Neurotransmitter signalling via NMDA receptors leads to decreased T helper type 1-like and enhanced T helper type 2-like immune balance in humans.

Given the pivotal roles that CD4+ T cell imbalance plays in human immune disorders, much interest centres on better understanding influences that regulate human helper T-cell subset dominance in vivo. Here, using primary CD4+ T cells and short-term T helper type 1 (Th1) and Th2-like lines, we investigated roles and mechanisms by which neurotransmitter receptors may influence human type 1 versus type 2 immunity. We hypothesized that N-methyl-d-aspartate receptors (NMDA-R), which play key roles in memory and learning, can also regulate human CD4+ T cell function through induction of excitotoxicity. Fresh primary CD4+ T cells from healthy donors express functional NMDA-R that are strongly up-regulated upon T cell receptor (TCR) mediated activation. Synthetic and physiological NMDA-R agonists elicited Ca2+ flux and led to marked inhibition of type 1 but not type 2 or interleukin-10 cytokine responses. Among CD4+ lines, NMDA and quinolinic acid preferentially reduced cytokine production, Ca2+ flux, proliferation and survival of Th1-like cells through increased induction of cell death whereas Th2-like cells were largely spared. Collectively, the findings demonstrate that (i) NMDA-R is rapidly up-regulated upon CD4+ T cell activation in humans and (ii) Th1 versus Th2 cell functions such as proliferation, cytokine production and cell survival are differentially affected by NMDA-R agonists. Differential cytokine production and proliferative capacity of Th1 versus Th2 cells is attributable in part to increased physiological cell death among fully committed Th1 versus Th2 cells, leading to increased Th2-like dominance. Hence, excitotoxicity, beyond its roles in neuronal plasticity, may contribute to ongoing modulation of human T cell responses.

Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

NMDA receptors mediate contractile responses in human airway smooth muscle cells.

Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-d-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.

Fragrance materials in asthma: a pilot study using a surrogate aerosol product.

OBJECTIVE: Many household products contain fragrances. Little is known about exposure to fragrances on human health, particularly within the airways. This study aimed to evaluate how common household fragrance products (i.e. air fresheners, cleaning products) affect people with asthma, who frequently report sensitivity to these products. Many of these products have volatile organic compounds or semi-volatile organic compounds. This study evaluated nine fragrance materials in an aerosol formulation to assess effects on airway physiology, airway inflammation and symptom perception in normal controls and those with asthma. METHODS: The effects of fragrances were evaluated in people without asthma, people with mild asthma and people with moderate asthma in a four-way crossover placebo-controlled study. Subjects were exposed twice to a fragranced aerosol and twice to a placebo aerosol (15 and 30 min each). Subjects completed a questionnaire for 29 symptoms during and up to 3 h after each exposure scenario. Spirometry was performed prior to and 3 h post-exposure; sputum induction was conducted 3 h post-exposure. RESULTS: Nasal symptoms showed the greatest frequency of response in all three subject groups, and moderate asthmatics reported the greatest symptom severity and symptom types. No significant differences were noted in physiology or cellular inflammation. CONCLUSION: A trend for increased symptoms was noted in moderate asthmatics, suggesting that asthma severity may play a factor in fragrance sensitivity.

Changes in thymic regulatory T-cell maturation from birth to puberty: differences in atopic children.

BACKGROUND: Characterization of regulatory immune pathways is a research priority for both the pathogenesis of allergic disease and potential therapeutic strategies. OBJECTIVE: The thymus is a rich source of regulatory T (Treg) cells, which offers a novel opportunity to document the maturation of these pathways beyond limited studies on small volumes of peripheral blood available from young children. METHODS: Thymus tissue was collected from children undergoing cardiac surgery (age, 1 week to 14 years), and skin prick testing was performed from 12 months of age. The ontogeny of Treg cell maturation and function was examined in atopic (n = 20) and nonatopic (n = 20) children by assessing their phenotype, enumeration, proliferation, and suppressive ability. RESULTS: Age-related changes in the thymic cytokine milieu paralleled the changes seen in peripheral immune function. Specifically, the thymic microenvironment is similarly T(H)2 skewed during the early postnatal period, and this undergoes age-related suppression as the T(H)1 (IFN-γ) response increased. We detected CD4(+)CD25(+)CD127(lo/-) forkhead box protein 3 (FOXP3)-positive Treg cells in the neonatal thymus. These cells suppressed the proliferative response to allogeneic stimulation of CD4(+)CD25(-) T cells dose dependently. In nonatopic children Treg cell turnover and suppressive function increased with age and paralleled the increase in global thymic FOXP3 mRNA expression, whereas in atopic children Treg cell maturation was significantly delayed compared with that seen in age-matched nonatopic children. CONCLUSION: These data suggest that the developmental changes in the thymus parallel the recognized changes in peripheral blood responses. There is also a developmental delay in the function of thymic regulatory cells in atopic compared with nonatopic children. These differences are fundamental to understanding early events that lead to immune dysregulation and might predispose to allergic disease.

Human versus mouse eosinophils: "that which we call an eosinophil, by any other name would stain as red".

The respective life histories of human subjects and mice are well defined and describe a unique story of evolutionary conservation extending from sequence identity within the genome to the underpinnings of biochemical, cellular, and physiologic pathways. As a consequence, the hematopoietic lineages of both species are invariantly maintained, each with identifiable eosinophils. This canonical presence nonetheless does not preclude disparities between human and mouse eosinophils, their effector functions, or both. Indeed, many books and reviews dogmatically highlight differences, providing a rationale to discount the use of mouse models of human eosinophilic diseases. We suggest that this perspective is parochial and ignores the wealth of available studies and the consensus of the literature that overwhelming similarities (and not differences) exist between human and mouse eosinophils. The goal of this review is to summarize this literature and in some cases provide experimental details comparing and contrasting eosinophils and eosinophil effector functions in human subjects versus mice. In particular, our review will provide a summation and an easy-to-use reference guide to important studies demonstrating that although differences exist, more often than not, their consequences are unknown and do not necessarily reflect inherent disparities in eosinophil function but instead species-specific variations. The conclusion from this overview is that despite nominal differences, the vast similarities between human and mouse eosinophils provide important insights as to their roles in health and disease and, in turn, demonstrate the unique utility of mouse-based studies with an expectation of valid extrapolation to the understanding and treatment of patients.

Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD).

BACKGROUND: Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiologic roles that eosinophils may serve in other human "eosinophil-associated" diseases remain obscure. OBJECTIVE: National Institutes of Health (NIH) Institutes and the Office of Disease Prevention assembled an international taskforce of clinical and basic scientists with the charge to propose and prioritize unmet research needs in eosinophil-associated diseases. METHODS: The taskforce used an organ system approach to identify the different and common themes of eosinophil cell involvement in these diseases. In early 2012, a draft document was circulated for review. The document was amended and the prioritizations were set at a NIH-organized workshop in June 2012. RESULTS: The taskforce identified significant research needs. These needs cross disease entities but some are disease specific. There are substantial shortcomings to the various preclinical animal models, as well as significant gaps in our epidemiologic, pathophysiologic, diagnostic, prognostic, and therapeutic knowledge. The taskforce recognized that recent efforts by patient advocacy groups have played instrumental roles in improving the identification and characterization of these disorders. However, communications among the eosinophil-interested communities, for example, governmental funding and regulatory agencies, and industry and clinician scientists need to be more comprehensive. CONCLUSIONS: Significant efforts are required to address our knowledge gaps to improve the outcomes of eosinophil-associated diseases. NIH Institutes, other federal agencies, lay organizations, and the pharmaceutical industry should consider the taskforce's recommendations in their future research activities.

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression.

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-ß(1) (TGF-ß(1)), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/ß, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/ß. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/ß on IMR-90 was not inhibited by TGF-ß(1). Lung epithelial cell-derived 14-3-3α/ß has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/ß, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.

Mouse and human eosinophils degranulate in response to platelet-activating factor (PAF) and lysoPAF via a PAF-receptor-independent mechanism: evidence for a novel receptor.

Platelet-activating factor (PAF [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine]) is a phospholipid mediator released from activated macrophages, mast cells, and basophils that promotes pathophysiologic inflammation. Eosinophil responses to PAF are complex and incompletely elucidated. We show in this article that PAF and its 2-deacetylated metabolite (lysoPAF) promote degranulation (release of eosinophil peroxidase) via a mechanism that is independent of the characterized PAFR. Specifically, we demonstrate that receptor antagonists CV-3988 and WEB-2086 and pertussis toxin have no impact on PAF- or lysoPAF-mediated degranulation. Furthermore, cultured mouse eosinophils from PAFR(-/-) bone marrow progenitors degranulate in response to PAF and lysoPAF in a manner indistinguishable from their wild-type counterparts. In addition to PAF and lysoPAF, human eosinophils degranulate in response to lysophosphatidylcholine, but not phosphatidylcholine, lysophosphatidylethanolamine, or phosphatidylethanolamine, demonstrating selective responses to phospholipids with a choline head-group and minimal substitution at the sn-2 hydroxyl. Human eosinophils release preformed cytokines in response to PAF, but not lysoPAF, also via a PAFR-independent mechanism. Mouse eosinophils do not release cytokines in response to PAF or lysoPAF, but they are capable of doing so in response to IL-6. Overall, our work provides the first direct evidence for a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, we document and define PAF and lysoPAF-mediated activities that are not dependent on signaling via PAFR, suggesting the existence of other unexplored molecular signaling pathways mediating responses from PAF, lysoPAF, and closely related phospholipid mediators.

Metabolomic profiling of asthma: diagnostic utility of urine nuclear magnetic resonance spectroscopy.

BACKGROUND: The ability to diagnose and monitor asthma on the basis of noninvasive measurements of airway cellular dysfunction is difficult in the typical clinical setting. OBJECTIVE: Metabolomics is the study of molecules created by cellular metabolic pathways. We hypothesized that the metabolic activity of children with asthma would differ from healthy children without asthma. Furthermore, children having an asthma exacerbation would be different compared with children with stable asthma in outpatient clinics. Finally, we hypothesized that (1)H-nuclear magnetic resonance (NMR) would measure such differences using urine samples, one of the least invasive forms of biofluid sampling. METHODS: Children (135 total, ages 4-16 years) were enrolled, having met the criteria of healthy controls (C), stable asthma in the outpatient clinic (AO), or unstable asthma in the emergency department (AED). Partial least squares discriminant analysis was performed on the NMR data to create models of separation (70 metabolites were measured/urine sample). Some NMR data were withheld from modeling to be run blindly to determine possible diagnostic accuracy. RESULTS: On the basis of the model of AO versus C, 31 of 33 AO samples were correctly diagnosed with asthma (94% accuracy). Only 1 of 20 C samples was incorrectly labeled as asthma (5% misclassification). On the basis of the AO versus AED model, 31 of the 33 AO samples were correctly diagnosed as outpatient asthma (94% accurate). CONCLUSION: This is the first report suggesting that (1)H-NMR analysis of human urine samples has the potential to be a useful clinical tool for physicians treating asthma.

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI. METHODS: Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry. RESULTS: EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy. CONCLUSION: The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.

Agonist activation of f-actin-mediated eosinophil shape change and mediator release is dependent on Rac2.

BACKGROUND: Tissue recruitment and activation of eosinophils contribute to allergic symptoms by causing airway hyperresponsiveness and inflammation. Shape changes and mediator release in eosinophils may be regulated by mammalian Rho-related guanosine triphosphatases. Of these, Rac2 is essential for F-actin formation as a central process underlying cell motility, exocytosis, and respiratory burst in neutrophils, while the role of Rac2 in eosinophils is unknown.We set out to determine the role of Rac2 in eosinophil mediator release and F-actin-dependent shape change in response to chemotactic stimuli. METHODS: Rac2-deficient eosinophils from CD2-IL-5 transgenic mice crossed with rac2 gene knockout animals were examined for their ability to release superoxide through respiratory burst or eosinophil peroxidase by degranulation. Eosinophil shape change and actin polymerization were also assessed by flow cytometry and confocal microscopy following stimulation with eotaxin-2 or platelet-activating factor. RESULTS: Eosinophils from wild-type mice displayed inducible superoxide release, but at a small fraction (4-5%) of human eosinophils. Rac2-deficient eosinophils showed significantly less superoxide release (p < 0.05, 26% less than wild type). Eosinophils lacking Rac2 had diminished degranulation (p < 0.05, 62% less eosinophil peroxidase) and shape changes in response to eotaxin-2 or platelet-activating factor (with 68 and 49% less F-actin formation, respectively; p < 0.02) compared with wild-type cells. CONCLUSION: These results demonstrate that Rac2 is an important regulator of eosinophil function by contributing to superoxide production, granule protein release, and eosinophil shape change. Our findings suggest that Rho guanosine triphosphatases are key regulators of cellular inflammation in allergy and asthma.

Eosinophil granules function extracellularly as receptor-mediated secretory organelles.

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-gamma, and G protein-coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Thymic indoleamine 2,3-dioxygenase-positive eosinophils in young children: potential role in maturation of the naive immune system.

Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils ( approximately 2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = -0.57, P = 0.02) and toll-like receptor 4 expression (r = -0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses.

Metabolomic biomarkers in a model of asthma exacerbation: urine nuclear magnetic resonance.

RATIONALE: Airway obstruction in patients with asthma is associated with airway dysfunction and inflammation. Objective measurements including sputum analysis can guide therapy, but this is often not possible in typical clinical settings. Metabolomics is the study of molecules generated by metabolic pathways. We hypothesize that airway dysfunction and inflammation in an animal model of asthma would produce unique patterns of urine metabolites measured by multivariate statistical analysis of high-resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy data. OBJECTIVES: To develop a noninvasive means of monitoring asthma status by metabolomics and urine sampling. METHODS: Five groups of guinea pigs were studied: control, control treated with dexamethasone, sensitized (ovalbumin, administered intraperitoneally), sensitized and challenged (ovalbumin, administered intraperitoneally, plus ovalbumin aerosol), and sensitized-challenged with dexamethasone. Airway hyperreactivity (AHR) to histamine (administered intravenously) and inflammation were measured. Multivariate statistical analysis of NMR spectra based on a library of known urine metabolites was performed by partial least-squares discriminant analysis. In addition, the raw NMR spectra exported as xy-trace data underwent linear discriminant analysis. MEASUREMENTS AND MAIN RESULTS: Challenged guinea pigs developed AHR and increased inflammation compared with sensitized or control animals. Dexamethasone significantly improved AHR. Using concentration differences in metabolites, partial least-squares discriminant analysis could discriminate challenged animals with 90% accuracy. Using only three or four regions of the NMR spectra, linear discriminant analysis-based classification demonstrated 80-90% separation of the animal groups. CONCLUSIONS: Urine metabolites correlate with airway dysfunction in an asthma model. Urine NMR analysis is a promising, noninvasive technique for monitoring asthma in humans.

CCL5 persists in RSV stocks following sucrose-gradient purification.

Respiratory syncytial virus (RSV) is associated with bronchiolitis in infancy and the later development of asthma. Research on RSV in vitro requires preparation of a purified RSV stock. The objective for this work was to develop best methods for RSV purification, while monitoring the samples for potential contaminating proinflammatory mediators. Using polyethylene glycol concentration, and sucrose-gradient ultracentrifugation, we collected samples at each step of purification and measured the values of RSV titer, total protein (µg/mL), and proinflammatory cytokines (ELISA). We analyzed the efficacy of each step in the purification procedure. In so doing, we also determined that despite optimal purification methods, a well-known chemokine in the field of allergic disease, CCL5 (RANTES), persisted within the virus preparations, whereas other cytokines did not. We suggest that researchers should be aware that CCL5 appears to co-purify with RSV. Despite reasonable purification methods, a significant level of CCL5 (RANTES) persists in the virus preparation. This is relevant to the study of RSV-induced allergic disease.

The effect of cationic charge on release of eosinophil mediators.

BACKGROUND: In patients with atopic diseases, cationic-charged eosinophil proteins are deposited in inflamed tissues. Although the role of cytokines in cell activation is well established, the presence of cationic-charged tissue can also be an important factor in inflammatory cell function. OBJECTIVES: We sought to determine whether increased cationic charge seen in an atopic microenvironment plays a role in the activation of eosinophils. METHODS: Human eosinophils were incubated with Sepharose beads coated with cationic or anionic compounds in the presence and absence of a cytokine cocktail (IL-3, IL-5, and GM-CSF) to simulate the milieu of inflammation. Eosinophil peroxidase and eosinophil-derived neurotoxin (EDN) release were compared with eosinophil morphology and expression of CD18, as determined by means of confocal microscopy. RESULTS: Cytokines with positively charged beads caused greater eosinophil peroxidase release (lysine coated, 44.2 nmol/L; compound 48/80, 40.0 nmol/L; or EDN coated, 49.1 nmol/L) than cytokines alone (14.9 nmol/L). Beads coated with heparin, dextran sulfate, and aspartic acid did not show this effect. EDN release was also induced by lysine-coated beads with cytokines (67.1 ng/100 microL) and blocked by heparin. Eosinophil incubation with wortmannin, genistein, and the src kinase inhibitor PP1 blocked cationic signaling. Eosinophils adherent to cationic-charged beads but not anionic-charged beads show polarization of CD18 expression toward the bead's surface. CONCLUSION: Cationic-charged surfaces induce increased eosinophil mediator release by increasing the density of CD18 expression available at the target surface.

Virus-induced eosinophil mediator release requires antigen-presenting and CD4+ T cells.

BACKGROUND: The most frequent trigger of asthma exacerbation is infection with common airway viruses; however, the precise mechanism regulating such severe reactions is not understood. The presence of airway eosinophil products is a unique feature detected in asthmatic airways. Using an animal model, we previously demonstrated that T cells play an important role in regulating an eosinophil-dependant pathway of virus-induced airway hyperreactivity. We hypothesize that human eosinophils respond to viruses, although only after interaction with T cells. OBJECTIVES: We sought to determine whether eosinophils can respond to airway viruses in vitro and determine the mechanism of response. METHODS: An in vitro coculture model of human eosinophils, antigen-presenting cells, and T cells was used with parainfluenza virus, respiratory syncytial virus, or rhinovirus. We measured release of eosinophil peroxidase (EPO) in concert with T-cell proliferation, cytokine release, and changes in T-cell phenotype. RESULTS: The viruses induced release of EPO when coincubated in the presence of antigen-presenting cells (dendritic cells or macrophages) and T cells. Virus-mediated release was associated with proliferation of CD3(+)CD4(+) T cells and release of cytokines. UV inactivation of the virus did not prevent virus-induced EPO release or T-cell proliferation. Proliferating CD4(+) T cells show increased expression of CD25 and CD45RO. CD8(+) T cells were not activated. CONCLUSION: Virus-induced EPO release can occur in the context of antigen presentation to CD4(+) T cells.

Differential expression and activation of Rab27A in human eosinophils: relationship to blood eosinophilia.

Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.