Xianglan Yao

p11, a Unique Member of the S100 Family of Calcium-binding Proteins, Interacts with and Inhibits the Activity of the 85-kDa Cytosolic Phospholipase A2

Using a two hybrid system screen of a human cDNA library, we have found that p11, a unique member of the S100 family of calcium-binding proteins, interacts with the carboxyl region of the 85-kDa cytosolic phospholipase A2(cPLA2). p11 synthesized in a cell-free system interacts with cPLA2 in vitro. The p11-cPLA2complex is detectable from a human bronchial epithelial cell line (BEAS 2B). Furthermore, p11 inhibits cPLA2 activity in vitro. Selective inhibition of p11 expression in the BEAS 2B cells by antisense RNA results in an increased PLA2activity as well as an increased release of prelabeled arachidonic acid. This study demonstrates a novel mechanism for the regulation of cPLA2 by an S100 protein.

Dexamethasone Alters Arachidonate Release from Human Epithelial Cells by Induction of p11 Protein Synthesis and Inhibition of Phospholipase A2 Activity

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2(cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11·cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10−6 m). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.

Proteomic Profiling of Human Plasma Exosomes Identifies PPARγ as an Exosome-associated Protein

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-gamma (PPARgamma), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARgamma as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

Apolipoprotein E Negatively Regulates House Dust Mite–induced Asthma via a Low-Density Lipoprotein Receptor–mediated Pathway

RATIONALE Distinct sets of corticosteroid-unresponsive genes modulate disease severity in asthma. OBJECTIVES To identify corticosteroid-unresponsive genes that provide new insights into disease pathogenesis and asthma therapeutics. METHODS Experimental murine asthma was induced by nasal administration of house dust mite for 5 days per week. Dexamethasone and apolipoprotein E (apo E) mimetic peptides were administered via osmotic minipumps. MEASUREMENTS AND MAIN RESULTS Genome-wide expression profiling of the lung transcriptome in a house dust mite-induced model of murine asthma identified increases in apo E mRNA levels that persisted despite corticosteroid treatment. House dust mite-challenged apo E⁻(/)⁻ mice displayed enhanced airway hyperreactivity and goblet cell hyperplasia, which could be rescued by administration of an apo E(130-149) mimetic peptide. Administration of the apo E(130-149) mimetic peptide to house dust mite-challenged apo E⁻(/)⁻ mice also inhibited eosinophilic airway inflammation, IgE production, and the expression of Th2 and Th17 cytokines. House dust mite-challenged low-density lipoprotein receptor (LDLR) knockout mice displayed a similar phenotype as apo E⁻(/)⁻ mice with enhanced airway hyperreactivity, goblet cell hyperplasia, and mucin gene expression, but could not be rescued by the apo E(130-149) mimetic peptide, consistent with a LDLR-dependent mechanism. CONCLUSIONS These findings for the first time identify an apo E-LDLR pathway as an endogenous negative regulator of airway hyperreactivity and goblet cell hyperplasia in asthma. Furthermore, our results demonstrate that strategies that activate the apo E-LDLR pathway, such as apo E mimetic peptides, might be developed into a novel treatment approach for patients with asthma.

5A, an Apolipoprotein A-I Mimetic Peptide, Attenuates the Induction of House Dust Mite-Induced Asthma

New treatment approaches are needed for patients with asthma. Apolipoprotein A-I (apoA-I), the major structural protein of high-density lipoproteins, mediates reverse cholesterol transport and has atheroprotective and anti-inflammatory effects. In this study, we hypothesized that an apoA-I mimetic peptide might be effective at inhibiting asthmatic airway inflammation. A 5A peptide, which is a synthetic, bihelical apoA-I mimetic, was administered to wild-type A/J mice via osmotic mini-pump prior to the induction of house dust mite (HDM)-induced asthma. HDM-challenged mice that received the 5A apoA-I mimetic peptide had significant reductions in the number of bronchoalveolar lavage fluid eosinophils, lymphocytes, and neutrophils, as well as in histopathological evidence of airway inflammation. The reduction in airway inflammation was mediated by a reduction in the expression of Th2- and Th17-type cytokines, as well as in chemokines that promote T cell and eosinophil chemotaxis, including CCL7, CCL17, CCL11, and CCL24. Furthermore, the 5A apoA-I mimetic peptide inhibited the alternative activation of pulmonary macrophages in the lungs of HDM-challenged mice. It also abrogated the development of airway hyperresponsiveness and reduced several key features of airway remodeling, including goblet cell hyperplasia and the expression of collagen genes (Col1a1 and Col3a1). Our results demonstrate that the 5A apoA-I mimetic peptide attenuates the development of airway inflammation and airway hyperresponsiveness in an experimental murine model of HDM-induced asthma. These data support the conclusion that strategies using apoA-I mimetic peptides, such as 5A, might be developed further as a possible new treatment approach for asthma.

Apolipoprotein A-I Attenuates Ovalbumin-Induced Neutrophilic Airway Inflammation via a Granulocyte Colony–Stimulating Factor–Dependent Mechanism

Apolipoprotein A-I (apoA-I) is a key component of high-density lipoproteins that mediates reverse cholesterol transport from cells and reduces vascular inflammation. We investigated whether endogenous apoA-I modulates ovalbumin (OVA)-induced airway inflammation in mice. We found that apoA-I expression was significantly reduced in the lungs of OVA-challenged, compared with saline-challenged, wild-type (WT) mice. Next, to investigate the role of endogenous apoA-I in the pathogenesis of OVA-induced airway inflammation, WT and apoA-I(-/-) mice were sensitized by intraperitoneal injections of OVA and aluminum hydroxide, followed by multiple nasal OVA challenges for 4 weeks. OVA-challenged apoA-I(-/-) mice exhibited a phenotype of increased airway neutrophils compared with WT mice, which could be rescued by an administration of a 5A apoA-I mimetic peptide. Multiple pathways promoted neutrophilic inflammation in OVA-challenged apoA-I(-/-) mice, including the up-regulated expression of (1) proinflammatory cytokines (IL-17A and TNF-α), (2) CXC chemokines (CXCL5), (3) vascular adhesion molecules (i.e., vascular cell adhesion molecule-1), and (4) granulocyte colony-stimulating factors (G-CSF). Because concentrations of G-CSF in bronchoalveolar lavage fluid (BALF) were markedly increased in OVA-challenged apoA-I(-/-) mice, we hypothesized that enhanced G-CSF expression may represent the predominant pathway mediating increased neutrophilic inflammation. This was confirmed by the intranasal administration of a neutralizing anti-G-CSF antibody, which significantly reduced BALF neutrophilia by 72% in OVA-challenged apoA-I(-/-) mice, compared with mice that received a control antibody. We conclude that endogenous apoA-I negatively regulates OVA-induced neutrophilic airway inflammation, primarily via a G-CSF-dependent mechanism. Furthermore, these findings suggest that apoA-I may play an important role in modulating the severity of neutrophilic airway inflammation in asthma.

Interferon-γ stimulates human Clara cell secretory protein production by human airway epithelial cells

Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-γ can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-γ was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-γ (300 U/ml) treatment for 8-36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-γ induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-γ-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-γ can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.

Apolipoprotein Mimetic Peptides: A New Approach for the Treatment of Asthma

New treatments are needed for severe asthmatics to improve disease control and avoid severe toxicities associated with oral corticosteroids. We have used a murine model of house dust mite (HDM)-induced asthma to identify steroid-unresponsive genes that might represent targets for new therapeutic approaches for severe asthma. This strategy identified apolipoprotein E as a steroid-unresponsive gene with increased mRNA expression in the lungs of HDM-challenged mice. Furthermore, apolipoprotein E functioned as an endogenous negative regulator of airway hyperreactivity and goblet cell hyperplasia in experimental HDM-induced asthma. The ability of apolipoprotein E, which is expressed by lung macrophages, to attenuate AHR, and goblet cell hyperplasia is mediated by low density lipoprotein (LDL) receptors expressed by airway epithelial cells. Consistent with this, administration of an apolipoprotein E mimetic peptide, corresponding to amino acids 130–149 of the LDL receptor-binding domain of the holo-apoE protein, significantly reduced AHR and goblet cell hyperplasia in HDM-challenged apoE−/− mice. These findings identified the apolipoprotein E – LDL receptor pathway as a new druggable target for asthma that can be activated by administration of apoE-mimetic peptides. Similarly, apolipoprotein A-I may have therapeutic potential in asthma based upon its anti-inflammatory, anti-oxidative, and anti-fibrotic properties. Furthermore, administration of apolipoprotein A-I mimetic peptides has attenuated airway inflammation, airway remodeling, and airway hyperreactivity in murine models of experimental asthma. Thus, site-directed delivery of inhaled apolipoprotein E or apolipoprotein A-I mimetic peptides may represent novel treatment approaches that can be developed for asthma, including severe disease.

Paradoxical Effects of Rapamycin on Experimental House Dust Mite-Induced Asthma

The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.

Tumor necrosis factor alpha stimulation of human Clara cell secretory protein production by human airway epithelial cells.

Abstract: Clara cell secretory protein (CCSP) or uteroglobin/CC10 is a product of epithelial cells in a variety of organs including the lung. CCSP has anti‐inflammatory properties and may act as an inhibitor of secretory phospholipase A2s. Tumor necrosis factor alpha (TNF‐α) is capable of inducing the expression of gene products including a variety of cytokines and chemokines in the airway epithelium that may upregulate the airway inflammatory response. Therefore, it was of interest to determine whether this proinflammatory cytokine might also induce the production of a counterregulatory protein such as CCSP, which might modulate the inflammatory response in the airway. Normal human tracheobronchial epithelial cells in primary culture and a human bronchial epithelial cell line (BEAS‐2B) were studied. CCSP mRNA levels in BEAS‐2B cells were detected by ribonuclease protection assay. CCSP mRNA levels increased in response to TNF‐α (20 ng/mL) stimulation after 8–36 h, with the peak increase at 18 h. Immunoblotting of CCSP released from BEAS‐2B cells into the culture media demonstrated that TNF‐α induced the synthesis and secretion of CCSP over 8 to 18 h. Similarly, TNF stimulated the release of CCSP from human tracheobronchial epithelial cells in primary culture at 8 and 18 h. The CCSP reporter gene including 801 bases 5′ of the transcription start site did not increase transcriptional activity in response to TNF‐α stimulation. A CCSP mRNA half‐life assay indicated that TNF‐α induced increases in CCSP mRNA at least in part at a posttranscriptional level. Therefore, TNF‐α induces airway epithelial cell expression of human CCSP and may modulate airway inflammatory responses in this manner.