Lack of demonstrable effect of cyclosporin A on human epidermal Langerhans cell function

Summary There is controversy about whether Cyclosporin A (CsA) affects antigen-presenting cell function. Within the skin, Langerhans cells (LC) are very potent antigenpresenting cells. We investigated the effect of CsA on alloantigen presentation by human LC using the in vitro mixed skin-cell lymphocyte reaction (MSLR). MSLR (6 day cultures) were performed in round-bottomed microplates and lymphocyte proliferation was assessed by ³H-thymidine incorporation during the final 18 h of culture. When CsA was added into the wells a dose-dependent inhibition of T-cell proliferation occurred. Similar results were obtained when crude or LC-enriched epidermal cells (EC) were incubated for 2 h in the presence of CsA and extensively washed. The inhibition caused by CsA treatment of EC was not overcome by the addition of indomethacin. However, when CsA-treated EC were added to a fresh MSLR, T-cell proliferation was impaired. Furthermore, supernatants from CsA-treated EC, that had been kept for 6 days in culture medium, were able to inhibit the T-cell proliferative assay. These supernatants were found to contain CsA by a radioimmunoassay. From these results, it is clear that inhibition of MSLR obtained after CsA pulsing of EC suspensions can be explained by a release of the drug into the supernatant and thus by a direct effect on T cells. These findings contrast with recent reports showing a direct effect of CsA on human LC function.     

Contact allergens, but not irritants, alter receptor-mediated endocytosis by human epidermal Langerhans cells

Allergic contact dermatitis is a T-cell-mediated inflammation, induced by contact with sensitizers and occurring through the release of epidermal cytokines and the activation of epidermal Langerhans cells (LCs). The aim of this study was to analyse early events of LC activation induced either by contact allergens or by irritants devoid of any contact allergenic properties. in order to obtain an in vitro method to discriminate between these two groups of molecules. Various contact sensitizers and irritants were studied for their effects on the endocytosis of major histocompatibility complex class II (MHC-II) molecules by freshly-isolated human epidermal LCs. As observed by flow cytometry, a spontaneous decrease in the surface expression of MHC-II (HLA-DR) molecules, linked to spontaneous internalization of the MHC-II molecules by LCs, was obtained by moving freshly-isolated LCs from 4 degrees C to 37 degrees C. Pre-incubation of LCs with either sensitizers or irritants increased the spontaneous internalization of HLA-DR molecules with a similar magnitude, but no clear discrimination between sensitizer and irritant effects was obtained by flow cytometry analysis. In contrast, confocal microscopy enabled discrimination between the effects of sensitizers and irritants: sensitizer-treated samples showed internalized HLA-DR molecules aggregated in large vesicles with very bright fluorescence; irritant-treated samples were not different from untreated controls and showed compact HLA-DR molecules in small vesicles with diffuse fluorescence, and mostly localized in the submembranous zone. Electron microscopy demonstrated that sensitizer-treated LCs internalized HLA-DR molecules preferentially in lysosomes collected near the nucleus, whereas the irritant-treated and non-treated LCs internalized these molecules in the prelysosomes only near the cell membrane. We conclude that contact allergens and irritants induce distinct patterns of HLA-I)R endocytosis, which may be useful for the development of in vitro screening tests.     

Association of basal-lamina defects with epidermal and dermal T6-positive cells: Evidence of Langerhans-cell migration

Summary We observed the apparent migration of Langerhans cells across the basal lamina of normal human skin by immunoelectron microscopy using monoclonal anti-T6 antibody. This technique made it possible to visualize cytoplasmic processes of Langerhans cells not normally detectable by routine transmission electron microscopy, and therefore facilitated the documentation of the migratory process. Although events early in the migratory sequence were not observed, perhaps as the result of the evanescent nature of this phase, the association of Langerhans cells with focal disruptions in the epidermal basal lamina was documented. The basal lamina adjacent to these langerhans cells was electron lucent, granular in character, and thinned, or intact, suggesting sequential reassembly after disruption. This study provides ultrastructural documentation supporting the hypothesis of ongoing migration of Langerhans cells across epidermal membranes, and suggests that this process is mediated by the disruption and reconstitution of the epidermal basal lamina.     

神经性皮炎皮损处神经纤维的数量及其与朗格汉斯细胞关系的研究

目的探讨神经性皮炎患者皮损处神经纤维的数量变化及其与朗格汉斯细胞接触的关系.方法用辣根过氧化物酶结合的链霉亲和素-生物素技术观察24例神经性皮炎患者皮损处神经纤维的表达及数量变化.用免疫荧光双标记及共聚焦激光扫描显微镜技术观察神经性皮炎患者皮损处神经纤维与朗格汉斯细胞接触数量关系.用实时定量PCR方法检测皮损处神经生长因子mRNA的表达情况.结果 神经性皮炎患者皮损处神经纤维长度显著增加,与皮损周边对照(t=6.90,P<0.001)及正常人对照(t=5.71,P<0.001)比较差异有统计学意义.皮损表皮内有神经纤维接触的朗格汉斯细胞占朗格汉斯细胞总数的百分数明显增多,与皮损周边对照(X²=43.91,P<0.001)及正常人对照(X²=46.11,P<0.001)比较差异有统计学意义.皮损处神经生长因子mRNA高表达,与皮损周边对照(t=3.25,P<0.01)及正常人对照(t=3.67,P<0.01)比较差异有统计学意义.结论 神经性皮炎患者皮损处存在高水平活性的NGF导致神经纤维增生.     

Reduction of the number of immunocompetent cells in the acute stage of herpes zoster

Summary Circulating and in situ mononuclear cell subsets were phenotypically characterized during both the acure and convalescent phase of herpes zoster infections in 14 patients. In peripheral blood a significant reduction in the absolute number of Leu 4⁺ T cells, Leu 2a⁺ suppressor/cytotoxic T cells, Leu 3a⁺ helper/inducer T cells, Leu 7⁺ killer cells, and B1⁺ B cells were found during the acute stage compared to convalescents and normal controls. In contrast no change in the absolute number of MO2⁺ monocytes was seen in the acute stage of the disease. During convalescence a return to normal values in the lymphocyte subsets and killer cells was seen within 1–2 months after the initial disease presentation.In skin biopsy specimens from 4 of the 14 patients with active herpes zoster lesions the cellular infiltrate consisted of T cells (Leu 4⁺) the majority being helper/inducer T cells (Leu 3a⁺). Most of the cells expressed HLA-DR (Ia) antigens and were according to this in an activated state.The observed changes in effector and regulatory cell numbers may have implications for the acquisition of Varicella-zoster virus infections, the immune deficiency state associated with the disease, and/or the immune response to resolve the infection.     

Alterations in human epidermal Langerhans cells by ultraviolet radiation: quantitative and morphological study

BACKGROUND: Ultraviolet (UV) exposure of human skin induces local and systemic immune suppression. This phenomenon has been well documented when UVB radiation (290-320 nm) is used. The mechanism is thought to involve Langerhans cells (LCs), the epidermal dendritic cells that play a crucial role in antigen presentation. A variety of studies have clearly demonstrated that UVB radiation decreases LC density and alters their morphology and immunological function, but little is known about the effects of the entire UV spectrum (ultraviolet solar simulated radiation, UV-SSR or UVB + UVA) or UVA (320-400 nm) radiation alone. OBJECTIVES: The purpose of this study was to analyse and compare the effects of a single exposure of human volunteers to UV-SSR, total UVA or UVA1 (340-400 nm) in the human epidermal LC density and morphology. METHODS: Immunohistochemistry on epidermal sheets with various antibodies and transmission electron microscopy (TEM) were used. RESULTS: Immunostaining for class II antigen revealed that a single UV-SSR exposure, corresponding to twice the minimal erythemal dose (MED), induced a significant reduction in LC density with only slight morphological alterations of remaining cells. After a single UVA exposure, LC density showed a dose-dependent reduction with a significant effect at 60 J cm(-2) (well above the MED). Moreover, the reduction of LC dendricity was also dose-dependent and significant for doses exceeding 30 J cm(-2). UVA1 radiation was as effective as total UVA for the later endpoint. As demonstrated by TEM, the location of Birbeck granules containing epidermal cells was modified in UVA-exposed areas. They were located in the spinous rather than in the suprabasal layer. In addition, the morphology of these cells was altered. We observed a rounding up of the cell body with a reduction of dendricity. Alterations of mitochondrial membrane and ridges were also seen. CONCLUSIONS: A single exposure of human skin in vivo to UV-SSR, UVA or UVA1 radiation results in different alterations of density and/or morphology of LCs. All these alterations may impair the antigen-presenting function of LCs leading to an alteration of immune response.     

Stimulation of T cells by autologous mononuclear leukocytes and epidermal cells in psoriasis

Summary Based on reports suggesting aberrant cell-mediated immunity and altered infiltration of immunocompetent cells into the skin in psoriasis, we studied the stimulation of T cells by autologous non-T mononuclear leukocytes (autologous mixed lymphocyte reaction, AMLR) and by epidermal cells isolated from lesional and clinically uninvolved skin in psoriasis (autologous mixed epidermal cell lymphocyte reaction, AMECLR). Age- and sex-matched individuals served as controls. We found that the AMLR in psoriasis (n=11) was similar to that in healthy controls (n=16); furthermore, cell proliferation was alike in the presence of either 5% AB-serum or autologous serum. By contrast, while the AMECLR in healthy controls (n=9) resembled that in psoriatics employing epidermal cells from univolved skin, epidermal cells from lesional sites (n=10) induced a significantly higher proliferation of autologous T cells in the AMECLR (P<0.01). We conclude that the in vitro stimulation of T cells by non-T mononuclear leukocytes is normal in psoriasis and is not regulated by autologous serum. Lesional psoriatic epidermal cells, however, are more active in stimulating autologous T cell proliferation than cells from univolved psoriatic or normal epidermis.     

Predominant cutaneous infiltration by OKT6- and OKT8-positive cells in a case of Sézary syndrome

Summary Using an immunoperoxidase (skin biopsy) and an immunofluorescence (peripheral blood, bone marrow punctate) technique, and monoclonal antibodies raised against peripheral mature lymphocytes, T helper subsets, T suppressor subsets, and Langerhans cells, we found a predominant dermal infiltration with lymphocytes of the suppressor phenotype and a predominant epidermal infiltration with Langerhans cells in a patient with Sézary syndrome (cutaneous T-cell lymphoma, CTCL). Repeated peripheral blood examinations showed an increased percentage of lymphocytes of the helper phenotype. A bone marrow examination revealed a ratio of suppressor/helper subsets of 1:4. The findings in the skin seem to be inconsistent with most of the results of previous studies in patients with CTCL; the significance of these findings is discussed.This study was partly supported by the Deutsche Forschungsgemeinschaft, Grant no. Lo 285/2-1This work is dedicated to Prof. Th. Nasemann on occasion of his 60th birthday     

Origin of Langerhans cells in normal skin and chronic GVHD after hematopoietic stem‐cell transplantation

Chronic graft‐versus‐host disease (cGVHD) is a common complication following allogeneic stem‐cell transplantation (SCT). Past studies have implicated the persistence of host antigen‐presenting cells (APCs) in GVHD. Our objective was to determine the frequency of host Langerhans cells (LCs) in normal skin post‐SCT and ask if their persistence could predict cGVHD. Biopsies of normal skin from 124 sex‐mismatched T‐cell‐replete allogenic SCT recipients were taken 100 days post‐transplant. Patients with acute GVHD and those with <9 months of follow‐up were excluded and prospective follow‐up information was collected from remaining 22 patients. CD1a staining and X and Y chromosome in‐situ hybridization were performed to label LCs and to identify their host or donor origin. At 3 months, 59 ± 5% of LCs were host derived. The density of LCs and the proportion of host‐derived LCs were similar between patients that did or did not develop cGVHD. Most LCs in the skin remained of host origin 3 months after SCT regardless of cGVHD status. This finding is in line with the redundant role of LCs in acute GVHD initiation uncovered in recent experimental models.     

Expression, but lack of calcium mobilization by high-affinity IgE Fcε receptor I on human epidermal and dermal Langerhans cells

Abstract In atopic dermatitis (AD) patients, IgE molecules are demonstrated on the surface of Langerhans cells (LC). FcεRI molecules, which are present on the surface of LC in AD patients as well as normal individuals, are responsible for this binding. In this study, we have investigated phenotypic and functional characteristics of FcεRI on epidermal and dermal cell populations. Epidermal and dermal cell suspensions were prepared enzymatically with dispase followed by either trypsin or collagenase treatment, respectively. Peripheral blood basophils were negatively selected by excluding other leukocytes with surface marker staining. Consistent with previous reports, both peripheral blood basophils and epidermal LC were positively stained with anti FcεRI monoclonal antibody. In addition, an FcεRI positive population was demon-strated among dermal HLA-DR positive cells. These cells express significant amounts of HLA-DR molecules (DR^Hi) and co-express CD la molecules, which identifies them as LC-like dendritie APC of the dermis. No other FcεRI positive population was found in the other dermal DR^Mid or DR populations, except for a minor DR^lo population, presumably mast cells. To analyze whether these FcεRI molecules are signal transducing for LC, intracellular calcium mobilization after crosslinking of FcεRI was measured with How cytometry. Following crosslinking, peripheral blood basophils clearly increased intracellular calcium. On the other hand, neither normal epidermal LC nor dermal DR^HiCD Ia⁺ cells changed their intracellular calcium level after FcεRI crosslinking. These data indicate that normal epidermal and dermal LC, but not basophils, are resistant to calcium flux following FcεRI engagement.     

Langerhans cells in various benign and malignant pigment-cell lesions of the skin

Summary We used immunohistochemistry to study Langerhans cells (LCs) and the composition of the dermal inflammatory infiltrate both in normal skin and in biopsies from various benign and malignant pigment-cell lesions. In normal skin and most benign pigment-cell lesions, epidermal LCs are regularly distributed. OKT₆-Positive cells outnumber the OKIa-positive cells. The inconspicuous dermal infiltrate studied in these biopsies was composed of helper and suppressor/cytotoxic T cells and some dermal LCs. More epidermal LCs with an abnormal cytologic presentation were found in a halo naevus and in the radial growth part of primary malignant melanomas. This finding was associated with a dermal infiltrate composed of suppressor/cytotoxic T cells, suggesting a defense mechanism of the host towards abnormal melanocytes. Epidermal LCs were rare in the central part of the biopsies which showed a primary malignant melanoma in its vertical growth. A dermal inflammatory infiltrate was absent in that area. These findings are interpreted as the morphologic expression of a damaged immune system.F. Facchetti is on leave from Istituto di Anatomia Patologica, Spedali Civili di Brescia, Brescia, Italy     

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-α and their degranulation is associated with expression of ICAM-1 in the epidermis

Abstract The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-α), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-α immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-α⁺ mast cells in lesional and nonlesional AD skin was 36 ± 22% and 21 ± 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 ± 25% and 15 ± 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-α antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-α and histamine. Received: 8 August 1997     

Langerhans cell markers CD1a and CD207 are the most rapidly responding genes in lesional psoriatic skin following adalimumab treatment

TNFα‐, IL‐23‐ and IL‐17‐targeting drugs are highly effective in the treatment of psoriasis. However, the precise molecular mechanism remains unknown. In psoriatic skin, the presence of Langerhans cells (LCs) is reduced, but the role of LC is poorly understood. The purpose of this study was to investigate the impact of TNFα and IL‐23/IL‐17 on the presence of LC in the skin during treatment. Therefore, psoriatic skin was investigated before and after 4 days of adalimumab or ustekinumab treatment. Furthermore, TNFα and IL‐17A stimulation was investigated in an ex vivo model of epidermis and dermis from healthy normal skin kept in cultures at an air‐liquid interphase for 4 days. In a gene array analysis, we found that the two LC markers, CD1a and CD207, were among the most up‐ or downregulated genes in psoriatic skin after anti‐TNFα therapy. Validation showed that both mRNA expression and protein level followed the same pattern and became significantly upregulated after 4 days of treatment. No changes were seen after ustekinumab treatment. In the ex vivo skin model, a decrease in the CD1a level was seen after TNFα stimulation and it was caused by LC migration from epidermis. No response in LC migration was seen after IL‐17A stimulation. Taken together, we demonstrated that changes in the LC level in epidermis precede the histological and clinical changes during adalimumab treatment in psoriatic skin. Furthermore, TNFα plays a prominent role in orchestrating LC migration in the skin. This seems not to be the true for the IL‐23/IL‐17A pathway.     

Cryopeeling versus trichloroacetic acid peeling in the treatment of solar lentigines: Effect on epidermal Langerhans cells

Trichloroacetic acid (TCA) peeling may be effective in solar lentigines, but with concerns regarding potential tumorigenesis. Cryopeeling would be better with improving the whole sun‐damaged skin. We aimed to compare the efficacy and safety of cryopeeling and TCA 35% peeling for treatment of solar lentigines and assess their influence on the number of epidermal Langerhans cells (LC). Twenty‐five patients were treated with TCA 35% and cryopeeling on the right and left hands, respectively. Two sessions were done 3 weeks apart. Evaluations were scheduled at weeks 0, 3, and 6. Skin biopsies, taken before and after treatment, were evaluated histologically and immunohistochemically for the number of CD1a + epidermal LCs. Lentigines decreased after cryopeeling from the first session (p < .001), but after the second session with TCA peeling (p = .004). Cryopeeling produced significant lightening, compared with TCA (p = .015). Blistering, hyper/hypopigmentation were reported with cryopeeling, whereas only hyperpigmentation was noted after TCA peeling. The LCs remained at about the pretreatment number after cryopeeling (p = .058), though they decreased after TCA (p = .002). Cryopeeling provided faster and superior improvement of lentigines compared with TCA peeling. Furthermore, TCA seems to suppress LCs raising the concern for carcinogenic potential.     

Effects of solar-simulated radiation dose fractionation on CD1a+ Langerhans cells and CD11b+ macrophages in human skin

BACKGROUND: There are few human studies investigating the immunosuppressive effects of exposure to solar-simulated radiation (SSR) and its relationship with sunburn/erythema, and few comparative data on the importance of SSR exposure regimens. OBJECTIVES: To evaluate whether SSR-induced erythema is a reliable end-point for assessing damage to antigen-presenting cells (APCs) in human skin. METHODS: We compared the relationship between SSR-induced erythema and alterations in epidermal CD1a+ Langerhans cells (LCs) and CD11b+ macrophages in human volunteers after single exposures to 0, 0.5, 1, 2 or 3 minimal erythema doses (MED). We also investigated whether SSR exposure leads to an accumulation or accommodation of the same end-points by comparing the effects of a relatively low cumulative SSR dose (3 MED) given in varying daily dose fractions (4 x 0.75 MED, 2 x 1.5 MED and 1 x 3 MED). RESULTS: Single SSR exposures induced a dose-dependent increase in erythema. CD1a+ LCs remaining in the irradiated epidermis showed a dose-dependent increase in cell size and altered morphology. Significant depletion of CD1a+ LCs and presence of CD11b+ macrophages only occurred in sites irradiated with 2 MED and 3 MED. Dose fractionation had no effect on the final erythemal response but the 4 x 0.75 MED and 1 x 3 MED protocols were better tolerated than 2 x 1.5 MED for alterations in CD1a+ LC and CD11b+ cell numbers. In contrast, dose fractionation protected against alterations in CD1a+ LC morphology or cell size. CONCLUSIONS: We found that erythema is a poor indicator of alterations in epidermal APCs and that dose fractionation is an important parameter in the immunological effects of ultraviolet radiation.