

Sheep vibrissa dermal papillae induce hair follicle formation in heterotypic skin equivalents

Cultured skin equivalents were constructed by combining keratinocytes, outer root sheath cells or isolated epidermis, in vitro, with a matrix composed of collagen and cultured fibroblasts. When equivalents were grafted on to host animals, the epidermis thickened considerably, and tongues of cells penetrated the dermis, giving the dermal/epidermal junction a deeply sculptured profile. No cutaneous appendages were found in these grafts. We explored the possibility of inducing hair follicles by incorporating ovine hair follicle dermal papillae into constructs composed of an isolated epidermal sheet and a contracted dermal equivalent. In vitro, no morphogenetic changes associated with follicle formation were observed in the recombinants, but when grafted on to nude mice, follicle-like structures were identified. The follicles were large, and had developed adjacent to the epidermis, indicating that the matrix environment of the induced follicles may not have been compatible with the downgrowth of the epidermal plugs normally observed during follicle formation in living skin. Nevertheless, in histological sections, the induced structures displayed many of the morphological characteristics of follicles in vivo, including the production of keratinized hairs. These results indicate that skin equivalents provide a useful model for the study of the chemical and structural features of matrices that facilitate hair follicle development.     

Wound healing of human skin transplanted onto the nude mouse after a superficial excisional injury: human dermal reconstruction is achieved in several steps by two different fibroblast subpopulations

Abstract It has been established that human skin grafted onto the nude mouse is able to regenerate after being subjected to a full-thickness wound. In the present work, we sought to determine the cells involved in the connective tissue repair process following superficial wounding. Two months after transplantation, superficial wounds were made at the center of the graft using mechanical dermabrasion. At various times thereafter, ranging from 2 days to 6 weeks, healing grafts were harvested and processed for immunohistological study with species-specific and cross-reacting antibodies directed against human or mouse antigens. The grafted human skin regenerated according to the following series of events. First, the human dermis underneath the scab became devoid of human fibroblasts while the surrounding human dermis preserved its own characteristics. The TUNEL reaction on early-phase healing wounds indicated that apoptosis occurred steadily within this area and could be the mechanism by which cells disappeared. Moreover, cell death was reduced when the wound was covered with an occlusive dressing. The human dermis beneath the wound was then invaded by mouse cells which deposited type I collagen on the human extracellular matrix and produced mouse granulation tissue at the surface above it. Human keratinocytes migrated over the mouse granulation tissue to reconstruct the epidermis. Eventually, the mouse granulation tissue was progressively invaded by human fibroblasts, which formed a human neodermis. The overall process appeared to depend upon several successive epithelial-mesenchymal interactions, which were not species-specific. This suggests that myofibroblasts arise from a specific subpopulation of fibroblasts, probably located at the interface between the dermis and adipose tissue, and that the granulation tissue is eventually remodeled by another population of fibroblasts present in the human dermis. Received: 31 March 1999 / Accepted after revision: 15 July 1999 / Accepted: 13 August 1999     

Development of human fetal skin transplanted to the nude mouse

Thirty-five human fetal skin (HFS) grafts were transplanted to nude mice for 7 to 70 d and evaluated histologically with 64 biopsies. The estimated gestational ages (EGA) of the grafts at the time of the transplantation ranged from 8 to 19 weeks. The maturation of the engrafted fetal skin was evaluated by assessing epidermal, dermal, and appendage development. Within the nude mouse, the HFS demonstrated progression in stratification and maturation of the epidermis. The dermis increased in depth, adding fibrovascular stroma and adipose tissue. The appendages demonstrated invagination, differentiation, and progression of organogenesis. Subcutaneously placed grafts showed the same rate of HFS development as HFS in utero. The grafts transplanted to the surface of the nude mice and exposed to air demonstrated an acceleration of development. We conclude that HFS transplanted to the nude mouse is an effective in vivo model for maintaining and altering HFS maturation.     

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.     

Recessive dystrophic epidermolysis bullosa phenotype is preserved in xenografts using SCID mice: development of an experimental in vivo model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a subgroup of hereditary blistering diseases characterized by repetitive wounding and healing with subsequent extensive scarring. The purpose of this study was to establish a xenograft model that retains the RDEB phenotype and thus might be used as an experimental in vivo model to explore the molecular and biochemical mechanisms of the chronically wounded phenotype of RDEB. Full-thickness, tumor-free RDEB skin tissues were grafted onto the dorsum of severe combined immunodeficiency (SCID) mice. At 4, 8, 12, and 24 weeks after grafting, the xenografts were removed for examination. Immunofluorescence studies were performed using species-specific antibodies to human class I antigen, mouse class I antigen, human type IV and VII collagens and with cross-reacting antibody against bullous pemphigoid antigen (BPA). Staining with the antibody to human class I antigen, W6/32, and with the antibody to mouse class I antigen, 20.8.4s, confirmed the species-specific results obtained with the type IV and type VII collagen and laminin antibodies. The RDEB grafts showed essentially no staining with the type VII collagen antibody. Antibodies against laminin and BPA showed normal staining patterns in RDEB grafts. There was an overall paucity of anchoring fibrils in the grafts when examined with electron microscopy. Blisters could be induced in these grafts with minor trauma and showed a sublamina densa separation by immunomapping and electron microscopy. As late as 24 weeks post-transplantation, the RDEB grafts remain human, are not significantly replaced by mouse cells, and retain the RDEB disease phenotype.     

Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice

We have previously demonstrated regrowth of hair in scalp skin grafts taken from patients with alopecia areata (AA) and alopecia universalis (AU) following engraftment on to nude mice. This present study was to determine whether serum from patients with AA and AU, has a role in the process of hair loss and the role of antibodies and complement. Forty mice were grafted with transplants obtained from seven patients. One group of the grafted mice was given patients' serum and another group normal serum. The mice were treated topically with cyclosporin (CyA), or olive oil. Hair growth was noted in most grafts and intravenous injections of serum did not prevent or inhibit this process. Immunofluorescence studies before grafting showed deposition of immunoglobulins and complement in hair follicles in both normal and affected scalp skin, but a more striking deposition was noted in the affected skin. Deposition of immunoreactants after grafting was observed only after the injection of serum from the patients but not with normal serum. Thus the sera from patients with AA or AU, when injected into nude mice with hair transplants from the scalp skin of patients with these disorders, does not alter the hair growth despite deposition of immunoreactants around the hair follicles.     

Ultrastructural immunogold labelling of human langerhans cells enriched epidermal cell suspension

Summary Colloidal gold particles are well suited as markers in electron microscopy. Indirect immunogold staining was used to identify cell membrane antigens defined by monoclonal antibodies OKT6 and BL6 on human Langerhans cells (LC) in suspensions. Isolated epidermal cells were obtained by skin trypsinization and enriched or depleted in OKT6 positive on BL6 positive LC using the panning method: incubation of OKT6 or BL6 preincubated cells on immunoglobulin coated dishes. Indirect immunogold staining was then performed after prefixation in 2% paraformaldehyde. In LC enriched suspensions, only LC exhibited a specific membrane labelling with OKT6 or BL6 recognized by the presence of small evently distributed gold granules. Neither Birbeck granules, nor other cytoplasmic organelles, were labelled. No other epidermal cells were found positive. In LC depleted suspensions, no labelling was observed. Immunogold labelling on LC enriched suspensions after panning is now in progress for the qualitative evaluation and the quantitative analysis of cell surface constituants and antigens expressed by human dendritic epidermal cells.Presented at the Society for Investigative Dermatology and the European Society for Dermatological Research (Joint International Meeting Washington, April 27, May 1, 1983)     

Grafting of venous leg ulcers. An intraindividual comparison between cultured skin equivalents and full-thickness skin punch grafts

Skin equivalents that consisted of a noncontracted collagen gel populated with allogeneic fibroblasts and covered with autologous cultured keratinocytes were used for grafting venous leg ulcers. The results were compared in the same patient with those obtained with a routinely used standard method of grafting with autologous full-thickness punch grafts. The skin equivalents and the punch grafts were grafted successfully in four of five patients. The median healing time of ulcers grafted with skin equivalents was 18 days whereas that of ulcers covered with punch grafts was 15 days. The cosmetic appearance of the skin equivalent-grafted ulcers was better than that of the punch-grafted ulcers.     

Langerhans cells in skin from patients with psoriasis: quantitative and qualitative study of T6 and HLA-DR antigen-expressing cells and changes with aromatic retinoid administration

Using a monoclonal antibody against human HLA-DR antigens and OKT6, we investigated by indirect immunofluorescence the distribution of Langerhans cells in normal human skin and involved and uninvolved skin from patients with psoriasis before, during, and after systemic aromatic retinoid administration. In parallel, enumeration of HLA-DR and of OKT6+ cells was also performed. In involved psoriatic epidermis the distribution of positive cells was disturbed; OKT6+ cells were reduced in number, as were HLA-DR+ cells which were seen in clusters. In control skin sections, a regular pattern of fluorescent dendritic epidermal cells was noted. In normal-appearing human skin, in nonlesional psoriatic skin, but not in diseased psoriatic skin, the number of OKT6+ cells per epidermal section surface unit was higher than that of HLA-DR expressing cells. Changes in the number and distribution of OKT6 and HLA-DR+ cells in psoriatic involved epidermis were corrected by oral retinoid treatment.     

Presence of somatostatin in normal human epidermis

Somatostatin (SOM) is a ubiquitous peptide which is responsible for the inhibition of numerous biological functions. SOM is described as an antiproliferative molecule and an inhibitor of exocrine or endocrine secretion from a variety of tissues, including pancreas, gastrointestinal tract, central and peripheral nervous system. Mediation of SOM effects can be indirect or direct, respectively, through other molecules or receptors on target cells. We have searched for the presence of SOM in the epidermis using immunofluorescence, confocal laser scanning microscopy, radioimmunoassay, and chromatography. Immunofluorescence and confocal laser scanning microscopy studies were performed using rabbit antiserum anti-SOM and mouse monoclonal antibody directed to CD1a Langerhans cell (LC) marker disclosed with fluorescein or tetramethylrhodamine isothiocyanate conjugates. SOM was extracted from whole skin or epidermal cell suspension or LC-enriched suspensions and analysed by radioimmunoassay. We used an antiserum which was reactive for the 6–11 portion of native SOM. Chromatographic columns were performed on extracts from whole skin. The epidermis was SOM immunoreactive. LC were immunoreactive for SOM and the staining was membranous. SOM was extracted from the whole skin at about 0·13±0·02 fmol/mg of tissue (mean±SEM). The SOM concentration in epidermal cell suspensions was 1·5±0·9 fmol/10⁶ cells. Data obtained with LC-enriched suspensions showed large variations between donors. Extracts from skin showed one peak with an elution profile like that of 14 amino acid SOM. This study demonstrates that 14 amino acid SOM is expressed in normal human epidermis.     

C-5 propyne-modified oligonucleotides penetrate the epidermis in psoriatic and not normal human skin after topical application

We have previously shown that antisense oligonucleotides effectively reduced insulin-like growth factor I receptor expression in human psoriatic skin grafted on to nude mice when injected intradermally. We therefore investigated the penetration of C-5 propyne modified antisense oligonucleotides into human normal and psoriatic skin after topical administration. Oligonucleotide (37.5 microg; 250 microM) was applied in aqueous solution or 5% methylcellulose gel for 24 h, prior to live confocal microscopy and fluorescence microscopy of fixed sections. We found that oligonucleotide could penetrate through the stratum corneum of psoriatic but not normal human skin over large regions of the epidermis. The oligonucleotide was localized to the nucleus of large parakeratotic cells in the psoriatic skin as well as smaller basal and suprabasal keratinocytes. In normal human skin, oligonucleotide was confined to the stratum corneum, with little or no oligonucleotide apparent in the viable epidermis. Electrophoresis of oligonucleotide recovered from treated psoriatic and normal skin revealed that the oligonucleotide remained intact over the 24 h period. In summary, we found that C-5 propyne modified antisense oligonucleotides could reach the target cells (in this case basal keratinocytes) after topical administration to psoriatic but not normal skin.