Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis by differential display

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-gamma stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.     

NR4A orphan nuclear receptor family in peripheral blood eosinophils from patients with atopic dermatitis and apoptotic eosinophils in vitro

To identify novel genes related to the clinical signs of atopic dermatitis (AD), differentially expressed genes were sought in peripheral blood eosinophils from both AD patients and healthy volunteers. RNA was prepared from eosinophils, expression of various genes was monitored using the Affymetrix GeneChip, and expression was quantified by real-time RT-PCR. Two genes, Nur77 and NOR1, members of NR4A orphan nuclear receptor family, were expressed at a significantly higher level in AD patients than in healthy volunteers. Expression of another gene in the NR4A receptor family, Nurr1, was also higher in AD patients than in healthy volunteers. When peripheral blood leukocytes from healthy volunteers were fractionated, NOR1 expression was highest in eosinophils, but expression of Nur77 and Nurr1 genes was not eosinophil-specific. Extremely intense apoptosis was induced in both eosinophils and an eosinophil cell line, AML14.3D10, by treatment with antibody (Ab) to both CD30 and Fas. Rapid expression of the genes for the NR4A receptor family was observed with anti-CD30 Ab treatment but not with anti-Fas Ab. The NR4A orphan nuclear receptor family gene expression and the subsequent eosinophil apoptosis were downregulated by the MAPK inhibitor, U0126. These results suggest that the expression of the NR4A receptor family genes through CD30 signaling may regulate eosinophil apoptosis in allergic conditions such as AD.     

Localization and content of nerve growth factor in peripheral blood eosinophils of atopic dermatitis patients

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing eczematous skin disorder characterized by eosinophilia. Nerve growth factor (NGF) modulates the allergic response through interactions with immune-inflammatory cells. Eosinophils have been reported to store NGF as a preformed mediator. OBJECTIVE: To gain further insight into the significance of eosinophils in association with NGF in the pathogenesis of AD, the localization of NGF within eosinophils and the difference of the eosinophil-derived NGF content in the peripheral blood of normal volunteers vs. AD patients were investigated. METHODS: We examined the localization of NGF within human eosinophils using the post-embedding immunoelectron microscopy and compared NGF content in freshly isolated eosinophil sonicates from the peripheral blood of 31 normal volunteers vs. 42 AD patients by immunoenzymatic assay. A possible correlation between the levels of NGF and major basic protein was also examined. RESULTS: Immunoelectron microscopic studies revealed that NGF was localized in the central core of normal eosinophil granules, where major basic protein is also present as a preformed mediator, in homogeneous granules and in intergranular ductal or vesicular structures adjacent to specific granules of eosinophils. NGF content in eosinophils was significantly increased in AD patients. Furthermore, there was a significant correlation between levels of NGF and major basic protein in eosinophils of AD patients. CONCLUSIONS: Increased levels of NGF contained in eosinophils of the peripheral blood from AD patients, when released with other mediators such as basic proteins, could promote inflammation and local tissue damage.     

Food hypersensitivity reactions and peripheral blood eosinophilia in patients suffering from atopic dermatitis

The aim of our study is to evaluate the count of eosinophils in peripheral blood in atopic dermatitis (AD) patients over 14 years of age and to compare it with the occurrence of food hypersensitivity (FH) reactions. Complete allergological and dermatological examination was performed in 212 patients included in the study (90 men, 122 women, average age 26.7 years, average SCORAD 32.9). According to our results, in AD patients the difference in count of eosinophils in patients with and without FH reactions is not statistically significant. When evaluating the occurrence of FH reactions to single foods, the count of eosinophils is significantly higher only in patients suffering from reactions to carrot.     

Interleukin-5, interleukin-3 and granulocyte-macrophage colony-stimulating factor prime actin-polymerization in human eosinophils: a study with hypodense and normodense eosinophils from patients with atopic dermatitis

Characteristic features of atopic diseases (AD) are immigration and local activation of eosinophils. Reorganization of the cytoskeleton modulates the function of leukocytes and is a prerequisite for the motility response. In this work, the regulation of actin polymerization has been investigated by flow cytometry using NBD-phallacidin and right angle light scatter measurements in purified eosinophils isolated from patients with atopic dermatitis and normal individuals. Stimulation of eosinophils with chemotaxins such as complement fragment C5a (C5a), CC chemokine RANTES/ CCL5 and platelet activating factor (PAF) induced a reversible polymerization of actin. Normodense eosinophils purified from patients with AD showed a decreased chemotaxin-induced actin response as compared to normodense eosinophils from healthy subjects and hypodense eosinophils from patients. Stimulation of eosinophils with Th2-cytokines such as interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF) did not exert a significant effect on actin polymerization. However, pretreatment with IL-3, IL-5 or GM-CSF potentiated the chemotaxin-induced actin polymerization and graded the differential responsiveness between normodense and hypodense eosinophils. We demonstrate a different actin responsiveness in eosinophils from atopic patients and healthy subjects which could be overcome by modulating effects of Th2-cytokines.     

Fc receptors for human, rabbit and pig antibodies on human eosinophils from normal persons and patients with atopic dermatitis

The presence of Fc receptors on eosinophils from normal persons and atopic patients was investigated using a rosetting technique. Normal eosinophils were found to react with pig antibody (32.5% rosettes), rabbit antibody (46,6% rosettes) and human antibody (29.3% rosettes). Eosinophils from atopic dermatitis patients also formed rosettes with these antibodies, but the percentage of rosettes with pig antibody was significantly higher (63,5%; p less than 0.001) than that given by normal eosinophils. A similar result was found with eosinophils from patients with other atopic diseases (asthma and hay fever) suggesting a similar pattern of eosinophil alteration in all atopic patients. On the other hand, eosinophils from patients with non-atopic eczemas did not differ from those of normals.     

60例遗传过敏性皮炎患者外周血淋巴细胞亚群的流式细胞仪检测结果

本文用OKT系列单克隆抗体以流式细胞仪对60例遗传过敏性皮炎(AD)患者(PAD14例、ADR15例、ADI12例及ADRI19例)外周血淋巴细胞亚群进行了检测。结果表明,总患者组T_3~ 、T_8~ 细胞百分比较正常人显著降低,T_4~ /T_8~ 比值升高。在AD分型观察中,ADR、ADI及ADRI等型患者T_8~ 细胞显著降低,T_4~ /T_8~ 比值升高。AD患者血清IgE显著高于正常人,但与T_8~ 细胞低下无相关性。提示T_8~细胞的低下与遗传过敏性背景有关。     

Differential up-regulation of neurotrophin receptors and functional activity of neurotrophins on peripheral blood eosinophils of patients with allergic rhinitis, atopic dermatitis and nonatopic subjects

Background Recent studies suggest that neurotrophins have a pivotal role in neuroimmune interactions. Indeed, in contrast to nonatopic subjects (NA), neurotrophins have been shown to be increased in atopic diseases such as allergic rhinitis (AR) and atopic dermatitis (AD).
Aim The aim of the study was to assess the functional role of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and -4 and the expression of pan-neurotrophin receptor (p75^NTR) and tyrosine kinase (trk)A, -B and -C on peripheral blood eosinophils in AR, AD and NA.
Methods Peripheral blood eosinophils of patients with AR, AD and NA were purified by CD16 negative selection (purity>98%). Neurotrophin receptor expression was analysed by FACS analysis. Apoptosis test (FACS analysis) and chemotactic index (modified Boyden chamber assay) were assessed after stimulation with BDNF, NT-3/-4 and NGF.
Results The expression of trkA-C and p75^NTR was significantly higher in AD>AR>NA ( P <0.05–0.001). Apoptosis was significantly inhibited by BDNF, NGF, NT-3 in AD ( P <0.05–0.001), by NT-3/-4 and NGF in AR ( P <0.05–0.01) and by NT-3 ( P <0.05–0.01) in NA eosinophils. Chemotaxis was significantly induced by BDNF and NT-3/4 ( P <0.01–0.001) in AD peripheral blood eosinophils.
Conclusion Neurotrophin receptor expression and neurotrophin functional activity was greatest in AD>AR>NA. AD eosinophils are pre-activated and may therefore better respond to neurotrophins. With this study, we provide new pathophysiologic insights into atopic diseases with a functional role of neurotrophins in peripheral blood eosinophils in AD and AR.     

Upregulation of IgE synthesis by staphylococcal toxic shock syndrome toxin-1 in peripheral blood mononuclear cells from patients with atopic dermatitis

Background Atopic dermatitis (AD) is a chronic skin disease associated with increased IgE synthesis and colonization with Staphyiococcus aureus secreting exotoxins, such as Toxic Shock Syndrome Toxin-1 (TSST-1). Objectives In this study, we were interested in determining the in vitro effects of TSST-1 on IgE synthesis in peripheral blood mononuclear cells from patients with AD. Methods We stimulated peripheral blood mononuclear cells (PBMC) from AD patients with a wide range of TSST-1 concentrations and measured IgE synthesis by enzyme-linked immunosorbent assay (ELISA) after 14 days. Results We show herein that TSST-1 produced antagonistic effects on IgE synthesis by PBMC from AD patients, depending on the concentration used: IgE synthesis was inhibited at 1000 pg/mL (P<0.05) and enhanced at 0.01 pg/mL (P<0.01) of toxin. TSST-1 was found to induce the production of much higher amounts of interferon-gamma (IFNγ) at 1000 pg/mL than at 0.01pg/mL of toxin (P= 0.0001). More importantly, immunoglobulin E (IgE) synthesis was enhanced by TSST-1 at 1 pg/mL in the presence of antibodies blocking IFN-γ activity. The other immunoglobulin (Ig) isotypes were also increased after TSST-1 stimulation suggesting that the enhanced IgE synthesis was secondary to a polyclonal B cell activation rather than an isotype switch. TSST-1-stimulated IgE synthesis was T cell-dependent because purified tonsil B cells were only able to synthesize increased amounts of IgE when small numbers of T cells were added to the cultures. Anti-HLA-DR and anti-LFA-1 monoclonal antibodies (MoAb) inhibited TSST-1-enhanced IgE synthesis, suggesting that the bridging of the T cell receptor (TCR) and major histocompalibilily complex (MHC) class II on B cells was necessary for activation of B cell differentiation. Conclusion These data indicate that staphylococcal superantigens are able, at concentrations inducing low amounts of IFNγ, to stimulate IgE synthesis by PBMC from AD patients, and suggest that staphylococcal toxins may contribute to elevated IgE synthesis in AD.     

The detection of IgE-secreting cells in the peripheral blood of patients with atopic dermatitis.

We report, for the first time, the identification of IgE-secreting cells in human peripheral blood with an ELISA plaque assay that detects the fingerprint of individual IgE-secreting cells. No IgE-secreting cells could be detected in the blood of normal individuals (IgE, less than 100 IU/ml) or atopic patients (IgE, less than 1000 IU/ml), but in patients with atopic dermatitis (AD) whose IgE was greater than 2000 IU/ml, there was an average of 49 +/- 9 IgE-secreting cells per 10(6) peripheral blood mononuclear cells (PBMNCs). The rate of IgE production per cell per day from the PBMNCs of patients with AD varied from 0.051 to 0.628 IU/ml, and the number of IgE-secreting cells was positively correlated with the serum-IgE levels of these subjects (r = 0.74; p less than 0.001) and the amount of IgE detected in the culture supernatant (r = 0.085; p less than 0.02). Secretion of IgE by these cells could be completely inhibited (96.2% +/- 3%) by the addition of 75 micrograms of cyclohexamide to the cultures. Preformed intracellular IgE comprised 10% of the IgE detected in the supernatants of 7-day cultures. PBMNCs from patients with AD depleted of monocytes by adherence and T cells by E rosetting, all contained some detectable IgE-secreting cells, whereas isolated T cells and monocytes did not, supporting the view that cells secreting IgE that were detected were indeed B cells.     

Serum eosinophil cationic protein in patients with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory disease of the skin, frequently associated with a family history of atopy, raised serum IgE levels and other immunological abnormalities. Both eosinophils and their basic proteins have been detected in the skin lesions of AD patients. We measured the levels of eosinophil cationic protein (ECP) in sera of 24 children with AD and found them to be increased, compared to nonatopic controls, both children and adults. High ECP values were also obtained in 3 patients with the hyper-IgE syndrome. However, no direct relationship between IgE and ECP serum levels could be established. We found no correlation between serum ECP and the number of circulating eosinophils, suggesting that part of ECP was produced by cells infiltrating the tissues. Measurement of ECP might represent a noninvasive tool to assess the activity of AD in relation to eosinophil involvement in this disease.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.     

CCR4 memory CD4+ T lymphocytes are increased in peripheral blood and lesional skin from patients with atopic dermatitis

BACKGROUND: Recent studies have reported that TH1 and TH2 cells express CXCR3 and CCR4, respectively. OBJECTIVE: Our goal was to assess the association of CCR4 and CXCR3 expression with TH2 and TH1 cells and association of CCR4 and CXCR3 expression with inflammation in patients with atopic dermatitis (AD). METHODS: Intracellular cytokine production and chemokine receptor expression in blood T cells were examined by flow cytometry. Immunohistochemical expression of chemokine receptors was also investigated in chronically lesional skin. RESULTS: CCR4+ and CXCR3+ CD4+ T cells predominantly produced IL-4 and IFN-gamma, respectively. Although the frequency of CXCR3+ cells among CD4+ CD45RO+ T cells was similar for patients with AD (n = 29) and healthy control subjects (n = 19), patients with severe AD (n = 14) had a reduced frequency of CXCR3+ cells. In contrast, the frequency of CCR4+ cells and the CCR4/CXCR3 ratio were higher in patients with AD (n = 22) than healthy control subjects (n = 16) and correlated with disease severity of AD. The frequency of CCR4+ cells correlated positively with eosinophil numbers and serum IgE levels, whereas the frequency of CXCR3+ cells correlated inversely with eosinophil numbers. The frequency of CCR4+ or CXCR3+ cells was similar in patients with psoriasis (n = 6) and healthy control subjects. Immunohistochemical analysis showed that the frequency of CCR4+ cells among CD4+ T cells in chronically lesional skin of patients with AD (n = 9) was higher than that of patients with psoriasis (n = 4). CONCLUSION: Our data suggest the association of CCR4 expression with TH2 cells, the predominance of CCR4+ cells in blood from patients with AD, and an important role of CCR4 in the migration of TH2 cells from blood into AD lesional skin.