Akiko Maekawa

Dectin-2 Recognition of House Dust Mite Triggers Cysteinyl Leukotriene Generation by Dendritic Cells

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.

Leukotriene E4–induced pulmonary inflammation is mediated by the P2Y12 receptor

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.

Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells

The cysteinyl leukotrienes (cys-LTs) LTC4, LTD4, and LTE4 are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC4, LTD4, and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC4, but only partially to LTD4. Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC4 and UDP (≈3 log10-fold shifts in dose-response for each agonist) over LTD4 (1.3 log10-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.

Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors.

The cysteinyl leukotrienes (cys-LTs) are a family of potent lipid mediators of inflammation derived from arachidonic acid. Activation of certain cell types results in the biosynthesis and export of leukotriene (LT) C4, which then undergoes extracellular metabolism to LTD4 and LTE4. LTE4, the most stable cys-LT, is only a weak agonist for the defined type 1 and type 2 cys-LT receptors (CysLT1R and CysLT2R, respectively). We had recognized a greater potency for LTE4 than LTC4 or LTD4 in constricting guinea pig trachea in vitro and comparable activity in eliciting a cutaneous wheal and flare response in humans. Thus, we hypothesized that a vascular permeability response to LTE4 in mice lacking both the CysLT1R and CysLT2R could establish the existence of a separate LTE4 receptor. We now report that the intradermal injection of LTE4 into the ear of mice deficient in both CysLT1R and CysLT2R elicits a vascular leak that exceeds the response to intradermal injection of LTC4 or LTD4, and that this response is inhibited by pretreatment of the mice with pertussis toxin or a Rho kinase inhibitor. LTE4 is ≈64-fold more potent in the CysLT1R/CysLT2R double-deficient mice than in sufficient mice. The administration of a CysLT1R antagonist augmented the permeability response of the CysLT1R/CysLT2R double-deficient mice to LTC4, LTD4, and LTE4. Our findings establish the existence of a third receptor, CysLTER, that responds preferentially to LTE4, the most abundant cys-LT in biologic fluids, and thus reveal a new target for therapeutic intervention.

Targeted Gene Disruption Reveals the Role of Cysteinyl Leukotriene 1 Receptor in the Enhanced Vascular Permeability of Mice Undergoing Acute Inflammatory Responses

The cysteinyl leukotrienes (cysLTs), leukotriene (LT) C4, LTD4, and LTE4, are proinflammatory lipid mediators generated in the mouse by hematopoietic cells such as macrophages and mast cells. There are two mouse receptors for the cysLTs, CysLT1 receptor (CysLT1R) and CysLT2R, which are 38% homologous and are located on mouse chromosomes X and 14, respectively. To clarify the different roles of the CysLT1R and CysLT2R in inflammatory responses in vivo, we generated CysLT1R-deficient mice by targeted gene disruption. These mice developed normally and were fertile. In an intracellular calcium mobilization assay with fura-2 acetoxymethyl ester, peritoneal macrophages from wild-type littermates, which express both CysLT1R and CysLT2R, responded substantially to 1 × 10−6 m LTD4 and slightly to 1 × 10−6 m LTC4, whereas the macrophages from CysLT1R-deficient mice did not respond to either LTD4 or LTC4. Plasma protein extravasation, but not neutrophil infiltration, was significantly reduced in CysLT1R-deficient mice subjected to zymosan A-induced peritoneal inflammation. Plasma protein extravasation was also significantly diminished in CysLT1R-deficient mice undergoing IgE-mediated passive cutaneous anaphylaxis as compared with the wild-type mice. Thus, the cysLTs generated in vivo by either monocytes/macrophages or mast cells utilize CysLT1R for the response of the microvasculature in acute inflammation.

Identification of GPR99 Protein as a Potential Third Cysteinyl Leukotriene Receptor with a Preference for Leukotriene E4 Ligand

Background: A most stable cysteinyl leukotriene, LTE4, mediates vascular permeability in mice lacking the two known receptors. Results: GPR99 deficiency abolishes LTE4-induced vascular permeability in mice also lacking the two known receptors. Conclusion: GPR99 is a potential third cysteinyl leukotriene receptor. Significance: GPR99 may be a therapeutic target for inflammatory diseases. The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4), a conjugation product of glutathione and eicosatetraenoic acid, and its metabolites, LTD4 and LTE4, are lipid mediators of smooth muscle constriction and inflammation in asthma. LTD4 is the most potent ligand for the type 1 cys-LT receptor (CysLT1R), and LTC4 and LTD4 have similar lesser potency for CysLT2R, whereas LTE4 has little potency for either receptor. Cysltr1/Cysltr2−/− mice, lacking the two defined receptors, exhibited a comparable dose-dependent vascular leak to intradermal injection of LTC4 or LTD4 and an augmented response to LTE4 as compared with WT mice. As LTE4 retains a cysteine residue and might provide recognition via a dicarboxylic acid structure, we screened cDNAs within the P2Y nucleotide receptor family containing CysLTRs and dicarboxylic acid receptors with trans-activator reporter gene assays. GPR99, previously described as an oxoglutarate receptor (Oxgr1), showed both a functional and a binding response to LTE4 in these transfectants. We generated Gpr99−/− and Gpr99/Cysltr1/Cysltr2−/− mice for comparison with WT and Cysltr1/Cysltr2−/− mice. Strikingly, GPR99 deficiency in the Cysltr1/Cysltr2−/− mice virtually eliminated the vascular leak in response to the cys-LT ligands, indicating GPR99 as a potential CysLT3R active in the Cysltr1/Cysltr2−/− mice. Importantly, the Gpr99−/− mice showed a dose-dependent loss of LTE4-mediated vascular permeability, but not to LTC4 or LTD4, revealing a preference of GPR99 for LTE4 even when CysLT1R is present. As LTE4 is the predominant cys-LT species in inflamed tissues, GPR99 may provide a new therapeutic target.

GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT1R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D4 with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT1R, the robust CysLT1R-mediated calcium response to LTD4 was abolished. The membrane expression of the CysLT1R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD4-elicited ERK phosphorylation and the specific binding of [3H]LTD4 to microsomal membranes were fully inhibited. CysLT1R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT1R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMΦs) increased both the membrane expression of CysLT1R protein by FACS analysis and the LTD4-elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT1R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT1R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT1R that suppresses CysLT1R-mediated function at the cell membrane.

The leukotriene E4 puzzle: Finding the missing pieces and revealing the pathobiologic implications

The intracellular parent of the cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), is formed by conjugation of LTA(4) and reduced glutathione by LTC(4) synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC(4) is converted to LTD(4) and LTE(4) through sequential enzymatic removal of glutamic acid and then glycine. Only LTE(4) 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. LTE(4) has received little attention because it binds poorly to the classical type 1 and 2 cysLT receptors and is much less active on normal airways than LTC(4) or LTD(4). However, early studies indicated that LTE(4) caused skin swelling in human subjects as potently as LTC(4) and LTD(4), that airways of asthmatic subjects (particularly those that were aspirin sensitive) were selectively hyperresponsive to LTE(4), and that a potential distinct LTE(4) receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE(4): P2Y(12), an adenosine diphosphate receptor, and CysLT(E)R, which was observed functionally in the skin of mice lacking the type 1 and 2 cysLT receptors. These findings prompt a renewed focus on LTE(4) receptors as therapeutic targets that are not currently addressed by available receptor antagonists.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans

Phospholipase A2 (PLA2) hydrolyzes the sn-2 position of cell membrane phospholipids to release fatty acids and lysophospholipids. We have previously reported that group V secretory PLA2 (sPLA2) translocates from the Golgi and recycling endosomes of mouse peritoneal macrophages to newly formed phagosomes and regulates the phagocytosis of zymosan, suggesting a role in innate immunity. Here we report that in macrophages lacking group V sPLA2, phagosome maturation was reduced 50–60% at early time points while the binding of zymosan was unimpaired. The ability of group V sPLA2 to regulate phagocytosis extended to phagocytosis of IgG- and complement-opsonized sheep RBC. Moreover, macrophages lacking group V sPLA2 had delays in phagocytosis, phagosome maturation, and killing of Candida albicans. Cytokine production and eicosanoid generation were not impaired by the lack of group V sPLA2. Furthermore, in a model of systemic candidiasis, mice lacking group V sPLA2 had an increased fungal burden in the kidney, liver, and spleen at day 7 postinfection and increased mortality. Thus, group V sPLA2 regulates phagocytosis through major phagocytic receptors and contributes to the innate immune response against C. albicans by regulating phagocytosis and killing through a mechanism that is likely dependent on phagolysosome fusion.