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-1i,

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

The idiopathic hypereosinophilic syndrome (IHES) is characterized by sustained peripheral blood eosinophilia associated with organ involvement in the absence of a defined etiology (1). The isolation ofeosinophils from patients with IHES has resulted in the identification of a population of eosinophils of a lower sedimentation density (hypodense) than that of eosinophils purified from healthy individuals (normodense) (2, 3). As compared with normodense eosinophils, hypodense eosinophils exhibit increases in calcium ionophore A23187-stimulated leukotriene C4 (LTC4) generation (4) and antibody-dependent helminthic cytotoxicity (3). The percentage of circulating hypodense eosinophils directly correlates with the degree ofperipheral blood eosinophilia (5), but the factors that may regulate the eosinophil phenotype in IHES have not been determined previously.A continuous exposure ofnormodense eosinophils to recombinant human granulocyte/macrophage colony-stimulating factor (rGM-CSF) (6), human rIL-3 (7), purified murine IL-5 or human rIL-5 (8) allows them to be maintained ex vivo for at least 2 wk. During this interval, the eosinophils become hypodense and exhibit both augmented calcium ionophore-stimulated LTC4 generation and antibody-dependent cytotoxicity against Schistosoma mansoni larvae . The similarity between hypodense eosinophils, which are generated in vitro by the exposure ofnormodense eosinophils to specific cytokines, and hypodense eosinophils, which are freshly isolated from the peripheral blood ofpatients with IHES, prompted an analysis of eosinophil phenotypes and cytokine activities in the peripheral blood of patients with IHES. Volume 170 July 1989 343-348 Materials and Methods Brief Definitive ReportPatients. Three patients underwent diagnostic studies to rule out collagen-vascular diseases, helminthic infections, neoplasia, drug reactions, atopy, or asthma, and each fulfilled

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with IL-la (5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of3.2 to a mean of8.2 pM (P < 0.05). Dexamethasone (100 nM) decreased the constitutive GM-CSF elaboration by 49% (P < 0.001) but did not diminish production by IL-lastimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-la-stimulated endothelial cell-conditioned medium (P < 0.05), which suggested viability antagonism by glucocorticoids. After 24 h of culture, eosinophil viability for replicate cells in enriched medium alone or with 1 pM GM-CSF decreased from means of 43 and 75% to means of 21 and 54%, respectively, when dexamethasone was included (P < 0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonuclease-specific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathobiologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentrations that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of select eosinophilic disorders to glucocorticoids. (J. Clin.

SummaryGranulocytes with the hybrid characteristics of eosinophils and basophils have been identified in the bone marrow and peripheral blood of humans with myeloid leukemias. We now describe a technique by which such hybrid granulocytes can be developed in vitro from normal cord blood precursors cultured in the presence of recombinant human interleukin (rhlL) 3 (350 pM) and rhlL-5 (200 pM) in a plastic vessel coated with Matrigel TM. After 14 d in culture, 90 +_ 3% (mean _+ standard error of the mean) of the nonadherent cells cultured in the Matrigel-coated flasks contained both eosinophil and basophil granules, as indicated by staining with Wright's and Giemsa stains. Of the nonadherent cells, 93 +_ 1% contained cyanide-resistant peroxidase, and 88 _+ 2% were toluidine blue-positive, characteristic of eosinophil and basophil granules, respectively. Transmission electron micrographs showed hybrid cells containing ultrastructurally distinct eosinophil granules with developing crystalline cores and basophil granules with reticular structures. These 14-d cord blood-derived cell cultures showed strong hybridization signals for eosinophil-derived neurotoxin by RNA blot analysis and contained 78 ng histamine per 106 cells. When the granulocytes were removed from cytokine-containing medium and suspended without Matrigel in RPMI 1640 medium containing 10% fetal calf serum (FCS), more than 80% of the granulocytes excluded trypan blue for as long as 5 d, and 93% had developed into eosinophils at 6 d. Conditioned medium prepared over 48 h from the 14-d cell cultures (hybrid granulocytes) sustained the 4-d viability in vitro of 78% of peripheral blood eosinophils from atopic donors. In comparison, 13 % survived in RPMI 1640 containing 10% FCS alone. This viability-sustaining activity was nearly completely neutralized by an anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody and was only minimally reduced by anti-IL-3 or -IL-5. Thus, cells possessing both eosinophil and basophil granules by both histochemical and ultrastructural analysis can be developed from normal progenitors in vitro in response to eosinophilopoietic cytokines and Matrigel. Their subsequent spontaneous development into mature eosinophils suggests that hybrid granulocytes are part of a normal developmental sequence during eosinophilopoiesis, Furthermore, these hybrid granulocytes are capable of autoregulation through elaboration of GM-CSF, which sustains their viability.