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Cutting Edge: Eotaxin Elicits Rapid Vesicular
Transport-Mediated Release of Preformed IL-4 from
Human Eosinophils
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of June 17, 2017.
Christianne Bandeira-Melo, Kumiya Sugiyama, Lesley J. Woods and
Peter F. Weller
J Immunol 2001; 166:4813-4817; ;
doi: 10.4049/jimmunol.166.8.4813
http://www.jimmunol.org/content/166/8/4813
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References
●
Cutting Edge: Eotaxin Elicits Rapid
Vesicular Transport-Mediated Release
of Preformed IL-4 from Human
Eosinophils1
Christianne Bandeira-Melo, Kumiya Sugiyama,
Lesley J. Woods, and Peter F. Weller2
E
osinophils, prominent in Th2-driven immune responses,
including asthma and allergic and parasitic diseases, may
have multiple roles in these diseases. As effector cells
eosinophils, based in part on their release of cationic granule proteins and lipid mediators, may contribute to the immunopathogenesis of allergic diseases (1). Additional functional roles for eosinophils are indicated by findings that eosinophils may exert
immunomodulatory activities via interactions with T and B lymphocytes. For these potential interactions with lymphocytes, human eosinophils express costimulatory surface proteins, including
CD40, CD28, CD86, and MHC class II Ags (2– 4), and produce
over two dozen cytokines, including the prototypical Th2 cytokine
IL-4 (5).
Like eosinophils, IL-4 is a hallmark of allergic and parasitic
disorders. IL-4 contributes to the polarization toward Th2 differentiation and promotes IgE class switching (6, 7). The actions of
IL-4 are not limited to the initiation of Th2 responses, but also may
stimulate other cellular responses that contribute to manifestations
of allergic diseases (6). Potential cellular sources of IL-4 include
CD4⫹ T cells (7), mast cells (8), basophils (9), NK1.1⫹ T cells
(10), ␥␦ T cells (11), and eosinophils (12–14). Within eosinophils,
unlike CD4⫹ T cells, IL-4 is stored as a preformed pool within
eosinophil-specific granules (13–17). A role for eosinophil-derived
IL-4 has been demonstrated in a murine system in which the i.p.
instillation of Schistosoma mansoni eggs led to the enhanced generation over 12 h of IL-4 derived from peritoneal exudate eosinophils (12). Because nothing is known about how IL-4 within human eosinophils might be mobilized, we have investigated stimuli
and mechanisms that lead to the extracellular release of IL-4 stored
within normal donor-derived eosinophils.
Materials and Methods
Purification of human eosinophils
Eosinophils were isolated from the blood of 18 healthy nonatopic donors
by negative selection using the MACS anti-CD16 immunomagnetic bead
procedure (Miltenyi Biotec, Auburn, CA) (18). Eosinophil purity and viability was ⬎99 and ⬎95%, respectively.
Detection of intracellular IL-4
Department of Medicine, Harvard Thorndike Laboratories, Charles A. Dana Research
Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
Massachusetts 02215
Received for publication December 14, 2000. Accepted for publication February
26, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants A20241, AI22571,
AI41995, and HL56386.
2
Address correspondence and reprint requests to Dr. Peter F. Weller, Beth Israel
Deaconess Medical Center, DA-617, 330 Brookline Avenue, Boston, MA 02215.
E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
●
Intracellular IL-4 was analyzed by 1) immunofluorescence microscopy of
cytospin preparations of paraformaldehyde-fixed/saponin-permeabilized eosinophils stained with Alexa546-labeled (Molecular Probes, Eugene, OR)
anti-IL-4 mAb (clone 3010.211) or isotype control mouse IgG1 (both obtained from R&D Systems, Minneapolis, MN) and viewed with a TE300
Nikon fluorescence microscope; 2) flow cytometry (FACScan, CellQuest
software; BD Biosciences, Mountain View, CA) of paraformaldehydefixed/saponin-permeabilized eosinophils stained with PE-conjugated antiIL-4 mAb (clone MP4-25D2) or isotype control rat IgG1 (both obtained
from BD PharMingen, San Diego, CA); and 3) ELISA (R&D kit) of postnuclear supernatants (14,000 ⫻ g for 20 min) of eosinophil lysates (in 1
mM DTT, 1 mM EDTA, 0.1% SDS and 1% Nonidet P-40, pH 7.5, 150 mM
NaCl, and 20 mM HEPES with protease inhibitors).
0022-1767/01/$02.00
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IL-4 release is important in promoting Th2-mediated allergic
and parasitic immune responses. Although human eosinophils
are potential sources of IL-4, physiologic mechanisms to elicit
its release have not been established. By flow cytometry and
microscopy, eosinophils from normal donors uniformly contained preformed IL-4. In contrast to cytolytic IL-4 release
from calcium ionophore-activated eosinophils, eotaxin and
RANTES, but not IFN-␥, elicited IL-4 release by noncytotoxic
mechanisms. With a dual Ab capture and detection immunofluorescent microscopic assay, IL-4 was released at discrete cell
surface sites. IL-5 enhanced eotaxin-induced IL-4 release,
which was mediated by G protein-coupled CCR3 receptors,
detectable as early as 5 min and maximum within 1 h. IL-4
release was not diminished by transcription or protein synthesis inhibitors, but was suppressed by brefeldin A, an inhibitor
of vesicle formation. Thus, CCR3-mediated signaling can rapidly mobilize IL-4 stored preformed in human eosinophils for
release by vesicular transport to contribute to immune
responses. The Journal of Immunology, 2001, 166: 4813– 4817.
4814
CUTTING EDGE
ELISA for detection of eosinophil-released IL-4
Eosinophils (2 ⫻ 10 cells in 1 ml) were incubated for 1 h (37°C) with
chemokines (R&D Systems) or A23187 (Sigma, St. Louis, MO) in RPMI
1640 medium containing 0.1% OVA. Eosinophil supernatant IL-4 was
measured by ELISA (sensitivity 10 pg/ml; R&D Systems).
6
EliCell assay for the detection of eosinophil-released IL-4
Results and Discussion
IL-4, a major mediator in the induction and regulation of allergic
and parasitic immune responses, is not produced exclusively by
activated Th2 lymphocytes. Earlier studies established that human
eosinophils synthesize and store IL-4 within their specific granules
and suggested, based on immunocytochemistry, that there might
be subpopulations of IL-4-positive and -negative eosinophils (13,
14). Our analyses of populations of eosinophils from normal donors extend these findings by demonstrating that all circulating
eosinophils contained preformed IL-4, as evidenced by a uniformly positive, unimodal pattern of intracellular IL-4 immunoreactivity by flow cytometry (Fig. 1A, top) and by high level, granule-associated eosinophil anti-IL-4 immunostaining (94.3 ⫾ 2.5%
positive cells; mean ⫾ SD, n ⫽ 4) by fluorescence microscopy
(images not shown). By ELISA of cell lysates, eosinophils contained more IL-4 (32.3 ⫾ 6.2 pg/2 ⫻ 106 cells) than did PBMCs
(2.3 ⫾ 0.9 pg/2 ⫻ 106 cells, means ⫾ SEM, n ⫽ 7) from the same
donors (Fig. 1A, bottom). Thus, in addition to the presence of IL-4
in tissue eosinophils in bronchial biopsies of asthmatic patients and
FIGURE 1. IL-5 enhances eotaxin-induced release of preformed IL-4 from eosinophils. A, Eosinophils from nonatopic donors contain preformed IL-4.
Top, Flow cytometry histogram of intracellular IL-4 in saponin-permeabilized eosinophils, with Alexa-546-labeled anti-IL-4 mAb (dashed line) and
nonimmune IgG1 (solid line). No anti-IL-4 staining was detected with nonpermeabilized eosinophils (data not shown). Result is representative of findings
from four donors. Bottom, ELISA-assayed quantities of preformed IL-4 in lysates of eosinophils and PBMCs from seven normal donors. Bars denote the
means. B, EliCell assays of IL-4 released extracellularly from eosinophils (captured with a biotinylated anti-IL-4 Ab and detected with Alexa546-labeled
anti-IL-4 mAb). Dose-responses of eotaxin-induced IL-4 release at 1 h with and without concomitant 2 nM IL-5 were expressed both as the average
fluorescence intensities for immunoreactive IL-4 around 50 individual eosinophils (top) and the percentages of eosinophils exhibiting extracellularly
released IL-4 (bottom). Results are means ⫾ SD from five donors. ⴱ and ⴱⴱ, p ⬍ 0.05 and p ⬍ 0.01, respectively, compared with unstimulated eosinophils.
C, Phase-contrast (left) and fluorescent (right) microscopic images of identical fields of eosinophils. Anti-IL-4 immunoreactive sites (red) are overlaid on
phase-contrast images to facilitate their localization. Images show representative eosinophils stimulated for 1 h with 2 nM IL-5, 6 nM eotaxin, or both.
Bottom image shows a representative brefeldin A (BFA; 1 ␮g/ml)-treated IL-5/eotaxin-stimulated eosinophil. Numerical values (right) are the fluorescent
intensities (in arbitrary units) of immunoreactive IL-4 released by each of the shown eosinophils.
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The EliCell assay, a gel-phase dual Ab capture and detection assay based
on microscopic observations of individual viable cells, was performed as
detailed (18) to enumerate the proportion of eosinophils releasing IL-4 and
to electronically quantitate the average relative amounts of IL-4 released
extracellularly. A biotinylated goat polyclonal anti-IL-4 Ab (20 ␮g/ml;
R&D Systems) was used as capturing Ab and an Alexa546-labeled antiIL-4 mAb (R&D Systems) was used (400 ␮l of 10 ␮g/ml) to detect released IL-4. Alexa546-labeled mouse IgG1 was included as a nonimmune
isotype control. An irrelevant biotinylated capture Ab was substituted in
combination with the Alexa546-labeled anti-IL-4 detection Ab to ascertain
that 1) cell permeabilization had not allowed detection of intracellular IL-4;
and 2) retention of surface-released IL-4 was dependent on the immobilized anti-IL-4 capturing Ab.
In some experiments, eosinophils were pretreated (37°C) for 30 min
with specific inhibitors: 1) a neutralizing anti-CCR3 mAb (clone
61828.111) or isotype-matched control rat IgG (both 10 ␮g/ml; R&D Systems); 2) pertussis toxin (10 or 100 ng/ml; Calbiochem, La Jolla, CA); 3)
actinomycin D and cycloheximide (both at 1 and 10 ␮M; Calbiochem); or
4) brefeldin A (0.1 and 1 ␮g/ml; Biomol, Plymouth Meeting, PA). Statis-
tical comparisons were made by ANOVA followed by Student NewmanKeuls t test with differences considered significant when p ⬍ 0.05.
The Journal of Immunology
4815
(data not shown). The latter condition assured that neither intracellular nor membrane-bound IL-4 was being detected in the nonpermeabilized eosinophils, and the punctate pattern of immunoreactive IL-4 released at discrete loci proximate to the cell surface
(Fig. 1C) (as confirmed by confocal microscopy; data not shown)
was compatible with a vesicular transport-mediated process of
IL-4 release.
IL-8, which does not stimulate normal eosinophils (27), did not
elicit IL-4 release (Table I). Of greater interest, IFN-␥, at a concentration that effectively elicited vesicular transport-mediated release of RANTES from eosinophils (18, 25), failed to elicit IL-4
release (Table I), suggesting that differential signaling may function to selectively mobilize at least the cytokine proteins stored
preformed in eosinophil granules.
IL-5 by itself did not elicit detectable IL-4 release from eosinophils (Fig. 1B), but IL-5 did enhance eotaxin-stimulated mobilization of IL-4. The simultaneous addition of IL-5 with even low
concentrations of eotaxin (0.06 and 0.6 nM) evoked release of IL-4
from significant proportions of eosinophils, whereas these eotaxin
concentrations alone did not stimulate detectable IL-4 release (Fig.
1B). IL-5 in concert with higher concentrations of eotaxin (6 and
60 nM) doubled the number of eosinophils releasing IL-4 and the
amounts of IL-4 released (Fig. 1B). IL-5 also accelerated eotaxininduced IL-4 release from eosinophils. Even as early as 5 min,
IL-5 in concert with eotaxin enhanced both the percentages of
eosinophils releasing IL-4 and the quantities of IL-4 being released
(Fig. 2A).
We next evaluated mechanisms underlying the IL-5/eotaxinelicited release of IL-4 from eosinophils. The release of IL-4 from
IL-5/eotaxin-stimulated eosinophils was mediated through the G
protein-linked CCR3 chemokine receptor. Pretreatment of eosinophils with a neutralizing anti-CCR3 receptor mAb reduced the
percentages of eosinophils releasing IL-4 (71 ⫾ 5% inhibition vs
0% with a control Ab) and the amounts of IL-4 released (98 ⫾ 2%
inhibition vs 11% with a control Ab) (both n ⫽ 3; p ⬍ 0.05).
Likewise, pertussis toxin pretreatment at 10 and 100 ng/ml inhibited eotaxin-elicited IL-4 release and reduced the percentages of
eosinophils releasing IL-4 by 56 ⫾ 10 and 89 ⫾ 3%, respectively
(both p ⬍ 0.05, n ⫽ 3). Neither actinomycin D, an inhibitor of
transcription, nor cycloheximide, a protein synthesis inhibitor, suppressed eosinophil IL-4 release induced by IL-5/eotaxin (Fig. 2B).
Therefore, the mechanisms underlying IL-5/eotaxin-elicited IL-4
release are not likely to require either the new synthesis of IL-4 or
of other proteins that contribute to the secretory process. In contrast, brefeldin A, an inhibitor of vesicle formation, substantially
suppressed the quantities of IL-4 released extracellularly around
eosinophils without diminishing the overall number of cells releasing IL-4 (Figs. 1C and 2B).
Table I. ELISA and EliCell assays of IL-4 released from human eosinophilsa
ELISA
EliCell
Stimulus
Dose
n
IL-4 (pg/ml)
% Eosinophils
releasing IL-4
IL-4 fluorescence
intensity ⫻ 106/cell
Medium
IFN-␥
Eotaxin
RANTES
IL-8
A23187
500 U/ml
6.0 nM
6.4 nM
6.2 nM
0.5 ␮M
4
4
4
3
3
4
1.6 ⫾ 0.3
ND
0.9 ⫾ 0.4
1.7 ⫾ 0.1
1.1 ⫾ 0.7
45.4 ⫾ 12.5**
1⫾1
1⫾1
44 ⫾ 11*
59 ⫾ 22*
0⫾0
89 ⫾ 5**
0.0 ⫾ 0.0
0.0 ⫾ 0.0
1.0 ⫾ 0.2*
0.5 ⫾ 0.3*
0.0 ⫾ 0.0
5.2 ⫾ 0.7**
a
Eosinophils were incubated with stimuli for 1 h. ELISA assessed IL-4 in 1-ml supernatants from 2 ⫻ 106 eosinophils. EliCell assessed both the percentages of eosinophils
exhibiting extracellular IL-4 and the average electronically measured immunofluorescent intensities of extracellular IL-4. Results are means ⫾ SD. *, p ⬍ 0.01 and **, p ⬍ 0.001
compared with medium. n, Number of different donors.
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in allergen-induced cutaneous late-phase reactions in atopic subjects (15, 19), human blood-derived eosinophils are a major potential source of IL-4.
Because eosinophils contain preformed IL-4, the mechanisms to
mobilize IL-4 from specific granule storage sites for its extracellular release need to be defined. Prior studies established the releasability of IL-4 from eosinophils activated with either serumcoated beads, IgA immune complexes, or calcium ionophore
A23187 (13, 14, 20), but these nonphysiological stimuli are not
selective and may be uniformly exocytotic and/or cytolytic (21,
22). Indeed with our methods, A23187 activation of eosinophils
led to the cytolytic release of IL-4. Not only were the amounts of
immunoreactive IL-4 released in eosinophil supernatants following A23187 stimulation (Table I) equivalent to the total preformed
IL-4 content of eosinophils (Fig. 1A, bottom), but microscopy of
eosinophils in EliCell assays demonstrated extensive IL-4 immunostaining of eosinophils (Table I) that exhibited morphological
signs of cell damage (data not shown, but as previously illustrated
for RANTES staining in A23187 stimulated eosinophils; Ref. 18).
An alternative mechanism to either exocytosis or cytolysis for
the release of eosinophil granule-derived proteins has been indicated by ultrastructural observations of tissue and blood eosinophils activated in vivo. At least for the major cationic protein components of eosinophil-specific granules, selective losses of the core
or matrix components of the granules and other findings suggest
that eosinophil-specific granule contents may be mobilized by selective incorporation into small vesicles that traffic to the cell surface and release these granule contents by a process of “piecemeal” degranulation based on vesicular transport (23, 24).
Recently, Lacy and coworkers identified IFN-␥ as a physiological
stimulus that in vitro induces piecemeal release of RANTES, a
chemokine also stored preformed in eosinophil-specific granules
(25); we have established a microscopic assay, the EliCell assay, to
study the piecemeal degranulation process in eosinophils (18).
We evaluated the capacity of several eosinophil agonists to elicit
IL-4 release from eosinophils. The C-C chemokines, eotaxin and
RANTES, did not elicit IL-4 release at levels detectable by ELISA
of supernatant fluids (Table I), consistent with a recent report that
these cytokines elicited ELISA-detectable “degranulation” only if
eosinophils were pretreated with cytochalasin B (26). In contrast
with the EliCell assay, both eotaxin and RANTES stimulated release of IL-4 detectable extracellularly (Table I). No IL-4 staining
was found with unstimulated eosinophils or when the Alexa546labeled anti-IL-4 detection Ab was replaced by an Alexa546-labeled isotype IgG1 nonimmune control. Moreover, no IL-4 was
detectable when the biotinylated anti-IL-4 capture Ab (which was
necessary to immobilize IL-4 at its extracellular sites of release;
Ref. 18) was substituted with a biotinylated irrelevant control Ab
4816
CUTTING EDGE
References
Our findings indicate that chemokines acting via CCR3-initiated
signaling pathways can very rapidly mobilize preformed stores of
IL-4 from within human eosinophils. The means of extracellular
release was by noncytotoxic, vesicular transport as indicated by the
microscopic patterns of focal cell surface IL-4 release, the absence
of required new protein synthesis, and the inhibition by a vesicle
formation inhibitor, brefeldin A. In support of this mechanism, we
have localized the vesicle-associated membrane protein-2 by immunogold electron microscopy not only at vesicles within eosinophils but also at the outer membrane of eosinophil-specific granules (28), indicating the capacity of secretory vesicles to traffic
from eosinophil granule membranes. Although released IL-4 concentrations in supernatant fluids were not sufficient to be detectable
by conventional ELISA, local concentrations of released IL-4 may
effectively stimulate responses in tissue sites of eosinophil localization. IL-4 released by eosinophils may augment effector actions
of eosinophils, including enhancing the IL-4-dependent generation
of airway mucous secretion (29) or even the elicitation of further
eotaxin generation (30). Moreover, because airway eosinophils can
traffic back to regional lymph nodes and effectively present airway-derived Ags to elicit proliferation of CD4⫹ T cells (31), local
IL-4 release by eosinophils within lymph nodes, the thymus (32),
or other sites may also modulate the local responses of lymphocytes. Thus, in contrast to CD4⫹ T cells in which IL-4 synthesis
needs to be transcriptionally induced, human eosinophils have the
capacity by means of vesicular transport to physiologically and
rapidly release their preformed stores of IL-4.
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FIGURE 2. IL-5 accelerates the eotaxin-induced release of IL-4 from
eosinophils, which is mediated by vesicular transport. With EliCell assays,
IL-4 released extracellularly from eosinophils was captured with anti-IL-4
Ab bound to a gel matrix and detected with Alexa546-labeled anti-IL-4
mAb. IL-4 release was measured both as the average fluorescence intensities for immunoreactive IL-4 around 50 individual eosinophils (top) and
the percentages of eosinophils exhibiting extracellularly released IL-4 (bottom). A, A representative (n ⫽ 4) time course of IL-4 release from eosinophils stimulated with medium (E), 6 nM eotaxin (䡺), 2 nM IL-5 (F), or
both eotaxin and IL-5 (f). B, Eosinophils were pretreated for 30 min with
actinomycin D (ActD), cycloheximide (Cycl), or brefeldin A (BFA) and
then stimulated with 6 nM eotaxin and 2 nM IL-5 for 1 h. Results are
means ⫾ SD from three donors. ⴱ and ⴱⴱ, p ⬍ 0.05 and p ⬍ 0.01, respectively, compared with no inhibitors.
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