doi: 10.

1093/intimm/dxg148
Impaired signaling via the high-afnity IgE
receptor in WiskottAldrich syndrome
protein-decient mast cells
Vadim I. Pivniouk
1,5
, Scott B. Snapper
2
, Alexander Kettner
1
, Harri Alenius
1
,
Dhafer Laouini
1
, Herve Falet
3
, John Hartwig
3
, Frederick W. Alt
4
and Raif S. Geha
1
1
Division of Immunology, Children's Hospital,
2
Division of Gastroenterology, Massachusetts General
Hospital,
3
Division of Hematology, Brigham and Women's Hospital, and
4
Howard Hughes Medical
Institute, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
5
Present address: Department of Cell Biology and Anatomy, University of Arizona, Tucson,
AZ 85724-5030, USA
Keywords: allergy and immunology, cell degranulation, receptor-mediated signal transduction,
WiskottAldrich syndrome
Abstract
WiskottAldrich syndrome protein (WASP) is the product of the gene decient in boys with
X-linked WiskottAldrich syndrome. We assessed the role of WASP in signaling through the high-
afnity IgE receptor (FceRI) using WASP-decient mice. IgE-dependent degranulation and cytokine
secretion were markedly diminished in bone marrow-derived mast cells from WASP-decient mice.
Upstream signaling events that include FceRI-triggered total protein tyrosine phosphorylation, and
protein tyrosine phosphorylation of FceRIb and Syk were not affected by WASP deciency.
However, tyrosine phosphorylation of phospholipase Cg and Ca
2+
mobilization were diminished.
IgE-dependent activation of c-Jun N-terminal kinase, cell spreading and redistribution of cellular
F-actin in mast cells were reduced in the absence of WASP. We conclude that WASP regulates
FceRI-mediated granule exocytosis, cytokine production and cytoskeletal changes in mast cells.
Introduction
Allergic reactions are mediated by products released from
mast cells and basophils following ligation of their high-afnity
receptor for IgE (FceRI) by allergen. These products include
preformed mediators stored in mast cell granules as well as
mediators and cytokines that are synthesized and released
following FceRI ligation. FceRI expressed on mast cells is
comprised of an IgE-binding a chain and two chains, b and g,
which function as signal-transducing subunits. Both of these
chains contain an immunoreceptor tyrosine-based activation
motif (ITAM) with paired tyrosine residues. The cross-linking of
IgE bound to FceRI with multivalent antigen (allergen) results
in activation of Src family protein tyrosine kinase Lyn (1), and in
phosphorylation of the tyrosines within ITAM motifs of FceRIb
and FceRIg chains (2). This creates docking sites for the
protein tyrosine kinase Syk and causes its activation. Lyn and
Syk phosphorylate a number of targets that include phospho-
lipase C (PLC)g, linker for activation of T cells (LAT), the
adaptor protein SLP-76, Vav-1 and Btk (26). PLCg activation
generates inositol triphosphate (IP
3
) and diacylglycerol, which
play a critical role in Ca
2+
mobilization and in activation of
protein kinase C, respectively. FceRI cross-linking results in
the activation of mitogen-activated protein kinase (MAPK)
pathways that include extracellular signal regulated kinase, c-
Jun N-terminal kinase (JNK) and p38 MAPK (79). JNK
activates transcription factors such as c-Jun that regulate the
expression of cytokine genes that include IL-6 and tumor
necrosis factor (TNF)-a (10).
Study of mice with targeted gene mutations has demon-
strated that mast cell degranulation and cytokine production
are decient in Syk
/
(11), btk
/
(3), SLP-76
/
(12), LAT
/
(13)
and Vav-1
/
mice (14). A candidate link between these
signaling molecules and downstream events that include
actin cytoskeleton reorganization is the WiskottAldrich syn-
drome protein (WASP). WASP is the product of the gene
decient in boys with X-linked WiskottAldrich syndrome and
is expressed in hematopoietic cells (15). WASP possesses a
WASP homology domain 1, a Cdc42/Rac GTPase-binding
domain, a proline-rich domain, a G-actin-binding verprolin
Correspondence to: R. S. Geha; E-mail: raif.geha@tch.harvard.edu
Transmitting editor: S. J. Galli Received 26 March 2003, accepted 2 September 2003
International Immunology, Vol. 15, No. 12, pp. 14311440 2003 The Japanese Society for Immunology

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homology domain, a colin homology domain and a C-terminal
acidic segment that can interact with the actin polymerizing
complex Arp2/3 (16,17). WASP may be linked to SLP-76 by the
adaptor protein Nck (18) and by Vav-1 by virtue of its ability to
bind Cdc42. WASP is thought to play an important role in
linking cell-surface antigen receptor signaling and actin
cytoskeleton reorganization (17). WASP is expressed in mast
cells (19), and its phosphorylation increases after FceRI cross-
linking and is facilitated by activated Cdc42 (19).
WASP-decient T cells proliferate poorly and are decient in
cap formation following TCR cross-linking (2022). The role of
WASP in mast cell development and activation remains
unknown. We have used mast cells derived from WASP-
decient mice to examine the role of WASP in FceRI signaling.
We show that degranulation, JNK activation, cytokine produc-
tion and spreading on IgE-coated surfaces following FceRI
ligation are decient in WASP
/
mast cells. These ndings
suggest that WASP plays an important role in linking FceRI to
granule exocytosis, JNK activation, cytokine secretion and the
actin cytoskeleton.
Methods
Mice
The generation of WASP
/
mice has been described (21).
WASP
/
mice were backcrossed onto the 129Sv background
for ve generations. WASP
+/+
and WASP
+/
littermates were
found to have indistinguishable responses from 129Sv age-
matched animals which were used as wild-type controls. Both
WASP
/0
male and WASP
/
female mice used in this study are
referred to as WASP
/
. Mice were housed under pathogen-
free conditions according to institutional regulations.
Reagents and antibodies
Anti-phosphotyrosine mAb 4G10 was from Upstate
Biotechnology (Waltham, MA). Antibodies against phospho-
SAPK/JNK, phospho-p38, SAPK/JNK and p38 were from New
England Biolabs (Beverly, MA). Anti-phospho-Erk and anti-Erk
antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). Polyclonal rabbit anti-phospho-Akt and anti-Akt anti-
bodies were from PharMingen (San Diego, CA). Cytochalasin
D was from Calbiochem-Novabiochem (San Diego, CA).
Enumeration of mast cells in ear skin
Ears from WASP
/
and control mice were xed in 2%
paraformaldehyde, processed into 3-mm thick, parafn-
Fig. 1. In vitro development of mast cells in the absence of WASP.
(A) Bone marrow cells from WASP
/
and wild-type mice were
examined for IgE binding by FACS analysis after 4 weeks of culture
in WCM. Cells were incubated with mouse IgE followed by
biotinylated anti-mouse IgE and streptavidinCyChrome (dashed
line). For control staining, cells were incubated with biotinylated anti-
mouse IgE and streptavidinCyChrome in the absence of IgE (solid
line). Similar results were obtained on four occasions using
independently derived lines of BMMC. (B and C) Western blot
analysis of WASP (B) and N-WASP expression (C) in BMMC.
Fig. 2. IgE-mediated degranulation in the absence of WASP. (A)
Plasma histamine levels in IgE-anti-DNP sensitized mice before and
1.5 min after DNP-HSA challenge in WASP
/
mice (n = 5) and wild-
type controls (n = 6). (B) Release of b-hexosaminidase by WASP
/
and wild-type BMMC sensitized with the indicated concentrations of
rat IgE and challenged with F(ab)
2
mouse anti-rat Ig (25 mg/ml). IO =
ionomycin (10 mM). Results represent the mean of indicated
numbers of mice (A) and the mean of ve experiments (B), each
performed in duplicate. Bars represent mean 6 SEM. **P < 0.01.
1432 WASP is required for the signaling via the FceRI

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embedded sections and stained with toluidine blue. Slides
were examined by light microscopy for determination of mast
cell numbers. Mast cells in six randomly chosen elds were
counted per slide (six slides total for both WASP
/
and control
mice).
Blood histamine measurements
WASP
/
mice and wild-type controls were sensitized with 3 or
10 mg of mouse IgE anti-DNP mAb SPE-7 (Sigma, St Louis,
MO) by i.v. injection in the retro-orbital vein. Twenty-four hours
later the mice were challenged with i.v. injection of HSA-DNP
(500 mg/mouse). Blood histamine levels were determined by
competitive radioimmunoassay (Immunotech, Brea, CA) on
100 ml of plasma 1.5 and 5 min after antigen challenge
according to the manufacturer's instructions.
Derivation and characterization of bone marrow-derived
mast cells (BMMC)
BMMC were derived as previously described (12). Briey,
bone marrow cells were cultured in WEHI-3 conditioned
medium (WCM) as a source of IL-3 (23). Passages were
made every week. To assess IgE binding, the cells were
incubated with 1 mg/ml of mouse IgE (SPE-7), followed by
biotinylated rat anti-mouse IgE and streptavidinCyChrome
(both from PharMingen). The cells were analyzed on a
FACSCalibur ow cytometer (Becton Dickinson, San Jose,
CA). Data on 510 3 10
5
viable cells (as determined by
forward versus side scatter) were collected for each sample.
b-Hexosaminidase release assay
b-Hexosaminidase release assay was performed as pre-
viously described (12). BMMC (1 3 10
6
) were incubated in
WCM containing the indicated concentrations of rat mono-
clonal IgE anti-DNP (clone LO-DNP-30; Serotec, Oxford, UK)
for 1 h on ice. After washing, pellets were resuspended in their
original volume with WCM with 25 mg/ml F(ab)
2
fragments of
mouse anti-rat Ig (Jackson ImmunoResearch, West Grove,
PA) and incubated for 230 min at 37C. The cell pellets were
lysed in their original volumes. Aliquots (5 ml) of supernatants
and cell lysates were incubated for 30 min with 80 ml of
Fig. 3. IgE-mediated cytokine release and gene expression by BMMC. WASP
/
and wild-type BMMC were sensitized and challenged as in
Fig. 2. Release of IL-6 (A) and TNF-a (B) by BMMC was measured after 24 h of culture. Results shown are representative of three
independent experiments, each performed in duplicate. (C) RT-PCR analysis of IL-6 mRNA. (D) Quantication of RT-PCR results from (C).
Levels of IL-6 mRNA were normalized to that of b
2
-microglobulin mRNA as described in Methods. IL-6 mRNA levels induced with 1 mM
ionomycin (IO) are shown for comparison. For (B) and (C), results are representative of four independent experiments.
WASP is required for the signaling via the FceRI 1433

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substrate solution (1.3 mg/ml p-nitrophenyl-b-D-2-acetamido-
2-deoxyglucopyranozide in 0.1 Mcitrate, pH4.5). The reaction
was stopped by the addition of 200 ml of 0.2 M glycine
(pH 10.7). OD was read at 405 nm in an ELISA reader. The
percent release values were calculated by the formula [S/(S +
P)] 3 100, where S and P are the b-hexosaminidase contents
of the supernatant (S) and pellet (P) from each sample.
Measurement of IL-6 and TNF-a secretion
BMMC (1 3 10
6
) preloaded for 1 h with the indicated
concentrations of rat IgE were incubated with 25 mg/ml
F(ab)
2
fragments of mouse anti-rat Ig, with ionomycin
(10 mM) or with ionomycin (1 mM) plus PMA (20 ng/ml) for
indicated periods of time in culture medium. Supernatants
were assayed for their content of IL-6 or TNF-a using an IL-6 or
TNF-a ELISA kit (both from R & D, Minneapolis, MN)
respectively.
RT-PCR analysis of IL-6 gene expression
cDNA was synthesized from 10 mg of total RNA using
Superscript II (Gibco/BRL, Carlsbad, CA) for 120 min at
37C. The primers used to amplify cDNA for b
2
-microglobulin
and IL-6 were as described previously (24). To quantify
mRNA, a xed amount of reverse-transcribed cellular mRNA
was co-amplied in the presence of serial dilutions of a
multispecic internal plasmid control, pMUS3 (24), which
contains nucleotide sequences of multiple cytokines. The
dilution at which pMUS3-derived and cDNA-derived signals
were of equivalent intensity was used to establish the relative
amount of cytokine mRNA. The results are expressed as a ratio
of cytokine cDNA to cDNA of the constitutively expressed
b
2
-microglobulin gene.
Western blotting and immunoprecipitation
Lysates prepared from 0.5 3 10
6
BMMC by boiling in SDS
loading buffer were separated on a 9% gel by PAGE and
transferred to nitrocellulose membrane. The blots were
developed using indicated antibodies followed by Protein G
linked to horseradish peroxidase (Bio-Rad, Hercules, CA) and
ECL (Amersham, Piscataway, NJ) or the Super Signal Ultra
system (Pierce, Rockford, IL) according to the manufacturer's
instructions.
For immunoprecipitation, cell lysates prepared in RIPA
buffer were precleared for 2 h with normal rabbit serum
coupled to Protein GSepharose beads, then immunopreci-
pitated overnight with rabbit antibody to Syk prepared in our
Fig. 4. Protein tyrosine phosphorylation in BMMC in response to FceRI cross-linking. WASP
/
and wild-type BMMC were sensitized with
5 mg/ml rat IgE, followed by cross-linking with 25 mg/ml F(ab)
2
mouse anti-rat Ig. Cell lysates were examined at the indicated time points (0, 5
or 0, 5 and 15 min) for tyrosine phosphorylation of FceRIb (A), Syk (B) and PLCg (C) by immunoprecipitatation with the indicated antibodies
followed by western blotting with mAb 4G10. (D) Akt phosphorylation was examined by western blotting with phospho-Akt specic antibody.
Equal loading was veried by western blotting with appropriate antibodies. Results shown are representative of at least two independent
experiments.
1434 WASP is required for the signaling via the FceRI

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laboratory or FceRIb mAb (kind gift from Dr J. P. Kinet, Beth
Israel Hospital, Boston) on Protein GSepharose beads. The
immunoprecipitates were resolved by SDSPAGE and trans-
ferred to nitrocellulose membranes. Blots were probed with
anti-phosphotyrosine mAb 4G10 and developed with ECL as
above. To ensure equal loading, blots were stripped and then
reprobed with the appropriate antibody.
Measurement of [Ca
2+
]
i
BMMC were loaded with Indo-1/AM as previously described
(25). Cells were sensitized with rat IgE (LO-DNP-30) while
loading with the dye and stimulated with anti-IgE antibody
while monitoring Ca
2+
content using a spectrouorimeter
(LS50; Perkin-Elmer Cetus, Norwalk, CT). Excitation wave-
length was 354 nm, and emission wavelengths were 405 and
485 nm.
Time-lapse video microscopy and immunouorescence
microscopy
Glass coverslips were coated with 2.5, 10 and 25 mg/ml of
mouse monoclonal IgE anti-ovalbumin (a gift of Dr Mamoru
Kiniwa, Taiho Pharmaceutical, Saitama, Japan) overnight at
4C. BMMC (1 3 10
6
/ml) were allowed to attach to the
coverslips for 30, 60 or 90 min at 37C. Unattached cells were
washed off. When indicated, BMMC were pre-incubated with
cytochalasin D at 10 mM for 1 h on ice, washed twice and
resuspended in culture medium. Coverslips with attached
BMMC in culture medium were transferred to the warm stage
of the Nikon Eclipse TE2000 microscope. Images were
acquired with a CCD-300-RC charge-coupled device (Dage-
MTI, Michigan City, IN) at 3400 magnication. Frames were
taken every 5 s for 20 min.
Immunouorescence
Cells were spun down onto the glass slides (controls) or were
allowed to attach to the IgE-coated coverslips for 60 min at
37C, and xed with 4% formalin solution (Sigma) in PBS at
room temperature for 20 min, washed twice with PBS and
permeabilized with 0.5% Triton X-100 in PBS at room
temperature for 5 min. Cells were washed twice with PBS
and blocked with 2% BSA (Gibco/BRL) in PBS for 10 min, and
Fig. 5. Ca
2+
mobilization in BMMC in response to IgE cross-linking.
Changes in the intracellular Ca
2+
concentration monitored in Indo-1
AM-loaded WASP
/
(dashed line) and wild-type (solid line) BMMC
sensitized with 5 mg/ml of rat IgE, and then challenged at the
indicated time points with 2.5 and 0.25 mg/ml F(ab)
2
mouse anti-rat
Ig. Results are expressed as ratio of uorescence intensities at 405
and 485 nm. Results shown are representative of four independent
experiments.
Fig. 6. Activation of MAPK in WASP
/
and wild-type BMMC following
FceRI cross-linking. BMMC were stimulated for 0, 5, 15 and 30 min
as described in the legend to Fig. 4, and lysed in the SDSPAGE
sample buffer. Aliquots of the same lysates were analyzed for
phosphorylation of Erk1/2 (A), p38 MAPK (B) and SAPK/JNK (C) by
western blotting with the corresponding phospho-specic
antibodies. Lower panels represent the same membranes stripped
and reprobed with kinase-specic antibodies as a control for the
loading. Insets show relative phosphorylation of MAPK determined
by densitometry and normalized for loading. Results shown are
representative of three independent experiments.
WASP is required for the signaling via the FceRI 1435

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then stained for actin. For actin staining, cells were incubated
with 1 mM TRITCphalloidin (Sigma) for 20 min at room
temperature. Photographs were taken under a uorescent
microscope (Nikon Eclipse E800) using a CCD-300-RC
camera (Dage-MTI). Images were processed using Adobe
Photoshop.
Results
BMMC develop normally in the absence of WASP
Mouse bone marrow cells cultured in the presence of IL-3
differentiate into mast cells (26). After 4 weeks of culture in
IL-3-containing WCM, similar numbers and percentages of
cells (~90%) from WASP
/
and wild-type mice were mast cells
as evidenced by their capacity to bind IgE (Fig. 1A).
Furthermore, the same proportion of cells contained meta-
chromatic granules when stained with toluidine blue (data not
shown). Expression of WASP and its homologue N-WASP was
examined by western blotting. Figure 1(B) conrms that
BMMC from WASP
/
mice do not express WASP. Both wild-
type and WASP
/
BMMC expressed N-WASP (Fig. 1C). The
kinetics of cell growth and differentiation of WASP
/
and wild-
type BMMC were similar (data not shown). We also examined
the number of tissue mast cells in WASP
/
mice. The number
of mast cells in the ear skin of WASP
/
mice was comparable
to that of controls [16.3 6 3.8 (n = 6) in WASP
/
mice versus
18.9 6 3.8 (n = 6) in controls].
IgE-mediated systemic histamine release is greatly
diminished in WASP
/
mice
Adoptive transfer of IgE antibodies to normal mice primes
them to undergo passive systemic anaphylactic reactions in
response to i.v. challenge with specic antigen. IgE-mediated
anaphylaxis is dependent on FceRI signaling, as it is virtually
absent in FceRIa-decient mice (27). IgE-mediated anaphy-
laxis is also dependent on mast cells, as it is diminished in
mast cell-decient W/W
v
mice (28,29) and virtually absent in
mast cell-decient Sl/Sl
d
mice (30). A major vasoactive
mediator released by activated mast cells and basophils is
histamine. Systemic anaphylaxis is associated with a dramatic
increase in plasma histamine levels (31).
To evaluate the role of WASP in IgE-dependent systemic
anaphylaxis, we passively sensitized six WASP
/
mice and six
controls with 3 mg mouse IgE anti-DNP mAb. This was followed
24 h later by challenge with DNP-HSA. Plasma histamine
content was determined before and 1.5 min after challenge
with antigen (Fig. 2A). The plasma histamine level prior to
antigen administration was comparable in wild-type and
WASP
/
mice. Following antigen challenge, there was a
marked rise in plasma histamine in control mice. The
antigen-induced increase in plasma histamine was signi-
cantly reduced in WASP
/
mice. Similar results were obtained
when 10 mg mouse IgE anti-DNP mAb was used for
sensitization with plasma histamine measured at 5 min after
DNP-HSA challenge (data not shown). These results suggest
that WASP is essential for optimal IgE-mediated mast cell
degranulation in vivo.
IgE-induced degranulation in vitro is impaired in
WASP-decient BMMC
Upon cross-linking of FceRI-bound IgE by antigen, mast cells
degranulate by releasing preformed mediators stored in their
granules. The capacity of WASP
/
BMMCto degranulate upon
FceRI cross-linking was examined. BMMC were sensitized
with rat IgE (0.055 mg/ml) for 1 h, washed and bound IgE was
cross-linked with mouse anti-rat Ig (25 mg/ml), and the release
of b-hexosaminidase, an enzyme found in mast cell granules
(32), was measured. b-Hexosaminidase release was lower in
WASP
/
BMMC compared to control wild-type BMMC at all
concentrations of IgE antibody used and at 2, 5, 15 and 30 min
(Fig. 2B and data not shown). Treatment of mutant BMMC with
ionomycin resulted in normal degranulation, demonstrating
that their cellular machinery for degranulation and their
b-hexosaminidase granule content are normal.
IgE-induced cytokine production requires WASP protein
In addition to degranulation, FceRI cross-linking induces the
secretion of a number of cytokines by mast cells, including
IL-6 (33) and TNF-a (10). To assess the role of WASP in FceRI-
mediated cytokine release, BMMC were sensitized and
challenged as above, and IL-6 and TNF-a release measured
by ELISA 24 h later. WASP
/
BMMC secreted markedly less
IL-6 and TNF-a than control BMMC at 6, 24 and 48 h after
FceRI cross-linking (Fig. 3A and B and data not shown).
Furthermore, RT-PCR analysis demonstrated that IL-6 mRNA
levels following FceRI ligation were substantially lower in
WASP
/
BMMC compared to controls (Fig. 3C and D).
Ionomycin or ionomycin plus PMA, which bypass receptor
signaling, induced equivalent levels of IL-6 expression, and
IL-6 and TNF-a secretion by both WASP
/
and wild-type
BMMC.
IgE-induced tyrosine phosphorylation of PLCg and Ca
2+
mobilization are impaired in BMMC from WASP
/
mice
Like other ITAM-containing receptors, FceRI signals through
associated protein tyrosine kinases of the src and syk families
(2). Both the pattern and the intensity of protein tyrosine
phosphorylation following FceRI ligation were comparable in
wild-type and WASP-decient BMMC (data not shown). More
importantly, protein tyrosine phosphorylation of the FceRIb
chain and Syk, both of which are thought to lie upstream of
WASP, was similar in WASP
/
BMMC and controls (Fig. 4A
and B). Protein tyrosine phosphorylation of SLP-76 and Vav
was also unaffected (data not shown). In contrast, tyrosine
phosphorylation of PLCg was diminished in WASP
/
BMMC
(Fig. 4C). FceRI ligation activates phosphatidylinositol-3-
kinase resulting in the generation of phosphatidylinositol-
3,4,5-tris phosphate and subsequent phosphorylation and
activation of Akt (34). Akt phosphorylation was similar in
WASP
/
BMMC and controls (Fig. 4D).
FceRI-induced phosphorylation and activation of PLCg
causes a rapid rise in [Ca
2+
]
i
(2), which is important for both
degranulation and cytokine release. Since mast cells of
WASP-decient mice are impaired in their capacity to release
various mediators and phosphorylate PLCg following FceRI
cross-linking, we examined Ca
2+
mobilization in these cells.
BMMC were sensitized with rat IgE, loaded with a Ca
2+
-
1436 WASP is required for the signaling via the FceRI

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Fig. 7. Spreading of BMMC on IgE-coated glass slides. (A) Quantication of spreading, as percentage of adherent cells at 30, 60 and 90 min.
Cells were counted in 10 elds/coverslip under high power (3400). Results represent the mean 6 SEM. P values shown were determined by
Student's t-test. (B) Representative frames of wild-type (left), cytochalasin D-treated wild-type (middle) and WASP
/
BMMC (right) incubated
for 1 h on IgE-coated coverslips. (C) F-actin distribution in wild-type (left panels) and WASP
/
BMMC (right panels) xed on uncoated
coverslips in cytospin preparations (upper panels), and after incubation for 1 h on coverslips coated with IgE (25 mg/ml). Results shown are
representative of three independent experiments.
WASP is required for the signaling via the FceRI 1437

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sensitive dye and then challenged with F(ab)
2
fragments of
anti-rat Ig. Ca
2+
mobilization in response to FceRI cross-linking
was clearly diminished in WASP
/
BMMC (Fig. 5).
IgE-induced phosphorylation of MAPK in WASP-decient
BMMC
FceRI cross-linking induces phosphorylation and activation of
the MAPK Erk, JNK and p38 in mast cells (2,8). The
phosphorylation status of the MAPK Erk, JNK and p38 in
WASP
/
BMMC following FceRI cross-linking was examined
by western blotting with antisera that recognize selectively
these kinases in their phosphorylated state. Erk 2 (p42) was
the major Erk isoform phosphorylated following FceRI ligation
in mast cells. Phosphorylation of Erk2 and p38 was minimally
affected in WASP
/
mast cells (Fig. 6A and B). In contrast,
phosphorylation of both p54 and p46 JNK isoforms was
severely reduced in WASP
/
mast cells (Fig. 6C).
Spreading of mast cells on IgE-coated slides require WASP
protein
T cells spread and extend protrusions when exposed to an
anti-TCRCD3-coated surface. This is dependent on actin
cytoskeleton reorganization which is mediated by WASP (35
37). FceRI ligation has been shown to induce actin cytoskeletal
changes in the RBL-2H3 mast cell line (38). We examined
whether normal murine BMMC spread and extend protrusions
when exposed to an IgE-coated surface. Wild-type mast cells
adhered to coverslips coated with 25 mg/ml IgE, but not to
untreated coverslips (data not shown). Following attachment,
wild-type BMMC spread and extended membrane protrusions
(Fig. 7A and B). Pre-incubation with cytochalasin D reduced
spreading (from 72 6 7.3 to 36 6 11% at 1 h) and inhibited
protrusion formation (Fig. 7B), suggesting that actin polymer-
ization is important in these processes. WASP
/
mast cells
adhered normally to IgE-coated coverslips (data not shown).
However, they had a signicantly reduced ability to spread
(Fig. 7A) and were impaired in their ability to form protrusions
(Fig. 7B). Similar data was obtained using concentrations of
coating IgE of 2.5 and 10 mg/ml (data not shown).
We compared the actin cytoskeleton of wild-type and
WASP-decient mast cells by intracellular staining for
F-actin. The morphology of resting wild-type and WASP
/
BMMC was similar (Fig. 7C). Following incubation for 1 h on
IgE-coated coverslips, wild-type mast cells spread and
exhibited prominent membrane rufes (Fig. 7C). In contrast,
the majority of WASP
/
mast cells failed to spread, and their
morphology and F-actin distribution remained similar to those
of resting cells. The small fraction of WASP
/
cells that spread
exhibited membrane rufes (data not shown).
Discussion
In this study we show that BMMC from WASP
/
mice are
impaired in their ability to secrete both early and late
mediators, and to spread following FceRI cross-linking. This
identies WASP as an important signaling molecule which
links FceRI to mediator release and actin cytoskeletal
changes.
Mouse mast cell development is known to be driven by IL-3
(26) and other cytokines such as stem cell factor (39), but is
thought to be independent of FceRI signals, as it proceeds
normally in FceRIa
/
mice (27). We found that WASP is not
essential for mast cell growth and differentiation as the
numbers of tissue mast cells in ears of WASP
/
mice and
the development of mast cells from bone marrow of WASP
/
mice in vitro were normal (Fig. 1A).
Following IgE antibody sensitization and antigen challenge
WASP
/
mice exhibited a signicantly reduced rise in plasma
histamine levels compared to wild-type controls (Fig. 2A).
Since IgE-mediated histamine release in vivo is dependent on
FceRI (27), our results strongly suggest that WASP is a critical
component of the FceRI signaling pathway. The residual
histamine response of WASP
/
mice in passive IgE-mediated
anaphylaxis could be due to residual signaling via FceRI. This
may possibly involve N-WASP, which we found to be
expressed by mast cells (Fig. 1C).
Degranulation and cytokine secretion are respectively early
and late events triggered by FceRI ligation (40). The signaling
pathways leading to the release of these mediators are likely to
be distinct. FceRI-mediated mast cell degranulation and
cytokine release were decient in WASP
/
BMMC. This was
evidenced by their decreased ability to release b-hexos-
aminidase (Fig. 2B), and produce IL-6 and TNF-a (Fig. 3)
following IgE cross-linking. These results place WASP in a
pivotal position in FceRI-mediated degranulation and cytokine
gene expression.
Our studies of early events in FceRI signaling revealed that
total protein, FceRIb, Syk, and Akt phosphorylation was
grossly intact in the absence of WASP, but that PLCg
phosphorylation was diminished (Fig. 4). This suggests that
WASP is acting downstream of Src and Syk, and of
phosphatidylinositol-3-kinase which generates phosphatidyl-
inositol-3,4,5-tris phosphate, a mediator that is essential for
activation of Akt. Consistent with the decreased PLCg phos-
phorylation, calcium mobilization in response to FceRI cross-
linking in WASP
/
BMMC was clearly diminished (Fig. 5). It is
likely that diminished calcium mobilization in WASP
/
BMMC
contributes to their impaired degranulation and cytokine
secretion. Activation of PLCg and subsequent calcium
mobilization are dependent on LAT, SLP-76 and Tec family
kinases. In T cells, WASP was reported to be a component of a
multimolecular complex that includes SLP-76, Nck and Vav
(41), and thus may be important for optimal activation of PLCg,
possibly by virtue of its ability to nucleate actin polymerization
which may provide a scaffold for a LATSLP-76Tec kinase
PLCg complex. Our observation that tyrosine phosphorylation
of SLP-76 and Vav was unaffected in WASP-decient BMMC
suggests that WASP may act downstream of these proteins.
FceRI cross-linking induces phosphorylation and activation
of the MAPK Erk, JNK and p38 in mast cells (79). We found
that FceRI-mediated JNK phosphorylation was decreased in
WASP
/
BMMC (Fig. 6C). Normal JNK activation following
TCR ligation has been reported in WASP
/
T cells (22). The
discrepancy between this result and ours may be due to
differences in cell types, receptors (FceRI versus TCR) and/or
mouse lines examined. WASP could potentially modulate JNK
activation via its effects on actin polymerization, as JNK
activation has been reported to be regulated by the actin
cytoskeleton (42). Activation of JNK plays an important role in
the expression of the cytokine IL-6 (43,44) and TNF-a (10)
1438 WASP is required for the signaling via the FceRI

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in mast cells. Thus, it is likely that impaired JNK activation in
WASP
/
BMMC may underlie their decreased ability to
produce IL-6 and TNF-a following FceRI cross-linking.
We have found that normal BMMC adhere to IgE-coated
coverslips, and undergo dramatic shape changes that include
spreading and formation of rufes. These changes are similar
to those observed in T cells incubated on anti-CD3-coated
surfaces (45) and are dependent on actin polymerization
since they are inhibited by cytochalasin D. WASP-decient
mast cells adhered to IgE-coated plates, but were severely
impaired in their ability to spread and form rufes (Fig. 7).
These results suggest that WASP links FceRI signaling to actin
cytoskeleton reorganization in mast cells. They also raise the
possibility that WASP may play a direct role in granule
movement and fusion with the plasma membrane. FceRI
signaling causes a redistribution of cellular F-actin with cortical
depolymerization and with centripetal accumulation of F-actin,
which is associated with granule exocytosis (46). Loss of the
Cdc42WASPactin polymerization pathway may result in
impaired F-actin redistribution and may contribute to defective
degranulation in WASP
/
BMMC. The fact that the calcium
ionophore ionomycin induced normal degranulation in these
cells does not rule out a role for WASP in granule movement,
because non-physiologically high calciumconcentrations may
circumvent a physiologic role for WASP in this process.
Our results demonstrate that WASP is essential for optimal
FceRI-induced degranulation and cytokine production in mast
cells. Since IgE-dependent mast cell mediator release plays a
critical role in the pathophysiology of allergic diseases, our
results also suggest a novel potential approach to the
treatment of these diseases.
Acknowledgements
We thank Drs Lewis Cantley and Lucia Rameh for helpful suggestions.
This work was supported by NIH grant AI-35714, and by grants from
Baxter Healthcare, Aventis and Olsten Corporations to R. S. G. F. W. A
is supported by a grant from the Howard Hughes Medical Institute.
Abbreviations
BMMC bone marrow-derived mast cells
FceRI high-afnity receptor for IgE
ITAM immunoreceptor tyrosine-based activation motif
IP
3
inositol 1,4,5-triphosphate
JNK c-Jun N-terminal kinase
LAT linker for activation of T cells
MAPK mitogen-activated protein kinase
PLC phospholipase C
TNF tumor necrosis factor
WASP WiskottAldrich syndrome protein
WCM WEHI-3 conditioned medium
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1440 WASP is required for the signaling via the FceRI

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