The Journal of Allergy and Clinical Immunology
Volume 125, Issue 2 , Pages 477-482, February 2010

Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

  • Josiane S. Neves, PhD

      Affiliations

    • Division of Allergy and Inflammation, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Mass
    • Institute of Biomedical Sciences (ICB), Federal University of Rio de Janeiro, UFRJ, Rio de Janeiro, Brazil
    • ,
  • Amy L. Radke, BS

      Affiliations

    • Division of Allergy and Inflammation, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Mass
    • ,
  • Peter F. Weller, MD

      Affiliations

    • Division of Allergy and Inflammation, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Mass
    • Corresponding Author InformationReprint requests: Peter F. Weller, MD, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, CLS 943, Boston, MA 02215.

Received 17 August 2009; received in revised form 27 October 2009; accepted 10 November 2009.

Article Outline

Background

Cysteinyl leukotrienes (cysLTs) are recognized to act via receptors (cysLTRs) expressed on cell surface plasma membranes. Agents that block cysLT1 receptor (cysLT1R) are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis, and urticaria, but have unknown functional capabilities.

Objective

We evaluated the expression of cysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules.

Methods

We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysLTs, and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and Western blot.

Results

We report that receptors for cysLTs, cysLT1R, cysLT2 receptor, and the purinergic P2Y12 receptor, are expressed on eosinophil granule membranes. Leukotriene (LT) C4 and extracellularly generated LTD4 and LTE4 stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12 receptor antagonist, inhibited cysLT-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of cysLT1R, inhibited eosinophil cationic protein release elicited by LTC4 and LTD4 as well as by LTE4.

Conclusion

These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysLT-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly.

Key words: Granules, cysteinyl leukotriene, eosinophil, allergy, asthma, montelukast

Abbreviations used: cysLT, Cysteinyl leukotriene, cysLTR, Cysteinyl leukotriene receptor, cysLT1R, Cysteinyl leukotriene 1 receptor, cysLT2R, Cysteinyl leukotriene 2 receptor, ECP, Eosinophil cationic protein, GPCR, G protein–coupled receptor, LT, Leukotriene, P2Y12R, P2Y12 receptor

 

Cysteinyl leukotrienes (cysLTs) constitute an important class of potent, proinflammatory mediators that are synthesized from membrane-derived arachidonic acid via the 5-lipoxygenase pathway leading to the formation of leukotriene (LT) A4 that is converted into LTC4 by the action of LTC4 synthase.1 Intracellular LTC4 is actively transported extracellularly, where it is enzymatically converted sequentially to LTD4 and then to LTE4.1 cysLTs are cell-membrane impermeant and are recognized to mediate their actions by engaging 2 heptahelical G protein–coupled receptors (GPCRs), designated cysLT1 receptor (cysLT1R) and cysLT2 receptor (cysLT2R), that are expressed on cell surface plasma membranes.1, 2 The rank order of affinities of cysLTs for human cysLT1R and cysLT2R, based on transfected cells, is LTD4>LTC4 = LTE4 and LTC4 = LTD4>LTE4, respectively.3, 4

Eosinophils, prominent leukocytes in allergic inflammation and anthelminthic responses,5 are characterized by an abundance of intracellular granules that contain preformed proteins including distinct cationic proteins, such as eosinophil cationic protein (ECP), and a wide range of preformed cytokines, chemokines, and growth factors.6, 7 Human eosinophils are major sources of cysLTs and express both cysLT1R and cysLT2R.8 cysLTs and their receptors have critical roles in allergic diseases and represent important therapeutic targets for the control of asthma and other pathophysiological conditions.9, 10 Medications, including those recognized to inhibit ligand-binding to cysLT1R, such as montelukast, are used in the management of asthma and related allergic diseases.1

In addition to its conventional plasma membrane expression, cysLT1R has been immunolocalized to nuclei in colorectal adenocarcinoma cells11 and in a human mast cell line,12 although the functions of nuclear cysLT1Rs have not been defined. We have recognized that some cytokine and chemokine receptors are richly present on eosinophil granules,13 and we have recently demonstrated that eosinophil granules, on extrusion from eosinophils, respond to a stimulating cytokine, IFN-γ, and a chemokine, eotaxin-1 chemokine (C-C motif) ligand 11 (CCL11), via cognate granule membrane-expressed receptors, to activate intragranular signaling pathways that elicit granule protein secretion.14 Intact membrane-bound eosinophil granules have long been recognized to be present extracellularly in tissues and secretions in many human eosinophil-enriched disorders, including asthma, rhinitis, urticaria, atopic dermatitis, eosinophilic esophagitis, and helminth infections.15, 16, 17, 18, 19, 20, 21 The capacity of cell-free human eosinophil granules to act via receptor-mediated responses to polypeptide agonists and secrete granule-derived cytokines and cationic proteins has indicated that cell-free eosinophil granules may be functionally significant.14 In the current study, we have evaluated whether receptors for cysLTs, in addition to their conventional plasma and nuclear membrane localizations, are expressed and functional on the surface membranes of cell-free human eosinophil granules. We investigated the efficacy of intracellular LTC4 and extracellular LTD4 and LTE4 as potential agonists of eosinophil granule secretion and the capacity of montelukast to inhibit cysLT-elicited eosinophil granule secretion.

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Methods 

Eosinophil purification and subcellular fractionation 

Eosinophils were purified from the blood of healthy and atopic donors as previously described.14, 22 Experiments were approved by the Beth Israel Deaconess Medical Center Committee on Clinical Investigation, and informed consent was obtained from all subjects. Subcellular fractionation and eosinophil granule isolation were performed as described.14, 22 Briefly, eosinophils were disrupted by nitrogen cavitation (600 psi, 10 minutes) and postnuclear supernatants were ultracentrifuged (100,000g, 1 hour at 4°C) in linear isotonic Optiprep (Axis-Shield, Oslo, Norway) gradients (0% to 45%). Purity of isolated granules free of plasma membranes or other contaminating structures was rigorously ascertained as previously documented.14, 22

Stimulation of isolated eosinophil granules 

Subcellular fractions containing isolated granules were mixed with RPMI + 0.1% ovalbumin (without phenol red; Sigma, St Louis, Mo) followed by centrifugation (2500g, 10 minutes). Granule pellets were resuspended in 250 μL of the same medium. Treatments with montelukast (0.1 and 1 μmol/L; Merck, Rahway, NJ) and MRS 2395 (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester, 1 and 10 μmol/L; Sigma), a selective P2Y12 receptor (P2Y12R) antagonist, were performed for 15 minutes before stimulation with LTC4, LTD4, or LTE4 for 30 minutes at 37°C. After centrifugation at 4°C (2500g, 10 minutes), granule supernatants were collected and stored at –80°C. Drugs were diluted in dimethyl sulfoxide at a final concentration <0.01%, which had no effect on granule secretion.

Assays of granule-secreted proteins 

The ECP levels in eosinophil granule supernatants were analyzed by a quantitative ECP ELISA kit (Medical & Biological Labs, Naka-ku Nagoya, Japan) according to the manufacturer's instructions. Stimulated ECP secretion represents ECP levels from stimulated samples minus ECP levels from unstimulated samples. Cytokines IL-4, IL-6, IFN-γ, IL-10, IL-12 (p70), IL-13, and TNF-α were quantified by using multiplex assays (Bio-Rad Laboratories, Inc, Hercules, Calif).

Flow cytometry of isolated granules and eosinophils 

Isolated granules or eosinophils were incubated for 1 hour with primary antibody or primary antibody premixed with blocking peptide. Then the granules were washed and incubated with the respective fluorescein isothiocyanate–conjugated secondary antibodies for 15 minutes on ice in the absence of granule fixation. After staining, granules were fixed in buffer containing 2% paraformaldehyde without methanol (Electron Microscopy Sciences, Fort Washington, Pa) for 5 minutes. Control or nonimmune antibodies were included for all. Analyses were performed on a FACScan with CELLQUEST software (BD Biosciences, San Jose, Calif).

Mouse antihuman P2Y12R polyclonal antibody (1:100) was purchased from Abnova Corp (Taipei, Taiwan). Goat polyclonal antibody against a peptide mapping the N-terminus domain of cysLT1R (N-20; 5 μg/mL) and the blocking peptide (20 μg/mL) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Rabbit polyclonal antibodies against a peptide mapping the C-terminus of the cysLT1R (5 μg/mL) and the N-terminus of the cysLT2R (5 μg/mL) and the blocking peptide (20 μg/mL) were purchased from Cayman Chemical (Ann Arbor, Mich). Fluorescein isothiocyanate–conjugated F(ab')2 goat antimouse, donkey antigoat, and goat antirabbit IgGs were used as secondary antibodies (1:100). Mouse, goat, and rabbit normal IgGs were used as control antibodies (Jackson Immuno Research Inc, West Grove, Pa).

Western blotting 

Granules and eosinophils were lysed in lithium dodecyl sulfate (LDS) sample reducing buffer (Nupage; Invitrogen, Carlsbad, Calif) and boiled for 5 minutes. Samples were loaded on 10% Bis-Tris gels (Invitrogen) and run using 3-(n-morpholino) propanesulfonic acid (MOPS) running buffer. Gels were transferred to nitrocellulose membranes (Thermo Fisher Scientific, San Jose, Calif), blocked with 5% milk for at least 1 hour, and probed with rabbit anti-P2Y12R polyclonal antibody (1:400; Alomone Labs, Jerusalem, Israel) or the antibody premixed with the blocking peptide overnight. Antirabbit antibody conjugated to horseradish peroxidase (1:15,000; Jackson Immuno Research Inc) was used as secondary Ab. Membranes were developed with West Femto chemiluminescence kits (Thermo Fisher Scientific).

Statistical analysis 

Secreted ECP levels, means of duplicates ± SDs, are ECP levels from stimulated granules minus ECP levels from unstimulated granules. Data are shown for 1 experiment representative of 3. Quantities of unstimulated and stimulated ECP secreted varied among replicate experiments, but the patterns of release and statistical differences were consistent in each of the replicate experiments. Results were analyzed by 1-way ANOVA, followed by the Newman-Keuls test. P values <.05 were considered significant (2-tailed test).

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Results 

Extracellular eosinophil granules express on their membranes amino-terminal, ligand-binding domains for cysLT receptors 

To evaluate whether secretory responses of eosinophil granules, as intracellularly resident or extracellularly released organelles, might be mediated by intracrine or paracrine acting cysLTs, we first analyzed by flow cytometry the expression of cysLT1R and cysLT2R proteins on the surface membranes of isolated human eosinophil granules. Without membrane permeabilization, granules displayed immunoreactivity for both cysLT1R (Fig 1, A) and cysLT2R (Fig 1, B) using polyclonal antibodies raised against epitopes present specifically in the nominally “extracellular,” ligand-binding regions of each cysLT receptor (cysLTR). Specificities of each polyclonal antibody for cysLT1R (Fig 1, A) and cysLT2R (Fig 1, B) were corroborated by complete neutralization of immunostaining through preincubation of the anti-cysLT1R and anti-cysLT2R polyclonal antibodies with their respective specific blocking peptide immunogens. In contrast, eosinophil granules exhibited no staining with a polyclonal antibody raised to an “intracellular” carboxyl-terminal domain of cysLT1R (Fig 1, C).

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  • Fig 1. 

    Isolated granules expressed extracellular domains of cysLT1R (A) and cysLT2R (B) but not the carboxy-terminal intracellular domain of CysLT1R (C). Shaded histograms represent staining with control antibody (Ab). Solid and dashed lines represent staining with polyclonal specific antibodies (pAb) and anti-cysLTR pAbs neutralized by absorption with their respective immunogen peptide. Data are from 1 experiment, representative of 3.

Extracellular eosinophil granules secrete ECP in response to cysLTs 

Given our recent finding that a cytokine and a chemokine could elicit receptor-mediated secretion by cell-free eosinophil granules,14 we evaluated whether cysLTs might elicit functional secretory responses by extracellular human eosinophil granules that express cysLTRs. Isolated eosinophil granules were stimulated with LTC4, LTD4, and LTE4, and all 3 cysLTs effectively stimulated secretion of ECP from within eosinophil granules (Fig 2). Notably, dose-responses to the 3 cysLTs differed. LTC4 (Fig 2, A) and LTE4 (Fig 2, C) elicited ECP secretion only in lower concentrations (including well below 30 nmol/L), fully compatible with physiologic signaling responses in vivo. Inhibited ECP secretion at higher LTC4 and LTE4 levels of 300 and 3000 nmol/L is consistent with the high-dose inhibition characteristic of GPCRs. Likewise, LTD4 (Fig 2, B) induced granule ECP secretion at significant levels at very low physiologic concentrations (0.3 and 0.03 nmol/L) and not at higher intermediate (3 and 30 nmol/L) concentrations. In contrast, LTD4 also elicited granule ECP secretion at higher concentrations (300 and 3000 nmol/L). This dose-response suggests engagements of 2 receptors for LTD4—the first responds to low LTD4 levels and then exhibits higher dose inhibition, and a second receptor putatively mediates secretion elicited by much higher concentrations of LTD4. Whereas a cytokine (IFN-γ) and a chemokine (eotaxin-1, CCL11) stimulated selective, differential secretion of specific cytokines, as well as ECP, from eosinophil granules,14 we evaluated whether cysLTs elicited cytokine secretion from within eosinophil granules. All 3 cysLTs failed to induce cytokine secretion of known eosinophil secretable cytokines,23 as measured in granule supernatants using cytokine multiplex assays for IL-4, IL-6, IFN-γ, IL-10, IL-12 (p70), IL-13, and TNF-α. Within the limits of cytokine detection, these findings suggest that cysLT stimulation of eosinophil granules may provide a means for selective mobilization and secretion of the granule cationic protein, ECP, without concomitant mobilization of eosinophil granule-stored cytokines.

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  • Fig 2. 

    Isolated granules were stimulated with different concentrations (0.03-3000 nmol/L) of LTC4 (A), LTD4 (B), and LTE4 (C), and ECP levels were measured in the supernatants. Data are shown for 1 experiment, representative of 3. +, Significantly increased ECP release (P < .05) compared with nonstimulated granules. M, mol/L.

A notable finding was the capacity of low concentrations of LTE4 to elicit granule ECP secretion (Fig 2, C). As a major extracellularly generated cysLT, the capacity of LTE4 to stimulate eosinophil granule secretory responses could be pertinent to the known, albeit often overlooked, presence of free membrane-bound eosinophil granules in human diseases, including allergic asthma and rhinitis, dermatitis, helminth infections, eosinophilic esophagitis, and urticaria.14 LTE4 is a weak stimulus for both human cysLT1R and cysLT2R.3, 4 Moreover, LTE4, in contrast to LTD4, has elicited airways responses in human beings not likely based on cysLT1R and cysLT2R.24, 25, 26

cysLT-induced ECP release is inhibited by MRS 2395 via eosinophil granule-expressed P2Y12Rs 

An additional human LTE4 receptor, the purinergic P2Y12R, was identified by in silico and in vitro methods.27 To assess whether ECP secretion induced by cysLTs on eosinophil granules might be mediated by P2Y12R, isolated granules were pretreated with MRS 2395, a P2Y12R antagonist. ECP secretion induced by LTC4 (Fig 3, A), LTD4 (Fig 3, B and C), and LTE4 (Fig 3, D) was dose-dependently inhibited by MRS 2395. Nonaka et al,27 in their in silico screening for P2Y12R ligands, identified both LTE4 and LTD4 (LTC4 not tested) as potential endogenous ligands for this receptor. In our assays, a P2Y12R antagonist effectively inhibited ECP secretion from eosinophil granules induced by all 3 cysLTs.

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  • Fig 3. 

    MRS 2395, a selective P2Y12R antagonist, dose-dependently inhibited the ECP release induced by LTC4 30 nmol/L (A), LTD4 0.3 nmol/L (B) and 300 nmol/L (C), and LTE4 30 nmol/L (D). + and ∗, P <.05 for ECP released compared with nonstimulated and leukotriene-stimulated granules, respectively. Eosinophil (E) and isolated granule surface membranes (F) expressed the P2Y12R. Shaded histograms and solid lines represent staining with control antibody and with an anti-P2Y12R specific polyclonal antibody (pAb), respectively. G, The expression of the P2Y12R was confirmed on eosinophil and isolated granules by Western blots. Specificity of immunodetection was confirmed by neutralization with the anti-P2Y12R pAb by absorption with its respective immunogen peptide. All data are shown for 1 experiment, representative of 3. Eos, Eosinophils; Gran, granules; M, mol/L.

Human eosinophils express several mRNAs that encode P2X and P2Y receptor subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y14, P2X1, P2X4, and P2X7.28 However, the expression of the subtype P2Y12R had not been recognized for human eosinophils. To ascertain that the inhibitory effects of MRS 2395 on granules were a result of its inhibitory actions on P2Y12R, we investigated the expression of this receptor on eosinophils and isolated granules. By flow cytometry, under membrane nonpermeabilizing conditions, eosinophils (Fig 3, E) and isolated, extracellular granules (Fig 3, F) exhibited immunostaining for P2Y12R. Moreover, expression of P2Y12R on isolated granules and eosinophils was confirmed by Western blots (Fig 3, G). The specificity of the polyclonal antibody was ascertained by complete neutralization through preincubation of the anti-P2Y12R polyclonal antibody with its respective specific blocking peptide immunogen.

Montelukast inhibits ECP secretion from cysLT-stimulated eosinophil granules 

To evaluate the therapeutic potential of montelukast, currently used clinically on the basis of its actions as a cysLT1R antagonist, we assessed the capacities of montelukast to inhibit LTC4-elicited, LTD4-elicited, and LTE4-elicited ECP secretion from human eosinophil granules. Notably, montelukast inhibited eosinophil granule ECP secretion induced by LTC4 (30 nmol/L; Fig 4, A), LTD4 (0.3 and 300 nmol/L; Fig 4, B and C), and LTE4 (30 nmol/L; Fig 4, D).

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  • Fig 4. 

    Montelukast dose-dependently inhibited ECP secretion induced by LTC4 30 nmol/L (A), LTD4 0.3 nmol/L (B) and 300 nmol/L (C), and LTE4 30 nmol/L (D). Data are shown for 1 experiment, representative of 3. + and ∗, P <.05 for ECP released compared with nonstimulated granules and leukotriene-stimulated granules, respectively. μ, mol/L.

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Discussion 

Our findings of functional receptors for cysLTs on cell-free extracellular human eosinophil granule membranes, sensitive to inhibition by montelukast and a P2Y12R antagonist, identify novel mechanisms whereby cysLTs may serve as intracrine and paracrine mediators of eosinophil granule-derived secretion. CysLTRs, heretofore, have been widely recognized to localize and function principally at cell plasma membranes.1 To date, recognition of intracellular sites of cysLTRs has been limited to their immunolocalization, without defined functional roles, on nuclei of a human mast cell line12 and colon adenocarcinoma cells.11 We now demonstrate that receptors for cysLTs are expressed and functional on the membranes of an intracellularly derived organelle, the granules of human eosinophils. With antibodies specific to each receptor, the protein receptor components immunologically demonstrated to be present on human eosinophil granules include those for cysLT1R, cysLT2R, and the purinergic P2Y12R. Notably, each cysLTR protein was expressed with its ligand-binding domain on the outer membranes of human eosinophil granules.

As is true of other GPCRs, there is the potential for individual protein chains of cysLTRs to form heterodimers or hetero-oligomers, which might influence their pharmacology and function.29 Previously in a human mast cell line, functionally significant heterodimeric combinations of cysLT1R and cysLT2R were demonstrated.12 We document that 3 protein components of cysLT receptors, cysLT1R, cysLT2R, and the purinergic P2Y12R, are expressed on human eosinophil granules. Whether the 3 candidate cysLTR protein chains function or interact as homodimers, heterodimers, or oligomers in human eosinophil granules can not be ascertained in these “primary” cells, in contrast with transfectable cell lines. Nevertheless, of potential clinical pertinence, whatever the interactions maybe among the cysLTR protein chains, cell-free human eosinophil granules, long recognized to be present as intact membrane bound structures in tissues and secretions associated with varied allergic (eg, asthma, rhinitis, urticaria) and other eosinophil-associated diseases,15, 16, 17, 18, 19, 20, 21 have the capacity to secrete ECP in response to low and even subnanomolar concentrations of the 3 cysLTs, including the 2 extracellularly generated cysLTs, LTD4 and LTE4.

Cysteinyl LT–elicited ECP secretion was inhibited by MRS 2395, a selective P2Y12R antagonist. Clinically, this receptor is blocked by clopidogrel. Moreover, isolated granule secretory responses to each of the cysLTs were blocked by montelukast. The 1 μmol/L concentration of montelukast that uniformly inhibited LTC4-elicited, LTD4-elicited, and LTE4-elicited granule secretion of ECP is in accord with levels of montelukast achieved in patients receiving this agent.30 Decreases in serum ECP levels have been demonstrated in subjects with asthma treated with montelukast.31 Montelukast is known as a potent selective cysLT1R antagonist effective as a therapeutic for asthma and other allergic conditions.32 Inhibitory actions of montelukast beyond those based mainly on inhibition of cysLT1R have been reported.33, 34, 35 Montelukast inhibits the recently deorphanized GPR1733 and several purinergic G protein–coupled receptors, suggesting that cysLT1R antagonists possibly interact functionally with signaling pathways of P2Y receptors.34 Besides, on human eosinophils, montelukast was suggested as a regulator of eosinophil protease activity through a leukotriene-independent mechanism.35 In part, because LTE4 is a weak agonist for cysLT1R, montelukast-sensitive mechanisms that are operative in stimulating eosinophil granule ECP secretion are likely acting other than singularly by cysLT1R blockage.

Within eosinophils, synthesis of LTC4 (but not of extracellularly formed LTD4 or LTE4) occurs at perinuclear membranes and cytoplasmic lipid bodies.8, 36, 37, 38 For granules, as intracellular organelles, the presence of functional membrane-expressed receptors might be indicative of intracrine roles for LTC4 as regulators of granule protein mobilization, sorting, and secretion. We previously recognized a role principally for LTC4, and not LTD4 or LTE4, as an intracrine mediator of CCR3 receptor-elicited, vesicular transport-mediated IL-4 secretion by human eosinophils.39 Such release of IL-4 via vesicular transport from within eosinophils13 likely involves more complex steps beyond intracellular granule secretion that may be regulated by actions of LTC4. That intracellular LTC4 can activate release of ECP from cysLT-responsive receptors expressed on eosinophil granule membranes helps in further delineating roles for LTC4 as an intracrine mediator of eosinophil secretory responses. For granules, as extruded extracellular “organelles,” the expression of functional cysLTRs on granule membranes capable of responding to the extracellularly formed cysLTs, LTD4 and LTE4, underscores the secretion-competence of cell-free eosinophil granules. Together, the recognition that cysLTRs are localized on the outer membranes of human eosinophil granules and mediate cysLT-elicited secretion from within these granules identifies new roles, amenable to therapeutic interventions, for cysLTs as mediators in eosinophil-associated diseases.

Clinical implications

cysLTs elicit cell-free eosinophil granule secretion, suggesting new roles, amenable to therapeutic interventions, for these lipid mediators in eosinophil-associated diseases.

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References 

  1. Peters-Golden M, Henderson WR. Leukotrienes. N Engl J Med. 2007;357:1841–1854
  2. Rovati GE, Capra V. Cysteinyl-leukotriene receptors and cellular signals. Sci World J. 2007;7:1375–1392
  3. Lynch KR, O'Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature. 1999;399:789–793
  4. Heise CE, O'Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem. 2000;275:30531–30536
  5. Blanchard C, Rothenberg ME. Biology of the eosinophil. Adv Immunol. 2009;101:81–121
  6. Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol. 2006;24:147–174
  7. Gleich GJ. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol. 2000;105:651–663
  8. Bandeira-Melo C, Weller PF. Eosinophils and cysteinyl leukotrienes. Prostaglandins Leukot Essent Fatty Acids. 2003;69:135–143
  9. Capra V, Thompson MD, Sala A, Cole DE, Folco G, Rovati GE. Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends. Med Res Rev. 2007;27:469–527
  10. Kanaoka Y, Boyce JA. Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses. J Immunol. 2004;173:1503–1510
  11. Nielsen CK, Campbell JI, Ohd JF, Morgelin M, Riesbeck K, Landberg G, et al. A novel localization of the G-protein-coupled CysLT1 receptor in the nucleus of colorectal adenocarcinoma cells. Cancer Res. 2005;65:732–742
  12. Jiang Y, Borrelli LA, Kanaoka Y, Bacskai BJ, Boyce JA. CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene dependent mitogenic responses of mast cells. Blood. 2007;110:3263–3270
  13. Spencer LA, Melo RC, Perez SA, Bafford SP, Dvorak AM, Weller PF. Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion. Proc Natl Acad Sci U S A. 2006;103:3333–3338
  14. Neves JS, Perez SA, Spencer LA, Melo RC, Reynolds L, Ghiran I, et al. Eosinophil granules function extracellularly as receptor-mediated secretory organelles. Proc Natl Acad Sci U S A. 2008;105:18478–18483
  15. Erjefalt JS, Persson CG. New aspects of degranulation and fates of airway mucosal eosinophils. Am J Respir Crit Care Med. 2000;161:2074–2085
  16. Watanabe K, Misu T, Inoue S, Edamatsu H. Cytolysis of eosinophils in nasal secretions. Ann Otol Rhinol Laryngol. 2003;112:169–173
  17. Cheng JF, Ott NL, Peterson EA, George TJ, Hukee MJ, Gleich GJ, et al. Dermal eosinophils in atopic dermatitis undergo cytolytic degeneration. J Allergy Clin Immunol. 1997;99:683–692
  18. Gutierrez-Pena EJ, Knab J, Buttner DW. Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine. Parasitol Res. 1998;84:607–615
  19. Aceves SS, Newbury RO, Dohil R, Bastian JF, Broide DH. Esophageal remodeling in pediatric eosinophilic esophagitis. J Allergy Clin Immunol. 2007;119:206–212
  20. Toyoda M, Maruyama T, Morohashi M, Bhawan J. Free eosinophil granules in urticaria: a correlation with the duration of wheals. Am J Dermatopathol. 1996;18:49–57
  21. Chehade M, Sampson HA, Morotti RA, Magid MS. Esophageal subepithelial fibrosis in children with eosinophilic esophagitis. J Pediatr Gastroenterol Nutr. 2007;45:319–328
  22. Neves JS, Perez SA, Spencer LA, Melo RC, Weller PF. Subcellular fractionation of human eosinophils: isolation of functional specific granules on isoosmotic density gradients. J Immunol Methods. 2009;344:64–72
  23. Spencer LA, Szela CT, Perez SA, Kirchhoffer CL, Neves JS, Radke AL, et al. Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially. J Leukoc Biol. 2009;85:117–123
  24. Lee TH, Woszczek G, Farooque SP. Leukotriene E(4): perspective on the forgotten mediator. J Allergy Clin Immunol. 2009;124:417–421
  25. Gauvreau GM, Parameswaran KN, Watson RM, O'Byrne PM. Inhaled leukotriene E(4), but not leukotriene D(4), increased airway inflammatory cells in subjects with atopic asthma. Am J Respir Crit Care Med. 2001;164:1495–1500
  26. Austen KF, Maekawa A, Kanaoka Y, Boyce JA. The leukotriene E(4) puzzle: finding the missing pieces and revealing the pathobiologic implications. J Allergy Clin Immunol. 2009;124:406–414
  27. Nonaka Y, Hiramoto T, Fujita N. Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods. Biochem Biophys Res Commun. 2005;337:281–288
  28. Ferrari D, la Sala A, Panther E, Norgauer J, Di Virgilio F, Idzko M. Activation of human eosinophils via P2 receptors: novel findings and future perspectives. J Leukoc Biol. 2006;79:7–15
  29. Milligan G. G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function. Br J Pharmacol. 2009;158:5–14
  30. Friesen CA, Neilan NA, Schurman JV, Taylor DL, Kearns GL, Abdel-Rahman SM. Montelukast in the treatment of duodenal eosinophilia in children with dyspepsia: effect on eosinophil density and activation in relation to pharmacokinetics. BMC Gastroenterol. 2009;9:32
  31. Kopriva F, Janostakova A, Jarmila S, Zapalka M, Hajduch M. Montelukast decreases plasma endothelin-1 and serum eosinophil cationic protein levels in paediatric atopic asthma. Clin Drug Investig. 2006;26:351–356
  32. Storms W. Update on montelukast and its role in the treatment of asthma, allergic rhinitis and exercise-induced bronchoconstriction. Expert Opin Pharmacother. 2007;8:2173–2187
  33. Ciana P, Fumagalli M, Trincavelli ML, Verderio C, Rosa P, Lecca D, et al. The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor. EMBO J. 2006;25:4615–4627
  34. Mamedova L, Capra V, Accomazzo MR, Gao ZG, Ferrario S, Fumagalli M, et al. CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. Biochem Pharmacol. 2005;71:115–125
  35. Langlois A, Ferland C, Tremblay GM, Laviolette M. Montelukast regulates eosinophil protease activity through a leukotriene-independent mechanism. J Allergy Clin Immunol. 2006;118:113–119
  36. Bozza PT, Yu W, Penrose JF, Morgan ES, Dvorak AM, Weller PF. Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation. J Exp Med. 1997;186:909–920
  37. Brock TG, Anderson JA, Fries FP, Peters-Golden M, Sporn PH. Decreased leukotriene C4 synthesis accompanies adherence-dependent nuclear import of 5-lipoxygenase in human blood eosinophils. J Immunol. 1999;162:1669–1676
  38. Bandeira-Melo C, Phoofolo M, Weller PF. Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils. J Biol Chem. 2001;276:22779–22787
  39. Bandeira-Melo C, Woods LJ, Phoofolo M, Weller PF. Intracrine cysteinyl leukotriene receptor-mediated signaling of eosinophil vesicular transport-mediated interleukin-4 secretion. J Exp Med. 2002;196:841–850

 Supported by National Institutes of Health grants AI020241, AI022571, and AI051645 and an investigator-initiated grant from Merck.

 Disclosure of potential conflict of interest: P. F. Weller has received research support from Merck and has provided legal consultation or expert witness testimony on the topic of drug-induced eosinophilic disease. The rest of the authors have declared that they have no conflict of interest.

PII: S0091-6749(09)01750-3

doi:10.1016/j.jaci.2009.11.029

The Journal of Allergy and Clinical Immunology
Volume 125, Issue 2 , Pages 477-482, February 2010

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