The role of eosinophils in the pathogenesis of atopic dermatitis - eosinophil granule proteins as markers of disease activity

Currently, there is a large body of evidence that atopic dermatitis (AD) has an Immunologic basis. Atopy-specific helper T cells (Th2-like T cells) may play a pathogenetic role by producing and releasing cytokines rele van t for the allergic inflammation, such as IL-4, IL-5, and other growth factors. Eosinophils are believed to be of major importance as effector cells mediating the pathogenetically rele van t late-phase reaction which is associated with a significant destruction of the surrounding tissue. Accordingly, a significant preactivation of peripheral blood eosinophils was detected in AD patients, leading to an enhanced susceptibility of these cells to distinct stimuli such as IL-5. Toxic proteins, such as eosinophil cationic protein (ECP), contained in the matrix and the core of secondary granules of eosinophils, may play an important role by propagating the allergic inflammatory process and by modulating the immune response. The pathogenetic role of eosinophils in AD is further supported by the detection of these proteins in the eczematous skin of patients. Furthermore, recent data point to a significant correlation between disease activity and deposition of eosinophil granule content: ECP serum levels were significantly increased in AD patients. In addition, ECP levels correlated with the disease activity. Moreover, clinical improvement was associated with a decrease of both the clinical score and serum ECP levels. These data clearly indicate that activated eosinophils may play a major role in the allergic inflammatory process of AD. Therefore, modulation of eosinophil activation could prove to be an important pharmacologic modality for the treatment of AD.     

Expression of a human SOCS protein, HSOCP-1, in peripheral blood eosinophils from patients with atopic dermatitis

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34(SEI-1) was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16(INK4), suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.     

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.     

Eosinophilic gastroenteritis: ultrastructural evidence for a selective release of eosinophil major basic protein.

Ultrastructural study of mucosal eosinophils in a case of eosinophilic gastroenteritis involving stomach, duodenum and ileum showed an altered structure in ulcerated duodenal areas. The electron core density of eosinophil granules was inverted or disappeared and tubulovesicular structures occurred. Using immunogold staining with specific antibodies, major basic protein was detected diffusely in the matrix of eosinophil granules and out of the granules in tight association with extragranular membrane formations. In contrast, eosinophil cationic protein and eosinophil peroxidase were normally distributed in the granule matrix. When compared with the eosinophils in macroscopically normal duodenal mucosa in the same patient, these changes support a role for major basic protein in tissue damage in eosinophilic gastroenteritis. The diffusion of one granule protein from the granules to the exterior of the cells favours the view of a selective release of eosinophil mediators.     

Relationship of serum brain-derived neurotrophic factor level with other markers of disease severity in patients with atopic dermatitis

Elevated serum levels of neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been reported in allergic and autoimmune diseases. The aim of this study was to assess serum levels of BDNF in patients with atopic dermatitis (AD) and to investigate the relationship of the BDNF level with other markers of disease severity. Serum BDNF concentration was significantly higher in patients with AD (n=62) compared to control subjects (n=20) (P<0.01). Stepwise multiple regression analysis showed a significant influence of the peripheral blood eosinophil counts (F=6.90) and the percentage of CD4(+)IL-4(+) (Th2) cells (F=6.61). Moreover, after remission of AD patients with conventionally treated AD patients (n=14), serum levels of BDNF, eosinophil counts and percentage of Th2 cells were decreased significantly. These results suggest that serum BDNF may be a useful marker of disease activity in AD and that both eosinophils and Th2 cells are major cellular sources of serum BDNF.     

Eosinophil major basic protein: first identified natural heparanase-inhibiting protein

BACKGROUND: Heparanase and eosinophils are involved in several diseases, including inflammation, cancer, and angiogenesis. OBJECTIVE: We sought to determine whether eosinophils produce active heparanase. METHODS: Human peripheral blood eosinophils were isolated by immunoselection and tested for heparanase protein (immunocytochemistry, Western blot), mRNA (RT-PCR) and activity (Na(2)[(35)S]O(4)-labeled extracellular matrix degradation) before and after activation. Heparanase intracellular localization (confocal laser microscopy) and ability to bind to eosinophil major basic protein (MBP) were also evaluated (immunoprecipitation). A model of allergic peritonitis resulting in eosinophilia was induced in TNF knockout and wild-type mice for in vivo studies. RESULTS: Eosinophils synthesized heparanase mRNA and contained heparanase in the active (50-kd) and latent (65-kd) forms. Heparanase partially co-localized with and was bound to MBP. No heparanase enzymatic activity was detected in eosinophils resting or activated with various agonists, including GM-CSF/C5a. Eosinophil lysates and MBP inhibited recombinant heparanase activity in a concentration-dependent manner (100%, 2 x 10(-7) mol/L). Eosinophil peroxidase and eosinophil cationic protein, but not myelin basic protein or compound 48/80, partially inhibited heparanase activity. Poly-l-arginine at very high concentrations caused an almost complete inhibition. In allergic peritonitis, heparanase activity in the peritoneal fluid inversely correlated with eosinophil number. CONCLUSIONS: MBP is the first identified natural heparanase-inhibiting protein. Its presence in the eosinophil granules might indicate a protective function of these cells in diseases associated with inflammation and cancer progression.     

Retrospective study on fluticasone propionate aqueous nasal spray efficacy in patients with allergic rhinitis: evaluation of clinical and laboratory parameters

BACKGROUND: In allergic rhinitis, allergenic stimulation causes the release of various mediators that induce symptoms and the development of chronic inflammation, which, in turn, is caused by cells involved in the late phase of inflammation, such as eosinophils. The eosinophils also cause damage at the mucosal level through the secretion of eosinophil cationic protein and other preformed factors contained in their granules. The objective was to verify the efficacy of fluticasone propionate aqueous nasal spray in patients with allergic rhinitis; in a retrospective study, we have evaluated mediators of inflammation, making correlations with the clinical symptoms score during and outside the pollen season. METHODS: Forty patients with allergic rhinitis and 15 normal controls were included in our study. Eosinophil cationic protein, eosinophil chemotactic activity, and blood and nasal lavage eosinophil count were evaluated as laboratory parameters. RESULTS: We found a significant increase in nasal lavage levels of eosinophil cationic protein in allergic patients, and this was strictly correlated with the clinical symptoms score. No differences were found in the eosinophil count of allergic patients and in the serum eosinophil cationic protein of patients sensitized to seasonal allergens in comparison with normal subjects. By contrast, an increase in serum eosinophil cationic protein level was found in patients sensitized to perennial allergens. After topical administration of fluticasone propionate aqueous nasal spray, a reduction in nasal lavage eosinophil cationic protein secretion was obtained with a reduction of eosinophil chemotactic activity at the local level. This reduction correlated with an improvement of clinical symptoms. CONCLUSIONS: The clinical improvement and reduction in nasal lavage eosinophil cationic protein and eosinophil chemotactic activity after administration of fluticasone propionate aqueous nasal spray further confirms the role of this treatment in allergic rhinitis.     

Arginine-rich cationic proteins of human eosinophil granules: comparison of the constituents of eosinophilic and neutrophilic leukocytes.

Several arginine-rich cationic proteins previously isolated from granules of leukemic myeloid cells have been found to reside primarily in human eosinophil leukocytes. The major component has a molecular weight of 21,000 and it contains approximately 2.6 moles of zinc per mole of protein. Velocity centrifugation of cytoplasm from leukocytes of patients with marked eosinophilia showed that this group of proteins is packaged in the crystalloid-containing large eosinophil granules. Approximately 30% of the protein content of eosinophil granules belonged to this group of cationic proteins. Bactericidal or esterolytic activities of the cationic proteins were not detected, nor did they inhibit guinea pig anaphylatoxin or histamine-induced contraction. The basic protein previously demonstrated in guinea pig eosinophils may be analogous to the group of basic proteins of human eosinophils but great differences are found for molecular weight and amino acid composition.     

Dermal deposition of eosinophil-granule major basic protein in atopic dermatitis. Comparison with onchocerciasis

Although blood eosinophilia is commonly present in atopic dermatitis, accumulation of tissue eosinophils is not prominent. To determine whether eosinophil degranulation occurs in lesions of atopic dermatitis, we analyzed tissues by immunofluorescence for the presence of the eosinophil-granule major basic protein. Twenty biopsy specimens from 18 patients with atopic dermatitis were studied, and all showed major basic protein staining outside eosinophils. In 18 specimens, the staining was fibrillar, was located in the upper half of the dermis, and was similar to the distribution of elastic fibers. Twelve specimens with fibrillar staining also showed major basic protein staining in the form of extracellular granules. One specimen from unaffected skin showed minimal faint, fine, fluorescing fibrils, but there was marked deposition of the protein in affected skin. The fibrillar pattern of major basic protein staining in atopic dermatitis was very similar to that seen in lichenified lesions of untreated onchocerciasis. These results suggest that eosinophils commonly release granule proteins in the dermis and that assessment of eosinophil involvement in disease cannot be based simply on numbers of eosinophils in tissue.     

维生素D3上调蛋白1在哮喘嗜酸粒细胞中的表达及其与细胞活化的关系

目的: 探讨维生素D3上调蛋白1（VDUP1）在哮喘患者外周血嗜酸粒细胞（EOS）中的表达及与EOS活化的关系。方法: 实验分为对照组、哮喘发作组和缓解组。抽取外周静脉血15 mL，计算诱导痰EOS%，测定第1秒最大呼气量占预计值百分比（FEV1.0%）和最大呼气中期流速占预计值百分比（PEF%）；ELISA法检测血清嗜酸粒细胞阳离子蛋白（ECP）浓度。RT-PCR及Western blotting方法检测外周血EOS VDUP1表达情况。IL-5 与EOS共孵育，检测EOS VDUP1表达情况，ELISA法检测培养上清液ECP浓度。结果: 哮喘发作组外周血EOS VDUP1表达均显著低于正常组及缓解组，与诱导痰EOS%及血清ECP浓度呈明显负相关；缓解组基因表达与正常对照组无显著差异，与上述指标也无明显相关。IL-5刺激下，EOS VDUP1表达明显降低，同时伴上清ECP明显升高。结论： 哮喘发作时外周血EOS VDUP1表达明显下调，EOS VDUP1表达与血清ECP及诱导痰EOS%呈明显负相关，IL-5促进EOS活化可能与VDUP1通路有关。     

Eosinophil involvement in rheumatoid arthritis as reflected by elevated serum levels of eosinophil cationic protein.

Circulating levels of eosinophil cationic protein (ECP), an eosinophil specific granule protein, and numbers of peripheral eosinophils were determined in 42 patients with rheumatoid arthritis. At the time of the investigation the patients were without drug treatment. They had normal blood counts of eosinophils but on average a five-fold increase of the serum ECP values compared with healthy subjects. The intracellular content of ECP in eosinophils isolated from 14 patients was normal. High serum levels of ECP were particularly observed in patients with a disease of rather short duration but with a more aggressive course. Other factors associated with high ECP values were blood eosinophil counts in the upper normal range, high rheumatoid factor titre and increased inflammatory activity as defined by elevated serum haptoglobin and blood platelet counts. No relation was found between serum ECP and circulating immune complexes or serum total IgE. Synovial fluids obtained from 14 patients with rheumatoid arthritis contained very high concentration of ECP; on average nine times higher than those in the circulation of the patients. During corticosteroid but not NSAID therapy serum ECP decreased on average about 50% compared with pre-treatment values. Although eosinophils are not a notable feature of the synovial membrane infiltrate or cellular joint exudate, data obtained indirectly indicates their participation in the inflammatory reaction in RA.     

Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to granules and intragranular crystals in mature human basophils

Human blood basophils have been shown to form Charcot-Leyden crystals (CLC) and to contain quantities of CLC protein comparable to the eosinophil (Ackerman SJ, Weil GJ, Gleich GJ, J Exp Med 155:1597, 1982). We examined the subcellular localization of CLC protein in human basophils from peripheral blood using an ultrastructural postembedding immunogold method and affinity chromatography-purified polyclonal primary antibody to purified human eosinophil CLC protein. We found CLC protein to be uniquely associated with the main, large, particle-filled granule population of human basophils and particularly within intragranular Charcot-Leyden crystals in these granules. Rarely, CLC protein was localized within small, smooth perigranular vesicles. Neutrophils, lymphocytes, and monocytes present in preparations stained for the CLC protein were negative as were controls for the specificity of the immunogold staining. Mature eosinophils contained small numbers of positive crystalloid-free primary granules, as previously reported (Dvorak AM, Letourneau L, Login GR, Weler PF, Ackerman SJ, Blood 72:150, 1988). The presence of CLC protein in hexagonal and pyramidal crystals regularly present in activated types II and III basophils suggests two possibilities currently being investigated. Like eosinophils, basophils could synthesize CLC protein, or eosinophil-derived CLC protein could be internalized and stored in basophil granules. Regardless of the mechanism for acquisition of CLC protein (lysophospholipase) by basophils, the identification and subcellular localization of this enzyme in basophils requires that it be considered in basophil cell biology as well as in the pathobiology of basophil-rich reactions.     

A basic study on the plasma and serum levels of eosinophil cationic protein (ECP) in bronchial asthma]

A basic study on the blood eosinophil cationic protein (ECP) as an index for eosinophil activation in bronchial asthma was performed. 1) ECP concentration in the serum collected 2 hours after blood sampling and the peripheral eosinophil count were significantly higher in asthmatic patients than in normal individuals. A correlation between these two parameters was observed in normal controls but not in asthmatic patients. These findings suggest that the degree of eosinophil activation, which is reflected by the blood ECP concentration, varies with the clinical condition in asthmatic patients. 2) The plasma and serum ECP levels were compared. The serum ECP was higher than the plasma ECP in both normal controls and asthmatic patients. In asthmatic patients, however, plasma ECP levels poorly correlated with serum levels: the serum levels were markedly elevated in some cases, and only the plasma levels were significantly elevated during symptomatic periods compared with stable periods. Therefore, attention must be paid not only to the serum ECP concentration, but also to the plasma ECP concentration and the plasma-serum difference. 3) This plasma-serum difference in ECP concentration may be due to the in vitro release of ECP during coagulation after blood sampling rather than the effect of alpha 1-macroglobulin concentration, because supernatants from stimulated platelets elicited ECP release from eosinophils.     

Circulating eosinophils in asthma, allergic rhinitis, and atopic dermatitis lack morphological signs of degranulation

BACKGROUND: In allergic diseases, eosinophils in affected tissues release granule proteins with cytotoxic, immunoregulatory, and remodelling-promoting properties. From recent observations, it may be assumed that eosinophils degranulate already in circulating blood. If degranulation occurs in the circulation, this could contribute to widespread systemic effects and provide an important marker of disease. OBJECTIVE: To determine the degranulation status of circulating eosinophils in common allergic diseases. METHODS: Using a novel approach of whole blood fixation and leucocyte preparation, the granule morphology of blood eosinophils from healthy subjects, non-symptomatic patients, symptomatic patients with asthma, asthma and Churg-Strauss syndrome, allergic rhinitis, and atopic dermatitis was evaluated by transmission electron microscopy (TEM) and eosinophil peroxidase (TEM) histochemistry. Plasma and serum levels of eosinophil cationic protein were measured by fluoroenzymeimmunoassay. Selected tissue biopsies were examined by TEM. RESULTS: Regardless of symptoms, circulating eosinophils from allergic patients showed the same granule morphology as cells from healthy subjects. The majority of eosinophil-specific granules had preserved intact electron-density (96%; range: 89-98%), while the remaining granules typically exhibited marginal coarsening or mild lucency of the matrix structure. Abnormalities of the crystalline granule core were rarely detected. Furthermore, granule matrix alterations were not associated with any re-localization of intracellular EPO or increase in plasma eosinophil cationic protein. By contrast, eosinophils in diseased tissues exhibited cytolysis (granule release through membrane rupture) and piecemeal degranulation (loss of granule matrix and core structures). CONCLUSION: In symptomatic eosinophilic diseases, circulating blood eosinophils retain their granule contents until they have reached their target organ.     

Structural, cytochemical, immunocytochemical and ultrastructural characterization of blood granulocytes of the roadside hawk Buteo magnirostris (Gmelin, 1788) (Avian, Falconiform)

In the peripheral blood of the roadside hawk, Buteo magnirostris, the following types of granulocytic leucocytes were identified: heterophil, eosinophil and basophil. The heterophils presented acidophilic and spindle shaped granules, the eosinophils possess spherical eosinophilic granules and the basophils showed spherical and basophilic granules. The heterophils and eosinophils presented positive cytochemical reaction to glycogen and basic polyaminoacid, while the eosinophils presented sudanophilic granules, which were also positive for myeloperoxidase. The heterophils, alone, presented positivity for acid phosphatase in some granules and immunoreactivity to TGF-beta1 was observed only in the cytoplasm of the eosinophils. Electron microscopy demonstrated the heterophil granules as predominantly spindle shaped, being strongly electron-dense, while the eosinophils had numerous uniformly electron-dense spherical granules and the basophils presented three different types of granules identified according to their electron-density and the aspect of their matrix.     

Intracellular expression and serum levels of eosinophil peroxidase (EPO) and eosinophil cationic protein in asthmatic children

BACKGROUND: Eosinophils are involved in the chronic inflammatory response in asthma and their basic proteins are thought to play a major pathophysiological role in this process. While serum levels of basic proteins have been used to monitor the ongoing allergic disease, little is known about the intracellular expression of these proteins in clinical situations. OBJECTIVE: The aim of the study was to determine the intracellular expression of eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in asthmatic children and control subjects and relate it to serum levels of both proteins, lung function tests and immunoglobulin (Ig)E levels. METHODS: Serum ECP and EPO concentrations were determined by immunoassays in 13 asthmatic children (mean age: 9 +/- 1 years, mean FEV1: 92 +/- 10% predicted, geometric mean PC20 histamine 0.5 mg/mL) and 10 age-matched, healthy control subjects. A flow cytometric single cell assay was employed to detect intracellular ECP and EPO in peripheral blood eosinophils. RESULTS: While serum concentrations of both ECP (asthma: median 15.0 microg/L [range 3.6-57.7] vs control: 5.9 microg/L [2.7-9.1]; P = 0.02) and EPO (22.9 microg/L [5.2-82.5] vs 7. 2 microg/L [2.5-12.7]; P = 0.008) were significantly elevated in asthmatics, the intracellular expression of ECP and EPO (measured as mean fluorescence intensity) was decreased (EG1: 55.3 [17.7-120.8] vs 100.3 [46.5-264.4]; P = 0.01; EG2: 80.2 [24.1-135.3] vs 133.7 [32. 1-244.9]; P = 0.04 and EPO: 49.7 [23.1-155.8] vs 94.9 [28.8-115.2]; P = 0.03). In asthmatics there was a significant correlation of FEV1 with intracellular ECP and of bronchial hyperresponsiveness with serum EPO and ECP. Furthermore, total IgE levels were positively correlated with serum EPO only. CONCLUSION: We conclude that in asthmatics the intracellular content of ECP and EPO in peripheral eosinophils is reduced possibly due to degranulation. Epitope masking in activated eosinophils or a shift to early bone marrow-derived progenitors with less granule proteins are further possible explanations.     

Atopic dermatitis: correlation of peripheral blood T cell activation, eosinophilia and serum factors with clinical severity

In the first part of this study peripheral blood lymphocyte subpopulations. Iheir activation slate and various serum parameters were measured in extrinsic and intrinsic atopic dermatitis (AD) patients compared to normal individuals. Beside the characteristic eosinophilia, significantly increased numbers of CD4⁺ T cells with increased expression of IL-2 receptors (IL-2R) and HLA-DR were noted in the AD patients. In addition, extrinsic AD patients showed increased numbers of CD23⁺ B cells and decreased numbers of CD16⁺ natural killer cells. Moreover, increased serum levels of eosinophil canonic protein (ECP) and soluble 1L-2R as well as soluble factors lhat prolong survival of eosinophils in vitro could be demonstrated. In the second section of this study we determine how these blood immunological parameters relate to the clinical severity of the skin lesions of AD, by weekly analysis of 12 AD patients attending a high altitude clinic for 3 to 6 weeks. The patients were divided into two groups on the basis of treatment with topical steroids, but during the observation period a significant improvement in clinical status was observed in all AD patients independent of topical steroid therapy. A progressive decrease in eosinophil and activated T cell numbers. soluble IL-2R levels and serum eosinophil survival prolonging activity could be demonstrated, which closely correlated with the clinical severity of the AD.