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Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Eosinophils contain several preformed cytokines within their specific granules. Therefore, without requiring them in de novo synthesis of cytokines, eosinophils can release quantities of granule-derived cytokines by highly regulated mechanisms. However, eosinophil “degranulation” is poorly understood, in part, because available methodologies did not appear appropriate for analyzing vesicular mobilization and transport of eosinophil granular contents. The EliCell assay is a microscopic methodology substantially modified from other techniques employed to detect cytokine release (i.e., ELISPOT). The method is a dual antibody capture/detection system in which viable eosinophils are incubated in a solid streptavidin-conjugated agarose matrix, which contains a biotinylated capture antibody against the cytokine of interest. Released cytokine is detected around non-permeabilized eosinophils with a separate fluorochrome-labeled detection antibody. Thus, the EliCell system captures and detects extracellular cytokines at the site of their release from eosinophils. As examples, we have used EliCell essays to detect the selective release of either IL-4 or IL-12 cytokines found preformed in eosinophils—from eotaxin- or anti-CD9-stimulated eosinophils, respectively. With appropriate pairs of antibodies, any preformed cytokine found into eosinophil granules could be studied and the mechanisms of their secretion evaluated by using the EliCell assay.

Eosinophil granules are notable for their content of preformed proteins whose secretion underlies many of the contributions eosinophils make in allergic and anthelminthic responses. We have recently shown that cell-free eosinophil granules respond to eotaxin and IFN-γ via cognate signaling-coupled granule membrane-expressed receptors, triggering phosphorylation of intragranular signaling molecules and eliciting specific secretion of granule-derived proteins. Thus, fully intact eosinophil granules deposited extracellularly within diseased tissues maintain a capacity to function as secretory competent organelles, demanding a fresh look at our understanding of the secretion of eosinophil granule-derived proteins, both intracellularly and extracellularly. Here, we review a series of our recent work revealing the unique ultrastructure of human eosinophil granules and mechanisms of intragranule sorting and mobilization of specific cytokine products from intact human eosinophils. Implications of these findings t...

The interference of azelastine with pleurisy induced by antigen was investigated in actively sensitized rats. The antigenic challenge (ovalbumin, 12 micrograms/cavity) caused early plasma leakage, which peaked within 4 h, accompanied by intense neutrophil infiltration. Pleural exudate decayed 24 h after antigen provocation, when a long-lasting increase in the number of resident eosinophils was observed. Oral pretreatment with azelastine (1-10 mg/kg) dose dependently inhibited the vasopermeation (ED50 = 4.2 mg/kg) and reduced the pleural exudate (ED50 = 6.8 mg/kg) induced by the antigen. In contrast, azelastine (10 mg/kg) failed to modify the neutrophil influx observed at 4 h and the eosinophil accumulation detected at 24 h. Azelastine was also effective against rat pleurisy induced by either platelet-activating factor (PAF-acether), histamine or serotonin. It reduced exudation and the increase in the number of mononuclear cells, neutrophils and eosinophils observed 6 h after PAF-acether. Nevertheless, antagonism of PAF-acether may not be relevant to the inhibition observed in the present model of allergic pleurisy, as the inhibition was refractory to three distinct PAF-acether receptor antagonists. In contrast, like azelastine, the histamine H1 receptor antagonist meclizine and the dual histamine and serotonin receptor antagonist cyproheptadine blocked antigen-induced exudation and failed to interfere with cell influx. We conclude that the anti-exudatory activity of oral azelastine on antigen-induced pleurisy is consistent with it exerting direct effects against vasoactive amines, but is not related to an effect against leucocyte infiltration nor to its ability to inhibit PAF-acether.

Platelet-activating factor (PAF) has been shown, in the rat model of pleural inflammation, to induce the generation of an intermediate proteic factor able to cause eosinophil proliferation in vitro. This study was undertaken to investigate the effect of the anti-mitotic compound doxorubicin on PAF-induced eosinophilia in rats, in order to evaluate the contribution of local cell proliferation to this phenomenon. The late eosinophil infiltration caused by another chemoattractant leukotriene B4 was used for comparison. We observed that local treatment with doxorubicin (20 and 40 microg/cavity), given 6 h after PAF (1 microg/cavity), suppressed the eosinophil accumulation within 24 h, whilst only the higher dose was effective when the drug was given 12 h post-PAF. An effect on chemotaxis was ruled out, since local doxorubicin (40 microg/cavity) failed to modify the eosinophil migration noted 24 h after leukotriene B4 (0.5 microg/cavity) and the neutrophil/eosinophil infiltration noted at 6 h after PAF injection. Transfer of the pleural fluids collected 6 h after PAF from donors to recipient rats caused significant eosinophil accumulation in the recipient rats, an effect which was inhibited by the co-administration of doxorubicin (40 microg/cavity). No inhibitory effect was noted when the drug was given 6 h after the pleural fluids were transferred. We also found no change in the number of blood or bone marrow eosinophils after PAF stimulation. We conclude that doxorubicin selectively impaired the late eosinophil accumulation triggered by PAF in the pleural cavity of rats, clearly indicating that local cell proliferation seems to contribute to the development of this inflammatory response.

In previous research, we have observed that intrathoracic administration of platelet-activating factor-acether (PAF) promoted a delayed eosinophilia in the pleural cavity of rats that lasted for at least 96 h. We investigated the ability of pleural washings from rats previously injected with PAF (1 micrograms/cavity) to stimulate in vitro murine hematopoietic eosinophil proliferation. We observed that pleural fluid sustained eosinophil proliferation but not differentiation, under conditions in which PAF itself had no effect. The phenomenon lasted for 3 days and was maximal on the 1st day of culture. Treatment with neutralizing antibodies against interleukin (IL)-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3, alone or in combination, did not modify the eosinophil proliferation induced by PAF pleural fluid, suggesting that these cytokines may not be involved in the studied phenomenon. We conclude that the rat pleural fluid obtained 6 h after PAF administration induces eosinophil proliferation in vitro by a mechanism probably independent of IL-5, GM-CSF or IL-3.

H. C. Castro-Faria-Neto , M. A. Martins , P. T. Bozza , S. A. C. Perez , A. C. V. Correa-Da-Silva , M. C. R. Lima , H. N. Cruz , R. S. B. Cordeiro , M. V. Sousa and L. Morhy . Pro-inflammatory activity of enterolobin: a haemolytic protein purified from seeds of the Brazilian tree Enterolobium contortisiliquum. Toxicon, 29, 1143–1150, 1991.—The pro-inflammatory activity of enterolobin, a haemolytic protein from Enterolobium contortisiliquum seeds, was investigated. In doses ranging from 1 to 20 μg/site, enterolobin induced a dose-dependent paw oedema and pleurisy in rats. The effect was apparent after 15 min, peaked at 6 hr and decreased 24 hr after enterolobin was administered. One hour after the intrathoracic injection of enterolobin, the total leukocyte content of the pleural cavity increased significantly, mainly due to mononuclear and neutrophil accumulation. At 24 hr, although the number of mononuclear and neutrophil cells tended to decrease, a great rise in eosinophil counts was noted. Intraperitoneal treatment with the dual lipoxygenase and cyclooxygenase blockers, BW 755c (25 mg/kg) and NDGA (50 mg/kg) or the corticosteroid dexamethasone (0.1 mg/kg) inhibited enterolobin-induced paw oedema by 35, 38 and 47% respectively, whereas indomethacin (2 mg/kg) was inactive. The H1 antagonist, meclizine (25 mg/kg), was also effective against enterolobin oedema while the PAF-antagonists WEB 2086 and PCA 4248 (20 mg/kg) did not modify the reaction. It was concluded that enterolobin is a potent inducer of pleural exudation, cellular infiltration and paw oedema. Furthermore, enterolobin-induced oedema is partially dependent on lipoxygenase metabolites and histamine, while PAF and prostaglandins did not seem to be important in this reaction.