Expression of stratum corneum chymotryptic enzyme in relation to other markers of epidermal differentiation in a skin explant model

The serine proteinase stratum corneum chymotryptic enzyme (SCCE) has been proposed to be involved in the degradation of intercellular cohesive structures in cornified squamous epithelia in the process of desquamation. Since SCCE is expressed late in epidermal differentiation and is found at all sites where there is a formation of cornified epithelia it also serves as a marker for terminal epidermal differentiation. Earlier studies have shown that the link between expression and the formation of cornified cells may be stronger for SCCE than for other well characterized markers of epidermal differentiation. In an attempt to further elucidate the regulation of SCCE expression we have in this study compared the expression of SCCE with the expression of keratin 10, filaggrin and involucrin in an in vitro model with skin explants cultured for various periods of time on de-epidermized dermis at the liquid-air interface. The markers were analysed by means of immunohistochemistry. We found that the expression of SCCE preceded the expression of keratin 10 and filaggrin. In contrast to involucrin, which was expressed by all suprabasal keratinocytes, SCCE was expressed only by high suprabasal cells. Our results indicate that the expression of SCCE may be regulated in a way that differs from the regulation of the expression of keratin 10, filaggrin and involucrin.     

Thy-1 antigen-bearing dendritic cells populate murine epidermis

Two distinct cell populations, melanocytes and Langerhans cells (LC), have been recognized previously to possess dendritic configuration in normal mammalian epidermis. Employing immunofluorescence microscopy with monoclonal antibodies against Thy-1.2 antigen to identify cells in whole mounts of murine epidermis, we have identified a third dendritic cell population which differs from both LC and melanocytes. Thy-1 antigen-bearing (Thy-1+) epidermal cells are primarily dendritic, although round and angular forms may be found. They are distributed relatively evenly across skin surfaces, although densities vary greatly from site to site and from strain to strain. Densities were highest in ear epidermis from the pigmented strain B10.A (580 cells/mm2), a value approaching that of epidermal LC, and were lowest in ear epidermis from the albino strain BALB/c (5 cells/mm2). Thy-1+ epidermal cells possess neither Ia antigens nor substantial amounts of melanin, and their surface distributions are disparate from those of both LC and mature melanocytes. We propose that at least some of these cells are T lymphocytes whose malignant counterparts account for cutaneous T-cell lymphomas.     

Epidermal hyperproliferation and decreased skin barrier function in mice overexpressing stratum corneum chymotryptic enzyme

Stratum corneum chymotryptic enzyme (SCCE; also known as kallikrein 7) is a serine protease that may have an important role in the skin desquamation process. We have recently described transgenic mice overexpressing human SCCE in suprabasal epidermal keratinocytes, leading to increased epidermal thickness, hyperkeratosis, dermal inflammation and signs of severe pruritus in older animals. In order to further evaluate the scce-transgenic mice as a potential disease model, we compared transgenic animals and wild-type littermates for patterns of epidermal keratin expression, in situ hybridization of scce-mRNA, scratching behaviour and measurements of transepidermal water loss (TEWL). In 3-day-old mice, despite readily detectable amounts of human scce-mRNA in the epidermis of transgenic animals, there were no histological differences in skin appearance, and no differences could be found in epidermal expression of the keratins 5, 6 and 10. In mice 7-8 weeks of age and older, there was strong suprabasal expression of keratins 5 and 6 in the epidermis of transgenic animals, suggesting that the thickened epidermis in these animals is the result of keratinocyte hyperproliferation. In transgenic animals 11 weeks of age and older there was an increased frequency of scratching, suggestive of pruritus, and also signs of a deteriorating skin barrier function, as reflected by an increased TEWL. There was no correlation between increased TEWL and increased frequency of scratching in individual animals, suggesting that the defect barrier function was not an effect of skin damage caused by scratching.     

Clear and Dark Basal Keratinocytes in Human Epidermis

Standardized techniques of stereologic cytology were applied to normal human epidermis, in order to comparatively describe the suprabasal and the dark and clear basal keratinocytes. The results, expressed mainly in surface and volume densities of epidermal components, showed that some important stereologic parameters of dark basal keratinocyte constituents exhibited values which could be interpreted as intermediate between those of the clear basal keratinocytes and those of the suprabasal keratinocytes. Although the degenerative nature of dark cells cannot be ruled out, the stereologic characterization of their cell architecture also could indicate that dark basal cells are already differentiating keratinocytes still remaining in the basal layer.     

Location of HLA-A,B,C antigens in dendritic cells of normal human skin: an immunoelectron microscopic study

The distribution of the major histocompatibility antigens HLA-A,B,C, (HLA) on dendritic cells of normal human skin was studied by immunoelectron microscopy and a 4-step immunoperoxidase technique utilizing monoclonal antibodies. Light microscopy revealed peripheral staining for HLA of epidermal and pilar infundibular keratinocytes. In the epidermis, the staining was present from the basal layer to the upper stratum spinosum. In the follicles below the level of the infundibulum, HLA was detected only on rare intraepithelial dendritic cells. These dendritic cells could not be identified in the epidermis due to the HLA staining of the surrounding keratinocytes. Similar cells stained diffusely with anti-T6 antibody; the keratinocytes did not. Immunoelectron microscopy demonstrated: (1) the presence of HLA staining of keratinocyte membranes from the stratum basalis to the level of the upper stratum spinosum and in the pilar infundibulum, (2) the possible absence of HLA on melanocytes, (3) the presence of focal HLA staining of the membranes of epidermal and follicular dendritic cells that contained Birbeck granules and were, therefore, Langerhans cells, (4) dendritic mononuclear cells within the follicular epithelium, which although devoid of Birbeck granules, exhibited similar reactivity with anti-HLA antibody. These findings suggest that HLA antigens are present on the membranes of Langerhans cells, but are not demonstrable on melanocytes in normal human skin.     

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.     

Human epidermal Langerhans cells express the ICAM-3 molecule. Immunohistochemical and immunoelectron microscopical demonstration

Summary Three ligands have been described for the leucocyte integrin LFA-1, namely intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAMs show differences in tissue distribution and inducibility. The recently described ICAM-3 is highly expressed on resting lymphocytes, monocytes and neutrophils. Here, we demonstrate that the whole human epidermal Langerhans cell (LC) population expresses this molecule. Immunohistochemical staining of skin sections with an anti-ICAM-3 monoclonal antibody displayed reactivity with dendritic epidermal cells regularly distributed along the epidermis. Highly-sensitive immunoelectron microscopy procedures, performed on freshly suspended epidermal cells both at transmission and scanning electron microscopic levels, enabled demonstration that the whole LC population expresses cell surface ICAM-3. In contrast, keratinocytes and melanocytes were consistently negative. The prominent expression of ICAM-3 by resident LC could imply a crucial part for this molecule in leucocyte-intercellular interactions in the skin.     

Specialized keratin expression pattern in human ridged skin as an adaptation to high physical stress

We have analysed the expression of keratins in the epidermis of normal human palm and sole skin (ridged skin) using immunohistochemistry and in situ hybridization. The epidermis of human ridged skin expresses a more complex pattern of keratins than thin skin, which is probably due to the greater stress that ridged skin has to withstand. In addition to keratin K9, we document specific expression patterns of keratins K6, K16 and K17 which are suggestive of regional adaptations of this epidermis to a high cell turnover rate. In particular, the sequestered location of nests of K17-positive cells at the bottom of the deep primary epidermal ridges supports the notion of functional heterogeneity of basal cells and suggests that the K17-positive sites may include stem cells. Expression of K6 and K16 in some basal and most suprabasal keratinocytes is compatible with a constitutively high proliferative activity of normal ridged epidermis, but may also reflect different physical properties of the suprabasal cells, in contrast with regions expressing K9. The distinct labelling patterns observed in primary and secondary epidermal ridges as well as epidermal layers above dermal papillae suggest the existence of local microenvironmental niches leading to differences in keratinocyte differentiation.     

Characterization of sunburn cells after exposure to ultraviolet light

Sunburn cells (SBCs) appear in the epidermis shortly after acute UV damage, especially after exposure to UVB light. As yet, the mode of their formation remains to be satisfactorily elucidated. In order to characterize these cells, the expression of various markers of epidermal differentiation following UV exposure was investigated using immunhistochemical procedures. These were applied to paraffin-embedded (microwave technique) and frozen specimens of human skin 24 h after irradiation with 4 times the minimal erythema doses(MED). Normal nonirradiated skin without irradiation served as the control. We used a battery of antibodies directed against the following: cytokeratins (CKs) 5, 10, 17, and 19, actin, cell-adhesion proteins (desmoplakins, desmogleins), markers of terminal epidermal differentiation (filaggrin, involucrin and loricrin), markers of proliferation (PCNA, MIB, K6,16), a marker of endocytosis (clathrin) and markers of cell growth, (transforming growth factor [TGF-α]) and B-cell leukemia/lymphoma-2 [bcl-2]. After UV irradiation it was found that CK 5, which is typically confined to basal keratinocytes, was also expressed in suprabasal keratinocytes. The CKs 1 and 10/11 exhibit a normal suprabasal localization, but suprisingly, SBCs were negative for these CKs. Although CK 6,16, and 17 are not usually found in normal epidermis, UVB exposure induced their expression in suprabasal keratinocytes, but again failed to elicit their expression in SBCs. Antibodies specific for markers of late epidermal differentiation (filaggrin, involucrin and loricrin), cell-junction proteins (desmogleins, desmoplakins), proliferation (PCNA and MIB), and endocytosis (clathrin) also failed to produce positive staining of SBCs. Even though TGF-α immunoreactivity became detectable in most keratinocytes after UV exposure, this was not the case for SBCs. The number of basally located dendritic cells, most probably melanocytes, exhibiting bcl-2 staining was markedly reduced 6 and 12 h after irradiation as compared with normal skin. SBCs do not express any late differentiation markers, but they do contain proteins typical of basal keratinocytes (CK 5). It can be concluded that SBCs do not develop beyond a more basal-like differentiation pattern, probably as a result of cell death and migration through the epidermis.     

Expression of Bmi-1 in epidermis enhances cell survival by altering cell cycle regulatory protein expression and inhibiting apoptosis

The polycomb group (PcG) genes are epigenetic suppressors of gene expression that play an important role in development. In this study, we examine the role of Bmi-1 (B-cell-specific Moloney murine leukemia virus integration site 1) as a regulator of human epidermal keratinocyte survival. We identify Bmi-1 mRNA and protein expression in epidermis and in cultured human keratinocytes. Bmi-1 is located in the nucleus in cultured keratinocytes, and in epidermis it is expressed in the basal and suprabasal layers. Adenovirus-delivered Bmi-1 promotes keratinocyte survival and protects keratinocytes from stress agent-mediated cell death. This is associated with increased levels of cyclin D1 and selected cyclin-dependent kinases, and reduced caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage. Bmi-1 may be involved in the maintenance of disease state, as Bmi-1 levels are elevated in transformed keratinocytes, skin tumors, and psoriasis. The presence of Bmi-1 in suprabasal non-proliferative cells of the epidermis and within a high percentage of cells within skin tumors suggests a non-stem cell pro-survival role for Bmi-1 in this tissue. Based on the suprabasal distribution of Bmi-1 in epidermis, we propose that Bmi-1 may promote maintenance of suprabasal keratinocyte survival to prevent premature death during differentiation. Such a function would help assure proper formation of the stratified epidermis.     

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.     

Transgenic mice over-expressing a serine protease in the skin: evidence of interferon gamma-independent MHC II expression by epidermal keratinocytes

Stratum corneum chymotryptic enzyme (SCCE; also known as kallikrein 7) is a serine protease that is preferentially expressed in cornifying epithelia and possibly involved in the desquamation process. We have recently described transgenic mice over-expressing human SCCE in the epidermis showing increased epidermal thickness, hyperkeratosis, and an apparent dermal inflammation with pruritus. This suggests that SCCE may be involved in the pathophysiology of inflammatory skin diseases. We therefore carried out a further characterization of the skin changes observed in scce-transgenic mice. An increase in number of dermal cells was verified by stereological measurements showing a more than twofold increase of the volume fraction of dermis occupied by cell nuclei. In some, but not all, animals the number of dermal mast cells was increased. The dermal cell infiltrate was shown to consist mainly of macrophages and granulocytes. The number of epidermal and dermal T-lymphocytes was not increased. Dermal changes were found in transgenic animals before the age they became pruritic. No increase in interferon-gamma expression could be detected in the skin of transgenic animals. In spite of this, keratinocytes of adult transgenic mice were found to express MHC II antigen. We suggest that increased expression and/or activity of epidermal SCCE may lead to skin changes that contribute to development and maintenance of inflammatory skin diseases.     

Antigen-presenting cells in essential fatty acid-deficient murine epidermis: keratinocytes bearing class II (Ia) antigens may potentiate the accessory cell function of Langerhans cells.

Essential fatty acid deficiency (EFAD) is a useful model for studying the role of (n-6) fatty acid metabolism in normal physiology. Because cutaneous manifestations are among the earliest signs of EFAD and because abnormalities in the distribution and function of tissue macrophages have been documented in EFAD rodents, we studied the distribution and function of Class II MHC (Ia) antigen-bearing cells in EFAD C57B1/6 mouse epidermis. Immunofluorescence studies revealed 1.9-9.6 (mean +/- SEM = 5.2 +/- 2.6) times more class II MHC (Ia) antigen-bearing epidermal cells in suspensions prepared from EFAD as compared to normal skin. Analysis of epidermal sheets demonstrated similar numbers of dendritic Ia+ and NLDC145+ cells in EFAD and normal epidermis, however. This discrepancy occurred because some keratinocytes in EFAD epidermal sheets expressed class II MHC (Ia) antigens, whereas keratinocytes in normal mouse epidermis did not. Two-color flow cytometry confirmed that all Ia+ cells in normal epidermis are Langerhans (Ia+ NLDC145+) cells, whereas Ia+ cells in EFAD epidermis are comprised of Langerhans cells and a subpopulation of keratinocytes (Ia+ NLDC145-). Similar levels of Ia antigens were expressed on EFAD and normal Langerhans cells. EFAD and normal epidermal cells were also compared in several in vitro assays of accessory cell function. Epidermal cells prepared from EFAD C57B1/6 mice present the protein antigen DNP-Ova to primed helper T cells more effectively than epidermal cells prepared from normal animals. EFAD epidermal cells are also more potent stimulators of T cells in primary and secondary allogeneic mixed lymphocyte-epidermal cell reactions than normal epidermal cells. The functional differences between EFAD and normal epidermal cells do not appear to result from increased cytokine release or decreased prostaglandin production by EFAD epidermal cells. In view of these findings and the observation that the antigen-presenting cell activity of EFAD epidermal cells correlates with the number of Ia+ keratinocytes in epidermal cell preparations, Ia+ keratinocytes (in the presence of Langerhans cells) may potentiate cutaneous immune responses in vitro and perhaps in vivo as well. These results also suggest that (n-6) fatty acids or metabolites of (n-6) fatty acids are involved in regulating the expression of class II MHC (Ia) antigens by keratinocytes in vivo.     

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker. When freshly isolated mouse and human epidermal cells were incubated with LDL-gold complexes, only keratinocytes with the morphologic characteristics of basal cells showed binding and uptake of LDL-gold. No LDL receptor activity was found on Langerhans cells, melanocytes or highly differentiated keratinocytes. Since cell separation techniques can destroy receptors, the staphylococcal epidermolytic toxin was utilized to produce intercellular and intra-epithelial splitting of the epidermis. In preparations of both normal mouse and human epidermis, LDL-gold binding was restricted to basal cells and a few suprabasal keratinocytes. In contrast, in psoriatic epidermis, and to a lesser extent, essential fatty acid-deficient mouse epidermis, cells in the stratum spinosum showed abundant LDL-gold binding. Thus LDL-gold may be a useful marker for epidermal differentiation.     

UVB-Dependent Modulation of Epidermal Cytokine Network: Roles in UVB-Induced Depletion of Langerhans Cells and Dendritic Epidermal T Cells

The epidermis of mice consists of three cellular components, i.e., keratinocytes, Langerhans cells (LC), and dendritic epidermal T cells (DETC). Each epidermal subpopulation produces a different set of cytokines, thereby forming a unique cytokine milieu. These cytokines, in turn, support the survival and growth of LC and DETC and regulate their immunological functions. LC and DETC play important, but distinct, effector roles in protective immunity against antigens that are generated in or penetrate into the epidermis. Acute or chronic exposure of mice to ultraviolet B (UVB) radiation is known to impair this cutaneous immunity, as evidenced by the failure to induce T cell-mediated immune reactions, by the generation of antigen-specific immunological unresponsiveness, and by the development of skin cancers. Importantly, these changes are associated with reduced densities of LC and DETC in UVB-exposed skin, suggesting that the deficiency in these epidermal leukocytes may account for some of the deleterious influences of UVB radiation on skin. Here I will review the recent advance in our understanding of the mechanisms by which UVB radiation may deplete LC and DETC from epidermis. More specifically, I will discuss the following possibilities: a) UVB-mediated suppression of the production of relevant growth factors for LC and DETC, b) UVB-induced abrogation of surface expression of growth factor receptors, and c) UVB-triggered apoptotic cell death in epidermal leukocytes.     

银屑病患者皮损表皮生长因子及其受体的检测

用抗表皮生长因子（ＥＧＦ）多克隆抗体、抗表皮生长因子受体（ＥＧＦＲ）单克隆抗体及ＡＢＣ免 疫酶标技术，对２０例寻常型银屑病患者皮损及１０例正常人皮肤进行观察。结果表明：①正常人皮肤及 银屑病非皮损区皮肤ＥＧＦ及ＥＧＦＲ主要分布在表皮基底细胞层及基底层上部。②银屑病进行期皮损 ＥＧＦ及ＥＧＦＲ分布于表皮各层，表皮中、上层含量明显升高。③经有效治疗消退期皮损ＥＧＦ及ＥＧＦＲ 从角质层开始消退，分布趋于正常。提示ＥＧＦ及ＥＧＦＲ对银屑病皮损角肮细胞过度增殖及异常分化起 重要作用。     

Abnormal distribution of epidermal protein antigens in psoriatic epidermis.

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.     

Fos and jun proteins are specifically expressed during differentiation of human keratinocytes

Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.     

Altered keratin expression in benign and malignant skin diseases revealed with monoclonal antibodies

We studied keratin expression in benign epidermal skin diseases, and in Bowen's disease by using three monoclonal cytokeratin antibodies. In adult normal skin, these antibodies bind only to the follicular epithelium (PKK1), the basal keratinocytes (PKK2), or the suprabasal cells in interfollicular epidermis (KA5). Additionally, in fetal epidermis, the PKK1 antibody reacts with basal keratinocytes. In psoriasis and lichen planus, the PKK2 antibody distinctly revealed all epidermal cell layers by immunostaining. However, a negative basal cell-like layer was revealed in both lesions with the KA5 antibody. In pityriasis rubra pilaris, the basal cell layer was uniformly stained with the PKK2 antibody, but only some keratinocytes in upper cell layers showed fluorescence and, in chronic eczema, the 3-4 lowest epidermal cell layers were reactive. The PKK1 antibody did not stain interfollicular keratinocytes in any of the benign proliferative skin diseases studied. In Bowen's disease, a heterogeneous staining pattern with varying intensity among individual cells was seen with all of the antibodies used. Our results suggest different changes in keratin expression in chronic benign and malignant epidermal diseases that may reflect the mechanisms behind these changes.     

Fluorescence microscopic and flow cytometric analysis of bone marrow-derived cells in human epidermis: a search for the human analogue of the murine dendritic Thy-1+ epidermal cell

Lymphoid cells with an affinity for the epidermis (epidermotropic lymphocytes) have been proposed to play a role in the immune functions of the epidermis. However, antigen-presenting Langerhans cells (LC) and indeterminate cells are presently the only cells in the human epidermis which have been demonstrated to originate in the bone marrow. Recent studies of murine epidermis have identified a population of bone marrow-derived cells which express Thy-1 antigen and which are present in a similar density to, but distinct from, LC. We therefore sought to identify the potential human analogue of the murine Thy-1+ epidermal cell utilizing a battery of antileukocyte reagents in immunohistochemical, flow cytometric, and cell sorting studies. A panel of antibodies failed to detect significant numbers of human Thy-1 antigen-bearing cells, T cells, B cells, monocytes/macrophages (other than LC), and natural killer cells in tissue sections, epidermal sheets, and epidermal cell (EC) suspensions. This was the case using EC suspensions either unfractionated or fractionated on Ficoll-Hypaque to enrich for leukocyte subpopulations. Since the nature of the murine Thy-1+ EC is uncertain, it is possible that antibodies directed against well-defined leukocyte subpopulations may not be of value in the detection of a potential human analogue. We therefore utilized double fluorescence staining with anti-HLe-1, an antibody which identifies all human leukocytes, and anti-HLA-Dr (Dr), which identifies epidermal LC, in order to demonstrate a potential population of HLe-1+ Dr- non-LC, bone marrow-derived cells. The vast majority of HLe-1+ cells were HLA-Dr+ LC; these were present at a density of 608 cells/mm2 in epidermal sheets. A minor population of HLe-1+ cells which did not express HLA-Dr (HLe-1+ Dr-) was observed in tissue sections, epidermal sheets, and EC suspensions. The nondendritic morphology and low density of these HLe-1+ Dr- EC in epidermal sheets (mean density of 4.2 +/- 1.6 cells/mm2) precluded their representing a strict human analogue of the murine Thy-1+ EC, since murine Thy-1+ EC are dendritic and are present in a density similar to that of LC. Purified preparations of the minor HLe-1+ Dr- EC population obtained by electronic cell sorting or panning and examined ultrastructurally were not enriched for any bone marrow-derived cell population. Thus, using currently available markers and sorting technology, we have been unable to identify a human analogue of the murine dendritic Thy-1+ epidermal cell.