Hajime Oyamada

Elevated serum levels of thioredoxin in patients with acute exacerbation of asthma

The pathogenesis of bronchial asthma is chronic airway inflammation caused by immune cells such as T lymphocytes and eosinophils. Eosinophils release cytotoxic products including reactive oxygen species at the site of inflammation, leading to epithelial damage. Human thioredoxin (TRX), a redox-regulating protein with antioxidant activity, is induced and secreted from cells by oxidative stress. This study was undertaken to investigate the clinical significance of TRX in the pathogenesis of asthma. We collected blood samples from 48 patients with bronchial asthma with or without attack, and measured serum ECP and pulmonary function as well as serum TRX. The serum TRX levels in patients with asthma were significantly increased in patients with mild (34.63 [28.40-42.73] ng/ml, medians with 25 and 75% interquartiles, P=0.0064) and moderate (38.83 [35.14-50.80] ng/ml, P=0.0017) asthma attacks compared with those during the asymptomatic period. The serum TRX levels were inversely correlated with FEV(1.0)% (r=-0.44, P=0.039) and %PEF (r=-0.49, P=0.020) during attack. There was a significant correlation between the serum TRX and the serum eosinophil cationic protein (rs=0.32, P=0.016). These findings suggest that serum TRX is related to the state of asthma exacerbation and allergic inflammation.

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.

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.

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.

Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils

Sexual dimorphism in asthma links the estrogen and allergic immune responses. The function of estrogen was classically believed to be mediated through its nuclear receptors, i.e., estrogen receptors (ERs). However, recent studies established the important roles of G-protein-coupled estrogen receptor (GPER/GPR30) as a novel membrane receptor for estrogen. To date, the role of GPER in allergic inflammation is poorly understood. The purpose of this study was to examine whether GPER might affect the functions of eosinophils, which play an important role in the pathogenesis of asthma. Here, we demonstrated that GPER was expressed in purified human peripheral blood eosinophils both at the mRNA and protein levels. Although GPER agonist G-1 did not induce eosinophil chemotaxis or chemokinesis, preincubation with G-1 enhanced eotaxin (CCL11)-directed eosinophil chemotaxis. G-1 inhibited eosinophil spontaneous apoptosis and caspase-3 activities. The anti-apoptotic effect was not affected by the cAMP-phospodiesterase inhibitor rolipram or phosphoinositide 3-kinase inhibitors. In contrast to resting eosinophils, G-1 induced apoptosis and increased caspase-3 activities when eosinophils were co-stimulated with IL-5. No effect of G-1 was observed on eosinophil degranulation in terms of release of eosinophil-derived neurotoxin (EDN). The current study indicates the functional capacities of GPER on human eosinophils and also provides the previously unrecognized mechanisms of interaction between estrogen and allergic inflammation.

Theophylline and Dexamethasone Induce Peroxisome Proliferator-Activated Receptor-γ Expression in Human Eosinophils

Eosinophils are major effector cells in allergic diseases including asthma. Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARγ and that stimulation with a synthetic agonist for PPARγ attenuated the factor-induced eosinophil survival and chemotaxis. However, the modulator of the eosinophil PPARγ expression has not yet been studied. In this study, we investigated the effect of theophylline and dexamethasone (widely used drugs in the treatment of asthma) on PPARγ expression in eosinophils. Purified human peripheral blood eosinophils were cultured, and therapeutic concentrations of theophylline and dexamethasone were added. Subsequently, PPARγ was measured using quantitative real-time RT-PCR and flow cytometry. Theophylline and dexamethasone markedly enhanced both mRNA and protein levels of PPARγ. These findings suggest that the increase in PPARγ expression on eosinophils may play a role in the anti-inflammatory effects of theophylline and dexamethasone.

RANTES and Eotaxin Enhance CD11b and CD18 Expression on Eosinophils from Allergic Patients with Eosinophilia in the Application of Whole Blood Flow Cytometry Analysis

Background: C-C chemokines and adhesion molecules expressed on eosinophils play an important role in the pathology of allergic inflammatory disease. C-C chemokines such as eotaxin or RANTES are involved in β2 integrin expression on purified eosinophils; so far we have no data on unpurified eosinophils in the peripheral blood. We measured β1 and β2 integrin activation after stimulation with eotaxin or RANTES in vitro using whole-blood flow-cytometric analysis. Methods: Heparinized whole blood obtained from allergic patients with eosinophilia or normal subjects was diluted with the same volume of RPMI 1640, and then cells were incubated in the presence or absence of PMA/ionomycin or chemokines for 45 min at 37°C. After hemolyzation with lysing solution, expression of CD11b, CD11a, CD18 and CD49d on eosinophils was measured using flow cytometry. Results: The expression of CD11b, CD11a and CD18 in allergic patients was significantly higher than that in normal subjects. CD11b and CD18 expression showed a significant increase after stimulation with C-C chemokines, which was remarkable in allergic patients. Conclusion: Eosinophils in the blood of allergic patients exhibited a higher expression of β2 integrins and were more sensitive to RANTES and eotaxin than those of normal subjects.

The Synthetic PPARγ Agonist Troglitazone Inhibits IL-5-Induced CD69 Upregulation and Eosinophil-Derived Neurotoxin Release from Eosinophils

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor that regulates lipid metabolism. Recently, PPARγ was reported to be a negative regulator in the immune system. Eosinophils also express PPARγ, however, the role of PPARγ in eosinophil functions is not well understood. Surface expression of CD69 and eosinophil-derived neurotoxin (EDN) release are well-known activation markers of eosinophils. We investigated the effect of a PPARγ agonist on human eosinophil functions such as IL-5-induced CD69 surface expression and EDN release. IL-5 significantly induced eosinophil CD69 surface expression analyzed using flow cytometry and EDN release measured by ELISA. IL-5-induced eosinophil CD69 surface expression and EDN release were significantly inhibited by the synthetic PPARγ agonist troglitazone, and these effects were reversed by a PPARγ antagonist. The PPARγ agonist troglitazone has a potent inhibitory effect on activation and degranulation of eosinophils, and it may be a therapeutic modality for the treatment of allergic diseases.