ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo

Abstract

The proteolytic activity of a disintegrin and metalloproteinase 10 (ADAM10) regulates cell-fate decisions in Drosophila and mouse embryos. However, in utero lethality of ADAM10(-/-) mice has prevented examination of ADAM10 cleavage events in lymphocytes. To investigate their role in B cell development, we generated B cell-specific ADAM10 knockout mice. Intriguingly, deletion of ADAM10 prevented development of the entire marginal zone B cell (MZB) lineage. Additionally, cleavage of the low affinity IgE receptor, CD23, was profoundly impaired, but subsequent experiments demonstrated that ADAM10 regulates CD23 cleavage and MZB development by independent mechanisms. Development of MZBs is dependent on Notch2 signaling, which requires proteolysis of the Notch2 receptor by a previously unidentified proteinase. Further experiments revealed that Notch2 signaling is severely impaired in ADAM10-null B cells. Thus, ADAM10 critically regulates MZB development by initiating Notch2 signaling. This study identifies ADAM10 as the in vivo CD23 sheddase and an important regulator of B cell development. Moreover, it has important implications for the treatment of numerous CD23- and Notch-mediated pathologies, ranging from allergy to cancer.

Figure 1.

ADAM10^Δ/ΔCD19cre^+/− mice are B cell–specific ADAM10 knockouts. (A) PCR products for exon 9 of ADAM10 performed on isolated DNA, using primers P1 and P2 (Fig. S1). Spleen B220⁺ B cells and B220⁻ non–B cells from the indicated mice were sorted via FACS. 955- and 217-bp bands represent full-length and recombined ADAM10, respectively. (B) RT-PCR for ADAM10 mRNA isolated from FO and T1 spleen B cells. FO B cells identified as B220⁺CD23^int/hiCD21/35^intIgM^int and T1 B cells identified as B220⁺CD23^low/−CD21/35^lowIgM^hi were isolated via FACS. (C) Flow cytometric analysis of ADAM10 expression on the surface of FO and T1 spleen B cells from the indicated mice. Sorted and analyzed cells from EYFP⁺ mice were identified as EYFP⁺ instead of B220⁺. Data are representative of three independent experiments.

Figure 2.

ADAM10 is essential for MZB lineage development. (A and B) Flow cytometric analysis of spleen B cells labeled for CD21/35 and IgM expression, gated on (A) B220⁺CD23^int/hi and (B) B220⁺CD23^low/− cells. (C) Analysis of PMZBs and MZBs labeled for CD1d and CD9 expression. Flow cytometric plots in A–C are representative of six experiments. (D) Levels of B cell subsets are expressed as the percentage of total spleen B cells (n = 6 except for T2 cells [n = 3]). Data are cumulative from six independent experiments for all subsets except for T2 cells, which are cumulative from three independent experiments. In A–D, FO and T1 cells were identified as in Fig. 1. MZBs, T2 cells, and PMZBs were identified as follows: T2 cells, B220⁺CD23⁺CD21/35^intAA4.1⁺; MZBs, B220⁺CD23^low/−CD21^hiIgM^hi; and PMZBs, B220⁺CD23^int/hiCD21/35^hiIgM^hi. Numbers on plots indicate the percentage of B220⁺ cells in boxes. Cells from ADAM10^Δ/Δcre^+/−EYFP⁺ mice were identified as EYFP⁺ instead of B220⁺. Error bars represent the SEM between samples. (E) Immunohistochemical labeling of spleen follicles for MOMA-1, IgM, and IgD expression. Data are representative of three independent experiments. Bar, 100 µm.

Figure 3.

ADAM10 is the primary in vivo sheddase of CD23. (A) Cell-surface expression of CD23 on spleen FO B cells from the indicated mice. Data are representative of six independent experiments. (B) Mean fluorescent intensity (MFI) of mCD23 expression by FO B cells (B220⁺CD21^intIgM^int; n = 6). (C) Immunohistochemical staining of spleen cryosections from the indicated mice. CD23-PE and MOMA-1–FITC labeling of metallophilic macrophages in the marginal sinus surrounding spleen follicles is shown. Data are representative of three independent experiments. Bar, 100 µm. (D) Serum sCD23 levels measured by ELISA (n = 4). (E and F) Cleavage of CD23 on FO B cells treated with 19G5 or C0H2 ex vivo. FO B cells isolated from spleens as in Fig. 1 B were cultured for 24 h with CD40L, IL-4, and 8:A3 to elevate mCD23 levels before washing and culture with 100 µg 19G5 or C0H2 with fresh cytokines. Cells and supernatants were collected 17 h later and analyzed for mCD23 expression via flow cytometry (E) and sCD23 via ELISA (F), respectively (n = 4). Data in D–F are representative and cumulative from four independent experiments. Error bars represent the SEM between samples.

Figure 4.

CD23 expression does not regulate MZB development. Flow cytometric analysis of spleen B cells from C57BL/6, CD23^−/−, CD23 transgenic (TG), and ADAM10^Δ/Δcre^+/−EYFP⁺ mice labeled for surface expression of IgM, IgD, and CD21/35. (A) Numbers indicate the percentage of B220⁺ or EYFP⁺ cells in boxes. (B) CD21/35 expression of IgM^hiIgD^low B cells boxed in A. Numbers indicate the percentage of B220⁺IgM^hiIgD^low cells within gates (T1, B220⁺IgM^hiIgD^lowCD21/35^low; MZB, B220⁺IgM^hiIgD^lowCD21/35^hi). Cells from ADAM10^Δ/Δcre^+/−EYFP⁺ mice were identified as EYFP⁺ instead of B220⁺. Data are representative of three independent experiments.

Figure 5.

CD21/35 expression is reduced in ADAM10^Δ/ΔCD19cre^+/− mice. (A) Cell-surface expression of CD21/35 on spleen FO B cells. Data are representative of four independent experiments. (B) Quantified mean fluorescence intensity (MFI) from A. (C) Serum-soluble CD21/35 measured by ELISA (n = 5; data are cumulative from five independent experiments). (D) Quantitative PCR of CD21/35 mRNA expression in B220⁺ spleen cells relative to 18S expression (n = 4; data are cumulative from four independent experiments). Error bars represent the SEM between samples. (E) Immunohistochemistry of spleen follicles labeled with IgM-AMCA and CD21/35-PE. Arrows indicate MZBs with overlay staining. Bars, 100 µm. (F) Higher magnification of intense CD21/35 labeling in the ADAM10^Δ/Δcre^+/− follicles shown in E. FDC immune complexes (ICs) are labeled with anti-IgG–AMCA. Bar, 50 µm. Immunohistochemistry is representative of three independent experiments.

Figure 6.

ADAM10 initiates Notch2 signaling. (A–C) Gene expression of targets of Notch2 signaling in primary B cells determined by quantitative real-time PCR. Data are cumulative from at least four independent experiments. T1 cells, FO cells, PMZBs, and MZBs identified as in Fig. 2 were sorted via FACS (A, n = 7; B, n = 4; and C, n = 4) Expression by PMZBs and MZBs from ADAM10^Δ/Δcre^+/−EYFP⁺ mice was not determined because of cell loss (Fig. 2). (D) Expression of Dtx1 in FO B cells stimulated with CD40L and Fc-Dll1 or control mouse IgG for 60 h, with expression relative to 18S expression (n = 3; data are cumulative from three independent experiments). Error bars represent the SEM between samples. n.d., not determined.