A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.     

Autoimmune vitiligo: detection of antibodies to melanin-producing cells.

Vitiligo, a disorder characterized by the destruction of melanocytes, is often associated with diseases in which there are increased frequencies of autoantibodies. For this reason we investigated two patients with vitiligo, alopecia universalis, mucocutaneous candidiasis, and multiple endocrine insufficiencies for antibodies to melanin-producing cells. Using direct immunofluorescence of normal and vitiliginous skin from both patients and indirect immunofluorescence with both patients' serum, we could not detect these antibodies. However, an immunofluorescent complement-fixation test demonstrated a circulating antibody that bound to melanocytes in human skin, nevus cells and melanoma cells. Specificity of cellular fluorescence for nevus and melanoma cells was shown on serial sections stained with hematoxylin and eosin and was inferred for melanocytes from their distribution in human skin and their presence when the normal but not vitiliginous skin of both patients was used as substrate. This antibody was characterized as an IgG that activated complement via the classical pathway.     

Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in situ and in cell culture.

The Bcl-2 proto-oncogene regulates cell survival by antagonizing events that lead to apoptotic cell death and has been reported to be expressed in situ in lymphoid tissues, glandular epithelium, neurons, and basal epidermal cells. When we performed immunostaining on cryostat sections of normal skin, anti-Bcl-2 reactivity was confined to scattered dendritic cells in the basal epidermal layer. Double-staining experiments showed that the Bcl-2+ cells were positive for vimentin but negative for cytokeratins, CD1a, and CD45 antigens, excluding keratinocytes and Langerhans cells as possible candidates for constitutive Bcl-2 expression. Bcl-2+ epidermal cells also reacted with the monoclonal anti-melanocyte antibody NKI/beteb, and were absent from lesional skin in vitiligo, confirming that they represented epidermal melanocytes. Western blot analysis of cultured melanocytes and melanoma cell lines revealed a 26-kd protein specifically reacting with the anti-Bcl-2 monoclonal antibody. Immunostaining of pigmented lesions revealed strong expression of Bcl-2 by five of five nevocellular nevi and seven of seven melanomas. Our observations demonstrate that, within normal human epidermis, melanocytes are the only cells that express Bcl-2 constitutively and that Bcl-2 is expressed in benign and malignant pigmented tumors of the skin in situ.     

Do Langerhans cells play a role in vulvar epithelium resistance to squamous cell carcinoma?

Introduction: Langerhans cells (LCs) are a very important part of the skin immune system. Materials and Methods: Skin biopsies taken from 13 women after the removal of vulvar squamous cell carcinoma (SCC) who had not been treated earlier for any vulvar diseases were investigated. The control group consisted of 12 women who underwent a plastic surgical operation of the vulva region. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues samples using antihuman CD1a antibody (NCL-CD1a-235, Novocastra). Results: This study showed a large decrease in LCs in vulvar SCC. Conclusions: It is postulated that the reduction in the number of LCs may be one of the reasons for a higher tendency of carcinogenesis in the vulvar region. Their role as a main element of the skin immune system in the initiation of this process needs further investigation. It is possible that research on LCs in the skin will cast a new light on their role and even contribute to the prophylaxis and treatment of skin and mucosa carcinomas.     

The usefulness of tyrosinase in the immunohistochemical assessment of melanocytic lesions: a comparison of the novel T311 antibody (anti-tyrosinase) with S-100, HMB45, and A103 (anti-melan-A)

AIM: To compare the sensitivity and staining pattern of the new immunohistochemical antibody to tyrosinase (T311) with S-100, HMB45, and the recently evaluated antibody to melan-A (A103) in a range of melanocytic lesions. METHOD: Archival, formalin fixed, paraffin wax embedded sections from 50 benign and malignant melanocytic lesions were stained immunohistochemically with anti-tyrosinase, A103, S-100, and HMB45. They were scored semiquantitatively for the distribution and intensity of staining. RESULTS: All melanomas, with the exception of desmoplastic melanoma, showed some staining with all four antibodies. Overall, T311 and A103 showed an intermediate sensitivity compared with that of S-100 and HMB45. T311 stained most benign and malignant lesions strongly and diffusely with minimal background staining. Immunoreactivity was found to be patchy in some naevi, with weak or absent staining of the mature melanocytes. A103 showed strong and diffuse staining of all benign lesions and most melanomas with minimal background staining. S-100 was the most sensitive, with diffuse staining of most lesions, including desmoplastic and metastatic melanoma, but lacked specificity. HMB45 was the least sensitive antibody, frequently demonstrating patchy staining with absent staining in some benign naevi. CONCLUSIONS: S-100 remains the most sensitive marker of melanocytes. However, because of its lack of specificity, it should be used with at least one other more specific antibody. HMB45 is more specific, but lacks sensitivity; T311 is a reliable marker of melanocytes in paraffin wax embedded sections and is worth consideration for use in a staining panel, although it shows no additional benefit over A103.     

The appearance, density and distribution of melanocytes in human embryonic and fetal skin revealed by the anti-melanoma monoclonal antibody, HMB-45

Summary The presence, densities, and patterns of distribution of melanocytes in the epidermis of human embryos and fetuses, ranging in age from 40 d to 140 d estimated gestational age (EGA), were studied using the HMB-45 monoclonal antibody that recognizes an antigen in melanoma cells and fetal melanocytes. Immunostained sections of skin and epidermal sheets revealed dendritic melanocytes within the basal or intermediate layers of 50 d EGA and older skin. Melanocytes could not be identified by immunostaining or electron microscopy in younger (40–50 d EGA) epidermis or in cultured epidermal cells from these specimens. However, skin from a 45 d EGA embryo grown in organ culture for 11 d stained positively with HMB-45, suggesting that melanocytes are present at that age either in the epidermis or dermis of the explant. Double-labeling experiments using ATPase and HMB-45 confirmed the specificity of HMB-45 for melanocytes and demonstrated that melanocytes and Langerhans cells are nonoverlapping populations. Melanocytes were present in the embryonic epidermis in relatively high numbers (mean value of 1050 cells/mm²); they increased in density to 2300 cells/mm² during the late first trimester and early second trimester, then declined during later stages of development to a density of 800 cells/mm², within the range of values for the newborn child and young adult. Equivalent numbers of melanocytes were recognized by silver staining and with the HMB-45 antibody in an 87 d EGA test sample, indicating that HMB-45 reacted with the total melanocytic population. Melanocytes appeared to be distributed in epidermal sheets in a regular pattern. Statistical tests used to evaluate the randomness of a population revealed a tendency toward a non-random distribution in specimens younger than 80 d EGA, just prior to appendage formation and epidermal stratification into multiple layers, however there was variability in the degree of randomness for any given age. The results of this study have closed the gap in timing between the conclusion of neural crest formation and migration (around 6 weeks) and the appearance of melanocytes in the skin between 40–50 d EGA.Portions of this work were presented at the annual meeting of the Society for Investigative Dermatology, May, 1987 and at the 37th Annual Symposium on the Biology of Skin, October, 1987     

Novel predictive assay for contact allergens using human skin explant cultures.

Contact allergens sensitize the immune system by the binding to and subsequent activation of Langerhans cells (LCs), the antigen-presenting cells of the skin. At present, new chemicals are usually tested for their contact allergenicity in animal models. To develop an animal-replacing predictive in vivo assay for the identification of potential contact allergens, we compared the effects of epicutaneous application of six known contact allergens, five known irritants and two dermatologically inactive chemicals on LCs in skin biopsy cultures from seven healthy donors. Immunohistochemical analysis of cryostat sections of all the biopsies treated with contact allergens showed 1) a large reduction in the number of LCs in epidermis, as evaluated by a decrease in human leukocyte antigens (HLA)-DR-expressing cells, and CD1a-expressing cells and 2) accumulation of the remaining LCs at the epidermal-dermal junction. In contrast, the irritants, inactive chemicals, and solvents did not induce these changes. Morphometrical analysis indicated that the contact allergen-induced reduction in the number of HLA-DR+ and CD1a+ LCs per millimeter of epidermis was significant and was dependent on the concentration of the contact allergens. Flow cytometric analysis of isolated epidermal cells confirmed the immunohistochemical findings. In combination, these results suggest that the culture of ex vivo human skin explants provides a promising model to predict potential allergenicity of newly produced chemical compounds and can therefore replace current animal models.     

Immunofluorescence staining of adenovirus in fixed tissues pretreated with trypsin.

We describe an immunofluorescence staining procedure for adenovirus in Formalin-fixed, paraffin-embedded tissue sections pretreated with a 0.25% trypsin solution. In trypsinized tissue sections stained with fluorescein-labeled antibody to adenovirus, viral antigen was brightly fluorescent and easily detected each time specimens were processed, and the nonspecific background fluorescence was always minimal. Viral antigen was nonfluorescent or only weakly fluorescent in tissue sections not pretreated with trypsin. Because pathology laboratories usually receive Formalin-fixed tissues for examination, this rapid and practical procedure can be routinely used to extend the diagnostic capability of conventional histopathology.     

Conversion of Amadori products of the Maillard reaction to N(epsilon)-(carboxymethyl)lysine by short-term heating: possible detection of artifacts by immunohistochemistry

Accumulation of advanced glycation end products (AGE) of the Maillard reaction increases by aging and in age-enhanced diseases such as atherosclerosis and diabetic complications. Immunohistochemical analysis has been used to demonstrate AGE in vivo. In immunochemistry, the heat-induced epitope retrieval technique is extensively used with formalin-fixed, paraffin-embedded tissue sections. Here we examined whether AGE could be formed artificially through the heating process. Normal rat skin and liver samples were divided into two groups, one rapidly frozen, the other formalin-fixed, paraffin-embedded and submitted to heat-induced epitope retrieval treatment. In heat-treated sections, the cytoplasm of rat epidermal cells and hepatocytes were strongly stained by monoclonal antibody against N(epsilon)-(carboxymethyl)lysine (CML), while the staining was negligible in either frozen sections or in paraffin-embedded but heat-untreated sections. To clarify the mechanism, we conducted heat treatment to glycated human serum albumin (HSA), a model Amadori protein, and generation of CML was determined by immunochemical and HPLC analysis. CML was generated from glycated HSA by heat treatment (above 80 degrees C) and increased in a time-dependent manner. In contrast, generation of CML from glycated HSA was significantly inhibited in the presence of NaBH4, a reducing agent, diethylenetriamine pentaacetic acid, a chelator of transition metal ion, or aminoguanidine, a trapping reagent for alpha-oxoaldehydes. Furthermore, heat-induced CML formation in rat liver samples determined by HPLC was markedly reduced by pretreatment with NaBH4. Reactive intermediates such as glucosone, 3-deoxyglucosone, methylglyoxal, and glyoxal were formed upon heat treatment of glycated HSA at 100 degrees C, indicating that these aldehydes generated from Amadori products by oxidative cleavage can contribute to further CML formation. CML generated by heating, directly from Amadori products or via these aldehydes, might serve as an artifact upon immunohistochemistry.     

Proliferative Activity of Epidermal Basal Cells after Wounding

Quantitative changes in nucleolus organizer regions (NORs) are known markers of proliferation that can be demonstrated by a specific silver staining technique on paraffin-embedded sections. Wounding of skin induces proliferation of basal epidermal cells at the wound margin. The degree of proliferation depends on the survival time and can be measured by morphometric assessment of argyrophilic NORs (AgNORs). Following incision wounding of the pinnae, rats were allowed to survive for different intervals (7 rats per interval) up to 120 hours. Before each sacrifice, biopsies were taken and incubated in a bromodeoxyuridine (BrdU) solution, embedded in paraffin, and stained with an antibody against BrdU. At the same time morphometric analysis of AgNOR counts was performed on sections made from the same material. BrdU incorporating nuclei were assessed by simple counting, whereas morphometric analysis of AgNOR counts was computer aided. Both methods revealed an increase in the number of proliferating cells, a plateau phase being reached after about 36 hours, followed by a decline after about 70 hours. Both methods thus allowed a reliable temporal classification of the skin injury according to survival time. The molecular background of the AgNOR changes in relation to the proliferation of cellular elements is discussed in detail.     

Epidermal melanocytes adjacent to melanoma and the field change effect

The presence of a field change, affecting epidermal melanocytes in the skin surrounding melanomas, has been cited as a justification for performing radical excision of these lesions. Twenty-five consecutive re-excisions of melanoma specimens were examined and melanocytes per 100 keratinocytes counted continuously across the width of each, in order to ascertain whether melanocyte counts decreased from the centre to the periphery, as would be expected if there were a field change effect associated with the tumour. Melanocyte counts did not appear to diminish with increasing distance from the tumour site and were within the range seen in a previous study in which we demonstrated increased numbers of basal melanocytes and melanocyte atypia in sun-exposed skin from a control population. Melanocyte atypia of a mild degree was present in five of the 25 cases studied, but in no case was this greater than that observed in sun-exposed forearm skin. We therefore suggest that the 'field change effect' in epidermal melanocytes adjacent to melanoma could be a result of chronic sun exposure alone.     

Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase

An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.     

Antibodies to intermediate filament proteins as molecular markers in clinical tumor pathology. Differentiation of carcinomas by their reaction with different cytokeratin antibodies

Antibodies to human and bovine epidermal prekeratin and antibodies to mouse liver cytokeratin component D (Mr 49 000) have been applied in indirect immunofluorescence microscopy on sections of human tumors of mammary gland and liver. In non-neoplastic mammary gland all epithelial cells were stained with these antibodies. In pre-invasive and invasive ductal and lobular carcinomas a cell population was observed which was not significantly stained with antibodies to epidermal prekeratin but did strongly react with antibodies to liver cytokeratin D. In the liver, the antibodies to epidermal prekeratin as well as those directed against liver cytokeratin D strongly decorated bile duct epithelia. In contrast, significant staining of the hepatocytes was only achieved with antibodies to liver cytokeratin D. This different staining reaction was maintained in liver tumors of hepatocellular and cholangiocellular origin. Antibodies to vimentin stained mesenchymal cells and tumors of mesenchymal derivation but reacted not significantly with any of the epithelial and carcinoma cells examined. The difference is of practical importance for the discrimination between anaplastic carcinomas and sarcomas of unknown origin. Cytokeratin could also be detected by antibody staining using the peroxidase-antiperoxidase (PAP) technique in formaldehyde-fixed and paraffin-embedded material of skin, gastrointestinal, respiratory, urinary and genital tract as well as various glands, liver and kidney. Examples of positive reactions were shown in a squamous cell carcinoma, a basalioma and a pleomorphic adenoma of the parotis. It is concluded that the immunohistochemical analysis of intermediate filament proteins has diagnostic potential in clinical pathology and may help to elucidate histogenesis and differentiation of tumors and possibly also prognosis of tumor growth. It is further suggested to use antibodies recognizing different subsets of proteins of the cytokeratin family in order to distinguish between different types of carcinomas.     

Intradermal injection of an anti‐Langerin‐HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine‐targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti‐Langerin‐HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti‐HIV immune response without requirement of an adjuvant. Although the co‐injection of the TLR‐7/8 synthetic ligand, R‐848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA‐DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine‐targeted epidermal LCs and R‐848.     

Innate immunity mediated by epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin

Skin is an immunological organ consisting of epidermal cells, i.e. keratinocytes and Langerhans cells (LCs, antigen-presenting dendritic cells), and both innate and acquired immune systems operate upon exposure of the skin to various external microbes or their elements. To explore the relationship between innate and acquired immunities in the skin, we investigated whether Toll-like receptor (TLR) ligation of epidermal cells enhances the ability of LCs to present a specific antigen to T cells in mice. LC-containing epidermal cells were incubated with CpG oligonucleotide (TLR9 ligand) modified with trinitrophenyl hapten, and cultured with hapten-primed CD4(+) T cells. TLR9 ligand was capable of enhancing the hapten-presenting ability of LCs when LC-enriched epidermal cells, but not purified LCs, were used as the LC source, suggesting that bystander keratinocytes play a role in the enhancement of LC function. Cultivation of freshly isolated epidermal cells with CpG promoted the expression of major histocompatibility complex (MHC) class II and CD86 molecules on LCs. CpG enhanced the production of interleukin (IL)-1alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha by primarily cultured keratinocytes. The addition of a cocktail of neutralizing antibodies against these cytokines abrogated the CpG-promoted, antigen-presenting ability of LC-enriched epidermal cells. Moreover, the addition of culture supernatants from CpG-stimulated keratinocytes restored the ability of purified LCs. Our study demonstrated that although the direct effect of CpG on LCs is minimal, LC function can be up-regulated indirectly by cytokines released by CpG-stimulated keratinocytes. This also implies that innate immunity evoked by TLR ligation of keratinocytes enhances acquired immunity comprising LCs and T cells.     

Extraction of human Langerhans cells: a method for isolation of epidermis-resident dendritic cells

Langerhans cells (LCs) are immature dendritic cells in the epidermis that play a central role in T-lymphocyte mediated skin immunity. Upon activation with antigenic stimuli, they differentiate drastically into mature dendritic cells while migrating from the epidermis to regional lymph nodes. Thus, in order to study biological details of immature LCs, it is crucial to isolate epidermis-resident, immature LCs without dermal dendritic cell contamination. Methods for extracting LCs from human skin as well as in vitro derivation of LC-like cells from hematopoietic progenitor cells have been described previously, but the cell preparations can potentially contain a significant number of dendritic cells that are not identical to epidermal LCs. Here, we describe a technique by which purely epidermis-resident LCs are extracted from human skin. Following digestion of human skin with dispase, the epidermis was separated mechanically without any attached dermal component. The trypsinized epidermal cells were then fractionated by centrifugation with a discontinuous density gradient composed of bovine albumin and sodium metrizoate. The LC-enriched preparation thus obtained contained 80% to >90% CD1a+, E-cadherin+ cells that expressed Birbeck granules and the Lag protein. Consistent with their being at an immature stage, the freshly isolated LCs lacked the expression of CD83, a marker for mature dendritic cells. The purified LCs were able to activate allogeneic T cells, indicating that the cells retained T-cell stimulation ability even after extraction. Thus, the present work offers an opportunity for precise in vitro studies of epidermal LCs.     

The use of MUC5B antibody in identifying the fungal type of fungal sinusitis

Fungal sinusitis is an opportunistic fungal infection. Candida albicans, Aspergillus spp, and Mucorales, the most common pathogenic fungi, differ in both prognosis and therapy. Early diagnosis and differentiation between the subtypes therefore are pivotal for adequate treatment. This report describes a diagnostic immunohistochemical method that is able to distinguish these types of fungi. Formalin-fixed paraffin-embedded blocks of 89 fungal sinusitis specimens (12 C albicans, 52 Aspergillus spp, and 25 Mucorales) and 21 cultures (5 C albicans, 11 Aspergillus spp, and 5 Mucorales) were stained with MUC2, MUC5AC, and MUC5B antibodies. The immunohistochemical staining results of paraffin-embedded samples for MUC5B were successful in 100% and 92.3% of the C albicans and Aspergillus spp samples, respectively. Only 4.0% of the Mucorales parafin sections were found positive, demonstrating a significant difference in detection from C albicans and Aspergillus spp (P < .001). MUC5B expressions for cultures showed that it stained 100% and 90.9% for C albicans and Aspergillus spp, respectively, but negative for Mucorales. The expressions of MUC2 and MUC5AC for both paraffin-embedded samples and cultures were negative. The present study demonstrates the ability of an MUC5B antibody to distinguish C albicans and Aspergillus spp from Mucorales and its use as a diagnostic tool in fungal sinusitis.     

Langerhans cells renew in the skin throughout life under steady-state conditions

Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.