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Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells. The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C4 were evaluated. A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1beta slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-gamma; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1beta-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C4 than control cells (872.4 and 103.9 pg x mL(-1), respectively). Endothelial cell pretreatment with IL-4 or IL-1beta further increased leukotriene C4 release (1,789.1 and 2,895.1 pg x mL(-1), respectively), whereas pretreatment with IFN-gamma decreased it (293.7 pg x mL(-1)). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis.

Background: Tissue eosinophils express more membrane receptors and release more mediators than blood eosinophils, suggesting that migration from blood to tissue modulates eosinophil phenotype and functions., Objective: We postulated that eosinophil passage through endothelial basement membrane, an important step of eosinophil migration into tissue, may be responsible for some of these changes., Method: We previously showed that 5-oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE) in combination with IL-5 promotes eosinophil migration through Matrigel, a mouse tumour cell-derived basement membrane. Using this model, we evaluated the effect of trans-Matrigel migration on purified human blood eosinophil expressions of CD44, CD69 and HLA-DR that either increase or appear on activated eosinophils, and releases of peroxidase (EPO), leukotriene (LT) C(4) and granulocyte-monocyte colony stimulating factor (GM-CSF)., Results: IL-5, but not 5-oxo-ETE, increased eosinophil expression of CD44 and CD69. Migration of eosinophils through Matrigel significantly increased CD44 expression level over the one induced by IL-5 (P = 0.0001). Migration through Matrigel did not modify CD69 expression compared with the one obtained in the presence of IL-5 alone; however, incubation of eosinophils on Matrigel decreased IL-5-induced CD69 (P = 0.0001). Trans-Matrigel migration did not modify HLA-DR expression, nor EPO, LTC(4) and GM-CSF releases., Conclusion: These data show that in vitro trans-Matrigel migration and Matrigel contact modulate eosinophil membrane receptor expression. Consequently, they suggest that migration through basement membrane mediates changes in cell-surface phenotype observed on activated eosinophils and probably prepares them for interactions with tissue components and cells.

Background: Blood eosinophils have mRNA for FcgammaRIIIB (CD16) but no or minimal spontaneous CD16 expression. Because IFN-gamma and chemotactic factors induce eosinophil CD16 expression in vitro, we postulated that blood eosinophils could express CD16., Objective: Blood of nonallergic controls and subjects with allergic rhinitis, allergic and nonallergic asthma, or hypereosinophilia of various etiologies were analyzed for leukocyte CD16 surface expression., Methods: CD16(+) eosinophils were identified on the basis of physico-optic characteristics, major basic protein, CD49b expression, and sorting by flow cytometry and microscope examination., Results: Subjects with allergic rhinitis and subjects with asthma had higher median percentages of CD16(+) eosinophils (8.1% [1% to 48.6%] and 7.3% [1.4% to 31.1%], respectively) than nonallergic controls and nonallergic asthmatics (3% [0% to 11%] and 4.6% [2.9% to 5.1%], respectively). In subjects with hypereosinophilia, CD16(+) eosinophils were increased only in a case of drug allergy. When subjects with mild allergic asthma were challenged with a relevant aeroallergen, blood CD16(+) eosinophils further increased during or after the late-phase response (6 to 48 hours after challenge; mean +/- SEM, 9.4% +/- 2.5% to 20.0% +/- 3.0%). CD16(+) eosinophils expressed more IL-5 receptor but less CD11b and IL-12p35 than did CD16(-) eosinophils., Conclusion: Upregulation of blood CD16(+) eosinophils in allergic conditions and its association with a modified phenotype suggest that CD16 receptor could play a role in eosinophil activation in allergy.

Basement membrane transmigration is an important step in tissue recruitment of eosinophils into inflamed tissue. Recent reports showed that this phenomenon is modulated by platelet-activating factor (PAF) in combination with cytokines and proteinases. We investigated the in vitro efficacy of 5-oxo-6,8,11, 14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid and known as a potent eosinophil chemotactic factor, in promoting the transmigration of blood eosinophils from normal and asthmatic subjects through a Matrigel basement membrane. 5-Oxo-ETE proved to be a more potent (> 10-fold) inducer of eosinophil transmigration than PAF, and this effect was similar in cells from normal and asthmatic subjects (82.0 +/- 3.7% and 88.1 +/- 3.7%, respectively). Moreover, 5-oxo-ETE was active in the absence of interleukin (IL)-5, although this cytokine amplified the effect of 5-oxo-ETE from 61.3 +/- 3.3% to 92.8 +/- 1.8% (p = 0.003). The membrane receptor for urokinase plasminogen activator (CD87), a serine protease, was observed on eosinophils, and its expression was increased by IL-5. The inhibition of both metalloproteinases (MMP) and plasmin/plasminogen complex with inhibitor or monoclonal antibodies decreased cell transmigration by about 50%. Combination of an MMP inhibitor with anti-CD87 antibodies had no additive effect. These data show that 5-oxo-ETE is an efficient promoter of eosinophil transmigration in vitro, and is much more potent in this respect than PAF. The data suggest that 5-oxo-ETE could play an important role in eosinophil recruitment in vivo. Moreover, they demonstrate that in addition to MMP, the plasmin/plasminogen system could be involved in eosinophil transmigration.

This study describes a method for flow cytometric (FCM) identification and analysis of eosinophils (EOS) in mixed granulocyte populations without the need of further cell purification. Granulocytes were prepared from 10 ml blood samples obtained from 14 non-atopic healthy subjects and 16 hypereosinophilic subjects. The cells were fixed in paraformaldehyde (PFA) and permeabilized with either saponin or n-octyl-beta-D-glucopyranoside (OG) before FCM. Side scatter light (SS) and depolarized sideward light scatterings (dSS) were measured before and after granulocyte fixation and permeabilization. Based on autofluorescence of cells, EOS were depicted in forward scatter (FS)-SS and FS-dSS cytograms. FS-dSS analysis of PFA-saponin-treated cells provided the best discrimination between EOS (high dSS) and neutrophils (low dSS) in normal and hypereosinophilic blood samples (n = 22). Subsequent sorting and differential staining of the two populations further confirmed cell identity in high dSS (97.2 +/- 3.0% EOS) and low dSS (99.6 +/- 0.5% neutrophils) PFA-saponin-treated cells (n = 5). Similar FS-dSS profiles were obtained for hypodense (HBSS-1.082 g/ml interface) and normodense (1.082-1.100 g/ml interface) EOS separated by centrifugation on discontinuous Percoll gradients and treated with PFA-saponin. Using this method, we successfully detected the cytokines interleukin-2 (IL-2), IL-4, IL-5, and interferon gamma (IFN gamma) in blood EOS of normal and hypereosinophilic subjects (n = 12). In conclusion, this method allows the analysis of EOS intracellular antigens in mixed granulocyte populations obtained from small blood samples. This analysis is not affected by the buoyant density of eosinophils, and can therefore be applied for the analysis of blood EOS in various types and severities of diseases.

To verify the hypothesis that eosinophils produce interleukin-2 (IL-2), a cytokine essential for lymphocyte activation, the expression of IL-2 was examined in peripheral blood eosinophils obtained from normal, atopic, asthmatic and hypereosinophilic subjects. Purified blood cell preparations were > 95% eosinophils, the remaining cells being neutrophils. Based on morphological observations and on CD3 expression, no lymphocytes were detected in these eosinophil preparations. The expression of IL-2 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in total RNA extracted from purified eosinophils stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF), with or without calcium ionophore (A23187). In-cell RT-PCR combined with in situ hybridization further confirmed that it was the eosinophils that expressed IL-2 mRNA. Moreover, in this experiment IL-2 mRNA expression increased upon costimulation with A23187 and GM-CSF suggesting that a steady-state level of IL-2 mRNA was inducible. Finally, IL-2 was detected in purified eosinophils by immunochemistry. These data, obtained by different techniques, demonstrate that eosinophils can express IL-2. An IL-2-mediated eosinophil-lymphocyte interaction could contribute to the chronic state of cell activation in inflamed tissues where these cells are implicated.

As there is much heterogeneity in the morphology and function of blood eosinophils, comparison of their properties between groups of subjects requires recovering the majority of these cells. In two currently used techniques to isolate eosinophils, blood granulocytes are processed either on Percoll gradients after an incubation of granulocytes with 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or on a magnetic cell sorter (MACS). In this study, these techniques were modified to increase the efficiency of eosinophil recovery. With the Percoll gradients, using 1.078 g/ml as the top gradient instead of 1.082 g/ml doubled the eosinophil recovery from 43 +/- 5.3% (mean +/- SEM) to 86.9 +/- 2.9%, without decreasing the purity (96.1 +/- 1.4% versus 96.2 +/- 0.9%). With a MACS, the neutrophils in granulocytes obtained on Ficoll-Paque (1.077 g/ml) instead of on Percoll gradient 1.082-1.094 g/ml, were tagged with anti-CD16 antibodies and eliminated by passing them through a magnetic field. When blood eosinophils of the same subjects were isolated using the two techniques, similar recovery and purity levels were obtained: Percoll gradients, 72.7 +/- 6.8% and 92.5 +/- 2.2%; MACS, 80.2 +/- 5.1% and 90.4 +/- 3.8%. Eosinophils isolated through the two techniques were also compared for their production of superoxide anion and leukotriene (LT) C4, with and without pre-incubation with cytokines interleukin-3, interleukin-5 and granulocyte-macrophage colony stimulating factor. The release of these products was similar between the two eosinophil preparations under all conditions tested except for interleukin-3 where eosinophils isolated with a MACS produced more LTC4. These results show that both techniques efficiently recover pure eosinophils. Furthermore, cell incubation with 10(-8) M fMLP did not enhance superoxide anion and LTC4 production nor modify the response to cytokines. The two modified techniques are therefore suitable for comparative studies of eosinophils from different groups of subjects.