Murine B cells regulate serum IgE levels in a CD23-dependent manner

Abstract

The manifestations of allergic disorders are closely tied to the biologic effects of IgE activation with Ag. In immediate hypersensitivity reactions, IgE effector function requires prior binding to innate immune cells, primarily mast cells and basophils, with the blood acting as a reservoir for unbound IgE. As the severity of allergic disease is proportional to the size of this unbound IgE pool, we hypothesized that cellular mechanisms exist to limit the size and/or enhance the clearance of free IgE molecules. We examined this in mice by engineering a reporter IgE molecule that allowed us to track the fate of IgE molecules in vivo. The absence of FcεRI-expressing cells did not affect serum IgE levels, but B cells regulated serum IgE by controlling the size of the free IgE pool. B cells captured IgE by direct binding to the low-affinity IgE receptor, CD23. These data indicate a mechanism regulating serum IgE and additionally clarify the role of CD23 in this process.

Figure 1

Construction and characterization of HA-Fcε. A) Schematic of the HA-Fcε construct. The Cε2–4 domains of the IgE heavy chain are positioned C-terminal of a V_kappa signal sequence and the HA epitope tag. B) Groups of 2–3 IgE-deficient 4getRag2^−/− mice received 1 μg of native IgE (■) or HA-Fcε (▲) at time 0. We then serially bled the mice to follow the fate of infused IgE. Error bars represent the standard error of the mean (SEM). In C) and D), 4getRag2^−/− mice received 1 μg of native IgE (C) or HA-Fcε (D) by tail vein injection. Twenty-four hours later, we analyzed splenic basophils (GFP⁺ CD49b⁺ SSC^lo) for the presence of both total IgE and HA (solid black lines). The gray histograms indicate the staining of negative control IgE-deficient mice. Data are representative of at least 3 independent experiments.

Figure 2

Uptake of HA-Fcε by splenic basophils and peritoneal mast cells. We infused 4get BALB/c mice with increasing amounts of HA-Fcε as listed at the top of the figure. Twenty-four hours later, we analyzed GFP⁺CD49b⁺SSC^lo splenic basophils (A) or GFP⁺c-kit⁺ peritoneal mast cells (B) for HA-Fcε using an anti-HA antibody. The percent of HA-Fcε⁺ cells is depicted with each histogram. Data are representative of 2 independent experiments.

Figure 3

B cells regulate serum IgE levels. A) Wild-type (■) or FcεRI^−/− (▲) mice received 2.5 μg of HA-Fcε by tail vein injection. We then analyzed HA-Fcε levels at the indicated time points. B) We analyzed wild-type (■) or μMT^−/− (▲) animals for HA-Fcε clearance at the indicated time points. C) Wild-type (■), Stat6^−/− (▲), or IL-4/13^−/− (▼) mice received 2.5 μg of HA-Fcε and were analyzed at the indicated time points. Data are representative of at least 2 independent experiments each. Wild-type and FcεRI^−/− groups had 3 mice each. μMT and Stat6^−/− groups had 4 mice each. The IL-4/13^−/− group had 2 animals. Error bars represent the SEM. *p<0.001, **p<0.007

Figure 4

Anti-CD23 blockade increases peak serum IgE levels and enhances IgE loading. A) BALB/c mice received either 100 μg of B3B4 (▲) or isotype (■) control antibody by tail vein injection. One day later, the mice received 2.5 μg of HA-Fcε and were serially bled to examine HA-Fcε serum levels. There were 5 mice in each group with error bars representing the SEM. *p<0.015, **p<0.005 In B) and C), the mice received B3B4 (solid black line) or isotype (dashed line) as above and were given 0.5 μg of HA-Fcε the next day. Surface HA-Fcε capture was assessed on B) Peritoneal mast cells and C) Splenic basophils twenty-four hours later. The mean fluorescence intensity is noted above the histograms. The shaded histograms represent the negative control. The data are representative of 2 independent experiments.

Figure 5

B cells bind HA-Fcε in a CD23-dependent manner. A) CD19⁺ splenocytes from BALB/c mice were stained with B3B4 (solid line) or isotype control (shaded histogram). B) BALB/c mice received either PBS or 2.5 μg of HA-Fcε. We harvested splenocytes 2 or 24 hours after HA-Fcε infusion and fixed the cells in 4% paraformaldehyde immediately. The timepoint at harvest is indicated in each of the histograms. We stained single cell suspensions with CD19, B220, and anti-HA antibodies. The solid lines in the each of the panels represent cell surface HA-Fcε on CD19⁺B220⁺ cells. The shaded histograms represent staining on B cells from mice that received PBS. C) Two hours after receiving 2.5 μg HA-Fcε, fixed B cells from mice that had received 100 μg of isotype control (left panel) or B3B4 (right panel) one day prior were examined as above. D) Two hours after receiving 2.5 μg HA-Fcε, fixed B cells from mice were stained for extracellular HA-Fcε (left panel) or intracellular HA-Fcε (right panel). The middle panel depicts extracellular HA-Fcε staining after collagenase digestion. Shaded histograms represent B cells from PBS infused animals. Data are representative of at least 2 independent experiments.

Figure 6

CD23 blockade enhances local hypersensitivity reactions. Mice received either 100 μg isotype control or B3B4 antibody on Day 0. On Day 1, the mice were loaded with 0.5 μg of anti-TNP IgE. On Day 3, the mice received 200 ul of 1% Evans Blue dye by tail vein injection and then a 1 μg intradermal injection of TNP₁₃-OVA into the ear. A non-IgE loaded group of mice served as a background control. Average values were determined from the ears from 2 mice in the non-loaded group, 3 mice in the isotype treated group, and 4 mice from the B3B4 treated group. The data are representative of 2 independent experiments. *p<0.01

Figure 7

CD23 blockade leads to an increase in the serum IgE pool. Groups of 3 BALB/c mice received 100 μg of either isotype control (■) or B3B4 (▲) antibody on Day 0. One, four, and seven days later, serum was collected from individual animals and serum IgE levels examined by ELISA. Error bars for each timepoint represent the SEM. Data are representative of 2 independent experiments. *p<0.01