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     

Immunophenotyping of inflammatory cells in lesional skin of the extrinsic and intrinsic types of atopic dermatitis

BACKGROUND: There is a subgroup of atopic dermatitis (AD) patients with normal total and specific IgE levels and negative skin tests towards common allergens. This form of the disease has been referred to as the 'intrinsic' form of AD. Although previous studies have demonstrated differences in the cytokine profile between the extrinsic and intrinsic subtypes, the pathogenesis of both subtypes of AD remains unclear. OBJECTIVES: To compare the inflammatory micromilieu in both forms of AD. METHODS: Immunophenotyping of the inflammatory cells was performed in lesional and nonlesional skin from 18 patients with extrinsic and 17 with intrinsic AD. RESULTS: Immunohistochemical analysis revealed a high proportion of CD4+ T cells in the dermis, with a similar CD4/CD8 ratio in the two groups. The expression levels of other T-cell markers and epidermal Langerhans cells were increased in both forms of AD. Although the T-cell repertoires in the two subtypes were similar, dermal infiltration of eosinophils and eosinophil granular proteins was more prominent in the extrinsic type than in the intrinsic type. Eotaxin immunoreactivity was also significantly higher in the extrinsic subtype. CONCLUSIONS: The data suggest that although the overall inflammatory microenvironment in the two subtypes appears to be similar, differences in T-cell cytokine production might contribute to the differential tissue eosinophilia in these subtypes.     

不同预后尖锐湿疣组织中Toll样受体3、9的表达

目的 探讨不同预后尖锐湿疣（CA）患者皮损局部Toll样受体3（TLR3）和Toll样受体9（TLR9）表达定位及TLR3 mRNA、TLR9 mRNA的表达情况。方法 免疫组化SP法及反转录聚合酶链反应（RT-PCR）法检测10例CA复发者、14例CA无复发者及10例包皮组织中TLR3 及TLR9表达情况。结果 TLR3、TLR9在无复发CA皮损中表达以棘层、颗粒层为主;在复发组CA皮损中表达以基底层、棘层为主。无复发组CA组织中TLR3mRNA表达较对照组及复发组均明显升高（P ＜ 0.01、P ＜ 0.05）,TLR9 mRNA较对照组明显升高（P ＜ 0.05）,较复发组差异无统计学意义。结论 表皮TLR3 及TLR9的表达部位和表达量的改变可能与CA预后有关,TLR3的影响可能更大。     

The role of lymphocytes, granulocytes, mast cells and their related cytokines in lesional skin of linear IgA bullous dermatosis

Linear IgA bullous dermatosis (LAD) is an acquired, heterogeneous, subepidermal blistering disease characterized by linear IgA deposits at the dermoepidermal basement membrane zone (BMZ), often with circulating IgA antibodies to the BMZ. The pathogenetic mechanism, possibly related to the immunophenotype of infiltrating cells, as well as the potential role of cytokines in determining bullous lesions, have not yet been elucidated. An immunohistochemical study was performed with a large panel of monoclonal antibodies [to CD3, CD4, CD8, CD25, CD1a, CD30, CD54, CD50, endothelial leucocyte adhesion molecule-1, vascular cell adhesion molecule-1, myeloperoxidase (MPO), eosinophil cationic protein EG1 and EG2, tryptase, HLA-DR, human interleukin (IL)-3, human IL-5, human IL-8, human IL-4, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma and granulocyte/macrophage colony-stimulating factor] using the alkaline phosphatase-antialkaline phosphatase procedure on lesional and perilesional skin of nine patients (one male, eight female; age range 8 months-80 years) with clinical, histological and immunofluorescent proven LAD. The predominant infiltrating cells, distributed mostly inside and below the bullae, were neutrophils and eosinophils which showed intense activation (MPO +, EG1 +, EG2 +). The lymphocytic infiltrate, consisting principally of CD4 +, HLA-DR + and CD30 + T cells, had a predominantly perivascular distribution. Proinflammatory cytokines, such as TNF-alpha and IFN-gamma, showed a moderate focal expression on the dermal perivascular sites; IL-8 was found to have a particularly intense staining on all the epidermal cell layers and at perivascular and vascular sites. Other cytokines, such as IL-4 and IL-5, showed a prevalent intracytoplasmic staining on some cells of the dermal infiltrate (probably mastocytes and lymphocytes), and at the dermal-epidermal separation sites there was also an intense scattered distribution of IL-5. The specific tissue lesions of LAD may be the consequence of the IgA deposits at the BMZ and also of the release of these cytokines together with tissue damage enzymes derived from neutrophils or eosinophils.     

Fucosyltransferase VII-positive, skin-homing T cells in the blood and skin lesions of atopic dermatitis patients

Abstract: Patients with atopic dermatitis (AD) have an abnormally increased frequency of cutaneous lymphocyte antigen (CLA)⁺ Th2 cells responsible for local inflammation; however, this is paradoxical, given the well-recognized defective capacity of Th2 cells to migrate to the skin sites of inflammation. These discrepant observations would stem from the ambiguity of CLA⁺ T cells, because CLA does not represent the epitope required for binding to E-selectin but the epitope generated by fucosyltransferase VII (Fuc-TVII) and because skin-homing T cells are composed of three distinct subpopulations; Fuc-TVII⁺ E-selectin ligand (ESL)⁺ CLA⁻, Fuc-TVII⁺ ESL⁺ CLA⁺ and Fuc-TVII⁻ ESL⁻ CLA⁺ cells. We therefore asked which subpopulations of skin-homing Th2 cells could be increased in the blood and skin lesions of AD. We analysed the frequencies of the three subpopulations in purified CD4⁺ peripheral blood T cells from AD patients and healthy controls by immunohistochemistry and flow cytometry. The Fuc-TVII⁺ CLA⁺ or CLA⁺ ESL⁺ CCR4⁺ cells were dramatically increased in frequency not only in the blood but also in the skin lesions of AD patients and this increase was related to the severity of the clinical symptoms. Our data indicate the clinical importance of identifying skin-homing T cells with the potent capacity to migrate into the skin by analysing their Fuc-TVII expression and E-selectin binding ability in patients with AD.     

Tacrolimus decreases the expression of eotaxin, CCR3, RANTES and interleukin-5 in atopic dermatitis

Background There is a lack of studies on the effect of tacrolimus on eosinophils and related molecules including eotaxin, CCR3, RANTES and interleukin (IL)‐5. Objectives To investigate the effects of tacrolimus on in vivo eosinophil counts and on the related molecules eotaxin, CCR3, RANTES and IL‐5 in patients with atopic dermatitis (AD). Methods Lesional skin specimens and sera were obtained from 15 patients with AD and from 15 normal controls. For 8 weeks, the patients with AD applied 0·03% tacrolimus ointment to all affected areas twice daily. Blood sampling and skin biopsies were then repeated. We evaluated serum eotaxin and IL‐5 levels, and tissue eotaxin, CCR3, RANTES and IL‐5 levels. Additionally, tissue levels of eotaxin and CCR3 mRNA were measured. Results After treatment with topical tacrolimus twice daily for 8 weeks, significant decreases were found in serum IL‐5 levels, immunoreactive cell counts of eotaxin, IL‐5, CCR3 and RANTES in AD skin, and tissue eosinophil counts. However, the change in the serum eosinophil count was not statistically significant, and mRNA levels of eotaxin and CCR3 were not decreased significantly after treatment. Conclusions Topical tacrolimus reduces the number of eosinophils in tissue and suppresses the expression of eotaxin, CCR3, RANTES and IL‐5 related to proliferation, recruitment, activation and survival of eosinophils.     

Lymphocytes and macrophages of the epidermis and dermis in lesional psoriatic skin,but not epidermal Langerhans cells,are depleted by treatment with cyclosporin A

Summary Since cyclosporin A (CsA) is an immuno-suppressive agent, its beneficial effect in psoriasis suggests that immune cells may play a role in the pathogenesis and resolution of psoriasis. To determine early effects of CsA in psoriasis, we quantitated immune cells using double immunofluorescence microscopy on biopsy specimens obtained prior to therapy and after 3,7, and 14 days of CsA therapy. CsA therapy resulted in significant reductions in the absolute number of immune cells (including T cells, monocytes/macrophages, and antigen presenting cells) contained within psoriatic skin. The effect was rapid, with over one-half of the reduction in the density of HLe1⁺ (human leukocyte antigen-1 positive or bone marrow derived) cells, including T cells, activated T cells, monocytes, and Langerhans cells (LCs), occurring within 3 days. Despite the overall reduction in the numbers of immunocytes in the skin, the proportion of T cells, Langerhans cells, and monocytes in relation to the total number of immune cells was unchanged with therapy, reflecting equally proportional losses of each subtype. Dermal CD1⁺DR⁺ cells (putative Langerhans cells), which are not found in normal skin but are present in lesional psoriasis skin, were virtually cleared from the papillary dermis after CsA therapy. Although absolute numbers of epidermal Langerhans cells, defined as cells expressing both CD1 (T6) and DR molecules (CD1⁺DR⁺), were also reduced after CsA, epidermal non-Langerhans CD1^-DR⁺ cells (macrophages, activated T cells, DR^- keratinocytes) demonstrated a proportionally greater decrease, with the ratio of CD1⁺DR⁺ Langerhans cells/non-Langerhans CD1^-DR⁺ epidermal cells changing from a mean of 0.82 at baseline to 1.92 at day 14. Thus, early in the course of therapy, CsA appears to be effective at clearing CD1^-DR⁺ cells while leaving LC relatively intact in the epidermis.This work was supported in part by the Babcock Foundation     

Increased interleukin‐36γ expression in skin and sera of patients with atopic dermatitis and mycosis fungoides/Sézary syndrome

Interleukin (IL)‐36γ is expressed by keratinocytes and functions as a key initiator of inflammation in the skin. IL‐36γ expression is enhanced by tumor necrosis factor‐α and IL‐17A, having a strong association with psoriasis. In this study, we examined the role of IL‐36γ in atopic dermatitis (AD) and mycosis fungoides (MF)/Sézary syndrome (SS). Serum levels of IL‐36γ in AD patients and MF/SS patients were elevated compared with those of healthy controls. Importantly, serum IL‐36γ levels in AD patients positively correlated with Eczema Area and Severity Index and those of MF/SS patients positively correlated with serum soluble IL‐2 receptor levels. IL‐36γ mRNA levels in AD skin and MF/SS skin were significantly higher than those of normal skin. IL‐36γ mRNA levels in MF/SS skin positively correlated with IL‐17A mRNA levels. Immunohistochemical staining revealed that IL‐36γ was highly expressed in keratinocytes in lesional skin of AD and MF/SS. Taken together, our study demonstrated that IL‐36γ expression was increased in sera and skin of patients with AD and MF/SS as was reported in psoriatic patients.     

生殖器疱疹患者皮损HSV-2病毒载量与外周血细胞免疫功能的关系

目的：研究生殖器疱疹（GH）患者皮损部位单纯疱疹病毒－Ⅱ（HSV－2）载量和外周血细胞免疫功能的相关性。方法：采用荧光定量聚合酶链反应（fluorescent quantitative polymerase chain reaction，FQ—PCR）定量检测28例现症GH患者皮损部位HSV－2DNA载量，采用流式细胞计数的方法监测28例现症GH患者外周血T淋巴细胞亚群、NK细胞及B细胞的比例，用线性相关和回归方法分析现症GH患者病毒载量与细胞免疫功能的相关性。结果：①GH患者皮损内疱液HSV－2检出率为100％，病毒载量为6190714±1962085拷贝／μgDNA。HSV－2病毒载量与患者复发次数呈显著正相关。②GH患者CD3^＋、CD4^＋、CD56细胞数及CD4^＋／CD8^＋比值均较对照组低（P〈0．01），但B细胞数与对照组相比差异无统计学意义（P〉0．05）。GH患者皮损HSV-2病毒载量与患者外周血CD3^＋、CD4^＋、CD8^＋、NK、CD4^＋／CD8^＋细胞呈显著的负相关。结论：GH患者的细胞免疫功能抑制可能是由于外周血T淋巴细胞亚群的变化和NK细胞减少，以及Th及Tc细胞亚群分化失衡造成，这种状态不利于机体抑制病毒复制和增殖，造成GH反复发作。     

Increased frequency of intracellular interleukin (IL)-13 and IL-10, but not IL-4, expressing CD4+ and CD8+ peripheral T cells of patients with atopic dermatitis

BACKGROUND: A number of studies exist demonstrating the increased expression of type 2 cytokines and decreased capacity to produce interferon-gamma (IFN-gamma) in peripheral blood mononuclear cells (PBMCs) of patients with atopic dermatitis (AD). OBJECTIVES: To clarify the results of recent studies concerning the role of interleukin (IL)-4 and IL-13 in PBMCs of AD patients, we analysed the activation status of lymphocyte subpopulations. METHODS: We measured the intracellular expression and serum levels of certain type 1 and type 2 cytokines, using cell surface and intracellular cytokine staining, flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: The frequency of IL-10 and IL-13 producing CD4+ and CD8+ T cells was significantly higher in patients with AD, while the frequency of IFN-gamma secreting helper and cytotoxic T cells was significantly lower in patients with AD than in control subjects. The serum levels of IL-10 and IL-13 were also significantly increased. There were no significant differences observed between the experimental groups in the frequency of IL-4 producing CD4+ and CD8+ cells. CONCLUSIONS: This study demonstrates a type 2 cytokine production in the CD4+ and CD8+ T cells of AD patients, which is characterized by an elevated IL-13, but not by IL-4 secretion, and by an increased level of the immunoregulatory IL-10, which can contribute to a decrease in IFN-gamma expression.     

Increased expression of PD1 and CD39 on CD3+CD4+ skin T cells in the elderly

Normal ageing is associated with an impaired systemic immune response contributing to an increased susceptibility to infectious diseases. The aim of this study was to compare the lymphocyte phenotype in human skin from old and young healthy subjects. Skin samples from donors were used for explant cultures before flow cytometric analysis. Our results depicted a higher proportion of CD4⁺ and a lower proportion of CD8⁺ among CD3⁺ T cells, a decreased proportion of CD45RA⁺ naive T cells (3.5 ± 1.9% vs 22.9 ± 11.1%, P ≤ 0.007) and an upregulation of the expression of CD39 and PD1 on CD3⁺CD4⁺ T cells (25.1 ± 8.5% vs 12.5 ± 8.5%, P ≤ 0.003, 68.8 ± 11.6% vs 50.0 ± 11.3%, P ≤ 0.01, respectively) in the skin of old subjects. These findings could explain a reduced generation of long‐lived memory T cells and an impaired antitumoral response in the skin of the elderly.     

Differential expression and in vivo secretion of the antimicrobial peptides psoriasin (S100A7), RNase 7, human beta‐defensin‐2 and ‐3 in healthy human skin

Antimicrobial peptides (AMP) are key players in the skin's defense system. Previous observations suggest a site‐ and age‐dependent expression of individual AMP. We investigated the expression and secretion patterns of four important AMP in a representative collective of healthy human skin samples. Levels of psoriasin, RNase 7 and hBD‐3 expression – assessed by immunohistochemistry – varied between different body localisations. Older individuals expressed hBD‐2 more frequently. No gender‐related expression was observed. The in vivo secretion of psoriasin, measured in skin washing fluids using ELISA, was related to body localisation and age, whereas RNase 7 secretion showed no significant differences regarding these variables. HBD‐2 and ‐3 secretion could not be detected. Our findings suggest the usage of control samples matching localisation and approximate age (in the case of hBD‐2) for comparative immunohistochemical analysis. To avoid bias through great interindividual differences, sufficient large collectives should be used for in vivo secretion analyses.     

Alteration in the production of IL-10 and IL-12 and aberrant expression of CD23, CD83 and CD86 by monocytes or monocyte-derived dendritic cells from atopic dermatitis patients

Atopic dermatitis (AD) is characterized by the presence of Th2-type cells in the skin infiltration as well as in the peripheral blood, although a predominant infiltration of interferon-gamma (IFN-gamma)-producing cells is also reported in the chronic skin lesions of AD. Recently it has become clear that the development of Th1 or Th2 is strongly influenced by factors produced by the antigen presenting cells (APCs). To clarify whether APCs from AD patients play a favorable role in the differentiation of Th2 cells, we compared the production of cytokines and the expression of co-stimulatory molecules by monocytes (MOs) and monocyte-derived DCs (MoDCs) after stimulations with various reagents between 13 AD patients and 13 age-matched healthy controls. We examined their production of IL-1 beta, IL-10, IL-12p40, and IL-12p70, and their expression of CD23, CD40, CD54, CD80, CD83, CD86 and HLA-DR. We stimulated them with superantigens, lipopolysaccharide (LPS), agonistic anti-CD40 antibody, phytohemagglutinins (PHA), IL-1beta/TNF-alpha, IL-4, or IFN-gamma. The following results were obtained (1): IL-10 production was significantly enhanced in AD MOs after LPS stimulation. In contrast, IL-12p40 production was significantly lower in AD MOs than in HC MOs after a variety of stimulations (2). IL-12p40 was also significantly lower in AD MoDCs after LPS stimulation (3). The induction of CD23 with IL-4 was significantly higher in AD MOs. and finally (4), AD MoDCs augmented the expression of CD83, CD86, and HLA-DR less significantly than HC MoDCs after anti-CD40 Ab stimulation. These data indicate that AD APCs show some responses different from those observed in HC APCs after several stimulations, such as LPS, IL-1 beta/TNF-alpha, IL-4, or anti-CD40 Ab, and that these responses might play a role in the polarizing process of helper T cells into Th2 cells as recognized in AD patients.     

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Please cite this paper as: Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes. Experimental Dermatology 2010; 19 : e44–e49. Abstract: Toll‐like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP‐1 and MMP‐9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose‐dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IκB‐α and activated the nuclear translocation of NF‐κB. MMP‐1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11‐7082 (NF‐κB inhibitor) or SP600125 (JNK inhibitor), whereas MMP‐9 expression was inhibited by pretreatment with BAY11‐7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP‐1 and MMP‐9 in human epidermal keratinocytes. In addition, NF‐κB or JNK mediated the MMP‐1 expression induced by TLR2, TLR3 and TLR5, whereas NF‐κB, JNK or p38 MAPK mediated the MMP‐9 expression induced by TLR2, TLR3 and TLR5.