Fc RI -ITAM Is Differentially Required for Mast Cell Function In Vivo
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首席医学网
2008年06月26日 22:18:33 Thursday
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作者：Daiju Sakurai,,, Sho Yamasaki,, Kanako Arase,, Seung Yong Park,, Hisashi Arase,, Akiyoshi Konno, and Takashi Saito,, 作者单位：Departments of 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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【摘要】 The cross-linking of IgE-bound Fc RI by Ags triggers mast cell activation leading to allergic reactions. The in vivo contribution of Fc RI signaling to IgE/Fc RI-mediated mast cell responses has not yet been elucidated. In this study Fc RI -/- mast cells were reconstituted with either wild-type or mutant Fc RI in transgenic mice and transfected mast cells in vitro. We demonstrate that Fc RI -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 Fc RI 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 Fc RI -immunoreceptor tyrosine-based activation motif. In contrast, the IgE-mediated up-regulation of Fc RI is independent of Fc RI signaling. These results indicate that Fc RI -mediated signals differentially regulate the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.
【关键词】 Differentially Required Function
Introduction
Mast cells play a central role in various types of hypersensitivity, particularly in immediate phase allergic reactions. The aggregation of IgE-bound Fc 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 Fc RI-mediated activation of mast cells has been thought to occur only upon cross-linking of Fc RI with IgE and Ag (IgE(+Ag)). However, recent reports have suggested that cell surface Fc RI expression and mast cell survival are regulated by IgE in the absence of Ag (IgE(-Ag)) ( 3, 4, 5 ). Although the surface Fc RI 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 Fc RI has been shown to be involved ( 4 ), the mechanism is largely unknown.
Fc RI is expressed on rodent mast cells as a tetrameric structure composed of,, and homodimers ( 6 ). The -chain (Fc RI ) is responsible for binding to IgE. The - and -chains (Fc RI, Fc RI ) possess immunoreceptor tyrosine-based activation motifs (ITAMs) 6 ( 7 ) within their cytoplasmic domains. The cross-linking of Fc RI 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 Fc RI, 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 Fc RI cross-linking ( 8 ). Although Fc RI is believed to play a role in the amplification of activation signals through Fc RI ( 9, 10 ), it has been shown that Syk binds to phospho-ITAMs of Fc RI as well as Fc RI ( 11 ) and that the cross-linking of Fc RI induces weak Ca 2+ influx ( 12 ). Therefore, it is possible that Fc RI -mediated signals may trigger some in vivo responses via ITAM.
Fc RI is thought to be a pivotal subunit of the Fc RI complex for intracellular signaling upon IgE(+Ag) stimulation ( 1, 13 ). We and another group have generated Fc RI -deficient mice ( -/- ) ( 14, 15 ) and have analyzed the in vivo function of Fc R and Fc R in various systems ( 14, 15, 16, 17, 18, 19, 20, 21 ). However, the in vivo function of Fc RI -mediated signals in Fc RI-mediated responses has not been elucidated, mainly because Fc RI is essential for cell surface expression.
In this study, -/- mast cells were reconstituted with mutant Fc RI in transgenic mice and bone marrow-derived mast cells (BMMCs), and the function of Fc RI -ITAM was analyzed. We demonstrate that most Fc RI-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 Fc RI -ITAM signaling. In contrast, we show that the up-regulation of surface Fc RI expression on mast cells by IgE is regulated independently of Fc RI -ITAM. Thus, we unveiled the differential requirement of Fc RI -mediated signals for the receptor expression, activation, and survival of mast cells and systemic anaphylaxis.
Materials and Methods
Generation of Fc RI transgenic mice
The cDNAs encoding murine wild-type Fc RI (WT) and two mutant Fc RI (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 Hin dIII site of pH-2/IV (murine H-2K d promoter) ( 22 ). After digestion with Pvu II and Sph I, 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 Fc RI -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-Fc RI 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-Fc RII/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-Fc RI (IC 51-64 ) Ab was prepared by immunizing rabbits with the synthetic peptide RKAAIASREKADAV corresponding to aa 51-64 of Fc RI (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 Fc RI (YF and CT) were subcloned into the Eco RI 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 Fc RI was prepared by fusing the signal sequence (1-18 aa)-deleted Fc RI to the Signaling lymphocyte activation molecule signal peptide-driven Flag sequence at the N terminus. Flag-Fc RI WT, YF, and CT were subcloned into pMX-neo.
Retroviral transfection
The cDNAs encoding wild-type and mutant Fc RI (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 10 6 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 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 Fc RI ( 26 ), and -actin as an internal control. Real-time fluorescent PCR was used to quantitate Fc RI 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 Fc RII/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 Fc RI, 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-Fc RI (IC 51-64 ) 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 Fc RI, 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 Fc RI 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 Tyrodes buffer (130 mmol/L NaCl, 5 mmol/L KCl, 1.4 mmol/L CaCl 2, 1 mmol/L MgCl 2, 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 Tyrodes 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 PGD 2
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 PGD 2 in the supernatant was measured using an enzyme immunoassay kit (Cayman, Ann Arbor, MI) according to the manufacturers 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.
Results
Generation of Fc RI Tg mice
To analyze the in vivo function of Fc RI, particularly of its ITAM, in IgE/Fc RI-mediated responses, we generated Tg mice expressing mutants of Fc RI. Two mutant Fc RI 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 Fc RI ( WT ) were subcloned into an expression vector containing an H-2K d promoter. Several Tg lines for each construct were established with the C57BL/6 background, and all Tg mice were crossed with Fc RI -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 Fc RI is not required for in vivo expression of Fc RI
BMMC from Tg mice, cultured in IL-3-containing medium for 4-8 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 Fc RI expression by flow cytometry. Whereas BMMCs from -/- mice did not express surface Fc RI as previously described ( 18 ), those from all three Tg mice expressed similar levels of surface Fc RI, but at lower (5-11%) levels than in normal mice (Fig. 1 A ). These results demonstrate that the cytoplasmic distal region of Fc RI is not essential for the surface expression of the Fc RI complex on mast cells in vivo. We then examined the efficiency of mutant Fc RI expression by comparing the cell surface expression level with the total amounts of Fc RI proteins. To detect these mutant Fc RI proteins, we produced an anti-Fc RI (IC 51-64 ) Ab specifically against the transmembrane-proximal region of Fc RI, which could even detect CT. This Ab precipitated both Flag-tagged WT and mutant Fc RI 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 Fc RI and found that the Fc RI protein expression level in BMMCs from each Tg mouse correlated with cell surface expression.
FIGURE 1. Expression of Fc RI ( -, -, and -chains) in BMMCs from Tg mice and retro-BMMC. A and B, Flow cytometric analysis of surface Fc RI expression on BMMCs. A, BMMCs from normal mice (+/+), -/- mice (-/-), and Fc RI Tg mice with each -/- background (WT, YF, and CT) 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; CT, 13. B, BMMCs from normal (+/+), and retrovirally transfected -/- bone marrow cells (mock, WT, YF, and CT) were pretreated with 2.4G2, and stained and analyzed as described in A. MFI: normal, 262; WT, 197; YF, 181; and CT, 160. C, Reactivity of anti-Fc RI (IC 51-64 ) Ab to mutant Fc RI. 293T cells were transiently transfected with expressible constructs of Flag-tagged Fc RI (WT) and mutants (YF or CT). Cell lysates were immunoprecipitated with anti-Fc RI (IC 51-64 ) Ab. Total lysates ( upper panel ) and immunoprecipitates ( lower panel ) were blotted with anti-Flag mAb. D, Expression level of total Fc RI in BMMCs from Tg mice. Lysates of BMMCs derived from normal mice (+/+), -/- mice (-/-), and Fc RI Tg mice with each -/- background (WT, YF, and CT) were biotinylated and immunoprecipitated with anti-Fc RI (IC 51-64 ) Ab (see Materials and Methods ). The precipitates were analyzed on nonreducing (NR) and reducing (R) two-dimensional gels to detect all Fc RI proteins. E, Expression of Fc RI in the cell surface Fc RI complex. BMMCs were incubated with IgE at 4°C for 30 min, washed, and lysed in 0.5% Triton X-100. The IgE-bound Fc RI complex in the lysates were immunoprecipitated with anti-IgE Ab and protein A-Sepharose. Immunoprecipitates were blotted with anti-Fc RI Ab ( upper panel ). Total cell lysates were also blotted with anti-Fc RIb Ab ( second panel ) and anti-Erk1/2 Ab ( third panel ) as controls. Note that although Fc RI precipitated from YF appears somewhat higher than WT or CT, this reflects a slightly higher level of Fc RI on the cell surface of YF as shown in A. RT-PCR analysis for Fc RI mRNA was performed using primers specific for Fc RI and normalized by -actin ( bottom panel ). Representative data of unsaturated amount of cDNA are shown. The results of additional real-time PCR are described in Results.
It has been well documented that Fc RI is also essential for the surface expression of murine Fc RI ( 6, 10, 30 ). To confirm the involvement of Fc RI in the reconstituted Fc RI, we performed biochemical analysis. Fig. 1 E shows that the amount of Fc RI associated with IgE-bound surface Fc RI correlated with the cell surface expression level of Fc RI. As the surface Fc RI expression on YF is a little higher than others, YF expressed a proportionally higher level of Fc RI in the complex. The apparent reduction of the Fc RI protein expression level in -/- BMMCs suggests that the Fc RI protein is susceptible to degradation in the absence of Fc RI, consistent with the observation that Fc RI -deficient cells express similar amounts of Fc RI mRNAs (Fig. 1 E, lanes 1 and 2 ) ( 31 ). Similar expression levels of Fc RI 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 Fc RI as normal mast cells, we took another approach by using gene-transfected BMMCs to generate BMMCs expressing normal levels of Fc RI with mutant Fc RI. 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 Fc RI expression. Whereas mock vector-transferred BMMCs from -/- mice (BMMC (mock)) failed to express cell surface Fc RI, gene-transferred BMMCs (WT, YF, and CT) expressed levels of Fc RI 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 Fc RI including ITAM is not required for surface expression of the Fc RI complex on BMMCs.
IgE(+Ag)-induced activation of mast cells depends on Fc RI -ITAM
To analyze the three major pathways triggered by the aggregation of IgE-bound Fc RI 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 Fc RI. In contrast, no significant degranulation was induced in BMMCs from YF, CT, or -/- mice (Fig. 2 A ). The degree of degranulation upon stimulation with Ca 2+ ionophore was similar among these mutant Fc RI -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 Fc RI cross-linking. Cytokine production was induced in BMMCs from WT and normal mice, but not from YF, CT, and -/- mice in response to Fc RI cross-linking with IgE(+Ag) (Fig. 2, B and C ). Thirdly, similar to degranulation and cytokine production, PGD 2 release was undetectable in mast cells from YF, CT, and -/- mice, whereas WT produced PGD 2 at a level comparable to that of normal mice (Fig. 2 D ).
FIGURE 2. IgE(+Ag)-induced activation of mast cells is mediated by Fc RI -ITAM. Ag-dependent activation of mast cells was analyzed in mast cells from Tg mice ( A-D ) and retrovirally transfected BMMCs ( E-H ) for -hexosaminidase release ( A and E ), secretion of TNF- ( B and F ) and IL-6 ( C and G ), and PGD 2 production ( D and H ). A-D, BMMCs from normal mice (+/+), -/- mice (-/-), and Fc RI Tg mice with each -/- background (WT, YF, and CT) were stimulated. E-H, BMMCs from normal mice (+/+) and retrovirally transfected -/- bone marrow cells (mock, WT, YF, and CT) 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 -hexosaminidase, PGD 2 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.
To confirm the results from Tg mice, we used retro-BMMC expressing similar levels of cell surface Fc RI as normal BMMCs. Degranulation, cytokine secretion, and PGD 2 production were similarly induced in normal and WT BMMCs upon Fc RI 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 Fc RI -ITAM is essential for all three major pathways that mediate the release of proinflammatory mediators upon Fc RI engagement, and Fc RI cannot replace this function of Fc RI.
Dependence of IgE-induced mast cell survival on Fc RI signaling
To investigate the requirement of Fc RI -ITAM-mediated signals for IgE(-Ag)-induced mast cell survival, we used retro-BMMC expressing similar levels of cell surface Fc RI as normal mast cells, similar to the previous analysis of Fc RI 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. 3 A ). These results clearly demonstrate a critical role of Fc RI -ITAM in IgE(-Ag)-induced mast cell survival.
FIGURE 3. IgE(-Ag) acts through Fc RI -ITAM to trigger mast cell survival and cytokine production. A, BMMCs derived from normal mice (+/+) and retrovirally transfected -/- 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- -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- -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).
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 Fc RI -ITAM for mast cell survival induced by another monoclonal IgE (H1 DNP- -26). As shown in Fig. 3 A, 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 Fc RI -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- -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 Fc RI -ITAM.
An Fc RI -ITAM-independent mechanism mediates the in vivo up-regulation of cell surface Fc RI expression by IgE
We next examined the requirement of Fc RI -ITAM in IgE(-Ag)-mediated up-regulation of cell surface Fc RI expression. YF - and CT -containing BMMCs from Tg mice showed an up-regulation of surface Fc RI expression regardless of whether the Fc RI was WT or mutant (Fig. 4 A ). The results were confirmed using retro-BMMC that expressed equal levels of Fc RI on the cell surface. Indeed, IgE increased the surface Fc RI expression equally in all mast cells expressing YF, CT, and WT (Fig. 4 B ). These results suggest that this regulation is independent of phosphorylation-mediated signals through Fc RI.
FIGURE 4. Up-regulation of cell surface Fc RI expression by IgE(-Ag) is mediated by an Fc RI -ITAM-independent mechanism. A, BMMCs derived from normal ( ), -/- ( ), and Fc RI Tg mice with each -/- background (WT (), YF ( ), and CT ( )); B, BMMCs from normal mice ( ) and retrovirally transfected -/- bone marrow cells (mock ( ), WT (), YF ( ), and CT ( )) were cultured in the presence of 5 µg/ml IgE for the indicated periods and assessed for surface Fc RI 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 Fc RI expression on peritoneal mast cells from -/- (-/-), and Fc RI Tg mice with each -/- background (WT, YF, and CT) under air-uncontrolled conditions. Serum total IgE levels were measured by ELISA, and surface Fc RI expression was analyzed by flow cytometry. The number in each panel indicates the correlation coefficient (R) as calculated by Pearsons correlation coefficient test.
We next analyzed the up-regulation of surface Fc 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 Fc RI expression on mast cells from reconstituted mice. Fig. 4 C shows the relationship between the mean fluorescence intensity (MFI) of the surface expression of Fc RI on peritoneal mast cells and total serum IgE. The results demonstrate that the in vivo surface expression levels of Fc RI on mast cells from YF, CT, and WT mice exhibit positive correlations with total serum IgE. In contrast to the increased surface Fc RI 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 Fc RI -ITAM-mediated signals.
IgE-mediated passive systemic anaphylaxis through Fc RI -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 Fc RI - and/or Fc RI -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 Fc RI -ITAM, which cannot be replaced by Fc RI -ITAM.
FIGURE 5. IgE(+Ag)-induced passive systemic anaphylaxis in vivo is dependent on Fc RI -ITAM. Rectal temperatures of normal ( ), -/- ( ), and Fc RI Tg mice with each -/- background (WT (), YF ( ), and CT ( )) during IgE(+Ag)-induced PSA were measured at the time indicated. Four normal, three -/-, five WT, nine YF, and three CT 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 -/- mice). The difference in rectal temperature between normal and WT mice was not statistically significant.
Discussion
In this study we have distinguished between Fc RI -ITAM-dependent and -independent responses in the Fc RI-mediated function of mast cells. We demonstrated that Fc RI -ITAM is essential for Fc RI-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 Fc RI -ITAM. In contrast, we found that IgE(-Ag)-mediated up-regulation of cell surface Fc RI expression is independent of Fc RI -ITAM.
Using Fc RI -reconstituted Tg mice, we showed expression of Fc RI with a mutant Fc RI lacking the cytoplasmic tail on the surface of mast cells in vivo, indicating that Fc RI -ITAM is not required for surface expression. Biochemical analyses of the reconstituted Fc RI complex on the cell surface reveal that the composition of Fc RI with -, -, and -chains is similar to that on normal mast cells, and the surface expression level correlates with the expression of Fc RI protein.
Until now, the in vivo function of Fc RI -ITAM in Fc RI-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 PGD 2 synthesis, upon IgE(+Ag) stimulation. These results indicate that the phosphorylation of Fc RI -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 Fc RI augment Fc RI -ITAM-mediated activation signals rather than transduces them independently of Fc RI ( 10 ).
We also provide new insights into mast cell function/signaling induced by IgE(-Ag), particularly surface Fc RI expression and cell survival. We have demonstrated that the surface Fc RI expression on mast cells is up-regulated by IgE(-Ag) regardless of the mutation/deletion of Fc RI -ITAM and is therefore regulated independently of Fc RI -ITAM. These results are consistent with early in vitro studies suggesting that the up-regulation of surface Fc RI expression by IgE(-Ag) is independent of Fc RI-mediated intracellular signals ( 36, 37 ). Taken together, the up-regulation of surface Fc RI expression by IgE(-Ag) appears to be regulated by receptor stabilization on the plasma membrane upon binding to IgE to Fc RI, without activation.
The recent finding that the binding of IgE(-Ag) to Fc RI 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 Fc RI -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, Fc RI-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 Fc RI 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 Fc RI -ITAM-dependent manner. Furthermore, both mAbs induced Fc RI -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 Fc RI -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 Fc RI up-regulates cell surface Fc RI 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 Fc RI -ITAM-dependent and independent manner. Targeting Fc RI on the basis of these results may provide a new approach for the prevention of allergic diseases.
Acknowledgments
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.
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