Terminally Differentiating Eosinophils Express Neutrophil Primary Granule Proteins as well as Eosinophil-specific Granule Proteins in a Temporal Manner

Neutrophils and eosinophils, 2 prominent granulocytes, are commonly derived from myelocytic progenitors through successive stages in the bone marrow. Our previous genome-wide transcriptomic data unexpectedly showed that genes encoding a multitude of neutrophil primary granule proteins (NPGPs) were markedly downregulated during the end period of eosinophilic terminal differentiation when cord blood (CB) cluster of differentiation (CD) 34+ cells were induced to differentiate toward the eosinophil lineage during a 24-day culture period. Accordingly, this study aimed to examine whether NPGP genes were expressed on the way to eosinophil terminal differentiation stage and to compare their expression kinetics with that of genes encoding eosinophil-specific granule proteins (ESGPs). Transcripts of all NPGP genes examined, including proteinase 3, myeloperoxidase, cathepsin G (CTSG), and neutrophil elastase, reached a peak at day 12 and sharply declined thereafter, while transcript of ESGP genes including major basic protein 1 (MBP1) attained maximum expression at days 18 or 24. Growth factor independent 1 (GFI1) and CCAAT/enhancer-binding protein α (C/EBPA), transactivators for the NPGP genes, were expressed immediately before the NPGP genes, whereas expression of C/EBPA, GATA1, and GATA2 kinetically paralleled that of eosinophil granule protein genes. The expression kinetics of NPGPs and ESGPs were duplicated upon differentiation of the eosinophilic leukemia cell line (EoL-1) immature eosinophilic cells. Importantly, confocal image analysis showed that CTSG was strongly coexpressed with MBP1 in differentiating CB eosinophils at days 12 and 18 and became barely detectable at day 24 and beyond. Our results suggest for the first time the presence of an immature stage where eosinophils coexpress NPGPs and ESGPs before final maturation.

Roles of RUNX1 and PU.1 in CCR3 Transcription

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.

Aspirin induces IL-4 production: augmented IL-4 production in aspirin-exacerbated respiratory disease

Aspirin hypersensitivity is a hallmark of aspirin-exacerbated respiratory disease (AERD), a clinical syndrome characterized by the severe inflammation of the respiratory tract after ingestion of cyclooxygenase-1 inhibitors. We investigated the capacity of aspirin to induce interleukin-4 (IL-4) production in inflammatory cells relevant to AERD pathogenesis and examined the associated biochemical and molecular pathways. We also compared IL-4 production in peripheral blood mononuclear cells (PBMCs) from patients with AERD vs aspirin-tolerant asthma (ATA) upon exposure to aspirin. Aspirin induced IL-4 expression and activated the IL-4 promoter in a report assay. The capacity of aspirin to induce IL-4 expression correlated with its activity to activate mitogen-activated protein kinases, to form DNA-protein complexes on P elements in the IL-4 promoter and to synthesize nuclear factor of activated T cells, critical transcription factors for IL-4 transcription. Of clinical importance, aspirin upregulated IL-4 production twice as much in PBMCs from patients with AERD compared with PBMCs from patients with ATA. Our results suggest that IL-4 is an inflammatory component mediating intolerance reactions to aspirin, and thus is crucial for AERD pathogenesis.

Hypereosinophilia-associated Diseases and the Therapeutic Agents in Development / 한양의대학술지

Eosinophil is one of the most enigmatic leukocytes that plays pleiotropic roles in initiation and propagation of inflammatory conditions, modulation of innate and adaptive immune responses, homeostasis, and remodeling and repair of diverse tissues in health and disease. Eosinophils arise from CD34+ hematopoietic cells in the bone marrow under the influence of transcription factors (C/EBPalpha and GATA-1) and hematopoietic cytokines (IL-5, IL-3, and GM-CSF). The unusually high numbers of eosinophils in blood and/or tissues, so-called hypereosinophilia, are often critically involved in pathophysiology of a wide variety of inflammatory diseases in many organs, including many allergic diseases (asthma, rhinitis, conjunctivitis, atopic dermatitis), gastrointestinal diseases (eosinophilic eosophagitis, ulcerative colitis, Crohn's disease, Duchenne's muscular dystrophy, idiopathic myositis), cancers (pancreas, bladder, liver, kidney, breast, melanoma, colon, glioblastoma, gastric, uterine, oral/nasal, lung), infectious diseases (helminth, bacteria, virus, fungi), transplantation rejection (lung, cardiac, corneal, skin, liver, and renal), reproduction, and autoimmune diseases. A dozen of therapeutic agents, notably including humanized anti-IL-5 monoclonal antibodies, that directly and indirectly target eosinophils have been developed and are studied extensively under clinical and preclinical trials. Some agents have been shown to have promising perspectives to hypereosinophilic diseases, especially against asthma exacerbations and hypereosinophilic syndromes. Further studies are required for discovery of the specific mechanisms of actions of the different eosinophil-targeted therapies, dosing strategies and treatment options with identification of biomarkers that can monitor and predict the responses.

Eosinophil Development, Regulation of Eosinophil-Specific Genes, and Role of Eosinophils in the Pathogenesis of Asthma

Eosinophils arise from hematopoietic CD34+ stem cells in the bone marrow. They acquire IL-5Ralpha on their surface at a very early stage during eosinophilopoiesis, and differentiate under the strong influence of interleukin (IL)-5. They then exit to the bloodstream, and enter the lung upon exposure to airway inflammatory signals, including eotaxins. In inflamed tissues, eosinophils act as key mediators of terminal effector functions and innate immunity and in linking to adaptive immune responses. Transcription factors GATA-1, CCAAT/enhancer-binding protein, and PU.1 play instructive roles in eosinophil specification from multipotent stem cells through a network of cooperative and antagonistic interactions. Not surprisingly, the interplay of these transcription factors is instrumental in forming the regulatory circuit of expression of eosinophil-specific genes, encoding eosinophil major basic protein and neurotoxin, CC chemokine receptor 3 eotaxin receptor, and IL-5 receptor alpha. Interestingly, a common feature is that the critical cis-acting elements for these transcription factors are clustered in exon 1 and intron 1 of these genes rather than their promoters. Elucidation of the mechanism of eosinophil development and activation may lead to selective elimination of eosinophils in animals and human subjects. Furthermore, availability of a range of genetically modified mice lacking or overproducing eosinophil-specific genes will facilitate evaluation of the roles of eosinophils in the pathogenesis of asthma. This review summarizes eosinophil biology, focusing on development and regulation of eosinophil-specific genes, with a heavy emphasis on the causative link between eosinophils and pathological development of asthma using genetically modified mice as models of asthma.

CpG methylation at GATA elements in the regulatory region of CCR3 positively correlates with CCR3 transcription

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.

Identification and functional characterization of an alternative splice variant within the fourth exon of human nanog

Nanog, a homeodomain (HD) transcription factor, plays a critical role in the maintenance of embryonic stem (ES) cell self-renewal. Here, we report the identification of an alternatively-spliced variant of nanog. This variant lacked a stretch of amino acids (residues 168-183) located between the HD and tryptophan-repeat (WR) of the previously-reported full length sequence, suggesting that the deleted sequence functions as a linker and possibly affects the flexibility of the C-terminal transactivation domain relative to the DNA binding domain. Expression of mRNA encoding the splice variant, designated as nanog-delta 48, was much lower than that of the full length version in human ES cells. The ratio of nanog-delta 48 transcript to full length transcript increased, however, in multipotent adult progenitor cells. EMSA analysis revealed that both forms of Nanog were able to bind a nanog binding sequence with roughly the same affinity. A reporter plasmid assay also showed that both variants of nanog modestly repressed transactivation of gata-4, whose expression is proposed to be inhibited by nanog, with comparable potency. We conclude that, despite the difference in primary structure and expression pattern in various stem cells, the alternatively-spliced variant of Nanog has similar activity to that of the full length version.

Proteomic Analysis of Differently Expressed Proteins in a Mouse Model for Allergic Asthma

Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Many proteins involved in allergic asthma have been identified individually, but complete protein profiles (proteome) have not yet been reported. Here we have used a differential proteome mapping strategy to identify tissue proteins that are differentially expressed in mice with allergic asthma and in normal mice. Mouse lung tissue proteins were separated using two-dimensional gel electrophoresis over a pH range between 4 and 7, digested, and then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS). The proteins were identified using automated MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. This approach identified 15 proteins that were differentially expressed in the lungs of mice with allergic asthma and normal mice. All 15 proteins were identified by MS, and 9 could be linked to asthma-related symptoms, oxidation, or tissue remodeling. Our data suggest that these proteins may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy.

Identification of cytokines inhibiting eosinophil activation and the inhibitory mechanism / 천식및알레르기

No abstract available.

The Differentiation of HL-60 Cells Causes to Lose their Ability to Express TNF mRNA / 대한면역학회지

It is well known that HL-60 cell, a human promyelocytic line, is differentiated into eosinophil-like cells in the presence of butyric acid, and thus the differentiated HL-60 cells have been used as a model system to study irnmunological properties of peripheral eosinophils which are thought to be terminally differentiated. To study whether HL-60 cells alter their capability of expressing cytokines during differentiation to eosinophil-like cells, we examined TNF mRNA levels in HL-60 cells treated with butyric acid by Ribonuclease Protection Assay (RPA). HL-60 cells were incubated for 3 days in the presence of butyric acid (0.5 mM), and stimulated with PMA and lipopolysaccharide (LPS). The levels of TNF mRNA decreased by 50 % and 95 % upon one and two days of post-treatment of butyric acid, respectively. The decreased pattern in TNF mRNA levels was also observed in HL-60 cells that have been treated with retinoic acid known as an inducer for differentiation of them. In accordance with these results, prominent azurophilic granules typical in eosinophils appeared in the cytoplasm of the differentiated HL-60 cells. The decreased expression of TNF mRNA was not attributable to the presence of serum, since increasing concentrations of serum had no effect. Furthermore, interleukin-5 (IL-5), which is known to be involved in activation and trafficking of eosinophils in vivo and in vitro, failed to affect TNF mRNA production when it was used in place of butyric acid. These data suggest that the differentiated HL-60 cells may have immunological resemblance to eosinophils in that they weakly produce the cytokine mRNA.

The Effects of Proinflammatory Cytokines and TGF-beta, on The Fibroblast Proliferation / 결핵

BACKGROUNDS: The injury of a tissue results in the infalmmation, and the imflammed tissue is replaced by the normal parenchymal cells during the process of repair. But, constitutional or repetitive damage of a tissue causes the deposition of collagen resulting in the loss of its function These lesions are found in the lung of patients with idiopathic pulmonary fibrosis, complicated fibrosis after diffuse alveolar damage (DAD) and inorganic dust-induced lung fibrosis. The tissue from lungs of patients undergoing episodes of active and/or end-stage pulmonary fibrosis shows the accumulation of inflammatory cells, such as mononuclear cells, neutrophils, mast cells and eosinophils, and fibroblast hyperplasia. In this regard, it appears that the inflammation triggers fibroblast activation and proliferation with enhanced matrix synthesis, stimulated by inflammatory mediators such as interleukin-1 (lL-1) and/or tumor necrosis factor (TNF). It has ken well known that TGF-beta enhance the proliferation of fibroblasts and the production of collagen and fibronectin, and inhibit the degradation of collgen. In this regard, It is likely that TGF-beta undergoes important roles in the pathogenesis of pulmonary fibrosis. Nevertheless, this single cytokine is not the sole regulator of the pulmonary fibrotic response. It is likely that the balance of many cytokines including TGF-beta, IL-1, IL-6 and TNF-alpha regulates the pathogenesis of pulmonary fibrosis. In this study, we investigate the interaction of 'PGF-beta, IL-1beta, IL-6 and TNF-alpha and their effect on the proliferation of fibroblasts. METHODS: We used a human fibroblast cell line, MRC-5 (ATCC). The culture of MRC-5 was confirmed by immunofluorecent staining. First we determined the concentration of serum in cuture medium, in which the proliferation of MRC-5 is supressed but the survival of MRC-5 is retained. Second, we measured optical density after staining the cytokine-stimulated cells with 0.5% naphthol blue black in order to detect the effect of cytokines on the proliferation of MRC-5. RESULT: In the medium containg 0.5% fetal calf serum, the proliferation of MRC-5 increased by 50%, and it was maintained for 6 days. IL-1beta, TNF-alpha and IL-6 induced the proliferation of MRC-5 by 45%, 160% and 120%, respectively. IL-1beta and TNF-a enhanced TGF-beta-induced proliferation of MRC-5 by 64% and 159%, but IL-6 did not affect the TGF-beta-induced proliferation. And TNF-alpha-induced proliferation of MRC-5 was reduced by IL-1beta in 50%. TGF-beta, TNF-alpha and both induced the proliferation of MRC-5 to 89%, 135% and 222 %, respectively. CONCLUSIONS: TNF-alpha TGF-beta and lL-1beta, in the order of the effectiveness, showed the induction of MRC-5 proliferation of MRC-5. TNF-alpha and IL-1beta enhance the TGF-beta-induced proliferation of MRC-5, but IL-6 did not have any effect. TNF-alpha-induced proliferation of MRC-5 is diminished by IL-i, and TNF-alpha and TGF-beta showed a additive effect.