Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio
Correspondence: Gurjit K. Khurana Hershey, M.D., Ph.D., Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati OH 45229. E-mail: Gurjit.Hershey{at}chmcc.org
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receptor 1 (FcRI) modulation in bone marrow-derived mast
cells (BMMCs) from me/me and wild-type mice. IL-4
down-regulated FcRI expression in wild-type BMMCs but had no effect
on FcRI expression in me/me BMMCs. Furthermore,
me/me mast cells did not exhibit enhanced or prolonged
IL-4-induced Stat6 activation compared with wild-type cells, indicating
that mast cells possess alternative tyrosine phosphatases that are
responsible for down-regulating Stat6 or can substitute for SHP-1.
Thus, SHP-1 is not a negative regulator of IL-4 signaling in BMMCs.
These results demonstrate the complexity and cellular specificity of
these signaling pathways and indicate a previously unrecognized role
for SHP-1 in murine mast cells.
Key Words: mast cells/basophils • protein kinases/phosphatases • signal transduction • cytokine receptors
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heavy-chain locus [1
] and genes encoding vascular cell
adhesion molecule-1 [2
], CD23 [3
] and
major histocompatibility complex class II [4
5
].Furthermore, studies have demonstrated that IL-4 is critical
for the development of T helper cell type 2 cells [6
7
]. IL-4 is also a potent regulator of mast cell phenotype,
growth, and differentiation [8
9
10
11
]. Mast cells have a
pivotal role in atopic reactions through the release of preformed and
newly synthesized mediators. Degranulation of mast cells occurs through
antigen-mediated cross-linking of the surface high-affinity
immunoglobulin (Ig) E Fc receptor 1 (FcRI). The molecular events
that regulate FcRI expression are poorly understood. IgE has been
shown to be a potent inducer of surface FcRI expression in human and
murine mast cells [12
13 ]. Recently, IL-4 has been
shown to inhibit murine mast cell FcRI expression
[14
]. Furthermore, IL-4 promotes intercellular adhesion
molecule-1 expression [15
] but inhibits c-kit
expression in mast cells [16
17
]. Thus, IL-4 may
function as both a positive and negative regulator of mast cells.
Biologic responses to IL-4 are mediated via two types of receptor
complexes. The type I receptor consists of an IL-4-binding 140-kDa 
chain (IL-4R) [18
19
20
] and the 
c chain, which is
common to multiple receptor systems [21
22
]. Upon the
binding of IL-4 to the chain, the c chain heterodimerizes with
the chain, and activation of associated Janus kinases JAK3 and
JAK1, respectively, occurs [23
24
25
26
]. JAK activation
leads to the phosphorylation of signal transducer and activator of
transcription-6 (Stat6), which mediates gene induction [27
28
]. In nonhematopoietic cells, IL-4 can signal in the absence
of the c chain through the type II receptor, which is composed of
IL-4R and IL-13R1 [29
30
]. Signaling via this
receptor also leads to activation of the JAK-Stat pathway.
Although much is known about the IL-4 signaling pathway, the molecular
mechanisms responsible for the dephosphorylation of key signaling
intermediates and for negative modulation of the IL-4 cascade remain to
be elucidated. Sulfhydryl 2 domain-containing tyrosine phosphatase-1
(SHP-1) dephosphorylates regulatory phosphotyrosine residues and has
been implicated in termination of signaling via many cytokine receptors
including erythropoietin, IL-2, IL-3, colony-stimulating factor, and
IL-4 receptor (IL-4R) [31
32
33
34
35
36
37
]. Recent studies
have shown that SHP-1 may also have a positive regulatory role in some
pathways including the mitogen-activated protein kinase pathway
[38
39
] and epidermal growth factor-induced Stat
activation [40
]. Motheaten mice (me/me) are
homozygous for a spontaneous frameshift mutation in the SHP-1 gene, and
they lack functional SHP-1 [41
]. Bone marrow-derived
macrophages and pre-B-cell lines derived from me/me mice
display enhanced IL-4 signaling when compared with their wild-type
counterparts, consistent with a negative regulatory role for SHP-1 in
IL-4 responses [37
]. These mice develop profound
abnormalities in their B- and T-cell function and die from hemorrhagic
pneumonitis within the first few months of life [42
].
The mice also have defects in natural killer cell and macrophage
function [42
43 ]. However, me/me mast cells
possess normal cell surface marker expression and degranulate normally
[44
45
]. In the present study, we examined IL-4
signaling and IL-4-dependent FcRI down-modulation in
me/me and wild-type mast cells to elucidate the role of
SHP-1 in IL-4 signaling and responsiveness in mast cells.
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Antibodies and cytokines
Recombinant murine IL-4 was obtained from R&D Systems
(Minneapolis, MN). Rabbit polyclonal anti-Stat6 antibody was the
generous gift of Ulrike Schindler (Tularik Inc., San Francisco, CA).
Murine IgE was obtained from PharMingen (San Diego, CA); fluorescein
isothiocyanate-labeled rat anti-mouse IgE was from Southern Biotech
(Birmingham, AL); and anti-murine CD16/CD23 (FCIII/II Receptor-Fc
block®) was from PharMingen.
Intracellular [Ca2+] measurement
BMMCs were incubated in the presence of 5 µg/mL of IgE for
1 h at 4°C, pelleted by centrifugation, and then 2 x
106 cells/mL were loaded with 5 µM Fura-2 AM (Molecular
Probes, Eugene, OR) in Hanks balanced saline solution containing 1%
fetal bovine serum for 30 min at 37°C in the dark. After washing with
flux buffer (145 mM NaCl, 4 mM KCl, 1 mM NaHPO4, 0.8 mM
MgCl2, 1.8 mM CaCl2, 25 mM HEPES, and 22 mM
glucose; pH 7.4), cells were resuspended at 2 x 106
cells/mL and maintained on ice. Next, cells were prewarmed to 35°C
and stimulated with 10 µL of anti-IgE antibody EM95, kindly provided
by Fred Finkelman (University of Cincinnati, Cincinnati, OH), in a
RatioMaster Fluorimeter (Photon Technology, Inc., South Brunswick, NJ).
Data were recorded as the relative ratio of fluorescence emitted at 510
nm after excitation at 340 and 380 nm (y-axis) over time
(x-axis).
Flow-cytometric analysis
BMMCs (5 x 105) were incubated in the presence
of 0.125µg of Fc block® (PharMingen) for 10 min at 4°C in a total
volume of 100 µL; 0.5 µg/10 µL of murine IgE was added; after 30
min, 0.5 µg/10 µL of anti-IgE-fluorescein isothiocyanate was added,
and the samples were incubated for 30 min, washed, and analyzed using
FACScan® (Becton Dickenson, San Diego, CA). Control samples were
stained in the absence of IgE.
Propidium iodide staining for determination of DNA content was performed as previously described [47 48 ].
Electrophoretic Mobility Shift Assay
BMMCs (2.5 x 106) were lysed in 10 mM HEPES
(pH 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetate (EDTA), 1.5 mM
MgCl2, 0.2% Nonidet P-40, 1.0 mM dithiothreitol (DTT), and
0.5 mM phenylmethylsulfonyl fluoride, and nuclei were reconstituted in
nuclear extract buffer (20 mM HEPES [pH 7.9], 420 mM NaCl, 0.1 mM
EDTA, 1.5 mM MgCl2, 25% glycerol, 1.0 mM DTT, and 0.5 mM
phenylmethylsulfonyl fluoride). Extracts (5 µg each) in equal volumes
of 2x electrophoretic mobility shift assay (EMSA) reaction buffer (24
mM HEPES [pH 7.9], 8 mM Tris, 50 mM KCl, 10 mM MgCl2,
24% glycerol, 0.08 µg/mL of poly dI-dC, 2 mM EDTA, and 2 mM DTT)
were incubated for 10 min on ice, and then 0.2 ng of end-labeled
double-stranded Stat6 oligonucleotide (Santa Cruz Biotechnologies,
Santa Cruz, CA) was added for 10 min on ice. A 100-fold excess of
unlabeled nucleotide (20 ng) was used in cold competition samples, and
1.0 µL of anti-Stat6 polyclonal antibody was added to supershift
samples. Extracts were resolved on a 5% polyacrylamide gel.
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RI expression in BMMCs did
not occur in me/me BMMCsRI
expression in BMMCs cultured in the presence of IL-3
[14
]. SHP-1 has been implicated as a negative regulator
of IL-4 responses in B cells [37
]. Thus, we hypothesized
that, in the absence of SHP-1-mediated down-regulation, SHP-1-deficient
me/me BMMCs would display augmented IL-4-dependent
inhibition of FcRI expression. IL-4 inhibited FcRI expression as
predicted in the wild-type mice (Fig. 1A
), but surprisingly, IL-4 did not down-regulate FcRI expression
in the me/me cells (Fig. 1B)
. There was no difference in
baseline FcRI expression between the wild-type and SHP-1-deficient
mast cells (Fig. 1)
. The decrease in FcRI expression by IL-4 has
been shown to require several days of stimulation [14
].
We examined whether the kinetics of IL-4-mediated FcRI modulation
were altered in the me/me BMMCs. The wild-type cells
displayed maximal IL-4-mediated inhibition at 96 h (Fig. 1A)
, but
the me/me BMMCs did not exhibit any change in FcRI
expression even when cultured in the presence of IL-4 for 144 h
(Fig. 1B)
. The results of two separate experiments are shown
graphically in Figure 1C
. We did not observe any effect of IL-4 on
FcRI expression even at doses of up to 100 ng/mL of IL-4 in
SHP-1-deficient mast cells (data not shown).
 View larger version (33K): [in a new window] |
Figure 1. Temporal responsiveness of IL-4-mediated down-modulation of FcRI
expression in me/me and wild-type murine mast cells. BMMCs
derived from wild-type (A) or me/me (B) mice were treated
for increasing time intervals with 20 ng/mL of IL-4 for 96 h and
then surface FcRI expression was analyzed by flow cytometry (dotted
line, anti-IgE alone; solid line, IgE and anti-IgE, in the absence of
IL-4; bold line, IgE and anti-IgE, in the presence of IL-4). (C)
Flow-cytometry data from two separate experiments using different lines
(me/me, squares; wt, circles)
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RI expression in the
me/me mast cells, it was possible that IL-4 signaling was
not occurring normally in these cells. Stat6 has been shown to be
required for IL-4-dependent inhibition of FcRI expression
[14
]. To determine whether IL-4 signaling was intact in
the SHP-1-deficient BMMCs, we examined IL-4-dependent Stat6 activation
in control and me/me BMMCs by EMSA (Fig. 2
). Stat6 activation occurred in the presence or absence of SHP-1,
and the magnitude of activation was enhanced with increasing doses of
IL-4. Both the wild-type and SHP-1-deficient cells exhibited identical
sensitivity of IL-4-dependent Stat6 activation, with maximal Stat6
activation occurring at a dose of 10 ng/mL of IL-4. Thus, Stat6
activation occurred in the me/me BMMCs and was not
sufficient for IL-4-dependent modulation of FcRI expression.
 View larger version (27K): [in a new window] |
Figure 2. IL-4-dependent Stat6 activation occurs equally in
me/me and wild-type murine mast cells. BMMCs derived from
wild-type or me/me mice were treated with the indicated
doses of IL-4 for 15 min. 10 µg of nuclear extracts were then
examined for the presence of activated Stat6 by EMSA. The identity and
specificity of the Stat6 band was illustrated by complete supershifting
of this band using an anti-Stat6 antibody; disappearance of this band
when the assay was carried out in the presence of an excess of
unlabeled probe containing the Stat6 recognition sequence; and the
absence of the band in unstimulated cells. The depicted experiment is
representative of three separate experiments.
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 View larger version (43K): [in a new window] |
Figure 3. IL-4-dependent Stat6 activation is not prolonged in the absence of
SHP-1. (A) BMMCs were stimulated in the presence or absence of IL-4 for
the designated time in hours. 10 µg of nuclear extracts were then
examined for the presence of activated Stat6 by EMSA. The identity and
specificity of the Stat6 band was illustrated by complete supershifting
of this band using an anti-Stat6 antibody; disappearance of this band
when the assay was carried out in the presence of an excess of
unlabeled probe containing the Stat6 recognition sequence; and the
absence of the band in unstimulated cells. (B) The data from panel A
and a repeat experiment were quantitated by densitometry, and the
relative mean amounts of activated Stat6 in densitometric units
detected in lysates from the wild-type cells (solid bars) and
me/me cells (hatched bars) are shown.
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RI expression in BMMCs was not
dependent on SHP-1RI
[49
]. To clarify whether the lack of an IL-4 effect on
FcRI expression in the me/me cells was specific to IL-4
or was due to a general abnormality in FcRI modulation, we next
examined IgE-dependent modulation of FcRI in wild-type and
me/me BMMCs. IgE is a potent inducer of FcRI expression
in murine mast cells [12
13
]. Both control and
me/me BMMCs displayed equivalent IgE-dependent up-regulation
of FcRI expression (Fig. 4
). Thus, FcRI modulation via this pathway is intact in the
me/me cells and does not require SHP-1.
 View larger version (25K): [in a new window] |
Figure 4. Equivalent IgE-dependent up-regulation of FcRI expression in
me/me and wild-type BMMCs. (A) BMMCs derived from wild-type
or me/me mice were incubated in the absence (left) or
presence (right) of 5 µg/mL of IgE for 5 days. (B) Surface FcRI
expression was then analyzed by flow cytometry. The flow-cytometry data
from three separate experiments (each bar represents the means and
SE of three separate experiments) are displayed graphically
in the bottom graph (wild type, filled bars; me/me, open
bars).
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 View larger version (14K): [in a new window] |
Figure 5. Similar DNA contents of me/me and wild-type BMMCs. Wild-type
(dotted line) and me/me (solid line) mast cells were stained
with propidium iodide and then analyzed by flow cytometry. Data are
representative of three separate experiments.
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[36
50
], and, recently, to negatively regulate IL-4 responses in
pre-B-cell lines derived from me/me mice
[37
]. Herein, we examined the role of SHP-1 in IL-4
responses in mast cells. Our data demonstrate that IL-4 was not a
negative regulator of IL-4 responses in murine BMMCs and support the
possibility that SHP-1 is a positive regulator of IL-4 responses in
mast cells. Pre-B-cell lines derived from me/me mice displayed enhanced and prolonged IL-4-induced Stat6 activation [37 ], supporting a role for SHP-1 in the deactivation of Stat6. In contrast, IL-4 induced Stat6 activation equally in the wild-type and me/me BMMC lines. Furthermore, the rate and magnitude of Stat6 deactivation were not altered in the absence of SHP-1. Thus, the role of SHP-1 in the regulation of IL-4 signal transduction in mast cells was quite distinct from that in B cells. In addition, mast cells must have alternative tyrosine phosphatases that are responsible for down-regulating Stat6 or can substitute for SHP-1. It is not surprising that different regulatory pathways may exist in mast cells since mast cells play a unique role in the immune response. Recently, IL-4 production in mast cells was shown to occur in the absence of c-maf, indicating that IL-4 transcription in mast cells is controlled by a more distinct mechanism than in T cells [51 ].
In BMMCs, IL-4 has been shown to inhibit FcRI expression through a
Stat6-dependent mechanism [14
]. Since SHP-1 is a
negative regulator of many cytokine pathways, we hypothesized that we
would observe an exaggerated response to IL-4 in me/me mast
cells. Surprisingly, despite normal IL-4-dependent Stat6 activation in
me/me BMMCs, IL-4-dependent negative modulation of FcRI
expression did not occur in the absence of SHP-1. Thus, Stat6
activation is not sufficient for IL-4-dependent modulation of FcRI
expression. Furthermore, our observations suggest that SHP-1 may also
act as a positive regulator and be required for the induction of
certain IL-4 responses. Recently, phosphatase activity was shown to be
required for the induction of IL-4-dependent induction of IL-4R in
lymphocytes [52
], supporting the possibility that
phosphatases can be positive regulators of IL-4 responses.
The mechanism of IL-4-mediated inhibition of FcRI expression and the
precise role of SHP-1 are unclear. Although Stat6 is required, it is
not sufficient. One possible mechanism is that IL-4 alters FcRI
expression by influencing the internalization and/or degradation
pathway of FcRI through a Stat6- and SHP-1-dependent mechanism.
SHP-1 has been shown to constitutively associate with FcRI and to
regulate tyrosine phosphorylation of the FcRIß and FcRI
subunits [49
]. The function of SHP-1 is unclear, but its
association with FcRI supports its role in FcRI physiology. It is
interesting that our data indicated that SHP-1 is not a negative
regulator of IL-4 signaling in BMMCs. Rather, SHP-1 is required for
IL-4-mediated FcRI down-modulation. These results demonstrate the
complexity and cellular specificity of these signaling pathways and
indicate a previously unrecognized role for SHP-1 in murine mast cells.
Received October 19, 2000; revised December 10, 2000; accepted December 12, 2000.
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receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells J. Immunol. 159,2911-2921[Abstract] receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc receptor I expression and mediator release J. Immunol. 162,5455-5465RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions J. Exp. Med. 185,663-672RI expression through a Stat6-dependent mechanism J. Immunol. 161,6915-6923 chain between receptors for IL-2 and IL-4 Science 262,1874-1877-stimulated activation of STAT transcription factors in HeLa cells J. Biol. Chem. 272,23376-23381 receptor I aggregation J. Immunol. 158,1353-1360[Abstract]-chain J. Immunol. 164,1211-1215This article has been cited by other articles:
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