Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin

Summary Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymaseand tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72–73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activitiy in the upper dermis may lead to an imbalance in the biochemical regulatory systems.     

Immunohistochemical analysis of sensory nerves and neuropeptides,and their contacts with mast cells in developing and mature psoriatic lesions

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p<0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions. Morphological contacts between mast cells and SP-containing nerves give further evidence to the view that SP is capable of amplifying the inflammatory reaction also through the axon-reflex mechanism.Part of this work was presented at the meeting of the European Society for Dermatological Research, London, UK, 4–7 April 1992     

Mast cell tryptase and chymase in developing and mature psoriatic lesions

The number and distribution of mast cells in nonlesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In nonlesional psoriatic skin, 70 ± 24% (mean ± SD) of the mast cells contained chymase enzyme activity, and 78 ± 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, ₁-antitrypsin and ₁-antichymotrypsin, localized immunohistochemically in mast cells of lesional and nonlesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27–38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis. Earliest alterations in tryptase-positive cells were observed on day 14 as increased mast cell contacts with the epidermis combined with only a slight increase in mast cell numbers in the upper dermis. During the development of a psoriatic lesion, TC mast cells (tryptase⁺, chymase⁺) increase in number in the upper dermis, but chymase becomes inactive at an early stage. The abundant presence of active trypase but inactive chymase in the upper dermis may have a potential role in psoriasis since both of these enzymes can process several biologically active peptides and proteins.     

Mast cell density in psoriatic skin. The effect of PUVA and corticosteroid therapy

Summary Numbers and volume fractions of mast cells in nonlesional and chronic lesional skin of psoriatic patients were compared with those of normal control skin. Mast cell densities were similar in psoriatic nonlesional and normal control skin. The superficial dermis of lesional psoriatic skin contained more mast cells than either normal or nonlesional psoriatic skin. Neither PUVA nor corticosteroid treatment for 3–4 weeks significantly reduced mast cell numbers or volume fractions in lesional skin, although both treatments clinically and histologically markedly improved the lesions. The results indicate that the initiation of the healing process in psoriatic plaques is not correlated with the mast cell density. The remaining high mast cell density may be normalized later, or after a longer therapy.     

银屑病患者皮损间充质干细胞环状RNA表达异常研究

目的 探讨环状RNA（circRNA）在银屑病发病中的作用。方法 分离、培养15例银屑病患者皮损及15例健康对照皮肤间充质干细胞（MSC）,用流式细胞仪及多向分化法进行鉴定。用RNA测序法检测circRNA表达,并进行详细的生物信息学分析。挑选7个差异表达的circRNA构建circRNA-microRNA相互作用网络,从中挑选3个与其相关的microRNA进行qRT-PCR验证。银屑病患者组和对照组qRT-PCR验证结果采用两独立样本t检验。结果 RNA测序结果显示,共检测到6 323个circRNA,其中3 227个为本实验首次发现。与对照组相比,患者组中有129个circRNA呈差异表达,其中123个表达上调,6个表达下调。挑选7个差异circRNA进行circRNA-microRNA相互作用预测,显示与这7个circRNA关系密切的银屑病相关microRNA,包括miR-17-5p、miR-30e-5p、miR-142-3p/5p、miR-369-3p、miR-184、miR-4490、miR-654-3p、miR-423-5p等。qRT-PCR也证实,这7个差异circRNA在患者组也表达上调。与对照组相比,挑选的3个microRNA在银屑病皮损中低表达（t值分别为3.993、3.217、2.918,均P < 0.05）。结论 银屑病皮损MSC circRNA表达异常,并可能参与了银屑病发病。     

Coculture of mast cells with psoriatic fibroblasts: an experimental system for studying the two cell interactions

In this study we established a coculture system for rat peritoneal mast cells (MC) with psoriatic (PSO) dermal fibroblasts. Rat MC adhered within five minutes to the fibroblasts monolayer and their attachment and viability was maintained for at least 2-3 days. Cocultured MC could be activated to release high percentages of histamine with compound 48/80 indicating that they retained their full functional activity. Attachment, viability and functional activity were similar for MC seeded on PSO fibroblasts or on normal human fibroblasts (NOR) used as a control. This would indicate that PSO fibroblasts altered biochemical characteristics do not interfere with these MC properties. We suggest that this coculture system is a suitable in vitro defined model to study mutual effects of MC and fibroblasts in psoriasis.     

银屑病患者的心理压力、神经内分泌系统与肥大细胞研究现状

银屑病中有相当比例的患者因心理压力而使病情恶化,但其机制尚不明确.心理压力能激活下丘脑-垂体-肾上腺轴和皮肤感觉神经,导致内分泌激素和神经介质释放.多种激素和神经介质能活化肥大细胞产生和释放一系列的炎症介质和细胞因子.银屑病患者中,一些激素、神经肽、感觉神经纤维和肥大细胞在表达水平和数量上增高.心理压力影响银屑病病情的机制之一可能与应激介质通过肥大细胞的作用有关.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.     

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin

Summary Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzemidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-antitrypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15,30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefullnes of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.     

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 μm serial sections. Ten sections, 30 μm apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy’s fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis. Received: 17 April 1996     

Cytokine expression in psoriatic skin lesions during PUVA therapy

To determine whether an improvement in skin lesions as a result of PUVA therapy may be correlated with changes in cytokine patterns, RT-PCR amplification was used to compare the levels of IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ cytokine mRNA expression in serial biopsies from three chronic plaque psoriatic patients. In each case, 3-mm punch biopsies were taken from lesional skin before and during 2–28 days of treatment with PUVA. Total mRNA was extracted from each biopsy, cDNA synthesized, and then amplified by 35 cycles of PCR using cytokine-specific primers. The specificity of the PCR products was confirmed by the Southern blot technique. Substantial levels of specific mRNA for each of the cytokines studied was present in the lesions prior to treatment. In two of the three patients who responded well to PUVA, a reduction in all the cytokines including IL-10 was observed compared with baseline levels. In contrast, PUVA proved to be ineffective in clearing the psoriasis of the third patient whose skin lesions worsened during the course of treatment. This was accompanied by an increase in IFN-γ but not of the other cytokines investigated, above the pretreatment level. This study showed an association between PUVA-induced resolution and decreases in the levels of various cytokines highly expressed in psoriatic lesions. Received:14 August 1995     

Mast cells and macrophages in early relapsing psoriasis

Summary Five patients with widespread plaque-type psoriasis were treated continuously with clobetasol under occlusion. Clinical healing was seen after 6–10 days of treatment. All plaques treated in this way clinically relapsed approximately 12 days later. During the period of remission, sequential biopsies were taken and prepared for light and electron microscopy. Histologically, the earliest indications of relapse were endothelial alterations (swelling, intercellular widening) followed by the appearance of mast cells around the postcapillary venules; these mast cells showed signs of degranulation. Hours later, activated macrophages showing pericellular edema were present, and these migrated into the epidermis soon after. Associated with the presence of macrophages, there was a complete loss of desmosome-tonofilament complexes. Later, lymphocytes and neutrophils were seen. Under these experimental conditions, the psoriatic-tissue alterations appear to have been initiated by degranulating mast cells as well as by macrophages which later invaded the epidermis.This paper was presented at the 14th Meeting of the ESDR (Amsterdam, April 1984)     

Quantitative analysis of contact sites between mast cells and sensory nerves in cutaneous psoriasis and lichen planus based on a histochemical double staining technique

Summary The aim of the present study was to test further our previous hypothesis that the inflammatory reaction in psoriasis is neurogenic. For this purpose, contact sites between mast cells and sensory nerves were morphometrically analysed in the basement membrane zone, papillary dermis and three dermal zones of lesional/non-lesional psoriatic and lichen planus skin as well as in healthy control skin. The analyses were made on sections stained with a histochemical double stain developed for this study. With the double stain, active mast cell tryptase was stained blue enzyme histochemically, and the sensory nerves black using specific monoclonal anti-neurofilament antibodies with immunogold. In psoriatic lesions, both mast cells and mast cell — nerve contacts were markedly more frequent in the basement membrane zone and in the papillary dermis when compared with the corresponding areas in the other groups. Mast cell numbers were increased in both lesional and symptom-free skin in lichen planus, but no increase was found in the mast cell — nerve contacts. Increased contacts between mast cells and sensory nerves indicate that the elements exist for neurogenic inflammation in psoriatic lesions. These increased contacts are not due to the extensive inflammatory reaction only, because they were not observed in lichen planus lesions.     

Heterogeneous distribution of lipoxygenase products in psoriatic skin lesions

Summary Several biologically active lipoxygenase products or arachidonic acid (AA) have been demonstrated in psoriatic skin lesions. The purpose of the present study was to determine the amounts of the different lipoxygenase products simultaneously in psoriatic skin. Slices of psoriatic skin were obtained at different levels with a keratome. Extracted lipids were identified by high performance liquid chromatography, UV-absorption spectrum, radioimmunoassay, and chemokinesis. Leukotriene B₄ (LTB₄), and 12- and 15-hydroxy-eicosatetraenoic acid (HETE) were detected in most psoriatic lesions. However, there was a remarkable variation from lesion to lesion. The biopsy specimens contained: 276.2±126.0 pg/g wet tissue of LTB₄, 3,130.0±2,898.0 ng/g wet tissue of 12-HETE, and 3,633.0±1,692.0 ng/g wet tissue of 15-HETE. No correlation was found between the levels of the different lipoxygenase products. The content of each of the identified lipoxygenase products was higher in the superficial part of the biopsy specimen consisting of approximately two-thirds of the epidermis plus papillary dermis than in the lower part consisting of approximately one-third of the epidermis plus some reticular dermis. Also, there was a great variation from one anatomical region to another within the same patient. Because these lipoxygenase products possess different biological activities, the variation in their occurrence may be important for understanding their potential role in psoriasis. To determine which lipoxygenase products may be of pathogenic importance, analysis of early psoriatic lesions is warranted.