Serum IgE clearance is facilitated by human FcεRI internalization (original) (raw)
The surface level of FcεRI is tightly regulated in BDCA1+ DCs compared with basophils. We recruited 11 healthy adult blood donors (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI68964DS1) and examined the correlation between serum IgE levels and surface FcεRI levels in basophils and BDCA1+ DCs (hereafter referred to as DCs). Serum IgE concentration was determined by ELISA. FcεRI surface levels were determined by flow cytometry using the antibody CRA-1, which binds to FcεRI irrespective of its binding to IgE (Figure 1A). IgE surface levels were also determined by flow cytometry using an anti-IgE antibody (Figure 1A). We found that FcεRI surface levels increased with serum IgE concentration in both basophils and DCs, but to a much lesser degree in DCs; when a line of best fit was calculated for each cell type, the slope for basophils was more than an order of magnitude greater than the slope for DCs (Figure 1B). The same trend was seen with surface IgE levels, which rose rapidly in basophils with serum IgE but much slower in DCs (Figure 1C). Thus, surface FcεRI levels in DCs correlated with serum IgE to a much lesser degree than those in basophils, indicating comparatively tight regulation of surface FcεRI in DCs.
DCs regulate FcεRI surface expression more tightly than basophils. (A) Gating strategy of human basophils and DCs, and histograms of surface FcεRI expression and surface IgE bound to the cells. Anti-hFcεRIα antibody (CRA-1) and anti-hIgE staining are shown in black, and isotype control antibody staining is shown in grey. (B) Surface FcεRI levels of human blood basophils and DCs in healthy donors. PBMCs were analyzed for FcεRI expression by flow cytometry, and serum was analyzed for IgE concentration by ELISA. Isotype control stain MFI was subtracted from anti-hFcεRIα antibody stain MFI. Data for each donor were plotted according to serum IgE level. Lines of best fit were calculated and drawn; slope and r2 values are shown. (C) Surface IgE levels for the same donors as in B with similarly calculated lines of fit. (D) IgE occupancy rate of FcεRI in DCs as compared with basophils. The MFI of IgE was divided by the MFI of FcεRI for DCs and basophils after subtracting appropriate isotype control MFI values to generate an occupancy index. DC occupancy index was divided by basophil occupancy index to generate an occupancy rate of DCs compared with basophils.
Previous studies have shown that DCs lack FcεRIβ (12) and that FcεRIβ promotes FcεRIα transport from the ER to the plasma membranes, thus enhancing FcεRI surface expression (2). Consistent with this idea, we found that DCs had much lower FcεRI surface levels than basophils in every donor examined (Figure 1B). IgE surface levels were also lower in DCs (Figure 1C). Interestingly, however, the degree of difference appeared greater for IgE than for FcεRI (see the histograms in Figure 1A as an example), suggesting that FcεRI on DCs might be occupied by IgE to a lesser degree than that on basophils. To quantitatively assess FcεRI occupancy in DCs relative to that of basophils, we divided the MFI of IgE by the MFI of FcεRI for DCs and basophils from each donor, and normalized the value of DCs to the value of basophils. Remarkably, we found that FcεRI occupancy in DCs was lower than that in basophils in 10 of 11 donors examined (Figure 1D). This was unexpected considering that these cells were exposed to the same IgE pool in the blood and because the IgE-binding portion of FcεRI, the α-chain (23), is present in both cell types. The relatively low occupancy of FcεRI in DCs raised the possibility that FcεRI on the surfaces of these cells may turn over in an active manner irrespective of IgE binding. Alternatively, DCs may turn over in the blood much faster than basophils. We sought to address the former possibility by comparatively examining FcεRI trafficking in DCs and basophils.
FcεRI is specifically localized in endolysosomes of DCs and monocytes but not of basophils. To determine the trafficking patterns of FcεRI, we examined FcεRI localization in blood basophils and DCs by confocal microscopy. In basophils, FcεRIα was localized in mesh-like structures (Figure 2A), whereas in DCs it was localized in vesicular compartments (Figure 2B). The FcεRI+ mesh-like structures in basophils were extensively labeled with calnexin, a marker of the ER, but not with Lamp1, a lysosome marker, indicating that basophil FcεRI was specifically localized in the ER (Figure 2A). In contrast, FcεRI+ compartments in DCs were labeled by neither calnexin nor TGN46 (a marker of Golgi), but partially labeled with EEA1 (an early endosome marker) and extensively labeled with Lamp1 and HLA-DR (lysosome markers) (Figure 2B), indicating that DC FcεRI is specifically localized in endolysosomal compartments. To further determine FcεRI endolysosomal localization in DCs, we incubated DCs with FITC-conjugated ovalbumin for 30 minutes at 37°C, washed extensively, and incubated for another 30 minutes. This method has been commonly used to label functional endolysosomes (24). We found that the anti-FcεRIα antibody colocalized strongly with FITC (Figure 2B), confirming that FcεRIα is localized in endolysosomes in DCs. To determine whether FcεRI lysosomal localization is restricted to blood DCs or a general feature of DCs regardless of tissue origin, we performed a similar microscopic analysis using BDCA1+ DCs isolated from the lungs. FcεRI expression in these lung DCs was confirmed by flow cytometry prior to microscopy (Supplemental Figure 1). Similar to blood DCs, lung DCs localized FcεRI in Lamp1+ lysosomes (Figure 2C). Last, we examined FcεRI localization in monocytes isolated from blood and found that it was also localized in lysosomes labeled by Lamp1 (Figure 2D). These findings indicate that FcεRIα is distinctively localized in endolysosomes in human DCs and monocytes.
FcεRI is localized in the lysosomes of DCs and monocytes. (A–D) Intracellular localization of hFcεRI in blood basophils (A), blood BDCA1+ DCs (B), lung BDCA1+ DCs (C), and blood monocytes (D). Each cell type was isolated as described in Methods, stained using the indicated antibodies, and examined by confocal microscopy. Basophil images are representative of at least 48 recorded images from at least 7 unique and representative donors. Blood DC images are representative of at least 25 recorded images from 2–6 unique and representative donors. The lung DC image is representative of 10 images from 4 unique and representative donors. The monocyte image is representative of 26 recorded images from 3 unique and representative donors. For all confocal images, original magnification is ×60, scale bars are 2.5 μm, and negligible staining by isotype control antibodies was confirmed (Supplemental Figure 2). (E) FcεRIα maturation state in basophils and DCs. FcεRIα was immunoprecipitated from blood basophil and blood BDCA1+ DC lysates. Half of the immunoprecipitates were treated with EndoH. The resulting samples were run on SDS-PAGE, transferred, and blotted with an FcεRIα antibody. The asterisk indicates EndoH that cross-reacted with FcεRIα antisera.
FcεRI in DCs is mature. Our confocal microscopy indicated that FcεRIα in basophils is localized in the ER, whereas FcεRIα in DCs and monocytes is mostly excluded from the ER. This finding suggests that a significant portion of FcεRIα is immature in basophils, whereas a majority of FcεRIα in DCs is mature. To confirm this, we directly examined the maturation status of FcεRIα in basophils and DCs by determining its sensitivity to endoglycosidase H (EndoH). EndoH cleaves the high-mannose residues that are attached to immature FcεRIα in the ER; mature α-chain has trimmed glycosylated residues that are resistant to EndoH cleavage (25). In FcεRIα immunoprecipitates from basophils, we found a sharp band at 45 kDa and a smear of bands between 50 and 75 kDa (Figure 2E), consistent with a previous report (17). The 45 kDa band completely disappeared after EndoH treatment, reflecting EndoH-sensitive immature FcεRIα, whereas the bands between 50 and 75 kDa were resistant to EndoH, reflecting mature FcεRIα. When the same experiments were performed with DCs, we saw a smear of bands between 40 and 55 kDa, and no extra band was observed. Furthermore, the smear did not disappear after EndoH treatment. This experiment was repeated with cells from another independent donor, and the result was similar (Supplemental Figure 3). This finding suggests that basophils contain both immature and mature FcεRIα, whereas DCs contain FcεRIα mainly in its mature form, with distinct carbohydrate moieties attached.
IgE bound to FcεRI on DCs is efficiently internalized. Our finding that FcεRIα is localized in endolysosomal compartments in DCs raised the possibility that FcεRI might be constitutively endocytosed from the plasma membrane in these cells. In this event, IgE bound to FcεRI at the DC surface would also be endocytosed, and if so, it would be detected inside DCs. To determine the presence of intracellular IgE, we fixed and permeabilized freshly isolated blood basophils and DCs and stained them using an anti-IgE antibody. We found that the anti-IgE antibody mainly labeled the plasma membranes of basophils, while it additionally labeled intracellular compartments in DCs (Figure 3A). To quantitatively determine intracellular IgE, we used flow cytometry to measure the fraction of total IgE left after surface IgE was stripped by an acid wash. We found that approximately 6% of total IgE remained after the stripping of basophils, while approximately 12% was left in DCs (Figure 3B). To examine the possibility that this acid-resistant IgE simply represents surface IgE that was not completely stripped, we determined the efficiency of the acid wash by measuring the fraction of surface IgE remaining after acid treatment. We found that approximately 3% and 4% of surface IgE was not stripped in basophils and DCs, respectively (Figure 3B), indicating that the acid wash could not completely remove surface IgE. Nevertheless, the acid-resistant fraction of total IgE was substantially greater than the acid-resistant fraction of surface IgE in both basophils and DCs (Figure 3B), indicating that both cell types have intracellular IgE, although DCs have significantly more.
IgE bound to FcεRI on DCs is efficiently internalized. (A) Intracellular IgE in basophils and DCs. The basophil image is representative of 83 recorded images from 7 unique and representative donors, and the DC image is representative of 121 recorded images from 9 unique and representative donors. Original magnification, ×60 and scale bars are 2.5 μm. (B) Intracellular IgE was quantified by flow cytometry. Isolated basophils and DCs were washed with acid (see Methods for detail) or PBS. Data are mean ± SEM for 8 representative donors. (C) Entry of hIgE-A647 into basophils and DCs of one representative donor. hIgE-A647 (0.5 μg/ml) was added to PBMCs. At each indicated time point, cells were treated with acid or PBS before permeabilization and analysis by flow cytometry. (D) IgE entry to basophils and DCs is IgE receptor mediated. PBMCs were incubated for 4 hours with hIgE-A647 (0.5 μg/ml) alone (left), with excess unlabeled IgE (middle), or IgG (40 μg/ml) (right). Cells incubated with hIgE-A647 alone were washed with acid or unwashed. Cells incubated together with excess IgE or IgG were all washed with acid. Then, the A647 MFI of acid-washed cells was divided by that of unwashed cells to comparatively determine intracellular IgE content. Data are mean ± SEM for 4 representative donors.
To more directly compare the ability of FcεRI on DCs and basophils to mediate IgE internalization, we incubated PBMCs with human IgE conjugated to the fluorophore Alexa Fluor 647 at 37°C for 1 or 4 hours. At each time point, the level of total and acid-resistant hIgE–Alexa Fluor 647 associated with DCs and basophils were determined by flow cytometry. We found that total IgE increased over time in both DCs and basophils (Figure 3C and Supplemental Figure 4). Remarkably, the amounts of IgE associated with DCs were comparable to or greater than those associated with basophils (Figure 3C and Supplemental Figure 4), despite FcεRI surface expression being much lower in DCs (Figure 1B), which is consistent with our earlier observation that FcεRI is less occupied by IgE in DCs than in basophils (Figure 1D). We found that acid-resistant IgE also increased over time in both cell types, but to a much higher level in DCs (Figure 3C and Supplemental Figure 4). In fact, up to 60% of total IgE in DCs was acid resistant at 4 hours of incubation, while a maximum of 10% was acid-resistant in basophils (Figure 3D), indicating that DCs internalize IgE more efficiently than basophils do. To verify that internalization is mediated by FcεRI, we performed the same experiment in the presence of excess amounts of unlabeled IgE or IgG. Unlabeled IgE but not IgG markedly inhibited entry of hIgE–Alexa Fluor 647 into both DCs and basophils (Figure 3D), indicating that the entry was indeed IgE receptor mediated. Since the low-affinity IgE receptor FcεRII is not expressed in blood DCs or basophils in the steady state (26, 27), it is FcεRI that mediates internalization of IgE in these cells.
IgE internalized by DCs is delivered to the lysosomes and degraded. Next, we determined the fate of the internalized IgE in DCs and basophils. First, we identified the subcellular localization of hIgE–Alexa Fluor 647 that had entered DCs or basophils by confocal microscopy. While IgE is readily denatured at a pH of 5 and below (28), Alexa Fluor 647 is stable and fluorescent in acidic environments such as lysosomes. We found that Alexa Fluor 647 colocalized with Lamp1 in DCs (Figure 4A), which indicates that IgE had been delivered to the lysosomes. No Alexa Fluor 647 was detected inside basophils (data not shown), consistent with limited entry of IgE into these cells. Second, we determined whether IgE internalized by DCs was degraded in the lysosomes. DCs were isolated from PBMCs and cultured in the presence or absence of chloroquine, an inhibitor of lysosomal acidification (29). After 8 hours of culture, cells were fixed, stained using anti-IgE antibody, and examined by microscopy. We found that chloroquine treatment enlarged the size of the anti-IgE–labeled intracellular compartments specifically in DCs (Supplemental Figure 5). To quantitatively determine the effect of chloroquine on the intracellular IgE pool, we imaged chloroquine-treated and untreated DCs and basophils by confocal microscopy. On the micrographs of each individual cells imaged, the regions of plasma membrane and cytosol were manually drawn (Figure 4B). The anti-IgE antibody signal density in each region was quantitated, and the ratio of cytosolic (“intracellular”) to plasma membrane region (“extracellular”) signal density was determined. We found that this ratio was significantly higher in chloroquine-treated DCs compared with untreated cells (Figure 4B), indicating that chloroquine treatment increased intracellular IgE in DCs. For comparison, the same experiment was performed using basophils isolated from the same donors. We found that chloroquine treatment did not increase or only slightly increased the intracellular IgE fraction (Figure 4B). Last, we employed flow cytometry to better quantitate the effect of chloroquine on the amount of intracellular IgE. We found that chloroquine treatment consistently increased the fraction of intracellular IgE in DCs (Figure 4C). Notably, intracellular IgE in basophils also increased (Figure 4C), indicating that they too contained some IgE in lysosomes, although markedly less than DCs.
IgE internalized by DCs is delivered to the lysosomes and degraded. (A) IgE traffics to lysosomes after entering DCs. DCs were incubated with 0.5 μg/ml hIgE-A647 for 4 hours before preparation for confocal microscopy. Data are representative of 17 images from two unique and representative donors. Original magnification, ×60; scale bars: 2.5 μm. (B) Effect of chloroquine on the intracellular IgE pool determined by confocal microscopy. DCs and basophils were incubated for 8 hours at 37°C with or without 0.5 μM chloroquine (Chlor) and stained with anti-IgE antibody before confocal microscopy. On each confocal micrograph, intracellular and cell membrane (extracellular) regions were identified manually (shown on the left; scale bar: 2.5 μm), fluorescence density was measured, and the signal ratio of intracellular to extracellular regions was determined. Shown are summarized data from 30 cells of each type in each condition for 2 donors. Data represent mean ± SEM and *P < 0.005. (C) Effect of chloroquine on intracellular IgE pool determined by flow cytometry. Basophils and DCs were incubated for 8 hours at 37°C with or without 2 mM chloroquine and washed with acid or PBS. The acid-resistant IgE fraction was determined by staining cells with anti-IgE after permeabilization and dividing the MFI of acid-washed cells by the MFI of unwashed cells as described for Figure 3B. Data are from 4 representative donors.
Human FcεRIα is specifically localized in the lysosomes of FCER1A-Tg mouse conventional DCs and monocytes. To obtain evidence that IgE entered DCs via FcεRI in vivo, we used human _FCER1A_-transgenic (_FCER1A_-Tg) mice, which lack mouse Fcer1a but express human FcεRIα (FCER1A) under transcriptional control of the human promoter (30). We found that these mice expressed hFcεRI on blood conventional DCs (cDCs), monocytes, and basophils, whereas B cells and other lymphocytes did not express it, as in humans (Figure 5A and not shown). hFcεRI in blood basophils of these mice did not colocalize with Lamp1 but did colocalize with calnexin (Figure 5B), similar to observations with human basophils (Figure 2A). We also found that hFcεRIα in blood monocytes of these mice colocalized strongly with Lamp1 (Figure 5B). hFcεRIα in blood cDCs of these mice could not be visualized by microscopy due to the relatively low expression of FcεRIα. Instead, we imaged CD11b+ cDCs (the mouse counterpart of human BDCA1+ DCs; ref. 31) cultured from bone marrow of the mice using Flt3L (ref. 32 and Supplemental Figure 6). hFcεRI in these BMDCs colocalized strongly with Lamp1 (Figure 5C). Thus the cell type–specific expression and trafficking of human FcεRIα was recapitulated in the _FCER1A_-Tg mice.
_FCER1A_-Tg mice express and localize hFcεRI in a similar manner to humans. (A) Surface expression of hFcεRI in _FCER1A_-Tg mouse blood leukocytes. Blood from adult hFcεRIα(+) transgenic (Tg+, black) and hFcεRIα(–) (Tg–, gray shaded) littermates was collected and stained with an anti-hFcεRIα antibody and cell type–specific antibodies after red blood cell lysis. Gating strategies are shown in Supplemental Figure 6A. (B) Intracellular localization of hFcεRI in Tg+ mouse blood basophils and monocytes. Sorted blood basophils and monocytes were stained for confocal microscopy as described for Figure 2. Images are representative of results from 3 independent experiments. Note that the calnexin signal has been switched from blue to cyan for ease of visualization in single-stain format, while it has not been altered for merged images. (C) Intracellular localization of hFcεRI in Tg+ mouse CD11b+ BMDCs. BMDCs were cultured using Flt3L, sorted for CD11b+ cDCs as described for Supplemental Figure 6B, and stained as in B. Images are representative results from 3 independent experiments. For all confocal images, magnification was ×60, scale bars are 2.5 μm, and negligible staining by isotype control antibodies was confirmed (Supplemental Figure 7).
IgE bound to hFcεRI on monocytes and cDCs is internalized in FCER1A-Tg mice. Having found that, like humans, _FCER1A_-Tg mice express and localize hFcεRI in the lysosomes of cDCs and monocytes, we used these mice to examine whether hFcεRI mediates cellular internalization of IgE in vivo. First, Tg+ and Tg– mice were i.v. injected with human IgE. It is noteworthy that the injected human IgE had been pre-tested and confirmed for the absence of aggregates that can potentially activate hFcεRI-expressing cells by crosslinking hFcεRI. Briefly, we added the human IgE preparation to mast cells cultured from _FCER1A_-Tg mouse bone marrow and measured degranulation of these cells by a hexosaminidase release assay (33). The addition of up to 10 μg/ml of IgE to the cells did not cause any release of hexosaminidase above the level of spontaneous release (Supplemental Figure 8). In contrast, addition of anti-hFcεRI antibody/secondary antibody complexes resulted in release of 20% of hexosaminidase. These data indicated that the human IgE that we injected to mice did not contain a substantially level of aggregates and would not cause hFcεRI crosslinking in vivo.
After injection of human IgE into Tg+ and Tg– mice, we bled the mice at multiple time points and determined the amount of human IgE bound to hFcεRI-expressing cells by flow cytometry. We found very little IgE bound to monocytes and basophils of Tg– mice at all time points examined (Figure 6A). In contrast, surface IgE levels increased for both basophils and monocytes in Tg+ mice having reached their maximum approximately 4 hours after injection and remained at least at that level for the next 4 hours (Figure 6A). At 24 hours after injection, however, monocytes had lost approximately 80% of IgE, while basophils retained approximately 100% (Figure 6A). We also monitored IgE on the surface of cDCs in the blood. Since cDCs are scarce in blood, we euthanized individual mice at each time point and performed flow cytometry using whole blood. Similar to monocytes, cDCs lost most of their surface IgE at 24 hours after injection (Figure 6B). In addition to the blood cells, we also examined monocytes and DCs in the lungs. They also captured substantial amounts of IgE but lost most of it as early as 12 hours after injection (Figure 6C). Last, we examined mast cells in the peritoneal cavity. Unlike monocytes and cDCs, these cells retained 100% of the peak surface IgE at 24 hours after injection, similar to basophils (Figure 6D). Notably, hFcεRI was not found in the lysosomes of mast cells but was found on the plasma membranes and ER, as was the case with basophils (Supplemental Figure 9). These findings indicate that human IgE injected into _FCER1A_-Tg mice initially binds to all hFcεRI-expressing cells, but gradually disappears from monocytes and cDCs.
Human IgE injected into _FCER1A_-Tg mice is internalized by cDCs and monocytes in the steady state. (A) Surface IgE levels on blood basophils and monocytes following human IgE injection. Before and at 1, 4, 8, and 24 hours after hIgE injection, Tg+ and Tg– mice were bled for flow cytometric analysis of surface IgE levels in basophils and monocytes. Data for Tg+ mice are shown in solid lines and for Tg– mice in dotted lines. Data for 3 mice from one representative experiment of 3 are presented with mean ± SEM. (B–D) Surface IgE levels on blood cDCs (B), lung DCs and monocytes (C), and peritoneal mast cells (D) of Tg+ mice following human IgE injection. At each time point following hIgE injection, mice were sacrificed and whole blood, lungs, or peritoneal lavage collected and analyzed by flow cytometry. Data for 9 mice (B), 8 mice (C), or 14 mice (D) from one representative experiment of 2 are presented with mean ± SEM. (E and F) Intracellular localization of human IgE in basophils and monocytes of _FCER1A_-Tg mice injected with hIgE. At 6 hours after injection, basophils and monocytes were isolated and examined for intracellular human IgE by confocal microscopy as described for Figure 3A. Original magnification, ×60; scale bars: 2.5 μm. (F) Intracellular IgE levels were quantified as in Figure 4B. Thirty images of Tg+ monocytes and basophils were analyzed. Resulting values are presented with mean ± SEM. *P < 0.05.
We reasoned that IgE bound to monocytes and cDCs of Tg+ mice could be subsequently internalized, which might explain the gradual loss of IgE from the surface of these cells while the IgE bound to basophils and mast cells remained. Therefore, we looked for intracellular hIgE in blood monocytes and basophils of hIgE-injected Tg+ mice by isolating each cell population at 6 hours after IgE injection, staining with an anti–human IgE antibody after permeabilization, and examining by confocal microscopy. We found that the human IgE was mostly associated with the plasma membranes in basophils, whereas it was detected both at the plasma membranes and in intracellular compartments in monocytes (Figure 6E). Quantification of micrograph images showed that monocytes had a significantly higher proportion of IgE intracellularly compared with basophils (Figure 6F). Taken together, these findings suggest that FcεRI expressed in cDCs and monocytes actively mediates IgE internalization in vivo.
Human FcεRI expressed by cDCs or monocytes contributes to serum IgE clearance in FCER1A-Tg mice. Previous studies have demonstrated that wild-type mice and mFcεRIα-deficient mice clear serum IgE at similar rates, indicating that mFcεRI is not involved in IgE clearance in mice (34, 35). Nevertheless, we hypothesized that human FcεRI could be involved in IgE clearance based on our finding that human FcεRI expressed in monocytes and cDCs mediated cellular internalization of IgE in a constitutive manner. To test this hypothesis, we injected human IgE into Tg+ and Tg– mice, bled them at various times after injection, and determined serum concentrations of human IgE by ELISA. We found that serum concentrations of human IgE were reduced over time in both strains of mice, but at a markedly accelerated rate in Tg+ mice (Figure 7A). Quantitative analysis showed that the half-life of human IgE in the Tg+ mice was approximately 4-fold shorter than in Tg– mice (Figure 7B), suggesting that hFcεRI significantly contributes to serum IgE clearance.
Human FcεRI expressed by DCs or monocytes significantly contributes to serum IgE clearance in _FCER1A_-Tg mice. (A) Serum IgE clearance in Tg+ and Tg– littermates. 2.5 μg hIgE was i.v. injected into mice, blood was collected following injection, and hIgE concentration was determined by ELISA. Data are representative of 3 independent experiments using 3 mice per strain. For each mean, a line of best fit with indicated r2 values was calculated. (B) Serum half-life of hIgE in Tg+ and Tg– mice. IgE half-life was calculated as the time for peak serum IgE to halve from lines of best fit calculated as in A. Symbols represent individual mice injected with 2.5 μg (squares) and 5 μg (circles). *P < 1.5 × 10–6. (C and D) hFcεRI-expressing hematopoietic cells are responsible for serum IgE clearance. Mixed bone marrow chimeras were made on Tg– recipient hosts, and serum hIgE clearance was examined as described for A and B. *P < 0.05. (E and G) Flt3L increases the frequency of DCs/monocytes, while IL-3 increases basophils. 10:90 (Tg+/Tg–) mixed chimeric mice were injected with Flt3L-expressing B16 cells (E) or with IL-3/anti–IL-3 antibody complexes (G). The percentage of [CD11b+ DCs + monocytes] or basophils in spleen was determined by flow cytometry. *P < 0.05. (F and H) Serum hIgE clearance is accelerated by an increase in DCs/monocytes but not by an increase in basophils. *P < 0.05. Data in C–H are representative of 2 independent experiments. The second experiment of E–H is shown in Supplemental Figure 11.
To verify that the rapid serum IgE clearance observed in Tg+ mice is directly attributed to FcεRI-expressing cells and not to some other features associated with genetic alteration of Tg+ mice, we reconstituted the hematopoietic compartment of Tg– mice with bone marrow that had been isolated from Tg– or Tg+ mice and mixed at specific ratios of Tg+ to Tg–. We found that mice reconstituted with Tg+ bone marrow cleared serum IgE at a much faster rate than those reconstituted with Tg– bone marrow (Figure 7C). We also found that mixed chimeric mice cleared serum IgE in rates proportional to the percentage of Tg+ bone marrow used (Figure 7C). Interestingly, however, the half-life of serum IgE in 50% Tg+ chimeric mice was comparable to that in 100% Tg+ chimeric mice (Figure 7D), suggesting that in this experimental setting, IgE is sufficiently cleared by 50% of the hematopoietic, hFcεRI-expressing cell compartment. Nevertheless, this study demonstrates that it was indeed hFcεRI-expressing cells that were responsible for the rapid serum IgE clearance in _FCER1A_-Tg mice.
Next, we examined the specific contribution of cDCs and monocytes compared with basophils to serum IgE clearance; the former cells internalize and degrade IgE, while the latter retain IgE at the cell surface. Since we found that the rate of IgE clearance increased in proportion to the number of hFcεRI-expressing cells only when these cells were present in limited numbers (Figure 7D), we increased the number of cDCs and monocytes or basophils in 10:90 (Tg+/Tg–) mixed bone marrow chimeric mice by implanting them with a melanoma cell line producing Flt3L. Flt3L stimulates proliferation and differentiation of the monocyte/DC common progenitor in mice (36, 37). Accordingly, injection of Flt3L or implantation with Flt3L-producing melanoma has been shown to markedly increase the number of monocytes and cDCs in mice (36–38). Consistent with this previous finding, Flt3L melanomas significantly increased the frequency of monocyte and CD11b+ cDC, but not basophil, populations (Figure 7E). Note that we did not include CD8+ cDCs in this analysis because these DCs expressed hFcεRI at extremely low levels in _FCER1A_-Tg mice (data not shown) and were thus irrelevant to our study. Remarkably, Flt3L melanoma–implanted mice cleared serum IgE at a significantly faster rate than untreated mice (Figure 7F). When the same experiment was performed using Tg– mice, however, no difference was observed (Supplemental Figure 10). This finding indicates that neither Flt3L, the melanoma, nor the increase in DCs and monocytes accelerates serum IgE clearance independently of hFcεRI. Next, we increased the frequency of basophils in a separate group of 10:90 (Tg+/Tg–) mixed chimeric mice by injecting IL-3/anti–IL-3 antibody complexes. As shown in previous studies (39, 40), this treatment increased the frequency of basophils by approximately 8-fold but did not increase the frequency of monocytes or DCs (Figure 7G). Furthermore, the rate of serum IgE clearance was not accelerated by injection of IL-3/anti–IL-3 antibody complexes (Figure 7H). Taken together, these findings indicate that hFcεRI-expressing cDCs or monocytes, but not basophils, significantly contribute to serum hIgE clearance in _FCER1A_-Tg mice.