Il Yup Chung

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-5Rα 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.

γ-Secretase Inhibitor Reduces Allergic Pulmonary Inflammation by Modulating Th1 and Th2 Responses

RATIONALE Gamma-secretase inhibitor (GSI) has been used to effectively block Notch signaling, which is implicated in the differentiation and functional regulation of T helper (Th) effector cells. In asthma, a subset of CD4(+) T cells is believed to initiate and perpetuate the disease. OBJECTIVES The aim of this study was to evaluate the therapeutic potential of GSI against allergic asthma. METHODS GSI was administered to an ovalbumin-sensitized mouse via an intranasal route at the time of ovalbumin challenge. MEASUREMENTS AND MAIN RESULTS The administration of GSI inhibits asthma phenotypes, including eosinophilic airway inflammation, goblet cell metaplasia, methacholine-induced airway hyperresponsiveness, and serum IgE production. GSI treatment of bronchoalveolar lavage cells stimulated via TCR or non-TCR pathways led to a decrease in Th2 cytokine production with a concomitant increase in Th1 cytokine secretion. Expression of Hes-1, a target of Notch signaling, was down-regulated in conjunction with a reduction of Notch intracellular domain and GATA-3 levels after GSI treatment of bronchoalveolar lavage cells. GSI treatment resulted in an inhibition of NF-kappaB activation, and combined treatment with GSI and an NF-kappaB inhibitor augmented IFN-gamma production in a synergistic manner. CONCLUSIONS These data suggest that GSI directly regulates Th1 and Th2 responses in allergic pulmonary inflammation through a Notch signaling-dependent pathway and that GSI is of high therapeutic value for treating asthma by inhibiting airway inflammatory responses.

S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor-κB pathways.

Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI‐H292 lung carcinoma cells in a dose‐dependent manner. A Toll‐like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end‐products (RAGE) inhibited only S100A12‐mediated production of MUC5AC. The S100 protein‐mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen‐activated protein kinases, nuclear factor‐κB (NF‐κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal‐regulated kinase (ERK) and nuclear translocation of NF‐κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF‐κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil‐dominant airway inflammation to mucin hyperproduction.

Association of interleukin-5 and eotaxin with acute exacerbation of asthma.

Background: Airway eosinophilia is frequently observed during acute exacerbation of asthma. Interleukin-5 (IL-5) and eotaxin are directly involved in the airway eosinophilia found in persistent asthma. Interrelation between these cytokines is expected to occur in acute exacerbation of asthma. Thus, we evaluated the relevance of interaction between eotaxin and IL-5 in the airway inflammation of acute exacerbation. Methods: We measured the number of inflammatory cells and the amount of eotaxin and IL-5 in sputum from 22 healthy subjects, 21 asthmatics with acute exacerbation and 16 patients with mild persistent asthma, and reassessed these values in 7 subjects with acute exacerbation after 7 days’ treatment with systemic steroid (2 mg/kg/day). Sources of IL-5 and eotaxin were investigated by immunohistochemical staining of sputum cells of 4 cases from each group. Results: Both IL-5 and eotaxin levels were higher in patients with acute exacerbation of asthma than in patients with persistent asthma and normal subjects. IL-5 and eotaxin levels were significantly correlated with eosinophil percentages in mild persistent asthma. Eotaxin staining was found mainly on macrophages and occasionally on eosinophils. Steroid treatment markedly decreased eosinophil percentages and IL-5 levels within 7 days but did not alter eotaxin levels. Conclusions: Both IL-5 and eotaxin are associated with acute exacerbation of asthma. IL-5 rather than eotaxin is effectively decreased by the inhibitory effect of steroid in acute exacerbation.

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway.

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.

Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity.

Objective Aspirin affects interleukin-4 (IL-4) synthesis; however, the genetic role of IL-4 has not been evaluated in asthmatics with aspirin hypersensitivity. The objective of the study was to examine the influence of single nucleotide polymorphisms (SNPs) in IL-4 gene on aspirin hypersensitivity in asthmatics at the genetic and molecular levels. Methods Aspirin-intolerant (AIA, n=103) and aspirin-tolerant asthmatics (n=270) were genotyped and functional promoter assays were performed. Results Of 15 SNPs tested, seven (−589T>C (rs2243250) in promoter, −33T>C (rs2070874) in the 5′-untranslated region, +4047A>G (rs2243266), +4144C>G (rs2243267), +4221C>A (rs2243268), +4367G>A (rs2243270), and +5090A>G (rs2243274) in introns) were significantly associated with AIA risk. The frequency of the rare allele (C) of −589T>C was higher in the AIA group than in the aspirin-tolerant asthmatic group (Pcorr=0.016), and a gene dose-dependent decline in forced expiratory volume in 1 s was noted after an aspirin challenge (P=0.0009). Aspirin unregulated IL-4 mRNA production in Jurkat T and K562 leukemia cells. A reporter plasmid assay revealed that aspirin augmented IL-4 promoter transactivation with the −589T>C C and −33T>C C alleles, compared with that bearing the −589T>C T and −33T>C T alleles. Further, electrophoretic mobility shift assay showed the formation of nuclear complexes with −33T>C and −589T>C allele-containing probes; this was augmented by aspirin. The complexes formed with the −33T>C and −589T>C probes were shifted by treatment with anti-CCAAT-enhancer-binding proteins &bgr; and anti-nuclear factor of activated T-cells antibodies, respectively, indicating the inclusion of these transcription factors. Conclusion Aspirin may regulate IL4 expression in an allele-specific manner by altering the availability of transcription factors to the key regulatory elements in the IL4 promoter, leading to aspirin hypersensitivity.

Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01

A novel lipase gene, lipase A, of Acinetobacter species SY-01 (A. species SY-01) was cloned, sequenced, and expressed in Bacillus subtilis 168. The deduced amino acid (aa) sequences for the lipase A and its chaperone, lipase-specific chaperone, were found to encode mature proteins of 339 aa (37.2 kDa) and 347 aa (38.1 kDa), respectively. The aa sequence of lipase A and lipase-specific chaperone shared high homology 82 and 67% identity with the lipase A and the lipase B of A. species RAG-1. This new lipase was defined as a group I Proteobacterial lipase family. The expressed lipase A was purified through sequential treatment with Q-Sepharose, Resource Q, and Superdex-S75 columns. The maximal activity was observed at 50 degrees C for hydrolysis of p-nitrophenyl monoesters and found to be stable at pH 9-11, with optimal activity at pH 10. Lipase A hydrolyzed wide range of fatty acid esters of p-nitrophenyl, but preferentially hydrolyzed short length acyl chains (C2 and C4). Moreover, lipase A from A. species SY-01 catalyzed hydrolysis of the two acetate isomers of cis-(+/-)-2-(bromomethyl)-2-(2,4-dichloro phenyl)-1,3-dioxolane-4-methyl acetate, an intermediate required for the synthesis of Itraconazole which was an anti-fungal drug, at different rate and yielded cis-(-)-isomer in 81.5% conversion with 91.9% enantiomeric excess.

Elevation of S100 calcium binding protein A9 in sputum of neutrophilic inflammation in severe uncontrolled asthma

BACKGROUND Neutrophilic airway inflammation is frequently observed in severe uncontrolled asthma (UA) and controlled asthma (CA). However, there is no sputum biomarker to differentiate the 2 conditions. OBJECTIVE To identify biomarkers of severe uncontrolled asthma with neutrophilic airway inflammation. METHODS Sputum with a neutrophil content larger than 70% was pooled from 5 patients with severe UA and from 10 patients with CA. Two-dimensional electrophoresis was adopted for differential display proteomics, and candidate proteins were identified using matrix-assisted laser adsorption/ionization-time of flight mass spectrometric analysis. S100 calcium binding protein A9 (S100A9) was identified by western blot and its level was measured in sputum from asthmatics with varying disease severity, patients with chronic obstructive lung disease, and normal controls using enzyme-linked immunosorbent assay. RESULTS Fourteen protein spots exhibited differences in relative intensity between patients with severe UA and those with CA. Matrix-assisted laser adsorption/ionization-time of flight/time of flight of these spots showed an increase in human neutrophil peptide-2, S100A9, β-amylase, neutrophil gelatinase-associated lipocalin, 4-aminobutyrate transaminase, and cystatin SA in patients with UA compared with patients with CA. There was a decrease in the plunc precursor, complement C3 component, immunoglobulin heavy-chain variable region, glial fibrillary acidic protein isoform-1, IgM κIIIb SON, MLL-AF4 der(11) fusion protein, cytokeratin-8, and recombinant IgG4 heavy chain. S100A9 was detected at a higher level in western blots of neutrophilic sputum from patients with severe UA vs CA. S100A9 levels were significantly increased, as measured by enzyme-linked immunosorbent assay, in neutrophilic UA compared with CA, eosinophilic UA and CA, and chronic obstructive lung disease. CONCLUSION S100A9 in sputum may be a biomarker of neutrophilic inflammation in severe UA.

A Single Nucleotide Polymorphism on the Promoter of eotaxin1 Associates with Its mRNA Expression and Asthma Phenotypes

Eotaxin1 plays a pivotal role in eosinophil-associated inflammation. Previously, we demonstrated 14 single-nucleotide polymorphisms (SNPs) in the human eotaxin1 gene and the association between the EOT+67G>A allele and the level of IgE. In this study, we investigated the association between the SNPs and plasma eotaxin1 levels, peripheral blood eosinophil counts, and PC20 methacholine values in normal and asthmatic subjects, and the effects of SNPs on the process of eotaxin1 production. The EOT−576C>T and EOT−384A>G polymorphisms and haplotypes (ht1 and ht4) were significantly associated with plasma eotaxin1 levels in the asthmatics (p < 0.001–0.040). The log [plasma eotaxin1] values correlated with the log [serum total IgE] values in the asthmatics and the normal controls (p = 0.012 and p = 0.004, respectively), and with the log [PC20 methacholine] values in the asthmatics (p = 0.014). A DNA-protein complex was formed with EOT−384A>G, but not with the other SNPs of the promoter. The interaction was stronger with the minor allele than with the common allele, and was reduced upon TNF-α exposure. TNF-α-stimulated PBMCs from the asthmatics with the minor allele homozygote expressed significantly lower levels of eotaxin1 mRNA than those from individuals with the common allele. The EOT+67G>A polymorphism, which substitutes alanine with threonine, did not affect eotaxin1 production or activity. Our data suggest that the EOT−384A>G SNP participates in the regulation of eotaxin1 expression by providing a potential binding site for a repressor, and that the ANOVA of EOT-384A>G may predict asthma phenotypes.

MUC5AC Expression through Bidirectional Communication of Notch and Epidermal Growth Factor Receptor Pathways

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ–dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)–mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.