gp49B1 Inhibits IgE-initiated Mast Cell Activation through Both
Immunoreceptor Tyrosine-based Inhibitory Motifs, Recruitment of
src Homology 2 Domain-containing Phosphatase-1, and
Suppression of Early and Late Calcium Mobilization*
Jennifer M.
Lu-Kuo
§¶,
David M.
Joyal§,
K. Frank
Austen
§, and
Howard R.
Katz
§
From the
Department of Medicine, Harvard Medical
School and the § Division of Rheumatology, Immunology and
Allergy, Brigham and Women's Hospital,
Boston, Massachusetts 02115
 |
ABSTRACT |
We define by molecular, pharmacologic, and
physiologic approaches the proximal mechanism by which the
immunoglobulin superfamily member gp49B1 inhibits mast cell activation
mediated by the high affinity Fc receptor for IgE (Fc
RI). In rat
basophilic leukemia-2H3 cells expressing transfected mouse gp49B1,
mutation of tyrosine to phenylalanine in either of the two
immunoreceptor tyrosine-based inhibitory motifs of the gp49B1
cytoplasmic domain partially suppressed gp49B1-mediated inhibition of
exocytosis, whereas mutation of both abolished inhibitory capacity.
Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1
and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone
marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with
gp49B1 within 1 min after coligation of gp49B1 with cross-linked
Fc
RI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of
gp49B1-mediated inhibition of Fc
RI-induced exocytosis at
concentrations of IgE providing optimal exocytosis, revealing a
central, but not exclusive, SHP-1 requirement in the counter-regulatory
pathway. Coligation of gp49B1 with cross-linked Fc
RI on mBMMCs
inhibited early release of calcium from intracellular stores and
subsequent influx of extracellular calcium, consistent with SHP-1
participation. Because exocytosis is complete within 2 min in mBMMCs,
our studies establish a role for SHP-1 in the initial
counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of Fc
RI-mediated exocytosis.
 |
INTRODUCTION |
The cross-linking of the high affinity Fc receptor for IgE
(Fc
RI)1 on the surface of
mast cells activates intracellular signaling pathways that lead to
exocytosis of preformed secretory granule components, generation of
membrane-derived lipid mediators, and elaboration of immunoregulatory
and proinflammatory cytokines. Through the release of their array of
bioactive mediators, mast cells have an unusually high proinflammatory
potential and contribute to host defense in animal models (1-3) and to
the pathogenesis of allergic reactions and bronchial asthma in humans.
In addition to Fc
RI, mouse mast cells express on their surface
gp49B1 (4, 5), an immunoglobulin (Ig) superfamily member with two
Ig-like domains (6) and two cytoplasmic immunoreceptor tyrosine-based
inhibition motifs (ITIMs), which have the amino acid sequence
(I/V/S)XYXX(L/V) (7-12). We have shown that when rat IgE bound to Fc
RI on mouse interleukin-3-dependent,
bone marrow-derived mast cells (mBMMCs) is both cross-linked to itself and coligated with rat anti-gp49B1 mAb bound to gp49B1, by means of
F(ab')2 mouse anti-rat IgG (heavy and light chain
reactive), the release of secretory granule- and lipid-derived
mediators is inhibited (9). As defined by ALIGN amino acid homology
analysis, gp49B1 belongs to a family of receptors that has
immunoregulatory potential (9, 13). In the mouse, the sole other member
of this homology-based family (14) that bears an ITIM is PIR-B (also
termed p91), which has six Ig-like domains and is expressed as mRNA
transcripts in mouse mast cells (15, 16). When chimeric receptors
possessing the cytoplasmic domain of PIR-B are expressed by
transfection in the rat mucosal mast cell-like cell line RBL-2H3 (17)
and coligated with cross-linked Fc
RI, the chimeric molecule becomes
tyrosine-phosphorylated and associates with the tyrosine phosphatases
SHP-1 and SHP-2. Early and late calcium fluxes, as well as exocytosis,
as assessed by the release of incorporated [3H]serotonin,
are inhibited (10, 11).
A number of human members of the family are encoded in a gene complex
on chromosome 19. Ig-like transcript 3 (ILT3, also termed HM18 and
leukocyte Ig-like receptor 5), was cloned based on its homology with
mouse gp49B1 (18) and by a separate strategy (19). Solid phase
anti-ILT3 induces the association of SHP-1 with ILT3, and
antibody-mediated coligation of ILT3 to certain cross-linked activating
receptors on monocytes inhibits early and late calcium fluxes (19). The
extracellular domain of another human relative of gp49B1, ILT2 (also
referred to as leukocyte Ig-like receptor 1 and monocyte-macrophage
Ig-like receptor 7) (20-22), interacts broadly with major
histocompatibility complex class I molecules derived from multiple
human leukocyte antigen loci (23), as does another gp49B1 relative,
ILT4/leukocyte Ig-like receptor 2 (24). Treatment of a B cell line with
the tyrosine phosphatase inhibitor pervanadate results in the
association of SHP-1 with ILT2, and antibody-mediated coligation of
ILT2 with cross-linked B cell antigen receptor inhibits early and late
calcium fluxes (25). In addition, co-immobilization of IgE and
anti-ILT2 on a solid phase inhibits serotonin release from RBL-2H3
transfectants expressing ILT2 (25).
Human killer cell inhibitory receptors (KIRs), which are primarily
expressed on natural killer (NK) cells and certain T cells, are also
ITIM-bearing members of the homology-defined family that includes
gp49B1 (9, 13). In contrast to ILT2, individual human KIRs bind major
histocompatibility complex class I molecules expressed by a particular
human leukocyte antigen locus. When KIRs bind major histocompatibility
complex class I on a target cell or are bound by anti-KIR Ig, they
become tyrosine-phosphorylated, recruit SHP-1, and impair both the
release of calcium from intracellular stores and the influx of
extracellular calcium (7, 26). Furthermore, active SHP-1 is required to
transduce KIR-mediated inhibition of antibody-dependent
cytotoxicity in NK cells (27). As predicted from the studies of NK
cells, ITIM-bearing cytoplasmic domains of KIRs transfected into B cell
or mast cell lines attenuate both phases of calcium mobilization when
coligated with the B cell receptor or Fc
RI, respectively, (28,
29).
We now demonstrate with site-directed mutagenesis that the ITIMs of
gp49B1 are critical to inhibition of Fc
RI-mediated mast cell
activation and that both tyrosines in the ITIMs of gp49B1 participate.
We also show that gp49B1, when tyrosine-phosphorylated, binds SHP-1 and
SHP-2 recruited from the cytoplasm of mBMMCs and that gp49B1-mediated
inhibition of Fc
RI-driven activation is reduced in
SHP-1-deficient mBMMCs. That inhibition of mast cell activation by
gp49B1 is accompanied by decreases in both the release of calcium from
intracellular stores and the subsequent influx of extracellular calcium
supports the role of SHP-1 in the proximal transmission of the
counter-regulatory signal.
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EXPERIMENTAL PROCEDURES |
Cells--
mBMMCs from BALB/c mice were grown as described (30).
mBMMCs from motheaten-viable and +/+ mice (The Jackson Laboratory) were
developed in methylcellulose before expansion in liquid culture as
described for mBMMCs derived from SHIP
/
mice (31). RBL-2H3 cells
were grown in Eagle's minimal essential medium (EMEM) with Earle's
salts supplemented with nonessential amino acids, 1 mM pyruvate, and 15% heat-inactivated fetal calf serum (enriched EMEM).
Antibodies--
Rat IgM anti-mouse gp49B1 mAb B23.1
(anti-gp49B1) was produced in ascites fluid induced in pristane-primed,
BALB/c nu/nu mice (The Jackson Laboratory) and was partially purified
by precipitation with ammonium sulfate. A rabbit antiserum directed to
a tyrosine-phosphorylated peptide consisting of amino acids 315-326 of
gp49B1, which encompasses the downstream ITIM
(anti-phosphogp49B1315-326), was prepared by QCB
(Hopkinton, MA). Rat IgE anti-dinitrophenyl mAb LO-DNP-30 (Zymed Laboratories Inc., South San Francisco, CA),
F(ab')2 mouse IgG anti-rat IgG (heavy and light chain
reactive), and fluorescein isothiocyanate-labeled and unlabeled goat
IgG anti-rat IgM (Jackson ImmunoResearch Laboratories, Avondale, PA),
mouse IgG anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake
Placid, NY), rabbit IgG anti-SHP-1 (Santa Cruz Biotechnology, Santa
Cruz, CA), rabbit IgG anti-SHP-2 (Santa Cruz Biotechnology), and rabbit
IgG anti-SHIP (gift of Dr. Gerald Krystal, British Columbia Cancer
Agency, Vancouver, British Columbia, Canada), horseradish peroxidase
(HRP)-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG
(Bio-Rad) were obtained as noted. Antibodies were coupled to
cyanogen bromide-activated Sepharose as described (5).
Preparation of Stable Transfectants of RBL-2H3
Cells--
Mutated gp49B1 cDNAs were generated by polymerase chain
reaction mutagenesis (32). The upstream and downstream primers
corresponded to nucleotides 1-21 and 1237-1257 of gp49B1,
respectively (33). The mutagenic primers possessed adenosine to
thymidine substitutions at nucleotides 920 and/or 986 to change
tyrosines to phenylalanines in the ITIMs. The wild-type and mutated
cDNAs were ligated into the pCR3 mammalian expression vector
(Invitrogen, San Diego, CA), and the sequences of all plasmid
constructions were determined by dideoxy sequencing to ensure that no
other mutations were introduced into the nucleotide sequences during
polymerase chain reaction. The cDNA constructs were released from
pCR3 with PmeI and XbaI and cloned into the
blunted SalI and native XbaI sites of pSR
neo (34).
For transfection, plasmid clones encoding gp49B1 with wild-type ITIMs
(clone W-8) or tyrosine-to-phenylalanine mutations in the upstream ITIM
(clone 1-6), downstream ITIM (clone 2-9), or both ITIMs (clone 3-20)
were linearized with ScaI. RBL-2H3 cells (5 × 106) were transfected with 20 µg of each linearized
plasmid by electroporation at 300 V and 960 microfarads in 0.5 ml of
EMEM. Two days after electroporation, transfected cells were selected
in enriched EMEM containing 0.75 mg/ml G418 (Life Technologies, Grand
Island, NY), and G418-resistant cells were cloned by limiting dilution
or with cloning cylinders. Once established, clones were maintained in enriched EMEM with 0.25 mg/ml of G418. Uncloned, bulk cultures of
transfectants were maintained in parallel in the same medium.
The expression of gp49B1 on the surface of each kind of transfectant
was assessed by flow cytometry. Cells (5 × 105) were
incubated in 50 µl of calcium- and magnesium-free Hanks' balanced
salt solution supplemented with 0.1% bovine serum albumin and 0.02%
sodium azide (HBA) alone or with a saturating amount of anti-gp49B1 for
30 min at 4 °C. Cells were washed with HBA, incubated for 30 min at
4 °C with fluorescein isothiocyanate-labeled F(ab')2
goat IgG anti-rat IgM, and washed again with HBA. For detection of
Fc
RI, cells were incubated with 5 µg/ml of mouse IgE and 5 µg/ml
of fluorescein isothiocyanate-labeled rat anti-mouse IgE mAb
(PharMingen, San Diego, CA) in the first and second steps, respectively. After staining, cells were resuspended in 2%
paraformaldehyde in Hanks' balanced salt solution and analyzed on a
Becton Dickinson FACSORT using logarithmic fluorescence amplification.
Fc
RI Cross-linking and Coligation with gp49B1 in RBL-2H3
Transfectants--
Duplicate samples of cells were suspended at 1 × 105/ml in enriched EMEM containing 0.5 µg/ml of rat
IgE and incubated overnight at 37 °C in 16-mm-diameter culture wells
(1 ml/well). The supernatant media were aspirated, and 0.3 ml of fresh
enriched EMEM containing the same concentrations of IgE, with a
saturating concentration of anti-gp49B1, was added to appropriate
wells. Cells were incubated on ice for 20 min, washed twice with PGC
buffer (0.025 M PIPES, 0.12 M NaCl, 0.005 M KCl, 0.04 M NaOH, 5.6 mM glucose,
0.1% bovine serum albumin, and 1 mM CaCl2),
and incubated with 0.3 ml of F(ab')2 mouse anti-rat IgG (25 µg/ml) for 40 min at 37 °C. The percentage of release of
-hexosaminidase (9, 35) elicited by the cross-linking of Fc
RI
alone and combined with coligation to gp49B1 was calculated for each
transfectant according to the formula (S/(S + P)) × 100, where S and P are the
mediator contents of the samples of each supernatant and cell pellet,
respectively. Cysteinyl leukotrienes were measured by reverse phase
high performance liquid chromatography as described (36).
Immunochemical Detection of gp49B1 Tyrosine Phosphorylation and
Association of Cytosolic Phosphatases--
Samples of mBMMCs (1 × 107) were pelleted by centrifugation and resuspended in
1 ml of enriched medium alone or containing 2.5 mM sodium
pervanadate (10 µl of a 1:1 mixture of 500 mM sodium orthovanadate and 3% hydrogen peroxide per ml). After a 10-min incubation at 37 °C, the cells were lysed in an extraction buffer containing 1% Nonidet P-40, 10 mM Tris, pH 7.4, 150 mM NaCl, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM pyrophosphate tetrasodium decahydrate, 5 mM EDTA, 4 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, and 20 µg/ml
N-acetyl-Leu-Leu-methioninal. Whole cell lysates were
centrifuged at 14,000 rpm for 20 min at 4 °C in a microcentrifuge to
sediment cell nuclei and debris, and the supernatants were incubated at
4 °C overnight with 70 µl of a 25% (v/v) solution of anti-gp49B1
coupled to Sepharose. The immunoprecipitates were washed four times
with extraction buffer, eluted from the Sepharose beads by boiling for
5 min in 150 µl of 1% SDS, and precipitated with 750 µl of cold
acetone. Precipitates were resuspended in 50 mM potassium
phosphate buffer, pH 7.0, containing 0.5% Nonidet P-40, 30 mM EDTA, 1 µM leupeptin, 1 µM
pepstatin, 0.2% SDS, and 2 units of N-glycosidase F
(Boehringer Mannheim) and were incubated overnight at 37 °C.
Reaction products were precipitated with cold acetone, and the pellets
were dried, resuspended in 25 µl of 1× SDS-polyacrylamide gel
electrophoresis (PAGE) sample buffer containing 5% 2-mecaptoethanol,
boiled for 5 min, and electrophoresed in 10% acrylamide gels under
reducing conditions. The resolved proteins were transferred to
Immobilon P membranes (Millipore, Bedford, MA) and immunoblotted
sequentially with anti-phosphotyrosine mAb 4G10 and
anti-phospho-gp49B1315-326, and immunoreactive proteins
were visualized with HRP-labeled goat anti-mouse IgG and HRP-labeled
goat anti-rabbit IgG, respectively, using chemiluminescence.
To assess the association of phosphatases with gp49B1 with and without
exposure of mBMMCs to pervanadate, gp49B1 was immunoprecipitated from
detergent lysates with anti-gp49B1 coupled to Sepharose as described
above for tyrosine phosphorylation of gp49B1, except that the
N-glycosidase F step was omitted. The non-deglycosylated immunoprecipitates were resuspended in SDS-PAGE sample buffer, boiled,
resolved on a 10% SDS-PAGE gel, transferred to Immobilon P membranes,
and immunoblotted sequentially with rabbit anti-SHP-1, anti-SHP-2, and
anti-SHIP. Immunoreactive bands were visualized with HRP-conjugated
goat anti-rabbit IgG and chemiluminescence.
To measure the association of enzymes with gp49B1 after its coligation
to Fc
RI, mBMMCs were incubated for 1 h at 4 °C with 3 µg/ml of rat IgE. A saturating amount of anti-gp49B1 was added for
the last 3 min before the cross-linking of Fc
RI and coligation to
gp49B1 with F(ab')2 mouse anti-rat IgG. Before and at
several times after the cross-linking and coligating step, samples of cells (1.5 × 107) were lysed in extraction buffer,
nuclei and debris were removed by centrifugation as described above,
and gp49B1 bound to anti-gp49B1 was immunoprecipitated from the lysates
with goat anti-rat IgM coupled to Sepharose. The immunoprecipitates
were processed as described above to resolve and visualize gp49B1 and
associated enzymes.
Calcium Measurements--
mBMMCs (1 × 107/ml)
were sensitized with 3 µg/ml rat IgE for 1 h at 4 °C and
incubated with 2.5 µM Fura-2 (Molecular Probes, Eugene,
OR) at 37 °C for 30 min. Medium alone or with a saturating amount of
anti-gp49B1 was added, and the cells were incubated for an additional
15 min at 4 °C. Cells were washed, resuspended in Hanks' balanced
salt solution containing 1 mM each CaCl2 and MgCl2, and placed in a fluorometer, and 25 µg/ml of
F(ab')2 mouse anti-rat IgG was added. In some experiments,
EGTA at a final concentration of 18 mM was added to
replicate samples before the addition of F(ab')2 mouse
anti-rat IgG to chelate extracellular calcium. Relative free cytosolic
calcium in the cells was measured by the ratio of fluorescence emission
intensities at 510 nm when the samples were exposed to excitation
wavelengths of 340 and 380 nm, respectively.
 |
RESULTS |
Analysis of the Requirement of ITIM Tyrosines in gp49B1-mediated
Inhibition of Mast Cell Activation--
RBL-2H3 cells were stably
transfected with cDNAs encoding either wild-type gp49B1 (YY) or
gp49B1 with tyrosine-to-phenylalanine mutations in the upstream (FY),
downstream (YF), or both ITIMs (FF) (Fig.
1A) to establish the
requirement for ITIMs in the inhibitory function of gp49B1. The
expression of gp49B1 and Fc
RI on transfected clones was assessed by
flow cytometry, and YY, FY, and YF clones were selected, for which the
net mean channel numbers of fluorescence for gp49B1 and Fc
RI ranged
from 43-48 and 78-81, respectively; the channel numbers were 84 and
147, respectively, for the closest available FF clone
(n = 2).

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Fig. 1.
Effects of tyrosine-to-phenylalanine ITIM
mutations on gp49B1-mediated inhibition of Fc RI exocytosis.
Clones of stable RBL-2H3 transfectants (1 × 107/ml)
expressing gp49B1 with either wild-type ITIMs (YY) or with
tyrosine-to-phenylalanine substitutions in the upstream
(FY), downstream (YF), or both (FF)
ITIMs (A) were activated by cross-linking 0.5 µg/ml rat
IgE bound to Fc RI with 25 µg/ml of F(ab')2 mouse
anti-rat IgG (heavy and light chain reactive) to establish a reference
value for uninhibited activation for each transfectant. Replicate cell
samples were coincubated with rat IgE and rat anti-mouse gp49B1 so that
added F(ab')2 mouse anti-rat IgG would both cross-link and
coligate receptors. B, the percentage of release of
secretory granule -hexosaminidase from each transfectant was
measured 40 min after Fc RI cross-linking with and without coligation
to gp49B1. Data are expressed as percentage of inhibition ± S.E.
of -hexosaminidase release obtained with coligation
(n = 3). The percentage of -hexosaminidase release
values for YY, FY, YF, and FF transfectants activated by cross-linking
IgE without gp49B1 coligation were 36 ± 6, 15 ± 2, 17 ± 6, and 25 ± 1%, respectively (mean ± S.E.,
n = 3).
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Cells were incubated with rat IgE (0.5 µg/ml), without or with a
saturating concentration of anti-gp49B1, and were washed. F(ab')2 mouse anti-rat IgG (25 µg/ml) was added both to
cross-link Fc
RI molecules sensitized with rat IgE to each other and
to coligate transfected gp49B1 molecules bearing rat IgM anti-gp49B1
with Fc
RI. The coligation of gp49B1 possessing wild-type ITIMs with cross-linked Fc
RI resulted in a 38 ± 10% (mean ± S.E.,
n = 3) inhibition of
-hexosaminidase release
compared with cells activated by Fc
RI cross-linking alone (Fig.
1B). The FY and YF mutations each partially suppressed
gp49B1-mediated inhibition of
-hexosaminidase release. In contrast,
mutation of both ITIM tyrosines abolished the ability of gp49B1 to
inhibit mediator release. Similar results were obtained with additional
clones expressing each type of gp49B1 (data not shown). Activation of
the cells with a 10-fold lower concentration of IgE afforded a greater
percentage of inhibition of exocytosis by the YY, FY, and YF clones,
but the FF clone remained fully noninhibitory (data not shown).
Tyrosine Phosphorylation of Native gp49B1 in
mBMMCs--
To demonstrate the tyrosine phosphorylation of endogenous
gp49B1 in mast cells, mBMMCs were incubated for 10 min at 37 °C in
enriched medium alone or containing sodium pervanadate, an inhibitor of
tyrosine phosphatases. Cell lysates were subjected to
immunoprecipitation with either anti-gp49B1 or a rat IgM negative control mAb, and precipitates were deglycosylated with
N-glycosidase F, resolved by SDS-PAGE, transferred to an
Immobilon P membrane, and immunoblotted sequentially with
anti-phosphotyrosine mouse mAb 4G10 and antibody from a rabbit
immunized with a tyrosine-phosphorylated peptide encompassing the
downstream ITIM of gp49B1 (anti-phospho-gp49B1315-326). In
the absence of pervanadate, a 37-kDa molecule immunoreactive with
anti-phospho-gp49B1315-326 (Fig.
2, lane 3, upper panel) but
not with anti-phosphotyrosine (Fig. 2, lane 3, lower panel) was present; 37 kDa is the size of the protein core of gp49B1 (5). When
cells had been treated with pervanadate, the apparent Mr of the band that was reactive with
anti-phospho-gp49B1315-326 increased to 40 kDa (Fig. 2,
lane 4, upper panel), concomitant with the acquisition of
immunoreactivity with anti-phosphotyrosine (Fig. 2, lane 4, lower
panel). Inasmuch as the 37- and 40-kDa bands were not detected in
negative control immunoprecipitates (Fig. 2, lanes 1 and
2, upper and lower panels), they represent nonphosphorylated and tyrosine-phosphorylated gp49B1, respectively.

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Fig. 2.
Effects of pervanadate on tyrosine
phosphorylation of gp49B1. mBMMCs (1 × 107/ml)
were incubated in medium with or without 2.5 mM pervanadate
for 10 min at 37 °C and were extracted in detergent buffer. gp49B1
was immunoprecipitated from extracts with anti-gp49B1 coupled to
Sepharose (lanes 3 and 4), and parallel extracts
were exposed to rat IgM mAb coupled to Sepharose as a negative control
(lanes 1 and 2). Precipitates were deglycosylated
with N-glycosidase F, resolved by SDS-PAGE, and transferred
to an Immobilon P membrane. The membrane was immunoblotted with mouse
anti-phosphotyrosine (anti-pY) mAb 4G10 and developed with
HRP-labeled goat anti-mouse IgG (lower panel). The membrane
was stripped and probed with rabbit
anti-phospho-gp49B1315-326 and developed with HRP-labeled
goat anti-rabbit IgG (upper panel). IP,
immunoprecipitation; IB, immunoblot.
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Binding of Phosphatases to gp49B1 ITIMs--
Phosphorylated
peptides encompassing each of the gp49B1 ITIM sequences bind the
tyrosine phosphatases SHP-1 and SHP-2, as well as the inositol
polyphosphate 5-phosphatase SHIP in detergent extracts of RBL-2H3 (37)
and mBMMCs (data not shown). To identify the preferred native
interaction(s), the ability of each enzyme to associate with
full-length gp49B1 expressed at the plasma membrane of mBMMCs was
assessed by stimulating tyrosine phosphorylation with pervanadate,
immunoprecipitating gp49B1 with anti-gp49B1 coupled to Sepharose,
resolving immunoprecipitates with SDS-PAGE, and immunoblotting with
each of the IgG anti-phosphatases. Pervanadate-mediated phosphorylation
resulted in the coprecipitation of the tyrosine phosphatases SHP-1
(Fig. 3, panel A, lane 1) and
SHP-2 (Fig. 3, panel B, lane 1) with gp49B1. Despite the
fact that mBMMCs contain SHIP (38), the enzyme did not
coimmunoprecipitate detectably with native gp49B1 after exposure of
mBMMCs to pervanadate (Fig. 3, panel C, lane 1).

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Fig. 3.
Binding of phosphatases to
tyrosine-phosphorylated, native gp49B1. mBMMCs (1 × 107/ml) were incubated in the absence (lanes 2 and 4) or presence (lanes 1 and 5) of
2.5 mM pervanadate for 10 min at 37 °C and extracted in
detergent buffer. gp49B1 was immunoprecipitated from extracts with
anti-gp49B1 coupled to Sepharose. Immunoprecipitates (lanes
1-3) and starting lysates (lanes 4 and 5)
were resolved by SDS-PAGE and transferred to an Immobilon P membrane;
the membrane was immunoblotted sequentially with rabbit anti-SHP-1
(A), anti-SHP-2 (B), and anti-SHIP
(C), using HRP-goat anti-rabbit IgG to visualize
immunoreactive bands in each sequential immunoblotting procedure.
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To extend these findings to the association of phosphatases with native
gp49B1 during physiologic inhibition of Fc
RI-mediated activation,
Fc
RI on mBMMCs were sensitized with rat IgE, and gp49B1 was bound
with rat anti-gp49B1; the cells were washed and extracted in detergent
before and at several times within 60 s of cross-linking and
coligation with F(ab')2 mouse anti-rat IgG. gp49B1 bound by
anti-gp49B1 was immunoprecipitated with goat anti-rat IgM-Sepharose,
eluted and resolved by SDS-PAGE, and analyzed for the presence of
coprecipitated phosphatases by immunoblotting. SHP-1 transiently
associated with gp49B1, with maximal association occurring at 30 s, after which there was a decrease at 45 and 60 s (Fig.
4). In four additional experiments,
maximal association of SHP-1 with gp49B1 occurred 15-45 s after
coligation. In all cases, the decrease in SHP-1 association at the
later time points was not the result of failure to immunoprecipitate
gp49B1, as judged by sequential immunoblotting with
anti-gp49B172-87 Ig (9)(data not shown). Several studies
with time points beyond 60 s were entirely negative for
coimmunoprecipitated SHP-1, and SHP-2 and SHIP were not detected in
association with gp49B1 at any of the time points within the first
60 s (data not shown).

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Fig. 4.
Association of SHP-1 with gp49B1 after
coligation with Fc RI. Five samples of
mBMMCs (1 × 107/ml) were incubated for 1 h with
3 µg/ml of rat IgE at 4 °C, and a saturating amount of anti-gp49B1
was added for the last 3 min. Cells were washed by centrifugation and
subjected to Fc RI cross-linking and coligation with
F(ab')2 mouse anti-rat IgG at 37 °C for 0-60 s. Samples
were extracted in detergent, and gp49B1/anti-gp49B1 complexes were
immunoprecipitated with goat anti-rat IgM coupled to Sepharose.
Immunoprecipitates were resolved by SDS-PAGE and transferred to an
Immobilon P membrane, which was immunoblotted with rabbit anti-SHP-1
and HRP-goat anti-rabbit IgG.
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Effects of SHP-1 Deficiency on gp49B1-mediated Inhibition of
Fc
RI-triggered Exocytosis in mBMMCs--
mBMMCs were cultured from
the bone marrow of motheaten-viable and +/+ mice and were activated by
the addition of F(ab')2 mouse anti-rat IgG to cross-link
and coligate the occupied Fc
RI and gp49B1 molecules. Coligation
suppressed the release of
-hexosaminidase from +/+ mBMMCs by
83-56% with increasing amounts of IgE, even though the percentage of
exocytosis was near or at a plateau level at all IgE concentrations
examined. For motheaten-viable mBMMCs, the gp49B1-mediated inhibition
fell from 60 to 0% in the presence of increasing IgE input, even
though the percentage of exocytosis in the absence of anti-gp49B1 was
near or at plateau levels throughout and indistinguishable from the +/+
mBMMCs (Fig. 5). Similar results were
obtained for the generation and release of the cysteinyl leukotriene,
LTC4, as measured by high performance liquid chromatography (n = 3; data not shown).

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Fig. 5.
Effects of SHP-1 deficiency on
gp49B1-mediated inhibition of exocytosis. mBMMCs generated from
motheaten-viable (me-v) (open symbols and
dotted lines) and wild-type (+/+) (closed symbols
and solid lines) mice were sensitized at 1 × 107/ml with incremental amounts of rat IgE alone or in the
presence of anti-gp49B1 and activated by adding F(ab')2
mouse anti-rat IgG to both cross-link and coligate the occupied
receptors, as described in Fig. 1. The data are expressed as the
percentage of -hexosaminidase released (mean ± S.E.;
n = 3 for 10 and 35 µg/ml and n = 4 for 15 and 25 µg/ml IgE).
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Effects of gp49B1/Fc
RI Coligation on the Mobilization of
Intracellular Calcium--
The cross-linking of rat IgE bound to
Fc
RI on the surface of mBMMCs with F(ab')2 mouse
anti-rat IgG elicited an early, steep rise in the intracellular calcium
level, beginning at 15 s and peaking at 90 s, followed by a
sustained period of elevated intracellular calcium (Fig.
6A). There was no response to
cross-linking gp49B1 in the absence of occupancy of Fc
RI by rat IgE
(data not shown). When Fc
RI was cross-linked and coligated with
gp49B1, there was no peak of calcium mobilization, and the level was
~30% of that achieved at 90 s with Fc
RI cross-linking alone
(Fig. 6A). In three experiments, including that depicted in
Fig. 6A, the peak early calcium flux after Fc
RI
cross-linking occurred at 97 ± 15 s (mean ± S.E.), and
cross-linking of Fc
RI with coligation to gp49B1 ablated the initial
peak and reduced the calcium flux at 97 s to 29 ± 2.7%
(mean ± S.E.) of that observed with cross-linking Fc
RI
alone.

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|
Fig. 6.
Effects of
Fc RI/gp49B1 coligation on intracellular
calcium mobilization in mBMMCs. A, mBMMCs (1 × 107/ml) were sensitized with rat IgE for 1 h at
37 °C in the absence and presence of a saturating amount of
anti-gp49B1, labeled with 25 µM Fura-2, and stimulated
with F(ab')2 mouse anti-rat IgG; the point of stimulation
is indicated by the closed triangle. Free cytosolic calcium
was detected by fluorometry. B, EGTA was added to replicate
cells (open triangles) to chelate extracellular calcium
before the addition of F(ab')2 mouse anti-rat IgG
(closed triangle).
|
|
In the presence of EGTA, the peak time of calcium increase with
cross-linking of Fc
RI alone occurred earlier (Fig. 6B) as a result of the inhibition of calcium influx (57 ± 3 s,
mean ± S.E., n = 3). Furthermore, cross-linking
of Fc
RI with coligation to gp49B1 in the presence of EGTA virtually
eliminated the increase in intracellular calcium that occurred with the
cross-linking of Fc
RI alone.
 |
DISCUSSION |
We have established that gp49B1 initiates a counter-regulatory
series of cellular responses within 60 s of coligation to
cross-linked Fc
RI on the surface of mast cells. Inasmuch as
exocytosis and lipid mediator generation are complete 120- 150 s
after Fc
RI cross-linking in mBMMCs (39), the rapid recruitment of
the tyrosine phosphatase SHP-1 to gp49B1 is consistent with the enzyme
initiating an effective silencing pathway for the immediate-onset
response of the mast cell to Fc
RI cross-linking. The known functions
of SHP-1 (26) are compatible with the capacity of gp49B1 to inhibit the
release of calcium from intracellular stores and the influx of
extracellular calcium. Taken together, our findings demonstrate that an
endogenous mast cell membrane protein can down-regulate IgE-mediated
mast cell activation in nontransformed cells via the actions of
recruited SHP-1.
The participation of gp49B1 ITIMs in the inhibition of mast cell
activation was established by mutagenesis of their tyrosine residues in
the context of native gp49B1. Inasmuch as the rat anti-gp49B1 does not
bind to RBL-2H3 cells, this rat mucosal mast cell line (17) was optimal
for transfection with wild-type and mutated mouse gp49B1 molecules. In
these transfectants, the native extracellular domain of wild-type and
mutant gp49B1 bound by anti-gp49B1 was coligated to Fc
RI sensitized
with rat IgE by means of F(ab')2 mouse anti-rat IgG, as
described previously for mBMMCs (9) and utilized here for studies of
calcium flux and SHP-1 deficiency in mBMMCs (Figs. 5 and 6). The
coligation of wild-type, transfected gp49B1 to cross-linked Fc
RI
inhibited the exocytosis achieved by the cross-linking of Fc
RI alone
(Fig. 1). Mutation of the tyrosines in both ITIMs abolished the ability
of coligated gp49B1 to inhibit
-hexosaminidase release, whereas
mutation of the tyrosines individually caused a partial loss of
inhibitory capacity (Fig. 1). When the PIR-B cytoplasmic domain was
expressed as a chimeric receptor by transfection of RBL-2H3 cells,
single tyrosine mutations in either of the two most downstream ITIMs
(Tyr-794 and Tyr-824) resulted in either no appreciable effect (10) or
partial loss of inhibitory function with each mutation (11); in yet
another profile of effects, there was a partial loss of inhibition of calcium flux with mutation of the Tyr-794 ITIM and no loss for mutation
of the Tyr-824 ITIM in a B cell line (12). The differences in these
findings, each of which resulted from studies that used a single
concentration of activating agonist, may relate to our findings that
both the inhibitory capacity of gp49B1 ITIMs bearing single ITIM
mutations and the SHP-1 independence of the native gp49B1
counter-regulatory mechanism decrease as the amount of IgE utilized for
activation via Fc
RI on the cell surface increases.
The ability of endogenous mast cell tyrosine kinases to phosphorylate
native gp49B1 in mBMMCs was established by inhibiting tyrosine
phosphatases pharmacologically with pervanadate (Fig. 2). When native
gp49B1 was phosphorylated by exposing mBMMCs to pervanadate,
SHP-1 and SHP-2 were coprecipitated detectably with tyrosine-phosphorylated gp49B1, as assessed by SDS-PAGE/immunoblotting (Fig. 3). Only SHP-1 coprecipitated detectably with gp49B1 after coligation with Fc
RI under conditions that inhibit mast cell activation (Fig. 4), although it is possible that SHP-2 and/or SHIP may
associate in amounts below their respective levels of immunochemical
detection. The association of SHP-1 with native gp49B1 was transient,
being detected only within 60 s of cross-linking of Fc
RI to
itself with coligation to gp49B1. Although time course data have not
been reported for the binding of SHP-1 to any of the human or mouse
relatives of gp49B1, their cross-linking to themselves, coligation to
an activating receptor, or binding to a natural ligand leads to their
association with SHP-1 when assessed 1-2 min later (7, 10, 19,
26-28). That the association of SHP-1 with gp49B1 is substantially
reduced from the maximum by 60 s after coligation with Fc
RI
(Fig. 4) may indicate that tyrosine-phosphorylated gp49B1 is a
substrate for SHP-1, inasmuch as certain phosphorylated ITIM peptides
can be substrates for SHP-1 in broken cell assays (40). The ability of
gp49B1 to inhibit Fc
RI-directed exocytosis (Fig. 5) in
SHP-1-deficient mBMMCs was absent at high inputs of IgE, indicating
that SHP-1 has a central role in mediating gp49B1-dependent inhibition of mast cell activation. However, there was substantial but
incomplete gp49B1-dependent inhibition in the
SHP-1-deficient mBMMCs at lower amounts of IgE that were still
sufficient for near-maximal or maximal IgE-dependent
exocytosis, which may reflect residual levels of SHP-1 activity but
more likely reveals that SHP-1 is not the only transmitter of
gp49B1-mediated inhibition of mast cell activation.
The intracellular calcium flux associated with the cross-linking of
Fc
RI to itself was markedly suppressed by coligation of Fc
RI to
gp49B1. Both the initial rate of release reflecting intracellular
stores and the magnitude of calcium increase were markedly attenuated
(Fig. 6A). In the presence of EGTA, the time of the peak
calcium increase after Fc
RI cross-linking decreased by ~40 s (Fig.
6B), and the magnitude of total increase in calcium was
markedly diminished, indicating that the peak at ~100 s in the
absence of EGTA reflects the sum of two overlapping curves representing
the release of intracellular calcium and the influx of extracellular
calcium, respectively. In the presence of EGTA, the cross-linking of
Fc
RI to itself with coligation to gp49B1 provided virtually no
calcium flux, again indicating that gp49B1 mediated inhibition of the
release of calcium from intracellular stores. The inhibition of NK
cell-mediated target cell lysis directed through KIRs and their
associated SHP-1 is also characterized by an absence of calcium release
from intracellular stores and lack of influx of extracellular calcium
(26).
Type IIb1 and IIb2 Fc receptors for IgG (Fc
RIIb1 and Fc
RIIb2),
which are the products of alternative splicing of mRNA from the
Fc
RII
gene (41), are Ig superfamily members that do not exhibit
significant amino acid sequence homology with gp49B1 (9) but that
nonetheless possess a single ITIM. We and others have shown that these
receptors are expressed on mouse mast cells (42, 43), and the
coligation of the Fc
RIIb species with cross-linked Fc
RI
suppresses mast cell mediator release through a process that depends on
ITIM phosphorylation (38, 44, 45). In contrast to gp49B1, the
inhibition of mBMMCs activation by Fc
RIIb1 and Fc
RIIb2 proceeds
through the inositol polyphosphate 5-phosphatase SHIP, resulting
primarily in inhibition of Fc
RI-induced sustained influx of
extracellular calcium, with little attenuation of the early increase in
free cytosolic calcium (38). This difference highlights a distinction
between the two inhibitory receptors in mast cells and may provide the
mast cell with a degree of selectivity in its inhibitory processes. In
addition, the fact that mouse mast cells express several ITIM-bearing
receptors of the Ig superfamily (gp49B1, Fc
RIIb1/b2, and PIR-B) is
consistent with the hypothesis that these cells are controlled by an
intricate network of inhibitory receptors. Inasmuch as gp49B1 and PIR-B
have two and six Ig-like domains, respectively, the specificity of
their SHP-1-transduced inhibition of exocytosis is likely regulated by
their different counterligands.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Lloyd Klickstein for advice
about transfection of RBL-2H3 cells, Dr. Cheryl Helgason regarding
development of mBMMCs in methylcellulose cultures, Dr. Bing K. Lam for
measuring sulfidopeptide leukotrienes, Dr. Andrew Scharenberg for
helpful discussions, and Jean Chambers and Nancy Xu for technical assistance.
 |
FOOTNOTES |
*
This work was supported by Grants AI-22531, AI-31599,
AI-41144, and HL-36110 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of a postdoctoral fellowship from the Arthritis Foundation.
To whom correspondence should be addressed: Brigham and
Women's Hospital, Div. of Rheumatology, Immunology and Allergy, Smith Bldg., Rm. 638A, 1 Jimmy Fund Way, Boston, MA 02115. Tel.:
617-525-1307; Fax: 617-525-1308; E-mail:
hrkatz{at}mbcrr.harvard.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
Fc
RI, high
affinity Fc receptor for IgE;
anti-gp49B1, rat IgM anti-mouse gp49B1
mAb B23.1;
EMEM, Eagle's minimal essential medium;
Fc
RIIb, Fc
receptors for IgG, type IIb;
Ig, immunoglobulin;
HRP, horseradish
peroxidase;
IL, interleukin;
ILT, Ig-like transcript;
ITIM, immunoreceptor tyrosine-based inhibition motif;
KIR, killer cell
inhibitory receptor;
mAb, monoclonal antibody;
mBMMC, mouse bone
marrow-derived mast cell;
NK, natural killer;
PAGE, polyacrylamide gel
electrophoresis;
SHIP, src homology 2 domain-containing
polyinositol 5-phosphatase;
SHP, src homology 2 domain-containing phosphatase;
RBL, rat basophilic leukemia.
 |
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