Hiroyuki Sano

Blockade of eosinophil migration and airway hyperresponsiveness by cPLA2-inhibition

We examined the role of a cytosolic phospholipase A2 (cPLA2) in antigen-induced eosinophil infiltration of airways and in airway hyperresponsiveness to methacholine. Inhibition of cPLA2, or blockade of the platelet-activating factor (PAF) receptor, blocked antigen-induced airway hyperresponsiveness and suppressed eosinophil infiltration. Neither cyclooxygenase nor 5-lipoxygenase inhibition had either effect. We show here that, in antigen-sensitized guinea pigs, cPLA2 inhibition prevents both eosinophilic infiltration and subsequent airway hyperresponsiveness after antigen challenge. We also show that this effect is mediated by first-step hydrolysis of membrane phospholipid into lysophospholipid rather than by prostanoid or leukotriene metabolites of arachidonate.

Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A2 (PLA2) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA2 activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC50 = 8.5 nM)- and time-dependent (t1/2 = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C4 (LTC4) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA2 (cPLA2) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A2 by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca2+-independent phospholipase A2 inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC4 release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1–5 min, and declined thereafter. fMLP stimulation also increased cPLA2 activity in eosinophils, which was inhibited completely by 30 μM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA2 activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA2 activation causes AA hydrolysis and LTC4 secretion. We also find that cPLA2 activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA2 isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC4.

Trichostatin A, a histone deacetylase inhibitor, down-regulates interleukin-12 transcription in SV-40-transformed lung epithelial cells.

Inhibition of histone deacetylation results in increased gene expression. Trichostatin (Ts)A, a specific histone deacetylase (HDAC) inhibitor, up-regulates transcription of some genes but represses expression of others. We quantified histone acetylation in SV-40-transformed lung epithelial cells using flow cytometry. Further, to evaluate the effect of TsA on transcription of genes associated with airway inflammation, we measured interleukin (IL)-8 production by enzyme-linked immunosorbent assay as well as IL-12 transcription by reverse transcription-polymerase chain reaction, in the transformed cells after stimulation with lipopolysaccharide (LPS) in the presence of TsA. Pretreatment of cells with TsA before LPS stimulation induced hyperacetylation of histones (especially in the S phase of the cell cycle), enhanced IL-8 production, and suppressed IL-12p35 and IL-12p40 mRNA accumulation. Thus we have demonstrated a useful way to detect hyperacetylation at the single-cell level, as well as the ability of an HDAC inhibitor to repress genes in epithelial cells.

Extracellular Signal-Regulated Kinase 1/2-Mediated Phosphorylation of Cytosolic Phospholipase A2 Is Essential for Human Eosinophil Adhesion to Fibronectin

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A2 (cPLA2) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 μg/ml FN at 30 min, and decreased after 60–90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA2 were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA2 inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA2 phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA2 phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA2 activation is essential for eosinophil adhesion to FN.

The Strategy for Predicting Future Exacerbation of Asthma Using a Combination of the Asthma Control Test and Lung Function Test

Background. Various factors have been reported to be useful for predicting future exacerbations. Objective. This study was intended to determine a usefulness of a combination of a patient-based questionnaire, such as the Asthma Control Test (ACT) score with objective assessments, such as forced expiratory volume in 1 second (FEV1) and/or exhaled nitric oxide (FENO), for predicting future exacerbations in adult asthmatics. Methods. We therefore enrolled 78 subjects with mild to moderate asthma, who were clinically stable for 3 months who all had been regularly receiving inhaled steroid treatment. All subjects underwent a routine assessment of asthma control including the ACT score, spirometry, and FENO, and then were followed up until a severe exacerbation occurred. The predictors of an increased risk of severe exacerbation were identified and validated using decision trees based on a classification and regression tree (CART) analysis. The properties of the developed models were the evaluated with the area under the ROC curve (AUC) (95% confidence interval [CI]). Results. The CART analysis automatically selected the variables and cut-off points, the ACT score ≤23 and FEV1 ≤ 91.8%, with the greatest capacity for discriminating future exacerbations within one year or not. When the probalility was calculated by the likelihood ratio of a positive test (LP), the ACT score ≤23 was identified with a 60.3% probability, calculated by 1.82 of LP, whereas the combined ACT score ≤23 and the percentage of predicted FEV1 ≤ 91.8% were identified with an 85.0% probability, calculated by an LP score of 5.43, for predicting future exacerbation. Conclusion. These results demonstrated that combining the ACT score and percentage of predicted FEV1, but not FENO, can sufficiently stratify the risk for future exacerbations within one year.

Elevated urinary 8‐hydroxydeoxyguanosine, a biomarker of oxidative stress, and lack of association with antioxidant vitamins in chronic obstructive pulmonary disease

Objective:  The purpose of this study was to investigate whether patients with COPD are under oxidative stress and to elucidate the relationship between the level of oxidative stress and antioxidant vitamins.

Characterization of monoclonal antibodies specific for 14-kDa human group V secretory phospholipase A2 (hVPLA2).

Secretory phospholipase A2 (PLA2) consists of several 14-kDa isoforms with extensive homology, which makes it difficult to identify a specific isoform. In this study, we have developed and characterized monoclonal antibodies (MAbs) directed specifically against human group V sPLA2 (hVPLA2) derived from cultured hybridomas. These hybridomas were produced from the fusion of BALB/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant hVPLA2. Three hybridomas secreting MAbs, MCL-3G1, MCL-2A5, and MCL-1B7, were selected and subcloned on the basis of their specificity to recognize hVPLA2 using solid-phase enzyme-linked immunoadsorbent assay (ELISA). The purified MAbs demonstrated a common pattern of immunoreactivity to hVPLA2, but not to human group IIa isoform (hIIaPLA2). Isotype analysis indicates that these hybridomas are of the IgG1 type. Under reducing conditions, MCL-3G1 sensitively detected hVPLA2 and demonstrated no cross-reactivity to either hIIaPLA2 or group IV cytosolic PLA2. Although specific for hVPLA2, a relatively modest signal was recognized with MCL-1B7 and MCL-2A5. These newly developed MAbs allow for determination of tissue distribution and cell-specific functions of hVPLA2.

Effects on asthma and induction of interleukin-8 caused by Asian dust particles collected in western Japan.

Abstract Objective: Asian dust storms (ADS) contain various airborne particles that may augment airway inflammation by increasing the level of interleukin-8. The objective of the study was to investigate the association of exposure to an ADS with worsening of symptoms of adult asthma and the effect of ADS particles on interleukin-8 transcriptional activity. Methods: The subjects were 112 patients with mild to moderate asthma who recorded scores for their daily upper and lower respiratory tract symptoms and measured morning peak expiratory flow (PEF) from March to May 2011. Interleukin-8 transcriptional activity was assessed in THP-G8 cells that were exposed to airborne particles collected during days of ADS exposure. Results: Of the 112 patients, 31 had comorbid allergic rhinitis (AR) and/or chronic sinusitis (CS), and had worsened scores for upper respiratory tract symptoms on ADS days compared to non-ADS days. Scores for lower respiratory tract symptoms during ADS days were higher than non-ADS days in all patients. Three patients also had unscheduled hospital visits for exacerbation of asthma on ADS days. However, there was no significant difference in daily morning PEF between ADS and non-ADS days. Airborne particles collected on ADS days induced interleukin-8 transcriptional activity in THP-G8 cells compared to the original soil of the ADS. Conclusion: Exposure to an ADS aggravates upper and lower tract respiratory symptoms in patients with adult asthma. ADS airborne particles may increase airway inflammation through enhancement of interleukin-8 transcriptional activity.

Decreased Pulmonary Function in School Children in Western Japan after Exposures to Asian Desert Dusts and Its Association with Interleukin-8

The objective of the study was to investigate the influence of Asian dust storms (ADS) on pulmonary function of school children and the relationship of this effect with interleukin-8. Morning peak expiratory flow (PEF) was measured daily in 399 children from April to May 2012 and in 384 of these children from March to May 2013. The data were analyzed for an association between ADS events and PEF by linear mixed models. Interleukin-8 transcriptional activity was assessed in THP-G8 cells stimulated by airborne particles collected on ADS days. Seven ADS days were identified: April 23 and 24, 2012; March 8 to 10, 2013; and March 19 and 20, 2013. Changes in PEF after ADS exposure were −8.17 L/min (95% confidence interval, −11.40 to −4.93) in 2012 and −1.17 L/min (−4.07 to 1.74) in 2013, and there was a significant difference between 2012 and 2013. Interleukin-8 transcriptional activity was significantly higher in 2012 at 10.6 ± 2.9-fold compared to 3.7 ± 0.4 in March 8 to 10, 2013, and 2.3 ± 0.2 in March 19 and 20, 2013. The influence of ADS events on pulmonary function of children differs with each ADS event and may be related to interleukin-8 production.

Augmentation of eosinophil degranulation and LTC4 secretion by integrin-mediated endothelial cell adhesion

We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C4 and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 ± 15.3 (BSA control) to 778 ± 225 ng/106 cells for eosinophils exposed to interleukin-1α-treated HUVECs ( P < 0.05) and also caused a twofold increase in stimulated LTC4secretion ( P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC4 from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to α4- or β2-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC4 and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion.We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C(4) and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 +/- 15.3 (BSA control) to 778 +/- 225 ng/10(6) cells for eosinophils exposed to interleukin-1alpha-treated HUVECs (P < 0.05) and also caused a twofold increase in stimulated LTC(4) secretion (P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC(4) from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to alpha(4)- or beta(2)-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC(4) and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion.