Yumiko Kamada

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.

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.

Expression of mRNA for RANTES in human eosinophils

RANTES has been considered to play an important role in various immune and allergic disorders since RANTES is a potent chemoattractant for various important inflammatory cells such as eosinophils. Eosinophils, on the other hand, are considered to be the major inflammatory cells in bronchial asthma since eosinophil-specific granule proteins can damage bronchial mucosal cells. The recent demonstration that eosinophils themselves could synthesize some cytokines such as IL-5 indicates the role of these cytokines not only in paracrine but also in autocrine regulation of eosinophil production, differentiation and activation. Therefore, in this study, we examined mRNA for RANTES and release of RANTES by human eosinophils. The present findings revealed that eosinophils obtained from asthmatic patients could express and release RANTES. Taken together, these findings suggest that eosinophils play a crucial role in the pathogenesis, particularly in eosinophil and T lymphocyte recruitment into the inflamed sites in asthma through RANTES production.

Possible novel receptor for PGD2 on human bronchial epithelial cells.

Prostaglandin D2 (PGD2), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. Recent studies have shown that PGD2 exerts its effects through two different G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper type-2 cells (CRTH2), expressed in various human tissues. The PGD2/CRTH2 system mediates the chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. We have reported that normal human bronchial epithelial cells (NHBE) and epithelial cell lines (NCI-H292) expressed CRTH2, and PGD2 induces production of IL-8 and GM-CSF. This review discusses the role of CRTH2/DP on epithelial cells and mentions a possible novel receptor for PGD2.

CCR3 mRNA Expression in Bronchial Epithelial Cells and Various Cells in Allergic Inflammation

Background: RANTES and eotaxin are important chemokines involved in the activation and migration of eosinophils and are considered to play a major role in allergic inflammation. Methods: In this study, we used RT-PCR to investigate the kinds of cells that express mRNA for CCR3, a common receptor of these chemokines, and eotaxin, a ligand for CCR3. Results: CCR3 mRNA was expressed in eosinophils, peripheral mononuclear cells, an eosinophilic cell line (EoL-1), a bronchial epithelial cell line (NCI-H292), human endothelial cells and nasal washings from patients with allergic rhinitis. Conclusion: These results suggest that the CCR3-eotaxin system plays an important role in generating inflammation, since these substances are expressed not only in cells implicated in activation or migration of eosinophils but also in various other cells involved in allergic inflammation.

The Synthetic PPARγ Agonist Troglitazone Inhibits Eotaxin-Enhanced Eosinophil Adhesion to ICAM-1-Coated Plates

Accumulation and activation of eosinophils in tissue are critical events in the allergic inflammatory response and adhesion molecules play important roles in this process. We previously demonstrated that human eosinophils expressed a nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ), and that stimulation with a PPARγ agonist attenuated cytokine/chemokine-induced eosinophil activation, such as survival, chemotaxis and degranulation. In the present study, we investigated the effect of troglitazone, a synthetic PPARγ agonist, on adherence to intercellular adhesion molecule-1 (ICAM-1). Eosinophils were purified from human peripheral blood, and the functional adherence to recombinant soluble ICAM-1-coated plates was examined. We found that in the presence of eotaxin, troglitazone inhibited eosinophil adherence in a concentration-dependent manner. This novel activity appears to be associated with modulation of qualitative change of integrins in response to eotaxin, because quantitative reduction of CD11a, CD11b and CD18 expression by troglitazone was not observed using flow cytometry. The PPARγ agonist troglitazone has a potent inhibitory effect on eosinophil adhesion to ICAM-1, and this may be a therapeutic modality for the treatment of eosinophil-related diseases including bronchial asthma.

Regulation of Peroxisome Proliferator-Activated Receptor-γ Expression in Human Eosinophils by Estradiol

Background: Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor that regulates not only adipogenesis but also immune reaction. We previously demonstrated that human eosinophils expressed functional PPARγ, although the modulator of PPARγ expression is less well understood. Because clinical studies have shown that the efficacy of PPARγ agonists as insulin sensitizers is stronger in women than in men, we investigated whether sex hormones caused any changes in eosinophil PPARγ expression levels. Methods: First, purified human peripheral blood eosinophils were cultured with 17β-estradiol for 24 h, followed by PPARγ measurement using a flow cytometer. Next, eosinophil PPARγ expression and serum estradiol were studied in 10 healthy women during the menstrual and follicular phases to identify the physiological significance of estradiol. Eosinophil PPARγ expression was also compared in 22 men, 21 non-pregnant women, and 15 pregnant women. Results: We observed that PPARγ protein expression in eosinophils was significantly enhanced by 10–6M 17β-estradiol. Although serum estradiol concentration was increased during the follicular phase, PPARγ expression levels were not affected by the menstrual cycle. In addition, no significant differences in PPARγ expression were observed in terms of sex and pregnancy. Conclusions: These findings suggest that estradiol potentially upregulates eosinophil PPARγ expression in vitro, although some other mechanisms might be involved in its regulation in vivo.

Hepatocyte Growth Factor Suppresses Production of Reactive Oxygen Species and Release of Eosinophil-Derived Neurotoxin from Human Eosinophils

Background:Reactive oxygen species (ROS) and eosinophilic granule proteins such as eosinophil-derived neurotoxin (EDN) are known to damage bronchial tissue and cause airway hyperresponsiveness (AHR) in asthma. Hepatocyte growth factor (HGF) regulates various biological activities and is known to be a multifunctional factor. In our previous study, we found that HGF suppressed allergic airway inflammation and AHR in a murine model of asthma. However, there have been few reports regarding the detailed mechanism of the anti-allergic effect of HGF in asthma. In this study, we investigated the potential of recombinant HGF to regulate the production of ROS and the release of EDN from human eosinophils. Methods:Eosinophils were isolated from subjects with mild eosinophilia by modified CD16-negative selection. We investigated the expression of CD69, an activation marker of eosinophils, on eosinophils, using flow cytometry. Further, ROS production from eosinophils was analyzed using luminol-dependent chemiluminescence, and EDN release was measured by ELISA. Results:Treatment with HGF suppressed interleukin-5-induced upregulation of CD69 expression, ROS production and EDN release from human eosinophils. Conclusion:Taken together, these data suggest that in asthma, HGF attenuates allergic airway inflammation and AHR through at least the suppression of ROS production and EDN release from eosinophils.