Article

An essential role for Rab27a GTPase in

eosinophil exocytosis
John Dongil Kim,* Lian Willetts,* Sergei Ochkur,† Nutan Srivastava,* Rudolf Hamburg,*
Anooshirvan Shayeganpour,* Miguel C. Seabra,‡ James J. Lee,†,1 Redwan Moqbel,§,1
and Paige Lacy*,1,2
*Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; †Division of
Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Arizona, USA; ‡Clinical
Neuroscience, Division of Neuroscience and Mental Health, National Heart and Lung Institute, Imperial College London,
London, UK; and §Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada
RECEIVED AUGUST 30, 2012; REVISED JULY 31, 2013; ACCEPTED AUGUST 1, 2013. DOI: 10.1189/jlb.0812431

ABSTRACT eosinophils, both in vitro and in vivo. In mouse models,

Eosinophil degranulation has been implicated in inflam-
matory processes associated with allergic asthma.
Rab27a, a Rab-related GTPase, is a regulatory intracel-
lular signaling molecule expressed in human eosino-
phils. We postulated that Rab27a regulates eosinophil
degranulation. We investigated the role of Rab27a in
eosinophil degranulation within the context of airway
inflammation. Rab27a expression and localization in eo-
sinophils were investigated by using subcellular frac-
tionation combined with Western blot analysis, and the
results were confirmed by immunofluorescence analy-
sis of Rab27a and the granule membrane marker
CD63. To determine the function of eosinophil Rab27a,
we used Ashen mice, a strain of Rab27a-deficient ani-
mals. Ashen eosinophils were tested for degranulation
in response to PAF and calcium ionophore by measur-
ing released EPX activity. Airway EPX release was also
determined by intratracheal injection of eosinophils into
mice lacking EPX. Rab27a immunoreactivity colocalized
with eosinophil crystalloid granules, as determined by
subcellular fractionation and immunofluorescence anal-
ysis. PAF induced eosinophil degranulation in correla-
tion with redistribution of Rab27a⫹ structures, some of
which colocalized with CD63⫹ crystalloid granules at
the cell membrane. Eosinophils from mice had signifi-
cantly reduced EPX release compared with normal WT
Abbreviations: BAL⫽bronchoalveolar lavage; bmEos⫽bone marrow– de-
rived eosinophils; CIHR⫽Canadian Institutes of Health Research;
DAPI⫽4=6=diamidino-2-phenylindole; EDN⫽eosinophil-derived neurotoxin;
eos. equiv./microliter⫽eosinophil equivalents per microliter; EPX⫽eosinophil
peroxidase; GTPase⫽guanosine triphosphatase; FLT3L⫽Fms-related ty-
rosine kinase 3 ligand; hE⫽human eotaxin; LDH⫽lactate dehydrogenase;
NIH⫽National Institutes of Health; NSF⫽N-ethylmaleimide-sensitive factor;
NWT⫽Northwest Territories; OPD⫽o-phenylenediamine; OVA⫽ovalbumin;
PAF⫽platelet-activating factor; Q-SNARE⫽glutamine-soluble SNARE;
RabGEF⫽Rab guanine nucleotide exchange factor; RPMI⫽Rosewell Park
Medical Institute; R-SNARE⫽arginine -soluble SNARE; SCF⫽stem cell fac-
tor; Slp/Slac2⫽synaptotagmin-like protein (Slp) homologue lacking C2 do-
mains; SNAP⫽soluble NSF attachment protein; SNARE⫽soluble NSF at-
tachment protein receptor; VAMP⫽ vesicle-associated membrane protein;
WT⫽wild-type
Ashen mice demonstrated reduced EPX release in BAL
fluid. These findings suggest that Rab27a has a key role
in eosinophil degranulation. Furthermore, these findings
have implications for Rab27a-dependent eosinophil de-
granulation in airway inflammation. J. Leukoc. Biol. 94:
1265–1274; 2013.
Introduction
Eosinophils are inflammatory granulocytes implicated in
asthma and parasitic helminth infections [1– 4]. They are
highly granulated white blood cells enriched in cationic gran-
ule proteins that are thought to contribute to tissue damage in
inflammatory airway diseases, including atopic asthma [5].
Granule proteins from eosinophils are associated with pheno-
types of asthma, which include, but are not limited to, hyper-
secretion of mucus, increased vascular permeability, bronchial
epithelial damage, and smooth muscle contraction [4]. In hu-
mans, however, a proinflammatory role for eosinophils in al-
lergic asthma remains controversial. Early studies using me-
polizumab, a humanized anti-IL-5 monoclonal antibody, pro-
posed a lack of efficacy in the asthma patients studied, thus
concluding that eosinophils may not play a direct effector role
in asthma [6 – 8]. However, 2 studies in 2009, using the same
antibody therapy, indicated that eosinophil ablation signifi-
cantly reduces the number of asthma exacerbations in patients
with sputum eosinophilia [9, 10]. Another 2 studies in eosino-
phil-deficient mice have supported a proinflammatory role for
eosinophils in asthma [11, 12]. On the basis of these studies
and others, a role for eosinophils in airway hyperresponsive-
ness and remodeling in atopic asthma continues to be the fo-
cus of intensive research. Furthermore, the precise contribu-
tion of Rab27a to eosinophil effector functions, principally de-
granulation, has not been definitively established.
1. These authors contributed equally to this work.
2. Correspondence: Pulmonary Research Group, 559 HMRC, Department of
Medicine, University of Alberta, Edmonton, AB, T6G 2S2, Canada. E-mail:
paige.lacy@ualberta.ca

0741-5400/13/0094-1265 © Society for Leukocyte Biology Volume 94, December 2013 Journal of Leukocyte Biology 1265
 In the midst of this ongoing debate, Coughlin et al. [13]
shed new light on a potential effector role for eosinophil
Rab27a in asthma. Rab27a, a Rab-related GTPase, is involved
in vesicular transport, budding, and movement, upstream of
docking and fusion [14]. This secretory GTPase is usually
membrane bound and becomes activated after displacement of
bound GDP and association with GTP by the action of Rab-
GEFs [15]. A rare, autosomal recessive disorder known as Gris-
celli syndrome type 2 is caused by a deficiency in Rab27a,
characterized by partial albinism and immunodeficiency result-
ing in early childhood death, unless a bone marrow transplant
is performed [16, 17]. This disorder is specifically associated
with deficient exocytosis from immune and other secretory
cells, including melanocytes. In our recent study, the expres-
sion and magnitude of activity of eosinophil Rab27a was com-
pared between patients with asthma and control subjects. We
reported significantly higher expression and activity of Rab27a
in eosinophils from those with asthma [13]. From our data, we
concluded that Rab27a and possibly eosinophil exocytosis are
important contributors to an asthma phenotype.
The Ashen mouse strain was used to examine the in vivo rel-
evance of this observation. The Ashen mouse is a Rab27a-defi-
cient strain that produces a truncated Rab27a protein lacking
critical GTP binding domains [18, 19]. These mice have di-
luted coat colors, related to reduced melanocyte secretion,
and exhibit a profound immunodeficiency due to a failure to
release cytolytic granules from CTLs and NK cells [20, 21].
Thus, many secretory cells from Ashen mice are characterized
by abnormal exocytosis. We hypothesized that Rab27a defi-
ciency reduces eosinophil exocytotic capacity. Eosinophils were
isolated from Ashen mice and compared for their degranula-
tion responses with IL-5 transgenic eosinophils. Ashen mice
were also crossed with IL-5/hE2 double-transgenic mice to de-
termine the role of Rab27a in in vivo eosinophil exocytosis.
IL-5/hE2 double-transgenic mice are characterized by high
expression of T-cell-derived IL-5, driven by the CD3 promoter,
along with hE2 from Clara cells residing in the lungs, by fu-
sion of the hE2 gene with the CC10 promoter [22]. These cy-
tokines contribute significantly to eosinophil differentiation
(IL-5) [23] and chemotaxis (hE2, including egress from bone
marrow) [24]. IL-5/hE2 double-transgenic mice exhibit eosin-
ophilia and spontaneously develop severe asthma-like symp-
toms with significant airway hyperresponsiveness. Thus, IL-5/
hE2 double-transgenic mice serve as an abundant source of
spontaneously degranulating eosinophils. In this study, IL-5/
hE2 mice were crossed with the Ashen strain [18, 19], which
then provided a Rab27a-deficient, eosinophil-producing
model.
MATERIALS AND METHODS
Animals
All animals in this study were on a C57BL/6 background, and C57BL/6
WT mice were purchased from Jackson Laboratory (Bar Harbor, ME,
USA). NJ.1638 IL-5 [23], hE2 [22], and IL-5/hE2 [22] transgenic mice
were bred in house. These strains were crossed with Ashen [19] to generate
IL-5/Ashen, hE2/Ashen, and IL-5/hE2/Ashen mice. The Ashen mice pos-
sessed similar numbers of total and differential peripheral blood eosino-
phils and leukocytes to that of WT C57BL/6 animals (Fig. 1). The IL-5/
hE2 mice were hemizygous for both transgenes and served as controls for
the IL-5/hE2/Ashen mice. Eosinophil peroxidase knockout (EPX⫺/⫺) mice
were generated that lacked EPX protein and activity [25]. (The acronym
EPX is used here to avoid confusion with EPO, denoting erythropoietin.)
IL-5/hE2 mice were also crossed with EPX⫺/⫺ mice to generate IL-5/hE2/
EPX⫺/⫺ mice. The mice were maintained in ventilated microisolator cages
housed in specific-pathogen–free environments at the University of Alberta
(Edmonton, AB, Canada) and Mayo Clinic Scottsdale (Scottsdale, AZ,
USA). All experiments were performed were in accordance with the guide-
lines of the University of Alberta’s Animal Care and Use Committee, the
Canadian Council of Animal Care, the National Institutes of Health, and
the Mayo Foundation.
Preparation of eosinophils
HL-60 clone 15 (HL-60c15) cells (ATCC, Manassas VA, USA), chosen for
this study based on their propensity to differentiate into highly granulated
eosinophil-like cells, were treated with 500 ␮M n-butyrate [26]. Human eo-
sinophils were purified from peripheral blood of atopic donors [27–29].
The purity and viability of human eosinophils was typically ⬎98%. Splenic
eosinophils were isolated from IL-5 transgenic mice by using a published
method [30, 31]. For peripheral blood mouse eosinophils, the cells were

A B C
ood Cells

100
10 3
WT
80 Ashen
8
o Total White Blo

2
6 60

4 40
1
2 20
% of

0 0 0
T

en
T

en

es

es

ils
ls
W

hi
sh

sh

yt

yt

ph
op
oc

oc
A

no
tr
ph

on

si
eu
m

Eo
N
Ly

Figure 1. Total and differential peripheral blood leukocyte counts were similar between WT C57BL/6 and Ashen mice. (A) Total cell counts from
the peripheral blood of WT C57BL/6 and Ashen mice were similar. (B) Percentages of peripheral blood eosinophils were identical in WT and
Ashen mice. (C) Differential counts of leukocytes in WT and Ashen mice showed similar proportions (n⫽3).

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recovered from the peripheral blood of IL-5 transgenic mice at ⬎98% pu-
rity [23]. Cultured, in vitro bmEos were also generated from WT C57BL/6
mice and differentiated in the presence of recombinant mouse SCF,
FLT3L, and IL-5 [32].
Subcellular fractionation and marker enzyme assays
Differentiated HL-60c15 cells and peripheral blood mouse eosinophils were
homogenized in a cell cracker (a ball-bearing– containing cell homoge-
nizer), and the resulting organelles were separated by a linear Nycodenz or
Histodenz (Sigma-Aldrich St. Louis, MO, USA) gradient (0 – 45%) [33].
Marker enzyme assays were used on fractionated samples. EPX activity in
the resulting fractions was measured by the reactivity with tetramethylbenzi-
dine substrate, and cytosol fractions were detected with LDH or protein
assay [33] (Bio-Rad, Hercules, CA, USA).
Immunofluorescence
Image analysis of the immunofluorescence of adherent eosinophils was per-
formed after plating of peripheral blood mouse eosinophils from IL-5
transgenic NJ.1638 mice or human eosinophils from atopic donors in
8-well chamber slides (Lab-Tek Chambered Coverglass System; Nunc, Roch-
ester, NY, USA) [34]. The cells were resuspended in serum-free RPMI me-
dium, added to the glass surfaces of the chamber slides, and incubated for
15 min at 37°C, to allow adhesion. PAF (5 ␮M) was added to each well for
5 or 15 min. Stimulation was terminated by removal of the medium and
fixation of the cells with 4% paraformaldehyde for 20 min. The cells were
permeabilized with 0.1% Triton X-100 in PBS for 3 min before staining.
The mouse eosinophils were then blocked for 20 min with 2% BSA⫹2%
goat serum in PBS, to reduce nonspecific binding by antibodies, whereas
the human eosinophils were blocked with 2% human IgG in PBS [35].
Mouse monoclonal anti-Rab27a (5 ␮g/ml; 20/RAB27 clone; BD Biosci-
ences, Inc., San Jose, CA, USA) was then added and detected with anti-
mouse IgG-Cy3, followed by fluorescein isothiocyanate (FITC)– mouse
monoclonal anti-CD63 (Serotec, Raleigh, NC, USA). After the cells were
washed with PBS, ProLong Gold mounting medium containing DAPI (In-
vitrogen-Molecular Probes, Eugene, OR, USA) was added to each well. Im-
munofluorescence labeling was imaged with a Deltavision OMX microscope
equipped with a ⫻60 objective (1.43 NA; Applied Precision, Issaquah, WA,
USA).
Gel electrophoresis and Western blot analysis
Proteins were separated by SDS-PAGE gels and transferred onto nitrocellu-
lose membranes. The membranes were blocked with Odyssey blocking buf-
fer (Li-Cor Bioscience, Lincoln, NE, USA) diluted 1:2 in PBS. Rab27a pro-
tein was probed with an anti-Rab27a rabbit antibody (Santa Cruz Biotech-
nology Inc., Dallas, TX, USA) [36]. Immunofluorescent protein bands were
quantified by using IRDye-conjugated secondary antibodies against primary
antibodies with a Li-Cor Odyssey infrared imaging system (Li-Cor Biosci-
ence).
In vitro stimulation of mouse eosinophils
Eosinophils (2⫻105) were stimulated with 0.1 ␮M PAF, 5 ␮M ionomycin,
or both. The cells were initially centrifuged at 400 g for 10 min, and then
supernatants from this step were centrifuged again at 10,000 g for 10 min
at 4°C to clarify the supernatants for the assay. The resulting supernatants
were placed in 96-well plates for detection of EPX by ELISA.
Preparation of BAL fluid
BAL fluid was recovered by introducing and collecting 1 ml of 2% FCS in
PBS. BAL fluid was initially centrifuged at 400 g for 10 min at 4°C, after
which the supernatant was recovered for a further centrifugation at 10,000
g for 10 min at 4°C to remove any remaining debris. Pellets from BAL sam-
ples were analyzed for total and differential cell counts.
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Kim et al. Rab27a in eosinophil exocytosis
Intratracheal instillation of mouse eosinophils
Peripheral blood eosinophils isolated from IL-5 transgenic mice were re-
constituted at 10 ⫻ 106 cells in 30 ␮l of PBS and adoptively transferred
into the lungs of lightly anesthetized mice (3% isoflurane). BAL samples
from these mice were collected 24 h after instillation of eosinophils.
ELISA of EPX
Quantification of EPX was achieved by an EPX ELISA method developed
by Mayo Clinic Arizona [37]. EPX amounts were quantified by comparing
them to the standard curve generated from measuring EPX in pure mouse
eosinophil lysates. The results are shown as eos. equiv./microliter, to indi-
cate the absorbance measured per eosinophil count.
Statistical analysis
Data were analyzed and graphed with Prism (version 5; GraphPad, San Di-
ego, CA, USA). The results are represented as the mean ⫾ se and analyzed
by 1-way ANOVA with Tukey’s post hoc analysis. For all experiments, P ⬍
0.05 was considered to be statistically significant.
RESULTS
Rab27a was expressed in eosinophils and colocalized
with EPX-containing granules
To determine whether mouse eosinophils express Rab27a,
bmEos and peripheral blood mouse eosinophils were tested
for expression of Rab27a mRNA and protein. We determined
that Rab27a mRNA and protein were expressed in bmEos
from WT C57BL/6 mice as well as bmEos and peripheral
blood eosinophils from IL-5 transgenic NJ.1638 mice (Fig. 2).
The levels of expression of Rab27a mRNA and protein, as well
as the molecular weight of Rab27a protein, in mouse eosino-
phils was similar to that in human eosinophils and butyrate-
treated HL-60c15 cells differentiated into an eosinophilic phe-
notype.
To determine the subcellular localization of Rab27a, the
eosinophils were subjected to lysis through a cell cracker, and
the resulting intact organelles were separated across a linear
density gradient consisting of Histodenz (0 – 45%). Peripheral
blood eosinophils from IL-5 Tg mice expressed high EPX ac-
tivity in crystalloid granule-containing fractions (high-density
fractions 6 –10) that substantially overlapped with fractions
containing Rab27a (fractions 6 –13) as determined by Western
blot analysis. Most of the Rab27a immunoreactivity appeared
in fractions enriched in membrane-bound organelles, which
typically entered the gradient and was separate from cytosolic
LDH-containing fractions above the gradient. The colocaliza-
tion of Rab27a with membrane-bound organelles in fraction-
ated eosinophils confirmed its membrane-associated proper-
ties, on the one hand [15]. Plasma membrane gp91phox expres-
sion, on the other hand, corresponded to lower density,
plasma membrane-containing fractions (fractions 11–15).
These findings suggest that Rab27a colocalizes with EPX-con-
taining crystalloid granules, as well as other lower density
membrane-bound organelles, in unstimulated eosinophils.
Next, we determined the effects of PAF stimulation on the
intracellular localization of Rab27a. PAF is a potent eosinophil
secretagogue that induces EPX release [38]. Upon stimulation
by PAF, both EPX activity and Rab27a expression shifted to a
Volume 94, December 2013 Journal of Leukocyte Biology 1267
 A B
R b27
Rab27a
Figure 2. Rab27a was expressed in mouse eosin- β-Actin
ophils and colocalized with crystalloid granule
fractions. (A) RT-PCR analysis of differentiated C Peripheral
p blood mouse IL-5 Tgg eosinophils
HL-60c15 cells and bmEos cultured from
C57BL/6 mice. (B) Western blot analysis of
Rab27a expression in eosinophils from mouse
and human sources. BmEos were isolated from
C57BL/6 mice (left) and bmEos, and peripheral
blood (PB) eosinophils were prepared from IL-5 30
Tg mice (right), to compare Rab27a expression
with that in human eosinophils and differenti-
ated HL-60c15 cells. A total of 20 ␮g protein
was loaded in each lane. (C) Subcellular frac-
20
tionation of peripheral blood eosinophils from
IL-5 Tg mice. An enzymatic assay was used to
detect EPX in fractions containing crystalloid
granules (fractions 6 –10), and LDH was assayed
to identify cytosolic fractions (fractions 16 –23). 10
Western blot analysis showed Rab27a (fractions
6 –13) and gp91phox (a marker for plasma mem-
brane, fractions 11–15).
0
0 5
Rab27a
gp91phox
slightly higher density fraction (fraction 6) compared with that
in the resting cells (Fig. 2C). The amount of total granule-as-
sociated EPX activity was also reduced in stimulated cells, sug-
gesting degranulation of EPX. However, the distribution of
Rab27a was not markedly altered after PAF stimulation and
exhibited expression in both low- and high-density fractions
(fractions 6 –13) similar to that of unstimulated cells.
We confirmed colocalization of Rab27a with eosinophil crys-
talloid granules by performing immunofluorescence analysis of
mouse and human eosinophils. CD63, used as a granule mem-
brane marker for eosinophil crystalloid granules [39], showed
that Rab27a colocalized with CD63⫹ granules in the cytoplasm
of mouse and human eosinophils (Fig. 3A, B). CD63 is a
membrane-bound tetraspanin molecule [39]. As expected, im-
munofluorescence for CD63 showed characteristic ring-like
structures in human eosinophils, suggesting granule mem-
brane localization of CD63. These intracellular CD63⫹ crystal-
loid granules were densely packed in human eosinophils and
showed minimal plasma membrane localization. In mouse eo-
sinophils, CD63⫹ ring structures were less prevalent, but simi-
lar to human eosinophils, CD63 was found only in intracellu-
lar structures resembling granules. In contrast, Rab27a was
more widely distributed throughout the cells, with punctate
1268 Journal of Leukocyte Biology Volume 94, December 2013
Rab27a 27 kDa
β-Actin 42 kDa
p
Plasma
Granules membrane Cytosol
EPX Resting
EPX Stimulated
LDH
10 15 20
Fraction #
Res
ng
PAF-S
mulated
staining indicating granular and vesicular localization, some of
which colocalized with CD63⫹ crystalloid granules. These find-
ings are in agreement with subcellular fractionation results.
Upon stimulation with PAF, several CD63⫹ granules redis-
tributed toward the periphery and plasma membrane in both
mouse (Fig. 3C) and human (Fig. 3D) eosinophils. Granule
polarization to leading edges in both human and mouse eosin-
ophils was evident within 5 min of stimulation with PAF, be-
fore EPX release. Previous studies showed that PAF at 5 ␮M
induces significant EPX release after 30 min of stimulation
[38]. PAF was applied to cell supernatants and not as a che-
motactic gradient, suggesting that polarization of granules oc-
curred within the cells and that degranulation from PAF-stimu-
lated eosinophils involves polarized exocytosis.
In parallel with that finding, immunofluorescence for
Rab27a showed colocalization with CD63⫹ granules in un-
stimulated cells (Fig. 3). However, consistent with subcellular
fractionation data, Rab27a was not exclusively localized to
CD63⫹ granules in mouse and human eosinophils, as other
Rab27a⫹ structures were evident that did not colocalize with
CD63. Upon stimulation of mouse eosinophils with PAF (5
min), Rab27a appeared to redistribute away from the CD63⫹
granules found within cytoplasmic sites to other locations in
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 Kim et al. Rab27a in eosinophil exocytosis

A Rab27 CD63 Merge

Figure 3. Rab27a colocalized with

crystalloid granules in mouse and
human eosinophils. (A) Mouse eo-
sinophils from the peripheral
blood of IL-5 Tg mice were plated
onto glass coverslips in 8-chamber
slides and allowed to adhere be-
B Rab27 CD63 Merge fore fixation and staining for
Rab27a and CD63, a membrane
marker for crystalloid granules.
Arrowhead indicates colocalization
of Rab27a and CD63 to the mem-
brane of a single crystalloid gran-
ule. (B) Human eosinophils (ar-
rowhead) from peripheral blood
were labeled similarly. (C) Mouse
IL-5 Tg eosinophils were treated
C 0 min 5 min 15 min
with PAF (5 ␮M) for 0, 5, and 15
min before fixation and staining.
Arrowhead and inset show strong
colocalization of Rab27a and CD63
at the cell periphery. (D) Human
peripheral blood eosinophils also
demonstrated cell-stretching, along
with granule polarization at the
leading edge, with colocalization of
D 0 min 5 min 15 min Rab27a and CD63 at the edges of
the cells during stimulation. Im-
ages of stimulated cells are repre-
sentative of 30 – 40% of cells in 10
high-powered fields each. Scale
bar ⫽ 5 ␮m.

the cell, including the cell membrane (Fig. 3C). This selective
redistribution of Rab27a⫹ organelles is suggestive of piecemeal
degranulation, in which small, rapidly mobilized secretory vesi-
cles shuttle granule products from the crystalloid granules to
the cell membrane, as previously shown in human eosinophils
[29]. However, at 15 min of PAF stimulation, Rab27a and
CD63 colocalized to highly focused, punctate regions at or
near the cell membrane in mouse eosinophils. In human eo-
sinophils, the pattern of Rab27a redistribution was distinct.
PAF induced a redistribution of Rab27a that colocalized with
CD63⫹ structures in polarized regions of cells, with relatively
little concentration at the cell membrane (Fig. 3D, arrow-
head).
Ashen eosinophils showed deficient EPX release
compared with WT eosinophils in vitro
Rab27a is a critical regulatory protein in granule exocytosis in
other secretory cells, and so we examined the role of Rab27a
in degranulation from eosinophils. We isolated eosinophils
from Ashen mice, a substrain of C57BL/6 mice that contains a
splicing mutation in Rab27a [18], that were crossed with IL-5-
overexpressing mice (NJ.1638) [23], to obtain a large number
of splenic and peripheral blood eosinophils lacking Rab27a
expression. We then compared degranulation responses of eo-
sinophils from the resulting IL-5/Ashen offspring with those of
the respective WT controls against the same background strain
(C57BL/6) as was crossed with IL-5 transgenic mice.
Peripheral blood eosinophils from IL-5/WT and IL-5/Ashen
mice were stimulated with the secretagogues PAF, ionomycin,
or PAF⫹ionomycin, with EPX release being quantified as eos.
equiv./microliter. This unit of measurement was used to en-
sure that peripheral eosinophils isolated from both types of
mice contained equal amounts of EPX, as measured by an in-
house ELISA [37]. Using this approach, we were able to detect
increased EPX release in supernatants from eosinophils stimu-
lated with PAF, the calcium ionophore ionomycin, and a
combination of PAF and ionomycin (Fig. 4). In contrast,
IL-5/Ashen exhibited significantly reduced EPX release in
response to all stimuli, compared with that in IL-5/WT eo-
sinophils (Fig. 4).
Ashen eosinophils exhibited defective EPX release
in vivo
We next confirmed that Ashen eosinophils are defective in
their degranulation responses when administered in vivo. WT
and Ashen mice were sensitized and challenged with OVA, and

www.jleukbio.org Volume 94, December 2013 Journal of Leukocyte Biology 1269

 Thus, we compared EPX levels from BAL supernatants of
IL-5 mice with intratracheal instillation of IL-5/WT and IL-5/Ashen
IL-5/Ashen peripheral blood eosinophils (Fig. 6). BAL from mice that re-
ceived IL-5/WT eosinophils exhibited robust EPX release. In
contrast, BAL from IL-5/Ashen eosinophil-instilled mice
showed significantly reduced amounts of EPX (P⬍0.001) (Fig.
6A). When differential cell counts were performed on these
samples, no differences were observed in total BAL cellularity
and eosinophil count (Fig. 6B, C). Thus, these results support
and confirm our data from EPX levels in BAL of OVA-treated
mice and suggest that Rab27a-deficient Ashen eosinophils have
defective degranulation responses in vivo.
Vehicle PAF +
PAF ionomycin We tested the apparent degranulation deficiency of Ashen
control ionomycin
eosinophils further in IL-5/hE2/WT and IL-5/hE2/Ashen
Figure 4. Eosinophils from IL-5/Ashen mice were deficient in EPX mice. These were generated by crossing double-transgenic IL-
release in vitro. In vitro degranulation was observed in purified pe- 5/hE2 mice with either WT C57BL/6 or Ashen mice and using
ripheral blood eosinophils from IL-5 Tg and IL-5/Ashen mice. Super- the offspring that were positive for the appropriate genotypes.
natants from stimulated cells were measured by EPX ELISA. Cells were For comparison, a third strain of IL-5/hE2/EPX⫺/⫺ mice was
stimulated with 50 ng/ml PAF and/or 1 ␮M ionomycin for 6 h. The
generated in which EPX expression was absent. BAL superna-
data are the mean ⫾ se (nⱖ10). *P ⬍ 0.05, ***P ⬍ 0.001.
tants from these 3 strains of mice (6 – 8 wk) were collected,
and spontaneously secreted EPX levels were measured (Fig. 7).
EPX release was determined by ELISA of BAL samples ob- Again, IL-5/hE2/Ashen mice showed markedly reduced EPX
tained on day 28 (Fig. 5). WT and Ashen mice treated with sa- levels in BAL than in IL-5/hE2/WT BAL. Taken together, our
line alone did not show any difference in EPX levels in their data demonstrate that eosinophils lacking Rab27a have a defi-
BAL. However, OVA sensitization and challenge of Ashen mice ciency in secretion of EPX-containing crystalloid granules.
led to a significant (P⬍0.001) decrease in EPX content in BAL
samples compared with those from OVA-treated WT mice. These
findings suggest that airway eosinophil degranulation is deficient
after OVA sensitization and challenge in Ashen mice.
However, upon further assessment of total BAL cellularity,
A B
we observed a reduced total number of cells and eosinophils
in the BAL of OVA-treated Ashen compared with that of simi-
larly treated WT mice, even though the percentages of eosino-
phils of total BAL cellularity were similar in both (Fig. 5B–D).
Thus, reduced EPX concentrations in BAL samples from Ashen
mice may be related to a decrease in eosinophils rather than a
degranulation defect.
Saline OVA Saline OVA Saline OVA Saline OVA
The question of whether the reduced BAL EPX quantities
Wild type Ashen Wild type Ashen
observed in OVA-treated Ashen mice was due to lower eosino-
phil counts was addressed in a novel ex vivo eosinophil de-
C D
granulation experiment. Peripheral blood eosinophils from
IL-5/WT and IL-5/Ashen mice were isolated, purified, and
adoptively transferred into the lungs of triple-transgenic IL-5/
hE2/EPX⫺/⫺ mice. These mice were generated to evoke maxi-
mally stimulating eosinophil degranulation conditions in vivo,
with a complete absence of endogenous EPX secreted by the
recipient animal. Typically, a large number of eosinophils
Saline OVA Saline OVA Saline OVA Saline OVA
transmigrate to the airway spaces in IL-5/hE2 double-trans-
Wild type Ashen Wild type Ashen
genic animals and spontaneously degranulate in response to
tissue-overexpressed IL-5 and eotaxin-2 [22]. Thus, intratrache-
ally introduced WT eosinophils would be expected to degranu- Figure 5. OVA-sensitized and -challenged Ashen mice exhibited de-
late in the airways of these animals (due to transgenic overex- creased BAL EPX release. (A) EPX was detected in BAL samples ob-
pression of eosinophil-specific activating cytokine IL-5 and tained on day 28 of OVA sensitization and challenge or saline controls
from WT and Ashen mice. EPX quantity was measured by EPX ELISA.
chemokine eotaxin-2). In this ex vivo degranulation assay sys-
(B) BAL cellularity, (C) number of BAL eosinophils, and (D) BAL
tem, any EPX that is detected in BAL samples would be exclu- percentage of eosinophils in saline- and OVA-challenged WT and
sively derived from donor eosinophils instilled into the trachea Ashen mice. The data are the mean ⴞ se (n>5). *P ⬍ 0.05;
of IL-5/hE2/EPX⫺/⫺ mice. **P ⬍ 0.01; ***P ⬍ 0.001.

1270 Journal of Leukocyte Biology Volume 94, December 2013 www.jleukbio.org


A B
PBS IT IL-5/WT IL-5/Ashen
Eos IT Eos IT
Figure 6. IL-5/Ashen eosinophils were defective in EPX release when injected intratracheally. (A) Purified peripheral blood eosinophils (107)
from IL-5/WT or IL-5/Ashen mice were intratracheally instilled into IL-5/hE2/EPX⫺/⫺ mice, and BAL was collected from euthanized mice 24 h
after instillation. (B) BAL cellularity and (C) BAL eosinophils after intratracheal injection of eosinophils into IL-5/hE2/EPX⫺/⫺ mice. The data
are the mean ⴞ se (n⫽4). ***P ⬍ 0.001.
DISCUSSION
This report describes, for the first time, a direct role for
Rab27a in eosinophil degranulation, identified in the Ashen
Rab27a gene knockout strain. The role of Rab27a in granule
exocytosis and the development of inflammatory processes has
been investigated in CTLs [20, 21, 40], NK cells [16, 41], and
neutrophils [42], whereas Rab27b has been implicated in mast
cells [43]. Our previous report on the expression and function
of Rab27a in human eosinophils demonstrated that eosino-
phils express and activate Rab27a in response to artificial ago-
nists (phorbol esters and calcium ionophore) as well as physio-
logical stimuli (fMLF and serum-coated zymosan) [13]. Hu-
man peripheral blood eosinophils expressed mRNA for Rab8a,
-8b, -10, -11a, -27a, and -37, with weak signals for Rab11b and
-13 and no detectable signal for Rab27b. We also found in-
creased expression of Rab27a protein, with greater activity ob-
served in eosinophils from patients with asthma compared
with normal subjects [13]. However, a direct role had not
IL-5/hE2/EPX-/- IL-5/hE2 IL-5/hE2/Ashen
Figure 7. Spontaneous release of EPX was reduced in BAL fluid from
IL-5/hE2/Ashen mice. IL-5/hE2/Ashen mice (6 – 8 wk) were generated
to determine spontaneous EPX release in BAL fluid in comparison
with that in age-matched IL-5/hE2 mice. EPX was measured by EPX
ELISA. The data are the mean ⴞ se (n⫽3– 4). ***P ⬍ 0.001.
www.jleukbio.org
Kim et al. Rab27a in eosinophil exocytosis
C
IL-5/WT IL-5/Ashen IL-5/WT IL-5/Ashen
Eos IT Eos IT Eos IT Eos IT
been shown for Rab27a in exocytosis of crystalloid granules in
eosinophils.
The function of Rab27a in granule exocytosis is dependent
on a wide range of specific effector molecules [44]. Proteins
containing an N-terminal Rab27a-binding region, known as
Slp/Slac2 proteins or exophilins, have been shown to bind
Rab27a and mediate respiratory burst in neutrophils [42, 45].
Rab27a may also bind directly to SNARE fusion proteins, to
initiate exocytotic membrane fusion between granules and the
plasma membrane. The R-SNAREs VAMP-2, -7, and -8, along
with their respective binding partners, the Q-SNAREs
SNAP-23 and syntaxin-4, are expressed in human eosino-
phils, and VAMP-7 has been shown to regulate exocytotic
release of the granule proteins EPX and EDN [46 – 49]. Fi-
nally, another putative effector molecule for Rab27a may be
the Sec/Munc family member Munc13-4, which has been
demonstrated in other cell types, including CTL cells, NK
cells, mast cells, and platelets, to regulate Rab27a-induced
exocytosis [50 –52].
The expression and function of Rab27a and its effector mol-
ecules have not yet been explored in eosinophils. These obser-
vations led to the current study with a view to further elucidate
the expression and function of Rab27a in eosinophils and to
determine how this protein may influence degranulation. Us-
ing Western blot analysis, subcellular fractionation, and immu-
nofluorescence analysis, we established that Rab27a was ex-
pressed in both mouse and human eosinophils and that it co-
localized with eosinophil crystalloid granules, as well as other
membrane-bound organelles. To pursue this finding, we chose
the Ashen strain, which exhibits a functional deficiency in
Rab27a, and derived mouse bmEos from those animals, to
confirm the function of Rab27a in mouse eosinophils and vali-
date ensuing mouse models of airway inflammation. Previous
studies in neutrophils have shown that Rab27a is mostly local-
ized to specific and gelatinase-enriched tertiary granules and is
not essential for myeloperoxidase release from azurophilic
granules [53]. In contrast, our results provide strong evidence
that Rab27a associates with eosinophil crystalloid granules con-
Volume 94, December 2013 Journal of Leukocyte Biology 1271
taining CD63 and EPX and that Rab27a is necessary for EPX
release.
To determine whether the localization of Rab27a and EPX
change with stimulation, eosinophils were treated with the po-
tent secretagogue PAF [38]. After PAF activation, the total
EPX level across subfractions was significantly reduced, sug-
gesting extracellular release of EPX, and EPX activity was split
between 2 fractions (6 and 8). A possible explanation for the
double peak of EPX activity in stimulated eosinophils is that
stimulated cells release EPX through piecemeal degranulation
[29, 54]. In confirmation of this finding, immunofluorescence
images showed that a proportion of Rab27a redistributed away
from CD63⫹ granules after 5 min of PAF stimulation in mouse
eosinophils. Some Rab27a immunoreactivity was found at the
cell membrane, colocalizing with a proportion of CD63⫹ gran-
ules in mouse eosinophils. However, at 15 min of PAF stimula-
tion, there was a redistribution of Rab27a⫹ structures colocal-
izing with CD63⫹ granules at the cell membrane in highly fo-
cused, punctate regions. This suggests that Rab27a may be
involved in piecemeal degranulation in early cell stimulation,
as well as crystalloid granule fusion with the cell membrane in
mouse eosinophils at later stages of degranulation (within 15
min of PAF stimulation).
In human eosinophils, PAF induced a marked polarization,
whereupon CD63⫹ granules mobilized to the leading edges of
the cells. Rab27a was found to colocalize with some CD63⫹
granules at the leading edge of the cells. Taken together,
these findings suggest that Rab27a coordinates fusion between
crystalloid granules and plasma membrane to regulate granule
exocytosis in eosinophils, although there were subtle differ-
ences in the manner in which Rab27a was redistributed after
PAF stimulation in mouse and human cells.
The role of Rab27a in eosinophil degranulation was further
investigated by stimulating peripheral blood eosinophils iso-
lated from WT and Ashen mice. In this system, a novel EPX
ELISA [37] was used to measure the amount of released
mouse EPX after stimulation. This EPX ELISA was developed
in house and provides a 10-fold higher sensitivity than does
the widely used OPD assay [55]. Some studies have suggested
that mouse eosinophils do not readily degranulate either in
vitro or in vivo [13, 56, 57]. However, we have recently re-
ported that mouse eosinophils degranulate in vitro in response
to at least 2 secretagogues: PAF and calcium ionophore
[34, 38].
Our in vivo findings confirmed that Rab27a plays an impor-
tant role in eosinophil exocytosis in association with EPX-con-
taining crystalloid granules. OVA-treated Ashen BAL samples
exhibited significantly diminished levels of EPX when com-
pared to OVA-treated WT BAL. However, when differential
cell counts were performed, OVA-treated Ashen mice had
lower BAL cellularity, despite having a percentage of eosino-
phils similar to that in OVA-treated WT mice. This result sug-
gests that the reduction of EPX observed in the BAL of OVA-
treated Ashen mice is related to a reduction in airway eosino-
phils. The finding was unexpected, but was not surprising,
given that global gene deletion of Rab27a may indirectly affect
eosinophil recruitment and accumulation in the lungs through
1272 Journal of Leukocyte Biology Volume 94, December 2013
cytokine and chemokine secretion from other cell types that
are dependent on Rab27a.
To further explore the possibility that Rab27a is involved in
eosinophil degranulation, a novel ex vivo degranulation assay
for eosinophils was designed in which eosinophils were intra-
tracheally injected into IL-5/hE2/EPX⫺/⫺ mice. The use of
IL-5/hE2/EPX⫺/⫺ mice provided a unique lung environment
where intratracheally instilled eosinophils are induced to de-
granulate by in vivo secretagogues generated from tissue
sources. These mice lack systemically expressed EPX, and any
detected EPX in BAL samples must therefore originate exclu-
sively from donor, not recipient, eosinophils. We found signifi-
cantly less EPX in BAL from IL-5/hE2/EPX⫺/⫺ mice with IL-
5/Ashen eosinophils instilled when compared with those with
IL-5/WT eosinophils instilled, and differential cell counts per-
formed on BAL from these 2 groups were not significantly dif-
ferent. These data provide supportive evidence for Rab27a in
eosinophil degranulation, using a model in which exogenously
administered eosinophils are introduced into a highly stimula-
tory airway environment. Consistent with these findings, dou-
ble-transgenic IL-5/hE2 mice crossed with Ashen mice to gen-
erate a large number of eosinophils lacking Rab27a also
showed a significant decrease in EPX in BAL samples from
IL-5/hE2/Ashen mice.
In conclusion, our findings provide strong evidence support-
ing a role for Rab27a in eosinophil degranulation, both in
vitro and in vivo, in the mouse model. The findings support
our study on Rab27a function in human eosinophils [13] and
suggest that eosinophils are dependent on Rab27a for degran-
ulation responses in allergic airway inflammation and asthma.
AUTHORSHIP
J.D.K., L.W., N.S., R.H., and A.S. conducted the experiments
and were supervised by J.J.L., R.M., and P.L. P.L. also con-
ducted experiments for the study. S.O. contributed to experi-
ments and training of users and was supervised by J.J.L. M.C.S.
provided the Ashen strain. This study was supervised by J.J.L.,
R.M., and P.L., who obtained the funding.
ACKNOWLEDGMENTS
This study was funded by CIHR (R.M., P.L.), the Lung Associa-
tion of Alberta and NWT (to J.D.K.), and U.S. National Insti-
tutes of Health, Bethesda, MD (J.J.L.). J.D.K. also received a
CIHR Banting & Best Canada Graduate Scholarship–Master’s
Award for this project. The authors thank Elizabeth Jacobsen
and others at the Mayo Clinic Scottsdale for lending excellent
technical support and expertise and Renjith Pillai, Caroline
Ethier, Dr. Francis Davoine, Vivek Gandhi, and Dr. Stephen
Ogg for support contributing to findings in this study. Images
were acquired using resources and expertise provided by the
Faculty of Medicine and Dentistry’s core imaging center at the
University of Alberta.
DISCLOSURES
The authors declare no conflicts of interest.
www.jleukbio.org

REFERENCES
1. Lacy, P., Moqbel, R. (2001) Immune effector functions of eosinophils in
allergic airway inflammation. Curr. Opin. Allergy Clin. Immunol. 1, 79 –84.
2. Adamko, D., Lacy, P., Moqbel, R. (2004) Eosinophil function in allergic
inflammation: from bone marrow to tissue response. Curr. Allergy Asthma
Rep. 4, 149 –158.
3. Rothenberg, M. E., Hogan, S. P. (2006) The eosinophil. Annu. Rev. Im-
munol. 24, 147–174.
4. Hogan, S. P., Rosenberg, H. F., Moqbel, R., Phipps, S., Foster, P. S.,
Lacy, P., Kay, A. B., Rothenberg, M. E. (2008) Eosinophils: biological
properties and role in health and disease. Clin. Exp. Allergy 38, 709 –750.
5. Jacobsen, E. A., Taranova, A. G., Lee, N. A., Lee, J. J. (2007) Eosino-
phils: singularly destructive effector cells or purveyors of immunoregula-
tion? J. Allergy Clin. Immunol.
6. Leckie, M. J., ten Brinke, A., Khan, J., Diamant, Z., O’Connor, B. J.,
Walls, C. M., Mathur, A. K., Cowley, H. C., Chung, K. F., Djukanovic, R.,
Hansel, T. T., Holgate, S. T., Sterk, P. J., Barnes, P. J. (2000) Effects of
an interleukin-5 blocking monoclonal antibody on eosinophils, airway
hyper-responsiveness, and the late asthmatic response. Lancet 356, 2144 –
2148.
7. Flood-Page, P. T., Menzies-Gow, A. N., Kay, A. B., Robinson, D. S.
(2003) Eosinophil’s role remains uncertain as anti-interleukin-5 only
partially depletes numbers in asthmatic airway. Am. J. Respir. Crit. Care
Med. 167, 199 –204.
8. Kips, J. C., O’Connor, B. J., Langley, S. J., Woodcock, A., Kerstjens,
H. A., Postma, D. S., Danzig, M., Cuss, F., Pauwels, R. A. (2003) Effect
of SCH55700, a humanized anti-human interleukin-5 antibody, in severe
persistent asthma: a pilot study. Am. J. Respir. Crit. Care Med. 167, 1655–
1659.
9. Nair, P., Pizzichini, M. M., Kjarsgaard, M., Inman, M. D., Efthimiadis, A.,
Pizzichini, E., Hargreave, F. E., O’Byrne, P. M. (2009) Mepolizumab for
prednisone-dependent asthma with sputum eosinophilia. N. Engl. J. Med.
360, 985–993.
10. Haldar, P., Brightling, C. E., Hargadon, B., Gupta, S., Monteiro, W.,
Sousa, A., Marshall, R. P., Bradding, P., Green, R. H., Wardlaw, A. J.,
Pavord, I. D. (2009) Mepolizumab and exacerbations of refractory eosin-
ophilic asthma. N. Engl. J. Med. 360, 973–984.
11. Lee, J. J., Dimina, D., Macias, M. P., Ochkur, S. I., McGarry, M. P.,
O’Neill, K. R., Protheroe, C., Pero, R., Nguyen, T., Cormier, S. A., Len-
kiewicz, E., Colbert, D., Rinaldi, L., Ackerman, S. J., Irvin, C. G., Lee,
N. A. (2004) Defining a link with asthma in mice congenitally deficient
in eosinophils. Science 305, 1773–1776.
12. Humbles, A. A., Lloyd, C. M., McMillan, S. J., Friend, D. S., Xanthou,
G., McKenna, E. E., Ghiran, S., Gerard, N. P., Yu, C., Orkin, S. H., Ge-
rard, C. (2004) A critical role for eosinophils in allergic airways remod-
eling. Science 305, 1776 –1779.
13. Coughlin, J. J., Odemuyiwa, S. O., Davidson, C. E., Moqbel, R. (2008)
Differential expression and activation of Rab27A in human eosinophils:
relationship to blood eosinophilia. Biochem. Biophys. Res. Commun. 373,
382–386.
14. Seabra, M. C., Mules, E. H., Hume, A. N. (2002) Rab GTPases, intracel-
lular traffic and disease. Trends. Mol. Med. 8, 23–30.
15. Seabra, M. C., Coudrier, E. (2004) Rab GTPases and myosin motors in
organelle motility. Traffic 5, 393–399.
16. Wood, S. M., Meeths, M., Chiang, S. C., Bechensteen, A. G., Boelens,
J. J., Heilmann, C., Horiuchi, H., Rosthoj, S., Rutynowska, O., Winiarski,
J., Stow, J. L., Nordenskjold, M., Henter, J. I., Ljunggren, H. G., Bryce-
son, Y. T. (2009) Different NK cell-activating receptors preferentially
recruit Rab27a or Munc13-4 to perforin-containing granules for cytotox-
icity. Blood 114, 4117–4127.
17. Arico, M., Zecca, M., Santoro, N., Caselli, D., Maccario, R., Danesino, C.,
de Saint Basile, G., Locatelli, F. (2002) Successful treatment of Griscelli
syndrome with unrelated donor allogeneic hematopoietic stem cell
transplantation. Bone Marrow Transplant. 29, 995–998.
18. Wilson, S. M., Yip, R., Swing, D. A., O’Sullivan, T. N., Zhang, Y., Novak,
E. K., Swank, R. T., Russell, L. B., Copeland, N. G., Jenkins, N. A.
(2000) A mutation in Rab27a causes the vesicle transport defects ob-
served in ashen mice. Proc. Natl. Acad. Sci. U. S. A. 97, 7933–7938.
19. Barral, D. C., Ramalho, J. S., Anders, R., Hume, A. N., Knapton, H. J.,
Tolmachova, T., Collinson, L. M., Goulding, D., Authi, K. S., Seabra,
M. C. (2002) Functional redundancy of Rab27 proteins and the patho-
genesis of Griscelli syndrome. J. Clin. Invest. 110, 247–257.
20. Haddad, E. K., Wu, X., Hammer, J. A., 3rd, Henkart, P. A. (2001) De-
fective granule exocytosis in Rab27a-deficient lymphocytes from Ashen
mice. J. Cell Biol. 152, 835–842.
21. Stinchcombe, J. C., Barral, D. C., Mules, E. H., Booth, S., Hume, A. N.,
Machesky, L. M., Seabra, M. C., Griffiths, G. M. (2001) Rab27a is re-
quired for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol.
152, 825–834.
22. Ochkur, S. I., Jacobsen, E. A., Protheroe, C. A., Biechele, T. L., Pero,
R. S., McGarry, M. P., Wang, H., O’Neill, K. R., Colbert, D. C., Colby,
T. V., Shen, H., Blackburn, M. R., Irvin, C. C., Lee, J. J., Lee, N. A.
(2007) Coexpression of IL-5 and eotaxin-2 in mice creates an eosino-
www.jleukbio.org
Kim et al. Rab27a in eosinophil exocytosis
phil-dependent model of respiratory inflammation with characteristics
of severe asthma. J. Immunol. 178, 7879 –7889.
23. Lee, N. A., McGarry, M. P., Larson, K. A., Horton, M. A., Kristensen,
A. B., Lee, J. J. (1997) Expression of IL-5 in thymocytes/T cells leads to
the development of a massive eosinophilia, extramedullary eosinophilo-
poiesis, and unique histopathologies. J. Immunol. 158, 1332–1344.
24. Garcia-Zepeda, E. A., Rothenberg, M. E., Ownbey, R. T., Celestin, J.,
Leder, P., Luster, A. D. (1996) Human eotaxin is a specific chemoattrac-
tant for eosinophil cells and provides a new mechanism to explain tissue
eosinophilia. Nat. Med. 2, 449 –456.
25. Denzler, K. L., Borchers, M. T., Crosby, J. R., Cieslewicz, G., Hines,
E. M., Justice, J. P., Cormier, S. A., Lindenberger, K. A., Song, W., Wu,
W., Hazen, S. L., Gleich, G. J., Lee, J. J., Lee, N. A. (2001) Extensive
eosinophil degranulation and peroxidase-mediated oxidation of airway
proteins do not occur in a mouse ovalbumin-challenge model of pulmo-
nary inflammation. J. Immunol. 167, 1672–1682.
26. Ishihara, K., Hong, J., Zee, O., Ohuchi, K. (2005) Mechanism of the
eosinophilic differentiation of HL-60 clone 15 cells induced by n-bu-
tyrate. Int. Arch. Allergy Immunol. 137(Suppl. 1), 77–82.
27. Lacy, P., Abdel-Latif, D., Steward, M., Musat-Marcu, S., Man, S. F., Moq-
bel, R. (2003) Divergence of mechanisms regulating respiratory burst in
blood and sputum eosinophils and neutrophils from atopic subjects. J.
Immunol. 170, 2670 –2679.
28. Levi-Schaffer, F., Lacy, P., Severs, N. J., Newman, T. M., North, J., Gom-
perts, B., Kay, A. B., Moqbel, R. (1995) Association of granulocyte-mac-
rophage colony-stimulating factor with the crystalloid granules of hu-
man eosinophils. Blood 85, 2579 –2586.
29. Lacy, P., Mahmudi-Azer, S., Bablitz, B., Hagen, S. C., Velazquez, J. R.,
Man, S. F., Moqbel, R. (1999) Rapid mobilization of intracellularly
stored RANTES in response to interferon-␥ in human eosinophils. Blood
94, 23–32.
30. Rothenberg, M. E., Luster, A. D., Leder, P. (1995) Murine eotaxin: an
eosinophil chemoattractant inducible in endothelial cells and in inter-
leukin 4-induced tumor suppression. Proc. Natl. Acad. Sci. U. S. A. 92,
8960 –8964.
31. Lacy, P., Willetts, L., Kim, J. D., Lo, A., Lam, B., MacLean, E. I., Moqbel,
R., Rothenberg, M. E., Zimmermann, N. (2011) Agonist activation of
F-actin-mediated eosinophil shape change and mediator release is de-
pendent on Rac2. Int Arch Allergy Immunol. 156, 137–147.
32. Dyer, K. D., Moser, J. M., Czapiga, M., Siegel, S. J., Percopo, C. M.,
Rosenberg, H. F. (2008) Functionally competent eosinophils differenti-
ated ex vivo in high purity from normal mouse bone marrow. J. Immu-
nol. 181, 4004 –4009.
33. Lacy, P., Levi-Schaffer, F., Mahmudi-Azer, S., Bablitz, B., Hagen, S. C.,
Velazquez, J., Kay, A. B., Moqbel, R. (1998) Intracellular localization of
interleukin-6 in eosinophils from atopic asthmatics and effects of inter-
feron ␥. Blood 91, 2508 –2516.
34. Lacy, P., Willetts, L., Kim, J. D., Lo, A. N., Lam, B., Maclean, E. I., Moq-
bel, R., Rothenberg, M. E., Zimmermann, N. (2011) Agonist activation
of f-actin-mediated eosinophil shape change and mediator release is de-
pendent on Rac2. Int. Arch. Allergy Immunol. 156, 137–147.
35. Mahmudi-Azer, S., Lacy, P., Bablitz, B., Moqbel, R. (1998) Inhibition of
nonspecific binding of fluorescent-labelled antibodies to human eosino-
phils. J. Immunol. Methods 217, 113–119.
36. Hume, A. N., Collinson, L. M., Rapak, A., Gomes, A. Q., Hopkins, C. R.,
Seabra, M. C. (2001) Rab27a regulates the peripheral distribution of
melanosomes in melanocytes. J. Cell Biol. 152, 795–808.
37. Ochkur, S. I., Kim, J. D., Protheroe, C. A., Colbert, D., Moqbel, R., Lacy,
P., Lee, J. J., Lee, N. A. (2012) The development of a sensitive and spe-
cific ELISA for mouse eosinophil peroxidase: assessment of eosinophil
degranulation ex vivo and in models of human disease. J. Immunol. Meth-
ods 375, 138 –147.
38. Dyer, K. D., Percopo, C. M., Xie, Z., Yang, Z., Kim, J. D., Davoine, F.,
Lacy, P., Druey, K. M., Moqbel, R., Rosenberg, H. F. (2010) Mouse and
human eosinophils degranulate in response to platelet-activating factor
(PAF) and lysoPAF via a PAF-receptor-independent mechanism: evi-
dence for a novel receptor. J. Immunol. 184, 6327–6334.
39. Mahmudi-Azer, S., Downey, G. P., Moqbel, R. (2002) Translocation of
the tetraspanin CD63 in association with human eosinophil mediator
release. Blood 99, 4039 –4047.
40. Bahadoran, P., Aberdam, E., Mantoux, F., Busca, R., Bille, K., Yalman,
N., de Saint-Basile, G., Casaroli-Marano, R., Ortonne, J. P., Ballotti, R.
(2001) Rab27a: a key to melanosome transport in human melanocytes.
J. Cell Biol. 152, 843–850.
41. Casey, T. M., Meade, J. L., Hewitt, E. W. (2007) Organelle proteomics:
identification of the exocytic machinery associated with the natural
killer cell secretory lysosome. Mol. Cell. Proteomics
42. Munafo, D. B., Johnson, J. L., Ellis, B. A., Rutschmann, S., Beutler, B.,
Catz, S. D. (2007) Rab27a is a key component of the secretory machin-
ery of azurophilic granules in granulocytes. Biochem. J. 402, 229 –239.
43. Mizuno, K., Tolmachova, T., Ushakov, D. S., Romao, M., Abrink, M.,
Ferenczi, M. A., Raposo, G., Seabra, M. C. (2007) Rab27b regulates mast
cell granule dynamics and secretion. Traffic 8, 883–892.
44. Fukuda, M. (2008) Regulation of secretory vesicle traffic by Rab small
GTPases. Cell. Mol. Life Sci. 65, 2801–2813.
Volume 94, December 2013 Journal of Leukocyte Biology 1273
45. McAdara Berkowitz, J. K., Catz, S. D., Johnson, J. L., Ruedi, J. M., Thon,
V., Babior, B. M. (2001) JFC1, a novel tandem C2 domain-containing
protein associated with the leukocyte NADPH oxidase. J. Biol. Chem. 276,
18855–18862.
46. Lacy, P., Logan, M. R., Bablitz, B., Moqbel, R. (2001) Fusion protein
vesicle-associated membrane protein 2 is implicated in IFN␥-induced
piecemeal degranulation in human eosinophils from atopic individuals.
J. Allergy Clin. Immunol. 107, 671–678.
47. Logan, M. R., Lacy, P., Bablitz, B., Moqbel, R. (2002) Expression of eo-
sinophil target SNAREs as potential cognate receptors for vesicle-associ-
ated membrane protein-2 in exocytosis. J. Allergy Clin. Immunol. 109,
299 –306.
48. Logan, M. R., Lacy, P., Odemuyiwa, S. O., Steward, M., Davoine, F.,
Kita, H., Moqbel, R. (2006) A critical role for vesicle-associated mem-
brane protein-7 in exocytosis from human eosinophils and neutrophils.
Allergy 61, 777–784.
49. Logan, M. R., Odemuyiwa, S. O., Moqbel, R. (2003) Understanding exo-
cytosis in immune and inflammatory cells: the molecular basis of media-
tor secretion. J. Allergy Clin. Immunol. 111, 923–932.
50. Shirakawa, R., Higashi, T., Tabuchi, A., Yoshioka, A., Nishioka, H., Fu-
kuda, M., Kita, T., Horiuchi, H. (2004) Munc13-4 is a GTP-Rab27-bind-
ing protein regulating dense core granule secretion in platelets. J. Biol.
Chem. 279, 10730 –10737.
51. Neeft, M., Wieffer, M., de Jong, A. S., Negroiu, G., Metz, C. H., van
Loon, A., Griffith, J., Krijgsveld, J., Wulffraat, N., Koch, H., Heck, A. J.,
Brose, N., Kleijmeer, M., van der Sluijs, P. (2005) Munc13-4 is an effec-
tor of rab27a and controls secretion of lysosomes in hematopoietic cells.
Mol. Biol. Cell 16, 731–741.
1274 Journal of Leukocyte Biology Volume 94, December 2013
52. Menager, M. M., Menasche, G., Romao, M., Knapnougel, P., Ho, C. H.,
Garfa, M., Raposo, G., Feldmann, J., Fischer, A., de Saint Basile, G.
(2007) Secretory cytotoxic granule maturation and exocytosis require
the effector protein hMunc13-4. Nat. Immunol.
53. Herrero-Turrion, M. J., Calafat, J., Janssen, H., Fukuda, M., Mollinedo,
F. (2008) Rab27a regulates exocytosis of tertiary and specific granules in
human neutrophils. J. Immunol. 181, 3793–3803.
54. Spencer, L. A., Melo, R. C., Perez, S. A., Bafford, S. P., Dvorak, A. M.,
Weller, P. F. (2006) Cytokine receptor-mediated trafficking of pre-
formed IL-4 in eosinophils identifies an innate immune mechanism of
cytokine secretion. Proc. Natl. Acad. Sci. U. S. A. 103, 3333–3338.
55. Adamko, D. J., Wu, Y., Gleich, G. J., Lacy, P., Moqbel, R. (2004) The
induction of eosinophil peroxidase release: improved methods of mea-
surement and stimulation. J. Immunol. Methods 291, 101–108.
56. Erjefalt, J. S., Greiff, L., Andersson, M., Adelroth, E., Jeffery, P. K.,
Persson, C. G. (2001) Degranulation patterns of eosinophil granulocytes
as determinants of eosinophil driven disease. Thorax 56, 341–344.
57. Stelts, D., Egan, R. W., Falcone, A., Garlisi, C. G., Gleich, G. J., Kreut-
ner, W., Kung, T. T., Nahrebne, D. K., Chapman, R. W., Minnicozzi, M.
(1998) Eosinophils retain their granule major basic protein in a murine
model of allergic pulmonary inflammation. Am. J. Respir. Cell Mol. Biol.
18, 463–470.
KEY WORDS:
allergic asthma 䡠 degranulation 䡠 subcellular fractionation 䡠 IL-5 trans-
genic mice 䡠 hE2 transgenic mice 䡠 Ashen mice
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