Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2436-2441, March 1996 Immunology

Constitutive production of /macrophage colony- stimulating factor by hypodense mononuclear developed in vitro from hybrid / (interleukin 5/interleukin 3) JOSHUA A. BOYCE*tt, DANIEL FRIEND§t, MICHAEL F. GURISH*t, K. FRANK AUSTEN*t, AND WILLIAM F. OWEN*t Departments of *Medicine and §Pathology, Harvard Medical School, and iDivision of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115 Contributed by K Frank Austen, November 21, 1995

ABSTRACT We recently described the development in and hypodense peripheral blood eosinophils maintain their vitro of cells with granules characteristic of eosinophils and viability in vitro in the presence of 1-10 pM eosinophilopoietic (hybrid granulocytes) from normal human cord cytokines interleukin (IL)-3, IL-5, or granulocyte/macrophage blood mononuclear cells cultured for 14 days with recombi- colony-stimulating factor (GM-CSF) (4, 9-12). Continuous nant human (rh) interleukin (IL)-3, rhIL-5, and a soluble exposure to these cytokines in the presence of 3T3 fibroblasts basement membrane, Matrigel. Hybrid granulocytes consti- converts normodense eosinophils to a hypodense phenotype tutively produced granulocyte/macrophage colony-stimulat- that is functionally similar to the hypodense eosinophils found ing factor (GM-CSF) and rapidly developed into eosinophils in vivo (9-12). Peripheral blood eosinophils, whether normo- after the exogenous cytokines and Matrigel were removed. To dense or hypodense, are protected from programmed cell characterize the developmental progression of hybrid granu- death only during their continuous exposure to an eosinophi- locytes, cells were maintained for an additional 14 days in lopoietic cytokine. medium containing rhIL-3, rhIL-5, and Matrigel. After 28 In comparison to normodense eosinophils, which are usually days, 73% ± 1% (mean + SEM; n = 6) ofthe nonadherent cells bilobed, freshly isolated hypodense eosinophils from patients were mononuclear eosinophils, 13% + 3% were eosinophils with IHES exhibit heterogeneous nuclear configurations, in- with two or more nuclear lobes, 13% + 4% were hybrid cluding nuclei that are hypersegmented or single lobed granulocytes, and 0.2% ± 0.1% were basophils. More than 90%Yo (termed mononuclear eosinophils) (1, 13, 14). In the late 19th ofthe mononuclear eosinophils were hypodense as determined century, eosinophils from the sputum of a child with asthma by centrifugation through metrizamide gradients. After an were noted to be predominantly mononuclear (15). Cutaneous additional 5 days of culture in medium without exogenous eosinophils elicited into skin windows of atopic patients have cytokines, 65% + 3% (n = 5) of the 28-day cells excluded a lower nuclear segmentation index than peripheral blood trypan blue. In contrast, 2% ± 1% of freshly isolated periph- eosinophils (16). Tissue specimens from patients with nonspe- eral blood eosinophils survived 5 days of culture without cific proctocolitis exhibit mixed nuclear morphologies of bi- exogenous cytokines (n = 5). Fifty percent conditioned me- lobed and single-lobed eosinophils (17). In an immunohisto- dium from in vitro derived 28-day mononuclear eosinophils chemical study of spontaneously expectorated sputum cells, and 14-day hybrid granulocytes maintained the survival of asthma was distinguished from chronic bronchitis on the basis 60% + 7% and 77% + 7%, respectively, of freshly isolated of the presence of eosinophils expressing GM-CSF, and some peripheral blood eosinophils for 72 h, compared with 20%o + of these cells appeared to have a single-lobed nucleus (18). 8% survival in medium alone (n = 3). The eosinophil viability- These observations suggest the existence of a lesion-associated sustaining activity of 50%o mononuclear eosinophil-condi- hypodense mononuclear eosinophil phenotype. tioned medium was neutralized with a GM-CSF antibody. A We have recently demonstrated that the 14-day culture of total of 88% of the 28-day cells exhibited immunochemical cord blood mononuclear cells in RPMI medium 1640 supple- staining for GM-CSF. Thus, during eosinophilopoiesis, both mented with 350 pM recombinant human (rh)IL-3 and 200 pM hybrid eosinophil/basophil intermediates and immature rhIL-5 on a layer of Matrigel provides a 90% pure population mononuclear eosinophils exhibit autocrine regulation of via- of hybrid eosinophil/basophil granulocytes that constitutively bility due to constitutive production of GM-CSF. produced GM-CSF (19). These hybrid granulocytes are meta- chromatic with toluidine blue staining, contain a cyanide- When centrifuged through discontinuous gradients of Percoll resistant peroxidase, and have ultrastructurally distinct eosin- or metrizamide, peripheral blood eosinophils segregate into ophil and basophil granules, identified by electron microscopy populations of normal (normodense) or relatively less (hypo- as developing crystalline cores and reticulated structures, dense) centrifugation density (1). Whereas normodense eo- respectively. Due to their production of GM-CSF, more than sinophils are the predominant physical phenotype found in the 80% of these hybrid granulocytes exclude trypan blue for 5 peripheral blood of healthy individuals, variable numbers of days when suspended in RPMI medium 1640, containing 10% hypodense eosinophils are found in the blood of patients with (vol/vol) fetal calf serum (FCS) and no exogenous cytokines, eosinophil-associated diseases, such as the idiopathic hyper- and more than 90% spontaneously progress to eosinophils. eosinophilic syndrome (IHES) (2-4). This physical diversity is These findings indicate that the eosinophil/basophil hybrid associated with functional heterogeneity (4-8). In comparison to normodense eosinophils, hypodense eosinophils exhibit Abbreviations: rh, recombinant human; IHES, idiopathic hypereosin- transiently enhanced ex vivo survival attributable to exposure ophilic syndrome; GM-CSF, granulocyte/macrophage colony- to eosinophilopoietic cytokines in vivo (4). Both normodense stimulating factor; IL, interleukin; FCS, fetal calf serum; BSA, bovine serum albumin. tTo whom reprint requests should be addressed at: Division of The publication costs of this article were defrayed in part by page charge Rheumatology and Immunology, Brigham and Women's Hospital, payment. This article must therefore be hereby marked "advertisement" in Seeley G. Mudd Building, Room 628,250 Longwood Avenue, Boston, accordance with 18 U.S.C. §1734 solely to indicate this fact. MA 02115. 2436 Downloaded by guest on September 27, 2021 Immunology: Boyce et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2437 granulocyte is a developmental intermediate during eosi- prepared on the basis of counts of 300 cells in each fraction. nophilopoiesis with autocrine and paracrine capabilities. We To generate density-gradient distribution plots, the total num- therefore sought to characterize a subsequently developing ber of eosinophils equilibrating at each interface was expressed immature eosinophil phenotype that arose by the continued as a percentage of the total eosinophil population (4). culture to 28 days of hybrid granulocytes in the presence of Determination of cell viability in culture in the absence of rhIL-3, rhIL-5, and Matrigel. These 28-day eosinophils are exogenous cytokines. To determine whether mononuclear eo- mononuclear and hypodense, constitutively elaborate GM- sinophils generated in vitro with rhIL-3 and rhIL-5 in the CSF, and exhibit enhanced viability in the absence of exoge- presence of Matrigel for 28 days rely on the continuous supply nous cytokines. This eosinophil phenotype may be related to of exogenous growth factors, the cells were extensively washed certain tissue-associated eosinophils observed in inflamma- with RPMI medium 1640 containing 10% (vol/vol) FCS either tory lesions (15-18). without exogenous cytokines or with incremental concentra- tions of rhIL-5 (0.01-100 pM) at a density of 106 cells per ml. The viability of the washed cells was determined every 24 h for MATERIALS AND METHODS 5 days on the basis of their capacity to exclude trypan blue dye. Cell Culture. Cord blood-derived hybrid granulocytes and Replicate 14-day hybrid granulocyte populations (19) were mononuclear eosinophils. Cord blood mononuclear cells were analyzed as a parallel control. isolated from umbilical cord blood obtained from the placenta To determine if the 28-day mononuclear eosinophils were after uncomplicated cesarean section deliveries as described elaborating a soluble, autocrine-active viability-sustaining fac- (19, 20). The cells were suspended in RPMI medium 1640 (Life tor, 48-h conditioned medium was prepared from these cells as Technologies, Gaithersburg, MD) containing 10% (vol/vol) described (19). To assess this conditioned medium for viability- FCS, 50 ,uM 2-mercaptoethanol, 200 pM rhIL-5 (R & D sustaining activity(s), freshly isolated human peripheral blood Systems, Minneapolis, MN), and 350 pM rhIL-3 (kindly pro- eosinophils were cultured in RPMI medium 1640 with 10% vided by Ryoji Matsumoto; expressed in a baculovirus system, (vol/vol) FCS alone; RPMI medium 1640 with 10% (vol/vol) PharMingen, San Diego) at a density of 0.5-1.0 x 106 cells per FCS supplemented with 0.01-100 pM GM-CSF; or RPMI ml and pipetted into plastic culture flasks coated with 0.5 ml medium 1640 with 10% (vol/vol) FCS enriched with 50%, of Matrigel (Collaborative Research, Burlington, MA) per cm2 25%, 10%, or 1% conditioned medium. The viability of the (19, 21-24). The medium containing the cytokines was eosinophils was assessed by their exclusion of trypan blue dye changed every 7 days. At defined days of culture, cytospin after 72 h of culture. For antibody-neutralization bioassays for slides of cells in suspension were stained with Wright's and GM-CSF, RPMI medium 1640 containing 50% conditioned Giemsa stains for differential cell counts to define the relative medium and 10% (vol/vol) FCS was incubated at 37°C for 2-4 purity of the eosinophil population. Typically, cells were h with a neutralizing rabbit polyclonal antibody directed harvested from culture after 14 days for hybrid granulocytes against human GM-CSF (Genzyme, Cambridge, Mass) before and after 28 days for mononuclear eosinophils. the eosinophils were added (4, 10, 19). The concentration of 50 Isolation ofperipheral blood eosinophils. For in vitro studies jig of antibody per ml of RPMI medium 1640 was selected on involving normal peripheral blood eosinophils, eosinophils the basis of the predetermined quantity required to neutralize were isolated from the peripheral blood of normal and atopic the viability-sustaining activity of a 10 pM dose of GM-CSF for donors by using immunomagnetic beads coupled to an anti- freshly isolated peripheral blood eosinophils after 72 h. Parallel CD16 antibody as described (25). The eosinophil fraction was dose-response curves were generated with GM-CSF at known quantitated by counting in a hemocytometer. Purity was concentrations (0.01-100 pM) to estimate the bioequivalency of assessed by differential cell counting on cytocentrifuge slides any viability-sustaining activity in the medium (4). stained with Wright's and Giemsa stains. The average purity of GM-CSF immunocytochemistry. To determine the propor- peripheral blood eosinophils used for this study was 98% ± tion of the 28-day cell population producing GM-CSF, these 1%. cells were subjected to immunocytochemical analysis. Slides of Characterization of Cord Blood-Derived Mononuclear Eo- 28-day cell preparations generated by cytocentrifugation were sinophils. Ultrastructural analysis of granules. Transmission incubated sequentially for 15 min at 37°C in 2 mM calcium electron microscopy was performed to determine the specific chloride, for 15 min at room temperature in phosphate- types in the mononuclear eosinophils. Cell pellets buffered saline (PBS) containing 0.05% Tween 20 and 0.1% containing 5-10 x 106 mononuclear eosinophils developed in bovine serum albumin (BSA), for 30 min at 37°C in PBS vitro with rhIL-5 and rhIL-3 in the presence of Matrigel for 28 containing 0.05% Tween 20 and 4% (vol/vol) normal goat days were fixed in 3% (vol/vol) glutaraldehyde/2% (wt/vol) serum, and overnight at 4°C in 4% (vol/vol) normal goat paraformaldehyde/0.1 M sodium cacodylate buffer, pH 7.4, serum containing either a 1:200 dilution of a rabbit anti-human for 2 h at room temperature. The fixed cells were washed in 0.1 GM-CSF polyclonal antibody (Genzyme) or a 1:200 dilution of M sodium cacodylate buffer, pH 7.4/5% sucrose. Samples a mouse monoclonal anti-human GM-CSF antibody (Gen- were postfixed with 1% OS04 in 0.1 M acetate veronal buffer, zyme). The samples were washed, incubated for 1.5 h at room pH 7.4, for 1 h at 4°C, en-bloc stained with 1% tannic acid (in temperature in PBS containing 0.5% Tween 20, 0.1% BSA, 0.05 M Tris buffer, pH 7.0) for 1 h at room temperature in the and containing either biotin-labeled goat anti-rabbit IgG or dark, and enhanced with 0.5% uranyl acetate in 0.1% acetate goat anti-mouse IgG, and washed again. The cytospin slides veronal buffer, pH 6.0, for 1 h at 37°C. The pellets were were washed twice in PBS containing 0.1% BSA and 0.05% dehydrated and embedded in Epon according to standard Tween 20, incubated for 40 min at room temperature in procedures (26). Grey to silver sections were cut with a Vectastin stain ABC-AP reagent (Vector Laboratories, Bur- Reichert-Jung Ultracut microtome (Leicha, Deerfield, IL), lingame, CA), and then incubated for 15 min in the dark at stained with uranyl and lead salts, and examined in a JEOL 100 room temperature in a blue alkaline phosphatase substrate CX microscope (JEOL) operating at 80 kV. solution (Vector Laboratories). Cover slips were applied with Density gradient sedimentation. Cord blood-derived cells at Immunomount (Shandon, Pittsburgh). Controls consisted of 28 days of culture were subjected to centrifugation through cytospin slides treated without primary antibody and cytospin discontinuous metrizamide gradients of 18-24% (wt/vol) (4) slides of purified peripheral blood eosinophils. Percentages of at 350 x g for 60 min. The cells at the interfaces were collected, cells exhibiting immunoreactivity were based on counts of 200 and cell counts were performed manually in a hemocytometer. cells on each slide. Cytocentrifugation slides were prepared and stained with Statistical analysis. The statistical significance of differences Wright's and Giemsa stains. Differential cells counts were between sample means for each set of cells was based on Downloaded by guest on September 27, 2021 2438 Immunology: Boyce et aL Proc. Natl. Acad. Sci. USA 93 (1996) comparison as determined by the two-tailed Student's t test. 28 d Culture Peripheral Blood Results are expressed as mean ± SEM. _ WV *_ Aa_ - RESULTS AND DISCUSSION After 14 days of culture in the presence of rhIL-3, rhIL-5, and Matrigel, 91% ± 1% of the nonadherent cell population were hybrid granulocytes on the basis of staining with Wright's and Giemsa stains, while 3% ± 1% were eosinophils, 4% ± 1% were mononuclear cells lacking visible granules, and 0.7% ± 0.2% were basophils. These findings were similar to our previously reported results with 14-day cells (19). By 28 days 1 ofculture, mononuclear eosinophils were the predominant cell type on the basis of staining with Wright's and Giemsa stains. F Of the nonadherent cell population, 73% ± 1% were mono- *4 nuclear eosinophils, 13% ± 3% were eosinophils with nuclear F-H4 i segmentation, 13% ± 4% were hybrid granulocytes, and 0.2% FIG. 1. Transmission electron micrographs showing granules with + 0.1% were basophils (n = 6), compared with 41% ± 1%, crystalline cores (arrows) of a mononuclear eosinophil developed in 22% ± 3%, 6% ± 2%, and 8% ± 1%, respectively, of cells vitro (a) and of a freshly isolated peripheral blood eosinophil (b). cultured in the presence of rhIL-3 and rhIL-5 without Matrigel (n = 3). Thus, with Matrigel, 100% of the cells developed in dense, compared with reference populations of peripheral vitro from cord blood precursors were granulocytes by day 28 blood eosinophils (2, 3, 4). (vs. 77% without Matrigel; n = 3; P < 0.01). Of these, 99% Cells cultured for 28 days (70% ± 1% mononuclear eosin- were eosinophils (vs. 69% without Matrigel; P < 0.001), and ophils, 11% ± 2% segmented eosinophils, 17% ± 3% hybrid 73% were mononuclear eosinophils (vs. 41% without Matrigel; granulocytes, 1% ± 0.3% basophils, and 0.2% ± 0.1% mono- P < 0.0001). Matrigel therefore favored eosinophilopoiesis nuclear cells lacking visible granules; n = 5) and cells cultured and the development of mononuclear eosinophils, when com- for 14 days (91% ± 1% hybrid granulocytes, 3% ± 1% pared with cell populations developed under the same condi- mononuclear eosinophils, 4% ± 1% mononuclear cells lacking tions but without Matrigel, after 28 days of culture. visible granules) were washed and placed in RPMI medium The mechanism by which Matrigel favors the predominance 1640 containing 10% (vol/vol) FCS and no exogenous cyto- of eosinophils is unknown. Both embryonic stem cells and kines. After an additional culture period of 5 days, 65% ± 3% mature eosinophils possess specific receptors for laminin, of the 28-day cells excluded trypan blue in the absence of fibronectin, and other matrix components (27-29). The recep- exogenous cytokines (Fig. 2), compared with 84% ± 3% of tor-mediated interaction of hematopoietic stem cells with 14-day cells cultured under identical conditions and 2% ± 1% matrix components may result in signal transduction and of peripheral blood eosinophils (n = 4). The inclusion of 100, expression of lineage-specific genes, as has been demonstrated 10, 1, and 0.1 pM rhIL-5 increased the proportion of the for mammary epithelium, hepatocytes, and other cell types mononuclear eosinophils excluding trypan blue from 65% + (21-24, 30). Alternately, the capacity of extracellular matrix 3% to 88% ± 3% (P < 0.05), 89% ± 2% (P < 0.05), 85% + proteins to specifically bind and stabilize growth factors may 2% (P < 0.05), and 84% ± 3% (P < 0.05), respectively, after ensure the exposure of stem cells to optimal local concentra- 5 days ofculture. There was no difference between the viability tions of cytokines (31). Furthermore, nerve growth factor, a of mononuclear eosinophils cultured in RPMI medium 1640 minor constituent of Matrigel, has synergistic activity with supplemented with 10% (vol/vol) FCS and 0.01 pM rhIL-5 GM-CSF (32) and with Mo-1 conditioned medium (33) in (66% ± 3%) or in RPMI medium 1640 with 10% FCS alone stimulating the growth and differentiation of granulocyte (65% ± 3%). This degree of survival independent of exoge- colonies, particularly of the eosinophil/basophil lineage. nous cytokines by the in vitro derived 28-day mononuclear The 28-day culture used to prepare the specimens for eosinophils exceeds that of hypodense eosinophils isolated by electron microscopy was 82% mononuclear eosinophils, as assessed by staining with Wright's and Giemsa stains. Of 50 1001 cells examined, 48 contained early eosinophil granules exclu- 90 sively (an amorphous or a homogeneous spherical core sur- 01) 80 rounded by a dense granule matrix) (Fig. la), consistent with CL 70 - eosinophilic myelocytes (34). The remaining cells that had 60- basophil-like nuclei also had only reticular structures, consis- 4) tent with basophil granules (data not shown). By comparison, 50- freshly isolated peripheral blood eosinophils had crystalline 0) 40* cores, consistent with crystallization of 0 30- (35) (Fig. lb). 200 Of the eosinophils used for the analysis of density gradient 90o distribution, 78% ± 3% were mononuclear, and 90% ± 1% of these mononuclear eosinophils segregated into the least dense 0 24 48 72 96 120 interfaces (supernatant/18%, 18%/20%, and 20%/21% me- Time in culture, h trizamide interfaces) (n = 3). Fewer than 4% of the mono- nuclear eosinophils were normodense. In contrast, 74% ± FIG. 2. Viability of 28-day mononuclear eosinophils (70% ± 1%) 18% of peripheral blood eosinophils obtained from healthy maintained in RPMI medium 1640 with 10% (vol/vol) FCS alone atopic donors as a reference population were normodense (-A-), or in RPMI medium 1640 with 10% (vol/vol) FCS and (23%/24% metrizamide interface and cell pellet), 23% ± 13% supplemented with 0.01 (-A,), 0.1 (-x-), 1 (4-), 10 (-*-), or 100 (+) pM rhIL-5. The viability of the cells was determined by their exclusion of were of intermediate density, and 3% ± 2% were hypodense trypan blue dye. Peripheral blood eosinophils maintained in RPMI (average ± 1/2 range; n = 2). Therefore, the eosinophils medium 1640 and 10% (vol/vol) FCS without exogenous cytokines differentiated in vitro for 28 days with rhIL-3, rhIL-5, and (+) are included as a reference. Standard errors are reported in the Matrigel were both predominantly mononuclear and hypo- text. Downloaded by guest on September 27, 2021 Immunology: Boyce et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2439 density centrifugation from patients with IHES; the latter Peripheral exhibited a 3-day half-life ex vivo (4). 28 d Culture Blood The presence of conditioned medium (50%, 25%, 10%, and 5,i 1%) derived from the 28-day cultures (68% + 2% mononu- clear eosinophils, 12% ±+ 3% segmented eosinophils, and 18% ± 3% hybrid granulocytes as determined by staining with Wright's and Giemsa stains) maintained the survival of 60% ± 7%, 54% + 8%, 48% ±+ 7%, and 32% ± 2%, respectively, of the freshly isolated peripheral blood eosinophils for 72 h (n = 3). Conditioned medium (50%, 25%, 10%, and 1%) from the 14-day cultures in the same three experiments (77% + 3% ot hybrid granulocytes, 7% ± 1% mononuclear eosinophils, 1% :: ± 1% segmented eosinophils, 3% + 2% basophils, and 11% + 3% mononuclear cells lacking identifiable granules) main- tained the survival of 73% ± 8%,59% ± 11%,52% ± 1%, and 38% ±- 1%, respectively, of peripheral blood eosinophils for 72 a 'I'" d h. Of the freshly isolated peripheral blood eosinophils, 20% ± 8% were viable in RPMI medium 1640 containing 10% I>~s- (vol/vol) FCS alone, and 96% ± 1% were viable in the presence of 10 pM GM-CSF. Parallel dose-response curves for a GM-CSF (data not shown) revealed that the eosinophil via- bility-sustaining bioactivity found in 50% 14-day hybrid gran- ulocyte-conditioned medium was equivalent to at least 500 fM GM-CSF and was -2-fold greater than that in the 28-day mononuclear eosinophil-conditioned medium. The addition of 50 jig of anti-GM-CSF antibody per ml reduced the viability- sustaining bioactivity of 28-day mononuclear eosinophil- conditioned medium by 98% ± 2%, of 14-day hybrid granu- locyte-conditioned medium by 79% ± 7%, and of 10 pM e GM-CSF by 91% ± 0% over 72 h of culture (Fig. 3). Thus, with progressive maturation, the production or release, or both, of GM-CSF by eosinophil progenitors declines. Cytocentrifugation slides prepared from 28-day cell popu- lations (68% ± 2% mononuclear eosinophils, 12% + 3% segmented eosinophils, 18% + 3% hybrid granulocytes, and 2% ±+ 1% basophils; n = 3) by staining with Wright's and Geimsa stains (Fig. 4a) contained 88% ±+ 10% cells with immunoreactivity for GM-CSF. Staining appeared to be mainly cytoplasmic (Fig. 4b). Replicate slides processed with- out primary antibody (Fig. 4 c and f) and freshly isolated peripheral blood eosinophils (n = 3 donors) did not exhibit c f i 100 10 jim 10 ulm 90 FIG. 4. Comparison of 28-day mononuclear eosinophils (a, b, and 80 c) with freshly isolated, peripheral blood eosinophils (d, e, andf). Cells CZ 70 are stained with Wright's and Giemsa stains (a and d) or with antibody against human GM-CSF (b and e). c and f show replicate cytocen- 60 0) trifugation slides processed without primary antibody as a negative 50 control. -0 40 immunochemical reactivity (Fig. 4e). It is therefore likely that 30 the enhanced viability of 28-day mononuclear eosinophils, 20. when compared to the viability of freshly isolated peripheral blood eosinophils, is due to their constitutive production of 10 GM-CSF. C.~~~~~~i Inasmuch as eosinophilopoiesis proceeds from agranular 14-d CM 28-d CM No CM mononuclear cells to a granulocyte acquiring progressive Conditioned medium degrees of nuclear segmentation and granulation (14), periph- FIG. 3. Viability of freshly isolated peripheral blood eosinophils eral blood and lesional mononuclear eosinophils may be an after 72 h of culture in RPMI medium 1640 with 10% (vol/vol) FCS immature eosinophil phenotype that is hypodense (13). Fur- supplemented with 50% conditioned medium (CM) from 14-day thermore, granulocytes having hybrid granule characteristics hybrid granulocytes and 28-day mononuclear eosinophils before (light of eosinophils and basophils described in the peripheral blood bars) and after (stippled bars) the addition of 50 ,g of anti-GM-CSF and of individuals with myelogenous leukemias per ml. For reference, replicate eosinophils were maintained for 72 h (36-39) may also be a normal eosinophil developmental in RPMI medium 1640 with 10% (vol/vol) FCS alone (black bar), or intermediate. Thus, both 14-day hybrid granulocytes and 28- in RPMI medium 1640 with 10% (vol/vol) FCS supplemented with 10 pM rhGM-CSF (diagonally striped bar) or with 10 pM GM-CSF + 50 day mononuclear eosinophils developed in vitro have counter- ,ug/ml of anti-GM-CSF (shaded). Standard errors are reported in the parts in vivo. It therefore seems reasonable to consider that the text. two cellular phenotypes recognized in this system of eosinophi- Downloaded by guest on September 27, 2021 2440 Immunology: Boyce et al. Proc. Natl. Acad. Sci. USA 93 (1996) lopoiesis in vitro are reflected in the limited in vivo observa- present in inflammatory lesions expressed GM-CSF, this tions. function might have both autocrine and paracrine actions. A subgroup of peripheral blood eosinophils from both individuals with asthma and unaffected individuals (21% and This work was supported by Grants HL-36110, AI-31599, DK-45656, 10% of peripheral blood eosinophils, respectively) exhibited AI-22531, HL-48598, AR-36308, and AI-07306 from the National immunoreactivity for GM-CSF in cytospin preparations (40). Institutes of Health, by a grant from the Allen and Hanburys Respi- GM-CSF localized to the eosinophil granule core as deter- ratory Institute, by an award from Jannsen Pharmaceutica, and by a mined by immunoelectron microscopy (40). It is noteworthy grant from the Charles H. Hood Foundation. that despite the presence of small intragranular quantities of 1. Owen, W. F. (1993) in Immunopharmacology ofEosinophils, ed. GM-CSF, normodense peripheral blood eosinophils do not Chapman, C. (Academic, London), pp. 57-72. survive ex vivo in the absence of an exogenous eosinophil- 2. Prin, L., Capron, M., Tonnel, A. B., Bletry, 0. & Capron, A. directed hematopoietin (9-12). Other studies reported that (1983) Int. Arch. Allergy Appl. Immunol. 72, 336-346. mature peripheral blood eosinophils have negligible GM-CSF 3. Prin, L., Charon, J., Capron, M., Gosset, P., Taelman, H., Tonnel, at baseline but generate both GM-CSF transcripts and protein A. B. & Capron, A. (1984) Clin. Exp. Immunol. 57, 735-742. after stimulation with calcium ionophore or interferon y (41) 4. Owen, W. F., Rothenberg, M. E., Petersen, J., Weller, P. F., and release both bioactive GM-CSF and IL-3 in response to Silberstein, D. S., Sheffer, A., Stevens, R. L., Soberman, R. J. & calcium ionophore (42) or to culture on fibronectin-coated Austen, K. F. (1989) J. Exp. Med. 170, 343-348. plates (43). The latter response depends upon a signal medi- 5. Sedgwick, J. B., Geiger, K. M. & Busse, W. W. (1990) Am. Rev. ated through the j31 integrin very late antigen (VLA)-4, Respir. Dis. 142, 120-125. suggesting that cell-matrix interaction might regulate the 6. Kajita, T., Yui, Y., Mita, H., Taniguchi, H., Saito, T., Mishima, T. & Shida, T. (1985) Int. Arch. Allergy Appl. Immunol. 78, production of GM-CSF by eosinophils (43). Furthermore, a 406-410. subgroup of lesion-associated eosinophils from patients with 7. Capron, M., Kazatchkine, M. D., Fischer, E., Joseph, M., But- nasal polyps and in airways of atopic patients after segmental terworth, A. E., Kusnierz, J. P., Prin, L., Papin, J. P. & Capron, antigen challenge express transcripts for GM-CSF (44, 45). It A. (1987) J. Immunol. 139, 2059-2065. was reported that the majority of cells found in eosinophil 8. Yukawa, T., Read, R. C., Kroegel, C., Rutman, A., Chung, K. F., colonies derived from peripheral blood progenitors from Wilson, R., Cole, P. J. & Barnes, P. J. (1990) Am. J. Respir. Cell atopic individuals were positive for GM-CSF, a finding in Mol. Bio. 2, 341-353. keeping with our observations (46). The possibility that the 9. Owen, W. F., Rothenberg, M. E., Silberstein, D. S., Gasson, J. C., constitutive production of GM-CSF in a subset of peripheral Stevens, R. L., Austen, K. F. & Soberman, R. J. (1987) J. Exp. blood or tissue-associated eosinophils is related to hemato- Med. 166, 129-141. poietic immaturity would be compatible with our findings of 10. 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(1989) in Blood Cells-A Practical Guide (Lippincott, peripheral blood and a lack of techniques for their purification, Philadelphia), pp. 50-51. little is known about their functional characteristics. In an 15. Aronson, H. & Philip, P. (1892) Dtsche. Med. Wochenschr. 21, ultrastructural study, cord blood-derived eosinophils differen- 48-49. 16. Sparrevohn, S. & Wulff, H. R. (1967) Acta Haematol. 37, 120-125. tiated in vitro by culture for 21-35 days in rhIL-5 and T-cell 17. Riis, P. & Anthonisen, P. (1964) Acta Med. Scand. 175, 85-89. conditioned medium were classified as myelocytes on the basis 18. Hoshi, H., Ohno, I., Honma, M., Tanno, Y., Yamauchi, K., of cell size, extensive Golgi structures, parallel cytoplasmic Tamura, G. & Shirato, K. (1995) Clin. Exp. Allergy 25, 720-728. arrays of distended cisternae of rough endoplasmic reticulum 19. Boyce, J. A., Friend, D., Matsumoto, R., Austen, K. F. & Owen, filled with lightly dense material, and features of granule W. F. (1995) J. Exp. 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