Epidermis and lymphocyte interactions during a tuberculin skin reaction

Summary Forty-one persons were tested for tuberculin skin reactivity. Epidermal cells (ECs) were isolated from the tuberculin reaction and from a contra lateral, non injected skin area. We found a significant increase of epidermal thymocyte activating factor (ETAF) in epidermis overlying a positive tuberculin reaction together with an increase of OKT6 and class II (HLA-DR) positive cells. Allogeneic lymphocytes proliferated significantly more when mixed with ECs from a positive tuberculin skin test. Injection of tuberculin per se or a negative reaction did not induce similar changes. The described model seems useful for functional studies of ECs and lymphocytes in patients with contact dermatitis.     

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.     

Delayed-type skin reaction to 2,4-dinitrophenylated epidermal cells in guinea pigs with contact sensitivity to 2,4-dinitrochlorobenzene

Summary Contact sensitivity (CS) induced by hapten has been thought to be analogous to delayed-type hypersensitivity, such as the Mantoux reaction, because of outstanding similarities between the two phenomena. It can be suggested that animals with CS respond also to intradermal injection of the conjugate of hapten and protein as well as to epicutaneous application of hapten. However, evidence against this has been reported. In the present experiments, delayed-type skin reaction (DSR) was successfully obtained in JY1 strain guinea pigs sensitized by painting the skin with 2,4-dinitrochlorobenzene using in vitro dinitrophenylated epidermal cell suspension (DNP-EC) as antigen for a delayed intradermal test. The experiment using anti-Ia alloantiserum and complement showed that the elicitation of DSR is due to the presence of Ia-positive cells (presumably Langerhans cells) among DNP-ECs. The delayed intradermal test with the conjugates such as haptenated ECs in the animals with CS is considered to be an experimentally useful way of analysing the antigen in the sensitivity.     

Inhibition of keratinocyte growth in cell culture and whole skin culture by mast cell mediators

Mast cells are suggested to participate in regenerative processes, but their influence on epithelialization and wound healing has not been well studied. Since mast cells can be found in contact with epidermis in chronic inflammatory skin diseases and venous ulcers, the effect of mast cells on keratinocyte growth was studied. Keratinocytes were cultured in serum-free conditions with (complete medium) or without (basal medium) epidermal growth factor (EGF) and bovine pituitary extract (BPE) to reach subconfluence in a 24-well plate, and the cells were treated with different mast cell mediators histamine, heparin and tryptase, or lysate from HMC-1 cells, a human leukemic mast cell line. Whole skin cultures were used as a model for in vitro wounds to study the effect of mast cells on epithelial outgrowth from skin specimens. Histamine inhibited 3H-thymidine incorporation of keratinocytes dose-dependently by 29% at 1 mM, and 89% at 5 mM histamine. In whole skin culture, histamine inhibited epithelial outgrowth dose-dependently by 64% already at 0.1 mM histamine and maximally (91%) at 1 mM histamine. Heparin inhibited 3H-thymidine incorporation dose-dependently by up to 33% at 2 microg/ml in the absence, but not in the presence, of EGF/BPE. In contrast, in whole skin culture, heparin first inhibited the epithelial outgrowth by up to 27% at 2 microg/ml, but then reversed the inhibition to 30% stimulation at 200 microg/ml. Skin tryptase (0.0285 to 2.85 microg/ml) with or without heparin (0.5 to 20 microg/ml) did not affect thymidine incorporation in keratinocytes. Lysate from HMC-1 cells, but not that from control, neuroblastoma cells, inhibited 3H-thymidine incorporation in keratinocytes dose-dependently, and maximal (47%) inhibition was reached with 16,700 lysed HMC-1 cells/ml. In whole skin culture, HMC-1 lysate inhibited the epithelial outgrowth by up to 36% at 67,000 lysed cells/ml. The results show that mast cells and their mediators are inhibitory to keratinocyte 3H-thymidine incorporation and epithelial outgrowth in vitro, although, the inhibitory effect of histamine was seen at high concentrations suggesting a requirement for close morphologic vicinity of mast cells to keratinocytes. Thus, mast cells are assumed to control epidermal regeneration and to impair epithelialization of chronic ulcers.     

Functional defect in cells involved in Langerhans cell histiocytosis

The characteristic cell type involved in Langerhans cell histiocytosis, LCH cells, express most of the enzyme histochemical and immunocytochemical markers of normal epidermal Langerhans cells. It is not known, however, whether these LCH cells express the functional characteristics of normal epidermal Langerhans cells. We studied the alloantigen-presenting activity of LCH cells derived from lesional sites of three patients with the disease. Lesional cells expressing the CD1a molecule were enriched using either fluorescein-activated cell sorting or negative selection with indirect immunomagnetic beads, and functional activity was assessed using the 6-day primary allogeneic mixed-cell reaction. Compared to epidermal Langerhans cells from healthy controls, LCH cells showed minimal alloantigen-presenting activity on a per-cell basis. The diminished activity was not reversed by exogenous prostaglandin synthetase inhibitor or recombinant human IL-1. This study confirms our previous report of a child, with fatal multi-system Langerhans cell histiocytosis suggesting that this disease represents a condition in which functionally defective cells of Langerhans cell phenotype accumulate and/or proliferate in various tissues. We postulate that the functional defect is a primary defect of these LCH cells that have acquired an as-yet-undetermined biological insult(s).This work was in part presented at the 22nd Annual Meeting of the European Society for Dermatology Research in April 1992     

Long-term tissue culture of epithelial-like cells from human skin (NCTC strain 2544)

Summary Human skin epithelial-like cells (NCTC strain 2544) were grown in NCTC 135 medium. Neuraminidase and hyaluronidase were added to the growth medium. Cells were incubated 96 h at 36° C. Growth rate and viscosity of cell suspensions were measured after forming single cells mechanically (mopping). With addition of neuraminidase and hyaluronidase, respectively, the growth rate remains unchanged. With neuraminidase a distinct raise in viscosity was achieved, whereas with hyaluronidase only a small effect was seen. The characteristic structure viscosity is maintained in all forms of the viscosity curves at different shearrates.Presented in part at the XV. International Congress of Dermatology, Mexico City, 1977     

白细胞介素8在银屑病发病机理中的作用

用酶免疫组化法１３例银屑病皮撷 、９例非皮损皮肤和１０例正常皮肤组织中ＩＬ－８的表达与分布，并研究ＩＬ－８对表皮细胞增殖的影响。结果表明９例皮损组织 表层层显著显示ＩＬ－８阳性反应，而患者非皮损皮肤及正常人皮肤表皮层均呈阴性反应。体外实验证实，ＩＬ－８能明显促进原代培养的人表皮细胞增国殖，并呈一定的剂量依赖性。     

Lymphocyte subsets in peripheral blood of patients with moderate-to-severe versus mild plaque psoriasis

In several studies peripheral blood T-cells have been quantified, yet few data are available on lymphocyte subsets in moderate-to-severe psoriasis (in terms of extent and activity of lesions) versus mild psoriasis. The objective is to compare lymphocyte subsets in peripheral blood of patients with moderate-to-severe disease (PASI-score ≥12) to patients with mild disease (PASI-score <12) and to healthy subjects. By means of flow cytometry method, lymphocytes in peripheral blood of 27 patients with psoriasis and 10 healthy controls were characterized. The absolute number of total lymphocytes was markedly decreased in patients with moderate-to-severe psoriasis as compared to patients with mild disease and normal subjects. Cellcounts of all analysed subsets were found to be increased in more severe psoriasis, except for CD8+CD45RO+ cells. The under-representation of CD8+CD45RO+ cells is compatible with the dynamics of acquired immunity, which requires a time log after the relapse of the lesions to differentiate from CD45RA+ naive cells.     

银屑病患者新鲜血细胞的天然免疫反应性研究

为研究银屑病患者血细胞的天然免疫功能状况，并探讨其在银屑病发病机制中的作用，采用分离新鲜血红细胞，淋巴细胞、粒细胞3系。测定其对肿瘤细胞的快速天然免疫反应。结果银屑病患者肿瘤红细胞花环率及肿瘤淋巴细胞花环率明显高于对照组。表明银屑病患者天然免疫反应能力增强。     

Jagged1蛋白在银屑病、基底细胞癌及皮肤鳞状细胞癌中的表达

目的探讨Jagged1蛋白在银屑病、基底细胞癌及皮肤鳞状细胞癌中的表达及意义。方法采用免疫组化Envision法检测Jagged1蛋白在银屑病、基底细胞癌及皮肤鳞状细胞癌皮损中的表达。结果 Jagged1蛋白在寻常性银屑病患者皮损中呈阴性表达,在基底细胞癌及皮肤鳞状细胞癌中的表达较正常人皮肤增强,差异有统计学意义(P<0.01);Jagged1蛋白在基底细胞癌及皮肤鳞状细胞癌中的表达较银屑病增强,差异有统计学意义(P<0.05)。结论 Jagged1蛋白在银屑病发病机制中与角质形成细胞异常增生及真皮乳头血管增生等组织病理变化可能不相关,提示此蛋白可能与皮肤恶性肿瘤的异常增生有关。     

Evaluation of lymphocyte activation in skin lesions of patients with mixed connective tissue disease and discoid lupus erythematodes

Summary Biopsy specimens from mixed connective tissue disease (MCTD) and discoid lupus erythematodes (DLE) skin lesions were stained with monoclonal antibodies to differentiation and activation antigens. In addition, the blast cells were studied by combining autoradiography with immunoperoxidase staining. In both disease conditions most of the inflammatory cells in situ were positive for T11 antigen, the CD4/CD8 ratio being low. Only a few of the cells were pan-B positive B cells. The expression of various activation antigens did not differ significantly between MCTD and DLE biopsy specimens; the number of T9, Tac, and 4F2 antigen carrying cells was relatively low, whereas Iapositive cells were more numerous. ³H-Thymidine incorporating T blasts comprised less than 1% of all inflammatory cells. T4 and T8 marker-carrying blast cells were present in about equal proportions. These findings suggest that Ia antigen-expressing T cells are important from the pathogenetic point of view in both MCTD and DLE. Because the local proliferation of T cells was extremely low according to the lack of interleukin-2 receptor and OKT9 markers and ³H-thymidine incorporation, it seems probable that most of the T cells are recruited from the circulation to the site of the inflammation.     

Lympho-epidermal interactions in patients receiving human Langerhans cell free cultured epidermal allografts as a skin substitute

Human Langerhans cell free epithelia can be cultured in vitro, and then can be used as epidermal allografts (EAG) without evidence of rejection. We studied the cellular basis of this phenomenon with mixed epidermal cell lymphocyte reactions (MELR). The capacity of donor-derived epidermal cells to stimulate allogeneic control or recipient cells was abolished when stimulatory cells were Langerhans cell-free cultured keratinocytes and respondors were obtained prior to grafting. Donor-type cultured keratinocytes were able to induce a low response by host-derived cells in assays conducted 2, 4 and 6 weeks after grafting, but not thereafter. They were unable to stimulate allogeneic cells unrelated to the recipient. The ability of crude epidermal suspensions with 2–4% Langerhans cells to stimulate host-derived cells did not increase with time after grafting. No secondary type MELR could be evidenced, suggesting that grafting of EAG did not induce an in vivo immunization to class I antigens expressed by cultured epidermal cells. Lastly, host-derived Langerhans cells were not able to restore the allostimulatory ability of Langerhans cell free epidermal cells from EAG donors when tested against host-derived cells. This suggests that host-derived Langerhans cells which colonize the grafts during the first few weeks following grafting cannot act in the presentation of foreign keratinocyte-bound antigens, which may account for the absence of rejection noted.     

Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cells

Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.     

Mast cell chymase in experimentally induced psoriasis

Mast cell chymase can have a pro‐inflammatory or an immunosuppressive function in psoriasis, but the outcome may depend on the level of chymase activity. Therefore, mast cells showing chymase activity (Chy_act) and immunoreactivity (Chy_prot) were studied during the Köbner reaction (0 days, 2 h, 1 day, 3 days and 7 days) of psoriasis induced by the tape‐stripping technique. Also, the effect of recombinant human chymase (rh‐chymase) or human LAD2 mast cells (LAD2) on the ³H‐thymidine uptake of psoriatic peripheral blood mononuclear cells (PBMC) or total T cells was studied. The Chy_act/Chy_prot ratio tended to be higher in all time‐point biopsies in the Köbner‐negative (n = 10) than ‐positive (n = 8) group (P = 0.073), although chymase activity decreased significantly at 2 h to 1 day only in the Köbner‐negative group. rh‐chymase (0.05–0.5 μg/mL) stimulated to a varying extent PBMC in eight out of nine cultures, but in all cultures 5 μg/mL rh‐chymase turned the stimulation towards inhibition. The effect of rh‐chymase on T cells varied from stimulation to inhibition, but in 11 of 15 cultures rh‐chymase, at least at 5 μg/mL, produced a change to inhibition. In co‐cultures, LAD2 inhibited PBMC in the absence of soybean trypsin inhibitor (SBTI). In the presence of SBTI, LAD2 stimulated PBMC in the majority of seven cultures. In summary, the psoriatic immunopathogenesis may be promoted at low, but controlled at high, activity status of chymase.     

Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin.

In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB.