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1.  Dectin-2 Regulates the Effector Phase of House Dust Mite-Elicited Pulmonary Inflammation Independently from its Role in Sensitization 
The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae (Df) through the generation of cysteinyl leukotrienes (cys-LTs) and pro-inflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. Here, we demonstrate that administration of the Dectin-2 mAb solely at the time of Df challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage (BAL) fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n−/−) sensitized with the adoptive transfer of Df-pulsed wild-type (WT) bone marrow-derived DCs (BMDCs) also had less Df-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n−/− mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production, and with no reduction in serum IgE. WT and Clec4n−/− mice recipients, sensitized with Df-pulsed WT BMDCs, had comparable levels of Df-elicited IL-6, IL-23, TNF-α, and cys-LTs in the lung. By contrast, Df-elicited CCL4 and CCL8 production from pulmonary CD11c+CD11b+Ly6C+ and CD11c+CD11b+Ly6C−CD64+ monocyte-derived DCs was reduced in Clec4n−/− recipients. Addition of CCL8 at the time of Df challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n−/− recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at Df challenge to promote the local inflammatory response.
doi:10.4049/jimmunol.1301809
PMCID: PMC4024442  PMID: 24453247
Knockout mice; Dendritic Cells; Lipid mediators; Allergy; Inflammation
2.  GPR17 Regulates Immune Pulmonary Inflammation Induced By House Dust Mite 
Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT1R) are efficacious for bronchoconstriction in humans with bronchial asthma; however, the clinical response to these drugs is heterogeneous. In particular, how CysLT1R expression and function are constitutively regulated in vivo is not known. Here we show that a 7-transmembrane receptor GPR17 negatively regulates the CysLT1R-mediated inflammatory cell accumulation in the bronchoalveolar lavage fluid and lung, the levels of IgE and specific IgG1 in serum, and Th2/Th17 cytokine expression in the lung after intranasal sensitization and challenge with house dust mite (Df) in mice. Sensitization of naïve wild-type recipients with Df-pulsed bone marrow-derived dendritic cells (BMDCs) of each genotype or sensitization of each genotype with Df-pulsed wild-type BMDCs and Df challenge revealed markedly increased pulmonary inflammatory and serum IgE responses for GPR17-deficient mice as compared to wild-type mice and reduced responses in the genotypes lacking CysLT1R. These findings reveal a constitutive negative regulation of CysLT1R functions by GPR17 in both the antigen presentation and downstream phases of allergic pulmonary inflammation.
doi:10.4049/jimmunol.1001131
PMCID: PMC4014011  PMID: 20574000
Knockout Mice; Lipid Mediators; Allergy; Inflammation; Lung
3.  Mast cells recruited to mesenteric lymph nodes during helminth infection remain hypogranular and produce IL-4 and IL-6 
Mast cells (MC) and basophils (Ba) share expression of the high affinity receptor for IgE (FcεRI) but can be distinguished by their divergent expression of KIT and CD49b. In BALB/c mice, MC lineage cells expressing high levels of FcεRI by flow cytometry were seen only in bone marrow (BM) while those expressing intermediate levels of FcεRI were present in BM and spleen of naïve mice and in mesenteric lymph nodes (mLN) of Trichinella spiralis-infected mice. These FcεRI+KIT+CD49b− cells had a membrane phenotype like intraperitoneal connective tissue-type MC, but were smaller and hypogranular by flow cytometry forward and side scatter profiles, respectively. Consistent with this, they lacked the prominent secretory granules identified by histochemistry and immunodetection for the MC-specific granule proteases that are readily seen in mature jejunal mucosal MC (MMC) that also are induced by the infection and present at the same time. The concentration of these MC lineage cells in mLN determined by flow cytometry was comparable to that of MC progenitors (MCp) measured by limiting dilution and clonal expansion with maturation. We observed upregulation of IL-4 mRNA levels by MCp in mLN and spleens of helminth-infected 4get mice, and demonstrated by intracellular cytokine staining production of IL-4 and IL-6 by the mLN MCp in helminth-infected mice. Furthermore, treatment of helminth-infected mice with anti-FcεRI mAb, a protocol known to deplete Ba, also depleted mLN MCp. Thus, this study identifies a hypogranular subset of MCp recruited to mLN by helminth infection that may be an important unrecognized source of cytokines.
doi:10.4049/jimmunol.1202567
PMCID: PMC3563837  PMID: 23319739
4.  Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food anaphylaxis 
Background
Sensitization to food antigen may occur through cutaneous exposure.
Objective
Test the hypothesis that epicutaneous (EC) sensitization with food antigen predisposes to IgE-mediated anaphylaxis upon oral allergen challenge.
Methods
BALB/c mice were EC sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks, or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks, then orally challenged with OVA. Body temperature was monitored and serum mouse mast cell protease 1 (mMCP-1) level was determined following challenge. Tissue mast cells (MCs) were examined by chloroacetate esterase (CAE) staining. Serum OVA-specific IgE and IgG1 antibodies, and cytokines in supernatants of OVA-stimulated splenocytes, were measured by ELISA. Serum interleukin-4 (IL-4) levels were measured using an in vivo cytokine capture assay (IVCCA).
Results
EC sensitized mice exhibited expansion of connective tissue MC in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis following oral challenge, as evidenced by decreased body temperature and increased serum mMCP-1 level. Intestinal MC expansion and anaphylaxis were IgE-dependent, as they did not occur in EC sensitized IgE−/− mice. Mice orally immunized with OVA+CT failed to increase serum IL-4 levels, expand their intestinal MCs, or develop anaphylaxis following oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable to those of EC sensitized mice.
Conclusion
EC sensitized mice, but not mice orally immunized with antigen+CT, develop expansion of intestinal MCs and IgE-mediated anaphylaxis following single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut may play an important role in the development of anaphylaxis.
Clinical Implications
The skin may be an important route of sensitization to food antigens. Avoidance of cutaneous sensitization may prevent the development of food anaphylaxis.
doi:10.1016/j.jaci.2012.11.032
PMCID: PMC3587010  PMID: 23374269
Food allergy; epicutaneous sensitization; IgE; mast cells; anaphylaxis
5.  Cysteinyl Leukotriene 2 Receptor on Dendritic Cells Negatively Regulates Ligand-Dependent Allergic Pulmonary Inflammation 
Cysteinyl leukotrienes (cys-LTs) can mediate Th2 immunity to the house dust mite, Dermatophagoides farinae (Df), via the type 1 receptor CysLT1R on dendritic cells (DCs). However, the role of the homologous type 2 receptor CysLT2R in Th2 immunity is unknown. Df sensitization and challenge of CysLT2R-deficient mice showed a marked augmentation of eosinophilic pulmonary inflammation, serum IgE, and Th2 cytokines. Wild-type (WT) mice sensitized by adoptive transfer of Df-pulsed CysLT2R-deficient bone marrow-derived DCs (BMDCs) also had a marked increase in Df-elicited eosinophilic lung inflammation and Th2 cytokines in restimulated hilar nodes. This response was absent in mice sensitized with Df-pulsed BMDCs lacking leukotriene C4 synthase (LTC4S), CysLT1R, or both CysLT2R/LTC4S, suggesting that CysLT2R negatively regulates LTC4S- and CysLT1R-dependent DC-mediated sensitization. CysLT2R-deficient BMDCs had increased CysLT1R-dependent LTD4-induced ERK phosphorylation, whereas N-methyl LTC4 activation of CysLT2R on WT BMDCs reduced such signaling. Activation of endogenously expressed CysLT1R and CysLT2R occurred over an equimolar range of LTD4 and N-methyl LTC4, respectively. Although the baseline expression of cell surface CysLT1R was not increased on CysLT2R-deficient BMDCs, it was upregulated at 24 h by a pulse of Df, as compared to WT or CysLT2R/LTC4S-deficient BMDCs. Importantly, treatment with N-methyl LTC4 reduced Df-induced CysLT1R expression on WT BMDCs. Thus, CysLT2R negatively regulates the development of cys-LT-dependent Th2 pulmonary inflammation by inhibiting both CysLT1R signaling and Df-induced LTC4S-dependent cell surface expression of CysLT1R on DCs. Furthermore, these studies highlight how the biologic activity of cys-LTs can be tightly regulated by competition between these endogenously expressed receptors.
doi:10.4049/jimmunol.1201865
PMCID: PMC3478400  PMID: 23002438
Knockout mouse; Lipid mediator; G protein-coupled receptor; Asthma; Lung
6.  Outbreak of Adverse Reactions Associated with Contaminated Heparin 
The New England journal of medicine  2008;359(25):10.1056/NEJMoa0806450.
BACKGROUND
In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions.
METHODS
Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case–control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants.
RESULTS
A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin.
CONCLUSIONS
Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause of the outbreak.
doi:10.1056/NEJMoa0806450
PMCID: PMC3810025  PMID: 19052120
7.  Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System 
The New England journal of medicine  2008;358(23):2457-2467.
Background
There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany.
Methods
Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine.
Results
The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin–kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine.
Conclusions
Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.
doi:10.1056/NEJMoa0803200
PMCID: PMC3778681  PMID: 18434646
8.  Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells1 
House dust mites are a significant source of airborne allergen worldwide, but there is little understanding of how they so potently generate allergic inflammation. We found that extracts from the house dust mites Dermatophagoides farinae (Df) and Dermatophagoides pteronyssinus and from the mold Aspergillus fumigatus stimulated a rapid and robust production of cysteinyl leukotrienes (cys-LTs), proinflammatory lipid mediators, from mouse bone marrow-derived dendritic cells (BMDCs). Con A affinity chromatography of the Df extract revealed that the relevant ligand is a glycan(s), suggesting stimulation via a dendritic cell (DC) lectin receptor. Cys-LT production in BMDCs from wild-type mice was inhibited by spleen tyrosine kinase (Syk) inhibitors and was abolished in BMDCs from FcRγ−/− mice, implicating either Dectin-2 or DC immunoactivating receptor. Transfection of each receptor in bone marrow-derived mast cells revealed that only Dectin-2 mediates cys-LT production by Df, Dermatophagoides pteronyssinus, and Aspergillus fumigatus. Lentiviral knockdown of Dectin-2 in BMDCs attenuated Df extract-elicited cys-LT generation, thereby identifying Dectin-2 as the receptor. Lung CD11c+ cells, but not peritoneal or alveolar macrophages, also generated cys-LTs in response to Df. These findings place Dectin-2 among the C-type lectin receptors that activate arachidonic acid metabolism and identify the Dectin-2/FcRγ/Syk/cys-LT axis as a novel mechanism by which three potent indoor allergens may activate innate immune cells to promote allergic inflammation.
PMCID: PMC3682801  PMID: 19124755
9.  Cysteinyl Leukotrienes Impair Hypoxic Pulmonary Vasoconstriction in Endotoxemic Mice 
Anesthesiology  2011;115(4):804-811.
Background
Sepsis impairs hypoxic pulmonary vasoconstriction (HPV) in patients and animal models contributing to systemic hypoxemia. Levels of cysteinyl leukotrienes are elevated in the bronchoalveolar lavage fluid of patients with sepsis, but the contribution of cysteinyl leukotrienes to the impairment of HPV is uncertain.
Methods
Wild-type mice, mice deficient in leukotriene C4 synthase, the enzyme responsible for cysteinyl leukotriene synthesis, and mice deficient in cysteinyl leukotriene receptor 1 were studied at 18 h after challenge with either saline or endotoxin. HPV was measured by the increase in left pulmonary vascular resistance induced by left mainstem bronchus occlusion. Levels of cysteinyl leukotrienes were determined in the bronchoalveolar lavage fluid.
Results
In the bronchoalveolar lavage fluid of all three strains, cysteinyl leukotrienes were not detectable after saline challenge; whereas endotoxin challenge increased cysteinyl leukotriene levels in wild-type mice and mice deficient of cysteinyl leukotriene receptor 1, but not in mice deficient of leukotriene C4 synthase. HPV did not differ between the three mouse strains after saline challenge (120±26, 114±16, and 115±24%, respectively; mean±SD). Endotoxin challenge markedly impaired HPV in wild-type mice (41±20%) but only marginally in mice deficient in leukotriene C4 synthase (96±16%, P<0.05 vs. wild-type mice), thereby preserving systemic oxygenation. While endotoxin modestly decreased HPV in mice deficient in cysteinyl leukotriene receptor 1 (80±29%, P<0.05 vs. saline challenge), the magnitude of impairment was markedly less than in endotoxin-challenged wild-type mice.
Conclusion
Cysteinyl leukotrienes importantly contribute to endotoxin-induced impairment of HPV in part via a cysteinyl leukotriene receptor 1-dependent mechanisms.
doi:10.1097/ALN.0b013e31822e94bd
PMCID: PMC3194098  PMID: 21934409
10.  Innate Immunity in Th2-type Airway Inflammation 
Immunity  2009;31(3):425-437.
Activated mast cells, eosinophils, and basophils infiltrate the airways of asthmatics as a result of an overexuberant T helper 2 (Th2) cell immune response that drives the production of IgE, primes mast cells and basophils, and promotes tissue eosinophilia and mast cell hyperplasia. Recent evidence demonstrates that these innate effectors can be activated outside of this classical Th2 cell paradigm and that they have additional roles in promoting the development of innate and adaptive pulmonary inflammation. There is also an appreciation for the role of airway epithelial cells in orchestrating allergic pulmonary inflammation. Emerging data from basic research highlights the involvement of many unique pathways in the inflammation triggered by complex native allergens and microbes at the airway mucosal surface. Here we review the role of effector cells and airway epithelial cells in augmenting and, at times, bypassing traditional Th2 cell-mediated allergic inflammation.
doi:10.1016/j.immuni.2009.08.014
PMCID: PMC3353417  PMID: 19766085
11.  The inflammatory response following an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5 
A second degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to non-muscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-KitW/KitWv mice which lack MCs in a context of other defects due to a mutation of the KIT receptor. To further address a MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury following a second degree scald burn while mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or the MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficent mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first h post burn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing non-redundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
doi:10.4049/jimmunol.1002803
PMCID: PMC3058366  PMID: 21076070
12.  Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes 
Dectin-2 expression on GM-CSF–cultured bone marrow cells is required for the generation of cysteinyl leukotrienes and Th2 cytokines in response to the house dust mite Dermatophagoides farinae in vivo.
The innate signaling pathways for Th2 immunity activated by inhaled antigens are not well defined. We previously identified Dectin-2 as a receptor for glycans in allergen extracts from the house dust mite Dermatophagoides farinae (Df) that mediates cysteinyl leukotriene (cys-LT) generation from pulmonary CD11c+ cells and from GM-CSF–cultured bone marrow cells (BMCsGM-CSF). Using lentiviral knockdown of Dectin-2 in BMCsGM-CSF and adoptive transfer of Df-pulsed BMCsGM-CSF to sensitize naive mice, we now report that Dectin-2 is critical for the development of Df-elicited eosinophilic and neutrophilic pulmonary inflammation and Th2 cytokine generation in the lungs and restimulated lymph nodes. Sensitization with Df-pulsed BMCsGM-CSF from LTC4 synthase (LTC4S)–deficient mice or type 1 cys-LT receptor (CysLT1R)–deficient mice demonstrated that both proteins were required for Df-elicited eosinophilic pulmonary inflammation and Th2 cytokine generation in the lungs and restimulated lymph nodes. Direct sensitization and challenge of Ltc4s−/− and Cysltr1−/− mice confirmed that cys-LTs mediate these parameters of Df-elicited Th2 pulmonary inflammation. Thus, the Dectin-2–cys-LT pathway is critical for the induction of Th2 immunity to a major allergen, in part through CysLT1R. These findings identify a previously unrecognized link between a myeloid C-type lectin receptor and Th2 immunity.
doi:10.1084/jem.20100793
PMCID: PMC3058587  PMID: 21357742
13.  The Role of the CCL2/CCR2 Axis in Mouse Mast Cell Migration In Vitro and In Vivo 
Tissue-resident mast cells (MCs) are important in allergic diseases. In a mouse model of allergic airways inflammation, an increase in peribronchiolar MCs was associated with increased concentrations of the chemokine CCL2 in lung lavage. MC progenitors (MCps) arising in bone marrow (BM) are recruited to tissues by transendothelial migration, and we found that CCL2 is chemotactic for MCps in freshly isolated BM in vitro. Immature, but not mature, BM-derived MCs migrated in response to CCL2 when cultured in IL-3+stem cell factor (SCF) but not when cultured in IL-3 alone. However, the cells under both culture conditions expressed mRNA for CCR2, the receptor for CCL2, and bound the radiolabeled chemokine with similar affinities, highlighting SCF as a key mediator in coupling CCR2 to downstream events, culminating in chemotaxis. Immature BM-derived MCs from IL-3 +SCF cultures, when administered i.v., accumulated at skin sites injected with CCL2 in vivo. MCp recruitment to the allergen-sensitized/challenged lung was significantly reduced in CCR2−/− and CCL2−/− mouse strains. However, reconstitution studies of sublethally irradiated and BM-reconstituted mice indicated that BM cells and stromal elements could provide CCL2, whereas the CCR2 function resided with stromal elements rather than BM cells. These experiments revealed a new function of SCF in chemokine receptor coupling, but they suggest a complex role of the CCL2/CCR2 axis in recruiting MCps during pulmonary inflammation.
doi:10.4049/jimmunol.0904177
PMCID: PMC2956277  PMID: 20427772
14.  The Catalytic Architecture of Leukotriene C4 Synthase with Two Arginine Residues* 
The Journal of Biological Chemistry  2011;286(18):16392-16401.
Leukotriene (LT) C4 and its metabolites, LTD4 and LTE4, are involved in the pathobiology of bronchial asthma. LTC4 synthase is the nuclear membrane-embedded enzyme responsible for LTC4 biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA4 as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA4 binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA4.
doi:10.1074/jbc.M110.150177
PMCID: PMC3091245  PMID: 21454538
Crystal Structure; Eicosanoid-specific Enzymes; Enzyme Mechanisms; Enzyme Structure; Membrane Proteins; LTC4S; Leukotriene C4 Synthase
15.  T Regulatory Cells Control Antigen-Induced Recruitment of Mast Cell Progenitors to the Lungs of C57BL/6 Mice 
In C57BL/6 mice, the recruitment of mast cell progenitors (MCps) to the lung is a feature of Ag-induced pulmonary inflammation that requires sensitization and challenge and is totally inhibited by the administration of anti-CD4 at the time of challenge. When mAb to TGFβ1 or to IL-10R was administered at the time of challenge, the recruitment of MCp/106 mononuclear cells (MNCs) to the lung was inhibited by 56.3 and 69.6%, respectively, whereas mAb to IL-4, IFN-γ, IL-6, IL-17A, and IL-17F had no effect. In sensitized and challenged C57BL/6 mice lacking TGFβRII on CD4+ cells, the recruitment of MCp/106 MNCs was reduced by 67.8%. The requirement for TGFβ1 and IL-10 suggested a role for CD4+CD25+ T regulatory cells. Mice treated with anti-CD25 at the time of Ag-challenge showed a reduction in the recruitment of MCp/106 MNCs by 77.2% without any reduction in MNC influx. These results reveal an unexpected role for T regulatory cells in promoting the recruitment of MCps to the lungs of C57BL/6 mice with Ag-induced pulmonary inflammation.
doi:10.4049/jimmunol.1001146
PMCID: PMC2951001  PMID: 20601599
16.  Mast Cell Protease 5 Mediates Ischemia-Reperfusion Injury of Mouse Skeletal Muscle1 
Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R2 = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C4 synthase, hemopoietic PGD2 synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.
PMCID: PMC2951006  PMID: 15905575
17.  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
18.  Antigen-Induced Increases in Pulmonary Mast Cell Progenitor Numbers Depend on IL-9 and CD1d-Restricted NKT Cells1 
Pulmonary mast cell progenitor (MCp) numbers increase dramatically in sensitized and aerosolized Ag-challenged mice. This increase depends on CD4+ T cells, as no MCp increase occurs in the lungs of sensitized wild-type (WT) mice after mAb depletion of CD4+ but not CD8+ cells before aerosol Ag challenge. Neither the genetic absence of IL-4, IL-4Rα chain, STAT-6, IFN-γ, or IL-12p40 nor mAb blockade of IFN-γ, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12p40, or IL-12p40Rβ1 before Ag challenge in WT mice reduces the pulmonary MCp increase. However, sensitized and Ag-challenged IL-9-deficient mice and sensitized WT mice given mAb to IL-9 just before Ag challenge show significant reductions in elicited lung MCp/106 mononuclear cells of 47 and 66%, respectively. CD1d-deficient mice and WT mice receiving anti-CD1d before Ag challenge also show significant reductions of 65 and 59%, respectively, in elicited lung MCp/106 mononuclear cells, revealing an additional requirement for MCp recruitment. However, in Jα18-deficient mice, which lack only type 1 or invariant NKT cells, the increase in the numbers of lung MCp with Ag challenge was intact, indicating that their recruitment must be mediated by type 2 NKT cells. Furthermore, anti-CD1d treatment of IL-9-deficient mice or anti-IL-9 treatment of CD1d-deficient mice does not further reduce the significant partial impairment of MCp recruitment occurring with a single deficiency. These findings implicate type 2 NKT cells and IL-9 as central regulators that function in the same pathway mediating the Ag-induced increase in numbers of pulmonary MCp.
doi:10.4049/jimmunol.0901471
PMCID: PMC2782612  PMID: 19783672
19.  Mast cell deficiency in KitW-sh mice does not impair antibody-mediated arthritis 
The Journal of Experimental Medicine  2007;204(12):2797-2802.
We previously reported that joint swelling, synovial thickening, and cartilage matrix depletion induced by the injection of anti-collagen monoclonal antibodies and lipopolysaccharide (LPS) in BALB/c mice are increased in the absence of inhibitory leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4; formerly gp49B1) in a neutrophil-dependent manner. Because both mast cells and neutrophils express LILRB4, we sought a mast cell requirement with mast cell–deficient mouse strains, but unexpectedly obtained full arthritis in KitW-sh mice and full resistance in KitW/KitW-v mice. KitW-sh mice were indeed mast cell deficient as assessed by histology and the absence of IgE/mast cell–dependent passive cutaneous anaphylaxis in the ear and joint as well as passive systemic anaphylaxis. Deletion of LILRB4 in KitW-sh mice exacerbated anti-collagen/LPS-induced joint swelling that was abolished by neutrophil depletion, establishing a counterregulatory role for LILRB4 in the absence of mast cells. Whereas blood neutrophil levels and LPS-elicited tissue neutrophilia were equal in KitW-sh and Kit+ mice, both were impaired in KitW/KitW-v mice. Although both strains are mast cell deficient and protected from IgE-mediated anaphylactic reactions, their dramatically different responses to autoantibody-mediated, neutrophil-dependent immune complex arthritis suggest that other host differences determine the extent of mast cell involvement. Thus, a conclusion for an absolute mast cell role in a pathobiologic process requires evidence from both strains.
doi:10.1084/jem.20071391
PMCID: PMC2118523  PMID: 17998392
20.  Neutrophil-derived leukotriene B4 is required for inflammatory arthritis 
Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
doi:10.1084/jem.20052371
PMCID: PMC2118292  PMID: 16567388
21.  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
22.  Acceptance of the Kober Medal It only gets better 
doi:10.1172/JCI200423465
PMCID: PMC522281  PMID: 15489967
23.  Prevention of Lipopolysaccharide-induced Microangiopathy by gp49B1 
The Journal of Experimental Medicine  2003;198(8):1243-1251.
gp49B1 is expressed on mast cells and inhibits immunoglobulin E–dependent activation and inflammation in vivo. We now show that gp49B1 is expressed on neutrophils and prevents neutrophil-dependent vascular injury in response to lipopolysaccharide (LPS). The intradermal (i.d.) injection of LPS into gp49B1-null (gp49B−/−) but not gp49B1-sufficient (gp49B+/+) mice elicited macroscopic hemorrhages by 24 h, which were preceded on microscopic analyses by significantly more intravascular thrombi (consisting of neutrophils, platelets, and fibrin) that occluded venules and by more tissue neutrophils than in gp49B+/+ mice. However, there were no differences in the number of intact (nondegranulating) mast cells or the tissue levels of mediators that promote neutrophil recruitment. Hemorrhage was prevented by depleting neutrophils, blocking β2 integrin–intercellular adhesion molecule 1 interactions, or inhibiting coagulation. These characteristics indicate that gp49B−/− mice are exquisitely sensitive to a local Shwartzman reaction (LSR) after a single i.d. injection of LPS, whereas in the classic LSR, a second exposure is required for increased β2 integrin function, intravascular neutrophil aggregation, formation of occlusive thrombi, and hemorrhage. Moreover, LPS increased gp49B1 expression on neutrophils in vivo. The results suggest that gp49B1 suppresses the LPS-induced increase in intravascular neutrophil adhesion, thereby providing critical innate protection against a pathologic response to a bacterial component.
doi:10.1084/jem.20030906
PMCID: PMC2194230  PMID: 14557414
Shwartzman reaction; thrombosis; hemorrhage; cell adhesion molecules; innate immunity
24.  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
25.  Intestinal Mast Cell Progenitors Require CD49dβ7 (α4β7 Integrin) for Tissue-specific Homing 
The Journal of Experimental Medicine  2001;194(9):1243-1252.
Mast cells (MCs) are centrally important in allergic inflammation of the airways, as well as in the intestinal immune response to helminth infection. A single lineage of bone marrow (BM)-derived progenitors emigrates from the circulation and matures into phenotypically distinct MCs in different tissues. Because the mechanisms of MC progenitor (MCp) homing to peripheral tissues have not been evaluated, we used limiting dilution analysis to measure the concentration of MCp in various tissues of mice deficient for candidate homing molecules. MCp were almost completely absent in the small intestine but were present in the lung, spleen, BM, and large intestine of β7 integrin-deficient mice (on the C57BL/6 background), indicating that a β7 integrin is critical for homing of these cells to the small intestine. MCp concentrations were not altered in the tissues of mice deficient in the αE integrin (CD103), the β2 integrin (CD18), or the recombination activating gene (RAG)-2 gene either alone or in combination with the interleukin (IL)-receptor common γ chain. Therefore, it is the α4β7 integrin and not the αEβ7 integrin that is critical, and lymphocytes and natural killer cells play no role in directing MCp migration under basal conditions. When MCp in BALB/c mice were eliminated with sublethal doses of γ-radiation and then reconstituted with syngeneic BM, the administration of anti-α4β7 integrin, anti-α4 integrin, anti-β7 integrin, or anti–MAdCAM-1 monoclonal antibodies (mAbs) blocked the recovery of MCp in the small intestine. The blocking mAbs could be administered as late as 4 d after BM reconstitution with optimal inhibition, implying that the MCp must arise first in the BM, circulate in the vasculature, and then translocate into the intestine. Inasmuch as MCp are preserved in the lungs of β7 integrin-deficient and anti-α4β7 integrin-treated mice but not in the small intestine, α4β7 integrin is critical for tissue specific extravasation for localization of MCp in the small intestine, but not the lungs.
PMCID: PMC2195984  PMID: 11696590
αE/CD103 integrin; β2/CD18 integrin; c-kit; stem cell factor; MAdCAM-1

Results 1-25 (69)