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The Journal of Immunology, 1999, 162: 4253-4259.
Copyright © 1999 by The American Association of Immunologists

Ligation of Fc{gamma}RII (CD32) Pivotally Regulates Survival of Human Eosinophils1

Jin-Tack Kim2, Andrew W. Schimming and Hirohito Kita3

Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The low-affinity IgG Fc receptor, Fc{gamma}RII (CD32), mediates various effector functions of lymphoid and myeloid cells and is the major IgG Fc receptor expressed by human eosinophils. We investigated whether Fc{gamma}RII regulates both cell survival and death of human eosinophils. When cultured in vitro without growth factors, most eosinophils undergo apoptosis within 96 h. Ligation of Fc{gamma}RII by anti-CD32 mAb in solution inhibited eosinophil apoptosis and prolonged survival in the absence of growth factors. Cross-linking of human IgG bound to Fc{gamma}RII by anti-human IgG Ab or of unoccupied Fc{gamma}RII by aggregated human IgG also prolonged eosinophil survival. The enhanced survival with anti-CD32 mAb was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated survival. In fact, mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb. In contrast to mAb or ligands in solution, anti-CD32 mAb or human IgG, when immobilized onto tissue culture plates, facilitated eosinophil cell death even in the presence of IL-5. Cell death induced by these immobilized ligands was accompanied by DNA fragmentation and was inhibited when eosinophil ß2 integrin was blocked by anti-CD18 mAb, suggesting that ß2 integrins play a key role in initiating eosinophil apoptosis. Thus, Fc{gamma}RII may pivotally regulate both survival and death of eosinophils, depending on the manner of receptor ligation and ß2 integrin involvement. Moreover, the Fc{gamma}RII could provide a novel mechanism to control the number of eosinophils at inflammation sites in human diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Eosinophils play important roles in the pathophysiology of allergic diseases, such as bronchial asthma and allergic rhinitis, and in host immunity to parasitic infections (reviewed in 1). During such inflammatory reactions, eosinophils release toxic granule proteins, which act as cytotoxins on a variety of cell types, including tracheal epithelial cells, pneumocytes, and parasites (reviewed in 2). Eosinophils are tissue-resident cells: >99% of human eosinophils reside in tissues (3). The number of tissue eosinophils is regulated by a discrete balance of various events, including recruitment from circulation, survival within the tissues, and elimination of dead cells by phagocytes (reviewed in Refs. 4 and 5). Recent studies have elucidated the mechanisms of eosinophil migration into the tissues (reviewed in 6). However, the precise mechanisms in tissue eosinophils for "choosing" survival or death are not fully understood. Cytokines, such as IL-3, IL-5, and granulocyte-macrophage-CSF (GM-CSF),4 or IFN-{gamma}, increase the life span of eosinophils by inhibiting apoptosis in vitro (7, 8, 9, 10). Increased expression of some of these cytokines by inflammatory cells, especially by T lymphocytes, has been reported in patients with allergic diseases (11) or parasitic infection (12). Therefore, the longevity of eosinophils is partly regulated by cytokines in the tissue microenvironment. In contrast, ligation of Fas by Fas-ligand (FasL) or administration of glucocorticoids accelerates eosinophil cell death, resulting in fewer tissue eosinophils (13, 14).

Much is known about the IgG Fc receptor (Fc{gamma}R) on lymphoid and myeloid cells and about the effector functions Fc{gamma}R mediates through interaction with the ligands, IgG and IgG/Ag complexes (reviewed in Refs. 15 and 16). However, little is known about the roles of Fc{gamma}R in proliferation, survival, and death of lymphoid and myeloid cells. In human NK cells, direct ligation of Fc{gamma}RIII (CD16) by mAb or IgG immune complex induces apoptosis (17, 18). In mice, ligation of Fc{gamma}R by anti-Fc{gamma}R mAb inhibits the development of eosinophil precursors and facilitates cell death through induction of apoptosis (19). Furthermore, mature murine eosinophils isolated from hepatic granulomas of Schistosoma mansoni-infected mice underwent apoptosis after Fc{gamma}R ligation (19). Thus, current information suggests that Fc{gamma}R is linked to apoptotic pathways in lymphoid and myeloid cells. In previous studies (20), we found that allergen-specific IgG1 and IgG3 induce degranulation of human eosinophils acting through Fc{gamma}RII (CD32), and that these interactions may be the mechanisms of eosinophil mediator release in the tissues of patients with allergic diseases. Because Fc{gamma}RII (CD32) is the major Fc{gamma}R expressed on mature human eosinophils with no or minimal expression of Fc{gamma}RI (CD64) or Fc{gamma}RIII (CD16) (21, 22), we wanted to know the role of Fc{gamma}RII (CD32) in human eosinophils. Therefore, we investigated the effects of ligation of Fc{gamma}RII (CD32) by mAb or their ligands on the survival of mature human eosinophils. We found, in contrast to current knowledge, that ligation of Fc{gamma}R by soluble mAb or its soluble ligands enhances eosinophil survival. In addition, ligation of Fc{gamma}R by mAb or ligands immobilized onto a solid surface lead to cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Anti-Fc{gamma}RII (CD32) mAb (IV.3, mIgG2b) was purchased from Medarex (West Lebanon, NH). F(ab')2 of goat anti-human IgG, human IgG, and mouse myeloma IgG2b were purchased from Cappel (Durham, NC). Mouse IgG was from Jackson Immunoresearch Laboratories (West Grove, PA). The mAb to GM-CSF, IL-5, and IL-3 were produced in our laboratory from hybridoma cell lines donated by Dr. J. S. Abrams (DNAX Research Institute, Palo Alto, CA). The specificities and potencies of these Abs have been described previously (23). Recombinant human IL-5 was a generous gift from the Schering-Plough Research Institute (Kenilworth, NJ). Heat-aggregated human IgG and mouse IgG were prepared by heating 10 mg/ml solutions of human IgG and mouse IgG in PBS for 30 min at 63°C. The preparations were centrifuged for 5 min at 12,000 x g and 4°C to remove particulate aggregates.

Eosinophil isolation

Eosinophils were isolated from peripheral blood of normal volunteers using a magnetic cell separation system (MACS) (Becton Dickinson, San Jose, CA), as described by Hansel et al. (24), with minor modifications. In brief, venous blood (90 ml) anticoagulated with 50 U/ml heparin was diluted with PBS at a 1:1 ratio. Diluted blood was overlayered on an isotonic Percoll solution (density, 1.085 g/ml) (Sigma, St. Louis, MO) and centrifuged at 200 x g at 4°C using a Beckman TJ-6 centrifuge. The supernatant and mononuclear cells at the interface were removed carefully. The inside wall of the centrifuge tube was wiped twice with sterile gauze to eliminate mononuclear cells adherent to the wall. Erythrocytes in the sediment were lysed by exposure to two cycles of sterile water. Isolated granulocytes were washed twice with PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, and 5.4 mM glucose, pH 7.4) containing 1% defined calf serum (DCS; HyClone Laboratories, Logan, UT), and an approximately equal volume of anti-CD16 Ab conjugated with magnetic particles (Miltenyi Biotic, Bergisch-Gladbach, Germany) was added to the cell pellet. After 60 min of incubation on ice, cells were loaded onto the separation column positioned in the MACS magnetic field. Cells were eluted three times with 5 ml of PIPES buffer with 1% DCS. The purity of eosinophils counted by Randolph’s stain was >98%. The contaminating cells were neutrophils, and no mononuclear cells or basophils were present. Purified eosinophils were used immediately for experiments.

Eosinophil survival assay

Freshly purified eosinophils were suspended at 0.5 x 106 cells/ml in Hybri-Care medium (American Type Culture Collection, Manassas, VA) supplemented with 50 µg/ml gentamicin and 10% DCS. One hundred microliter aliquots of cell suspension were mixed with 100 µl of serial dilutions of anti-CD32 mAb (IV.3), F(ab')2 of goat anti-human IgG, aggregated human IgG, or control Ig in 96-well flat-bottom tissue culture plates and cultured in the presence or absence of 100 pg/ml IL-5 for 4 days at 37°C and 5% CO2. In certain experiments, mAb or ligands were immobilized onto 96-well flat-bottom tissue culture plates by incubation of serial dilutions of anti-CD32 mAb (IV.3) or 50 µg/ml human IgG in the plates overnight at 4°C and blocking with 2.5% human serum albumin (HSA) for 2 h at 37°C; a cell suspension would then be incubated in a total volume of 200 µl. After the 4-day culture, the entire cell suspension was transferred to 12 x 75 mm polystyrene round-bottom tubes. An equal volume (200 µl) of propidium iodide (PI) solution was added to the cell suspensions to provide a final concentration of 0.50 µg/ml PI. Cell counting was performed by flow cytometry (FACScan, Becton Dickinson). At least 5000 cells were analyzed from each sample, and viable cells were calculated as the percentage of intact cells not stained with PI divided by the total number of intact cells. To examine cytokines involved in eosinophil survival, optimal concentrations (10 µg/ml) of anti-IL-3, anti-IL-5, or anti-GM-CSF mAb were included in the survival assay. The potencies and specificities of these mAb have been described elsewhere (23).

Quantification of DNA fragmentation

DNA fragmentation in cultured eosinophils was quantified by measuring cytoplasmic oligonucleosomal DNA using an ELISA kit (Cell Death Detection ELISA, Boehringer Mannheim, Mannheim, Germany), according to the manufacturer’s recommendations. Briefly, eosinophils were cultured in 96-well flat-bottom tissue culture plates coated with human IgG in the presence or absence of IL-5 for different periods. After incubation, cells were washed with PBS and treated with lysing solution for 30 min at 4°C. Cell lysates were centrifuged 13,000 x g for 4 min at 4°C, and oligonucleosomal DNA fragments in the supernatants were measured by sandwich ELISA using anti-histone mAb and anti-DNA mAb (25). In some experiments, fragmentation of DNA was confirmed by agarose gel DNA electrophoresis.

Detection of GM-CSF gene expression by RT-PCR

Detection of GM-CSF mRNA in eosinophils stimulated with soluble anti-CD32 mAb (IV.3) was performed, as previously described, with minor modifications (26). Briefly, eosinophils suspended in RPMI 1640 medium supplemented with 10 mM HEPES and 10% DCS (2 x 106 cells/sample) were incubated in 48-well tissue culture plates with 2.5 µg/ml of anti-CD32 mAb (IV.3) for up to 18 h at 37°C and 5% CO2. Cells were lysed and total cellular RNA was extracted using Trizol (Life Technologies, Gaithersburg, MD), as recommended by the manufacturer. The isopropanol-precipitated RNA was washed with 75% ethanol, dried, resuspended in 8 µl of oligo(dT) (Mayo Clinic Molecular Biology Core Facility, Rochester, MN) solution, and incubated at 65°C for 6 min. The mRNA was reverse transcribed to first strand cDNA in a final volume of 20 µl of reverse transcriptase (RT) mix. The RT mix contained 25 U of avian myeloblastosis virus RT, RT buffer (50 mM Tris-HCl, pH 8.5, 30 mM KCl, 8 mM MgCl2), 2.5 mM each of deoxynucleoside triphosphate (dNTP) (dATP, dCTP, dGTP, and dTTP), and 20 U of RNase inhibitor (Boehringer Mannheim). This mixture was incubated at 42°C for 2 h and stored at -70°C until used for PCR. cDNA was amplified from a 20-µl reaction mixture in a thin-wall polycarbonate 96-well plate (Corning Costar, Cambridge, MA) containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, dNTP mixture (0.2 mM of each dNTP), 0.2 M each of the primers for ß2-microglobulin (ß2m; Mayo Clinic Molecular Biology Core Facility) or GM-CSF (Clontech, Palo Alto, CA), and 1 U of Taq DNA polymerase (Boehringer Mannheim) and sample cDNA. The primer sequences for ß2m were 5'-CTCGCGCTACTCTCTCTTTCTGG-3' (5 primer) and 5'-GCTTACATGTCTCGATCCCACTTAA-3' (3 primer). Samples were overlayered with a drop of mineral oil (Sigma) and transferred to a thermal cycler (Omnigene, Hybaid Limited, Middlesex, U.K.). Thirty-five-cycle PCR was performed using 94°C for 45 s, 60°C for ß2m or 65°C for GM-CSF for 45 s, and 72°C for 2 min. At the end of PCR, a primer extension period of 7 min at 72°C was included. PCR products were electrophoresed on 3% agarose gel (NuSieve, FMC Bioproducts, Rockland, ME), and the gel was stained with ethidium bromide (Life Technologies) and photographed under UV light.

Assay for eosinophil-derived neurotoxin (EDN)

As an indicator of eosinophil activation, levels of an eosinophil granule protein, EDN, in the sample supernatants were measured. Eosinophils suspended in RPMI 1640 medium supplemented with 10 mM HEPES and 10% DCS were incubated with anti-CD32 mAb or isotype-matched control for 24 h as described above. Supernatants were collected, and the concentrations of EDN in the supernatants were measured by RIA. The RIA is a double-Ab competition assay using radiolabeled EDN, rabbit anti-EDN Ab, and burro anti-rabbit IgG, as described elsewhere (27). All assays were conducted in duplicate.

Statistical analysis

Data are presented as mean ± SEM from the numbers of experiments indicated. Statistical significance of the differences between various treatment groups was assessed with the paired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ligation of Fc{gamma}RII (CD32) by soluble mAb or soluble ligands on eosinophil survival

Unlike neutrophils or monocytes, Fc{gamma}RII (CD32) is the major Fc{gamma}R expressed on eosinophils with no or minimal expression of Fc{gamma}RI (CD64) or Fc{gamma}RIII (CD16) (21, 22). Earlier, we also reported that eosinophil degranulation induced by allergen-specific IgG is dependent on Fc{gamma}RII (CD32), but not on Fc{gamma}RIII (CD16) (28). To investigate whether Fc{gamma}RII (CD32) is involved in eosinophil survival, in addition to its known effects on cell activation, we incubated isolated human eosinophils with anti-CD32 mAb in the presence or absence of IL-5. When cells were incubated without mAb or IL-5, most cells underwent apoptosis and only 10% were alive after 4 days (Fig. 1GoA), consistent with previous findings (29, 30). When anti-CD32 mAb was added to the culture, eosinophil survival was enhanced in a concentration-dependent manner. Murine IgG2b, as a control, showed no effect on eosinophil viability. In contrast to its effect on enhancing eosinophil survival, soluble anti-CD32 mAb did not induce effector functions of eosinophils, as examined by release of an eosinophil granule protein, EDN (Table IGo). In an earlier report on NK cells, cell death induced by anti-Fc{gamma}R mAb (e.g., anti-CD16) required cell exposure to cytokines such as IL-2 (17). Because IL-5 is a critical cytokine to maintain viability of tissue eosinophils in patients with allergy (31), we examined whether ligation of Fc{gamma}RII affects eosinophil survival in the presence of IL-5. As shown in Fig. 1GoB, IL-5 enhanced eosinophil survival; anti-CD32 mAb or control Ig did not affect survival of eosinophils enhanced by IL-5. Thus, in contrast to previous reports in human NK cells (17) and murine eosinophils (19), ligation of Fc{gamma}R enhances survival of human eosinophils in the absence of exogenous cytokines.



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  		FIGURE 1. Viability of eosinophils incubated with soluble anti-Fc{gamma}RII (CD32) mAb. Eosinophils were cultured for 4 days with serial dilutions of anti-Fc{gamma}RII (CD32) mAb (•) or isotype-matched control (mouse IgG2b; {circ}) in the absence (A) or presence (B) of 100 pg/ml IL-5. After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as mean percent of viable cells ± SEM from five separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of Ab.

 

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  		Table I. Effects of two forms of anti-Fc{gamma}RII (CD32) mAb on eosinophil degranulation1

 
To ensure that occupancy of the receptor with more physiologically relevant ligands results in similar findings, two approaches were undertaken. Fc{gamma}RII (CD32) is a low-affinity IgG receptor, and some of these receptors on freshly isolated eosinophils are occupied by IgG; most of the receptors are unoccupied (28). As shown in Fig. 2GoA, ligation of unoccupied Fc{gamma}RII by the addition of aggregated human IgG, 25 µg/ml or higher, significantly enhanced eosinophil survival (p < 0.05). Aggregated mouse IgG showed no effect. Furthermore, ligation of receptor-bound IgG with F(ab')2 of goat anti-human IgG enhanced eosinophil survival (Fig. 2GoB); control Ig was ineffective. Thus, ligation of unoccupied or preoccupied Fc{gamma}RII (CD32) with its ligand, IgG, enhanced eosinophil survival similar to the effects of anti-CD32 mAb.



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  		FIGURE 2. Viability of eosinophils incubated with physiologic ligands for Fc{gamma}RII. A, Eosinophils were cultured for 4 days with serial dilutions of aggregated human IgG (Agg-hIgG) (•) or aggregated mouse IgG (Agg-mIgG) ({circ}). B, Eosinophils were cultured for 4 days with serial dilutions of F(ab')2 of goat anti-human IgG (•) or control Ab (goat IgG; {circ}). After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as mean percent of viable cells ± SEM from five (A) or four (B) separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of Ab.

 
Roles of autocrine cytokines for eosinophil survival induced by Fc{gamma}RII ligation

Cytokines, such as IL-3, IL-5, and GM-CSF, increase the life span of eosinophils by inhibiting apoptosis in vitro (7, 8, 9). Therefore, we investigated whether enhanced eosinophil survival after ligation of Fc{gamma}RII by anti-CD32 mAb is due to direct activation of the cell’s survival mechanisms or due to induction of eosinophil-active cytokines in the eosinophils. To this end, we performed blocking experiments using neutralizing Abs against various cytokines. As shown in Fig. 3GoA, survival of eosinophils incubated with anti-CD32 mAb was inhibited significantly by anti-GM-CSF mAb to the baseline level generated in the absence of anti-CD32 mAb. In contrast, anti-IL-5 mAb or anti-IL-3 mAb showed no effect. Combinations of mAb containing anti-GM-CSF plus anti-IL-3 or anti-IL-5 were no more effective than anti-GM-CSF alone, suggesting that the cytokine providing anti-CD32-induced eosinophil survival is mainly GM-CSF. Furthermore, as shown in Fig. 3GoB, by RT-PCR, we could detect mRNA for GM-CSF in eosinophils cultured with anti-CD32 mAb for 18 h. No mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb for 4 h, although we could detect mRNA for a housekeeping gene, ß2m, in the same samples. Altogether, ligation of Fc{gamma}RII by soluble anti-CD32 mAb likely enhances survival of human eosinophils through autocrine production of GM-CSF.



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  		FIGURE 3. Roles of autocrine cytokines in eosinophil survival induced by soluble anti-Fc{gamma}RII (CD32) mAb. A, Effects of anti-cytokine Ab on eosinophil survival induced by anti-Fc{gamma}RII (CD32) mAb. Eosinophils were cultured for 4 days with ({blacksquare}) or without ({square}) 2.5 µg/ml of anti-Fc{gamma}RII (CD32) mAb in the presence or absence of anti-GM-CSF mAb (anti-GM), anti-IL-3 mAb, anti-IL-5 mAb, or their combinations. After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as mean percent of viable cells ± SEM from three separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of anti-cytokine Ab. B, Expression of mRNA for GM-CSF in eosinophils incubated with anti-Fc{gamma}RII (CD32) mAb. Eosinophils were incubated with 2.5 µg/ml of anti-Fc{gamma}RII (CD32) mAb for indicated periods. Cells were then lysed and the expression of mRNA for GM-CSF or ß2m was examined by RT-PCR and specific primers as described in the Materials and Methods. Lanes 1 and 5 show cDNA for GM-CSF and ß2m, respectively. Lanes 4 and 8 are without cDNA. Lanes 2, 3, 6, and 7 are cDNA from eosinophils. The results of a representative experiment from three experiments are shown.

 
Effects of ligation of Fc{gamma}RII (CD32) by immobilized ligands on eosinophil survival

Targets with large surfaces, such as S. mansoni larvae (32) or Sepharose 4B beads (33), which have been sensitized with IgG, are often used to induce eosinophil effector functions. Furthermore, as shown in Table IGo (see above), anti-CD32 mAb immobilized onto tissue culture plates, but not the soluble anti-CD32 mAb, induced activation of eosinophils as monitored by the release of a granule protein, EDN. Therefore, we next examined whether ligands, which had been immobilized onto a large surface, would also induce eosinophil survival. As shown in Fig. 4GoA, only 11~13% of eosinophils were alive after 4 days in the absence of IL-5 or anti-CD32 mAb, consistent with the findings of Fig. 1Go. Furthermore and in contrast to soluble anti-CD32 mAb, immobilized anti-CD32 mAb did not enhance, but slightly inhibited, eosinophil survival. As shown in Fig. 4GoB, in the presence of IL-5, ~70% of cells were alive after 4 days; this enhanced eosinophil survival was inhibited down to <20% by immobilized anti-CD32 mAb, but not by immobilized control Ig.



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  		FIGURE 4. Viability of eosinophils incubated with immobilized anti-Fc{gamma}RII (CD32) mAb. Ninety-six-well tissue culture plates were coated with the indicated concentrations of anti-Fc{gamma}RII (CD32) mAb (•) or isotype-matched control (mouse IgG2b; {circ}). Eosinophils were added to the plates and incubated for 4 days in the absence (A) or presence (B) of 100 pg/ml IL-5. After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as mean percent of viable cells ± SEM from five separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of Ab.

 
The findings were confirmed by using the physiological ligand for Fc{gamma}RII. As shown in Fig. 5GoA, human IgG immobilized onto tissue culture plates significantly inhibited the 4-day survival of eosinophils incubated with IL-5 (p < 0.05, n = 4). Because eosinophil cell death induced by the removal of growth factor or administration of glucocorticoids involves apoptotic mechanisms (29, 34), we next determined if cell death induced by immobilized IgG also involves degradation of genomic DNA. Cells were incubated for 24 h with IL-5 and immobilized IgG; then the quantities of fragmented DNA in the cytosol were measured by ELISA. As shown in Fig. 5GoB, large amounts of fragmented DNA were detected when cells were cultured in the absence of IL-5, consistent with previous findings (29, 34). IL-5 dramatically inhibited the DNA fragmentation, and this inhibitory effect was abolished when immobilized IgG was added to the culture. In a kinetics study using IL-5, DNA fragmentation induced by immobilized IgG became apparent at 18 h or later in culture (data not shown). Overall, these data indicate that ligation of Fc{gamma}RII by immobilized mAb or its ligand enhances eosinophil cell death, while soluble mAb or ligand enhances eosinophil survival.



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  		FIGURE 5. Eosinophil cell death induced by immobilized human IgG. A, Comparison of cell viability in wells coated with human IgG ({blacksquare}) or without ({square}). Eosinophils were added to the plates and incubated for 4 days in the absence or presence of 100 pg/ml IL-5. After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as the mean percent of viable cells ± SEM from four separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of IgG. B, Comparison of DNA fragmentation in wells coated with human IgG ({blacksquare}) or without ({square}). Eosinophils were added to the plates and incubated for 24 h in the absence or presence or 100 pg/ml IL-5. After incubation, cells were lysed, and fragmentation of DNA was quantitated by ELISA as described in Materials and Methods. Results are presented as mean ± SEM from three separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of IgG.

 
Roles of ß2 integrin for eosinophil cell death induced by immobilized IgG

How then do the forms of mAb, either soluble or immobilized, determine eosinophil survival or death? In addition to Fc{gamma}RII, we previously reported on another cell surface molecule, ß2 integrin, and its involvement in eosinophil interaction with immobilized IgG (28). Therefore, we hypothesized that engagement of ß2 integrin may serve as a switch leading to cell death in eosinophils. We tested this hypothesis by adding anti-CD18 mAb, which blocks ß2 integrin (28), to eosinophils cultured with immobilized IgG. As shown in Fig. 6Go, in the absence of IL-5, immobilized IgG or anti-CD18 mAb showed minimal effects on eosinophil viability, suggesting that anti-CD18 mAb by itself does not affect eosinophil survival. In the presence of IL-5, eosinophil viability was enhanced up to 95%, but it was inhibited strikingly by immobilized IgG down to 26%. This inhibitory effect was reversed almost entirely by anti-CD18 mAb, but not by control Ig. These findings suggest that ß2 integrin is critically involved in eosinophil cell death induced by immobilized IgG.



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  		FIGURE 6. Effects of blocking the ß2 integrin on viability of eosinophils incubated with immobilized human IgG. Ninety-six-well tissue culture plates were coated with or without 50 µg/ml of human IgG. Eosinophils were preincubated with 10 µg/ml of anti-ß2 integrin (CD18) mAb or control Ig (mouse IgG1), added to the plates, and incubated for 2 days in the absence or presence of 100 pg/ml IL-5. After incubation, cell viability was determined by staining cells with PI and using flow cytometry. Results are presented as mean percent of viable cells ± SEM from three separate experiments. *, Statistically significant difference (p < 0.05) compared with cells cultured in the absence of IgG and in the presence of IL-5. **, Statistically significant difference (p < 0.05) compared with cells cultured with immobilized IgG in the presence of mouse IgG1 and IL-5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The new finding in this report is that ligation of Fc{gamma}RII (CD32) pivotally regulates both survival and cell death of mature human eosinophils; the form of the ligand is crucial to the regulation. This conclusion is based on several observations: 1) ligation of Fc{gamma}RII by soluble anti-CD32 mAb enhanced survival of eosinophils through autocrine production of GM-CSF in the absence of exogenous cytokines; 2) similarly, ligation of Fc{gamma}RII by physiologically relevant ligands in solution, including anti-human IgG and aggregated human IgG, also enhanced eosinophil survival; 3) in contrast, ligation of Fc{gamma}RII by anti-CD32 mAb or human IgG immobilized onto tissue culture plates inhibited eosinophil survival in the presence of IL-5; and 4) eosinophil cell death induced by immobilized human IgG was accompanied by fragmentation of genomic DNA. Thus, Fc{gamma}RII (CD32) may serve as a "two-edged sword" in the survival of mature human eosinophils. These observations may have important implications for the regulation of eosinophil numbers at the sites of inflammation in patients with allergy or parasitic infection. For example, in grass pollen hay fever, allergen-specific IgG is produced locally by plasma cells in the nasal mucosa (35). In patients with schistosome infection, circulating immune complexes involving several Ags have been demonstrated (36). Therefore, it is conceivable that soluble IgG immune complexes formed in vivo in circulation or mucosal tissues may cross-link Fc{gamma}RII on eosinophils, resulting in longevity of the tissue resident eosinophils. Alternatively, Ag-specific IgG is often immobilized on the surfaces of large organisms or cells in vivo, as most typically seen with schistosomula of S. mansoni (37). Moreover, when they are stimulated with immobilized IgG1- or IgG3-immune complexes, human eosinophils release granule proteins (28). Therefore, when Ab immobilized onto the "targets" at the sites of inflammation cross-links the Fc{gamma}RII on eosinophils, eosinophil activation and subsequent apoptotic cell death may occur, even in the presence of the normally survival-enhancing IL-5 (31). Thus, Fc{gamma}RII may play a pivotal role in regulating tissue eosinophil numbers; that is, it can maintain eosinophil viability before "target" encounter and eliminate cells after "target" interaction.

An unexpected observation in this study is the enhancement of eosinophil survival after ligation of Fc{gamma}R by soluble mAb or soluble ligands. Earlier reports demonstrated that direct ligation of Fc{gamma}RIII (CD16) by mAb or IgG immune complexes induced apoptotic cell death in human NK cells (17, 18). In addition, ligation of Fc{gamma}RII (CD32) by anti-Fc{gamma}R mAb inhibited the development of eosinophil precursors and promoted cell death through induction of apoptosis in mice (19). Therefore, previous studies suggested that Fc{gamma}R is linked to apoptotic pathways. Although the exact reason(s) for the difference between these previous observations and those described in this manuscript are unknown, heterogeneity in Fc{gamma}R and intracellular signaling molecules associated to Fc{gamma}R likely play roles. For example, in mice, Fc{gamma}RII occurs in two isoforms, ß1 and ß2, both of which contain a phosphotyrosine motif, designated the immunoreceptor tyrosine-based inhibitory motif (ITIM), that inhibits the immunoreceptor tyrosine-based activation motif (ITAM)-induced signal transduction (38, 39). In humans, Fc{gamma}RII is encoded by three genes, and two (Fc{gamma}RIIA and Fc{gamma}RIIC) contain ITAM sequences, while the other (Fc{gamma}RIIB) contains an ITIM sequence (reviewed in 16). Analysis with PCR suggests that granulocytes express Fc{gamma}RIIA and Fc{gamma}RIIC, but not Fc{gamma}RIIB (40). We also found by RT-PCR that human eosinophils express both Fc{gamma}RIIA/C and Fc{gamma}RIIB forms (data not shown). Furthermore, in mouse eosinophils ligation of Fc{gamma}RII (CD32) by soluble mAb induced production of superoxide anion (19). In contrast, ligation of Fc{gamma}RII (CD32) by soluble anti-CD32 or soluble anti-human IgG did not activate effector functions by human eosinophils (Ref. 28 and this manuscript). Thus, there are potential differences in signals and outcomes following ligation of Fc{gamma}RII in humans and mice. Alternatively, differences in the mAb used to ligate Fc{gamma}RII and/or amounts of cytokines used during incubation with mAb may explain the discrepancy in the results of this study and those of de Andrés et al. (19). In addition, ligation of Fc{gamma}RIII (CD16) on human NK cells by soluble ligands leads to activation of several intracellular signaling molecules (41), including Lck, ZAP-70, and Syk, which are not observed with human granulocytes activated with Fc{gamma}RII (CD32). Therefore, the differences among species, experimental conditions, and Fc{gamma}R may need to be taken into account before generalizing the roles of Fc{gamma}R for survival and apoptosis of myeloid and lymphoid cells.

The findings that immobilized mAb or ligands for Fc{gamma}RII induced eosinophil cell death, whereas soluble mAb or ligands for Fc{gamma}RII enhanced eosinophil survival, suggest the involvement of another molecule that may energize death pathways in eosinophils interacting with immobilized ligands. In fact, we found that blocking eosinophil ß2 integrin with anti-CD18 mAb inhibits cell death induced by immobilized IgG in the presence of IL-5 (Fig. 6Go). Because anti-CD18 mAb did not enhance eosinophil survival in the absence of IL-5 (see Fig. 6Go), this rescue effect of anti-CD18 mAb is not likely due to the direct ligation by anti-CD18 mAb of ß2 integrin or to the contamination of Ab preparation with eosinophil growth factors. Therefore, engagement of ß2 integrin, in addition to ligation of Fc{gamma}RII, may play a key role in inducing cell death in otherwise surviving eosinophils. Interestingly, a similar observation has been made with neutrophils in mice selectively deficient in a ß2 integrin molecule, CD11b/CD18 (42). In these mice, thioglycollate-induced neutrophil accumulation in the peritoneal cavity was paradoxically increased compared with wild-type animals, in spite of their impaired ability to adhere to endothelial cells. This local neutrophilia was associated with a significant delay in apotosis of CD11b/CD18-deficient neutrophils, suggesting a role for CD11b/CD18 in neutrophil apoptosis. Thus, ß2 integrin may play a novel homeostatic role in inflammation by accelerating apoptosis of granulocytes in vitro and in vivo.

At present, the key question is how would engagement of ß2 integrins induce eosinophil cell death? Our preliminary studies showed that FasL (CD95/APO-1) was not detectable on fresh or cultured eosinophils (data not shown), suggesting that the Fas/FasL system is not involved in the mechanism. Recent studies indicate that multiple intracellular events and/or molecules, such as Bax, reactive oxygen species, and increases in the intracellular calcium concentrations ([Ca2+]i), activate caspases, which are enzymes critically involved in cell apoptosis (reviewed in 43). Also, in eosinophils, we reported that ligation of a ß2 integrin, {alpha}Mß2 (Mac-1, CD11b/CD18), by mAb triggers activation of a number of signaling events, such as tyrosine phosphorylation of intracellular proteins, phosphoinositide turnover, and increased [Ca2+]i (44). Furthermore, blocking of ß2 integrins by anti-CD18 mAb inhibited both inositol phosphate production and the respiratory burst in eosinophils stimulated with immobilized IgG (28). Thus, ß2 integrins are likely not simply adhesion molecules but also critically involved in intracellular signaling pathways of eosinophils. Conceivably, engagement of ß2 integrins leads to activation of signaling pathways involved in eosinophil apoptosis. Studies are under way in our laboratory to identify the relevant intracellular molecule(s).

In summary, our study identifies a novel role for Fc{gamma}RII. In addition to its known role in activating eosinophil effector functions, ligation of Fc{gamma}RII can lead eosinophils to two opposite destinies, namely survival and apoptosis, depending on the manner of receptor ligation. Earlier information suggested that eosinophil survival at inflammation sites is regulated by cytokines, such as IL-5 and GM-CSF, in the tissue microenvironment. We now propose that the fate of tissue-dwelling eosinophils is closely regulated by cytokines, IgG Ab, and the availability of ligands for ß2 integrin.


   Acknowledgments
 
We thank Dr. Gerald J. Gleich for critical reading of this manuscript, Ms. Linda H. Arneson for secretarial assistance, and Ms. Cheryl Adolphson for editorial assistance.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health, AI 34486 and AI 34577, and by the Mayo Foundation. Back

2 Current address: Department of Pediatrics, Catholic University Medical College, Seoul, Korea. Back

3 Address correspondence and reprints requests to Dr. Hirohito Kita, Department of Immunology, Mayo Clinic Rochester, MN 55905. E-mail address: Back

4 Abbreviations used in this paper: GM, granulocyte-macrophage; ß2M, ß2-microglobulin; [Ca2+]i, intracellular calcium concentration; DCS, defined calf serum; EDN, eosinophil-derived neurotoxin; FasL, Fas ligand; Fc{gamma}R, IgG Fc receptor; HSA, human serum albumin; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; MACS, magnetic cell separation system; PI, propidium iodide; RT, reverse transcriptase; dNTP; deoxynucleoside triphosphate. Back

Received for publication July 14, 1998. Accepted for publication January 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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