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1.  The P2Y6 Receptor Inhibits Effector T Cell Activation in Allergic Pulmonary Inflammation1 
We show that the P2Y6 receptor, a G-protein-coupled receptor with high affinity for the nucleotide uridine diphosphate, is an important endogenous inhibitor of T cell function in allergic pulmonary inflammation. Mice conditionally deficient in P2Y6 receptors [p2ry6 (flox/flox);cre/+ mice] exhibited severe airway and tissue pathology relative to P2Y6-sufficient [p2ry6 (flox/flox)] littermates (+/+ mice) when treated intranasally with an extract (Df) of the dust mite Dermatophagoides farinae. P2Y6 receptors were inducibly expressed by lung, lymph node and splenic CD4+ and CD8+ T cells of Df-treated +/+ mice. Df-restimulated P2Y6-deficient lymph node cells produced higher levels of Th1 and Th2 cytokines, and polyclonally-stimulated P2Y6-deficient CD4+ T cells proliferated faster than comparably stimulated P2Y6-sufficient cells. The absence of P2Y6 receptors on CD4+ cells, but not antigen presenting cells, was sufficient to amplify cytokine generation. Thus, P2Y6 receptors protect the lung against exuberant allergen-induced pulmonary inflammation by inhibiting the activation of effector T cells.
doi:10.4049/jimmunol.1003669
PMCID: PMC3140636  PMID: 21724990
2.  Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics 
Background
Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders.
Methods
The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes.
Results
Supplementation with several EO/BO combinations increased circulating 20–22 carbon (20–22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%.
Conclusions
This study shows that dietary supplementation with BO/EO alters 20–22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.
doi:10.1186/1476-511X-12-141
PMCID: PMC3851449  PMID: 24088297
Asthma; Gammalinolenic acid; Stearidonic acid; Inflammation; Leukotrienes; Borage oil; Echium oil
3.  Pathogenesis of Aspirin-Exacerbated Respiratory Disease and Reactions 
doi:10.1016/j.iac.2012.11.006
PMCID: PMC3781366  PMID: 23639708
Cysteinyl leukotriene; Cyclooxygenase; Prostaglandin E2; Thromboxane; Eosinophil; Mast cell; Platelet; AERD
4.  The Translational Repressor T-cell Intracellular Antigen-1 (TIA-1) is a Key Modulator of Th2 and Th17 Responses Driving Pulmonary Inflammation Induced by Exposure to House Dust Mite 
Immunology letters  2012;146(1-2):8-14.
T-cell Intracellular Antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1−/−) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1−/− mice also had higher levels of Df-specific IgE and IgG1 in serum and ex vivo restimulated Tia-1−/− lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.
doi:10.1016/j.imlet.2012.04.001
PMCID: PMC3407291  PMID: 22525013
T-cell Intracellular Antigen-1; allergen-mediated pulmonary inflammation; cytokines; translation
5.  STAT1 negatively regulates lung basophil IL-4 expression induced by respiratory syncytial virus infection1 
IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3−CD49b+ cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3−CD49b+FcεRI+c-kit−). Because STAT1−/− mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1−/− mice. RSV infection resulted in significantly more IL-4-expressing CD3−CD49b+ cells in the lungs of STAT1−/− mice than in BALB/c mice. CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1−/− mice. Depletion of basophils in RSV-infected STAT1−/− mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.
doi:10.4049/jimmunol.0803167
PMCID: PMC3755459  PMID: 19587017
basophils; cytokines; viral; lung
6.  Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells 
PLoS ONE  2013;8(8):e71536.
Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT1R and CysLT2R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT1R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD4 and LTE4-induced calcium influx through CysLT1R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT1R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.
doi:10.1371/journal.pone.0071536
PMCID: PMC3744564  PMID: 23977066
7.  Prostaglandin E2 Exerts Homeostatic Regulation of Pulmonary Vascular Remodeling in Allergic Airway Inflammation 
Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE2 in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE2 synthase (mPGES)-1. Mice lacking mPGES-1 (ptges−/− mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE2 in the bronchoalveolar lavage fluids of Der f-treated ptges−/− mice were ~80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges−/− mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1-derived PGE2 was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges−/− mice. These vascular changes were suppressed by the administration of the stable PGE2 analog 16, 16-dimethyl PGE2, or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE2. Thus, mPGES-1 and its product, PGE2, protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.
doi:10.4049/jimmunol.0902835
PMCID: PMC3721536  PMID: 20028661
8.  Gene-by-environment effect of house dust mite on purinergic receptor P2Y12 (P2RY12) and lung function in children with asthma 
Clinical and Experimental Allergy  2012;42(2):229-237.
Background
Distinct receptors likely exist for leukotriene(LT)E4, a potent mediator of airway inflammation. Purinergic receptor P2Y12 is needed for LTE4-induced airways inflammation, and P2Y12 antagonism attenuates house dust mite-induced pulmonary eosinophilia in mice. Although experimental data support a role for P2Y12 in airway inflammation, its role in human asthma has never been studied.
Objective
To test for association between variants in the P2Y12 gene (P2RY12) and lung function in human subjects with asthma, and to examine for gene-by-environment interaction with house dust mite exposure.
Methods
19 single nucleotide polymorphisms (SNPs) in P2RY12 were genotyped in 422 children with asthma and their parents (n=1266). Using family-based methods, we tested for associations between these SNPs and five lung function measures. We performed haplotype association analyses and tested for gene-by-environment interactions using house dust mite exposure. We used the false discovery rate to account for multiple comparisons.
Results
Five SNPs in P2RY12 were associated with multiple lung function measures (P values 0.006–0.025). Haplotypes in P2RY12 were also associated with lung function (P values 0.0055–0.046). House dust mite exposure modulated associations between P2RY12 and lung function, with minor allele homozygotes exposed to house dust mite demonstrating worse lung function than those unexposed (significant interaction P values 0.0028–0.040).
Conclusions and clinical relevance
P2RY12 variants were associated with lung function in a large family-based asthma cohort. House dust mite exposure caused significant gene-by-environment effects. Our findings add the first human evidence to experimental data supporting a role for P2Y12 in lung function. P2Y12 could represent a novel target for asthma treatment.
doi:10.1111/j.1365-2222.2011.03874.x
PMCID: PMC3353543  PMID: 22010907
Purinergic receptor; leukotriene; asthma; house dust mite; lung function
9.  NIAID-Sponsored 2010 Guidelines for Managing Food Allergy: Applications in the Pediatric Population 
Pediatrics  2011;128(5):955-965.
Data from many studies have suggested a rise in the prevalence of food allergies during the past 10 to 20 years. Currently, no curative treatments for food allergy exist, and there are no effective means of preventing the disease. Management of food allergy involves strict avoidance of the allergen in the patient's diet and treatment of symptoms as they arise. Because diagnosis and management of the disease can vary between clinical practice settings, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored development of clinical guidelines for the diagnosis and management of food allergy. The guidelines establish consensus and consistency in definitions, diagnostic criteria, and management practices. They also provide concise recommendations on how to diagnose and manage food allergy and treat acute food allergy reactions. The original guidelines encompass practices relevant to patients of all ages, but food allergy presents unique and specific concerns for infants, children, and teenagers. To focus on those concerns, we describe here the guidelines most pertinent to the pediatric population.
doi:10.1542/peds.2011-0539
PMCID: PMC3208961  PMID: 21987705
food allergy; food hypersensitivity; infants; children; guidelines; anaphylaxis
10.  Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI 
Background
Studies of human mast cells are constrained by the paucity of functional cell lines, the expense of maintaining mast cells in culture, and technical complexities.
Objective
We derived and characterized a human mast cell line that arose spontaneously from a culture of non-transformed hematopoietic progenitor cells.
Methods
CD34+-enriched mononuclear cells derived from a donor with aspirin exacerbated respiratory disease were cultured for 8 weeks with stem cell factor and interleukin-6 and with interleukin-3 for the first week only. The cells (termed LUVA cells) survived and proliferated without further addition of any growth factors and have been maintained in culture for ~2 years.
Results
LUVA cells possess metachromatic cytoplasmic granules that are immunoreactive for tryptase, cathepsin G, and carboxypeptidase A3. They express transcripts encoding genes for FcεRI, c-kit, chymase, tryptase, histidine decarboxylase, carboxypeptidase A3, and the type 1 receptor for cysteinyl leukotrienes. Flow cytometry confirmed uniform expression of FcεRI, c-kit and FcγRII. FcεRI cross-linkage induced the release of β-hexosaminidase, prostaglandin D2, thromboxane A2, and macrophage inflammatory protein-1β. Immortalization was not associated with either a known genomic mutation of c-kit in the donor or a somatic mutation of c-kit within the cells, and it was not associated with c-kit autophosphorylation.
CONCLUSIONS
LUVA cells are an immortalized human mast cell line that can be maintained without stem cell factor and display high levels of normally signaling c-kit and FcεRI. These cells will prove valuable for functional human mast cell studies.
doi:10.1016/j.jaci.2010.12.1101
PMCID: PMC3052637  PMID: 21281958
Mast cell; SCF; FcεRI; c-Kit; Tryptase; CPA3
11.  Group V secretory phospholipase A2 reveals its role in house dust mite-induced allergic pulmonary inflammation by regulation of dendritic cell function 
We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. Here we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae (Df) had markedly reduced pulmonary inflammation and goblet cell metaplasia compared to wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to Df compared to WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by Df had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of Df-challenged mice. Adoptively transferred Df-loaded Pla2g5-null BMDCs were less able than Df-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null Df-loaded BMDCs exhibited significantly reduced local inflammatory responses to Df, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APC regulates Ag processing and maturation of dendritic cells, and contributes to pulmonary inflammation and immune response against Df. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by Df and whose function is also regulated by group V sPLA2.
doi:10.4049/jimmunol.1001384
PMCID: PMC3003260  PMID: 20817863
12.  Fas-activated serine/threonine phosphoprotein promotes immune-mediated pulmonary inflammation 
We have generated Fas activated serine threonine phosphoprotein-deficient mice (FAST−/−) to study the in vivo role of FAST in immune system function. In a model of house dust mite (HDM)-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes and neutrophils. FAST−/− mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST−/− mice compared to wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-α, IL-6 and IL-23) and chemoattractants (MIP-2 and KC) in bronchoalveolar lavage fluids. As FAST−/− neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g. pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a pro-inflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.
doi:10.4049/jimmunol.1000104
PMCID: PMC2858774  PMID: 20363972
13.  LPA5 Is Abundantly Expressed by Human Mast Cells and Important for Lysophosphatidic Acid Induced MIP-1β Release 
PLoS ONE  2011;6(3):e18192.
Background
Lysophosphatidic acid (LPA) is a bioactive lipid inducing proliferation, differentiation as well as cytokine release by mast cells through G-protein coupled receptors. Recently GPR92/LPA5 was identified as an LPA receptor highly expressed by cells of the immune system, which prompted us to investigate its presence and influence on mast cells.
Principal Findings
Transcript analysis using quantitative real-time PCR revealed that LPA5 is the most prevalent LPA-receptor in human mast cells. Reduction of LPA5 levels using shRNA reduced calcium flux and abolished MIP-1β release in response to LPA.
Conclusions
LPA5 is a bona fide LPA receptor on human mast cells responsible for the majority of LPA induced MIP-1β release.
doi:10.1371/journal.pone.0018192
PMCID: PMC3065470  PMID: 21464938
14.  The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications 
The intracellular parent of the cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C4, is formed by conjugation of LTA4 and reduced glutathione by LTC4 synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC4 is converted to LTD4 and LTE4 by sequential enzymatic removal of glutamic acid and then glycine. Only LTE4 is sufficiently stable to be prominent in biologic fluids, such as urine or bronchoalveolar lavage fluid of asthmatic individuals and at sites of inflammation in animal models. LTE4 has received little attention because it binds poorly to the classical receptors, CysLT1R and CysLT2R, and was much less active on normal airways than LTC4 or LTD4. However, early studies indicated that LTE4 caused skin swelling in humans as potently as LTC4 and LTD4, that airways of asthmatic subjects (particularly those that were aspirin-sensitive) were selectively hyperresponsive to LTE4, and that a potential distinct LTE4 receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE4; P2Y12, an ADP receptor, and CysLTER, observed functionally in skin of mice lacking CysLT1R and CysLT2R. These findings prompt a renewed focus on LTE4 receptors as therapeutic targets that are not currently addressed by available receptor antagonists.
doi:10.1016/j.jaci.2009.05.046
PMCID: PMC2739263  PMID: 19647860
Leukotriene E4; G protein-coupled receptor; bronchial asthma; inflammation; knockout mouse
15.  Leukotriene E4–induced pulmonary inflammation is mediated by the P2Y12 receptor 
The Journal of Experimental Medicine  2009;206(11):2543-2555.
Of the potent lipid inflammatory mediators comprising the cysteinyl leukotrienes (LTs; LTC4, LTD4, and LTE4), only LTE4 is stable and abundant in vivo. Although LTE4 shows negligible activity at the type 1 and 2 receptors for cys-LTs (CysLT1R and CysLT2R), it is a powerful inducer of mucosal eosinophilia and airway hyperresponsiveness in humans with asthma. We show that the adenosine diphosphate (ADP)–reactive purinergic (P2Y12) receptor is required for LTE4-mediated pulmonary inflammation. P2Y12 receptor expression permits LTE4 -induced activation of extracellular signal-regulated kinase in Chinese hamster ovary cells and permits chemokine and prostaglandin D2 production by LAD2 cells, a human mast cell line. P2Y12 receptor expression by LAD2 cells is required for competition between radiolabeled ADP and unlabeled LTE4 but not for direct binding of LTE4, suggesting that P2Y12 complexes with another receptor to recognize LTE4. Administration of LTE4 to the airways of sensitized mice potentiates eosinophilia, goblet cell metaplasia, and expression of interleukin-13 in response to low-dose aerosolized allergen. These responses persist in mice lacking both CysLT1R and CysLT2R but not in mice lacking P2Y12 receptors. The effects of LTE4 on P2Y12 in the airway were abrogated by platelet depletion. Thus, the P2Y12 receptor is required for proinflammatory actions of the stable abundant mediator LTE4 and is a novel potential therapeutic target for asthma.
doi:10.1084/jem.20091240
PMCID: PMC2768854  PMID: 19822647
16.  Advances in Mechanisms of Asthma, Allergy, and Immunology in 2008 
This review summarizes selected articles appearing in 2008 in the Journal of Allergy and Clinical Immunology (JACI). Papers chosen include those improving our understanding of mechanisms of allergic diseases by focusing on human basophil, mast cell and eosinophil biology; IgE and its high affinity receptor on various cells; novel properties of omalizumab; airways remodeling; and genetics. Papers from other journals have been included to supplement the topics being presented.
doi:10.1016/j.jaci.2009.01.041
PMCID: PMC2671063  PMID: 19281904
17.  P2Y6 Receptors Require an Intact Cysteinyl Leukotriene Synthetic and Signaling System to Induce Survival and Activation of Mast Cells1 
Cysteinyl leukotrienes (cys-LTs) induce inflammatory responses through type 1 (CysLT1R) and type 2 (CysLT2R) cys-LT receptors, and activate mast cells (MCs) in vitro. We previously demonstrated that cys-LTs cross-desensitized interleukin (IL)-4-primed primary human MCs (hMCs) to stimulation with the nucleotide uridine diphosphate (UDP). We now report that hMCs, mouse bone marrow-derived mast cells (mBMMCs), and the human MC line LAD2 all express UDP-selective P2Y6 receptors that cooperate with CysLT1R to promote cell survival and chemokine generation by a pathway involving reciprocal ligand-mediated cross-talk. LTD4, the most potent CysLT1R ligand, and UDP both induced phosphorylation of extracellular signal regulated kinase (ERK) and prolonged the survival of cytokine-starved hMCs and mBMMCs. ERK activation and cytoprotection in response to either ligand were attenuated by treatment of the cells with a selective P2Y6 receptor antagonist (MRS2578), which did not interfere with signaling through recombinant CysLT1R. Surprisingly, both UDP and LTD4-mediated ERK activation and cytoprotection were absent in mBMMCs lacking CysLT1R and the biosynthetic enzyme LTC4 synthase (LTC4S), implying a requirement for a cys-LT-mediated autocrine loop. In IL-4-primed LAD2 cells, LTD4 induced the generation of macrophage inflammatory protein 1β (MIP-1β) by IL-4-primed LAD2 cells, a response blocked by short hairpin RNA (shRNA)-mediated knockdown of CysLT1R or of P2Y6 receptors, but not of CysLT2R. Thus, CysLT1R and P2Y6 receptors, which are co-expressed with on many cell types of innate immunity, reciprocally amplify one another’s function in MCs through endogenous ligands.
PMCID: PMC2615395  PMID: 19124756
Mast Cells/Basophils; Lipid Mediators; Allergy; inflammation
18.  No audible wheezing 
The Journal of Experimental Medicine  2005;201(12):1869-1873.
Mouse models of T helper type 2 (Th2) cell–biased pulmonary inflammation have elucidated mechanisms of sensitization, cell traffic, and induced airway hyperresponsiveness (AHR). Nonetheless, most mice lack intrinsic AHR, a central property of human asthma, and disparities persist regarding the contributions of eosinophils and mast cells and the sensitivity to induced AHR in the commonly used mouse strains. We suggest that these discordances, reflecting methodological and genetic differences, may be informative for understanding heterogeneity of human asthma.
doi:10.1084/jem.20050584
PMCID: PMC2212041  PMID: 15967817
19.  Cysteinyl Leukotrienes and Uridine Diphosphate Induce Cytokine Generation by Human Mast Cells Through an Interleukin 4–regulated Pathway that Is Inhibited by Leukotriene Receptor Antagonists 
We previously reported that interleukin (IL)-4 upregulates the expression of leukotriene C4 synthase (LTC4S) by human cord blood–derived mast cells (hMCs), augments their high-affinity Fc receptor for IgE (FcεRI)-dependent generation of eicosanoids and cytokines, and induces a calcium flux in response to cysteinyl leukotrienes (cys-LTs) and uridine diphosphate (UDP) that is blocked by cys-LT receptor antagonists. We speculated that this IL-4–dependent, receptor-mediated response to the cys-LTs and UDP might induce cytokine generation by hMCs without concomitant exocytosis. Unlike hMCs maintained in cytoprotective stem cell factor (SCF) alone, hMCs primed for 5 d with IL-4 responded to UDP (1 μM), LTC4 (100 nM), and LTD4 (100 nM) by producing IL-5, tumor necrosis factor (TNF)-α, and especially large quantities of macrophage inflammatory protein (MIP)-1β de novo at 6 h, preceded by the induced expression of the corresponding mRNAs. Cys-LT– and UDP-mediated cytokine production by the primed hMCs occurred without histamine release or PGD2 generation and was inhibited by the CysLT1 receptor antagonist MK571. Additionally, pretreatment of hMCs with MK571 or with the cys-LT biosynthetic inhibitor MK886 decreased IL-5 and TNF-α production in response to IgE receptor cross-linkage, implying a positive feedback by endogenously produced cys-LTs. Cys-LTs and UDP thus orchestrate a novel, IL-4–regulated, non-IgE–dependent hMC activation for cytokine gene induction that could be initiated by microbes, cellular injury, or neurogenic or inflammatory signals; and this pathobiologic event would not be recognized in tissue studies where hMC activation is classically defined by exocytosis.
doi:10.1084/jem.20020044
PMCID: PMC2193768  PMID: 11877481
mast cells; cytokines; leukotrienes; uridine diphosphate; receptors
20.  Human Mast Cell Progenitors Can Be Infected by Macrophagetropic Human Immunodeficiency Virus Type 1 and Retain Virus with Maturation In Vitro 
Journal of Virology  2001;75(22):10808-10814.
Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit+ CD13+ cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.
doi:10.1128/JVI.75.22.10808-10814.2001
PMCID: PMC114662  PMID: 11602722
21.  T Helper Cell Type 2 Cytokines Coordinately Regulate Immunoglobulin E–Dependent Cysteinyl Leukotriene Production by Human Cord Blood–Derived Mast Cells 
Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE–stimulated hMCs elaborated minimal cys-LT (0.1 ± 0.1 ng/106 hMCs) and abundant prostaglandin (PG)D2 (16.2 ± 10.3 ng/106 hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE–dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD2 (by 2.5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE–mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD2 generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C4 synthase (LTC4S) mRNA within 6 h, and increased the expression of LTC4S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC4S biosynthetic pathway (induction of LTC4S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.
PMCID: PMC2195887  PMID: 11136826
eicosanoids; asthma; allergy; prostaglandin D2; FcεRI
22.  T Helper Cell Type 2 Cytokine–Mediated Comitogenic Responses and Ccr3 Expression during Differentiation of Human Mast Cells in Vitro 
Mast cells (MCs) arise in situ from circulating stem cell factor (SCF)-dependent committed progenitors (PrMCs) and accumulate at sites of allergic mucosal inflammation. We hypothesized that human (h)PrMCs and their mature counterparts might share overlapping patterns of chemokine and cytokine receptor utilization with eosinophils, basophils, and T helper type 2 (Th2) lymphocytes for their homing and allergy-associated hyperplasia. We have characterized committed hPrMCs and fully mature hMCs derived in vitro from cord blood for their functional responses to chemokine and cytokine agonists germane to allergic inflammation and for their maturation-related expression of the corresponding receptors. After 4 wk of culture in the presence of recombinant stem cell factor (SCF), interleukin (IL)-6, and IL-10, the cells were characterized as hPrMCs based upon their uniform surface expression of c-kit and CD13, low-level expression of Fc∈RIα, absence of CD14 and CD16 expression, and immunoreactivity for MC chymase in >80%, and about half were immunoreactive for tryptase and metachromatic with toluidine blue. By week 9, the cells had matured into hMCs, identified by higher levels of c-kit, continued expression of CD13 and low-level Fc∈RIα, uniform toluidine blue metachromasia, and uniform immunoreactivity for both tryptase and chymase. The 4-wk-old hPrMCs expressed four chemokine receptors (CXCR2, CCR3, CXCR4, and CCR5). Each receptor mediated transient rapid calcium fluxes in response to its respective ligand. Both recombinant human eotaxin and stromal cell–derived factor 1α elicited chemotaxis of hPrMCs. Only CCR3 was retained on the mature 9-wk-old hMCs from among these chemokine receptors, and hMCs responded to eotaxin with a sustained calcium flux but without chemotaxis. The Th2 cytokines IL-3, IL-5, IL-6, IL-9, and granulocyte/macrophage colony-stimulating factor each augmented the SCF-dependent proliferation of hPrMCs and hMCs. In contrast, the prototypical Th1 cytokine, interferon γ, suppressed SCF-driven proliferation of both hPrMCs and hMCs. Thus, throughout their development in vitro, hMCs obey SCF-dependent, cytokine-driven mitogenic responses that reflect a Th2-type polarization characteristic of allergy and asthma. Furthermore, committed hPrMCs have a unique profile of chemokine receptor expression from among reported hematopoietic cells, including CCR3, which is shared with the other cells central to allergic inflammation (eosinophils, basophils, and Th2 lymphocytes).
PMCID: PMC2195573  PMID: 10432289
chemokines; asthma; HIV; calcium flux; stem cell factor
23.  Human Peripheral Blood Eosinophils Express a Functional c-kit Receptor for Stem Cell Factor that Stimulates Very Late Antigen 4 (VLA-4)–mediated Cell Adhesion to Fibronectin and Vascular Cell Adhesion Molecule 1 (VCAM-1) 
We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell–derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0– 3.6-fold, average 2.8 ± 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 μg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 ± 1.4-, 5.3 ± 3.3-, and 5.4 ± 0.2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 μg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 ± 9.2-, 3.8 ± 2.5-, and 1.7 ± 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the α4 and β1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4–mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.
PMCID: PMC2198988  PMID: 9221761

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