BACKGROUND & AIMS Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low level... more BACKGROUND & AIMS Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha. METHODS Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. RESULTS TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this...
Airway epithelial cells represent the primary cell target of Respiratory Syncytial Virus (RSV) in... more Airway epithelial cells represent the primary cell target of Respiratory Syncytial Virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines, which recruit and activate leukocytes. RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted) is a member of the CC chemokine subfamily and is strongly chemotactic for T-lymphocytes, monocytes, basophils and eosinophils, all cell types which are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression following RSV infection has not been determined. In this study we examined the role of extracellular signal-regulated kinase (ERK) and p38, both members of the mitogen-activated protein ...
Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized con... more Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized contributors in the pathogenesis of lung diseases. It evokes a dramatic increase of phosphorylation events in the inflammatory cells after stimulation. Lipoxin A4 (LxA4) is a novel lipid mediator with putative proresolution properties that appear to function as inhibitory signal during the time course of inflammation. We investigated the influence of LxA4 on intracellular events during stimulation with GM-CSF. Methods: 2D electrophoresis was used to resolve whole-cell proteome, subcellular compartments, and GM-CSF signaling-specific proteome, using biotinylated ligand immunoprecipitation and GM-CSF receptor immunoprecipitation as well. Overall protein phosphorylation was assessed on the gels with phospho-specific stain Pro-Q-Diamond and on the Western blot using anti-phosphotyrosine immunostaining. Phosphorylation of ERK, SHP-2, and STAT-5 was revealed with specific antibodies. Protein inte...
Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 19... more Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 1939–1967 Former Editors: P. Morawitz (1934–1936), R. Staehelin (1937–1943), A. Hurst (1940–1945), W. Löffler (1943–1961), T.C. Hunt (1947–1967), N. Henning (1953–1962), B. Ihre (1953–1967), H. Bartelheimer (1963–1967), M. Demole (1963–1971), H. Kapp (1968–1970), R. Lambert (1972–1978), W. Creutzfeldt (1979–1992), R. Arnold (1993–2003), C. Beglinger (2004–2011), B. Göke (2004–2014), Y. Shinomura (2012–2015)
Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus a... more Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were emp...
Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump... more Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.
Apoptosis : an international journal on programmed cell death, 2016
The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the... more The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apopt...
IgE production by B cell is T cell dependent. Induction of human IgE synthesis requires IL-4 and ... more IgE production by B cell is T cell dependent. Induction of human IgE synthesis requires IL-4 and T/B cell physical interactions. The pivotal role of cytokines and of Fc epsilon R2 and its soluble fragments in regulation of IgE synthesis are presented.
Advances in experimental medicine and biology, 2014
In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a ... more In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work ...
of GMR with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophi... more of GMR with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMR with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMR. Importantly, we found that inhi- bition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2008
Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of a... more Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimul...
BACKGROUND & AIMS Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low level... more BACKGROUND & AIMS Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn's disease. We investigated the roles of transcription factors Sp1 and nuclear factor kappa B (NF-kappaB) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNFalpha. METHODS Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. RESULTS TNFalpha treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-kappaB inhibition before, but not after, 1 hour of TNFalpha-stimulation blocked the expression of ICAM-1. TNFalpha significantly enhanced Sp1/DNA binding. Sp1 bound to the 3' flanking region of a variant kappaB site in the -192/-172 region of ICAM-1 promoter. Mutation of this...
Airway epithelial cells represent the primary cell target of Respiratory Syncytial Virus (RSV) in... more Airway epithelial cells represent the primary cell target of Respiratory Syncytial Virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines, which recruit and activate leukocytes. RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted) is a member of the CC chemokine subfamily and is strongly chemotactic for T-lymphocytes, monocytes, basophils and eosinophils, all cell types which are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression following RSV infection has not been determined. In this study we examined the role of extracellular signal-regulated kinase (ERK) and p38, both members of the mitogen-activated protein ...
Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized con... more Rationale: Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of the recognized contributors in the pathogenesis of lung diseases. It evokes a dramatic increase of phosphorylation events in the inflammatory cells after stimulation. Lipoxin A4 (LxA4) is a novel lipid mediator with putative proresolution properties that appear to function as inhibitory signal during the time course of inflammation. We investigated the influence of LxA4 on intracellular events during stimulation with GM-CSF. Methods: 2D electrophoresis was used to resolve whole-cell proteome, subcellular compartments, and GM-CSF signaling-specific proteome, using biotinylated ligand immunoprecipitation and GM-CSF receptor immunoprecipitation as well. Overall protein phosphorylation was assessed on the gels with phospho-specific stain Pro-Q-Diamond and on the Western blot using anti-phosphotyrosine immunostaining. Phosphorylation of ERK, SHP-2, and STAT-5 was revealed with specific antibodies. Protein inte...
Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 19... more Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 1939–1967 Former Editors: P. Morawitz (1934–1936), R. Staehelin (1937–1943), A. Hurst (1940–1945), W. Löffler (1943–1961), T.C. Hunt (1947–1967), N. Henning (1953–1962), B. Ihre (1953–1967), H. Bartelheimer (1963–1967), M. Demole (1963–1971), H. Kapp (1968–1970), R. Lambert (1972–1978), W. Creutzfeldt (1979–1992), R. Arnold (1993–2003), C. Beglinger (2004–2011), B. Göke (2004–2014), Y. Shinomura (2012–2015)
Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus a... more Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were emp...
Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump... more Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.
Apoptosis : an international journal on programmed cell death, 2016
The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the... more The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apopt...
IgE production by B cell is T cell dependent. Induction of human IgE synthesis requires IL-4 and ... more IgE production by B cell is T cell dependent. Induction of human IgE synthesis requires IL-4 and T/B cell physical interactions. The pivotal role of cytokines and of Fc epsilon R2 and its soluble fragments in regulation of IgE synthesis are presented.
Advances in experimental medicine and biology, 2014
In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a ... more In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work ...
of GMR with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophi... more of GMR with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMR with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMR. Importantly, we found that inhi- bition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2008
Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of a... more Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimul...
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