Differential expression of RNA-binding proteins in bronchial epithelium of stable COPD patients.

PURPOSE: Inflammatory gene expression is modulated by posttranscriptional regulation via RNA-binding proteins (RBPs), which regulate mRNA turnover and translation by binding to conserved mRNA sequences. Their role in COPD is only partially defined. This study evaluated RBPs tristetraprolin (TTP), human antigen R (HuR), and AU-rich element-binding factor 1 (AUF-1) expression using lung tissue from COPD patients and control subjects and probed their function in epithelial responses in vitro. PATIENTS AND METHODS: RBPs were detected by immunohistochemistry in bronchial and peripheral lung samples from mild-to-moderate stable COPD patients and age/smoking history-matched controls; RBPs and RBP-regulated genes were evaluated by Western blot, ELISA, protein array, and real-time PCR in human airway epithelial BEAS-2B cell line stimulated with hydrogen peroxide, cytokine combination (cytomix), cigarette smoke extract (CSE), and following siRNA-mediated silencing. Results were verified in a microarray database from bronchial brushings of COPD patients and controls. RBP transcripts were measured in peripheral blood mononuclear cell samples from additional stable COPD patients and controls. RESULTS: Specific, primarily nuclear immunostaining for the RBPs was detected in structural and inflammatory cells in bronchial and lung tissues. Immunostaining for AUF-1, but not TTP or HuR, was significantly decreased in bronchial epithelium of COPD samples vs controls. In BEAS-2B cells, cytomix and CSE stimulation reproduced the RBP pattern while increasing expression of AUF-1-regulated genes, interleukin-6, CCL2, CXCL1, and CXCL8. Silencing expression of AUF-1 reproduced, but not enhanced, target upregulation induced by cytomix compared to controls. Analysis of bronchial brushing-derived transcriptomic confirmed the selective decrease of AUF-1 in COPD vs controls and revealed significant changes in AUF-1-regulated genes by genome ontology. CONCLUSION: Downregulated AUF-1 may be pathogenic in stable COPD by altering posttranscriptional control of epithelial gene expression.

Altered expression of TIAM1 in endotoxin-challenged airway epithelial cells and rodent septic models.

BACKGROUND: In sepsis, reorganization of the actin cytoskeleton in the epithelium during inflammation will lead to a breakdown of epithelial barrier integrity, and contribute to the pathogenesis of sepsis, but the exact changes of various components regulating the actin cytoskeleton pathway remain unclear. METHODS: We used lipopolysaccharide (LPS) challenged primary epithelial cells cultured at the air-liquid interface (ALI) to mimic epithelial barrier dysfunction during sepsis. Then we detected differential expression of T-lymphoma invasion and metastasis 1 (TIAM1) gene in lung epithelial cells and septic models. RESULTS: LPS induced barrier dysfunction in human tracheobronchial epithelial cells (HTBEs) as measured by statistically significant changes in ionic and macromolecular permeability. We observed differential expression of TIAM1 gene. The protein expression of TIAM1 was decreased after LPS challenge, in human bronchial epithelial cells. Furthermore, the expression levels of both TIAM1 mRNA and protein were decreased in lungs of septic rodent models. CONCLUSIONS: Given that expression levels of TIAM1 have been associated with mortality among sepsis patients, our findings have the potential for the development of diagnostic and treatment strategies relevant for patient management.

A genome-wide survey of CD4(+) lymphocyte regulatory genetic variants identifies novel asthma genes.

BACKGROUND: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. OBJECTIVE: We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma. METHODS: eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. RESULTS: Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. CONCLUSIONS: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

Coordinated post-transcriptional regulation of the chemokine system: messages from CCL2.

The molecular cross-talk between epithelium and immune cells in the airway mucosa is a key regulator of homeostatic immune surveillance and is crucially involved in the development of chronic lung inflammatory diseases. The patterns of gene expression that follow the sensitization process occurring in allergic asthma and chronic rhinosinusitis and those present in the neutrophilic response of other chronic inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD) are tightly regulated in their specificity. Studies exploring the global transcript profiles associated with determinants of post-transcriptional gene regulation (PTR) such as RNA-binding proteins (RBP) and microRNAs identified several of these factors as being crucially involved in controlling the expression of chemokines upon airway epithelial cell stimulation with cytokines prototypic of Th1- or Th2-driven responses. These studies also uncovered the participation of these pathways to glucocorticoids' inhibitory effect on the epithelial chemokine network. Unmasking the molecular mechanisms of chemokine PTR may likely uncover novel therapeutic strategies for the blockade of proinflammatory pathways that are pathogenetic for asthma, COPD, and other lung inflammatory diseases.

Aryl hydrocarbon receptor controls murine mast cell homeostasis.

We propose that the aryl hydrocarbon receptor (AhR), a unique chemical sensor, is critical in controlling mast cell differentiation, growth, and function in vitro and in vivo. In antigen-stimulated mast cells, exposure to AhR ligands resulted in a calcium- and reactive oxygen species (ROS)-dependent increase of reversible oxidation in and reduced activity of SHP-2 phosphatase, leading to enhanced mast cell signaling, degranulation, and mediator and cytokine release, as well as the in vivo anaphylactic response. Surprisingly, significant mast cell deficiency was noted in AhR-null mice due to defective calcium signaling and mitochondrial function, concomitant with reduced expression of c-kit and cytosolic STAT proteins, as well as enhanced intracellular ROS and apoptosis. Consequently, AhR-null mast cells responded poorly to stimulation, demonstrating a critical role of AhR signaling in maintaining mast cell homeostasis.

Chronic helminth infection reduces basophil responsiveness in an IL-10-dependent manner.

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.

The impact of self-identified race on epidemiologic studies of gene expression.

Although population differences in gene expression have been established, the impact on differential gene expression studies in large populations is not well understood. We describe the effect of self-reported race on a gene expression study of lung function in asthma. We generated gene expression profiles for 254 young adults (205 non-Hispanic whites and 49 African Americans) with asthma on whom concurrent total RNA derived from peripheral blood CD4(+) lymphocytes and lung function measurements were obtained. We identified four principal components that explained 62% of the variance in gene expression. The dominant principal component, which explained 29% of the total variance in gene expression, was strongly associated with self-identified race (P<10(-16)). The impact of these racial differences was observed when we performed differential gene expression analysis of lung function. Using multivariate linear models, we tested whether gene expression was associated with a quantitative measure of lung function: pre-bronchodilator forced expiratory volume in one second (FEV(1)). Though unadjusted linear models of FEV(1) identified several genes strongly correlated with lung function, these correlations were due to racial differences in the distribution of both FEV(1) and gene expression, and were no longer statistically significant following adjustment for self-identified race. These results suggest that self-identified race is a critical confounding covariate in epidemiologic studies of gene expression and that, similar to genetic studies, careful consideration of self-identified race in gene expression profiling studies is needed to avoid spurious association.

Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes.

Genome-wide association studies of human gene expression promise to identify functional regulatory genetic variation that contributes to phenotypic diversity. However, it is unclear how useful this approach will be for the identification of disease-susceptibility variants. We generated gene expression profiles for 22 184 mRNA transcripts using RNA derived from peripheral blood CD4+ lymphocytes, and genome-wide genotype data for 516 512 autosomal markers in 200 subjects. We screened for cis-acting variants by testing variants mapping within 50 kb of expressed transcripts for association with transcript abundance using generalized linear models. Significant associations were identified for 1585 genes at a false discovery rate of 0.05 (corresponding to P-values ranging from 1 × 10(-91) to 7 × 10(-4)). Importantly, we identified evidence of regulatory variation for 119 previously mapped disease genes, including 24 examples where the variant with the strongest evidence of disease-association demonstrates strong association with specific transcript abundance. The prevalence of cis-acting variants among disease-associated genes was 63% higher than the genome-wide rate in our data set (P = 6.41 × 10(-6)), and although many of the implicated loci were associated with immune-related diseases (including asthma, connective tissue disorders and inflammatory bowel disease), associations with genes implicated in non-immune-related diseases including lipid profiles, anthropomorphic measurements, cancer and neurologic disease were also observed. Genetic variants that confer inter-individual differences in gene expression represent an important subset of variants that contribute to disease susceptibility. Population-based integrative genetic approaches can help identify such variation and enhance our understanding of the genetic basis of complex traits.

TRPV2 has a pivotal role in macrophage particle binding and phagocytosis.

Macrophage phagocytosis is critical for defense against pathogens. Whereas many steps of phagocytosis involve ionic flux, the underlying ion channels remain ill defined. Here we show that zymosan-, immunoglobulin G (IgG)- and complement-mediated particle binding and phagocytosis were impaired in macrophages lacking the cation channel TRPV2. TRPV2 was recruited to the nascent phagosome and depolarized the plasma membrane. Depolarization increased the synthesis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)), which triggered the partial actin depolymerization necessary for occupancy-elicited phagocytic receptor clustering. TRPV2-deficient macrophages were also defective in chemoattractant-elicited motility. Consequently, TRPV2-deficient mice showed accelerated mortality and greater organ bacterial load when challenged with Listeria monocytogenes. Our data demonstrate the participation of TRPV2 in early phagocytosis and its fundamental importance in innate immunity.

Cultured peripheral blood mast cells from chronic idiopathic urticaria patients spontaneously degranulate upon IgE sensitization: Relationship to expression of Syk and SHIP-2.

Recently, signaling changes in the FcvarepsilonRI pathway involving inositol lipid phosphatases have been identified in the basophils of chronic idiopathic urticaria (CIU) subjects. Based on the profile of basophil FcvarepsilonRI-mediated histamine degranulation, we have segregated CIU subjects into two groups, CIU Responder (CIU R) or CIU Nonresponder (CIU NR). In the present study, we compared expression of SHIP-1, SHIP-2, and Syk protein to histamine release (HR) from mast cells (MC) cultured from the peripheral blood of CIU R, CIU NR, and normal subjects. The MC of CIU R donors contained significantly increased Syk and decreased SHIP-2 as compared to CIU NR (Syk: p=0.038, SHIP-2: p=0.038) and normals (Syk: p=0.042, SHIP-2: p=0.027). Spontaneous HR from CIU donors was increased two-fold compared to normals (p=0.04). In summary, our results suggest a possible predilection for urticarial MC to spontaneously degranulate upon IgE sensitization contributing to the increased pruritus associated with CIU.

New concepts in chronic urticaria.

Chronic urticaria is a common skin disease without a clear etiology in the vast majority of cases. The similarity of symptoms and lesion pathology to allergen-induced skin reactions supports the idea that skin mast cell and blood basophil IgE receptor activation is involved; however, no exogenous allergen trigger has been identified. The presence of serum IgG autoantibodies targeting IgE or the IgE receptor in approximately 40% of CIU cases supports the theory of an autoimmune basis for the disease. However, issues remain with the assays to detect autoantibodies among other serum factors, the relationship of autoantibodies to CIU disease activity, and the occurrence of autoantibodies in healthy subjects. Other studies have identified altered IgE receptor degranulation that reverts in disease remission and is accompanied by changes in signaling molecule expression and function in mast cells and basophils in active CIU subjects. The arrival of therapies targeting IgE and the IgE receptor pathway elements has potential use in CIU.

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models.

Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6-8 weeks and basophils with IL-3 (6.7 ng/ml) for 2-3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 +/- 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 +/- 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.

Distinct characteristics of signal transduction events by histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced priming and activation of human basophils.

We previously identified a negative correlation between histamine release to histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) in basophils. We have also demonstrated that HRF/TCTP primes basophils to release mediators. The purpose of this study was to begin characterization of signal transduction events directly induced by HRF/TCTP and to investigate these events when HRF/TCTP is used as a priming agent for human basophil histamine release. Highly purified human basophils were examined for surface expression of bound HRF/TCTP, changes in calcium, and phosphorylation of Akt, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), Syk, and FcepsilonRIgamma. Results showed that basophils from all donors bound HRF/TCTP. There was a biphasic calcium response to HRF/TCTP, which corresponded to the magnitude of histamine release. Furthermore, those donors who have direct histamine release when exposed to HRF/TCTP (HRF/TCTP responder [HRF/TCTP-R] donors) have phosphorylation of Syk, Akt, MEK, and ERK. Remarkably, basophils from HRF/TCTP-nonresponder (HRF/TCTP-NR) donors do not show phosphorylation of these molecules. This finding is different from IL-3, which also primes basophils for histamine release, but does show phosphorylation of these events. We conclude that priming induced by HRF/TCTP is distinct from that induced by IL-3.

Basophil FcepsilonRI histamine release parallels expression of Src-homology 2-containing inositol phosphatases in chronic idiopathic urticaria.

BACKGROUND: Basophils are implicated in the pathogenesis of chronic idiopathic urticaria (CIU). Autoantibodies to the IgE receptor (FcepsilonRI) and serum histamine releasing activity have been detected in some subjects with CIU, although their role in vivo is unclear. Basophils of patients with CIU have altered FcepsilonRI-mediated histamine release (HR); however, the mechanism is unknown. In the basophil FcepsilonRI signaling pathway, protein levels of Src-homology 2-containing-5'-inositol phosphatase (SHIP)-1 are inversely correlated with the release of mediators or releasability. A related phosphatase, SHIP-2, is a negative regulator of monocyte IgG receptor (FcgammaR) signaling . We hypothesized that SHIP levels are altered in CIU basophils. METHODS: Blood basophils were isolated from cold urticaria, CIU, or normal donors, and FcepsilonRI-dependent and independent HR were quantified. Protein levels of SHIP-1, SHIP-2, spleen tyrosine kinase, and phosphorylated Akt were determined by Western blotting. Subjects' serum was tested for serum histamine releasing activity and anti-FcepsilonRIalpha antibodies. RESULTS: CIU basophils displayed a bimodal response to anti-IgE activation. One half of CIU subjects' basophils had reductions in anti-IgE-induced HR and were designated nonresponders (CIU NR). CIU NR basophil HR remained diminished at 10-fold to 30-fold higher doses of anti-IgE. CIU anti-IgE responder basophils had HR similar to normal subjects. SHIP-1 and SHIP-2 proteins were increased in CIU NR basophils and were linked to reduced phosphoAkt after anti-IgE stimulation. CIU basophil anti-IgE response was not related to the presence of serologic factors. CONCLUSION: In CIU basophils, the observed changes in FcepsilonRI signaling pathway molecule expression may underlie changes in releasability. CLINICAL IMPLICATIONS: Patients with CIU can be segregated on the basis of basophil functional phenotype.

Regulation of rat basophilic leukemia-2H3 mast cell secretion by a constitutive Lyn kinase interaction with the high affinity IgE receptor (Fc epsilon RI).

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.

Basophils and mast cells in chronic idiopathic urticaria.

Chronic idiopathic urticaria (CIU) is diagnosed in patients when urticarial eruptions recur for more than 6 weeks, and no specific cause is determined. Given that urticaria resembles the lesions induced by injection of histamine or allergen into the skin, a role for mast cells or basophils has been proposed in the generation of localized urticarial lesions. However, currently, the exact mechanisms governing regional mast cell or basophil activation are unknown. In the past decade, there has been mounting interest in viewing CIU as an autoimmune disease, given the presence of circulating autoantibodies to IgE or the alpha subunit of the high-affinity IgE receptor (FceRI) in a subset of patients. In this review, we propose that in addition to autoantibodies, specific differences in the expression of FceRI-signaling molecules in the basophils or mast cells of CIU patients may contribute to the generation of urticarial eruptions.

Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as eElongation factor-1B beta).

The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions. To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library. We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1delta) encoding for amino acids 12 to 281. This interaction was confirmed by co-immunoprecipitation experiments. Previously, both HrHRF and EF-1delta have been isolated and identified in association with malignancy in numerous studies. EF-1delta is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis. Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone. Our findings of an interaction between HrHRF and EF-1delta taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.

Inhibition of cytokine gene transcription by the human recombinant histamine-releasing factor in human T lymphocytes.

Human recombinant histamine-releasing factor (HrHRF) preincubation enhances the secretion of histamine, IL-4, and IL-13 from FcepsilonRI-stimulated human basophils. In GM-CSF-primed human eosinophils, HrHRF increases IL-8 production. Our recent experiments were designed to evaluate the effects of HrHRF on human T cell cytokine production. Purified T cells were preincubated with GST-tagged HrHRF, followed by stimulation with PMA and A23187 overnight. A partial inhibition of IL-2 and IL-13 production (30 and 75%, respectively) was detected compared with that in cells treated with PMA/A23187 alone. However, the production of IFN-gamma was similar in PMA/A23187 stimulated cells with or without HrHRF. The inhibition of cytokine protein production was dose dependent and specific to the HrHRF portion of GST-HrHRF. The inhibition was not due to endotoxin, since preincubation with polymyxin B and HrHRF gave similar results to that with HrHRF alone. The same pattern and specificity of cytokine regulation were replicated in the Jurkat T cell line as for primary T cells. The PMA/A23187-stimulated activity of a proximal promoter IL-13, IL-4, or IL-2 luciferase construct transfected into Jurkat cells was partially inhibited (60, 32, or 70%, respectively) upon GST-HrHRF preincubation, suggesting that HrHRF functions to inhibit cytokine production in Jurkat cells by preventing gene transcription. The inhibition of IL-2 promoter activation was specific to the HrHRF portion of GST-HrHRF. We conclude that HrHRF, in addition to functioning as a histamine-releasing factor, can differentially modulate the secretion of cytokines from human basophils, eosinophils, T cells, and murine B cells, suggesting that it may induce a complex array of responses at sites of allergic inflammation.