| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RII (CD32) Pivotally Regulates Survival of Human Eosinophils1
Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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
FcRII 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 FcRII 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
FcRII by anti-human IgG Ab or of unoccupied FcRII 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, FcRII may pivotally regulate both survival and
death of eosinophils, depending on the manner of receptor ligation and
ß2 integrin involvement. Moreover, the FcRII could
provide a novel mechanism to control the number of eosinophils at
inflammation sites in human diseases. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, 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 (FcR) on lymphoid and
myeloid cells and about the effector functions FcR 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 FcR in
proliferation, survival, and death of lymphoid and myeloid cells. In
human NK cells, direct ligation of FcRIII (CD16) by mAb or IgG
immune complex induces apoptosis (17, 18). In mice, ligation of FcR
by anti-FcR 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 FcR ligation (19). Thus, current information
suggests that FcR 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 FcRII (CD32), and that these interactions
may be the mechanisms of eosinophil mediator release in the tissues of
patients with allergic diseases. Because FcRII (CD32) is the major
FcR expressed on mature human eosinophils with no or minimal
expression of FcRI (CD64) or FcRIII (CD16) (21, 22), we wanted to
know the role of FcRII (CD32) in human eosinophils. Therefore, we
investigated the effects of ligation of FcRII (CD32) by mAb or their
ligands on the survival of mature human eosinophils. We found, in
contrast to current knowledge, that ligation of FcR by soluble mAb
or its soluble ligands enhances eosinophil survival. In addition,
ligation of FcR by mAb or ligands immobilized onto a solid surface
lead to cell death.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anti-FcRII (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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RII (CD32) by soluble mAb or soluble
ligands on eosinophil survival
Unlike neutrophils or monocytes, FcRII (CD32) is the major
FcR expressed on eosinophils with no or minimal expression of
FcRI (CD64) or FcRIII (CD16) (21, 22). Earlier, we also reported
that eosinophil degranulation induced by allergen-specific IgG is
dependent on FcRII (CD32), but not on FcRIII (CD16) (28). To
investigate whether FcRII (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. 1
A), 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 I). In an earlier report on NK cells, cell death induced
by anti-FcR 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 FcRII affects eosinophil survival in
the presence of IL-5. As shown in Fig. 1B, 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
FcR enhances survival of human eosinophils in the absence of
exogenous cytokines.
|
|
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. 2A, ligation of unoccupied FcRII 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. 2B); control Ig was
ineffective. Thus, ligation of unoccupied or preoccupied FcRII
(CD32) with its ligand, IgG, enhanced eosinophil survival similar to
the effects of anti-CD32 mAb.
|
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
FcRII 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. 3A, 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. 3B, 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 FcRII by soluble anti-CD32 mAb likely enhances
survival of human eosinophils through autocrine production of GM-CSF.
|
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 I
(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. 4A, 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. 1. 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. 4B, 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.
|
RII. As shown in Fig. 5A, 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. 5B,
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 FcRII by immobilized
mAb or its ligand enhances eosinophil cell death, while soluble mAb or
ligand enhances eosinophil survival.
|
How then do the forms of mAb, either soluble or immobilized,
determine eosinophil survival or death? In addition to FcRII, 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. 6, 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.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 FcRII 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 FcRII by physiologically relevant ligands
in solution, including anti-human IgG and aggregated human IgG,
also enhanced eosinophil survival; 3) in contrast, ligation of FcRII
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, FcRII (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 FcRII 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 FcRII on eosinophils, eosinophil activation and
subsequent apoptotic cell death may occur, even in the presence of the
normally survival-enhancing IL-5 (31). Thus, FcRII 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 FcR by soluble mAb or soluble
ligands. Earlier reports demonstrated that direct ligation of FcRIII
(CD16) by mAb or IgG immune complexes induced apoptotic cell death in
human NK cells (17, 18). In addition, ligation of FcRII (CD32) by
anti-FcR mAb inhibited the development of eosinophil precursors
and promoted cell death through induction of apoptosis in mice (19).
Therefore, previous studies suggested that FcR 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 FcR and intracellular
signaling molecules associated to FcR likely play roles. For
example, in mice, FcRII 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, FcRII is
encoded by three genes, and two (FcRIIA and FcRIIC) contain ITAM
sequences, while the other (FcRIIB) contains an ITIM sequence
(reviewed in 16). Analysis with PCR suggests that granulocytes
express FcRIIA and FcRIIC, but not FcRIIB (40). We also found
by RT-PCR that human eosinophils express both FcRIIA/C and FcRIIB
forms (data not shown). Furthermore, in mouse eosinophils ligation of
FcRII (CD32) by soluble mAb induced production of superoxide anion
(19). In contrast, ligation of FcRII (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
FcRII in humans and mice. Alternatively, differences in the mAb used
to ligate FcRII 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
FcRIII (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 FcRII (CD32). Therefore, the differences among
species, experimental conditions, and FcR may need to be taken into
account before generalizing the roles of FcR for survival and
apoptosis of myeloid and lymphoid cells.
The findings that immobilized mAb or ligands for FcRII induced
eosinophil cell death, whereas soluble mAb or ligands for FcRII
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. 6). Because
anti-CD18 mAb did not enhance eosinophil survival in the absence of
IL-5 (see Fig. 6), 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 FcRII, 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, 
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 FcRII. In addition
to its known role in activating eosinophil effector functions, ligation
of FcRII 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 |
|---|
|
Footnotes |
|---|
2 Current address: Department of Pediatrics, Catholic University Medical College, Seoul, Korea.
3 Address correspondence and reprints requests to Dr. Hirohito Kita, Department of Immunology, Mayo Clinic Rochester, MN 55905. E-mail address:
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; FcR, 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.
Received for publication July 14, 1998. Accepted for publication January 8, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R induces c-myc-dependent apoptosis in IL-2-stimulated NK cells. J. Immunol. 154:491.[Abstract]
RII (CD32) is linked to apoptotic pathways in murine granulocyte precursors and mature eosinophils. Blood 90:1267.RII induce eosinophil degranulation. J. Clin. Invest. 95:2813.
and CD11/CD18 receptor expression on normal density and low density human eosinophils. Immunology 69:264.[Medline]
requires transcriptional arrest. J. Immunol. 153:1778.[Abstract]
RIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation. Immunity 3:635.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. Yoon, J. U. Ponikau, C. B. Lawrence, and H. Kita Innate Antifungal Immunity of Human Eosinophils Mediated by a {beta}2 Integrin, CD11b J. Immunol., August 15, 2008; 181(4): 2907 - 2915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. von Gunten, S. Yousefi, M. Seitz, S. M. Jakob, T. Schaffner, R. Seger, J. Takala, P. M. Villiger, and H.-U. Simon Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment Blood, August 15, 2005; 106(4): 1423 - 1431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Watanabe, M. Okano, H. Hattori, T. Yoshino, N. Ohno, N. Ohta, Y. Sugata, Y. Orita, T. Takai, and K. Nishizaki Roles of Fc{gamma}RIIB in Nasal Eosinophilia and IgE Production in Murine Allergic Rhinitis Am. J. Respir. Crit. Care Med., January 1, 2004; 169(1): 105 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Esnault and J. S. Malter Hyaluronic Acid or TNF-{alpha} Plus Fibronectin Triggers Granulocyte Macrophage-Colony-Stimulating Factor mRNA Stabilization in Eosinophils Yet Engages Differential Intracellular Pathways and mRNA Binding Proteins J. Immunol., December 15, 2003; 171(12): 6780 - 6787. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Nutku, H. Aizawa, S. A. Hudson, and B. S. Bochner Ligation of Siglec-8: a selective mechanism for induction of human eosinophil apoptosis Blood, June 15, 2003; 101(12): 5014 - 5020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kobayashi, K. Iijima, and H. Kita Marked Airway Eosinophilia Prevents Development of Airway Hyper-responsiveness During an Allergic Response in IL-5 Transgenic Mice J. Immunol., June 1, 2003; 170(11): 5756 - 5763. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Schettini, G. Salamone, A. Trevani, S. Raiden, R. Gamberale, M. Vermeulen, M. Giordano, and J. R. Geffner Stimulation of neutrophil apoptosis by immobilized IgA J. Leukoc. Biol., October 1, 2002; 72(4): 685 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Esnault and J. S. Malter Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-alpha plus fibronectin-activated peripheral blood eosinophils Blood, May 13, 2002; 99(11): 4048 - 4052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Hoontrakoon, H. W. Chu, S. J. Gardai, S. E. Wenzel, P. McDonald, V. A. Fadok, P. M. Henson, and D. L. Bratton Interleukin-15 Inhibits Spontaneous Apoptosis in Human Eosinophils via Autocrine Production of Granulocyte Macrophage-Colony Stimulating Factor and Nuclear Factor-kappa B Activation Am. J. Respir. Cell Mol. Biol., April 1, 2002; 26(4): 404 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Matsumoto, M. Hirashima, H. Kita, and G. J. Gleich Biological Activities of Ecalectin: A Novel Eosinophil-Activating Factor J. Immunol., February 15, 2002; 168(4): 1961 - 1967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Esnault and J. S. Malter Granulocyte Macrophage-Colony-Stimulating Factor mRNA Is Stabilized in Airway Eosinophils and Peripheral Blood Eosinophils Activated by TNF-{{alpha}} Plus Fibronectin J. Immunol., April 1, 2001; 166(7): 4658 - 4663. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 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.
) or without (
) 2.5 µg/ml of anti-Fc
