The cross-linking of IgE-bound FcεRI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcεRIγ signaling to IgE/FcεRI-mediated mast cell responses has not yet been elucidated. In this study FcεRIγ−/− mast cells were reconstituted with either wild-type or mutant FcεRIγ in transgenic mice and transfected mast cells in vitro. We demonstrate that FcεRIγ-immunoreceptor tyrosine-based activation motif is essential for degranulation, cytokine production, and PG synthesis as well as for passive systemic anaphylaxis. Recent reports have suggested that cell surface FcεRI expression and mast cell survival are regulated by IgE in the absence of Ag, although the molecular mechanism is largely unknown. We also found that the promotion of mast cell survival by IgE without Ags is mediated by signals through the FcεRIγ-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcεRI is independent of FcεRIγ signaling. These results indicate that FcεRIγ-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.

Mast cells play a central role in various types of hypersensitivity, particularly in immediate phase allergic reactions. The aggregation of IgE-bound FcεRI, the high affinity receptor for IgE, on mast cells by multivalent Ags triggers the activation of three major signaling pathways: 1) degranulation of preformed granules containing such chemical mediators as histamine and β-hexosaminidase, 2) generation of arachidonic acid metabolites such as PG, and 3) transcription of multiple cytokine genes such as IL-4 and IL-6. The secreted mediators are responsible for allergic inflammatory reactions (1, 2).

The FcεRI-mediated activation of mast cells has been thought to occur only upon cross-linking of FcεRI with IgE and Ag (IgE(+Ag)). However, recent reports have suggested that cell surface FcεRI expression and mast cell survival are regulated by IgE in the absence of Ag (IgE(−Ag)) (3, 4, 5). Although the surface FcεRI expression on mast cells increases upon binding to IgE (3), the mechanism underlying this regulation has not been fully elucidated. More importantly, two recent reports have revealed that mast cell survival is promoted by IgE(−Ag) (4, 5). Although FcεRI has been shown to be involved (4), the mechanism is largely unknown.

FcεRI is expressed on rodent mast cells as a tetrameric structure composed of α, β, and γ homodimers (6). The α-chain (FcεRIα) is responsible for binding to IgE. The β- and γ-chains (FcεRIβ, FcεRIγ) possess immunoreceptor tyrosine-based activation motifs (ITAMs)6 (7) within their cytoplasmic domains. The cross-linking of FcεRI with IgE(+Ag) initiates an activation signal cascade via the tyrosine phosphorylation of these ITAMs by Lyn. Syk is then recruited to the phospho-ITAMs of FcεRIγ, where it is activated to phosphorylate various substrates in the downstream cascade (1, 2). Recently, it has been reported that Fyn is also involved in the induction of alternative activation signals upon FcεRI cross-linking (8). Although FcεRIβ is believed to play a role in the amplification of activation signals through FcεRIγ (9, 10), it has been shown that Syk binds to phospho-ITAMs of FcεRIβ as well as FcεRIγ (11) and that the cross-linking of FcεRIβ induces weak Ca2+ influx (12). Therefore, it is possible that FcεRIβ-mediated signals may trigger some in vivo responses via ITAM.

FcεRIγ is thought to be a pivotal subunit of the FcεRI complex for intracellular signaling upon IgE(+Ag) stimulation (1, 13). We and another group have generated FcεRIγ-deficient mice (γ−/−) (14, 15) and have analyzed the in vivo function of FcεR and FcγR in various systems (14, 15, 16, 17, 18, 19, 20, 21). However, the in vivo function of FcεRIγ-mediated signals in FcεRI-mediated responses has not been elucidated, mainly because FcεRIγ is essential for cell surface expression.

In this study, γ−/− mast cells were reconstituted with mutant FcεRIγ in transgenic mice and bone marrow-derived mast cells (BMMCs), and the function of FcεRIγ-ITAM was analyzed. We demonstrate that most FcεRI-mediated mast cell activation and in vivo passive systemic anaphylaxis (PSA) by IgE(+Ag) as well as the promotion of mast cell survival by IgE(−Ag) are dependent on FcεRIγ-ITAM signaling. In contrast, we show that the up-regulation of surface FcεRI expression on mast cells by IgE is regulated independently of FcεRIγ-ITAM. Thus, we unveiled the differential requirement of FcεRIγ-mediated signals for the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.

The cDNAs encoding murine wild-type FcεRIγ (WT) and two mutant FcεRIγ (YF, tyrosines at positions 65 and 76 within ITAM were replaced with phenylalanines; ΔCT, the last 65–86 aa of the cytoplasmic domain were deleted), constructed by recombinant PCR, were subcloned into the HindIII site of pH-2/IV (murine H-2Kd promoter) (22). After digestion with PvuII and SphI, the inserted fragment was used for injection. Transgenic (Tg) mice were generated by microinjection into fertilized mouse embryos derived from C57BL/6. All Tg mice were crossed with FcεRIγ-deficient (γ−/−) mice (14).

C57BL/6 mice were purchased from the Shizuoka Laboratory Animal Corp. (Hamamatsu, Japan). γ−/− mice were established with the C57BL/6 background by the use of the C57BL/6 ES cell line (14). All mice were bred and maintained in our own animal facility under specific pathogen-free (SPF) conditions.

IgE anti-DNP mAb (SPE-7) was purchased from Sigma-Aldrich (St. Louis, MO). IgE anti-DNP mAb (H1 DNP-ε-26) and anti-FcεRIβ mAb (JRK) were provided by Dr. F. Liu (University of California, Davis, CA) (23) and Dr. J. Rivera (National Institutes of Health, Bethesda, MD) (24), respectively. FITC-conjugated, biotinylated, and unlabeled anti-mouse IgE mAb (R35-72) and anti-FcγRII/III mAb (2.4G2) were purchased from BD PharMingen (San Diego, CA). PE-conjugated anti-c-Kit mAb (2B8) was purchased from eBioscience (San Diego, CA). Anti-FcεRIγ(IC51–64) Ab was prepared by immunizing rabbits with the synthetic peptide RKAAIASREKADAV corresponding to aa 51–64 of FcεRIγ (Asahi Technoglass, Chiba, Japan).

For preparation of BMMCs, femoral bone marrow cells from C57BL/6 mice were cultured in RPMI 1640 medium containing 10% FCS and 10% of the culture supernatant of IL-3-secreting X63 cells (the IL-3 medium; provided by Dr. H. Karasuyama, Tokyo Medical and Dental University, Tokyo, Japan). Nonadherent cells were harvested and resuspended in the IL-3 medium weekly. More than 98% of the cells became c-Kit-positive after 4–8 wk of culture.

The cDNAs encoding wild-type and mutant FcεRIγ (YF and ΔCT) were subcloned into the EcoRI site of the retroviral vector, pMX-internal ribosome entry site (IRES)-green fluorescence protein (GFP; provided by Dr. Toshio Kitamura, Tokyo University, Tokyo, Japan). Flag-tagged FcεRIγ was prepared by fusing the signal sequence (1–18 aa)-deleted FcεRIγ to the Signaling lymphocyte activation molecule signal peptide-driven Flag sequence at the N terminus. Flag-FcεRIγ WT, YF, and ΔCT were subcloned into pMX-neo.

The cDNAs encoding wild-type and mutant FcεRIγ (YF and ΔCT) in pMX-IRES-GFP were transiently transfected into the packaging cell line Phoenix (from Dr. G. Nolan, Stanford University, Stanford, CA) using Lipofectamine Plus (Life Technologies, Gaithersburg, MD). The supernatants were collected 24 h later and used as viral supernatants. For infection, bone marrow cells from 10- to 15-wk-old γ−/− mice that had been injected with 5-fluorouracil (150 mg/kg i.p.; Sigma-Aldrich) 4 days previously were stained with FITC-conjugated anti-Sca-1 mAb (BD PharMingen) and anti-FITC microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by cell sorting using MACS (Miltenyi Biotec). The cells were incubated (1 × 106 cells/ml) for 3 days in 0.5 ml of viral supernatant and 0.5 ml of RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin/streptomycin, and a cytokine mixture (20 ng/ml murine IL-3, 100 ng/ml murine stem cell factor (Genzyme Techne, Minneapolis, MN), 100 ng/ml human IL-6 (from IL-6-producing X63 cells provided by Dr. H. Karasuyama, Tokyo Medical and Dental University) (25), and 10 μg/ml polybrene (Sigma-Aldrich)). The mixture and the viral supernatant were added again 24 h later, and the cells were incubated for an additional 72 h. GFP-positive cells were sorted by FACStar Plus (BD Bioscience; purity, >96%) and cultured in the IL-3 medium.

Total RNA was isolated from BMMCs and reverse transcribed using random primers and the Superscript preamplification system (Life Technologies). The titrated amount of cDNA was amplified by PCR using primers specific for FcεRIβ (26), and β-actin as an internal control. Real-time fluorescent PCR was used to quantitate FcεRIβ expression using SYBR Green fluorogenic probe (Bio-Rad). Fold changes in mRNA levels were calculated as 2−x, where x is the difference between the β-actin-normalized threshold cycle number values of each sample.

BMMCs and peritoneal mast cells were preincubated with 2.4G2 mAb to prevent nonspecific binding to FcγRII/III, and then stained with mouse IgE anti-DNP mAb (10 μg/ml) at 4°C for 1 h, followed by FITC-conjugated anti-mouse IgE mAb (5 μg/ml) or biotinylated anti-mouse IgE mAb (5 μg/ml) and allophycocyanin-streptavidin (BD PharMingen) at 4°C for 30 min. Cells were also stained with PE-conjugated anti-c-Kit mAb. Cells were analyzed on a FACSCalibur (BD Bioscience) using CellQuest software (BD Bioscience).

BMMCs were lysed in 0.5% Triton X-100, 150 mM NaCl, 5 mM EDTA, 5 mM sodium fluoride, 1 mM sodium vanadate, 10 μg/ml pepstatin A, 5 μg/ml leupeptin, and 10 μg/ml aprotinin. For analyzing the expression of protein level of FcεRIγ, total cell lysates were biotinylated with 0.25 mg/ml NHS-biotin (Pierce, Rockford, IL), incubated at 4°C for 30 min, and immunoprecipitated with anti-FcεRIγ(IC51–64) Ab and separated on two-dimensional nonreducing and reducing SDS-PAGE (27). Proteins were visualized by streptavidin-peroxidase (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) and chemiluminescent substrate (UltraSignal; Pierce, Rockford, IL). For analysis of surface FcεRIβ, cells were incubated with 10 μg/ml anti-DNP IgE (Sigma-Aldrich) at 4°C for 30 min and lysed in 0.5% Triton X-100 lysis buffer (28). Lysates were immunoprecipitated with anti-IgE Ab (BD PharMingen) and blotted with anti-mouse FcεRIβ mAb (JRK) (24).

The degranulation assay was performed as previously described (18). Briefly, mast cells were incubated overnight with 1 μg/ml mouse anti-DNP IgE at 37°C, followed by challenge with graded amounts of DNP-human serum albumin (DNP-HSA; Sigma-Aldrich) for 30 min in Tyrode’s buffer (130 mmol/L NaCl, 5 mmol/L KCl, 1.4 mmol/L CaCl2, 1 mmol/L MgCl2, 5.6 mmol/L glucose, 10 mmol/L HEPES, and 0.1% BSA, pH 7.4). Nonsensitized mast cells stimulated with 200 ng/ml A23187 (Wako, Osaka, Japan) were used as positive controls. The supernatants were collected, and the cell pellets were lysed in 0.5% Triton X-100-containing Tyrode’s buffer for measurement of β-hexosaminidase. Supernatants and cell lysates were incubated with a substrate (1.3 mg/ml p-nitrophenyl-N-acetyl β-d-glucosaminide in 0.1 mol/L sodium citrate, pH 4.5) for 2 h at 37°C. The reaction was stopped by adding 0.2 mol/L glycine (pH 10.7), and OD at 405 nm was measured. The percentage of specific β-hexosaminidase release was calculated as follows: 100 × supernatant activity/(supernatant activity + cell lysate activity).

BMMCs were cultured and stimulated with anti-DNP IgE and DNP-HSA as described above. TNF-α and IL-6 secreted into the supernatant were measured using an ELISA kit (Genzyme Techne) for TNF-α and a standard ELISA using anti-IL-6 mAbs (MP5-20F3; BD PharMingen) for coating and biotinylated anti-IL-6 mAbs (MP5-32C11; BD PharMingen) for detection. Nonsensitized mast cells stimulated with 200 ng/ml A23187 were used as positive controls. The released PGD2 in the supernatant was measured using an enzyme immunoassay kit (Cayman, Ann Arbor, MI) according to the manufacturer’s protocol.

The PSA experiment was performed as previously described (29). Mice were sensitized by i.v. injection of 20 μg of mouse anti-DNP IgE in 200 μl of PBS, followed by i.v. challenge with 1 mg of DNP-HSA in 200 μl of PBS 24 h later. Body temperature was monitored using a rectal probe before and at various intervals after Ag challenge.

To analyze the in vivo function of FcεRIγ, particularly of its ITAM, in IgE/FcεRI-mediated responses, we generated Tg mice expressing mutants of FcεRIγ. Two mutant FcεRIγ were constructed: YF, in which two tyrosines (Y65 and Y76) within ITAM were replaced with phenylalanines (γYF), and ΔCT, in which we deleted the distal region of its cytoplasmic domain including ITAM (aa 65–86; γΔCT). These two mutants as well as the WT FcεRIγ (γWT) were subcloned into an expression vector containing an H-2Kd promoter. Several Tg lines for each construct were established with the C57BL/6 background, and all Tg mice were crossed with FcεRIγ-deficient (γ−/−) mice and described as Tg mice. One representative line for each construct is described in this study. All Tg mice were born normally and were as healthy as normal mice.

BMMC from Tg mice, cultured in IL-3-containing medium for 4–8 wk, became >98% c-Kit-positive (data not shown). All BMMC developed normally, with no obvious difference in the kinetics of generation and the number of mast cells between the mice. We analyzed surface FcεRI expression by flow cytometry. Whereas BMMCs from γ−/− mice did not express surface FcεRI as previously described (18), those from all three Tg mice expressed similar levels of surface FcεRI, but at lower (5–11%) levels than in normal mice (Fig. 1,A). These results demonstrate that the cytoplasmic distal region of FcεRIγ is not essential for the surface expression of the FcεRI complex on mast cells in vivo. We then examined the efficiency of mutant FcεRIγ expression by comparing the cell surface expression level with the total amounts of FcεRIγ proteins. To detect these mutant FcεRIγ proteins, we produced an anti-FcεRIγ(IC51–64) Ab specifically against the transmembrane-proximal region of FcεRIγ, which could even detect γΔCT. This Ab precipitated both Flag-tagged WT and mutant FcεRIγ equally well (Fig. 1,C). As this Ab can be used for immunoprecipitation, but not immunoblotting, the total cell lysate of each cell was subjected to biotinylation, and biotinylated proteins were immunoprecipitated with this Ab, followed by analysis on nonreducing-reducing, two-dimensional gels. As shown in Fig. 1 D, using this Ab we detected all mutant proteins of FcεRIγ and found that the FcεRIγ protein expression level in BMMCs from each Tg mouse correlated with cell surface expression.

FIGURE 1.

Expression of FcεRI (α-, β-, and γ-chains) in BMMCs from Tg mice and retro-BMMC. A and B, Flow cytometric analysis of surface FcεRI expression on BMMCs. A, BMMCs from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were pretreated with 2.4G2 to prevent nonspecific binding, stained with IgE (10 μg/ml) at 4°C for 1 h, followed by FITC-anti-IgE mAb for 30 min, and analyzed by FACSCalibur. The solid line indicates control staining. MFI: normal, 212; WT, 11; YF, 24; ΔCT, 13. B, BMMCs from normal (+/+), and retrovirally transfected γ−/− bone marrow cells (mock, WT, YF, and ΔCT) were pretreated with 2.4G2, and stained and analyzed as described in A. MFI: normal, 262; WT, 197; YF, 181; and ΔCT, 160. C, Reactivity of anti-FcεRIγ(IC51–64) Ab to mutant FcεRIγ. 293T cells were transiently transfected with expressible constructs of Flag-tagged FcεRIγ (WT) and mutants (YF or ΔCT). Cell lysates were immunoprecipitated with anti-FcεRIγ(IC51–64) Ab. Total lysates (upper panel) and immunoprecipitates (lower panel) were blotted with anti-Flag mAb. D, Expression level of total FcεRIγ in BMMCs from Tg mice. Lysates of BMMCs derived from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were biotinylated and immunoprecipitated with anti-FcεRIγ(IC51–64) Ab (see Materials and Methods). The precipitates were analyzed on nonreducing (NR) and reducing (R) two-dimensional gels to detect all FcεRIγ proteins. E, Expression of FcεRIβ in the cell surface FcεRI complex. BMMCs were incubated with IgE at 4°C for 30 min, washed, and lysed in 0.5% Triton X-100. The IgE-bound FcεRI complex in the lysates were immunoprecipitated with anti-IgE Ab and protein A-Sepharose. Immunoprecipitates were blotted with anti-FcεRIβ Ab (upper panel). Total cell lysates were also blotted with anti-FcεRIb Ab (second panel) and anti-Erk1/2 Ab (third panel) as controls. Note that although FcεRIβ precipitated from YF appears somewhat higher than WT or ΔCT, this reflects a slightly higher level of FcεRI on the cell surface of YF as shown in A. RT-PCR analysis for FcεRIβ mRNA was performed using primers specific for FcεRIβ and normalized by β-actin (bottom panel). Representative data of unsaturated amount of cDNA are shown. The results of additional real-time PCR are described in Results.

FIGURE 1.

Expression of FcεRI (α-, β-, and γ-chains) in BMMCs from Tg mice and retro-BMMC. A and B, Flow cytometric analysis of surface FcεRI expression on BMMCs. A, BMMCs from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were pretreated with 2.4G2 to prevent nonspecific binding, stained with IgE (10 μg/ml) at 4°C for 1 h, followed by FITC-anti-IgE mAb for 30 min, and analyzed by FACSCalibur. The solid line indicates control staining. MFI: normal, 212; WT, 11; YF, 24; ΔCT, 13. B, BMMCs from normal (+/+), and retrovirally transfected γ−/− bone marrow cells (mock, WT, YF, and ΔCT) were pretreated with 2.4G2, and stained and analyzed as described in A. MFI: normal, 262; WT, 197; YF, 181; and ΔCT, 160. C, Reactivity of anti-FcεRIγ(IC51–64) Ab to mutant FcεRIγ. 293T cells were transiently transfected with expressible constructs of Flag-tagged FcεRIγ (WT) and mutants (YF or ΔCT). Cell lysates were immunoprecipitated with anti-FcεRIγ(IC51–64) Ab. Total lysates (upper panel) and immunoprecipitates (lower panel) were blotted with anti-Flag mAb. D, Expression level of total FcεRIγ in BMMCs from Tg mice. Lysates of BMMCs derived from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were biotinylated and immunoprecipitated with anti-FcεRIγ(IC51–64) Ab (see Materials and Methods). The precipitates were analyzed on nonreducing (NR) and reducing (R) two-dimensional gels to detect all FcεRIγ proteins. E, Expression of FcεRIβ in the cell surface FcεRI complex. BMMCs were incubated with IgE at 4°C for 30 min, washed, and lysed in 0.5% Triton X-100. The IgE-bound FcεRI complex in the lysates were immunoprecipitated with anti-IgE Ab and protein A-Sepharose. Immunoprecipitates were blotted with anti-FcεRIβ Ab (upper panel). Total cell lysates were also blotted with anti-FcεRIb Ab (second panel) and anti-Erk1/2 Ab (third panel) as controls. Note that although FcεRIβ precipitated from YF appears somewhat higher than WT or ΔCT, this reflects a slightly higher level of FcεRI on the cell surface of YF as shown in A. RT-PCR analysis for FcεRIβ mRNA was performed using primers specific for FcεRIβ and normalized by β-actin (bottom panel). Representative data of unsaturated amount of cDNA are shown. The results of additional real-time PCR are described in Results.

Close modal

It has been well documented that FcεRIβ is also essential for the surface expression of murine FcεRI (6, 10, 30). To confirm the involvement of FcεRIβ in the reconstituted FcεRI, we performed biochemical analysis. Fig. 1,E shows that the amount of FcεRIβ associated with IgE-bound surface FcεRI correlated with the cell surface expression level of FcεRI. As the surface FcεRI expression on YF is a little higher than others, YF expressed a proportionally higher level of FcεRIβ in the complex. The apparent reduction of the FcεRIβ protein expression level in γ−/− BMMCs suggests that the FcεRIβ protein is susceptible to degradation in the absence of FcεRIγ, consistent with the observation that FcεRIγ-deficient cells express similar amounts of FcεRIβ mRNAs (Fig. 1 E, lanes 1 and 2) (31). Similar expression levels of FcεRIβ mRNA in the BMMCs were also confirmed by quantitative real-time RT-PCR: 1.00 ± 0.15, 0.98 ± 0.16, 1.17 ± 0.16, 1.31 ± 0.32, and 0.83 ± 0.11 for +/+, −/−, WT, YF, and ΔCT, respectively.

As we could not establish mutant Tg mice expressing similar levels of surface FcεRI as normal mast cells, we took another approach by using gene-transfected BMMCs to generate BMMCs expressing normal levels of FcεRI with mutant FcεRIγ. To this end, these mutants in a retrovirus vector containing IRES-GFP were transfected into bone marrow cells from γ−/− mice. After 6–8 wk, BMMCs (>98% c-Kit-positive) were generated (retro-BMMC), and GFP+ cells were analyzed for surface FcεRI expression. Whereas mock vector-transferred BMMCs from γ−/− mice (BMMC (mock)) failed to express cell surface FcεRI, gene-transferred BMMCs (WT, YF, and ΔCT) expressed levels of FcεRI on the cell surface similar to those of normal BMMCs (Fig. 1 B). These results confirm the finding in Tg mice that the cytoplasmic tail of FcεRIγ including ITAM is not required for surface expression of the FcεRI complex on BMMCs.

To analyze the three major pathways triggered by the aggregation of IgE-bound FcεRI on mast cells by Ag degranulation, arachidonic acid metabolism, and cytokine production, BMMCs from Tg mice were sensitized with anti-DNP IgE mAb and stimulated with DNP-HSA.

Firstly, the degranulation, as measured by the release of β-hexosaminidase, was induced in BMMCs from WT and normal mice upon IgE(+Ag) stimulation, although the degree of degranulation in WT mast cells was much lower than that in normal mast cells, as expected from the surface expression of FcεRI. In contrast, no significant degranulation was induced in BMMCs from YF, ΔCT, or γ−/− mice (Fig. 2,A). The degree of degranulation upon stimulation with Ca2+ ionophore was similar among these mutant FcεRIγ-expressing cells, indicating that the downstream machinery for the degranulation in these cells is intact. Secondly, we measured the production of IL-6 and TNF-α as the representative cytokines secreted from mast cells upon FcεRI cross-linking. Cytokine production was induced in BMMCs from WT and normal mice, but not from YF, ΔCT, and γ−/− mice in response to FcεRI cross-linking with IgE(+Ag) (Fig. 2, B and C). Thirdly, similar to degranulation and cytokine production, PGD2 release was undetectable in mast cells from YF, ΔCT, and γ−/− mice, whereas WT produced PGD2 at a level comparable to that of normal mice (Fig. 2 D).

FIGURE 2.

IgE(+Ag)-induced activation of mast cells is mediated by FcεRIγ-ITAM. Ag-dependent activation of mast cells was analyzed in mast cells from Tg mice (A–D) and retrovirally transfected BMMCs (E–H) for β-hexosaminidase release (A and E), secretion of TNF-α (B and F) and IL-6 (C and G), and PGD2 production (D and H). A–D, BMMCs from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were stimulated. E–H, BMMCs from normal mice (+/+) and retrovirally transfected γ−/− bone marrow cells (mock, WT, YF, and ΔCT) were used. BMMCs were sensitized with 1 μg/ml anti-DNP IgE for 12 h and stimulated with DNP-HSA at the indicated concentrations (A–C: 0 (□), 5 (▦), 15 (▦), and 50 (▪) ng/ml; D–H: 0 (□) and 15 (▪) ng/ml) or with A23187 (A and E). Cells were stimulated for 30 min (A and E), 15 min (D and H), or 18 h (B, C, F, and G). The release of β-hexosaminidase, PGD2 and cytokines was measured as described in Materials and Methods. Data are presented as the mean ± SD of triplicate determinations and are representative of two experiments.

FIGURE 2.

IgE(+Ag)-induced activation of mast cells is mediated by FcεRIγ-ITAM. Ag-dependent activation of mast cells was analyzed in mast cells from Tg mice (A–D) and retrovirally transfected BMMCs (E–H) for β-hexosaminidase release (A and E), secretion of TNF-α (B and F) and IL-6 (C and G), and PGD2 production (D and H). A–D, BMMCs from normal mice (+/+), γ−/− mice (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) were stimulated. E–H, BMMCs from normal mice (+/+) and retrovirally transfected γ−/− bone marrow cells (mock, WT, YF, and ΔCT) were used. BMMCs were sensitized with 1 μg/ml anti-DNP IgE for 12 h and stimulated with DNP-HSA at the indicated concentrations (A–C: 0 (□), 5 (▦), 15 (▦), and 50 (▪) ng/ml; D–H: 0 (□) and 15 (▪) ng/ml) or with A23187 (A and E). Cells were stimulated for 30 min (A and E), 15 min (D and H), or 18 h (B, C, F, and G). The release of β-hexosaminidase, PGD2 and cytokines was measured as described in Materials and Methods. Data are presented as the mean ± SD of triplicate determinations and are representative of two experiments.

Close modal

To confirm the results from Tg mice, we used retro-BMMC expressing similar levels of cell surface FcεRI as normal BMMCs. Degranulation, cytokine secretion, and PGD2 production were similarly induced in normal and WT BMMCs upon FcεRI cross-linking. In contrast, none of these responses was triggered in BMMCs expressing γYF and γΔCT or mock vector (Fig. 2, E–H).

These results demonstrate that FcεRIγ-ITAM is essential for all three major pathways that mediate the release of proinflammatory mediators upon FcεRI engagement, and FcεRIβ cannot replace this function of FcεRIγ.

To investigate the requirement of FcεRIγ-ITAM-mediated signals for IgE(−Ag)-induced mast cell survival, we used retro-BMMC expressing similar levels of cell surface FcεRI as normal mast cells, similar to the previous analysis of FcεRI expression and activation. We found that whereas survival was significantly promoted by IgE (SPE-7) alone under the IL-3-depleted condition in normal and WT BMMCs, the survival of both BMMCs expressing γYF and γΔCT and vector alone was completely abrogated (Fig. 3 A). These results clearly demonstrate a critical role of FcεRIγ-ITAM in IgE(−Ag)-induced mast cell survival.

FIGURE 3.

IgE(−Ag) acts through FcεRIγ-ITAM to trigger mast cell survival and cytokine production. A, BMMCs derived from normal mice (+/+) and retrovirally transfected γ−/− bone marrow cells were subjected to IL-3 depletion and incubated with anti-DNP IgE (upper panels, 10 μg/ml SPE-7; lower panels, 5 μg/ml H1 DNP-ε-26; •) or without IgE (○) in the absence of IL-3 for the indicated periods (0–4 days). Cells were stained with propidium iodide and analyzed by flow cytometry. The percentages of cells that were not stained by propidium iodide are plotted. B, BMMCs were cultured with medium alone (□), 5 μg/ml H1 DNP-ε-26 (▦), or SPE-7 (▪) in the absence of IL-3 for 3 days. IL-6 concentrations in the culture supernatants were determined by ELISA. ∗, Undetectable level (<0.1 ng/ml).

FIGURE 3.

IgE(−Ag) acts through FcεRIγ-ITAM to trigger mast cell survival and cytokine production. A, BMMCs derived from normal mice (+/+) and retrovirally transfected γ−/− bone marrow cells were subjected to IL-3 depletion and incubated with anti-DNP IgE (upper panels, 10 μg/ml SPE-7; lower panels, 5 μg/ml H1 DNP-ε-26; •) or without IgE (○) in the absence of IL-3 for the indicated periods (0–4 days). Cells were stained with propidium iodide and analyzed by flow cytometry. The percentages of cells that were not stained by propidium iodide are plotted. B, BMMCs were cultured with medium alone (□), 5 μg/ml H1 DNP-ε-26 (▦), or SPE-7 (▪) in the absence of IL-3 for 3 days. IL-6 concentrations in the culture supernatants were determined by ELISA. ∗, Undetectable level (<0.1 ng/ml).

Close modal

Differences in the molecular mechanism involved in IgE(−Ag)-induced survival signaling have been proposed by using different IgE clones (4, 5). We examined the requirement of FcεRIγ-ITAM for mast cell survival induced by another monoclonal IgE (H1 DNP-ε-26). As shown in Fig. 3,A, the two IgEs equally induced the survival of BMMCs expressing WT, but not YF and ΔCT. Thus, these results indicate that IgE(−Ag)-mediated mast cell survival is triggered by FcεRIγ-ITAM-dependent signals regardless of the IgE mAb used. Furthermore, both IgE mAbs induced substantial IL-6 production in the absence of Ag in our systems (Fig. 3 B). It is noteworthy, however, that the magnitude of the response by H1 DNP-ε-26 is approximately one-fifth of that by SPE-7. This quantitative difference may reflect the difference in results between two previous reports that detectable cytokine secretion was induced by SPE-7 (5), but not by H1 DNP-ε-26 (4). Taken together, these findings suggest that IgE(−Ag) can induce responses through FcεRIγ-ITAM.

We next examined the requirement of FcεRIγ-ITAM in IgE(−Ag)-mediated up-regulation of cell surface FcεRI expression. γYF- and γΔCT-containing BMMCs from Tg mice showed an up-regulation of surface FcεRI expression regardless of whether the FcεRIγ was WT or mutant (Fig. 4,A). The results were confirmed using retro-BMMC that expressed equal levels of FcεRI on the cell surface. Indeed, IgE increased the surface FcεRI expression equally in all mast cells expressing γYF, γΔCT, and γWT (Fig. 4 B). These results suggest that this regulation is independent of phosphorylation-mediated signals through FcεRIγ.

FIGURE 4.

Up-regulation of cell surface FcεRI expression by IgE(−Ag) is mediated by an FcεRIγ-ITAM-independent mechanism. A, BMMCs derived from normal (▪), γ−/− (□), and FcεRIγ Tg mice with each γ−/− background (WT (•), YF (▴), and ΔCT (▵)); B, BMMCs from normal mice (▪) and retrovirally transfected γ−/− bone marrow cells (mock (□), WT (•), YF (▴), and ΔCT (▵)) were cultured in the presence of 5 μg/ml IgE for the indicated periods and assessed for surface FcεRI expression by flow cytometry. Data are presented as the mean ± SD of triplicate determinations and are representative of two experiments. C, Correlation of serum total IgE level with the surface FcεRI expression on peritoneal mast cells from γ−/− (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) under air-uncontrolled conditions. Serum total IgE levels were measured by ELISA, and surface FcεRI expression was analyzed by flow cytometry. The number in each panel indicates the correlation coefficient (R) as calculated by Pearson’s correlation coefficient test.

FIGURE 4.

Up-regulation of cell surface FcεRI expression by IgE(−Ag) is mediated by an FcεRIγ-ITAM-independent mechanism. A, BMMCs derived from normal (▪), γ−/− (□), and FcεRIγ Tg mice with each γ−/− background (WT (•), YF (▴), and ΔCT (▵)); B, BMMCs from normal mice (▪) and retrovirally transfected γ−/− bone marrow cells (mock (□), WT (•), YF (▴), and ΔCT (▵)) were cultured in the presence of 5 μg/ml IgE for the indicated periods and assessed for surface FcεRI expression by flow cytometry. Data are presented as the mean ± SD of triplicate determinations and are representative of two experiments. C, Correlation of serum total IgE level with the surface FcεRI expression on peritoneal mast cells from γ−/− (−/−), and FcεRIγ Tg mice with each γ−/− background (WT, YF, and ΔCT) under air-uncontrolled conditions. Serum total IgE levels were measured by ELISA, and surface FcεRI expression was analyzed by flow cytometry. The number in each panel indicates the correlation coefficient (R) as calculated by Pearson’s correlation coefficient test.

Close modal

We next analyzed the up-regulation of surface FcεRI expression on mast cells by IgE(−Ag) in vivo. The Tg mice produced no detectable IgE under SPF conditions in which they were maintained in a laminar filter-air flow enclosure in a bioclean room. However, when mating pairs of SPF mice were moved to an air-uncontrolled conventional room, high titers of IgE were detected in sera at 6 wk of age in all progeny, as reported for other mouse strains (32, 33). We used this system and examined the serum total IgE levels and the surface FcεRI expression on mast cells from reconstituted mice. Fig. 4 C shows the relationship between the mean fluorescence intensity (MFI) of the surface expression of FcεRI on peritoneal mast cells and total serum IgE. The results demonstrate that the in vivo surface expression levels of FcεRI on mast cells from YF, ΔCT, and WT mice exhibit positive correlations with total serum IgE. In contrast to the increased surface FcεRI expression, the number of peritoneal c-Kit-positive mast cells did not change in these Tg mice (data not shown). These results further demonstrate that the IgE-mediated up-regulation is independent of FcεRIγ-ITAM-mediated signals.

Finally, we examined in vivo allergic responses in these Tg mice. Although IgE-mediated passive anaphylaxis is abolished in γ−/− mice (15), it remains unclear whether the failure of IgE-mediated passive anaphylaxis in vivo is dependent on the lack of FcεRIγ- and/or FcεRIβ-mediated signals. To address this issue, Tg and normal mice as well as γ−/− mice were injected with anti-DNP IgE and challenged 24 h later with DNP-HSA as an Ag, and their body temperatures were monitored. As shown in Fig. 5, a rapid decrease in rectal temperature was observed in WT and normal mice 20–40 min after the Ag challenge. In contrast, there was no significant change in the rectal temperature of ΔCT, YF, and γ−/− mice. These results indicate that in vivo PSA is mediated mainly by signals through FcεRIγ-ITAM, which cannot be replaced by FcεRIβ-ITAM.

FIGURE 5.

IgE(+Ag)-induced passive systemic anaphylaxis in vivo is dependent on FcεRIγ-ITAM. Rectal temperatures of normal (▪), γ−/− (□), and FcεRIγ Tg mice with each γ−/− background (WT (•), YF (▴), and ΔCT (▵)) during IgE(+Ag)-induced PSA were measured at the time indicated. Four normal, three γ−/−, five WT, nine YF, and three ΔCT mice were injected with 20 μg of anti-DNP IgE i.v. All animals were then challenged i.v. with 1 mg of DNP-HSA 24 h later. Data are shown as the mean ± SD. ∗, p < 0.01; ∗∗, p < 0.001 (compared with γ−/− mice). The difference in rectal temperature between normal and WT mice was not statistically significant.

FIGURE 5.

IgE(+Ag)-induced passive systemic anaphylaxis in vivo is dependent on FcεRIγ-ITAM. Rectal temperatures of normal (▪), γ−/− (□), and FcεRIγ Tg mice with each γ−/− background (WT (•), YF (▴), and ΔCT (▵)) during IgE(+Ag)-induced PSA were measured at the time indicated. Four normal, three γ−/−, five WT, nine YF, and three ΔCT mice were injected with 20 μg of anti-DNP IgE i.v. All animals were then challenged i.v. with 1 mg of DNP-HSA 24 h later. Data are shown as the mean ± SD. ∗, p < 0.01; ∗∗, p < 0.001 (compared with γ−/− mice). The difference in rectal temperature between normal and WT mice was not statistically significant.

Close modal

In this study we have distinguished between FcεRIγ-ITAM-dependent and -independent responses in the FcεRI-mediated function of mast cells. We demonstrated that FcεRIγ-ITAM is essential for FcεRI-mediated cell activation and anaphylactic responses in vivo upon IgE(+Ag) stimulation. Moreover, we found that the promotion of mast cell survival by IgE(−Ag) is also mediated by signals through FcεRIγ-ITAM. In contrast, we found that IgE(−Ag)-mediated up-regulation of cell surface FcεRI expression is independent of FcεRIγ-ITAM.

Using FcεRIγ-reconstituted Tg mice, we showed expression of FcεRI with a mutant FcεRIγ lacking the cytoplasmic tail on the surface of mast cells in vivo, indicating that FcεRIγ-ITAM is not required for surface expression. Biochemical analyses of the reconstituted FcεRI complex on the cell surface reveal that the composition of FcεRI with α-, β-, and γ-chains is similar to that on normal mast cells, and the surface expression level correlates with the expression of FcεRIγ protein.

Until now, the in vivo function of FcεRIγ-ITAM in FcεRI-induced mast cell activation upon stimulation with IgE(+Ag) had not been analyzed. We have demonstrated that γ−/− mice expressing γYF and γΔCT with the ITAM mutation/deletion fail to exhibit IgE-mediated PSA, whereas their mast cells showed complete abrogation of all three activation pathways, degranulation, cytokine secretion, and PGD2 synthesis, upon IgE(+Ag) stimulation. These results indicate that the phosphorylation of FcεRIγ-ITAM is essential for mast cell activation in vivo, which agrees with previous observations in established cell lines in vitro (9, 34, 35). In addition, our results are consistent with the idea that FcεRIβ augment FcεRIγ-ITAM-mediated activation signals rather than transduces them independently of FcεRIγ (10).

We also provide new insights into mast cell function/signaling induced by IgE(−Ag), particularly surface FcεRI expression and cell survival. We have demonstrated that the surface FcεRI expression on mast cells is up-regulated by IgE(−Ag) regardless of the mutation/deletion of FcεRIγ-ITAM and is therefore regulated independently of FcεRIγ-ITAM. These results are consistent with early in vitro studies suggesting that the up-regulation of surface FcεRI expression by IgE(−Ag) is independent of FcεRI-mediated intracellular signals (36, 37). Taken together, the up-regulation of surface FcεRI expression by IgE(−Ag) appears to be regulated by receptor stabilization on the plasma membrane upon binding to IgE to FcεRIα, without activation.

The recent finding that the binding of IgE(−Ag) to FcεRI on mast cells promotes survival in the absence of IL-3 (4, 5) provides insights into the physiology of mast cell function. However, whether the signaling pathway induced by IgE(−Ag) is similar to that by IgE(+Ag) remains to be determined. Our data clearly show that IgE(−Ag)-induced mast cell survival is also mediated by FcεRIγ-ITAM. IgE(−Ag) can promote mast cell survival, but cannot induce degranulation and leukotriene synthesis (5), whereas IgE(+Ag) triggers degranulation and leukotriene synthesis. In Btk/Lyn doubly-deficient mast cells, FcεRI-induced degranulation and leukotriene release upon cross-linking with IgE(+Ag) are almost abrogated (38), whereas IgE(−Ag) treatment of BMMCs from these mice are reported to promote survival (4). These observations suggest a difference in the downstream signaling through FcεRI upon stimulation by IgE(−Ag) and IgE(+Ag). The recent finding that Fyn-mediated signaling is involved in IgE(+Ag)-induced degranulation may be relevant (8). With regard to cytokine production by IgE(−Ag), two groups obtained apparently discrepant results using different systems, including cell culture conditions (4, 5, 39). One difference was the IgE clone used: H1 DNP-ε-26 or SPE-7. In the present study both mAbs induced mast cell survival in an FcεRIγ-ITAM-dependent manner. Furthermore, both mAbs induced FcεRIγ-ITAM-dependent IL-6 production in the absence of Ag in our system, although H1 DNP-ε-26 induced much lower responses. Collectively, our data suggest that IgE(−Ag) acts through the FcεRIγ-ITAM-dependent signaling pathway for the induction of cytokine production and mast cell survival. We are now investigating the further downstream pathway in IgE(−Ag)-induced survival.

The binding of IgE to FcεRI up-regulates cell surface FcεRI expression, which enhances sensitivity to IgE, and triggers signals required for mast cell survival. Such an amplification circuit enables continuous sensitization with IgE and in the immediate and robust responses upon challenge by allergens (39). Our study reveals that this allergy-promoting system is maintained by multiple mechanisms in both an FcεRIγ-ITAM-dependent and independent manner. Targeting FcεRIγ on the basis of these results may provide a new approach for the prevention of allergic diseases.

We thank Drs. H. Ohno and S. Taki for discussion; M. Sakuma, R. Shiina, E. Ishikawa, and M. Kohno for technical assistance; and H. Yamaguchi and Y. Kurihara for secretarial assistance.

1

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology and from the Ministry of Health, Labor, and Welfare, Japan.

6

Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; BMMC, bone marrow-derived mast cell; GFP, green fluorescence protein; HSA, human serum albumin; IRES, internal ribosome entry site; MFI, mean fluorescence intensity; PSA, passive systemic anaphylaxis; SPF, specific pathogen free; Tg, transgenic; WT, wild type.

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