Shigeharu Ueki

Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Allergic airway hyperresponsiveness, inflammation, and remodeling do not develop in phosphoinositide 3-kinase γ–deficient mice

BACKGROUND Bronchial asthma is characterized by chronic airway inflammation caused by inflammatory cells. Phosphoinositide 3-kinases (PI3Ks) are known to play a prominent role in fundamental cellular responses of various inflammatory cells, including proliferation, differentiation, and cell migration. PI3Ks therefore are expected to have therapeutic potential for asthma. Although some investigations of the involvement between the pathogenesis of asthma and PI3K have been performed, it is unknown whether PI3Kgamma, a PI3K isoform, is involved in the pathogenesis of asthma. OBJECTIVE We investigated the role of PI3Kgamma in allergen-induced allergic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling with PI3Kgamma-deficient mice. METHODS After ovalbumin (OVA) sensitization, wild-type (WT) and PI3Kgamma-deficient mice were exposed to aerosolized OVA 3 days per week for 5 weeks. RESULTS In OVA-sensitized and OVA-challenged (OVA/OVA) PI3Kgamma-deficient mice, levels of airway inflammation, AHR, and airway remodeling were significantly decreased compared with those in OVA/OVA WT mice. On the other hand, no significant differences were detected in serum OVA-specific IgE and IgG1 levels and CD4/CD8 balance in bronchoalveolar lavage fluid between OVA/OVA WT mice and OVA/OVA PI3Kgamma-deficient mice. To determine in which phase of allergic responses PI3Kgamma plays a role, we transferred splenocytes from OVA-sensitized WT or PI3Kgamma-deficient mice to naive mice of either genotype. Similar increased levels of eosinophils were induced in both WT recipient mice but not in both PI3Kgamma-deficient recipient mice. CONCLUSION PI3Kgamma might be involved in allergic airway inflammation, AHR, and airway remodeling by regulating the challenge/effector phase of allergic responses.

Physiological Levels of 15-Deoxy-Δ12,14-Prostaglandin J2 Prime Eotaxin-Induced Chemotaxis on Human Eosinophils through Peroxisome Proliferator-Activated Receptor-γ Ligation

15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), mainly produced by mast cells, is known as a potent lipid mediator derived from PGD2 in vivo. 15d-PGJ2 was thought to exert its effects on cells exclusively through peroxisome proliferator-activated receptor-γ (PPARγ) and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), which are both expressed on human eosinophils. However, the physiological role of 15d-PGJ2 remains unclear, because the concentration generated in vivo is generally much lower than that required for its biological functions. In the present study we found that low concentrations (picomolar to low nanomolar) of 15d-PGJ2 and a synthetic PPARγ agonist markedly enhanced the eosinophil chemotaxis toward eotaxin, and the effect was decreased in a dose-dependent manner. Moreover, at a low concentration (10−10 M), 15d-PGJ2 and troglitazone primed eotaxin-induced shape change and actin polymerization. These priming effects were completely reversed by a specific PPARγ antagonist, but were not mimicked by CRTH2 agonist 13,14-dihydro-15-keto-PGD2, suggesting that the effects were mediated through PPARγ ligation. The effect exerted by 15d-PGJ2 parallels the enhancement of Ca2+ influx, but is not associated with the ERK, p38 MAPK, and NF-κB pathways. Furthermore, the time course and treatment of eosinophils with actinomycin D, an inhibitor of gene transcription, indicated that the transcription-independent pathway had a role in this process. PPARγ might interact with an eotaxin-induced cytosolic signaling pathway, because PPARγ is located in the eosinophil cytosol. Taken together with current findings, these results suggest that under physiological conditions, 15d-PGJ2 contributes to allergic inflammation through PPARγ, which plays a role as a biphasic regulator of immune response.

Peroxisome Proliferator-Activated Receptor γ Regulates Eosinophil Functions: A New Therapeutic Target for Allergic Airway Inflammation

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that regulates lipid metabolism and glucose homeostasis. PPARγ is not only highly expressed in adipose tissue but also in cells involved in the immune system, and it exerts anti-inflammatory activities. We showed that eosinophils, a major inflammatory cell in allergic inflammation, express PPARγ. PPARγ negatively modulates eosinophil functions, such as survival, chemotaxis, antibody-dependent cellular cytotoxicity and degranulation. Recently, three independent groups have demonstrated that PPARγ agonists inhibit airway inflammation in an animal model of asthma. This evidence suggests that PPARγ agonists may be a new therapeutic modality for the treatment of allergic diseases including asthma.

Expression of PPARγ in eosinophils and its functional role in survival and chemotaxis

Eosinophils play a pivotal role in the mechanism of allergic diseases including asthma. Interleukin-5 (IL-5) and eotaxin are critical cytokines/chemokines for eosinophil activation. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates lipid metabolism. Recent evidence has suggested that PPARgamma serves as a negative regulator in the immune system. In the present study, we investigated the expression of PPARgamma and effect of PPARgamma agonist on human eosinophils. We demonstrated that purified eosinophils and Eol-1 cells express PPARgamma at the mRNA and protein levels. The PPARgamma agonist troglitazone reduced the IL-5-stimulated, but not spontaneous, eosinophil survival in a concentration-dependent manner. Moreover, the eotaxin-directed eosinophil chemotaxis was dose-dependently inhibited by troglitazone. Our results suggest that the administration of the PPARgamma agonists thiazolidinediones could be a new therapeutic modality for the treatment of allergic diseases such as asthma.

C-reactive protein levels in the serum of asthmatic patients.

BACKGROUND Asthma is a chronic airway inflammatory disease caused by immune cells such as T lymphocytes and eosinophils. Recently, highly sensitive C-reactive protein (hs-CRP) assays have become available for detecting small changes in CRP levels within the reference range, allowing for the evaluation of clinical inflammation. OBJECTIVE To investigate the relationship between hs-CRP levels and bronchial asthma. METHODS We collected blood samples from 109 patients with bronchial asthma, with or without attacks, and measured serum eosinophil cationic protein levels, pulmonary function, and serum CRP levels using an hs-CRP assay. RESULTS Mean serum hs-CRP levels were significantly higher in patients without attacks (0.473 mg/L) and with attacks (0.908 mg/L) (P < .001 for both) than in controls (0.262 mg/L). Serum hs-CRP levels were inversely correlated with forced expiratory volume in 1 second/forced vital capacity in asthmatic patients (r = -0.4915; P < .01). CONCLUSION Serum hs-CRP levels may be related to the state of asthma exacerbation and allergic inflammation.

Retinoic acids are potent inhibitors of spontaneous human eosinophil apoptosis.

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10−6M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.

Leptin has a priming effect on eotaxin-induced human eosinophil chemotaxis.

Background: Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Systemic inflammation caused by adipose tissue in obesity via production of adipokines such as leptin has been attracting attention recently as a contributor to exacerbation of allergic immune reactions. In this study, we examined whether leptin might affect eosinophil chemotactic responses. Methods: Peripheral blood eosinophils were purified, and the effect of leptin on eosinophil migration was investigated using in vitro systems. Results: High concentrations of leptin induced eosinophil chemotaxis and rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase but not calcium mobilization. We also found that pretreatment of eosinophils with physiological concentrations of leptin amplified the chemotactic responses to eotaxin. This leptin-primed chemotaxis appears to be associated with increased calcium mobilization but not with ERK1/2 and p38 pathways. Conclusions: These results indicate that leptin has both direct and indirect effects on eosinophil chemotaxis and intracellular signaling. In physiological settings, leptin may maintain eosinophil accumulation at allergic inflammatory foci.

Gender difference in allergic airway remodelling and immunoglobulin production in mouse model of asthma

Epidemiological studies have shown that the prevalence of adult asthma and severe asthma is higher in women. It has also been reported that female mice are more susceptible than males to the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The influence of gender difference in the pathogenesis of severe asthma, especially airway remodelling in an animal model, has been studied rarely. We investigated gender difference in the development of airway remodelling using a long‐term antigen‐challenged mouse asthma model.

Prostaglandin D2 induces IL-8 and GM-CSF by bronchial epithelial cells in a CRTH2-independent pathway

Background: Prostaglandin D2 (PGD2), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. PGD2 demonstrates its effects through two G-protein-coupled receptors, DP and CRTH2. The PGD2/CRTH2 system mediates chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. Although recent studies have shown that the specific receptors for PGD2, DP, and CRTH2 are expressed in various human tissues, the role of PGD2 is unknown in human bronchial epithelial cells. In this study, we investigated the expression and function of CRTH2/DP on NCI-H292 and NHBE cells. Method: The CRTH2/DP expression was examined by RT-PCR and flow-cytometric analysis. NCI-H292 and NHBE cells were cultured in the presence of various stimulants. The resulting supernatants were measured by ELISA. Results: We demonstrated that PGD2 induced production of IL-8 and GM-CSF in NCI-H292 and NHBE cells. DK-PGD2 (CRTH2 agonist) and latanoprost (FP, a prostaglandin F receptor, agonist) failed to augment the production of these cytokines. Pretreatment with ramatroban (CRTH2 antagonist) and AL8810 (FP antagonist) did not reduce the production of these cytokines. The PGD2-induced cytokine production was inhibited by pertussis toxin or specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). Conclusion: These results suggest that PGD2 is a potent inducer of IL-8 and GM-CSF production with MAP/ERK and p38 MAP kinase activation, but this is independent of CRTH2 activation.