Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils.

BACKGROUND AND PURPOSE: Prostaglandin (PG) D(2) has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD(2) induces leukotriene (LT) C(4) secretion from eosinophils. Here, we have characterized how PGD(2) signals to induce LTC(4) synthesis in eosinophils. EXPERIMENTAL APPROACH: Antagonists and agonists of DP(1) and DP(2) prostanoid receptors were used in a model of PGD(2) -induced eosinophilic inflammation in vivo and with PGD(2) -stimulated human eosinophils in vitro, to identify PGD(2) receptor(s) mediating LTC(4) secretion. The signalling pathways involved were also investigated. KEY RESULTS: In vivo and in vitro assays with receptor antagonists showed that PGD(2) -triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP(1) and DP(2) receptors. DP(1) and DP(2) receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC(4) synthesis, but not LTC(4) transport/export, was activated by PGD(2) receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP(1) /DP(2) receptor-driven LTC(4) synthesis. Although not sufficient to trigger LTC(4) synthesis by itself, DP(1) receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD(2) -induced LTC(4) synthesis. Similarly, concurrent DP(2) receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD(2) -induced LTC(4) synthesis. CONCLUSIONS AND IMPLICATIONS: Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD(2) receptors described here, our data suggest that both DP(1) and DP(2) receptor antagonists might be attractive candidates for anti-allergic therapies.

Cutting edge: eotaxin elicits rapid vesicular transport-mediated release of preformed IL-4 from human eosinophils.

IL-4 release is important in promoting Th2-mediated allergic and parasitic immune responses. Although human eosinophils are potential sources of IL-4, physiologic mechanisms to elicit its release have not been established. By flow cytometry and microscopy, eosinophils from normal donors uniformly contained preformed IL-4. In contrast to cytolytic IL-4 release from calcium ionophore-activated eosinophils, eotaxin and RANTES, but not IFN-gamma, elicited IL-4 release by noncytotoxic mechanisms. With a dual Ab capture and detection immunofluorescent microscopic assay, IL-4 was released at discrete cell surface sites. IL-5 enhanced eotaxin-induced IL-4 release, which was mediated by G protein-coupled CCR3 receptors, detectable as early as 5 min and maximum within 1 h. IL-4 release was not diminished by transcription or protein synthesis inhibitors, but was suppressed by brefeldin A, an inhibitor of vesicle formation. Thus, CCR3-mediated signaling can rapidly mobilize IL-4 stored preformed in human eosinophils for release by vesicular transport to contribute to immune responses.

Extranuclear lipid bodies, elicited by CCR3-mediated signaling pathways, are the sites of chemokine-enhanced leukotriene C4 production in eosinophils and basophils.

Eosinophils and basophils, when activated, become major sources of cysteinyl leukotrienes, eicosanoid mediators pertinent to allergic inflammation. We show that the C-C chemokines, eotaxin and RANTES (regulated upon activation normal T cell expressed and secreted), activate eosinophils and basophils for enhanced leukotriene C(4) (LTC(4)) generation by distinct signaling and compartmentalization mechanisms involving the induced formation of new cytoplasmic lipid body organelles. Chemokine-induced lipid body formation and enhanced LTC(4) release were both mediated by CCR3 receptor G protein-linked downstream signaling involving activation of phosphoinositide 3-kinase, extracellular signal-regulated kinases 1 and 2, and p38 mitogen-activated protein kinases. Chemokine-elicited lipid body numbers correlated with increased calcium ionophore-stimulated LTC(4) production; and as demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid, lipid bodies were the predominant sites of LTC(4) synthesis in both chemokine-stimulated eosinophils and chemokine-primed and ionophore-activated eosinophils. Eotaxin and RANTES initiated signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases both elicits the formation of lipid body domains and promotes LTC(4) formation at these specific extranuclear sites.

Ultrastructural localization of vesicle-associated membrane protein(s) to specialized membrane structures in human pericytes, vascular smooth muscle cells, endothelial cells, neutrophils, and eosinophils.

Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculo-vacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells.

EliCell: a gel-phase dual antibody capture and detection assay to measure cytokine release from eosinophils.

Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individual viable eosinophils embedded in an agarose matrix that contains immobilized antibody to the protein of interest. Following stimulation of eosinophils, released protein is bound by the capture antibody at its site of release and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma-induced release of RANTES from eosinophils. Extracellularly released RANTES was visualized as focal immunoflourescent staining and was quantitated by scoring the numbers of eosinophils releasing RANTES and by measuring the fluorescent intensity over individual eosinophils. In comparison with ELISA assays of RANTES released into supernatant fluids by interferon-gamma-stimulated eosinophils, EliCell assays were more sensitive enabling detection of RANTES release at earlier times and at lower levels of interferon-gamma stimulation. The EliCell assay provides a sensitive method to study the regulated release of eosinophil-derived cytokines, chemokines and other granule proteins.

Cutting edge: lipoxin (LX) A4 and aspirin-triggered 15-epi-LXA4 block allergen-induced eosinophil trafficking.

Tissue eosinophilia prevention represents one of the primary targets to new anti-allergic therapies. As lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 (ATL) are emerging as endogenous "stop signals" produced in distinct pathologies including some eosinophil-related pulmonary disorders, we evaluated the impact of in situ LXA4/ATL metabolically stable analogues on allergen-induced eosinophilic pleurisy in sensitized rats. LXA4/ATL analogues dramatically blocked allergic pleural eosinophil influx, while concurrently increasing circulating eosinophilia, inhibiting the earlier edema and neutrophilia associated with allergic reaction. The mechanisms underlying this LXA4/ATL-driven allergic eosinophilia blockade was independent of mast cell degranulation and involved LXA4/ATL inhibition of both IL-5 and eotaxin generation, as well as platelet activating factor action. These findings reveal LXA4/ATL as a novel class of endogenous anti-allergic mediators, capable of preventing local eosinophilia.

Cyclooxygenase-2-derived prostaglandin E2 and lipoxin A4 accelerate resolution of allergic edema in Angiostrongylus costaricensis-infected rats: relationship with concurrent eosinophilia.

In noninfected rats, challenge with allergen following local IgE sensitization induced a pleurisy marked by intense protein exudation that plateaued from 30 min to 4 h after challenge, reducing thereafter. Infection of rats with Angiostrongylus costaricensis induced a 5-fold increase in blood eosinophil numbers by 25 days postinfection, whereas the numbers of eosinophils in the pleural cavity ranged from normal to a weak increase. In infected rats, identically sensitized, challenge with Ag induced a much shorter duration of pleural edema with complete resolution by 4 h, but no change in the early edema response. In parallel, infection increased the number of eosinophils recovered from the pleural cavity at 4 h, but not at 30 min, following allergen challenge. Pretreatment with IL-5 (100 IU/kg, i.v.) also increased eosinophil numbers in blood and, after allergen challenge, shortened the duration of the pleural edema and increased pleural eosinophil numbers. There were increases in the levels of both PGE2 and lipoxin A4 (LXA4) in pleural exudate. Selective cyclooxygenase (COX)-2 inhibitors, NS-398, meloxicam, and SC-236, did not alter pleural eosinophilia, but reversed the curtailment of the edema in either infected or IL-5-pretreated rats. Pretreatment of noninfected animals with the PGE analogue, misoprostol, or two stable LXA4 analogues did not alter the magnitude of pleural exudation response, but clearly shortened its duration. These results indicate that the early resolution of allergic pleural edema observed during A. costaricensis infection coincided with a selective local eosinophilia and seemed to be mediated by COX-2-derived PGE2 and LXA4.

Suppressive effect of distinct bradykinin B2 receptor antagonist on allergen-evoked exudation and leukocyte infiltration in sensitized rats.

1. Bradykinin is suggested to play a role in the pathophysiology of several acute and chronic diseases, including allergic disorders such as asthma. In the present study, we have investigated the importance of bradykinin in mediating allergic inflammation in rats. 2. To this end we have tested the effects of the B2 receptor antagonists Hoe 140, FR173657 or FR172357 on the pleural inflammatory response triggered by intrapleural (i.pl.) injection of allergen (ovalbumin, 12 microg cavity(-1)) in 14 day-actively sensitized Wistar rats. Analysis of the pleural fluid effluent revealed a sequence of mast cell-dependent inflammatory events, including early protein exudation and neutrophilia and late pleural eosinophil influx. 3. Local treatment with Hoe 140 (0.1 and 1 microg cavity(-1)), FR173657 (1 and 10 microg cavity(-1)) or FR172357 (1 and 10 microg cavity(-1)) inhibited dose-dependently allergen-induced mast cell activation with impairment of pleural plasma leakage, neutrophil accumulation and late eosinophil influx. 4. Moreover, the B2 receptor antagonists also dose-dependently inhibited the allergic like inflammatory pleurisy triggered by bradykinin (50 microg cavity(-1)), which is characterized by acute mast cell degranulation, protein leakage and pleural eosinophil infiltration. 5. Taken together, our findings provide substantial evidence to suggest that bradykinin acting on its B2 receptors play a critical role in mediating allergic mast cell-dependent inflammation in rats, and suggest that B2 receptor antagonists may be useful therapeutically to control allergic dysfunction.

Systemic and local dexamethasone treatments prevent allergic eosinophilia in rats via distinct mechanisms.

We have studied the effect of local and systemic treatment with dexamethasone for prevention of the pleural eosinophilia triggered by allergen in actively sensitised Wistar rats. Parallel changes in blood and marrow eosinophil numbers were assessed for comparison. The intrapleural (i.pl.) injection of ovalbumin into ovalbumin-sensitised animals led to a long-lasting pleural fluid eosinophilia which peaked from 24 to 72 h post-challenge. At these time points, there was a significant 2- to 3-fold increase in the blood eosinophil numbers, whereas the bone marrow number of mature eosinophils remained unaltered. Systemic treatment with dexamethasone (0.05-0.5 mg/kg, i.p.) abolished the pleural and blood eosinophilia observed 24 and 48 h post-challenge, also causing a significant reduction in marrow eosinophil numbers. Despite being unable to alter blood and bone marrow eosinophil numbers, the local i.pl. administration of dexamethasone (2.5-10 microg/cavity) inhibited dose dependently the allergen-induced pleural eosinophil influx at 24 h but not at 48 h post-challenge. This treatment also shortened the time course of eosinophil accumulation in the pleural space from the 48 h time point on. We conclude that the effect of systemic but not of local treatment with dexamethasone on allergen-induced eosinophil recruitment is well correlated with the inhibition of eosinophil production in bone marrow. In contrast, low amounts of dexamethasone injected into the pleural space seem to affect locally eosinophil recruitment and survival.

Modulatory role of eosinophils in allergic inflammation: new evidence for a rather outdated concept.

The eosinophilic response has been identified as a key alteration in the pathogenesis of asthma and other allergic diseases. A close-correlation between disease severity and eosinophilia, and the eosinophil ability to provide toxic and pro-inflammatory agents are the major elements supporting the interpretation that there is indeed a causal relationship between these phenomena. Nevertheless, controversy still persists since some studies have clearly demonstrated that eosinophil infiltration is not necessarily accompanied by tissue damage or hyperresponsiveness. In addition, there are some examples in the literature in which such alterations are not modified following abrogation of eosinophil influx. In this review it will be argued, based on a model of IgE-dependent pleurisy, that eosinophil infiltration can be associated with down-regulation of allergic inflammatory response. The potential mechanism by which eosinophils could be acting as a immunomodulatory cells in this particular system will also be assessed.

Involvement of prostaglandins in the down-regulation of allergic plasma leakage observed in rats undergoing pleural eosinophilia.

1. Recent evidence has implicated eosinophils in the inhibition of allergen-induced rat pleurisy, but the mechanism of this negative modulation is not completely understood. This study was undertaken in order to define the potential role of prostaglandins in this phenomenon. 2. Wistar rats were actively sensitized by subcutaneous injection of a mixture of ovalbumin and AI(OH)3 and challenged with an intrapleural (i.pl.) injection of ovalbumin (12 micrograms/cavity) 14 days later. 3. Analysis of the pleural fluid effluent revealed a massive mast cell degranulation and plasma protein extravasation 4 h post-challenge. We confirmed that concurrently with selective pleural fluid eosinophilia caused by platelet-activating factor (PAF), the pleural cavity became hyporesponsive to allergen-induced protein exudation and to the parallel reduction in the number of intact mast cells. 4. These hyporesponsive animals presented a significant augmentation in the pleural effluent level of prostaglandin E2 (PGE2), which increased with increasing numbers of eosinophils in the pleural cavity. Furthermore, pretreatment with either indomethacin or aspirin failed to modify allergen-induced exudation but reversed the exudatory hyporesponsiveness associated with eosinophil recruitment. 5. Allergic exudation was clearly down-regulated by the following pretreatments: (i) PGE2 (10 micrograms/cavity, i.pl.) plus rolipram (40 micrograms/cavity, i.pl.), (ii) misoprostol (200 micrograms kg-1, p.o.) or (iii) dibutyryl cyclic AMP (80 micrograms/cavity, i.pl.). 6. We conclude that prostaglandins may be implicated in the eosinophil-mediated inhibition of allergic pleurisy, probably acting via cyclic AMP signalling pathway activation.

Pleural fluid eosinophils suppress local IgE-mediated protein exudation in rats.

Eosinophils are supposed to play a critical role in the pathology of several allergic diseases because after activation they can release toxic and proinflammatory agents. In this study we have investigated whether IgE-mediated rat pleurisy could be affected by an ongoing pleural eosinophilic inflammatory response. IgE-passively sensitized rats were challenged with an intrapleural (i.pl.) injection of allergen (dinitrophenylated bovine serum albumin, 1 microgram/cavity) and exudation assessed by measuring the amount of protein extravasated into the pleural cavity within 4 h. We have confirmed that lipopolysaccharide (LPS) stimulation (250 ng/cavity i.pl.) was followed by a marked pleural neutrophilia, apparent at 3 h, which was followed by an eosinophil accumulation noted within 48-72 h postchallenge. We have also confirmed that a boiled sample of LPS pleural washing (LPS-PW, 200 microliters i.pl.) caused selective eosinophilia in recipient rats. Pleural exudation remained unaltered when the allergenic challenge was performed 3 h after LPS in a condition of intense pleural fluid neutrophilia. In contrast, this was significantly reduced (P < .001) when the challenge occurred 72 h after LPS or 24 h after LPS-PW in selective pleural fluid eosinophilia. In another series of experiments repeated daily i.pl. injections of platelet-activating factor (PAF; 1 microgram/cavity) resulted in a progressive increase in eosinophil number recovered from the pleural cavity. The values were 1.2 +/- 0.2, 3.0 +/- 0.2, and 5.8 +/- 0.5 x 10(6) eosinophils/cavity (mean +/- SEM) after 0, 1, and 4 injections, respectively. Allergen challenge performed after 0, 1, or 4 PAF stimulations led to pleural protein levels of 88.6 +/- 5.7, 33.7 +/- 0.7, and 19.4 +/- 2.3 mg/cavity, respectively, indicating that the allergic pleurisy is inhibited in a manner dependent on the magnitude of eosinophil accumulation. Furthermore, the impairment of PAF-induced eosinophil accumulation by cetirizine (30 mg/kg i.p.) restored the exudatory response. Exudation triggered by compound 48/80 (25 micrograms/cavity), histamine (200 micrograms/cavity), or 5-hydroxytryptamine (100 micrograms/cavity) was not affected by four previous PAF daily injections. The findings indicate that allergen-induced exudation is selectively down-regulated in the eosinophil-enriched pleural space of rats, a suppression that increased with increasing eosinophil number and disappeared after chemical impairment of the eosinophilia.