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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.

Amplified Expression Profiling of Platelet Transcriptome Reveals Changes in Arginine Metabolic Pathways in Patients With Sickle Cell Disease

Background— In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy. Methods and Results— Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54 000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by >10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified ≈100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. Conclusions— The present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants. Crude extracts from isolated EO granules also stimulated RGC release, suggesting that a granular protein might be responsible. Three proteins derived from EO granules, EO-derived neurotoxin, EO cationic protein (ECP), and major basic protein (MBP) were separated by sequential sizing and affinity chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release.

Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages

PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat–containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

Functional Characterization of Human Cysteinyl Leukotriene 1 Receptor Gene Structure

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT1R). The cysLT1R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT1R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT1R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT1R promoter region. IL-4 stimulation increased cysLT1R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD4 and LTC4) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT1R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT1R/cysLT2R inhibitor BAY-u9773, implying a cysLT1R-mediated mechanism. Thus, cysLTs signaling through cysLT1R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.

The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A2 (cPLA2) and lipid mediators produced from cPLA2 activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA2 and cyclooxygenase 2 (COX‐2), and cause prostaglandin E2 (PGE2) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA2 via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA2 activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA2, COX‐2 and production of PGE2 as a cPLA2 inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA2 product arachidonic acid and PGE2 also play an anti‐inflammatory role. This effect of PGE2 is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE2 because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE2.

Dexamethasone reduces rat tracheal goblet cell hyperplasia produced by human neutrophil products.

An experimental model leading to goblet cell hyperplasia was developed in order to examine the possible preventive effects of glucocorticosteroids. Human peripheral blood neutrophils were isolated and either freeze-thaw lysed or activated with serum opsonized zymosan. Supernatants from these neutrophil preparations were then instilled transorally into the tracheas of male Sprague-Dawley rats. After 7-35 days, the rats were sacrificed; the lower tracheas were excised, fixed in formalin, and stained with periodic acid-Schiff and alcian blue; and the goblet cells and total epithelial cells were counted. Supernatants from both lysed neutrophils and zymosan-activated neutrophils stimulated a 50% and 42% (respectively) increase in goblet cells after 3 weeks as compared to controls. Purified human neutrophil or porcine pancreatic elastase also caused goblet cell hyperplasia, while sham challenge, challenge with buffer, or challenge with lysates of human mononuclear cells failed to affect goblet cell number. The increase in goblet-cell number was maximal by three weeks and persisted through 35 days. Treatment of animals with glucocorticosteroids administered by the addition of dexamethasone to the drinking water (2 mg/l for 1 week followed by 0.2 mg/l for 2 weeks) ablated the goblet cell hyperplasia produced by neutrophil lysates or elastase alone. We conclude that goblet cell hyperplasia may be induced with insufflated neutrophil products and that this action can be inhibited by treatment with corticosteroids.

Endothelin-1 induces GM-CSF, IL-6 and IL-8 but not G-CSF release from a human bronchial epithelial cell line (BEAS-2B)

Endothelin (ET) is a powerful vasoconstrictor and bronchoconstrictor peptide that may be involved in the pathogenesis of bronchial asthma. We have investigated the effect of ET on the secretion of IL-6, IL-8, GM-CSF and G-CSF in a bronchial epithelial cell line (BEAS-2B). Incubation of BEAS-2B cells with ET-1 (10(-13) to 10(-7) M) for 4 h caused dose-related increases in the release of IL-8 (68% increase above control, P < 0.001) and IL-6 (43% increase above control, P < 0.001), compared to untreated control cells. After 48 h incubation, ET-1 also increased the release of IL-8 by 35% (P < 0.001) and GM-CSF by 38% (P < 0.01). ET-1 had no significant effect on G-CSF release. ET-1 did not induce cell proliferation at 24 or 48 h. Since ET-immunoreactive materials are expressed in epithelial cells in asthma, it is possible that ET-1 of epithelial origin may act in a paracrine or autocrine fashion on airway epithelial ET receptors to stimulate IL-8, IL-N6 and GM-CSF release. Thus, ET-1 may play a role in the regulation of the cytokine responses involved in inflammation of the airway mucosa.

Abnormal Nasal Nitric Oxide Production, Ciliary Beat Frequency, and Toll-like Receptor Response in Pulmonary Nontuberculous Mycobacterial Disease Epithelium

RATIONALE Pulmonary nontuberculous mycobacterial (PNTM) disease has increased over the past several decades, especially in older women. Despite extensive investigation, no consistent immunological abnormalities have been found. Using evidence from diseases such as cystic fibrosis and primary ciliary dyskinesia, in which mucociliary dysfunction predisposes subjects to high rates of nontuberculous mycobacterial disease that increase with age, we investigated correlates of mucociliary function in subjects with PNTM infections and healthy control subjects. OBJECTIVES To define ex vivo characteristics of PNTM disease. METHODS From 2009 to 2012, 58 subjects with PNTM infections and 40 control subjects were recruited. Nasal nitric oxide (nNO) was determined at the time of respiratory epithelial collection. Ciliary beat frequency at rest and in response to Toll-like receptor (TLR) and other agonists was determined using high-speed video microscopy. MEASUREMENTS AND MAIN RESULTS We found decreased nNO production, abnormally low resting ciliary beat frequency, and abnormal responses to agonists of TLR2, -3, -5, -7/8, and -9 in subjects with PNTM compared with healthy control subjects. The low ciliary beat frequency in subjects with PNTM was normalized ex vivo by augmentation of the NO-cyclic guanosine monophosphate pathway without normalization of their TLR agonist responses. CONCLUSIONS Impaired nNO, ciliary beat frequency, and TLR responses in PNTM disease epithelium identify possible underlying susceptibility mechanisms as well as possible avenues for directed investigation and therapy.

Substance P receptor-mediated secretion of respiratory glycoconjugate from feline airways in vitro

The effect of substance P (SP) and other tachykinins on respiratory glycoconjugate (RGC) release was studied in a feline tracheal organ culture system. SP in concentrations of 10(-5) and 10(-6) M stimulated an increase in RGC release of 35 +/- 8% and 18.5 +/- 5%, respectively. The addition of the protease inhibitor aprotinin or the enkephalinase inhibitor thiorphan to the cultures had no effect on the baseline secretion of RGC but markedly potentiated the activity of SP. SP in the presence of aprotinin or thiorphan was active at 10(-8) -10(-9) M concentrations and was more potent at each concentration studied (in the presence of peptidase inhibitors). Among other tachykinins studied, only physalaemin in the presence of aprotinin had a clear stimulatory effect on RGC release at 10(-6) M concentration (26% +/- 5% increase above control, n = 4, p less than 0.02); kassinin, neurokinin A, and neurokinin B had little or no effect on RGC secretion in concentrations of 10(-6) M or less. Autoradiographic studies of [125I]SP binding revealed SP receptor expression in the submucosal glands of the feline trachea. [125I]SP binding was inhibited in the presence of excess unlabeled SP. We conclude that SP receptors are present in the feline tracheal submucosal glands and that binding to SP receptors results in RGC secretion.