Normodense Eosinophils Research Articles

Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation.

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.

Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3.

Human eosinophils were cultured in the presence of recombinant human IL-3 for up to 14 d and their biochemical, functional, and density properties were assessed. After 3 d of culture in 10 pM IL-3, eosinophils had a viability of 70% compared with only 10% in enriched medium alone. Neither IL-1 alpha, IL-2, IL-4, tumor necrosis factor, basic fibroblast growth factor, nor platelet-derived growth factor maintained eosinophil viability. The 7- and 14-d survival of the cultured eosinophils was 55 and 53%, respectively. No other cell type, including neutrophils, was present after culture. After 7 d of culture, the normodense eosinophils were converted to hypodense cells as assessed by density centrifugation. Eosinophils exposed to 1,000 pM IL-3 for 30 min or cultured in 10 pM IL-3 for 7 d generated approximately threefold more leukotriene C4 (LTC4) in response to calcium ionophore than freshly isolated cells. Furthermore, whereas freshly isolated eosinophils killed only 14% of the antibody-coated Schistosoma mansoni larvae, these eosinophils killed 54% of the larvae when exposed to 100 pM IL-3. The enhanced helminth cytotoxicity was maintained for 7 d when eosinophils were cultured in the presence of both 10 pM IL-3 and 3T3 fibroblasts, but not when eosinophils were cultured in the presence of IL-3 alone. IL-3 thus maintains the viability of eosinophils in vitro, augments the calcium ionophore-induced generation of LTC4, enhances cytotoxicity against antibody-sensitized helminths, and induces the eosinophils to become hypodense cells. These phenotypic changes in the eosinophil may be advantageous to host defense against helminthic infections but may be disadvantageous in conditions such as allergic disease.

Ultrastructural study of eosinophils from patients with the hypereosinophilic syndrome: a morphological basis of hypodense eosinophils

We investigated the ultrastructural characteristics and the granule major basic protein (MBP) content of hypodense eosinophils from patients with the hypereosinophilic syndrome who had at least 90% hypodense eosinophils in their peripheral blood and compared these cells to normodense eosinophils from normal persons. The hypodense cells (density less than 1.082) contained significantly less MBP than normodense (density greater than 1.082) eosinophils (P less than .001) as measured by radioimmunoassay (RIA). Electron microscopic examination demonstrated a mean of 25.0 +/- 4.4 (X +/- 1 SD) granules per hypodense cell, compared to 30.6 +/- 8.4 granules per cell in the normodense group (P less than .1). The most striking difference between the hypodense and normodense eosinophils was the small individual granule size (X = .14 +/- .05 v .26 +/- .05 micron 2, respectively, P less than .001), and the smaller total granule area (3.2 +/- 1.8 vs 7.7 +/- 3.1 micron 2, respectively, P less than .001). Because the cytoplasmic areas were similar in the two groups, the mean percent area of cytoplasm occupied by granules was significantly lower in the hypodense group (P less than .001). The finding of consistently smaller granules in the presence of equal or fewer granules per cell in the hypodense eosinophils may explain the lower MBP content and thus provide a morphologic basis for the low density of eosinophils in patients with the hypereosinophilic syndrome.

Ultrastructural Study of Eosinophils From Patients With the Hypereosinophilic Syndrome: A Morphological Basis of Hypodense Eosinophils

We investigated the ultrastructural characteristics and the granule major basic protein (MBP) content of hypodense eosinophils from patients with the hypereosinophilic syndrome who had at least 90% hypodense eosinophils in their peripheral blood and compared these cells to normodense eosinophils from normal persons. The hypodense cells (density less than 1.082) contained significantly less MBP than normodense (density greater than 1.082) eosinophils (P less than .001) as measured by radioimmunoassay (RIA). Electron microscopic examination demonstrated a mean of 25.0 +/- 4.4 (X +/- 1 SD) granules per hypodense cell, compared to 30.6 +/- 8.4 granules per cell in the normodense group (P less than .1). The most striking difference between the hypodense and normodense eosinophils was the small individual granule size (X = .14 +/- .05 v .26 +/- .05 micron 2, respectively, P less than .001), and the smaller total granule area (3.2 +/- 1.8 vs 7.7 +/- 3.1 micron 2, respectively, P less than .001). Because the cytoplasmic areas were similar in the two groups, the mean percent area of cytoplasm occupied by granules was significantly lower in the hypodense group (P less than .001). The finding of consistently smaller granules in the presence of equal or fewer granules per cell in the hypodense eosinophils may explain the lower MBP content and thus provide a morphologic basis for the low density of eosinophils in patients with the hypereosinophilic syndrome.

Functional role of the alpha-chain of complement receptor type 3 in human eosinophil-dependent antibody-mediated cytotoxicity against schistosomes.

The participation of complement receptor type 3 (CR3) in antibody-dependent effector function of human eosinophils against parasites was studied by using monoclonal antibodies directed against various surface molecules. Both adherence and cytotoxicity of hypodense eosinophils to IgE-coated schistosomula of Schistosoma mansoni were strongly inhibited by anti-CR3 antibodies (OKM1 or Mo1). The specificity of the inhibitory effect for the alpha-chain of CR3 was shown by the lack of inhibition of anti-beta-chain or anti-LFA1 alpha-chain monoclonal antibodies, although these antigens were expressed on human eosinophils. These results associated to previous works on IgE receptors demonstrate that both receptor for Fc fragments of IgE and CR3 are essential in IgE-dependent cytotoxicity of human eosinophils. Flow microfluorometry analysis revealed that hypodense eosinophils were more intensively stained by OKM1 antibodies than the normodense populations. In the case of IgG-mediated cytotoxicity by normodense eosinophils, only the enhancement of cytotoxicity due to monokine activation was inhibited by anti-CR3 alpha-chain antibodies. These findings suggest an increased expression of CR3 on eosinophils after activation either in vivo or in vitro. The participation of CR3 in IgE-mediated cytotoxicity against schistosomes was also required in the case of blood monocytes but not for platelet-mediated killing, which does not require prior adherence. The biologic role of CR3 is therefore extended to effector mechanisms involving eosinophils and two different isotypes of antibodies and possibly implied in immunity against schistosomes.

Eosinophils cocultured with endothelial cells have increased survival and functional properties.

Human peripheral blood eosinophils, cells often associated with allergic and parasitic diseases, were maintained in vitro for at least 14 days when they were cocultured with bovine endothelial cells and for at least 7 days when cultured with either bovine or human endothelial cell-derived conditioned medium. The cocultured eosinophils became hypodense and generated about three times as much leukotriene C4 upon activation with calcium ionophore and killed about three times as many antibody-coated larvae of Schistosoma mansoni as freshly isolated normodense eosinophils. That these cells can be maintained in vitro by coculture with endothelial cells, and the surprising finding that the cocultured eosinophils have biochemical, cytotoxic, and density properties similar to those of eosinophils in patients with allergic and other disorders, will facilitate investigation of the regulation and role of these cells in health and disease.

Effects of platelet activating factor on the chemotaxis of normodense eosinophils from normal subjects

Highly purified eosinophils were obtained from normal subjects, and their chemotactic responses to platelet activating factor (PAF) were evaluated. PAF induced both eosinophil chemotactic and chemokinetic responses, and was 100 fold more potent eosinophil chemotactic factor as compared to eosinophil chemotactic factor of anaphylaxis. On the other hand, leukotriene B 4 did not show any eosinophil chemotactic activity. A selective PAF antagonist, BN52021, inhibited eosinophil chemotaxis in a dose dependent manner. Preincubation of eosinophils with PAF induced the deactivation of eosinophils for further chemotactic responses to PAF. These findings suggest that PAF is a potent chemotactic factor for normal eosinophils.

Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages.

An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE-dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2).

Human eosinophils express CR1 and CR3 complement receptors for cleavage fragments of C3

The functional and antigenic characteristics of C3 receptors expressed on human eosinophils were investigated using rosette assays with sheep erythrocytes coated with C3 fragments and flow cytometric analysis of cells stained with anti-receptor antibodies. Purified peripheral blood eosinophils from 13 patients with hypereosinophilia expressed CR1 antigens. In 8 patients, a mean of 14 + 9.5% eosinophils formed C3b-dependent rosettes that were inhibited by F(ab′) 2 anti-CR1 antibodies. This number increased to 33% following stimulation with leukotriene B4 (LTB4) (10 −7 M). Similar numbers of C3b rosettes were formed by hypodense and normodense eosinophils. Eosinophils from 2 patients from this group expressed 20,000 125I-labeled monoclonal anti-CR1 antibody binding sites/cell. In another group of patients, 55 ± 9% eosinophils spontaneously formed C3b-dependent rosettes that could not be enhanced by LTB4. In all patients, a mean of 16 ± 9% eosinophils formed cation-dependent rosettes with C3bi-bearing intermediates that were inhibited by anti-CR3 antibody OKM1. All eosinophils stained with monoclonal antibodies against the a chain of CR3. There was no C3d-dependent rosette formation with eosinophils and no eosinophils stained with monoclonal anti-CR2 antibody. Thus, human eosinophils express CR1 and CR3. Since CR3 is required for the adhesion of granulocytes to surfaces and antibody-dependent cellular cytotoxicity of neutrophils, the interaction of C3 fragments with CR3 and CR1 on eosinophils may be of importance in eosinophil-mediated damage of opsonized targets.