IL-3 Expands Pre-Basophil and Mast Cell Progenitors by Upregulating the IL-3 Receptor Expression

Abstract

Basophils and mast cells play a critical role in allergic inflammation and provide protective immunity against certain types of parasitic infections. Expansion of basophils and mast cells to the critical numbers is believed to be an essential step in enabling basophils and mast cells to carry out their protective functions. However, factors that drive basophil and mast cell expansion are still incompletely understood. We tested the roles of cytokines and growth factors IL-3, TSLP, GM-CSF, IL-5, SCF, IL-7, IL-25, and IL-33 in promoting the differentiation of pre-basophil and mast cell progenitors (pre-BMPs)in vitro.We found that while GM-CSF only expanded basophils, IL-3 promoted the differentiation of pre-BMPs into both basophils and mast cells. We found that IL-3 expanded the number of pre-BMPsin vivo. We showed that IL-3 upregulatedIl3ramRNA and protein expression on pre-BMPs, supporting that IL-3 expands pre-BMPs in part by upregulating the IL-3 receptor expression. Although Gata2 mRNA expression was upregulated by IL-3 treatment in pre-BMPs, it is dispensable for IL-3-mediated upregulation of IL-3 receptor expression. Our study reveals a novel mechanism through which IL-3 expands basophil and mast cells.

Fig. 1.

IL-3 greatly increases the number of pre-BMP-derived cells in vitro. (A) FACS sorting gates for FACS-sorting pre-BMPs. c-Kit and FcεR1α expression of the FACS-sorted pre-BMPs before they were cultured. (B) Total number of cells recovered from each culture. Pre-BMPs were FACS-sorted from bone marrow cells pooled from ten mice. The sorted pre-BMPs (11,400 cells/well) were cultured in complete IDMEM under the indicated conditions (20 ng/ml for IL-3, IL-7, IL-25, and IL-33; 1 μg/ml for TSLP; 50 ng/ml for GM-CSF, IL-5, and SCF) for 5 days. Error bars represent mean ± SEM, n=3 cultures from three independent experiments with one culture from each experiment. (C) FACS analysis on pre-BMP-derived cells at day 5. (D) The total number of pre-BMP-derived basophils and mast cells (E) under different culture conditions for five days. Error bars represent mean ± SEM, n=3 cultures from three independent experiments with one culture from each experiment.

Fig. 2.

IL-3 expands pre-BMPs in vivo. FACS analysis of (A) pre-BMPs, (B) pro-BMPs, and (C) GMPs from IL-3c or PBS-injected mice (left panels). The total number of (A) pre-BMPs, (B) pro-BMPs, and (C) GMPs in two femur bones per mouse (right panels). Data represent means ± SEM (n=5 mice for pre-BMPs, n=3 mice for pro-BMPs and GMPs from three independent experiments (one or two mice were used for each experiment). (D) FACS analysis of pre-BMPs from TSLP or PBS-injected mice (left panels) and the total number of pre-BMPs in two tibias and two femur bones per mouse (right panels). Error bars represent mean ± SEM, n=3 mice from three independent experiments (one mouse was used for each experiment). Statistical significance was analyzed with a two-tailed Student’s t-test.

Fig. 3.

IL-3 upregulates the IL-3Rα expression on pre-BMPs. (A) qPCR analysis of the Il3ra and Il4ra mRNA expression in FACS-sorted pre-BMPs or GMPs from pooled bone marrow cells of ten mice treated with PBS or IL-3c for each experiment. Error bars represent mean ± SEM, n=3 experiments. Statistical significance was analyzed with a two-tailed student’s t-test. (B) Flow analysis of the IL-3Rα expression on GMPs or pre-BMPs treated with PBS or IL-3c. MFIs of the gated populations were indicated. Data represent means ± SEM, n=3 mice from three independent experiments (one mouse for each experiment). Statistical significance was analyzed with a two-tailed Student’s t-test.

Fig. 4.

IL-3 upregulates the Gata2 mRNA expression in pre-BMPs. qPCR analysis of the Gata2, Cbfa2t3 and Atf4 mRNA expression in the FACS-sorted pre-BMPs and GMPs from pooled barrow cells of ten mice treated with PBS or IL-3c. Data were pooled from three independent experiments (means ± SEM). Statistical significance was analyzed with a two-tailed Student’s t-test.

Fig. 5.

GATA2 is dispensable for the upregulation of the IL-3Rα chain expression on cells derived from pre-BMPs. (A) Pre-BMPs were FACS-sorted from the bone marrow cells of individual Gata2^f/f Rosa^YFP/YFP Tg(creER)^hemi mice or control Gata2^+/+ Rosa^YFP/YFP Tg(creER)^hemi mice and cultured in the presence of 25 nM 4-HT for three days. YFP⁺ cells in the cultured Gata2^f/f Rosa^YFP/YFP Tg(creER)^hemi pre-BMPs (Gata2^−/−) and Gata2^+/+ Rosa^YFP/YFP Tg(creER)^hemi (Gata2^+/+) were FACS sorted using gates shown. Gata2 mRNA in the FACS-sorted YFP⁺ cells was analyzed by qPCR. DAPI was used to gate out dead cells. (B) IL-3Rα expression on the cultured YFP⁺ Gata2^−/− or the control YFP+ Gata2^+/+ cells was analyzed using gates shown in (A). 4HT Days indicated days after 4HT treatment at the time of FACS analysis. ΔMFIs were calculated as the MFIs of receptor staining minus the MFI of IgG isotype control. The numbers indicate the fold of difference in the ΔMFI of IL-3Rα between YFP⁺Gata2^−/− and YFP⁺Gata2^+/+ cells. (C) FcεR1α expression on YFP⁺Gata2^+/+ and YFP⁺Gata2^−/− cells. (D) c-Kit expression on YFP⁺Gata2^+/+ and YFP+Gata2^−/− cells. Mean ± SEM were calculated from two independent experiments (two mice for each experiment for total of n=4 mice). Statistical significance in receptor expression between YFP⁺ Gata2^+/+ and YFP⁺ Gata2^−/− cells was analyzed with a two-tailed Student’s t-test.