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RI
-ITAM Is Differentially Required for Mast Cell Function In Vivo1
Departments of
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Molecular Genetics and
Otorhinolaryngology, Graduate School of Medicine, Chiba University, Chiba, Japan;
PRESTO, Japan Science and Technology, Kawaguchi, Japan; and
Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of FcRI
signaling to IgE/FcRI-mediated mast cell responses has not yet been elucidated. In this study FcRI-/- mast cells were reconstituted with either wild-type or mutant FcRI in transgenic mice and transfected mast cells in vitro. We demonstrate that FcRI-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 FcRI 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 FcRI-immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of FcRI is independent of FcRI signaling. These results indicate that FcRI-mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, 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 
-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 FcRI-mediated activation of mast cells has been thought to occur only upon cross-linking of FcRI with IgE and Ag (IgE(+Ag)). However, recent reports have suggested that cell surface FcRI expression and mast cell survival are regulated by IgE in the absence of Ag (IgE(-Ag)) (3, 4, 5). Although the surface FcRI 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 FcRI has been shown to be involved (4), the mechanism is largely unknown.
FcRI is expressed on rodent mast cells as a tetrameric structure composed of 
, , and homodimers (6). The -chain (FcRI) is responsible for binding to IgE. The - and -chains (FcRI, FcRI) possess immunoreceptor tyrosine-based activation motifs (ITAMs)6 (7) within their cytoplasmic domains. The cross-linking of FcRI 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 FcRI, 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 FcRI cross-linking (8). Although FcRI is believed to play a role in the amplification of activation signals through FcRI (9, 10), it has been shown that Syk binds to phospho-ITAMs of FcRI as well as FcRI (11) and that the cross-linking of FcRI induces weak Ca2+ influx (12). Therefore, it is possible that FcRI-mediated signals may trigger some in vivo responses via ITAM.
FcRI is thought to be a pivotal subunit of the FcRI complex for intracellular signaling upon IgE(+Ag) stimulation (1, 13). We and another group have generated FcRI-deficient mice (-/-) (14, 15) and have analyzed the in vivo function of FcR and FcR in various systems (14, 15, 16, 17, 18, 19, 20, 21). However, the in vivo function of FcRI-mediated signals in FcRI-mediated responses has not been elucidated, mainly because FcRI is essential for cell surface expression.
In this study, -/- mast cells were reconstituted with mutant FcRI in transgenic mice and bone marrow-derived mast cells (BMMCs), and the function of FcRI-ITAM was analyzed. We demonstrate that most FcRI-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 FcRI-ITAM signaling. In contrast, we show that the up-regulation of surface FcRI expression on mast cells by IgE is regulated independently of FcRI-ITAM. Thus, we unveiled the differential requirement of FcRI-mediated signals for the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.
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Materials and Methods |
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RI transgenic mice
The cDNAs encoding murine wild-type FcRI (WT) and two mutant FcRI (YF, tyrosines at positions 65 and 76 within ITAM were replaced with phenylalanines; 
CT, 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 FcRI-deficient (-/-) mice (14).
Mice
C57BL/6 mice were purchased from the Shizuoka Laboratory Animal Corp. (Hamamatsu, Japan). -/- 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.
Antibodies
IgE anti-DNP mAb (SPE-7) was purchased from Sigma-Aldrich (St. Louis, MO). IgE anti-DNP mAb (H1 DNP--26) and anti-FcRI 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-FcRII/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-FcRI(IC51–64) Ab was prepared by immunizing rabbits with the synthetic peptide RKAAIASREKADAV corresponding to aa 51–64 of FcRI (Asahi Technoglass, Chiba, Japan).
Cell preparation
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.
DNA construction
The cDNAs encoding wild-type and mutant FcRI (YF and CT) 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 FcRI was prepared by fusing the signal sequence (1–18 aa)-deleted FcRI to the Signaling lymphocyte activation molecule signal peptide-driven Flag sequence at the N terminus. Flag-FcRI WT, YF, and CT were subcloned into pMX-neo.
Retroviral transfection
The cDNAs encoding wild-type and mutant FcRI (YF and CT) 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 -/- 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 x 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.
RT-PCR
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 FcRI (26), and -actin as an internal control. Real-time fluorescent PCR was used to quantitate FcRI 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 -actin-normalized threshold cycle number values of each sample.
Flow cytometric analysis
BMMCs and peritoneal mast cells were preincubated with 2.4G2 mAb to prevent nonspecific binding to FcRII/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).
Biotinylation, immunoprecipitation, and Western blotting
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 FcRI, 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-FcRI(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 FcRI, 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 FcRI mAb (JRK) (24).
Degranulation assay
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 -hexosaminidase. Supernatants and cell lysates were incubated with a substrate (1.3 mg/ml p-nitrophenyl-N-acetyl -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 -hexosaminidase release was calculated as follows: 100 x supernatant activity/(supernatant activity + cell lysate activity).
Measurement of cytokines and PGD2
BMMCs were cultured and stimulated with anti-DNP IgE and DNP-HSA as described above. TNF- and IL-6 secreted into the supernatant were measured using an ELISA kit (Genzyme Techne) for TNF- 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.
Passive systemic anaphylaxis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI Tg mice
To analyze the in vivo function of FcRI, particularly of its ITAM, in IgE/FcRI-mediated responses, we generated Tg mice expressing mutants of FcRI. Two mutant FcRI were constructed: YF, in which two tyrosines (Y65 and Y76) within ITAM were replaced with phenylalanines (YF), and CT, in which we deleted the distal region of its cytoplasmic domain including ITAM (aa 65–86; CT). These two mutants as well as the WT FcRI (WT) 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 FcRI-deficient (-/-) 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.
The cytoplasmic distal region of FcRI is not required for in vivo expression of FcRI
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 FcRI expression by flow cytometry. Whereas BMMCs from -/- mice did not express surface FcRI as previously described (18), those from all three Tg mice expressed similar levels of surface FcRI, but at lower (5–11%) levels than in normal mice (Fig. 1A). These results demonstrate that the cytoplasmic distal region of FcRI is not essential for the surface expression of the FcRI complex on mast cells in vivo. We then examined the efficiency of mutant FcRI expression by comparing the cell surface expression level with the total amounts of FcRI proteins. To detect these mutant FcRI proteins, we produced an anti-FcRI(IC51–64) Ab specifically against the transmembrane-proximal region of FcRI, which could even detect CT. This Ab precipitated both Flag-tagged WT and mutant FcRI equally well (Fig. 1C). 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. 1D, using this Ab we detected all mutant proteins of FcRI and found that the FcRI protein expression level in BMMCs from each Tg mouse correlated with cell surface expression.
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RI is also essential for the surface expression of murine FcRI (6, 10, 30). To confirm the involvement of FcRI in the reconstituted FcRI, we performed biochemical analysis. Fig. 1E shows that the amount of FcRI associated with IgE-bound surface FcRI correlated with the cell surface expression level of FcRI. As the surface FcRI expression on YF is a little higher than others, YF expressed a proportionally higher level of FcRI in the complex. The apparent reduction of the FcRI protein expression level in -/- BMMCs suggests that the FcRI protein is susceptible to degradation in the absence of FcRI, consistent with the observation that FcRI-deficient cells express similar amounts of FcRI mRNAs (Fig. 1E, lanes 1 and 2) (31). Similar expression levels of FcRI 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 CT, respectively.
As we could not establish mutant Tg mice expressing similar levels of surface FcRI as normal mast cells, we took another approach by using gene-transfected BMMCs to generate BMMCs expressing normal levels of FcRI with mutant FcRI. To this end, these mutants in a retrovirus vector containing IRES-GFP were transfected into bone marrow cells from -/- mice. After 6–8 wk, BMMCs (>98% c-Kit-positive) were generated (retro-BMMC), and GFP+ cells were analyzed for surface FcRI expression. Whereas mock vector-transferred BMMCs from -/- mice (BMMC (mock)) failed to express cell surface FcRI, gene-transferred BMMCs (WT, YF, and CT) expressed levels of FcRI on the cell surface similar to those of normal BMMCs (Fig. 1B). These results confirm the finding in Tg mice that the cytoplasmic tail of FcRI including ITAM is not required for surface expression of the FcRI complex on BMMCs.
IgE(+Ag)-induced activation of mast cells depends on FcRI-ITAM
To analyze the three major pathways triggered by the aggregation of IgE-bound FcRI 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 -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 FcRI. In contrast, no significant degranulation was induced in BMMCs from YF, CT, or -/- mice (Fig. 2A). The degree of degranulation upon stimulation with Ca2+ ionophore was similar among these mutant FcRI-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- as the representative cytokines secreted from mast cells upon FcRI cross-linking. Cytokine production was induced in BMMCs from WT and normal mice, but not from YF, CT, and -/- mice in response to FcRI 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, CT, and -/- mice, whereas WT produced PGD2 at a level comparable to that of normal mice (Fig. 2D).
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RI as normal BMMCs. Degranulation, cytokine secretion, and PGD2 production were similarly induced in normal and WT BMMCs upon FcRI cross-linking. In contrast, none of these responses was triggered in BMMCs expressing YF and CT or mock vector (Fig. 2, E–H).
These results demonstrate that FcRI-ITAM is essential for all three major pathways that mediate the release of proinflammatory mediators upon FcRI engagement, and FcRI cannot replace this function of FcRI.
Dependence of IgE-induced mast cell survival on FcRI signaling
To investigate the requirement of FcRI-ITAM-mediated signals for IgE(-Ag)-induced mast cell survival, we used retro-BMMC expressing similar levels of cell surface FcRI as normal mast cells, similar to the previous analysis of FcRI 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 YF and CT and vector alone was completely abrogated (Fig. 3A). These results clearly demonstrate a critical role of FcRI-ITAM in IgE(-Ag)-induced mast cell survival.
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RI-ITAM for mast cell survival induced by another monoclonal IgE (H1 DNP--26). As shown in Fig. 3A, the two IgEs equally induced the survival of BMMCs expressing WT, but not YF and CT. Thus, these results indicate that IgE(-Ag)-mediated mast cell survival is triggered by FcRI-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. 3B). It is noteworthy, however, that the magnitude of the response by H1 DNP--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--26 (4). Taken together, these findings suggest that IgE(-Ag) can induce responses through FcRI-ITAM.
An FcRI-ITAM-independent mechanism mediates the in vivo up-regulation of cell surface FcRI expression by IgE
We next examined the requirement of FcRI-ITAM in IgE(-Ag)-mediated up-regulation of cell surface FcRI expression. YF- and CT-containing BMMCs from Tg mice showed an up-regulation of surface FcRI expression regardless of whether the FcRI was WT or mutant (Fig. 4A). The results were confirmed using retro-BMMC that expressed equal levels of FcRI on the cell surface. Indeed, IgE increased the surface FcRI expression equally in all mast cells expressing YF, CT, and WT (Fig. 4B). These results suggest that this regulation is independent of phosphorylation-mediated signals through FcRI.
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RI 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 FcRI expression on mast cells from reconstituted mice. Fig. 4C shows the relationship between the mean fluorescence intensity (MFI) of the surface expression of FcRI on peritoneal mast cells and total serum IgE. The results demonstrate that the in vivo surface expression levels of FcRI on mast cells from YF, CT, and WT mice exhibit positive correlations with total serum IgE. In contrast to the increased surface FcRI 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 FcRI-ITAM-mediated signals.
IgE-mediated passive systemic anaphylaxis through FcRI-ITAM
Finally, we examined in vivo allergic responses in these Tg mice. Although IgE-mediated passive anaphylaxis is abolished in -/- mice (15), it remains unclear whether the failure of IgE-mediated passive anaphylaxis in vivo is dependent on the lack of FcRI- and/or FcRI-mediated signals. To address this issue, Tg and normal mice as well as -/- 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 CT, YF, and -/- mice. These results indicate that in vivo PSA is mediated mainly by signals through FcRI-ITAM, which cannot be replaced by FcRI-ITAM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-ITAM-dependent and -independent responses in the FcRI-mediated function of mast cells. We demonstrated that FcRI-ITAM is essential for FcRI-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 FcRI-ITAM. In contrast, we found that IgE(-Ag)-mediated up-regulation of cell surface FcRI expression is independent of FcRI-ITAM.
Using FcRI-reconstituted Tg mice, we showed expression of FcRI with a mutant FcRI lacking the cytoplasmic tail on the surface of mast cells in vivo, indicating that FcRI-ITAM is not required for surface expression. Biochemical analyses of the reconstituted FcRI complex on the cell surface reveal that the composition of FcRI with -, -, and -chains is similar to that on normal mast cells, and the surface expression level correlates with the expression of FcRI protein.
Until now, the in vivo function of FcRI-ITAM in FcRI-induced mast cell activation upon stimulation with IgE(+Ag) had not been analyzed. We have demonstrated that -/- mice expressing YF and CT 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 FcRI-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 FcRI augment FcRI-ITAM-mediated activation signals rather than transduces them independently of FcRI (10).
We also provide new insights into mast cell function/signaling induced by IgE(-Ag), particularly surface FcRI expression and cell survival. We have demonstrated that the surface FcRI expression on mast cells is up-regulated by IgE(-Ag) regardless of the mutation/deletion of FcRI-ITAM and is therefore regulated independently of FcRI-ITAM. These results are consistent with early in vitro studies suggesting that the up-regulation of surface FcRI expression by IgE(-Ag) is independent of FcRI-mediated intracellular signals (36, 37). Taken together, the up-regulation of surface FcRI expression by IgE(-Ag) appears to be regulated by receptor stabilization on the plasma membrane upon binding to IgE to FcRI, without activation.
The recent finding that the binding of IgE(-Ag) to FcRI 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 FcRI-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, FcRI-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 FcRI 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--26 or SPE-7. In the present study both mAbs induced mast cell survival in an FcRI-ITAM-dependent manner. Furthermore, both mAbs induced FcRI-ITAM-dependent IL-6 production in the absence of Ag in our system, although H1 DNP--26 induced much lower responses. Collectively, our data suggest that IgE(-Ag) acts through the FcRI-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 FcRI up-regulates cell surface FcRI 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 FcRI-ITAM-dependent and independent manner. Targeting FcRI on the basis of these results may provide a new approach for the prevention of allergic diseases.
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Acknowledgments |
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Footnotes |
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2 D.S. and S.Y. contributed equally to this study.
3 Current address: Laboratory of Microbiology and Immunology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.
4 Current address: Southern Tohoku Research Institute for Neuroscience, Kooriyama, 963-8563, Japan.
5 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics (H1), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: saito{at}med.m.chiba-u.ac.jp
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.
Received for publication August 18, 2003. Accepted for publication December 10, 2003.
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), and 50 (
) ng/ml; D–H: 0 (
) 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 (
), and 
)); B, BMMCs from normal mice (