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Drastic Up-Regulation of FcRI on Mast Cells Is Induced by IgE Binding Through Stabilization and Accumulation of FcRI on the Cell Surface¹

Shuichi Kubo^2,*,, Kunie Matsuoka^*,,, Choji Taya^*, Fujiko Kitamura, Toshiyuki Takai, Hiromichi Yonekawa^* and Hajime Karasuyama^*,,¶

Departments of ^* Laboratory Animal Science and Immunology, Tokyo Metropolitan Organization for Medical Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Japan Science and Technology Corporation, Tokyo, Japan; Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; and ^¶ Department of Immune Regulation, Tokyo Medical and Dental University, Graduate School, Tokyo, Japan

	Abstract

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It has been shown that IgE binding to FcRI on mast cells results in increased FcRI expression, which in turn enhances IgE-dependent chemical mediator release from mast cells. Therefore, prevention of the IgE-mediated FcRI up-regulation would be a promising strategy for management of allergic disorders. However, the mechanism of IgE-mediated FcRI up-regulation has not been fully elucidated. In this study, we analyzed kinetics of FcRI on peritoneal mast cells and bone marrow-derived mast cells. In the presence of brefeldin A, which prevented transport of new FcRI molecules to the cell surface, levels of IgE-free FcRI on mast cells decreased drastically during culture, whereas those of IgE-bound FcRI were stable. In contrast, levels of FcRIII on the same cells were stable even in the absence of its ligand, indicating that FcRI -chain, but not - and -chains, was responsible for the instability of IgE-free FcRI. As far as we analyzed, there was no evidence to support the idea that IgE binding to FcRI facilitated synthesis and/or transport of FcRI to the cell surface. Therefore, the stabilization and accumulation of FcRI on the cell surface through IgE binding appears to be the major mechanism of IgE-mediated FcRI up-regulation.

	Introduction

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The high-affinity receptor for IgE, FcRI, expressed on mast cells and basophils is a critical component in allergic responses. Cross-linking of IgE-bound FcRI by allergens results in activation of these cells and release of a range of preformed and newly generated chemical mediators and cytokines responsible for allergic inflammatory reactions (1, 2, 3, 4, 5, 6, 7). Thus, the binding of IgE produced against a given Ag confers specific reactivity to that Ag on these cells. Interestingly, IgE binding to FcRI also induces up-regulation of FcRI expression on these cells. In the late 1970s, it was noted that there was a good correlation between the density of IgE receptors on circulating basophils and the serum IgE titer (8). It was demonstrated later that FcRI expression on a rat mast cell line RBL-2H3 was up-regulated 2-fold by culturing cells with IgE in vitro (9, 10, 11, 12, 13). This FcRI up-regulation by IgE was found to be insensitive to cycloheximide, indicating its lack of dependence on protein synthesis (11). Therefore, it was proposed that the mechanism of this up-regulation could be the inhibition of degradation of FcRI by IgE binding.

It was recently reported that levels of FcRI expression on mast cells freshly isolated from IgE-deficient mice were extremely low (20% of normal level) (14). However, FcRI expression could be up-regulated up to 32-fold by in vitro incubation of mast cells with IgE or by injection of IgE in vivo (14, 15). Therefore, IgE-mediated FcRI up-regulation is not an artifact observed in cultured cell lines. This is also true for human mast cells (16, 17) as well as human and mouse basophils (18, 19). Importantly, the IgE-mediated FcRI up-regulation was shown to result in critical enhancement of effector functions of those cells. Both serotonin and cytokine release were substantially enhanced in terms of the sensitivity and the intensity of the response (14, 16, 20). This could be an important mechanism in facilitating host defense against parasites, while it could accelerate allergic inflammatory responses to allergens. Therefore, prevention of the IgE-mediated FcRI up-regulation would be a promising strategy for therapy of allergic disorders. However, the mechanism by which IgE binding up-regulates FcRI expression on mast cells and basophils has not been fully elucidated.

A question to be addressed is whether the drastic up-regulation (up to 32-fold) of FcRI on mast cells (14) can be explained by the inhibition of FcRI degradation proposed for the 2-fold increase of FcRI observed in RBL-2H3 cells (9, 10, 11, 12, 13). In the in vitro study with bone marrow-derived mast cells (BMMCs),³ two components of the FcRI up-regulation by IgE were identified: an early cycloheximide-insensitive phase as observed in RBL-2H3 cells, followed a few hours later by a more sustained component that was highly sensitive to cyclohexaminde (14). It is very difficult to clearly distinguish two mechanisms of the FcRI up-regulation from these results: the inhibition of degradation of FcRI vs the enhancement of synthesis and/or transport of FcRI. In the present study, to address this issue we directly examined dynamics of IgE-free vs IgE-bound FcRI expressed on mast cells and BMMCs by culturing cells with brefeldin A (BFA), which completely inhibited the transport of new FcRI molecules to the cell surface. We also compared transcription of FcRI subunits as well as the supply rate of new FcRI to the surface of cells that expressed FcRI at basal levels vs at highly up-regulated levels. Furthermore, kinetics of up-regulation of mouse and human FcRI expressed on the same cell was analyzed to explore possible signal transduction through IgE-bound FcRI. From these experiments we concluded that the stabilization and accumulation of FcRI on the cell surface through IgE binding is the major mechanism of IgE-mediated FcRI up-regulation. Physiological and pathological roles of IgE-mediated FcRI up-regulation will also be discussed.

	Materials and Methods

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Animals

BALB/c and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). µm^-/- mice (21) were kindly provided by Dr. K. Rajewsky (University of Cologne, Cologne, Germany) and Dr. D. Kitamura (Tokyo Science University, Tokyo, Japan), and were maintained in our own animal facility. FcRIIB^-/- mice were described previously (22). All the experiments in this study were performed according to the "Guidelines for Animal Use and Experimentation" as set out by our institutions.

Human FcRI -chain transgenic mice were established in our laboratory. A 17-kbp BamHI-BamHI human genomic DNA fragment covering the entire structural gene with five exons, a 2-kbp promoter region, and an 8-kbp 3' flanking region was microinjected into fertilized eggs of BALB/c mice, followed by transfer of viable eggs into the oviducts of pseudopregnant Slc:ICR mice (Japan SLC, Hamamatsu, Japan). Two founder lines of transgenic mice were established.

Antibodies

Mouse anti-trinitrophenyl (TNP) IgE mAb (C38-2), anti-mouse IgE^a mAb (UH297, rat IgG1), anti-mouse IgE^b mAb (JKS-6, rat IgG2a), anti-mouse CD23 mAb (B3B4), FITC-conjugated anti-mouse IgE mAb (R35-72), FITC-conjugated anti-mouse IgG2b (R12-3), FITC-conjugated anti-rat IgG1/2a mAb (G28-5), FITC-conjugated anti-FcRII/III mAb (2.4G2), FITC-conjugated rat IgG1 (R3-34), biotinylated anti-mouse c-kit mAb (2B8), PE-conjugated anti-mouse c-kit mAb (2B8), and allophycocyanin-conjugated streptavidin were purchased from BD PharMingen (San Diego, CA). Anti-human FcRI mAb (CRA1) was purchased from Kyokuto Pharmaceutical (Tokyo, Japan), and purified human IgE was purchased from Yamasa Shoyu (Chosin, Japan). Mouse anti-TNP IgE^a mAb (IGELa2), mouse anti-TNP IgE^b mAb (IGELb4), and anti-Fc mAb (2.4G2) were described previously (23, 24).

Cell preparation and flow cytometry

Peritoneal cells were isolated from mice and were depleted of RBCs by using hypotonic lysis buffer for culture and staining. BMMCs were generated by culturing femoral bone marrow cells in medium containing rIL-3 as described previously (25). Peritoneal cells and BMMCs were cultured in RPMI 1640 (Iwaki, Funabashi, Japan) with 10% FCS (JRH Bioscience, Lenexa, KS) with or without IgE in the presence or absence of BFA (Epicentre Technologies, Madison, WI). During culture of peritoneal mast cells, no stimulators such as stem cell factor were added. For flow cytometric analysis, freshly isolated or cultured cells were preincubated with 2.4G2 mAb (rat IgG2b) at 4°C for 15 min to prevent nonspecific binding of other Abs. To detect IgE-bound FcRI on the cell surface, cells were stained with FITC-anti-IgE mAb R35-72. To detect total (IgE-bound plus IgE-free) FcRI, cells were stained with FITC-anti-IgE mAb after incubation at 4°C with excess amounts of IgE (IGELb4 or C38-2) to saturate FcRI with IgE. To detect IgE^a- and IgE^b-bound FcRI, cells were stained with anti-IgE^a mAb and anti-IgE^b mAb, respectively, followed by FITC-conjugated anti-rat IgG1/2a mAb. To determine levels of human FcRI -chain expression, cells were stained with anti-human FcRI -chain mAb CRA1 followed by FITC-conjugated anti-mouse IgG2b mAb R12-3. To detect FcRIII, BMMCs derived from FcRIIB^-/- mice were stained with FITC-conjugated 2.4G2. For cultured cells, cells were also stained with propidium iodide and biotinylated anti-c-kit mAb followed by allophycocyanin-streptavidin. Propidium iodide^-c-kit⁺ cells were analyzed as live mast cells for the expression of FcRI by using FACSCalibur (BD Biosciences, Mountain View, CA). For freshly prepared peritoneal cells, autofluorescent cells (primarily macrophages) were rejected to clearly identify c-kit⁺ mast cells (14). The geo mean value of fluorescence intensity was converted to the linear scale number by the number of molecules of equivalent soluble fluorochrome units (MESF) using Quantum 25 microbeads (Flow Cytometry Standards, San Juan, PR), as per the specifications of the manufacturer. MESF was calculated by subtracting MESF of control staining from MESF of sample.

Northern blot analysis

Total cellular RNA was isolated from BMMCs cultured with or without IgE by using Isogen (Nippon Gene, Toyama, Japan) separated on a formaldehyde gel and transferred to nylon membranes (Hybond-N+, Amersham-Pharmacia Biotech, Piscataway, NJ). Transcripts of mouse FcRI, , , and actin were detected by specific probes. The radioactive bands were visualized by the phosphor imager Fuji BAS2000 (Fuji Photo Film, Tokyo, Japan).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Drastic change of FcRI expression on mast cells in vivo in correlation with serum IgE levels

Levels of FcRI expression on c-kit⁺ peritoneal mast cells were examined in three different mouse strains, B cell-deficient µm^-/- mice (21), normal BALB/c mice, and IgE-transgenic BALB/c mice (26). Their serum IgE levels were undetectable at 1.3 and 30 µg/ml, respectively. Representative results of flow cytometry analyzing IgE-bound and total (IgE-bound plus IgE-free) FcRI on peritoneal mast cells are shown in Fig. 1A. To compare levels of FcRI expression accurately, the geo mean values of fluorescence intensity were converted to the numbers of MESF, as shown in Fig. 1B. Although no IgE-bound FcRI was detected on mast cells from B cell-deficient µm^-/- mice as expected, total FcRI (IgE-free FcRI) was detectable on their surface. However, its expression levels were 20% of those on mast cells from normal BALB/c mice, which is consistent with the previous observation in mast cells from IgE-deficient mice (14). In contrast, levels of total FcRI on peritoneal mast cells from IgE-transgenic BALB/c mice were 5–6 times as high as those on mast cells from normal BALB/c mice. Furthermore, eventually all the FcRI molecules on mast cells from IgE-transgenic mice were occupied by IgE. Even in normal BALB/c mice, 80% of FcRI molecules on mast cells were occupied by IgE. Thus, levels of FcRI on mast cells can be altered in vivo at least by 25-fold in correlation with serum IgE levels and IgE binding to FcRI.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis of FcRI expression on peritoneal mast cells in three mice strains with different serum IgE levels. Peritoneal cells were isolated from µm-/-, normal BALB/c, and IgE transgenic mice and pretreated in vitro with anti-FcRII/III mAb 2.4G2 to prevent nonspecific binding of Abs. Cells were then stained with FITC-anti-IgE mAb R35-72 to detect IgE-bound FcRI on the cell surface. To detect total (IgE-bound plus IgE-free) FcRI, cells were stained with FITC-anti-IgE mAb after incubation at 4°C with excess amounts of IgE to saturate FcRI with IgE. In both cases, cells were also stained with PE-anti-c-kit mAb, and c-kit+ mast cells were analyzed for the expression of FcRI. Representative results are shown in A as histograms overlayed with control staining (dotted lines). B, To compare levels of FcRI expression accurately, the geo mean values of fluorescence intensity in A were converted to MESF, and MESF was calculated in each case as detailed in Materials and Methods. MESF values of IgE-bound FcRI () and total FcRI () are shown. Data shown are representative of five repeated analyses.

IgE-bound FcRI stays on the cell surface for a much longer time than IgE-free FcRI

To clarify the mechanism by which FcRI expression on mast cells is drastically up-regulated upon association with IgE, we first analyzed dynamics of FcRI on BMMCs. When BMMCs were cultured in vitro with 1 µg/ml IgE for 24 h, a 5- to 6-fold increase of FcRI expression was observed (Fig. 2A). An inhibitor of intracellular protein transport, BFA, added in culture inhibited this up-regulation of FcRI expression in a dose-dependent manner. Because addition of 1–10 µg/ml BFA resulted in complete inhibition, 3 µg/ml BFA was used for additional experiments. In the next experiment BMMCs were first preincubated with excess amounts of IgE at 4°C to saturate all FcRI molecules on the cell surface with IgE. After unbound IgE was washed away, BMMCs were cultured without adding IgE at 37°C for 16 h in the presence or absence of BFA. In the absence of BFA, levels of IgE-bound FcRI did not change during the culture, whereas levels of total FcRI increased 2-fold (Fig. 2B). Therefore, the increase of total FcRI appears to be due to the addition of IgE-free FcRI, namely the transport of new FcRI molecules from the cytoplasm to the cell surface. Indeed, BFA completely inhibited the increase of total FcRI. These results indicated that the half-life of IgE-bound and IgE-free FcRI expressed on the cell surface can be estimated by monitoring levels of FcRI during the culture in the presence of BFA, which blocks new supply of FcRI to the cell surface.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. BFA inhibits IgE-mediated FcRI up-regulation by preventing appearance of new FcRI molecules to the cell surface. A, BMMCs prepared from BALB/c mice were cultured in vitro with or without 1 µg/ml mouse IgE at 37°C for 24 h. In some cases BFA was also included in culture at various concentrations as indicated. After 24 h culture, levels of total FcRI on their surface were determined as in Fig. 1. B, BMMCs prepared from BALB/c mice were first incubated with IgE at 4°C to saturate FcRI, and then unbound IgE was washed away. Subsequently, cells were cultured without adding IgE at 37°C in the presence or absence of 3 µg/ml BFA for 16 h. Levels of IgE-bound and total FcRI were determined and are displayed as in Fig. 1. Data shown are representative of three repeated analyses.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. FcRI is stabilized by IgE binding and stays for longer time on the cell surface. A, BMMCs prepared from BALB/c mice were incubated with excess amounts of IgE at 4°C to saturate FcRI with IgE. After unbound IgE were washed away, the cells were cultured in the presence of 3 µg/ml BFA at 37°C for 24 h. At indicating time points during culture, the cells were harvested and stained with anti-IgE mAb to determine levels of IgE-bound FcRI on the cell surface (). In parallel, untreated BMMCs were cultured in the same way. Levels of FcRI (IgE-free) at indicated time points during culture were determined by staining cells with anti-IgE mAb after incubation at 4°C with excess amounts of IgE to saturate FcRI with IgE (). Data are shown as MESF. Mean ± SD (n = 4). B, Peritoneal mast cells freshly prepared from µm-/- mice were cultured in the presence of 3 µg/ml BFA at 37°C for 16 h with or without preincubation with excess amounts of IgE as in A. Levels of FcRI (IgE-free or IgE-bound) were determined as in A before (filled bars) and after 16 h culture (hatched bars). In parallel, freshly prepared peritoneal mast cells from C57BL/6 mice were cultured in the same way, and levels of IgE-bound FcRI were determined before and after the culture. Data shown are representative of three repeated analyses. C, BMMCs prepared from BALB/c mice were cultured in the presence of 5 µg/ml IgE at 37°C for 0, 24, or 48 h followed by incubation with excess amounts of IgE at 4°C. After unbound IgE was washed away, cells were further cultured without adding IgE in the presence of 3 µg/ml BFA at 37°C for 16 h. Levels of IgE-bound FcRI were determined before (filled bars) and after 16 h culture (). Mean ± SD (n = 3).

New FcRI molecules are supplied to the cell surface independent of levels of FcRI expression

We next examined whether the appearance of new FcRI molecules to the cell surface was altered during IgE-mediated FcRI up-regulation. BMMCs were cultured for 24 h with or without the b allotype of IgE (IgE^b), followed by saturation of their FcRI with IgE^b. After unbound IgE^b was washed away, the cells were cultured with the a allotype of IgE (IgE^a) for an additional 6 h, and levels of total, IgE^b-bound and IgE^a-bound FcRI expression were determined by flow cytometric analysis with IgE-specific and IgE allotype-specific mAbs, respectively. A 6-fold difference in levels of FcRI expression was observed after the 24-h incubation with vs without IgE^b (Fig. 4, upper panel, ). Levels of IgE^b-bound FcRI did not change during the following 6-h culture with IgE^a, while levels of total FcRI increased in both cases (Fig. 4, upper and middle panels, ). Therefore, the degree of FcRI up-regulation during the 6-h culture was determined as the amount of IgE^a-bound FcRI. Interestingly, that was comparable in the two cases (Fig. 4, bottom panel, ) despite the big difference in total amounts of FcRI on the cell surface (Fig. 4, upper panel, ). These results suggested that the input rate of new FcRI molecules to the cell surface was consistently independent of the total number of surface FcRI, at least within the range we examined.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. New FcRI molecules are supplied to the cell surface independent of levels of FcRI expression. BMMCs prepared from BALB/c mice were cultured with or without 10 µg/ml IgEb at 37°C for 24 h followed by incubation at 4°C with excess amounts of IgEb to saturate FcRI with IgE. After unbound IgEb was washed away, cells were further cultured with 10 µg/ml IgEa at 37°C for 6 h. Before () and after the 6 h culture with IgEa (), levels of total FcRI were determined by staining cells with FITC-labeled anti-IgE mAb (R35-72) while those of IgEa-bound and IgEb-bound FcRI were determined by staining cells with anti-IgEa mAb (UH297, rat IgG1) and anti-IgEb mAb (JKS-6, rat IgG2a), respectively, followed by FITC-labeled anti-rat IgG1/2a mAb. Data are shown as MESF. Mean ± SD (n = 3).

Levels of FcRI transcripts in BMMCs are not altered by IgE binding to FcRI

Northern blot analysis was performed to examine the possibility that IgE binding to FcRI transduces signals to increase transcripts of FcRI subunits. BMMCs prepared from BALB/c mice were cultured with or without IgE for 8 h. Although levels of FcRI expression on the cell surface increased up to 2.5-fold by culture with IgE (Fig. 5, upper panel), no significant difference in levels of transcripts of FcRI subunits (-, -, and -chains) was detected at any time point (2, 4, and 8 h) of culture between cells cultured with IgE and those without IgE (Fig. 5, lower panel). Thus, IgE binding to FcRI did not alter levels of FcRI transcripts.

View larger version (53K): [in this window] [in a new window]   FIGURE 5. Levels of FcRI transcripts in BMMCs are not altered by IgE binding to FcRI. BMMCs derived from BALB/c mice were cultured with or without 10 µg/ml IgE at 37°C for 8 h. At the indicated time points during the culture, cells were harvested and analyzed for surface expression of FcRI by flow cytometry (upper panel) as well as for transcripts of FcRI subunits and -actin by Northern blot (lower panel).

Human IgE up-regulates human FcRI, but not mouse FcRI expressed on the same cell

To explore the possibility that IgE binding to FcRI induces signals to accelerate synthesis and/or transport of FcRI to the cell surface, we used BMMCs derived from human FcRI -chain transgenic mice. Two-color flow cytometric analysis confirmed that mouse and human FcRI -chains were simultaneously expressed on BMMCs (data not shown). Both mouse and human FcRI -chains were associated with mouse - and -chains, as shown previously (27, 28, 29). The chimeric FcRI complex composed of human -chain and mouse - and -chains was competent to transduce signals because cross-linking of human -chains on mast cells with specific Ab in vivo resulted in systemic anaphylaxis in the transgenic mice (data not shown). BMMCs prepared from the transgenic mice were cultured with human IgE for 16 h. Human IgE binds to human FcRI, but not mouse FcRI (30). Levels of human FcRI increased 6-fold during the culture (Fig. 6). If signaling via - and -chains of FcRI is involved in this up-regulation through acceleration of synthesis and/or transport of FcRI to the cell surface, one may expect that levels of mouse FcRI expression on the same cells were also up-regulated. However, no significant alteration was observed in levels of mouse FcRI expression during the culture. Therefore, it is unlikely that IgE binding to FcRI facilitates synthesis and/or transport of FcRI to the cell surface.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Human IgE up-regulates human FcRI but not mouse FcRI expressed on BMMCs derived from human FcRI -chain transgenic mice. BMMCs prepared from human FcRI -chain transgenic mice were cultured with 1 µg/ml human IgE at 37°C for 16 h, and levels of human and mouse FcRI -chains were compared before and after the culture. To determine levels of human FcRI -chain expression, cells were stained with anti-human FcRI -chain mAb. To determine levels of mouse FcRI -chain expression, cells were first incubated at 4°C with human IgE to prevent mouse IgE for binding to human FcRI -chains and then incubated with mouse IgE followed by staining with anti-mouse IgE mAb. Data are shown as MESF. Mean ± SD (n = 3).

Difference of -chain in FcRI and FcRIII in correlation with their different stability on the cell surface

To know which subunit of FcRI determines the stability of FcRI on the cell surface, we compared kinetics of surface FcRI and FcRIII, which carry different -chains, but share - and -chains. Because 2.4G2 mAb reacts with both FcRIIB and FcRIII, BMMCs derived from FcRIIB-deficient mice were used for this purpose. Levels of FcRI on BMMCs were reduced to 14% of normal level after a 16-h culture in the presence of BFA, whereas those of FcRIII detected by 2.4G2 were unchanged during the culture (Fig. 7). Therefore, the difference of -chains in these two receptors appears to account for their different stability on the cell surface in the absence of ligands.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Comparison of FcRI and FcRIII in their stability on the cell surface. BMMCs derived from FcRIIB-/- mice were cultured in the presence of 3 µg/ml BFA at 37°C for 16 h, and levels of FcRI and FcRIII were compared before and after the culture. Levels of FcRI was determined as in Fig. 3 while those of FcRIII were determined by staining cells with FcRIIB/FcRIII-specific mAb 2.4G2. Data are shown as MESF. Mean ± SD (n = 3).

Efficient acquisition of Ag specificity through IgE-mediated FcRI up-regulation

To understand the physiological role of IgE-mediated FcRI up-regulation, BALB/c mice were treated with i.v. administration of 300 µg of monoclonal IgE^b specific to hapten TNP or an equivalent volume of PBS twice at a 3-day interval. Three days after the second treatment, the expression of IgE^a-bound and IgE^b-bound FcRI as well as IgE-free FcRI on their peritoneal mast cells was determined by flow cytometry with IgE allotype-specific mAbs. Relative amounts of each FcRI on mast cells prepared from IgE^b-treated and PBS-treated mice are shown in Fig. 8. In PBS-treated mice, only 20% of FcRI on mast cells were free of IgE, and the rest of them were occupied by endogenous IgE^a. In IgE^b-treated mice, total amounts of FcRI increased 3-fold, 80% of which were occupied by exogenous IgE^b. The rest were occupied by endogenous IgE^a. Thus, IgE-mediated FcRI up-regulation appears to facilitate acquisition of new Ag specificity in a short period.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Efficient acquisition of Ag specificity through IgE-mediated FcRI up-regulation. BALB/c mice were treated i.v. twice with 300 µg of IgEb or PBS each at 3 days interval. Three days after the second treatment, the expression of total, IgE-bound, IgEa-bound, and IgEb-bound FcRI on their peritoneal mast cells was determined as in Fig. 4. Relative amounts of IgE-free, IgEa-bound, and IgEb-bound FcRI on mast cells were calculated, and levels of total FcRI on peritoneal mast cells from PBS-treated mice are set as 100%. Data shown are calculated on basis of four independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A recent study demonstrated that IgE-mediated FcRI up-regulation on mouse BMMCs has two components: an early cycloheximide-insensitive phase, followed by a later and more sustained component that is highly sensitive to inhibition by cycloheximide (14). These findings were interpreted that IgE could up-regulate FcRI during the first hours simply by protecting against degradation of FcRI without being dependent on FcRI synthesis. Later, when the pool of available FcRI was fully used, further accumulation of FcRI by the same mechanism became dependent on protein synthesis (6). Though the data were not inconsistent with the interpretation, they could not rule out other mechanisms of FcRI up-regulation such as enhancement of synthesis and transport of FcRI complex. In the present study we first used BFA, an inhibitor of intracellular protein transport, to study kinetics of FcRI on mast cells. The up-regulation of FcRI expression on BMMCs cultured with IgE was completely inhibited by BFA. As expected, BFA inhibited the supply of new FcRI molecules to the cell surface, while it had no significant effect on the expression of pre-existing IgE-bound FcRI on the cell surface. Therefore, the fate of surface FcRI molecules can be investigated simply by monitoring levels of FcRI expression during culture in the presence of BFA. Under such culture conditions, levels of IgE-free FcRI on BMMCs decreased very quickly, whereas those of IgE-bound FcRI were fairly stable. This was also true for FcRI on peritoneal mast cells. These results clearly indicate that surface FcRI is unstable and quickly removed from cell surface unless IgE binds to FcRI. Upon association with IgE, FcRI is stabilized and stays on the surface for a much longer time than IgE-free FcRI. With excess amounts of IgE in culture without BFA, every new FcRI supplied to the cell surface is loaded with IgE, stabilized, and accumulated on the cell surface, leading to the up-regulation of surface FcRI expression.

Is the prevention of FcRI degradation on the cell surface only the mechanism of IgE-mediated up-regulation? One may assume that the binding of monomeric IgE to FcRI could trigger the transducing of signals for enhancement of synthesis and/or transport of FcRI. Because BFA inhibits the appearance of new FcRI molecules to the cell surface, it is impossible to rule out this possibility from the results of experiments using BFA. To address this issue we first examined levels of FcRI transcripts in BMMCs when cultured with or without IgE. Northern blot analysis revealed that IgE binding to FcRI did not increase transcripts of any subunit composed of FcRI even though it increased levels of surface FcRI. We next examined kinetics of FcRI on BMMCs derived from human FcRI -chain transgenic mice in which both mouse and human FcRI -chains are expressed on the cell surface. Mouse - and -chains are associated as signal transducing subunits with both mouse and human FcRI -chains (27, 28, 29). Therefore, the outcome of signal transduction via mouse FcRI and human FcRI is most likely the same. Indeed, both receptors were competent for inducing allergic reactions in vivo when cross-linked with specific mAbs (our unpublished observations). If IgE binding to FcRI triggers the transduction of signals, leading to acceleration of synthesis and/or transport of FcRI, it is expected that both mouse and human FcRI are up-regulated regardless of which receptor is fired by IgE binding. Culturing BMMCs with human IgE induced up-regulation of human FcRI as expected, but the up-regulation of mouse FcRI expressed on the same cell was not observed. Therefore, it seems unlikely that IgE binding to FcRI facilitates synthesis and/or transport of FcRI to the cell surface. Furthermore, the rate of appearance of new FcRI to the cell surface was found to be comparable between cells expressing the basal level of FcRI and cells expressing highly up-regulated FcRI. This favors the idea that IgE binding does not accelerate the transport of new FcRI to the cell surface. The level of FcRI expression on peritoneal mast cells is relatively low in normal mice, even though 80% of FcRI are occupied with IgE. This also supports the above idea. Taken together, we would like to conclude that the stabilization of FcRI through IgE binding followed by accumulation of IgE-bound FcRI on the cell surface is the major mechanism of IgE-mediated FcRI up-regulation in mast cells.

The in vivo experiments using different allotypes of IgE indicated that mast cells could easily acquire new Ag specificity through IgE-mediated FcRI up-regulation in a short period. Up to 80% of FcRI on mast cells are occupied with IgE even in normal mice with basal level of serum IgE, and IgE-bound FcRI molecules are stable on the cell surface. Therefore, only limited space is available for newly produced IgE if IgE-mediated FcRI up-regulation is not induced. In case of T and B cells, each cell has only one specificity to a given Ag. Therefore, the population reactive to a particular Ag is extremely small, and each clone needs to expand to protect host against foreign Ag. In contrast, large numbers of mast cells can simultaneously acquire new Ag specificity without expansion of cells through FcRI up-regulation induced by newly produced IgE. Mast cells can also acquire multiple Ag specificity by binding large numbers of different IgE species with distinct Ag specificities. Furthermore, the stable expression of large amounts of Ag-specific IgE on mast cells enables mast cells to keep their immunological memory for a fairly long period (memory of mast cells). This should help with protection from repeated reinfections of pathogens (31). Thus, IgE-mediated FcRI up-regulation appears to benefit the host in the environment where infection of pathogens such as parasites prevails.

It has been shown that IgE-dependent up-regulation of FcRI expression significantly enhances the ability of mouse mast cells to release serotonin, IL-6, IL-4, and vascular permeability factor/vascular endothelial cell growth factor in response to challenge with specific Ag (14, 20). Similar enhancement was also observed in human basophils (16). The augmentation of the sensitivity and the intensity of those responses would be a critical mechanism to facilitate host defense. Unfortunately, the same mechanism could also increase the severity of allergic disorders. In other words, the prevention of IgE-mediated FcRI up-regulation can be a promising strategy to manage the disorders. Indeed, the i.v. administration of nonanaphylactogenic anti-IgE Ab to atopic patients resulted in down-regulation of FcRI on basophils in parallel with the reduction of mediator release from activated basophils (32, 33). In this context, it is essential to know the exact mechanism by which IgE binding stabilizes FcRI. In humans, it has been reported that the FcRI expression on monocytes was higher in atopic subjects when compared with normal subjects (34). FcRI on human monocytes is composed of only - and -chains in contrast to FcRI (₂ tetramer) on mast cells and basophils (35, 36, 37, 38, 39, 40). Therefore, -chains of FcRI appear to be nonessential for IgE-mediated FcRI up-regulation. The comparison of FcRI and FcRIII stability in the present study suggested that FcRI -chains, but not - and -chains, are responsible for the instability of IgE-free FcRI. IgE binding to FcRI -chain might induce some conformational change of -chain to make FcRI resistant to degradation. Elucidation of this switching mechanism should facilitate the development of a new type of therapy for allergic disorders.

	Acknowledgments

 
We thank Dr. K. Rajewsky (University of Cologne) and Dr. D. Kitamura (Tokyo Science University) for µm^-/- mice, and Dr. C. Ra (Juntendo University, Tokyo, Japan) and Dr. H. Matsuda (Tokyo University of Agriculture and Technology, Tokyo, Japan) for their advice.

	Footnotes

 
¹ This work was supported by a grant for Specially Promoted Research on Atopic Disorders from the Tokyo Metropolitan Government; by grants-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japanese Ministry of Health, Labor and Welfare; and by Sankyo Company. T.T. was supported by Core Research for Engineering, Science, and Technology Program of Japan Science and Technology Corporation.

² Address correspondence and reprint requests to Dr. Shuichi Kubo, Department of Laboratory Animal Science, Tokyo Metropolitan Organization for Medical Science, Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan. E-mail address: skubo{at}rinshoken.or.jp

³ Abbreviations used in this paper: BMMCs, bone marrow-derived mast cells; BFA, brefeldin A; MESF, molecules of equivalent of fluorescence intensity; TNP, trinitrophenyl.

Received for publication May 2, 2001. Accepted for publication July 16, 2001.

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