Fc receptor ß subunit is required for full activation of mast cells through Fc receptor engagement
Shuichi Hiraoka,
Yasuko Furumoto,
Haruhiko Koseki1,
Yohtaro Takagaki2,
Masaru Taniguchi1,
Ko Okumura and
Chisei Ra
Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
1 Division of Molecular Immunology, Center for Biomedical Science, School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ku, Chiba-City 260, Japan
2 Department of Immunolgy, Mitubishi Kasei Institute of Life Sciences 11 Minami-ooya Machida-City, Tokyo 194, Japan
Correspondence to:
C. Ra
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Abstract
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The high-affinity IgE receptor (Fc
RI) and the low-affinity IgG receptor (Fc
RIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common ß subunit (FcRß) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRß is essential for the cell surface expression of the Fc
RI. In humans, the FcRß gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRß in vivo still remains ambiguous. To elucidate the functions of FcRß, we developed the mice lacking FcRß [FcRß(/)]. The FcRß(/) mice lacked the expression of the Fc
RI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRß(/) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRß(/), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRß accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues.
Keywords: FcRß knock-out mouse, Fc
RI, Fc
R, immunoreceptor tyrosine-based activation motif, mast cell
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Introduction
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Activated mast cells through the Fc
RI and the Fc
RIII on the cell surface release various inflammatory mediators and cytokines, leading to the typical allergic reaction. Both receptors share the common ß (FcRß) (16) and the common
(FcR
) (7,8) subunits which contain the signal-transducing motif called the immunoreceptor tyrosine-based activation motif (ITAM) in their intracellular C-terminal domains (9,10). The expression of FcRß is restricted in mast cells and basophils, while FcR
is also found on a variety of other cells (7). Thus the FcRß seems to play some specific roles in mast cells and basophils. In rodents, FcRß was shown to be essential for the cell surface expression of Fc
RI by studies to reconstitute functional receptors in Fc
RI cells (3). In humans, the FcRß gene is nominated as one of the candidate genes causing atopic diseases based on family analyses at the genetic level (11,12). Some alleles deducing the substitutions of amino acids in FcRß were also detected (13,14) although the relationship between these substitutions and the pathogenesis of atopic diseases is not yet as clear as the function of the FcRß.
While several approaches to elucidate the functions of FcRß in the Fc
RI complex have been performed, little has been carried out for the FcRß in the Fc
RIII complex on mast cells. The analyses of the reconstituted functional Fc
RI, and the analyses of the chimeric receptors containing C-terminal of FcRß and FcR
were reported, but they could not explain the functions of FcRß sufficiently. The human Fc
RI without FcRß still have the potential for cell activation, although a defect of FcR
causes loss of the cell surface expression of Fc
RI and cell activation (15). The chimeric molecules of FcRß containing intact ITAM could not activate cells by their aggregation (16,17). The detection of the FcRß-associated molecules led to different conclusions (1824). One model proposed on signal transduction via Fc
RI (18) that Lyn (a src family protein tyrosine kinase) bound to the ITAM of FcRß is activated by dephoshporylation of its C-terminal phosphorylated regulatory tyrosine on cross-linking of the Fc
RI, following the phosphorylation of the ITAM of FcRß and FcR
, and the activation of the other protein kinase, Syk, which is essential for mast cell activation (19,20). One reconstitution model to clarify the functions of FcRß in the Fc
RI complex indicated that FcRß augmented Syk phosphorylation and the authors concluded that its major function was the amplification of signals mediated by the Fc
RI
(21). However, the above-mentioned models are not sufficient to explain the roles of FcRß for the following reasons. Bone marrow-derived cultured mast cells (BMMC) from Lyn-deficient mice displayed almost normal phenotypes upon Fc
RI engagement, although the tyrosine phosphorylation of the intracellular proteins and the flux of Ca2+ in these cells were impaired (22). Furthermore, some molecules associated with FcRß, such as SH-2-bearing protein tyrosine phosphatase (SHP-1 and SHP-2) (23), which dephosphorylates the phosphorylated ITAM, and SH2-bearing inositol-5'-phosphatase (SHIP) inhibit cell activation through Fc
RI (24).
To clarify the functions of FcRß, we generated targeted-mutant mice for FcRß which did not produce any fragments of FcRß. The mice showed abnormal phenotypes in mast cells and anaphylaxis via Fc receptors. In this study, we tried to clarify the specific roles of FcRß in the Fc
R complex on mast cells.
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Methods
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Generation of FcRß-deficient mice
To produce the complete null mutant of the FcRß we chose the first exon as the targeting locus which contains the translation initiation codon (see Fig. 1
). The BamHIPstI genomic fragment containing from the second to a part of the fifth exon and the fragment of EcoRIEcoRV located 5' upstream of the translation initiation codon were isolated from the 129/sv genomic library (Stratagene, La Jolla, CA). Then, each fragment was attached to the 5' and the 3' flanks of the PGK-neo expression cassette (Neo) (25) in pBluescript SK (Stratagene). The HSV-tk expression cassette (26) was inserted into its SalI site and the BamHI site of Neo was disrupted by blunting. The obtained plasmid was used as the targeting vector.

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Fig. 1. Targeted inactivation of the FcRß gene by deletion of the first exon. (A) Genomic structure and restriction map of the FcRß locus. Exons are denoted by rectangles and protein-encoding sequences are filled. The restriction sites are: Ba, BamHI; Bg, BglII; Ec, EcoRI; Ev, EcoRV; P, PstI. Probe A is a 0.4 kb BglIIEcoRV fragment of the 5' upstream of start codon, and probe B is a 0.3 kb fragment of the PCR product between the fifth and the sixth exon (see Methods). (B) Targeting vector. The 0.5 kb fragment between the EcoRV site 5' upstream of the translation initiation site and the BamHI site of 3' down stream that was replaced with the PGK-neo expression cassette (Neo). The HSV-tk gene was added to its flanking SalI site in the cloning site of the plasmid pBluescript SK in which the deletion construct was cloned. (C) The predicted structure of the FcRß locus following homologous recombination with the targeting construct. (D) Southern blot analysis of the targeted ES cell clones. Genomic DNA (10 µg) from clone 126, 74 and from control R1 ES cells was digested with BglII and hybridized with probe B (left panel). The 5.5 kb band represents the wild-type allele and the 7.7 kb band is the mutant allele. Genomic DNA was digested with BamHI and hybridized with probe A (right panel). The 2.8 kb band is the wild-type allele and the 13.5 kb band is the targeted allele. (E) Southern blot analysis of the tail DNA. Genomic DNA was isolated from the tail tips of offspring of heterozygous intercrosses. The analysis of DNA was performed the same as in (D).
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Embryonic stem (ES) cell culture and electroporation were performed as previously described (27). The linearized targeting vector was introduced into one ES cell line, R1 (28), by electroporation. The clones resistant to G418 sulfate (Gibco/BRL, Tokyo, Japan) and gancyclovir (Syntex Research, Palo Alto, CA) were obtained after 9 day cultivation, and then their genomic DNA was extracted and analyzed by Southern blotting. The homologous recombinant clones were developed to chimeric mice through aggregation with eight cells from BDF1 mice and homozygous mice were obtained by breeding of their offspring as described previously (28).
Southern blot analysis
Genomic DNA was prepared from ES cells and tail biopsies (29). Probe B was obtained by PCR using one primer from within the fifth exon and another primer from within the sixth exon. Primer sequences were: fifth exon primer, 5'-TGTAACCGAAGACGACGGCTGCT, and sixth exon primer, 5'-TATAGATAGTGAACAACACAGC.
Immunoblot analysis
Total cell lysates of BMMC and control cells were prepared as previously described (30). They were electrophoretically resolved on a 10% SDSpolyacrylamide gel and transferred to PVDF membranes. FcRß of Fc
RI was probed by an anti-rat FcRß mAb (JRK) which also recognizes mouse FcRß (30). The membranes were developed by a horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody using the ECL detection system (Amersham, Amersham, UK).
Northern blot analysis
Total RNA (20 µg) extracted from BMMC was resolved by electrophoresis and transferred to a membrane (BIODYNE®, Pall. BioSuport, New York, NY) as previously described (29). The blots were probed with 32P-labeled cDNA of FcRß as previously described (3).
Generation of BMMC and connective tissue-type mast cells (CTMC)
BMMC were generated from bone marrow cells as described previously (31). Briefly, the bone marrow cells were obtained from 12- to 16-week-old mice. BMMC were induced from them by culturing in enriched RPMI medium supplemented with 10% FCS, pyruvate, non-essential amino acids and 10% of the culture supernatant of WEHI-3. After culture for 5 weeks, ~95% of the non-adherent cells were mast cells as judged by Toluidine blue staining.
Co-cultured mast cells were obtained following the method of Katz et al. (32). Briefly, 1x107 BMMC were seeded on the confluent 3T3 fibroblasts and co-cultured for 23 weeks. The cells were replated and non-adherent cells were recovered and used for analyses. PT18 and P815 cells were maintained as described previously (15).
Antibodies and cell surface staining
Anti-FcRß mAb (JRK) was kindly gifted from Dr Juan Rivera (NIH, Bethesda, MD). Rat mAb 2.4G2 (anti-mouse low-affinity IgG receptors, i.e. Fc
RII and Fc
RIII), biotinylated and FITC-labeled 2.4G2, rat mAb R35-72 (anti-mouse IgE), mouse IgG1 (anti-TNP), and FITC-labeled monoclonal rat anti-mouse c-kit antibody were purchased from PharMingen (San Diego. CA). Polyclonal goat anti-rat IgG was obtained from Cappel Organon Teknica (West Chester, PA). Mouse monoclonal IgE antibody (anti-DNP), SPE-7, was obtained from ATCC (Rockville, MD) and labeled with FITC.
Mast cells derived from mice of both genotypes were stained with FITC-labeled SPE-7 for Fc
RI and FITC-labeled 2.4G2 for Fc
R. The cells were also stained with FITC-labeled anti-c-kit antibody. The fluorescence intensity and number of the cells were measured on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The dead cells judged as propidium iodide-positive were eliminated in the analyses.
Serotonin and ß-hexosaminidase release assay
Degranulation of the BMMC via Fc receptors was estimated by serotonin release. BMMC (5x105 cells/ml) from FcRß(+/+) and FcRß(/) were labeled with the 2 mCi/ml of [3H] serotonin for 12 h. Cells were washed and saturated with anti-DNP IgE or 2.4G2 in Tyrode's () buffer (10 mM HEPES buffer, pH 7.4, 130 mM NaCl, 5 mM KCl, 5.6 mM glucose and 0.1% BSA) for 30 min. After removal of excess antibody, cells were resuspended at 1x106/ml in Tyrode's (+) buffer which is Tyrode's () buffer supplemented with 1 mM CaCl2 and 0.6 mM MgCl2, and exposed to 500 ng/ml of rat anti-mouse IgE antibody or to the indicated amount of goat anti-rat IgG antibody respectively. For positive control, 10 ng/ml of phorbol myristate acetate and 100 ng/ml ionomycin were added to the cell suspension. After 30 min incubation at 37°C, the reaction was terminated by centrifugation at 4°C. Supernatants were then collected and the equal amount of the cells for one reaction were lysed with 1% NP-40 to prepare total cell lysates. The radioactivity of the [3H]serotonin in the cell lysates and the supernatants was counted, and percent of the released radioactivity was calculated following the formula: percent release = [(supernatant background)/(total cell lysates background)]x100.
Degranulation of co-cultured mast cells via Fc
R was performed as previously described (33). Co-cultured mast cells from mice of both genotypes were suspended at the concentration of 5x104 cells in 100 µl of Tyrode's () buffer and saturated with 10 µg/ml of biotinylated 2.4G2 on ice for 30 min. The cells were washed and resuspended in 100 µl of Tyrode's (+) buffer. The cells were then triggered with the indicated amount of streptavidin at 37°C for 30 min. The supernatants and the cell lysates were prepared in the same way as in serotonin assay. The ß-hexosaminidase activity in the supernatant was assayed with p-nitrophenyl-N-acetyl-ß-D-glucopyranoside (Sigma, St Louis, MO) as ß-hexosaminidase substrate and the extent of degranulation is expressed as the percentage of the total cellular ß-hexosaminidase activity as previously described (34).
Measurement of the adhesion to fibronectin (FN)
Adhesion assay of BMMC was performed as previously described (31,35). Briefly, 96-well plates were coated with 20 µg/ml of FN for 12 h at 4°C and then blocked with PBS containing 1% BSA at room temperature for 2 h. BMMC from mice of both genotypes were saturated with 2.4G2 (2 µg/ml) for 30 min. Then 4x104 cells were placed in each well and adhered by stimulation with the indicated amount of anti-rat IgG for 30 min at 37°C. Non-adherent cells were washed out and adherent cells were loaded with 10% Alamer Blue (Biosource, Camarillo, CA). After 12 h, adherent cells were estimated using a photometer at the wavelength of 560 nm. EGTA was added to check the Ca2+ dependency of the adhesion. The percent values for each adhesion assay were calculated by the formula = [(S B)/(T B)]x100, where S, T and B are the absorbance of adhesion cells, total cells and blank respectively.
Induction of passive cutaneous anaphylaxis (PCA)
For induction of IgE-mediated PCA, mice of both genotypes were intradermally injected in the foot pads with 50 ng of anti-DNP mouse IgE or saline (50 µl). After 2 h, each mouse was injected i.v. with 200 µl of a solution containing 100 µg of DNP(3040)human serum albumin (Sigma) and 1% Evans blue. After 30 min, extravazation was visualized by blue staining of the foot pad as an indicator for a positive anaphylatic response. For induction of IgG-mediated PCA, mice were intradermally injected at the basolateral side with various amounts of mouse IgG1 (in 50 µl saline), which had been aggregated by incubation at 63°C for 3 h. After 5 h, each mouse was injected with 200 µl of 1% Evans blue. After 30 min, the injection sites at reverse side of the skin sections were analyzed.
Measurement of Ca2+ flux
BMMC were saturated with 2.4G2 and then loaded with 2 µM fura-2AM for 45 min at 37°C in a buffer (pH 7.4) containing 135 mM NaCl, 5 mM KCl, 1 mM CaCl2 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES and 0.1% BSA. Cells were analyzed in a cuvette on a spectrofluorometer model F2000 (Hitachi, Tokyo, Japan) with extraction set at 340 and 380 nm, and emission at 500 nm. Ca2+ concentration was calculated using the published value for the Kd of fura-2 at 37°C (36).
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Results
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Generation of the null mutant mice for FcRß
Because various truncated forms of the FcRß affect surface expression and functions of Fc
RI (15,21), in order to elucidate roles of the FcRß in vivo, analyses of the mouse model of the null mutant for FcRß is required. We subcloned the genomic fragments of the FcRß isolated from the 129/Ola
library and mapped them by digestions with restriction enzymes (Fig. 1A
). The sequence and size of each exon and intron was determined by sequencing analyses (Hiraoka et al., unpublished). We constructed a targeting vector which includes the FcRß locus deleted in the translation initiation codon and the splicing donor site by replacing the first exon with a neomycin-resistance gene (Neo) (Fig. 1B
). We obtained five mutant clones of homologous recombinants through introducing the vector to ES cells, and screening them with antibiotics selection and gene analyses (Fig. 1D
). Two ES clones (clone 74 and 126) were developed to chimeric mice. About 50% of the chimeras transmitted the mutant allele to their offspring and we obtained homozygous mutant animals by breeding them (Fig. 1E
). The homozygous mutant mice appeared healthy and were fertile. We examined whether these homozygous mutants expressed no FcRß at the RNA and protein levels. Northern blot and Western blot analyses of BMMC from homozygous mutant mice showed that no signals of mRNA and no fragments of FcRß protein were detected (Fig. 2
). We concluded that the targeted mutation resulted in a mutant allele that produces no FcRß protein.

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Fig. 2. Abolishment of FcRß in BMMC from targeted mice. (A) Northern blot analysis. Total RNA (20 µg) was analyzed from FcRß(+/+) and FcRß(/) mast cells. The 1.8 kb band is responsible for the FcRß message. (B) Western blot analysis. Total cell lysates from 5x105 cells were analyzed. PT18 cells and P815 cells are FcRß positive and negative mast cells respectively as shown previously (3). The 31 kDa band detected corresponds to FcRß.
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Expression of Fc receptors on BMMC
To evaluate whether the loss of FcRß affects the surface expression of Fc receptors, BMMC from FcRß(/) and FcRß(+/+) were stained with mouse monoclonal IgE, with monoclonal anti-mouse low-affinity IgG receptor antibody (2.4G2) which recognizes both Fc
RII and Fc
RIII (37), and with monoclonal anti-mouse c-kit antibody. The expression of the Fc
RI on the BMMC was not detected with IgE staining (Fig. 3
, upper panel), as was expected from the reconstitution studies of Fc
RI (3). The intensity of the 2.4G2 staining of BMMC from FcRß(/) was increased in contrast to FcRß (+/+) (Fig. 3
, middle panel). The metachromatic nature of BMMC from FcRß(/) was the same as those from FcRß (+/+) (data not shown). We concluded that FcRß was essential for expression of Fc
RI on the mouse mast cells
Functions of BMMC from FcRß(/)
We investigated the responses of BMMC from FcRß(/) mice via IgE and IgG receptors. BMMC from FcRß(/) and FcRß(+/+) were sensitized with monoclonal mouse IgE and 2.4G2 which is monovalent for Fc
R recognition (38) and cannot cross-link IgG receptors without some second cross-linker (31,33), and then triggered with anti-mouse IgE antibody or anti-rat IgG antibody respectively. Degranulation from mast cells was estimated by [3H]serotonin release. As expected, IgE-dependent degranulation of mast cells from FcRß(/) was completely abolished (Fig. 4A
), although that of FcRß(+/+) was well observed. IgG receptor-dependent degranulation of mast cells was not detected in mast cells from both genotypes, like previous reports that indicated that BMMC are immature and do not degranulate by cross-linking IgG receptors (33). However, adhesion of FcRß(/) BMMC was substantially increased as compared to that of FcRß(+/+) (Fig. 4B
). When BMMC saturated with 2.4G2 were triggered by second antibody, the adhesion of FcRß(/) BMMC was enhanced in comparison with that of FcRß(+/+) BMMC. This adhesion was completely inhibited by ablation of Ca2+ with EGTA. The Ca2+ influx in FcRß(/) BMMC upon IgG receptor engagement was larger than that of FcRß(+/+), in accordance with the results of the adhesion assay (Fig. 5
), suggesting that an increase of IgG receptor expression on BMMC led to the augmented responses for Ca2+ influx and cell adhesion.

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Fig. 4. Functional analyses of mast cells from FcRß(+/+) and FcRß(/) mice. (A) Assay for serotonin release from mast cells. Open and closed bars represent FcRß(+/+) and FcRß(/) respectively. BMMC were loaded with [3H]serotonin and then incubated with the monoclonal mouse IgE (SPE-7) or anti-IgG receptor (2.4G2). [3H]Serotonin released into the supernatant was quantified after exposure to monoclonal anti-mouse IgE or to anti-rat IgG. The cells were stimulated also with phorbol myristate acetate and ionomycin as a positive control. The percent values for each experimental condition were calculated by the formula = [(S B)/(T B)]x100 (see text). Data is presented as the mean ± SD (N = 3) and the values without error bars mean that the error range is <2%. (B) Adhesion of mast cells to FN through IgG receptor engagement. Open and closed bars represent FcRß (+/+) and FcRß(/) respectively. BMMC were saturated with 2.4G2 for 30 min. After removal of the excess antibody, anti-rat IgG was added to cross-link the IgG receptor bound with 2.4G2 and the cells were subsequently adhered to a 96-well plate precoated with FN. After 30 min, non-adherent cells were washed out and adherent cells were loaded with Alamer Blue. After 12 h, adherent cells were estimated using a photometer at the wavelength of 560 nm. EGTA was added to check the Ca2+ dependency of the adhesion. The percent values for each adhesion assay were calculated by the formula = [(S B)/(T B)]x100 (see text). Data is presented as the mean ± SD (N = 3) and the values without error bars mean that the error range is <2%.
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Fig. 5. Evaluation of Ca2+ flux in mast cells. BMMC from FcRß(+/+) (upper panel) and FcRß(/) (lower panel) mice were saturated with 2.4G2 and loaded with fura-2AM. Their capacity to respond to stimulation with the second antibody was estimated by a spectrofluorometer. Calcium concentration was calculated from photon counts using the published value of 2.24x107 for the Kd for fura-2AM at 37°C.
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IgE- and IgG-mediated PCA reaction is impaired in FcRß(/) mice
Since the increased expression of IgG receptor and augmented responses via the IgG receptor were observed in FcRß(/) BMMC, we next examined IgG-dependent anaphylaxis in vivo. Surprisingly, the PCA reaction triggered with heat-aggregated IgG was decreased in FcRß(/) mice (Fig. 6B
), while the IgE-dependent PCA was absent, as expected (Fig. 6A
). It implies that the response to IgG of FcRß(/) mast cells in the skin was decreased in spite of the increased expression of Fc
R on the cell surface.

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Fig. 6. IgE- and IgG-mediated PCA. (A) FcRß(+/+) (upper) and FcRß(/) (lower) mice were intradermally injected into foot pads with 50 ng of mouse anti-DNP IgE. After 2 h, 100 µg of DNP(3040)human serum albumin with 0.5% Evans blue was injected i.v. After 30 min, positive anaphylatic reaction was visualized by extravazation of blue dye. (B) FcRß(+/+) (wild) and FcRß(/) (mutant) mice were intradermally injected in the dorsal skin, from the bottom right to the upper left, with 0.3, 0.6, 1.3, 2.5, 5 and 0 µg (saline) of mouse IgG1 which was aggregated by incubation at 63°C for 3 h. After 5 min, 1% Evans blue was injected. After 30 min, the injected skins were analyzed for dye extravazation.
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Analyses of mature mast cells co-cultured with fibroblasts
CTMC are known to show a different metachromatic nature from those of BMMC and degranulate when stimulated with the IgG immune complex (33,39). Mast cells co-cultured with 3T3 fibroblasts represent CTMC phenotypes in metachromagy and have potential to degranulate on engagement of the IgG receptors (31,32,40). To explain the difference of responses via Fc
R found between BMMC and CTMC, we co-cultured BMMC with 3T3 cells to generate CTMC. The intensity of 2.4G2 staining of mast cells from mice of both genotypes increased significantly during co-culture with 3T3 fibroblasts, but the tendency of higher expression of the IgG receptor in FcRß(/) relative to FcRß(+/+) was not changed (Fig. 7A
). Subsequently, we evaluated the degranulation of the CTMC via Fc receptors (Fig. 7B
). The CTMC of FcRß(+/+) degranulated only when saturated with biotinylated 2.4G2 and triggered with avidin as second cross-linker in the same way as the adhesion to FN observed in BMMC; however, CTMC of the mutant did not exhibit significant degranulation (Fig. 7B
). IgE-mediated degranulation was just abolished because of the deficiency of the Fc
RI expression on the cell surface (data not shown). These observations are in accordance with impaired PCA in FcRß(/) mice. Taken together, we concluded that the FcRß is required for the full activation of mast cells, at least for FcR-mediated degranulation.

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Fig. 7. Functional analyses of mature mast cells. (A) IgG receptor expression. BMMC from mice of both genotypes were co-cultured with 3T3 cells for 3 weeks. After being dispersed by incubation with trypsin and EDTA, the cells were replated on dishes. After 2 h, mast cell-enriched fractions were recovered by collecting the nonadherent cells. These cells were stained with 2.4G2 and analyzed as in Fig. 3 . Upper panels and lower panels indicate BMMC and co-cultured BMMC respectively. (B) ß-Hexosaminidase release from mast cells co-cultured with 3T3 cells upon IgG receptor engagement. Open and closed bars represent FcRß(+/+) and FcRß(/) respectively. The co-cultured mast cells were obtained in the same way as in (A). Cells (5x104) were saturated with 10 µg of biotinylated 2.4G2 and then excess antibody was washed out. They were subsequently triggered with avidin for 30 min. Released hexosaminidase activity was evaluated by the ratio of enzyme activity of the supernatant to that of the total cell lysate. Data indicate mean ± range from duplicate analysis. Three independent experiments were performed and the same tendency of the lower response in the FcRß(/) mast cells relative to that in the FcRß(+/+) mast cells was observed. Data from one representative experiment are shown.
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Discussion
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FcRß is essential for the cell surface expression of rodent Fc
RI
It was demonstrated that some deletion mutants of FcRß affected the cell surface expression and functions of Fc
RI in reconstitution studies of the functional receptors in Fc
RI cells (15,21). In the rodent system, it was elucidated that FcRß is essential for the surface expression of Fc
RI. However, one T cell line established from mouse splenocytes was reported to express Fc
RI without FcRß (41). A candidate molecule which can substitute FcRß was also discovered and was detected in a mast cell line (42). Therefore analyses of the null mutant were necessary to clarify if FcRß is really essential for the expression of Fc
RI on native mast cells. We constructed one targeting vector which contains no translation initiation site and the first splicing site by deletion of the first exon (Fig. 1B
). The obtained mutant produced no fragments of FcRß as expected, resulting in the abolition of the Fc
RI expression on mast cells (Figs 2 and 3
). We concluded that FcRß is essential for the surface expression of Fc
RI on mouse mast cells.
Phenotypes of BMMC from the mutant mice
The abnormal expression and functions of Fc receptors on BMMC were observed in the FcRß(/) mice. Fc
R expression was increased in mutant BMMC (Fig. 3
), and Fc
R-dependent adhesion to FN and Ca2+ flux were also enhanced (Figs 4B and 5
). In spite of that, Fc
R-mediated degranulation was not observed in BMMC from mutant and wild-type mice (Fig. 4A
). A previous report documented that this adhesion was dependent on the FcR
and Fc
RIII on the cell surface (31). Therefore, the increased Fc
RIII on the mutant mast cells might augment the adhesion.
FcRß is essential for the full activation of mast cells via Fc receptors
In the mutant mice, IgE-mediated PCA disappeared because of the loss of Fc
RI expression on mast cells (Fig. 6A
). IgG-mediated PCA also decreased and mast cells co-cultured with 3T3 fibroblasts demonstrated impaired degranulation. It is thus concluded that FcRß is necessary for the full activation of mature mast cells through IgE and IgG receptors.
Why and how did the loss of FcRß cause the defective degranulation. CTMC represent different metachromatic natures from those of BMMC (39,43), and they degranulate and release the lipid mediators by Fc
R engagement (33). In FcRß-deficient mice, peritoneal mast cells and mast cells co-cultured with 3T3 cells displayed the same metachromatic nature as observed in FcRß(+/+) (data not shown). IgG receptors on FcRß(/) mast cells also increased during co-culture with the fibroblasts like FcRß(+/+) mast cells, suggesting that the decrease of Fc
R-mediated degranulation cannot be explained by abnormal development of immature mast cells to CTMC.
One model reported on the signal-transducing cascades elicited upon Fc
RI aggregation that Lyn associated with FcRß initiates the activation cascades by phosphorylation of ITAM of FcRß and FcR
, leading to the phosphorylation and activation of Syk, which is essential for mast cell activation (19,20). One typical reconstitution study of Fc
RI indicated that FcRß amplifies the Syk phosphorylation on the engagement of the Fc
RI (21). On the contrary, it is suggested that inhibitory signals may arise from FcRß because some molecules that inhibit cell activation such as SHIP, SHP-1 and SHP-2 can associate with FcRß. In addition, Lyn is considered to be essential also for the inhibitory effects of Fc
RII co-cross-linked with Fc
RI (44). BMMC from Lyn-deficient mice showed impaired tyrosine phosphorylation and Ca2+ flux on Fc
RI aggregation, but other cell responses including degranulation, etc., were mostly normal (22). Recently, the functions of FcRß for FcR signal transduction were analyzed by using one targeted mouse model in which the FcRß gene was disrupted by the insertion of a neomycin-resistance gene at the fourth exon (46). Although it was not described whether an alternative form of FcRß was produced by the modification of the FcRß gene or not, humanized Fc
RI without FcRß reconstituted on BMMC through introducing the human Fc
RI
gene to mouse Fc
RI
-deficient mice induced a lower cell response by their aggregation relative to that of complete Fc
RI with FcRß and, therefore, it was concluded that the function of the FcRß is an amplifier of signals through Fc
RI aggregation. Comparing responses of BMMC containing intact or disrupted FcRß under the Fc
RI
-deficient background, BMMC with disrupted FcRß could not degranulate and synthesize cytokine by addition of only 2.4G2, in contrast to the mast cells with normal FcRß. However, in our case, BMMC from both FcRß(/) and wild-type mice could not degranulate by the engagement of the Fc
R, whereas CTMC could degranulate by their aggregation, as in a previous report (33). Although the exact reason why the different responses of BMMC via Fc
R were observed in our and their analyses is unknown, it may be due to the difference of genetic background, i.e. Fc
RI
-deficient or not. These observations on mast cell responses and anaphylaxis via Fc
R in FcRß-deficient mice might suggest that the FcRß also amplifies the signals elicited via Fc
RIII. However, we cannot prove this hypothesis at present because we cannot exclude increased negative effect(s) of the Fc
RII co-ligated with Fc
RIII, whenever mast cells are triggered by engagement of the IgG receptor. Analyses of FcRß and Fc
RII double-deficient mice (47) are required to resolve this question.
Does the genetic variation of the FcRß gene contribute to the cause of atopic disease
In humans, the genomic locus for the FcRß was mapped as one of the candidate genes causing atopic diseases (11,13) and some alleles with amino acid substitutions were also detected (12,14). If the abnormalities of FcRß expression and/or its functions lead to the atopic diseases, genetic variation in the FcRß or its controlling elements would increase the effector cell responses in atopic patients. The amino acid substitutions of Ile181 to Leu and of Val183 to Leu in the fourth membrane segment of the FcRß, which were detected in British families (12), might affect each subunit association in the Fc
RI and Fc
RIII complex. The substitution of Glu237 to Gly detected in the C-terminal intracellular part of the FcRß (14) might alter its association molecules in the signal-transducing pathway and cause the abnormal cell responses via FcRs. However, abnormalities of mast cell functions associated with these alleles have not been reported, probably because the source of mast cells and basophils from atopic patients were limited and not sufficient for detailed analyses. Our targeted mutant mouse model may be useful to investigate the effects of these alleles because the mutant does not produce any fragments of intrinsic FcRß which affect functions of Fc receptors. Introduction of the human FcRß and its mutant alleles into our FcRß-deficient mice by gene transfer would enable us to answer this question.
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Acknowledgments
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We are very grateful to Nobukata Shinohara (Mitubishi-kasei Institute of Life Sciences) for his support and guidance. We also thank Dr Keiko Kawamoto and Hironori Matuda for their help of the analyses of PCA reaction. This work was supported in part by Grants in Aid from Ministries of Education, Health and Welfare, and Science and Technology Agency (CREST), Japan.
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Abbreviations
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BMMC | bone marrow-derived cultured mast cells |
CTMC | connective tissue-type mast cells |
ES | embryonic stem |
FcRß | Fc receptor ß subunit |
FcR | Fc receptor subunit |
FN | fibronectin |
ITAM | immunoreceptor tyrosine based activation motif |
PCA | passive cutaneous anaphylaxis |
SHP | SH-2 bearing protein tyrosine phosphatase |
SHIP | SH-2-bearing inositol-5-phosphatase |
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Notes
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Transmitting editor: K. Arai 
Received 20 August 1998,
accepted 20 October 1998.
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References
|
---|
-
Kinet, J.-P, Blank, U., Ra, C., White, K., Metzger, H. and Kochan, J. 1988. Isolation and characterization of cDNAs coding for the ß subunit of the high-affinity receptor for immunoglobulin E. Proc. Natl Acad. Sci. USA 85:6483.[Abstract]
-
Blank, U., Ra, C., Miller, L., White, K., Metzger, H. and Kinet, J.-P. 1989. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 337:187.[ISI][Medline]
-
Ra, C., Jouvin, M. H. and Kinet, J.-P. 1989. Complete structure of the mouse mast cell receptor for IgE (Fc
RI) and surface expression of chimeric receptors (ratmousehuman) on transfected cells. J. Biol. Chem. 264:15323.[Abstract/Free Full Text]
-
Küster, H., Zhang, L., Brini, A. T., MacGlashan, D. W. and Kinet, J.-P. 1992. The gene and cDNA for the human high affinity immunoglobulin E receptor ß chain and expression of the complete human receptor. J. Biol. Chem. 267:12782.[Abstract/Free Full Text]
-
Kurosaki, T., Gander, I., Wirthmueller, U. and Ravetch, J. V. 1992. The ß subunit of the Fc
RI is associated with the Fc
RIII on mast cells. J. Exp. Med. 175:447.[Abstract]
-
Dombrowicz, D., Flamand, V., Miyajima, I., Ravetch, J. V., Galli, S. J. and Kinet, J.-P. 1997. Absence of Fc
RI a chain results in upregulation of Fc
RIII-dependent Mast cell degranulation and anaphylaxis. J. Clin. Invest. 99:915.[Abstract/Free Full Text]
-
Ra, C., Jouvin, M. H., Blank, U. and Kinet, J.-P. 1989. A macrophage Fc
receptor and the mast cell receptor for IgE share an identical subunit. Nature 341:752.[ISI][Medline]
-
Takai, T., Li, M., Sylvestre, D., Clynes, R. and Ravetch, J. V. 1994. FcR ß chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[ISI][Medline]
-
Reth, M. G. 1989. Antigen receptor tail clue. Nature 338:383.[ISI][Medline]
-
Cambier, J. C. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL). Immunol. Today 16:110.[ISI][Medline]
-
Cookson, W. O., Sharp, P. A., Faux, J. A. and Hopkin, J. M. 1989. Linkage between immunoglobulin E responses underlying asthma and rhinitis and chromosome 11q. Lancet i:1292.
-
Shirakawa, T., Li, A., Dubowitz, M., Dekker, J. W., Shaw, A. E., Faux, J. A., Ra, C., Cookson, W. O. and Hopkin, J. M. 1994. Association between atopy and variants of the ß subunit of the high-affinity immunoglobulin E receptor. Nature Genet. 7:125.[ISI][Medline]
-
Shirakawa, T., Hashimoto, T., Furuyama, J., Takeshita, T. and Morimoto, K. 1994. Linkage between severe atopy and chromosome 11q13 in Japanese families. Clin. Genet. 46:228.[ISI][Medline]
-
Shirakawa, T., Mao, X. Q., Sasaki, S., Enomoto, T., Kawai, M., Morimoto, K. and Hopkin, J. 1996. Association between atopic asthma and a coding variant of Fc
RI ß in a Japanese population. Hum. Mol. Genet. 5:959.[Abstract/Free Full Text]
-
Alber, G., Miller, L., Jelsema, C. L., Varin, B. N. and Metzger, H. 1991. Structurefunction relationships in the mast cell high affinity receptor for IgE. Role of the cytoplasmic domains and of the ß subunit. J. Biol. Chem. 266:22613.[Abstract/Free Full Text]
-
Jouvin, M. H., Adamczewski, M., Numerof, R., Letourneur, O., Valle, A. and Kinet, J.-P. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269:5918.[Abstract/Free Full Text]
-
Wilson, B. S., Kapp, N., Lee, R. J., Pfeiffer, J. R., Martinez, A. M, Platt, Y., Letourneur, F. and Oliver, J. M. 1995. Distinct functions of the Fc
R1
and ß subunits in the control of Fc
R1-mediated tyrosine kinase activation and signaling responses in RBL-2H3 mast cells. J. Biol. Chem. 270:4013.[Abstract/Free Full Text]
-
Kihara, H. and Siraganian, R. P. 1994. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. Chem. 269:22427.[Abstract/Free Full Text]
-
Costello, P. S., Turner, M. and Walters, A. E., Cunningham, C. N., Bauer, P. H., Downward, J. and Tybulewicz, V. L. 1996. Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells. Oncogene 13:2595.[ISI][Medline]
-
Zhang, J., Berenstein, E. H., Evans, R. L. and Siraganian, R. P. 1996. Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells. J. Exp. Med. 184:71.[Abstract]
-
Lin, S., Cicala, C., Scharenberg, A. M. and Kinet, J.-P. 1996. The Fc
RIß subunit functions as an amplifier of Fc
RIß-mediated cell activation signals. Cell 85:985.[ISI][Medline]
-
Nishizumi, H. and Yamamoto, T. 1997. Impaired tyrosine phosphorylation and Ca2+ mobilization, but not degranulation, in lyn-deficient bone marrow-derived mast cells. J. Immunol. 158:2350.[Abstract]
-
Kimura, T., Zhang, J., Sagawa, K., Sakaguchi, K., Appella, E. and Siraganian, R. P. 1997. Syk-independent tyrosine phosphorylation and association of the protein tyrosine phosphatases SHP-1 and SHP-2 with the high affinity IgE receptor. J. Immunol. 159:4426.[Abstract]
-
Kimura, T., Sakamoto, H., Appella, E. and Siraganian, R. P. 1997. The negative signaling molecule SH2 domain-containing inositol-polyphosphate 5-phosphatase (SHIP) binds to the tyrosine-phosphorylated ß subunit of the high affinity IgE receptor. J. Biol. Chem. 272:13991.[Abstract/Free Full Text]
-
McBurney, M. W., Sutherland, L. C., Adra, C. N., Leclair, B., Rudnicki, M. A. and Jardine, K. 1991. The mouse Pgk-1 gene promoter contains an upstream activator sequence. Nucleic. Acids Res. 19:5755.[Abstract]
-
Mansour, S. L, Thomas, K. R. and Capecchi, M. R. 1988. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348.[ISI][Medline]
-
Joyner, A. L. 1992. Gene Targeting. Oxford University Press, Oxford.
-
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. C. 1993. Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc. Natl Acad. Sci. USA. 90:8424.[Abstract/Free Full Text]
-
Maniatis, T., Fritsh, E. F. and Sambrook, J. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
-
Rivera, J., Kinet, J.-P., Kim, J., Pucillo, C. and Metzger, H. 1988. Studies with a monoclonal antibody to the ß subunit of the receptor with high affinity for immunoglobulin E. Mol. Immunol. 25:647.[ISI][Medline]
-
Dastych, J., Hardison, M. C. and Metcalfe, D. D. 1997. Aggregation of low affinity IgG receptors induces mast cell adherence to fibronectin: requirement for the common FcRß-chain. J. Immunol. 158:1803.[Abstract]
-
Katz, H. R., Arm, J. P., Benson, A. C. and Austen, K. F. 1990. Maturation-related changes in the expression of Fc
RII and Fc
RIII on mouse mast cells derived in vitro and in vivo. J. Immunol. 145:3412.[Abstract/Free Full Text]
-
Katz, H. R., Raizman, M. B., Gartner, C. S., Scott, H. C., Benson, A. C. and Austen, K. F. 1992. Secretory granule mediator release and generation of oxidative metabolites of arachidonic acid via FcIgG receptor bridging in mouse mast cells. J. Immunol. 148:868.[Abstract/Free Full Text]
-
Yasuda, M., Hasunuma, Y., Adachi, H., Sekine, C., Sakanishi, T., Hashimoto, H., Ra, C., Yagita, H. and Okumura, K. 1994. Expression and function of fibronectin binding integrins on rat mast cells. Int. Immunol. 7:251[Abstract]
-
Noto, K., Kato, K., Okumura, K. and Yagita, H. 1995. Identification and functional characterization of mouse CD29 with a mAb. Int. Immunol. 7:835.[Abstract]
-
Millard, P. J. Ryan, T. A., Webb, W. W. and Fewtrell, C. 1989. Immunoglobulin E receptor cross-linking induce oscillations in intracellular free ionized calcium in individual tumor mast cells. J. Biol. Chem. 264:19730.[Abstract/Free Full Text]
-
Benhamou, M., Bonnerot, C., Fridman, W. H. and Däeron, M. 1990. Molecular heterogeneity of murine mast cell Fcß receptors. J. Immunol. 144: 3071.[Abstract/Free Full Text]
-
Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150:580.[Abstract]
-
Hazenbos, W., L, Gessner, J. E., Hofhuis, F. M., Kuipers, H., Meyer, D., Heijnen, I. A, Schmidt, R. E., Sandor, M., Capel, P. J., Däeron, M., van de Winkel, J. G. and Verbeek, J. S. 1996. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc
RIII (CD16) deficient mice. Immunity 5:181.[ISI][Medline]
-
Levi-Schaffer, F., Austen, K. F., Gravallese, P. M. and Stevens, R. L. 1986. Coculture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells. Proc. Natl Acad. Sci. USA 83:6485.[Abstract]
-
Louahed, J., Kermouni, A., Snick, J. V. and Renauld, J.-C. 1995. IL-9 induces expression of granzymes and high-affinity IgE receptor in murine T helper clones. J. Immunol. 154:5061.[Abstract/Free Full Text]
-
Adra, C. N., Lelias, J. M., Kobayashi, H., Kaghad, M., Morrison, P., Rowley, J. D. and Lim, B. 1994. Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the ß subunit of the high-affinity IgE receptor: evidence for a family of proteins with four membrane-spanning regions. Proc. Natl Acad. Sci. USA 91:10178.[Abstract/Free Full Text]
-
Kitamura, Y. 1989. Heterogeneity of mast cells and phenotypic change between subpopulations. Annu. Rev. Immunol. 7:59.[ISI][Medline]
-
Malbec, O., Fong, D. C., Turner, M., Tybulewicz, V. L., Cambier, J. C., Fridman, W. H. and Däeron, M. 1998. Fc
receptor I-associated lyn-dependent phosphorylation of Fc
receptor IIB during negative regulation of mast cell activation. J. Immunol. 160:1647.[Abstract/Free Full Text]
-
Dombrowicz, D., Flamand, V., Brigman, K. K., Koller, B. H. and Kinet, J.-P. 1993. Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor ß chain gene. Cell 75:969.[ISI][Medline]
-
Domobrrowcicz, D., Lin, S., Flammand, V., Brini, A. T., Kollwer, B. H. and Kinet, J.-P. 1998. Allergy-associated FcRß is a molecular amplifier of IgE- and IgG-mediated in vivo responses. Immunity 8:517.[ISI][Medline]
-
Takai, T., Ono, M., Hikida, M., Ohmori, H. and Ravetch, J. V. 1996. Augmented humoral and anaphylatic responses in Fc
RII-deficient mice. Nature 379:346.[ISI][Medline]