Human Langerhans cells in epidermal cell culture,in vitro skin explants and skin grafts onto “nude” mice

Summary In order to find a model system which best preserves human Langerhans cells (LC) outside of the human body, three possibilities were examined: epidermal cell culture, skin explants, and skin grafts onto nude mice. Using OKT-6 and anti-HLA-DR monoclonal antibodies, we quantified LC in epidermal sheets or epidermal cell cultures. All observations were carried out over a period of 4 weeks. We found that under epidermal cell culture conditions, LC rapidly disappeared, to the extent that after 10 days only rare HLA-DR-positive cells could be observed. In contrast, in the presence of intact dermis (explants and grafts), 60%–80% of the original number of LC, morphologically unchanged, dendritic and OKT-6 and HLA-DR-positive, were seen. These findings suggest that human LC are either a long-lived cell population or else can proliferate locally. The systems studied may be a useful tool for future investigation of LC function.     

Epidermal Langerhans cells--a cycling cell population

The limited number of Langerhans cells (LC) in human epidermis and the resultant technical difficulties have left open the question of LC kinetics. In the present study using flow cytometry (FCM) we have applied 3 methods to estimate LC-DNA distribution: (1) FCM-DNA measurement on highly enriched LC suspensions, (2) FCM-correlated analysis of DNA and OKT-6(+) cells in total epidermal cell suspensions, (3) LC-enriched suspensions (70-90%) were FACS (fluorescence-activated cell sorter) sorted on microscopic slides, and stained with the Feulgen technique, and DNA was measured densitometrically. In the latter method, contaminating keratinocytes were counterlabeled with antikeratin serum to eliminate them from LC-DNA estimation. All 3 in vitro analyses clearly showed that human LC are a cycling cell population in the epidermis. The number of LC in S (1.3-3.3%) and G2/M (1.0-2.5%) phase compares with those found for keratinocytes. Assuming that this percentage of keratinocytes in S and G2/M phases is sufficient to maintain the structural integrity of the epidermis, it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis.     

Langerhans cells in S-phase in normal skin detected by simultaneous analysis of cell surface antigen and BrdU incorporation

We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase. After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h. Studies of BrdU labeled cells included the determination of their cell surface phenotype. Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase. We observed 6% of the epidermal LC in S-phase. This is a proof that LC are able to proliferate in the epidermal microenvironment. This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.     

Differential distribution of Langerhans cells in organ culture of human skin

Epidermal Langerhans cells (LCs) function as the antigen-presenting cells in such cutaneous cell-mediated immune responses as contact hypersensitivity and in the mixed epidermal cell-lymphocyte reaction. They have also been implicated in the immune response in skin allograft rejection. Since organ culture of thyroid and pancreas has been shown to prolong allograft survival, presumably through the loss of antigen-presenting cells, we examined the effect of skin explant culture on LC survival. Human skin explants were placed in organ culture and examined serially as whole mounts of epidermis for the presence of LCs as judged by ATPase activity, and OKT-6 and HLA-DR antigens. Although we observed morphologic changes and an absolute reduction in the number of positively stained cells, culture for up to 28 days failed to deplete explants of these cells. Langerhans cells were also sought in the epidermal outgrowths that develop peripheral to the original explants. They were never seen in the area beyond 0.3 mm from the explant edge. Organ culture of skin thus provides a means to explore the contribution of LCs to skin allograft rejection by comparing the immunogenicity of epidermal portions of the explant with the epidermal outgrowth.     

Migration of Langerhans cells into the epidermis of human skin grafted into nude mice.

In a previous study, it was demonstrated that human Langerhans cells (LC) are preserved in human skin grafted onto a nude mouse. Moreover, although it was observed that mouse LC of the host invade skin grafts from allogeneic mouse or rat, they do not penetrate in human skin grafts. In most of the human skin equivalent systems produced in vitro, LC appear to be lost. The present study was designed to investigate whether the mouse LC will repopulate a human skin equivalent. For this purpose, two different systems of skin equivalent have been grafted into the nude mouse. They were composed of human keratinocytes deposited on dead human dermis, or on lattice composed of human fibroblasts embedded in type I collagen. At different times after grafting, the presence of LC in the transplants was assayed either by indirect immunofluorescence or by electron microscopy. Indirect immunofluorescence was performed on frozen sections or on epidermal sheets with anti-Ia, anti-HLA-DR, or OKT6 antibodies. It was observed that, at 2 months after grafting, Ia(+) HLA-DR(-) OKT6(-) cells are present in grafted human epidermis. Moreover, LC with typical Birbeck granules are also detected by electron microscopy. It could be concluded, from this study, that mouse LC can repopulate human epidermis devoid of human LC.     

The steady-state turnover of murine epidermal Langerhans cells

We have investigated the steady-state turnover of murine epidermal Langerhans cells (LCs) using an X-irradiation model, 3H-thymidine autoradiography and cultured epidermal sheet explants, and by assessing the LC population in normal mice. The LC density after whole-body irradiation without any cutaneous shielding was not significantly different from that in skin shielded during whole-body irradiation (P > 0.05), indicating that the additional irradiation to the skin did not contribute to a decrease in LC density. In both instances, the LC number gradually decreased in a linear fashion. The results indicate that epidermal LCs continuously leave the epidermis and are continually replaced by circulating precursor cells from the bone marrow at a steady rate. Autoradiographic studies after a pulse injection of 3H-thymidine showed a labelling index of 0.013%, indicating that local mitosis is not an important contributor to the maintenance of the epidermal LC population. Although local X-irradiation resulted in temporary reduction of LC density, epidermal sheet explant culture obtained immediately after local X-irradiation showed no difference in LC density as compared with control unirradiated skin, indicating that the decrease in LC density was not due to significant LC destruction. From these data, we calculated that the half-life of murine LCs in the epidermis is approximately 9 days.     

Use of in situ detection of histone mRNA in the assessment of epidermal proliferation: comparison with the Ki67 antigen and BrdU incorporation

The labelling index is commonly used as a measure of proliferation. However, the use of tritiated thyraidine or BrdU labelling of S-phase cells is limited to prospective samples. We have employed an oligonucleotide cocktail complementary to the mRNA species encoding the repiication-dependent histones H2B, H3 and H4 for non-isotopic in situ hybridization (NISH), and have compared the resultant proliferation indices in normal skin with those obtained by bromodeoxyuridine (BrdU) incorporation and by Kift 7 immunohistochemistry (MC) using the monoclonal antibody MIB1. In addition, we compared the staining characteristics of histone NISH and Kif>7 IHC in a further 25 samples from a variety of inflammatory dermatoses and neoplastic conditions, as well as from normal skin. In normal skin, S-phase (histone NISH and BrdU) and cycling (Ki67) cells were conflned to the basal and Jow suprabasal layers. The labetling indices determined by histone NISH and BrdU incorporation were similar, whereas that of Ki67 IHC was four times greater. In biopsies from hyperproliferative dermatoses and dysplastic or malignant lesions. the number of histone NISH- and Ki67 IHC-positive cells was generally elevated; in accordance with the differential expression of these two markers during the cell cycle, M1B1 consistently gave higher results. The advantage of histone NISH over Ki67 MC is that it is a marker of the same part of the cell cycle as BrdU incorporation. However, the combined use of both histone NISH and Ki67 MC to measure two cell cycle parameters, namely S-phase and the number of cycling cells, aliows more detailed retrospective study of epidermal proliferation than has been possible previously.     

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.     

Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice

BACKGROUND: It is recognized that UV radiation produced apoptotic cells (sun burn cells) in the epidermis of mice. However, the relationship between apoptosis and cell proliferation after UV exposure in the skin of hairless mice are still unclear. OBJECTIVE: To investigate the effects of ultraviolet (UV) radiation on molecular events associated with apoptosis and proliferation in SKH1-hr mouse skin. METHODS: Mice were irradiated with daily UVB exposure of 0.1 or 0.25 J/cm(2) for 14 days. The skin tissues were analyzed at 2 and 24 h after the end irradiation for the presence of apoptotic cells and Bromodeoxyuridine (BrdU)-positive cells. We measured the expression of p53, p21, bcl-2, bax and E2F-1. RESULTS: The results indicated that UVB irradiation caused to increase apoptotic cells in the epidermis of mice. The expression of p53 and p21 was increased at 2 and 24 h after irradiation compared with the control. UV radiation induced high levels of bax at 2 and 24 h after irradiation with a concomitant decrease in bcl-2 expression. The expression of E2F-1 in the skin was also increased at 2 and 24 h after irradiation. Coinciding with these changes, BrdU positive cells increased at 2 and 24 h after UVB exposure at the epidermis of hairless mice, which observed the apoptotic expression. CONCLUSION: These results suggest that UVB irradiation of mouse skin induces apoptosis and is mediated by the p53/p21/E2F-1/bax pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia.     

In vitro HIV-1 entry and replication in Langerhans cells may clarify the HIV-1 genome detection by PCR in epidermis of seropositive patients.

Being dendritic antigen-presenting cells in skin and mucous membrane, Langerhans cells (LC) occur in areas at risk for inoculation by human immunodeficiency virus (HIV), and the question whether LC act as a target, reservoir, or vector for transmission of HIV has given rise to much controversy. To address this question, we first analyzed the epidermal compartment of skin from patients seropositive for HIV DNA. Second, we tested the susceptibility of each cell type normally found in this compartment to in vitro infection by HIV-1. A non-denatured DNA was obtained from epidermal sheets after a thermochemical treatment of biopsies (0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.5 at 60 degrees C for 90 seconds). Optimization of amplification of viral genome was performed with three primer pairs derived from gag, env, and pol sequences. Polymerase chain reaction (PCR) products were analyzed by Southern blot. Viral genome was found in five of 11 HIV-seropositive patients. To control the permissivity of epidermal cell population for HIV, cells isolated from the epidermal sheet of normal skin by trypsinization were co-cultured with HIV-1-carrying promonocytic cells (U937) and observed by electron microscopy. After 3-6 h of co-culture, numerous virions were either tightly bound or apparently engaged in the process of internalization through receptor-mediated endocytosis. At day 4 of co-culture, some infected LC appeared to release mature viral particles through bud formation. The in vitro HIV-1 entry and replication in LC may confirm the presence of the HIV-1 genome by PCR in epidermis of seropositive patients. The consequences of the permissivity of LC for HIV on the antigen-presenting function remain to be determined.     

Number of Langerhans immune cells in painful and non-painful human skin after shingles

During injury or inflammation, paracrine sensitization of peripheral sensory neurons by immune cells contributes to the sensation of pain. It is less clear whether this neural sensitization contributes to neuropathic pain after neural injury as well. Shingles (herpes zoster) is a common disease that leaves some patients with prolonged neuropathic pain known as postherpetic neuralgia (PHN). Sensitization of cutaneous neurons has been hypothesized to contribute to PHN. Langerhans cells (LC), the Ia(+) macrophages of the skin, contact epidermal neurites and, when activated, synthesize molecules with the ability to sensitize axons. For these reasons, we examined morphological evidence for activation of LC in subjects with established PHN. We also evaluated the relationship between numbers of LC and nociceptive epidermal nerve endings; these are markedly reduced in PHN. We used design-based stereology to estimate the number of CD1a(+) LC in biopsies of painful and non-painful skin from ten adults with or without PHN after shingles on the torso. There were no differences in the number of LC in previously shingles-affected and normal-control skin biopsies. The number of LC also remained at normal levels in biopsies with near-loss of innervation from shingles. LC numbers were unrelated to the presence or severity of pain. These data suggest that neuropathic pain in established PHN is not associated with increased numbers of cutaneous macrophages, and that the number of cutaneous macrophages in skin from the human torso is independent of the number of epidermal nerve endings.     

Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model: a novel method for identifying potential contact allergens

The accelerated migration of Langerhans cells (LCs) out of the epidermis and up-regulation of maturation markers, upon treatment with subtoxic concentrations of chemicals, were used as the criteria to determine the potential of allergenic chemicals capable of inducing a hapten-specific delayed-type hypersensitivity reaction. Here we report the findings of a study in which seven chemicals, coded and tested in a blind fashion, were classified as contact allergens or non-allergens using the human organotypic skin explant culture (hOSEC) model. All chemicals that were identified as a contact sensitizer on decoding induced a definite decrease in the number of CD1a and HLA-DR-positive epidermal LCs in the epidermis of the skin explants, as determined by both semiquantitative immunohistochemistry and quantitative flow cytometric analysis. A significant increase in the number of CD83(+) cells was accompanied by up-regulation of activation molecules in the epidermis of hOSEC exposed specifically to contact allergens. In contrast, there were only minor alterations in epidermal LC numbers, expression of CD83 and other activation markers by LCs when the biopsies were treated with non-toxic concentrations of non-allergenic irritants and vehicles. The data suggest that an increased epidermal LC migration and maturation accompanied by increased expression of activation markers could be used as end-point determinants to screen allergens in a non-animal alternative hOSEC model.     

Migration of Langerhans cells in an in vitro organ culture system: IL-6 and TNF-alpha are partially responsible for migration into the epidermis

Although it is well established that epidermal Langerhans cells (LC) originate from bone marrow, little is known about the mechanism of this migration into the epidermis from bone marrow. In order to clarify the mechanism of this migration, we constructed an in vitro model. LC were depleted by daily topical application of clobetazole propionate (CP) solution onto the ear of Balb/c mice. Seven days later, ear skin was cut off, separated and co-cultured dermal-side-up with syngeneic (Balb/c), semisyngeneic ((C3H x Balb/c)F1), or allogeneic (C3H) epidermal cells (EC) for 3 days. We found (1) that a marked migration of donor LC into the recipient epidermis was observed in the LC-depleted skin, (2) that only syngeneic LC actively migrated into the recipient epidermis; however, the migration of semisyngeneic and allogeneic LC was detected at very low levels, (3) that the migratory capacity of donor LC was directly proved by a biolabeling technique using donor EC labeled with PKH-26, and (4) that anti-IL-6 and anti-TNF-alpha antibodies inhibited the migration of donor LC into the recipient epidermis. These data demonstrate that the resident LC have the potential to traffic through the dermis into the epidermis in a highly syngeneic-specific fashion, and that IL-6 and TNF-alpha are partially responsible for promoting this migration.     

Defined in situ enumeration of T6 and HLA-DR expressing epidermal Langerhans cells: morphologic and methodologic aspects

An essential prerequisite for the in situ enumeration of epidermal Langerhans cells (LCs) is the unequivocal identification of the desired cell type. We have examined over 250 cryostat sections of normal human skin to analyze morphologic and methodologic problems underlying the quantification of epidermal LCs, defined by anti-T6 (OKT6) and anti-HLA-DR (OKIal) immunoperoxidase staining. Our findings show that OKT6 reactivity of dendritic processes in cross-sectioned epidermis yields microscopic images which are not easy to analyze objectively. The morphology that we find leads us to categorize dendritic cells into 3 arbitrary types of T6+ LC profiles. In addition we describe criteria for the assessment of OKT6 staining patterns relating to the dendritic state of epidermal LCs. Preliminary quantitative data on this issue are discussed in relation to: epidermal thickness; the thickness of skin tissue sections; and the discrepancy between the number of T6+ and HLA-DR+ LCs. We hope that the principles outlined in this report may serve to overcome potential methodologic problems with quantitation of T6+ epidermal LCs in skin sections.     

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.     

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.     

Epidermal Langerhans cells in erythrokeratodermia variabilis

Summary The distribution of epidermal Langerhans cells (LC) in erythrokeratodermia keratodermia variabilis (EKV) was investigated using enzyme-histochemical (ATPase) and immunohistochemical (anti-T6 and anti-HLA-Dr) techniques. Biopsy specimens from lesional skin of five patients were examined before and 8 weeks after treatment with etretinate (RO 10-9359). In addition, electron microscopy studies were carried out in two of these cases.The number of ATPase-positive dendritic cells in lesional epidermis appeared to be remarkably reduced in comparison with normal skin from healthy subjects. After treatment, the number of ATPase-positive dentritic cells had increased but still remained below normal values. Similar but less striking results were found for anti-T6-stained specimens. The HLA-Dr antigen appeared to be unsuitable as a marker for comparative quantitative studies because of the highly variable expression of this antigen in the control specimens. Electron microscopy studies revealed LC in the basal and suprabasal layers of the lesional epidermis. Both before and after treatment, the LC exhibited a normal ultrastructure.In view of the clinical and histologic normalization of the skin lesions during treatment, these findings suggest that the decreased number of epidermal LC may be related to the abnormal keratinization that occurs in EKV.     

Quantitative analysis of the proliferation of epidermal cells using a human skin organ culture system and the effect of DbcAMP using markers of proliferation (BrdU, Ki-67, PCNA)

Abstract The process of re-epithelialization of a wound in the epidermis comprises the following steps: proliferation of epidermal basal cells, migration of epidermal cells to the wound surface, and cell differentiation. In the present study, we evaluated the proliferation of epidermal basal cells, an important process in wound healing, in the wound margin using a human skin organ culture system and immunohistochemical labeling with bromodeoxyuridine (BrdU), Ki-67, and proliferating cell nuclear antigen (PCNA), as markers of cell proliferation. Dibutyryl cyclic adenosine monophosphate (DbcAMP) is a derivative of cAMP and has been shown to modulate human keratinocyte proliferation. The proliferation of keratinocytes was promoted by DbcAMP and particularly strong effects in terms of BrdU labeling index, Ki-67-positive ratio, and PCNA-positive ratio, were seen at 10^–5 M. The skin organ culture system presented here uses adult preputial skin and is a simple technique that uses easily available materials. In addition to identifying S phase cells using BrdU as an index of cell proliferation, the immunohistochemical method for evaluating the expression of Ki-67 and PCNA is very simple. Accordingly, the method described here seems to be useful for evaluating cell dynamics in wound healing. Received: 6 June 2000 / Revised: 21 July 2000 / Accepted: 23 November 2000     

Antigenic thymus-epidermis relationships. Reactivity of a panel of anti-thymic cell monoclonal antibodies on human keratinocytes and Langerhans cells

We have investigated on human skin the reactivity of a panel of 42 anti-thymus monoclonal antibodies (MCA) supplied by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens, Oxford, 1986. MCA of the first cluster of differentiation (CD1) define a group of surface molecules expressed by cortical thymocytes. Some of them (OKT6, M241 and Na1/34) have been shown to react in normal human skin with the epidermal Langerhans cells (LC). Twenty-two CD1 MCA were investigated in the present study. On normal human skin, 13 MCA reacted with LC in situ. This result suggests and confirms the heterogeneity of CD1 MCA. Recently, some of them were shown to recognize biochemically different molecules and/or epitopes of thymocytes. In addition, 20 anti-thymic epithelium MCA were tested on human skin. The MCA which only reacted with the thymic epithelial cell network (except Hassall's corpuscles) decorated only the epidermal basal cell layer. The MCA which reacted with all the thymic epithelial cells (including Hassall's corpuscles) decorated all the epidermal cell layers. These results confirm the heterogeneity of the thymic epithelial microenvironment and underline the antigenic similarities between the thymic epithelial structures and the different epidermal cell layers. The existence of bone-marrow-derived CD1-positive cells (thymocytes or LC) in an epithelial cell network (the thymus and the epidermis) focus the speculation around the immunological role of the epidermal basal cell layer in the T cell education and the exact lineage of the epidermal LC.     

Effect of granulocyte macrophage-colony stimulating factor on Langerhans cells in normal and healthy atopic subjects

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a multipotent cytokine produced by many cutaneous cell types including keratinocytes. Langerhans cells (LC) represent the major antigen-presenting cells in skin, and in vitro studies demonstrate that GM-CSF is of pivotal importance in LC. Healthy volunteers ( n  = 3 non-atopic, n  = 3 with atopy) received recombinant human GM-CSF (0.05 μg/mL) by intradermal injection for 3 days to the same site. Diluent was injected in a similar manner as control. Biopsies were taken 24 h after the final injection and examined immunohistochemically for LC and inflammatory cell markers. Compared with control sites, intradermal GM-CSF resulted in shortening of dendritic cell processes and redistribution of LC in the epidermis; numbers of CD1a + cells in the epidermis were significantly decreased ( P  < 0.005), while those in the dermis were significantly increased ( P  < 0.05) following intradermal GM-CSF when compared with controls. Double labelling studies on epidermal CD1a + cells indicated de novo expression of intercellular adhesion molecule (ICAM)-1 and increased expression of HLA-DR following GM-CSF ( P  < 0.005, P  < 0.005, respectively). Additional findings included a marked mixed inflammatory cell infiltrate in the dermis and increased expression of the endothelial cell adhesion molecules E-selectin and ICAM-1. These data indicate that in normal human skin, GM-CSF induces changes in the phenotype and distribution of CD1a + cells consistent with LC functional maturation and exit from the epidermis to the dermis. As these events are central to the initiation of cutaneous inflammation, GM-CSF may potentially play a critical role in the pathogenesis of inflammatory dermatoses.