By
From the Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
Signaling through the high affinity receptor for immunoglobulin E (FcRI) results in the coordinate activation of tyrosine kinases before calcium mobilization. Receptors capable of interfering with the signaling of antigen receptors, such as Fc
RI, recruit tyrosine and inositol phosphatases that results in diminished calcium mobilization. Here, we show that antibodies
recognizing CD81 inhibit Fc
RI-mediated mast cell degranulation but, surprisingly, without
affecting aggregation-dependent tyrosine phosphorylation, calcium mobilization, or leukotriene synthesis. Furthermore, CD81 antibodies also inhibit mast cell degranulation in vivo as
measured by reduced passive cutaneous anaphylaxis responses. These results reveal an unsuspected calcium-independent pathway of antigen receptor regulation, which is accessible to engagement by membrane proteins and on which novel therapeutic approaches to allergic diseases could be based.
Crosslinking of high affinity receptor for immunoglobulin E (Fc In addition to activation events, receptor-activated PTKs
initiate the regulation of antigen receptor signaling by
phosphorylating tyrosine-based motifs on membrane receptors known as inhibitory receptors (19, 20). These proteins
bind SH2-containing tyrosine phosphatases (SHP-1 and
SHP-2), and the polyphosphatidylinositol (3,4,5) 5 In this report, we isolated mAbs that inhibited Fc Cell Culture, Reagents, and Antibodies.
The rat basophilic leukemia cell line (RBL-2H3) was cultured in Eagle's minimum essential medium supplemented with 16% heat-inactivated FCS, 2 mM
L-glutamine, and penicillin (100 U/ml)/streptomycin (50 µg
ml Production of Anti-RBL-2H3 Antibodies and Flow Cytometry.
Spleens from BALB/c immunized with whole RBL-2H3 cells were
fused with NS-1 myeloma cells and plated onto normal BALB/c
spleen feeder cells. To enhance the development of the hybridomas, Saccharomyces typhimurium mitogen (Ribi ImmunoChem Research, Inc., Hamilton, MT) was included in the culture medium
from days 0-10. Hybridoma supernatants were tested after day 14 by flow cytometry for binding to RBL-2H3 using FITC-conjugated
goat anti-mouse F(ab Serotonin Release and Leukotriene C4 Assays.
RBL-2H3 cells were
loaded with [3H]5-hydroxytryptamine ([3H]serotonin; 0.3 µCi/
105 cells) and saturated with DNP-specific IgE in 96-well microtiter tissue culture plates (105 cells well
RI)1-IgE complexes on mast cells and basophils by multivalent antigens initiates a signaling cascade
characterized by tyrosine kinase activation, calcium release
and influx and, later, by degranulation and release of inflammatory mediators (1). Like the B and T cell antigen
receptors, Fc
RI lacks endogenous signaling capacity and
uses tyrosine phosphorylation to recruit signaling effector molecules. Receptor aggregation leads to phosphorylation
and/or activation of several protein tyrosine kinases
(PTKs), Lyn, Syk, Btk, Itk, Fer, and FAK (1, 6), as
well as protein kinase C isoenzymes (9), MAP kinase (10),
and other signaling molecules such as Cbl and Shc (11, 12).
The precise role of many of these proteins in degranulation
remains undefined. However, it is clear that Fc
RI-mediated calcium mobilization, degranulation, and leukotriene
and cytokine synthesis depend on early tyrosine kinase activation events, especially the activation of the PTK Syk.
Fc
RI signaling is initiated by tyrosine phosphorylation of
immunoreceptor tyrosine-based activation motifs (ITAM;
defined by the sequence [D/E]x2Yx2Lx6-7Yx2[L/I]; references 13, 14), found in Fc
RI
and FcR
chains upon receptor aggregation (1, 3, 4). The primary function of
Fc
RI
is to amplify FcR
signals, as it has no autonomous
signaling capacity (4). Phosphorylated ITAMs facilitate
binding of src homology (SH) domain-containing proteins
to Fc
RI (15, 16). The dimeric FcR
phosphorylated ITAMs bind Syk via its tandem SH2 domains, leading to
Syk phosphorylation and activation (3, 4, 15, 16). The importance of Syk recruitment to calcium mobilization, degranulation, and leukotriene synthesis has been demonstrated
in mast cells lacking Syk expression or by introduction of
dominant negative Syk proteins. Fc
RI-mediated calcium
mobilization and degranulation are absent in Syk-negative mast cells despite the Fc
RI-mediated tyrosine phosphorylation of receptor subunits (17). In addition, expression of kinase-inactive Syk blocks Fc
RI-induced calcium release
from endoplasmic reticulum (ER) stores (3) and introduction
of kinase-negative Syk SH2 domains inhibits both degranulation and leukotriene release in Fc
RI-stimulated cells (18).
phosphatase (SHIP), upon coengagement with antigen or growth
factor receptors. Although the molecular targets are still being defined, phosphatase recruitment to inhibitory receptors has one of two general effects on signaling. Engagement of inhibitory receptors that preferentially bind SHIP,
such as the low affinity receptor for IgG (Fc
RIIb1; references 21, 22), results in selective inhibition of calcium influx with little or no effect on receptor-mediated calcium
release or tyrosine phosphorylation. On the other hand,
killer cell inhibitory receptors (KIR) bind SHP-1 upon receptor costimulation, resulting in reduced tyrosine phosphorylation, calcium release from the ER, and calcium influx (23, 24). In both mechanisms, calcium mobilization is
inhibited along with downstream signaling events.
RI-induced mast cell degranulation. Through protein isolation,
peptide sequencing, cloning, and gene expression, we have
identified CD81 as a novel inhibitory receptor for Fc
RI.
Anti-CD81 mAbs also inhibited passive cutaneous anaphylaxis (PCA) reactions, a model of IgE-dependent, mast cell
activation in vivo.
1) (Biofluids, Rockville, MD). NS-1 myeloma cells were cultured in RPMI-1640 supplemented with 20% FCS, glutamine,
and antibiotics. C1.MC/C57.1 cells were cultured as described
(25). DNP-human serum albumin (DNP-HSA) (30-40 mol
DNP/mol albumin) was purchased from Sigma Chemical Co.
(St. Louis, MO). DNP-specific IgE supernatants were used to saturate Fc
RI as described (26). For PCA experiments, MOPC 31c
(IgG1) and anti-DNP mouse IgE (clone SPE-7) were purchased from Sigma Chemical Co. and anti-rat
2 integrin (anti-LFA-1
, CD18; clone WT.3) was purchased from PharMingen (San Diego, CA). MOPC 31c and anti-DNP IgE were dialyzed to remove sodium azide before in vivo injections. Anti-rat CD81
(5D1, IgG1) was purified from ascites on protein G-Sepharose
(Pharmacia, Uppsala, Sweden).
)2-specific antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and analyzed by flow
cytometry on a FACScan® flow cytometer (Becton Dickinson, San
Jose, CA). From three separate fusions, a total of 2,160 wells were
plated and 622 supernatants from wells with hybridoma growth
were screened by FACS® for reactivity with RBL-2H3 cells. In
all, 283 of 622 (45%) elicited detectable reactivity by FACS® with
membrane antigens of RBL-2H3 (data not shown). The screening of RBL-2H3-reactive mAbs by serotonin release assay lead to
the identification of 1A12 (IgG2b) and 5D1 (IgG1), which were
characterized further. Rat CD81 transfectants of C1.MC/C57.1
cells were stained with purified 1A12 and 5D1 (1 µg/106 cells),
counterstained with goat anti-mouse F(ab
)2-specific antibody, and
analyzed by flow cytometry on a FACScan® flow cytometer.
1, 37°C, 5% CO2) as described (27, 28). Monolayers were washed three times with buffer
(glucose-saline, Pipes buffer (pH 7.2) containing (in mM) 25 Pipes, 110 NaCl, 5 KCl, 5.6 glucose, 0.4 MgCl2, 1 CaCl2, and
0.1% BSA; and 25 µl of a dilution of purified antibody was added
to the monolayers and plates were incubated for 30 min (or as indicated) at room temperature. Triggering of Fc
RI was performed
by the addition of DNP-HSA (final concentration 10-250 ng
ml
1) and plates were incubated at 37°C (30 min except as indicated in Fig. 1 D) with control samples present on each plate. Degranulation was stopped by placing the plates on ice and by the
addition of 150 µl of cold culture medium/well. 100-µl aliquots
were taken from replicate wells for scintillation counting. Total
cellular incorporation was determined from 1% SDS, 1% NP-40
lysates.
Fig. 1.
Effect of preincubation of purified mAb 5D1 on FcRI-mediated degranulation in RBL-2H3 cells. (A-D) Degranulation of IgE-saturated, RBL-2H3 cells after incubation with buffer (closed circles) or purified 5D1 mAb (mouse IgG1) at 2.5 ng (closed squares), 25 ng (closed triangles), or 50 ng (closed inverted triangles) (A, C, D) or with 100 ng (B) of 5D1
mAb per 105 cells before triggering with the indicated concentrations of DNP-HSA (A), 50 ng/ml
1 DNP-HSA (C and D), or with PMA and
ionomycin (B). Cells were preincubated with 5D1 or buffer for 30 min
(A, B, D) or for the indicated times (C) at room temperature before triggering for 30 min (A-C; 37°C, 5% CO2) or as indicated (D). The data
shown are representative of >10 experiments with the 5D1 mAb. Data
are expressed as mean dpm ± standard deviation or as percentages of control (no antibody) mean dpm. Statistical significance versus untreated controls was determined using an unpaired Student's t test: *, P <0.05; **, P
<0.01; ***, P <0.001 for A. All data points in C and D were found to be
significantly different from controls (P <0.02) with the exception of the
5-min preincubation time point with 2.5 ng mAb 5D1 (C, P = 0.067).
[View Larger Version of this Image (39K GIF file)]
1 DNP-HSA. Supernatants were stored at
80°C until measurement of LTC4 by specific enzyme immunoassay (Cayman Chemical, Ann Arbor, MI).
Immunoaffinity Chromatography, Electrophoresis, and Western Blotting.
RBL-2H3 cells were cultured in routine culture medium
in spinner flasks to a cell density of ~106/ml, harvested by centrifugation, and washed twice with cold PBS. Washed cells (1010
RBL-2H3 cells were obtained from spinner flasks [12 l]) were extracted in 0.5 M K2HPO4 (pH 7.5) with proteinase inhibitors (10 µg/ml pepstatin, 5 µg/ml leupeptin, and 10 µg/ml aprotinin) at
5.0 × 107 ml1 (60 min, 4°C) with frequent mixing. n-octylglucoside (10 mM) was added during the extraction to ensure protein solubility. Postnuclear lysates were prepared by centrifugation at 15,000 g (20 min, 4°C), desalted, and passed several times
over protein G-Sepharose coupled to 1A12 (2 mg ml
1 bed volume), washed with PBS (10 mM n-octylglucoside), and eluted with 0.2 M glycine (pH 2.3). Tris-neutralized, concentrated extracts were reduced with
-mercaptoethanol, resolved on 12.5%
preparative SDS-PAGE, and transferred to ImmobilonSQ (Millipore, Bedford, MA). The membrane was stained with amido black and the Mr 25-kD band was excised, eluted, alkylated, and digested overnight with Lys-C. Peptides were separated by reverse phase-HPLC and the peptide peak eluting at 36 min was sequenced.
Construction and Screening of RBL-2H3 cDNA Library in UNI-ZAPTM. Poly (A)+ messenger RNA was isolated from RBL-2H3, reverse-transcribed into cDNA, size-fractionated on Sephacryl S-500 spin columns, and ligated into UNI-ZAP-XR lambda vector according to the instructions of the manufacturer (Stratagene, La Jolla, CA). After rescue of the cDNA inserts and appropriate restriction enzyme digests, it was determined that 96% of the plamids contained inserts, with an average size of 1.7 kB. 5 × 105 plaques were screened with 32P-labeled mouse CD81 cDNA probe. After hybridization, nitocellulose filters were washed one time with 2× SSC containing 0.1% SDS (room temperature) and three times with 0.5× SSC containing 0.1% SDS at 50°C. Filters were autoradiographed and plaques picked and eluted. Candidate plaques were subjected to three additional rounds of plaque purification before rescue the cDNA inserts into pBluescript. Sequencing was performed on eleven isolates and all were found to align with EMBL/GenBank/DDBJ accession number U19894 isolated from rat brain (29).
Transfections. Rat CD81 cDNA from two isolates was subcloned into the pBJ1neo expression vector (4) and 20 µg of ethanol-precipitated DNA was used for electroporation of C1.MC/ C57.1 cells (1,050 µF, 270 V). Selection of stable transfectants was initiated 48 h later by replating at 500-10,000 cells/well with 2 mg/ml G418 (GIBCO BRL, Gaithersburg, MD).
Confocal Microscopy.
After overnight adherence and saturation
of FcRI with DNP-specific IgE, RBL-2H3 cells were washed
with buffer and incubated with 3 µM fluo3/AM (Molecular
Probes, Eugene, OR) and 0.2 mg/ml Pluronic (Molecular Probes)
at 37°C for 30 min (5% CO2) in a buffer containing 140 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose,
and 1 mM Na-Hepes (pH 7.4). Dye-loaded cells were then washed once with the same buffer before preincubation (30 min, room temperature) with buffer (± 5D1, 1 µg/chamber/105 cells)
and triggering with 100 ng/ml DNP-HSA. Ca2+ measurements
in single cells were monitored using a laser-scanning confocal microscope (LSM4, Zeiss, New York) equipped with an argon/kryton laser to excite the dye at 488 nm. Fluorescence emission
above 510 nm was measured after placing a long pass filter in
front of the photomultiplier tube. The confocal system was used
in slow scan mode and fluorescence images were collected every
5 s. Fluo-3 fluorescence measurements were normalized by dividing the average fluorescence intensity (F) occurring during the
course of the experiment to the average fluorescence intensity determined at the beginning of the experiment (F0). All measurements were performed at 22-24°C.
Passive Cutaneous Analphylaxis in Rats.
Male Wistar rats (275-
300 g) were used in these experiments. Rats were first anesthetized with ether, back skin hair was shaved, and rats were injected
intradermally with 50 µl containing 100 ng anti-DNP IgE or 25 ng anti-DNP-IgE mixed with 50 µg of MOPC 31c (mouse
IgG1, specificity unknown) or 5D1 (mouse IgG1; anti-rat
CD81). Control sites received buffer alone (PBS containing 10 µg/ml1 mouse serum albumin; Sigma Chemical Co). Sites were
marked on the skin for orientation and rats that received 100 ng
anti-DNP injections received a second injection 21 h later with
50 µg of 5D1 or anti-rat LFA-1
(CD18; mouse IgG1) into previously injected sites. Sites receiving IgE and IgG1 were injected
in triplicate on the same rat. 24 h after IgE injections animals received 1 ml of 1 mg/ml DNP-HSA containing 1% Evan's blue
dye injected intravenously under ether anesthesia. 30 min after
intravenous injection, rats were killed and punch biopsies (2.5 cm2) were obtained, minced, and extracted three times with hot
formamide (80°C, 3 h) (32). Pooled samples from tissue sites were
centrifuged and absorbance at 610 nm was measured. A610 values
were converted to micrograms of Evan's blue dye based on a
standard curve of dilutions of Evan's blue in formamide.
To identify membrane proteins capable of regulating
FcRI signaling, we produced mAbs to the rat basophilic
leukemia (RBL-2H3) cell line and identified antibodies
that inhibited Fc
RI-mediated degranulation. As shown in
Fig. 1 A, pretreatment of anti-DNP IgE-saturated RBL-2H3 cells with purified mAb 5D1 inhibited Fc
RI-mediated degranulation by 75% as measured by release of granule-stored [3H] serotonin. Blockage of serotonin release
was significant (*, P <0.05) even at subsaturating concentrations of 5D1 (2.5 ng mAb/105 cells; Fig. 1 A; data not
shown). 5D1-mediated inhibition was specific for Fc
RI
signaling as degranulation induced by PMA and calcium
ionophore ionomycin was unaffected (Fig. 1 B). Furthermore, maximal inhibition of Fc
RI-mediated degranulation by mAb 5D1 required only brief periods of preincubation (Fig. 1 C) and inhibition was sustained for at least 1 h
of antigen stimulation (Fig. 1 D).
Next, we sought to identify the protein recognized by
the degranulation-inhibitory 5D1 mAb. 5D1 and a second
degranulation-inhibitory mAb (designated 1A12) recognized proteins of Mr 25 kD (Fig. 2 A; data not shown).
5D1 and 1A12 blocked each others' binding to RBL-2H3
cells, although neither mAb inhibited IgE binding and, conversely, saturation of FcRI with IgE had no effect on
1A12 binding (data not shown), suggesting that (a) 1A12
and 5D1 recognized the same protein (see Fig. 2 C) and (b)
Fc
RI and the 1A12/5D1 antigen were not colocalized on
the cell membrane. Because mAb 1A12 was more effective
at immunoprecipitation and on Western blots, it was used
for protein purification. Batch preparations of RBL-2H3
extracts were immunoprecipitated with mAb 1A12, resolved on preparative SDS-PAGE, and transferred to nitrocellulose for protein sequencing. In Fig. 2B, peptide sequence
obtained from Lys-C digests of 1A12 immunoprecipitates is
shown aligned with homologous sequences from mouse
and human CD81. Based on these data, we cloned rat
CD81 from a RBL-2H3 cDNA library using mouse CD81
cDNA as a probe and expressed the cDNA in the mouse
mast cell line C1.MC/C57.1 (25). As shown in Fig. 2 C,
both degranulation-inhibitory mAbs 1A12 and 5D1 recognized rat CD81.
CD81 belongs to the transmembrane 4 superfamily
(TM4SF) which includes CD9, CD53, CD63, and CD82
(33). It is broadly expressed on hematopoietic cells (T and
B lymphocytes, granulocytes, monocytes) and on some
nonlymphoid tumors. The function of CD81 (or other TM4SF proteins) is incompletely understood although
CD81 appears to modulate the signaling of other membrane receptors. CD81 is found in the CD19-CD21 complex on B cells and mAbs to CD81 or CD19 have been reported to reduce the threshold for B cell receptor signaling
(34) and enhance B cell adhesion via VLA4 (35). Consistent with a costimulatory role in B cell receptor signaling, CD81 /
mice express lower levels of CD19 on B cells,
which is proposed to contribute to a defect in humoral immunity (36). For T lineage cells, both stimulatory and inhibitory activities for anti-CD81 mAbs have been reported
(37). CD81 ligation enhances IL-4 production from
antigen-specific CD4+ T cells (37), and integrin activation
and IL-2-dependent proliferation in human thymocytes
(38). Alternatively, CD81 was originally called TAPA-1
(target of antiproliferative antibody) based on inhibition of
proliferation in human T cell lines by CD81 antibodies (39). Some of these pleiotropic effects may stem from the
potential signaling molecules with which CD81 has been
reported to associate, including CD4, CD8, MHC class II,
other TM4SF proteins, integrin VLA4, and phosphatidylinositol 4 kinase (33, 41).
To target the site of CD81 inhibition of degranulation,
we next examined the effect of CD81 antibodies on the
earliest events of FcRI signal transduction, i.e., tyrosine
phosphorylation of proteins by activated, nonreceptor tyrosine kinases including Lyn and Syk, and calcium mobilization (1). In these experiments, IgE-saturated RBL-2H3 cells were pretreated with purified anti-CD81 before
triggering with DNP-HSA for the indicated periods of
time, followed by extraction and immunoprecipitation of
total tyrosine-phosphorylated proteins. As shown in Fig. 3
A, no major changes in the pattern of Fc
RI-induced tyrosine phosphorylation were detected with anti-CD81 treatment before antigen triggering. Although not shown
in Fig. 3 A, incubation of RBL-2H3 cells with 5D1 alone
(no antigen triggering) did not induce detectable tyrosine
phosphorylation. This observation in RBL-2H3 cells contrasts with anti-CD81-induced tyrosine phosphorylation induced in B cells, the latter probably signaling through the CD19-CD21 complex (45).
The effect of anti-CD81 on FcRI-induced calcium
mobilization was monitored on individual, adherent RBL-2H3 cells by confocal microscopy in cells loaded with calcium dye fluo-3. As shown in Fig. 3 B, no inhibition of
Fc
RI-induced calcium mobilization in anti-CD81-treated
versus controls was observed by confocal microscopy, despite inhibition of degranulation under these conditions (Fig. 3 C). Anti-CD81 pretreatment had no effect on calcium release from intracellular stores in cells triggered in
Ca2+-free buffer containing 0.5 mM EGTA or on pretriggering baseline values (data not shown). Similar results
were also obtained with RBL-2H3 triggered through
Fc
RI in suspension using a spectrophotometer (data not
shown). In separate experiments, anti-CD81 mAb 5D1 did not inhibit leukotriene C4 (LTC4) production induced by
DNP-HSA/IgE stimulation (Fig. 3 D). LTC4 production
is dependent on activation of phospholipase A2 (tyrosine
kinase and calcium dependent) and is regulated by PMA-sensitive, protein kinase C isozymes (46, 47). These data
suggest that CD81 acts independently of early tyrosine
phosphorylation and calcium mobilization events that are
critical for mast cell degranulation.
These results were unexpected in light of the reported
modes of action of other inhibitory receptors. These proteins fall into two major classes: type I, transmembrane proteins that are members of the Ig superfamily (FcRIIb1,
KIR, CTLA-4, CD22, gp49b1, paired Ig-like receptors,
signal-regulatory proteins) and type II, transmembrane,
C-type lectins (e.g., Ly-49, NKG2A, mast cell function-
associated protein) (21, 48). Inhibitory receptors share
a cytoplasmic motif, the immunoreceptor tyrosine-based
inhibitory motif (ITIM consensus sequence V/Ix2Yx2I/L;
references 20, 48), which is a target for tyrosine phosphorylation during receptor activation. Phosphorylated ITIMs
bind the phosphatases SHP-1, SHP-2, or SHIP, and physical associations with these phosphatases and/or functional
evidence of these associations (tyrosine dephosphorylation and/or decreased calcium mobilization) have been demonstrated in all inhibitory receptors that have been characterized (21, 48, 49, 52).
CD81 differs from these inhibitory receptors in three
important ways. First, unlike other inhibitory receptors,
CD81 inhibits FcRI-mediated degranulation while leaving both tyrosine phosphorylation and calcium mobilization apparently unaffected. Although these results cannot
exclude a very selective inhibition of kinase activity by
CD81 antibodies, it is clear that no detectable effect is
found on tyrosine kinase-sensitive calcium mobilization or
LTC4 production. Second, CD81 belongs to a different
structural class of proteins than the other inhibitory receptors. CD81 is a TM4SF protein with four transmembrane
spanning segments, two extracellular loops, two short cytoplasmic tails, and a short intracellular loop between transmembrane segments 2 and 3 (33). Third, the cytoplasmic tails of CD81 lack ITIM motifs. Although there is an
ITIM-like sequence (GCYGAI) in the short intracellular
loop between transmembrane segments 2 and 3, there is no
evidence that this site is phosphorylated by tyrosine kinases
or capable of binding to SH2 domains.
To assess the activity of anti-CD81 in FcRI signaling in
normal mast cells, we chose the PCA model, a classical system for studying mast cell activation in vivo (32, 56). In
these experiments, rats were injected intradermally with
anti-DNP IgE mixed with anti-CD81 mAb 5D1 (IgG1) or
with class-matched mouse IgG1 as controls (Fig. 4 A). Additional rats received anti-DNP IgE alone into the skin at
time 0, followed by a second injection (buffer, 5D1, or anti-rat LFA-1
[IgG1]) (Fig. 4 B) into IgE-injected sites
21 h after IgE injections. 24 h after IgE priming, rats received 1 mg of antigen intravenously (DNP-HSA containing 1% Evan's blue dye). Mast cell activation through
Fc
RI in PCA results in the release of several vasoactive
substances, which act to increase vascular permeability, a
property that is quantified by local accumulation of the
Evan's blue dye from the vasculature into the sites of IgE
injections. These results are expressed as micrograms of
Evan's blue dye converted from A610 measurements of formamide-extracted tissue biopsies (32). As shown in Fig. 4
A, coinjection of anti-CD81 mAb 5D1 during IgE priming
significantly inhibited IgE-dependent PCA reactions (P = 0.024) compared with class-matched controls. To limit the
possibility of nonspecific suppression of PCA reactions due
to tissue deposition of IgG1 mAbs, we repeated these experiments by injecting anti-CD81 mAb 5D1 or anti-LFA-1
(CD18) into the IgE-injected sites 3 h before antigen administration. LFA-1
is expressed on mast cell lines including
RBL-2H3, whereas anti-LFA-1
has no effect on Fc
RI-mediated degranulation in RBL-2H3 cells (57; data not
shown). Similar to coinjection of IgE and IgG1 mAbs, separate injections of anti-CD81 yielded significant inhibition of PCA reactions compared with anti-LFA-1
controls
(Fig. 4 B).
In conclusion, we have demonstrated that CD81 is a
novel inhibitory receptor for FcRI. The observation that
CD81 acts on calcium-independent events required for
mast cell degranulation distinguishes CD81 from previously
described inhibitory receptors, such as Fc
RIIb1 and KIR,
that act upstream of calcium influx. Anti-CD81 mAbs also
inhibited IgE-dependent PCA reactions, which suggests
the CD81 pathway is present in normal mast cells and capable of being engaged to inhibit mast cell responses in
vivo. Therefore, the pathway engaged by CD81 is a candidate for therapeutic strategies aimed at intervention of allergic responses.
Received for publication 7 July 1997.
Address all correspondence to Dr. Jean-Pierre Kinet, Department of Pathology, Laboratory of Allergy and Immunology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215. Phone: 617-667-1324; FAX: 617-667-3616; E-mail: jkinet{at}mercury.bidmc.harvard.edu. The present address of F. Van Laethem is Free University of Brussels, 1180 Brussels, Belgium.We thank Dr. Shoshana Levy for mouse CD81 cDNA, Dr. Lan Bo Chen for use of the confocal microscope, and Dr. Marie-Hélène Jouvin for critical reading of the manuscript.
This work was supported in part by USPHS grants CA/AI-72074 and AI/CA-23990 (S.J. Galli) and GM-53950 (J.-P. Kinet, T.J. Fleming, by fellowships from the Human Frontier Science Program (E. Donnadieu), the Korea Science and Engineering Foundation (C.H. Song), and the Beth Israel Hospital Pathology Foundation.
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