Ontogeny of Ia-positive and Thy-1-positive leukocytes of murine epidermis

Murine epidermis harbors 2 populations of dendritic leukocytes: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy-1 +DEC). In the adult mouse these cell types are morphologically distinct and display highly characteristic phenotypes. LC bear Ia-antigens and a group of markers typical for mononuclear phagocytes: Fc- and C3bi-receptors, macrophage-specific antigen F4/80, and membrane ATPase. Thy-1 +DEC, in contrast, lack these markers but express high levels of Thy-1 and asialo-GM1 (asGM1) antigen. Since LC and Thy-1 +DEC share a common origin from the bone marrow we expected to gain insight into their relationship by studying their ontogenetic development. Epidermal sheets from fetal and newborn C3H/He and C57B1/6 mice obtained at defined ages from day 17 of gestation up to day 30 of postnatal life were monitored for the emergence of the above-mentioned markers for LC and Thy-1 +DEC. In double-labeling experiments LC markers were first detected by visualizing the monoclonal antibodies by a sensitive triple-layer rhodamine-immunofluorescence technique; in a second step, after appropriate blocking procedures, Thy-1 and asGM1 antigens were demonstrated by direct and indirect immunofluorescence. We found that in fetal epidermis, only few cells expressed either Thy-1 or Ia (4 and 1 cells/mm2, respectively, on day 18 of gestation). The bulk of Thy-1 +DEC and Ia +EC appeared only after birth. Adult proportions of Thy-1 +DEC and Ia +EC were reached at around 1 month of postnatal life. In contrast, all the other LC markers were expressed on a substantial number of fetal dendritic cells (280 cells/mm2 on day 18 of gestation), indicating the presence of phenotypically immature Ia-negative LC in fetal epidermis. By day 4 of postnatal life all F4/80 +EC and ATPase +EC (i.e., LC) had acquired Ia-antigens. Surprisingly, LC also bore asGM1 antigens, which in the adult epidermis are strictly confined to Thy-1 +DEC, up to day 5 of postnatal life. Thus, LC in fetal and early newborn epidermis are not yet fully differentiated. As they differentiate, they acquire Ia antigens and lose asGM1 antigens. In contrast, a phenotypically immature Thy-1 +DEC population could not be traced with the markers used. Thy-1 +DEC appear to be characterized by a stable phenotype (Thy-1+/asGM1+) throughout their lifetime.     

Regional development of Langerhans cells and formation of Birbeck granules in human embryonic and fetal skin.

The regional development of Langerhans cells (LC) and the formation of Birbeck granules (BG) were examined in human embryonic and fetal skin. Samples were obtained from multiple anatomic sites and stained with anti-CD36, anti-CD1a, and anti-HLA-DR antibody as well as Lag antibody specifically reactive to BG and some vacuoles of human LC. In the first trimester, CD36+ dendritic epidermal cells were identified before the appearance of CD1a+ cells and Lag+ cells. Some of the former co-expressed HLA-DR antigens but not CD1a antigens. In the second trimester, regional variations in LC development were observed. Epidermal LC of palms and soles reached a peak in number in the first trimester but were rarely detected after 18 weeks estimated gestation age (EGA), whereas, in other regions, their number increased with age. In the second trimester, CD1a+ cells and Lag+ cells were also identified in the epidermis, although Lag+ cells appeared later than CD1a+ cells. The Lag+ cells until 17 weeks EGA showed a variety of staining intensities and immunoelectron microscopy revealed that they contained various amounts of Lag-reactive BG. Flow cytometric analysis showed that relative amounts of Lag antigens in LC increased during the second trimester and that fetal LC of 18 weeks EGA expressed the same amounts of HLA-DR, CD1a, and Lag antigens as did adult human LC. In the dermis, in the second trimester, numerous CD36+ cells and HLA-DR+ cells were found, whereas CD1a+ cells and Lag+ cells were rarely detected. Taken together, it is suggested that HLA-DR+ dendritic cells acquire CD1a+ antigens first and then form BG after migration to the epidermis and that fetal LC are phenotypically mature in the second trimester.     

Differential effects of benzo[a]pyrene and dimethylbenz[a]-anthracene on Langerhans cell distribution and contact sensitization in murine epidermis

The exposure of murine skin to potent chemical carcinogens induced distinctive effects on the distribution of epidermal Langerhans cells (LC). Our previous finding that weekly applications of 7,12-dimethylbenz[a]anthracene deplete the numbers of adenosine triphosphatase (ATPase)-positive LC was extended to show that LC are also depleted on Ia and beta-glucuronidase staining. In contrast, application of the tobacco-derived carcinogen, benzo[a]pyrene (BP), caused a significant increase in Ia-positive LC density within 2 weeks and elevated levels were maintained for up to 6 months with continuous treatment. The tobacco-derived cocarcinogenic agent, catechol, also enhanced the numbers of epidermal LC. The LC in carcinogen treated epidermis were morphologically abnormal; after BP and catechol treatment LC appeared smaller with shorter dendrites, whereas in DMBA treated epidermis LC were enlarged with elongated dendrites. Application of the contact sensitizing agent, dinitrofluorobenzene, to skin treated with BP induced hyporesponsiveness rather than contact sensitivity upon subsequent antigen challenge. Hence, the function of the large number of morphologically altered LC in BP treated skin was impaired. We conclude that carcinogen-induced alterations of LC are associated with impaired immunocompetence, although different carcinogens probably operate via different mechanisms to induce such phenomena.     

Differentiation markers in fetal epidermis: transglutaminase and transpeptidase.

Two members of the transpeptidase family of enzymes, transglutaminase and gamma glutamyl transpeptidase, were assayed histochemically and biochemically in developing rat epidermis from day 15 of gestation through postnatal day 5. Electron microscopic examination of serial skin biopsies enabled precise dating of fetal epidermis and periderm and correlation of ultrastructural details of the cells with marker enzyme activities. Transglutaminase activity appeared histochemically in surface epidermis and in hair follicle inner root sheath on day 18 and day 21 of gestation, respectively, concomitant with the onset of terminal keratinization in these tissues. Enzyme activity was biochemically detectable 2 days before the histochemical stain became positive. Transpeptidase was active in fetal epidermis prior to keratinization but was only detectable in basal cells thereafter. Subsequent to birth, enzyme activity rose geometrically in hair follicles undergoing initial differentiation, and was thereafter found in all anagen hairs. Transglutaminase is active only in cells approaching terminal keratinization, while transpeptidase is associated with early phases of epidermal proliferation and differentiation.     

Lymphocyte subsets and Langerhans cells/indeterminate cells in erythema multiforme

A peroxidase-antiperoxidase study using monoclonal antibodies directed against T and B lymphocytes and Langerhans cells/indeterminate cells (LC/IC) was undertaken in order to understand more clearly the changes observed in erythema multiforme. At the various stages of development, from normal skin to target lesions, the quantity of inflammatory cells differed, but in each case the number of T8+ (cytotoxic/suppressor) cells was greater than the number of T4+ (helper/inducer) cells in the epidermis, whereas the latter exceeded the former in the dermis. Concomitant with the initial epidermis changes, there was an increase in the number of T6+ (LC/IC) cells in the upper and lower epidermis. With slight to moderate basal unit destruction, the number of LC/IC in the upper epidermis exceeded those in the lower epidermis. With severe basal unit destruction, there was a loss of LC/IC in the lower epidermis as detected by T6 reactivity. In fully formed blisters, the LC/IC in the upper half of the epidermis were decreased in parallel with the degree of epidermal necrosis. The character of the lymphocytic inflammatory infiltrate and redistribution in LC/IC are similar to those findings described in allergic contact dermatitis. The clinical, histologic, and immunopathologic changes in erythema multiforme appear to be due in part to cellular immune mechanisms with the lymphocyte as the predominant effector cell, and our data suggest a possible role for LC/IC in this disorder.     

Characterization of angiotensin-converting enzyme expression during epidermis morphogenesis in humans: a potential marker for epidermal stem cells

Background  Recent evidence has revealed that angiotensin-converting enzyme (ACE) participates in cutaneous wound healing and contributes to the pathophysiological process of some skin diseases. However, little is known about the role of ACE in epidermis morphogenesis during development.
Objective  To clarify the expression pattern of ACE during embryonic development of human skin.
Methods  Skin samples were obtained from aborted fetuses at different gestational ages and from healthy individuals. Localization of ACE, together with β₁-integrin, keratin 19 (K19) and p63 was examined by immunofluorescence and immunohistochemical staining.
Results  In human fetal skin, at 11–13 weeks of gestation, ACE-positive cells were observed in the primitive epidermis. As the fetuses developed, ACE-positive cells appeared in all the epidermal layers. From 21 weeks of gestation, ACE expression was largely restricted to the basal layer of the fetal epidermis. In contrast, ACE-positive cells were found only in the adult skin basal layer which harbours epidermal stem cells. To explore the possible link between ACE and epidermal stem cells, we further examined the expression of β₁-integrin, K19 and p63, the putative markers for epidermal stem cells. Consistent with the results of ACE expression, from 21 weeks of gestation, the expression of β₁-integrin, K19 and p63 was mainly confined to the basal layer. Immunofluorescent double labelling revealed that ACE-positive cells substantially overlapped with β₁-integrin-, K19- and p63-positive cells.
Conclusions  Our results suggest that ACE may play a role in human epidermis morphogenesis during fetal life and serve as an unrecognized marker for keratinocyte progenitor cells.     

Sequential observation of morphogenesis of cutaneous nerve fibers in fetal rat skin: an immunohistochemical and electron microscopic study

Skin of DA/Slc rat fetuses removed on the fetal days 11 to 20 and on delivery day (21 days post-fertilization) was used. In the immunohistochemistry, the anti-neurofilament 69 kDa (NF68)-positive fibers appeared in the subepidermal dermis on the fetal day 12. The subepidermal and intraepidermal NF68-positive fibers reached to a maximum number on the fetal day 14. They disappeared completely on the fetal day 16. After the fetal day 19, a small number of the subepidermal and intraepidermal NF68-positive fibers were seen again. As for anti-nerve growth factor (NGF), intermediate cells, spinous cells and granular cells of the epidermis showed strongly positive stain. In the electron microscopy, there were bundles of immature axons which were packed by abundant microtubules and neurofilaments during the entire fetal period. On the fetal days 16 and 17, a considerable number of axons showed severe degenerative changes. From these findings, the subepidermal and intraepidermal nerve fibers in the fetal rat skin increased temporarily to a maximum number, and soon after they disappeared completely, followed by another period of re-appearance until delivery day. Such a dynamic change of the number of the cutaneous nerve fibers was not synchronous with expression of NGF of the epidermal cells.     

Suppressor cell activation and enhanced skin allograft survival after tumor promotor but not initiator induced depletion of cutaneous Langerhans cells

During chemical carcinogenesis Langerhans cells (LC) are depleted from the epidermis, disrupting the normal immunological functions of the skin. Tumor promotors but not initiators, have been shown to deplete adenosine triphosphatase (ATPase)-positive LC from the skin and therefore the cutaneous immune system may be impaired during tumor promotion but not initiation. The present study shows that the tumor promotor 12-O-tetradecanoylphorbol 13-acetate (TPA) but not the initiator urethane depletes Ia-positive LC from BALB/c murine ear epidermis, and beta-glucuronidase-positive LC from C57BL mouse tail skin. Sensitization with 2,4-dinitrofluorobenzene (DNFB) through urethane-treated skin resulted in a normal contact sensitivity response when the mice were challenged 5 days later. In contrast, tolerance resulted from sensitization through TPA-treated skin as a result of the generation of suppressor cells. In addition, TPA but not urethane-treated C57BL mouse tail skin survived for an extended time when grafted onto histoincompatible BALB/c mice. Therefore, impairment of the normal immunological functions of skin resulted from treatment with the tumor promotor TPA but not the tumor initiator urethane, which suggests that a loss of LC during tumor promotion may impair immunological protection against skin tumors.     

An ultrastructural study of a sclerosing epithelial hamartoma

The histological and ultrastructural features of a sclerosing epithelial hamartoma are described. By electron microscopy, epithelial cords formed by basal-like cells and epithelial cysts with a pattern of keratinization similar to that of normal epidermis or of follicular infundibula were seen. By conventional light microscopy, continuities were found between the epithelial cords and the overlying epidermis. The stroma was found to be fibrous and contained some fibroblasts or possibly myofibroblasts with bundles of microfilaments. The data are interpreted with respect to the origin of the neoplasm, its line of differentiation, and its sclerosing behavior.     

Clear cells of Toker in the developing anogenital region of male and female fetuses

The clear cells of Toker are a mysterious population of intra-epidermal glandular cells. They were originally described in nipples, but were recently observed in the vulva as well. It was hypothesized that intra-epidermal embryonic remnants or underlying glands were a potential source. The embryological aspects were investigated by studying specimens of the anogenital region of 18 male and 15 female fetuses between 12 and 39 weeks gestation. The search for Toker cells was enhanced by cytokeratin (CK) 7 immunohistochemistry. The investigation showed that Toker cell elements are a normal, though highly variable constituent of the developing anogenital region. The study revealed the following: (1) single intra-epidermal glandular vesicles near follicular anlages in interlabial sulcuses of female fetuses of 15 and 16.5 weeks gestation; (2) CK7+ solitary cells, clusters, and vesicles which were related to developing intra-epidermal follicular canal tracks and tended to disperse inside the epidermis in fetuses of approximately 18 weeks gestation; (3) dispersed CK7+ cells in fetuses of 19-23 weeks gestation; (4) characteristic CK7+ Toker cell proliferations in fetuses more than 23 weeks gestation. These observations indicate that in the anogenital region, primordial follicular cells programmed to participate in the formation of apocrine and mammary-like glands, become displaced into the epidermis where they disperse, and proliferate into Toker cell populations. However, the proximity of Toker cells to CK7+ cells in excretory ducts of late fetal apocrine and mammary-like glands suggested a possible additional source. Consequences for Toker cells of the breast and primary Paget disease are discussed.     

Study of the skin of a new hairless rat mutant

Histological and immunological examinations were performed on the skin of a newly established strain of hairless rat (Hirosaki hairless rat; HHR). The appearance of the mutant rat skin was marked by resembles to human skin with wrinkles and scarce vellus hair. The presence of scattered dilated follicular cysts in the mid-dermis was the most predominant microscopic change. Electron microscopically, some of the basal cells contained many oval organelles in the cytoplasm, although their role was not clear. Langerhans cells were observed, but melanocytes were lacking in the epidermis. Immunofluorescence technique demonstrated Ia-positive cells (presumably Langerhans cells) in epidermal sheets from the soles of the mutants, which also developed contact sensitivity to dinitrochlorobenzene (DNCB). HHRs may be a useful experimental model for studies of allergic as well as nonallergic dermatitis requiring gross observation and wrinkled skin.     

Effects of ultraviolet irradiation on surface marker expression by epidermal immunocompetent cells and contact sensitization to dinitrofluorobenzene in mice

We studied the effects of ultraviolet (UV) irradiation on murine epidermal Ia-positive Langerhans cells (Ia + LC) and Thy-I-positive dendritic epidermal cells (Thy-I + dEC). We also studied contact hypersensitivity to dinitrofluorobenzene (DNFB) introduced through UV-treated epidermis. C3H/HeN mice were exposed to UVB or 8-methoxypsoralen plus UVA (PUVA). UVB and PUVA treatment led to a dramatic reduction in surface marker expression of both Ia + LC and Thy-I + dEC. High-dose UVB irradiation (360 J/m2) interfered with contact hypersensitivity to DNFB; the density of Ia + LC may thus be related to the sensitizing potential. In contrast, low-dose UVB (120 J/m2) and PUVA treatment had little effect on contact hypersensitivity despite a marked reduction in Ia + LC. The density of Thy-I + dEC appeared not to be associated with contact hypersensitivity. These results suggest that there may be a Langerhans cell density-independent mechanism for the induction of contact hypersensitivity.     

Occurrence of IgE-bearing epidermal Langerhans cells in atopic eczema: a study of the time course of the lesions and with regard to the IgE serum level

Uninvolved and lesional skin of untreated and treated patients with atopic eczema has been investigated immunohistochemically to determine the conditions in which IgE-bearing CD1a+ Langerhans cells/indeterminate cells (LC/IC) occur in this disease. IgE-bearing epidermal dendritic cells were present in patients with elevated IgE serum level (greater than 300 UI/ml) and the staining pattern was stronger in lesional skin. On double immunostaining, a subpopulation of CD1a+ LC/IC was found not to bear IgE molecules as determined by the ratio IgE+/CD1a+ cells on serial sections as well. The ratio IgE+/CD1a+ cells decreased in patients who underwent a local therapy with glucocorticosteroids. These results suggest that the expression of IgE receptors and/or binding of IgE molecules on epidermal LC/IC in atopic eczema may be controlled by a complex network of mediators from the epidermis or the inflammatory infiltrate, or both, and that this phenomenon could be down regulated by glucocorticosteroids.     

Expression of the basal cell adhesion molecule (B-CAM) in normal and diseased human skin

The basal cell adhesion molecule (B-CAM) is a 90-kD cell surface glycoprotein with a characteristic immunoglobulin domain structure. The pattern of B-CAM expression in cultured cells suggests that the molecule is associated with a substrate-adherent growth pattern in some lineages. We investigated the expression of B-CAM in normal and diseased human epidermis by means of immunohistochemistry employing a single batch of high-titer mouse monoclonal antibody G253. Snap-frozen biopsy material from normal skin (n = 8), psoriasis (n = 5), contact dermatitis (n = 6), basal cell carcinoma (n = 5) and fetal skin (n = 6) was studied. In normal human skin, B-CAM was found in varying degrees throughout the epidermis with a preference for suprabasal expression, hair follicles were regularly of a B-CAM-positive phenotype. There were no qualitative differences with regard to the B-CAM expression pattern in normal skin in comparison to psoriasis and contact dermatitis. In contrast, fetal skin (15th to 18th week of gestation) was characterized by B-CAM-positive cells in the basal layer of the epidermis as well as in the outer root sheath of hair follicles. Basal cell carcinomas also regularly expressed high levels of B-CAM. A strong B-CAM-positive phenotype can be found in the outer root sheath of hair follicles of adult and fetal human skin as well as in fetal basal keratinocytes.     

Dynamic changes in epidermal Ia-positive cells in allergic contact sensitivity reactions in mice

In mice sensitized with trinitrochlorobenzene, serial changes in epidermal Ia-positive cells were studied at various times after challenge. Until 3 days post-challenge, the Ia-positive cells consisted only of dendritic Langerhans cells; their number was decreased but they were significantly enlarged, with extending dendrites. Some Langerhans cells were also found surrounding a hair follicle, extending their dendrites toward the follicle like the spokes of a wheel. From 3 to 9 days after challenge, keratinocytes also began to express Ia antigens in the epidermis in addition to Langerhans cells, whose size diminished. This suggests that there are two phases in the response of the epidermal Ia antigens in contact sensitivity reactions, i.e. an early phase in which enlarged Langerhans cells are the only Ia-positive cells in the epidermis, and a late phase in which keratinocytes take over as the major Ia-positive cells, while Langerhans cells resume their original size. Ia antigen expression on keratinocytes in this late phase probably plays a crucial role in completely eliminating allergens deposited on the keratinocytes.     

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.     

Accessory and adhesion molecules expressed on murine epidermal Langerhans cells and the modulation by cytokines.

Langerhans cells (LC) are MHC class II (Ia)-positive dendritic cells that act as an antigen-presenting cells for T cell-dependent immune responses. LC originate from cells in bone marrow and migrate into the epidermis through blood vessels. LC also migrate from the epidermis to regional lymph nodes after antigen stimulation where they present antigens to T cells. These are the essential features of LC. The morphological and functional properties of LC are modulated by external stimuli or various cytokines. In this review we focus on the accessory and adhesion molecules on LC and describe how these molecules are modulated by cytokines. We also describe the molecules participating in the migration of LC into and from the epidermis. Moreover, we introduce our data obtained from purified murine epidermal LC and from the transgenic mice overexpressing several cytokines in the epidermis.     

Expression of cyclooxygenase isozymes during morphogenesis and cycling of pelage hair follicles in mouse skin: precocious onset of the first catagen phase and alopecia upon cyclooxygenase-2 overexpression

Cyclooxygenase (COX)-1 and -2 catalyze the key reaction in prostaglandin biosynthesis. Whereas COX-1 is found in most tissues, COX-2, with a few exceptions, is not expressed in normal tissues but becomes transiently induced in the course of inflammatory reactions. In many neoplastic epithelia, COX-2 is constitutively overexpressed. Here we show that COX isozymes are spatiotemporally expressed during morphogenesis of dorsal skin epithelium of NMRI mice. COX-1 and COX-2 mRNA and protein were detected in embryonic and postnatal epidermal tissue by RT-PCR, northern blot, and immunoblot analysis indicating that both isoforms may contribute to prostaglandin production. Being barely detectable in interfollicular epidermis and resting hair follicles of adult mice, COX-2 protein appeared in embryonic skin first in epidermal precursor cells and later on in the basal cells and the peridermal layer of the stratified epidermis. In the course of pelage hair follicle morphogenesis, COX-2 remained expressed in the basal interfollicular compartment and, in addition, became apparent in elongated hair germs and hair pegs and later on in the outer root sheath cells of the distal and proximal hair follicles as well as in basal sebaceous gland cells. During the subsequent synchronous phases of hair cycling, COX-2 expression declined in catagen, was barely detectable in telogen, and was reinduced in the basal outer root sheath and basal sebaceous gland cells of anagen hair follicles. COX-1 immunosignals were detected predominantly in the interfollicular spinous and granular layers of the developing, neonatal, and adult epidermis but not in follicular epithelial cells of developing or cycling hair follicles. Dendritic cells in the interfollicular epidermis and distal hair follicles were also COX-1-positive. Transgenic overexpression of COX-2 under the control of a keratin 5 promoter in basal cells of the interfollicular and follicular epidermis induced a precocious entry into the first catagen stage of postnatal hair follicle cycling and a subsequent disturbance of hair follicle phasing. Furthermore, transgenic mice developed an alopecia. Inhibition of transgenic COX-2 activity by feeding the specific COX-2 inhibitor valdecoxib suppressed the development of alopecia, indicating that COX-2-mediated prostaglandin synthesis is involved in hair follicle biology.     

Development of the hair in the rat: in vivo and in transplanted tissue

Differentiation of the hair was examined in the head skin of rat embryos aged from days 12.5 to 18.5 of gestation. In 12.5-day-old embryos, the skin consisted of single layered epidermis and underlying loose connective tissue. On day 17.5 of gestation, the epidermis appeared to have a two or three layered epithelium, and the subepidermal connective tissue gave rise to an overlying dense tissue, into which blood capillaries arose from the deeper layer. The epidermis showed partial thickenings, where basal cells were arranged perpendicular to the surface of the epidermis. Beneath the epidermal thickenings, a number of connective tissue cells appeared to have accumulated and occasionally were accompanied by blood capillaries. In sequential development of the skin, the thickenings protruded as cords to the underlying dermal tissue and formed hair follicles covered with a connective tissue sheath. Skin obtained from 12.5-day-old embryos was transplanted singly or with cerebral cortex into the third ventricle of adult male rats. Thirty days after the transplantation, hairs and sebaceous glands had developed from the grafted skin. Hair growth tended to be more prominent in the cotransplanted skin grafts. However, no nerve fibers immunoreactive for substance P or calcitonin gene-related peptide (CGRP) were found in the grafted skin, although these fibers appeared in the dermis at the end of gestation in vivo. These results showed that the skin of 12.5-day-old embryos was able to induce hair growth in the third ventricle without accompanying peripheral neuronal fibers or blood capillaries, although the effects of connective tissue cells could not be ruled out.