IL-3 Induces Basophil Expansion In Vivo by Directing - Progenitors to Differentiate into Basophil Lineage-Restricted Progenitors in the Bone This information is current as Marrow and by Increasing the Number of of September 29, 2021. Basophil/ Progenitors in the Spleen Keitaro Ohmori, Yuchun Luo, Yi Jia, Jun Nishida, Zhengqi Wang, Kevin D. Bunting, Demin Wang and Hua Huang Downloaded from J Immunol 2009; 182:2835-2841; ; doi: 10.4049/jimmunol.0802870 http://www.jimmunol.org/content/182/5/2835 http://www.jimmunol.org/ References This article cites 41 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/182/5/2835.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-3 Induces Basophil Expansion In Vivo by Directing Granulocyte-Monocyte Progenitors to Differentiate into Basophil Lineage-Restricted Progenitors in the and by Increasing the Number of Basophil/Mast Cell Progenitors in the Spleen1

Keitaro Ohmori,2* Yuchun Luo,* Yi Jia,* Jun Nishida,* Zhengqi Wang,† Kevin D. Bunting,† Demin Wang,‡§ and Hua Huang3*¶

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, Downloaded from basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in ϳ4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on

STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor http://www.jimmunol.org/ expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R␣-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen. The Journal of Immunology, 2009, 182: 2835–2841.

ecent work has established important roles for basophils dramatically in number after infection with intestinal nematode, in initiating (1–3) and augmenting Th2-type immune Nippostrongylus brasiliensis (11). They also increase in number responses to challenges and parasitic infection after allergen challenge (2). ϩ R by guest on September 29, 2021 (4–6), mediating (7), and maintaining memory IL-3, produced primarily by CD4 T cells (12–14), has been responses (8). However, due to the difficulty of obtaining a suffi- shown to be pivotal in expanding basophils. In vitro, IL-3 has been cient numbers of basophils, basophil biology has not yet been stud- demonstrated to induce basophil differentiation and to enhance ied extensively. acute IL-4 production in mouse basophils (15–17). Galli and col- Basophils are a minor cell population, constituting Ͻ1% of pe- leagues (18) reported that mice lacking IL-3 failed to show in- ripheral and bone marrow cells. Basophils are normally gen- creased numbers of basophils and failed to expel nematode Strong- erated from GMPs, which also give rise to , , lyoides. Nonetheless, mechanisms underlying basophil expansion , and mast cells. These cells share many common char- remain obscure. Mature basophils have a short half-life and re- acteristics with mast cells, such as expression of a high affinity spond to IL-3 stimulation with a limited proliferation capacity (13, immunoglobin (IgE) receptor (Fc␧RI), and contain many of the 19). Thus, it is likely that IL-3 might induce basophil expansion by granules that can be found in mast cells (9, 10). Basophils expand enhancing basophil lineage commitment. In this study, we report that administrating the IL-3 complex (IL-3 plus anti-IL-3 Ab) in vivo greatly facilitated the differenti- *Division of and Immunology, Departments of Medicine, National Jewish 4 Health, Denver, CO 80206; †Departments of Medicine, Case Western Reserve Uni- ation of GMPs into basophil lineage-restricted progenitors (BaPs) versity, Cleveland, OH 44106; ‡Blood Research Institute, Blood Center of Wisconsin, specifically in a STAT5 signaling-dependent manner and increased § Milwaukee, WI 53226; Department of Microbiology and Molecular Genetics, Med- the number of basophil/mast cell progenitors (BMCPs) in the ical College of Wisconsin, Milwaukee, WI 53226; and ¶Integrated Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80206 spleen. We showed that GMPs, but not common myeloid progen- Received for publication August 29, 2008. Accepted for publication December itors (CMPs), expressed low levels of IL-3 receptor. The IL-3 re- 22, 2008. ceptor expression was dramatically up-regulated in BaPs, but not The costs of publication of this article were defrayed in part by the payment of page eosinophil lineage-restricted progenitors (EoPs). We showed that charges. This article must therefore be hereby marked advertisement in accordance ϳ38% of BMCPs expressed the IL-3 receptor. The IL-3 receptor with 18 U.S.C. Section 1734 solely to indicate this fact. expression patterns might explain why IL-3 specifically expanded 1 This work is supported by grants from the National Institutes of Health R01 AI48568 and R01 AI068083 (H.H.), R01 HL073284 and R01 AI079087 (D.W.), Scholar Award basophils in vivo. from the & Society (D.W.), and R01DK059380 (K.B.). 2 Current address: Tokyo University of Agriculture and Technology, 3-5-8 Saiwai- cho, Fuchu, Tokyo, Japan. 4 Abbreviations used in this paper: BaP, basophil lineage-restricted progenitor; 3 Address correspondence and reprint requests to Dr. Hua Huang, Departments of BMCP, basophil/mast cell progenitor; CMP, common myeloid progenitor; EoP, eo- Medicine and Immunology, National Jewish Health and University of Colorado sinophil lineage-restricted progenitor. Health Sciences Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802870 2836 BASOPHIL DEVELOPMENT AND EXPANSION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 1. IL-3 dramatically and specifically increases the percentage and number of basophils. A, C57BL/6J mice were or were not injected with IL-3 alone, anti-IL-3 Ab alone or IL-3 mixed with anti-IL-3 Ab (IL-3 complex) i.v. via a tail vein. Three days after injection, the bone marrow cells were isolated and stained with PE-labeled anti-Fc␧RI␣ and FITC-labeled anti-c- Abs. Basophils were defined as Fc␧RI␣ϩc-kitϪ. Data are representative of five independent experiments. B, A time course analysis of basophil numbers in the bone marrow. Two to five mice were used for each time point. Error bars show the SD from two to five mice. C, C57BL/6J mice were or were not injected with the IL-3 complex. Three days after injection, the bone marrow, peripheral blood, spleen, and peritoneal cavity cells were isolated and stained with PE-labeled anti-Fc␧RI␣ and FITC-labeled anti-c-kit Abs for basophil or mast detection or with PE-labeled anti-Siglec-F Ab for eosinophils detection. Mast cells were defined as Fc␧RI␣ϩc-kitϩ cells. Eosinophils were marked as Siglec-Fϩ cells. D, C57BL/6J mice were or were not injected with the IL-3 complex. The total number of basophil, eosinophil, and mast cell were shown. Error bars show the SD from two to five mice. p values were analyzed using Student’s t test. Data are representative of three independent experiments.

Materials and Methods with FITC-labeled anti-c-kit (2B8), biotin-labeled anti-Fc␧RI␣ (MAR-1), and Mice PE-labeled Siglec-F (E50–2440) Abs, followed by streptavidin-allophyco- cyanin. Basophils were further characterized with PE-labeled anti-Thy1.2 C57BL/6J and B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice were purchased (30-H12) or PE-labeled anti-CD49b (DX5) Abs. Basophils were defined as from The Jackson Laboratory. A pair of STAT5ϩ/Ϫ breeder mice (20) was Fc␧RI␣ϩc-kitϪ, while mast cells were defined as Fc␧RI␣ϩc-kitϩ cells. Eo- provided by Dr. Lothar Hennighausen of the National Institutes of Health sinophils were marked as Siglec-Fϩ or Siglec-Fϩ and CCR3ϩ cells (22, (Bethesda, Maryland). These mice have been bred and maintained under 23). For detecting donor-derived basophils, Pacific Blue-labeled anti- pathogen-free conditions at the animal facility of National Jewish Health. A CD45.2 Ab was used (clone 104, Biolegend). Ϫ/Ϫ pair of IL-3 mice (18) was provided by Dr. Glenn Dranoff of Dana-Farber CMPs, GMPs, BaPs, BMCPs, and EoPs were FACS sorted or analyzed Cancer Institute (Boston, MA) via Dr. Shigeo Koyasu of Keio University according to the published protocols (24–27) with some of the Abs labeled Ϫ/Ϫ School of Medicine (Tokyo, Japan). IL-3 , CD45.1, and C57BL/6J mice with different fluorochromes. CMPs and GMPs were identified as LinϪIL- Ϫ/Ϫ were used at 6–14 wk of age. E14.5 STAT5 fetuses were used for gener- 7R␣ϪSca-1Ϫc- kitϩCD34ϩFc␥RII/IIIlow and Fc␥RII/IIIhigh cells, respectively. ating mice chimera. Animals were handled according to protocols approved by For , bone marrow cells were incubated with biotin-labeled lineage the National Jewish Health Animal Care and Use Committee. markers (CD3 (CT-CD3), CD4 (RM4–5), CD8 (53–6.7), B220 (RA3–6B2), injection Gr-1 (RB6–8C5), and CD19 (1D3), biotin-labeled IL-7R␣ (A7R34)), fol- lowed by streptavidin-PE-Cy5 (BD Biosciences). PE-labeled anti-Fc␥RII/III, IL-3 (10 ␮g) was mixed with either anti-IL-3 Ab (5 ␮g; MP2–8F8, BD allophycocyanin-Cy7-labeled anti-c-kit, Alexa Fluor 647-labeled anti-CD34 Pharmingen) at room temperature for 1 min based on published methods (21). (RAM34), and Pacific Blue-labeled anti-Sca-1 (D7) mAbs were also added to The cytokine and Ab mixture (in 0.2–0.3 ml of PBS) was injected into mice the staining mix. EoPs were defined as LinϪSca-1ϪCD34ϩc-kitlowIL-5R␣ϩ via the lateral tail vein. Three days after injection, mice were killed and cells cells. Staining was conducted by incubating bone marrow cells with 2.4G2 at were obtained for flow cytometric analysis. 4°C for 10 min, before adding the biotin-labeled lineage markers (followed by ␣ ⌻ FACS analysis and sorting streptavidin-PE-Cy5), followed by PE-labeled anti-IL5R ( 21.2), allophy- cocyanin-Cy7-labeled anti-c-kit, Alexa Fluor 647-labeled anti-CD34 For flow cytometric analysis of basophils, mast cells, and eosinophils, cells (RAM34), and Pacific Blue-labeled anti-Sca-1 (D7) mAbs were also added to were incubated with anti-Fc␥RII/III (2.4G2) at 4°C for 10 min and stained the staining mix. BMCPs were identified as LinϪc-kitϩ␤7highFc␥RII/IIIhigh The Journal of Immunology 2837 Downloaded from http://www.jimmunol.org/

FIGURE 2. Further characterization of Fc␧RI␣ϩ c-kitϪ cells. A, C57BL/6J FIGURE 4. Isolation of CMPs, GMPs, and BaPs. A, FACS sorting gates mice were injected with the IL-3 complex. Three days after injection, bone are shown. CMPs and GMPs were identified as LinϪIL-7R␣Ϫ Sca-1Ϫc- marrow cells were isolated and stained with biotin-labeled anti-Fc␧RI␣ Ab, kitϩCD34ϩ Fc␥RII/IIIlow and Fc␥RII/IIIhigh cells, respectively. BaPs were followed by streptavidin-conjugated APC, FITC-labeled anti-c-kit Ab, and PE- Ϫ ϩ Ϫ ϩ ϩ Ϫ identified as Lin CD34 c-kit Fc␧RI␣ cells. The numbers next to the labeled anti-CD49b or PE-labeled anti-Thy1.2 Ab. B,Fc␧RI␣ c-kit cells sorting gates indicate the percentage of GMPs, CMPs, or BaPs in total bone were isolated by FACS sorting and stained with Giemsa. Cells were analyzed ϫ marrow cells (left panel) or within the gated populations (right panel). B,

by light microscopy ( 100) or by transmission electron microscopy (a Philips by guest on September 29, 2021 Sorted progenitors (1000 cells) were cultured in complete modified CM-10 electron microscope: Philips) as reported previously (42). Iscove’s medium containing 1% methylcellulose in the presence of IL-3 for 6 days. The numbers of colonies were counted. The percentage of ba- sophils was determined by FACS analysis. cells. For staining of BMCPs, spleen cells were incubated with biotin-labeled lineage markers, followed by streptavidin-PE-Cy5 (BD Biosciences). PE-la- beled anti-Fc␥RII/III (BD Biosciences), allophycocyanin-labeled anti-c-kit (BD Biosciences), and FITC-labeled anti-␤7 (Biolegend, FIB504) mAbs were also added to the staining mix. The number of BMCPs was cal- LinϪc-kitϩ␤7highFc␥RII/IIIhigh cells. BaPs were identified as LinϪ- culated by multiplying the total number of spleen cells by the percent of CD34ϩFc␧RI␣ϩc-kitϪ cells. Staining was conducted by incubating bone mar- row cells with 2.4G2 at 4°C for 10 min, before adding the biotin-labeled lineage markers (followed by streptavidin-PE-Cy5), allophycocyanin-Cy7-la- beled anti-c-kit, Alexa Fluor 647-labeled anti-CD34, and PE-labeled anti- Fc␧RI␣ Abs. Cells were acquired by CyAn (DakoCytomation) and analyzed using FlowJo software (Tree Star). The number of CMPs and GMPs were calculated by multiplying the total number of bone marrow cells by the percent of LinϪSca-1ϪIL-7R␣Ϫc-kitϩ and the percent of CMP or GMP. The number of BaPs was calculated by multiplying the total number of bone marrow cells by the percent of LinϪCD34ϩ and the percent of BaPs. For detecting donor-derived BaPs, the procedure was the same as that described above for analyzing BaPs, except that Pacific Blue-labeled anti- CD45.2 Ab was also used. For IL-3 receptor staining on CMPs, GMPs, and BaPs, bone marrow cells were stained using the same procedures as described above, except that FITC-labeled anti-IL-3 receptor Ab was also added. Cell sorting was conducted using a MoFlo machine (DakoCytomation). CMPs, GMPs, BaPs, and basophils were sorted using the surface pheno- FIGURE 3. STAT5 is critical for basophil development and IL-3-driven Ϫ/Ϫ type defined above, except that PE-Cy5-labeled anti-Sca-1 Ab instead of basophil expansion. WT or STAT5 FL cell reconstituted mice were or Pacific Blue-labeled anti-Sca-1 Ab was used for sorting CMPs and GMPs. were not injected with the IL-3 complex. Three days after injection, the All Abs used for flow cytometry analysis and sorting were purchased from percentage of donor-derived basophils was analyzed by FACS. The num- BD Pharmingen, eBioscience, or Caltag Laboratories. bers indicate the percentage of basophils within donor-derived bone mar- row cells. The total number of donor-derived basophils was calculated by Generation of mice chimera multiplying the total number of donor-derived bone marrow cells in the Single cell suspensions were prepared from E14.5 fetal livers of STAT5ϩ/ϩ reconstituted mice by the percentage of donor-derived basophils. Three to and STAT5Ϫ/Ϫ mice. Cells (2 ϫ 106) were injected into sublethally irra- five chimera mice were used for each group. Data are representative of one diated CD45.1 congenic recipient mice (600 rad) i.v. Mice were kept under of three independent experiments with similar results. pathogen-free conditions with water-containing antibiotics. Four to six 2838 BASOPHIL DEVELOPMENT AND EXPANSION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. IL-3 greatly expands the number of BaPs and BMCPs. A, C57BL/6J mice were or were not injected with the IL-3 complex i.v. Three days after injection, CMPs, GMPs, and BaPs in the bone marrow were analyzed by FACS. BMCPs from spleen were also analyzed. CMPs and GMPs were identified as LinϪIL-7R␣ϪSca-1Ϫc-kitϩCD34ϩFc␥RII/IIIlow and Fc␥RII/IIIhigh cells, respectively. BaPs were identified as LinϪCD34ϩFc␧RI␣ϩc-kitϪ cells. BMCPs were identified as LinϪc-kitϩ␤7highFc␥RII/IIIhigh Cells. The percentages indicate the percent of cells with defined phenotypes within the LinϪsca-1ϪIL-7R␣Ϫc-kitϩ or the linϪCD34ϩ or linϪc-kitϩ cell population as indicated. B, The table summarizes the total number of CMP, GMP, and BaPs in the bone marrow or BMCPs in the spleen per mouse with or without IL-3 injection. The data are average of seven mice in the no-injection group and nine mice in the IL-3 injected group. SDs are shown. The calculation methods are described in the Materials and Methods section. C, Bone marrow cells from C57BL/6J mice that were or were not injected with the IL-3 complex were analyzed for EoPs. EoPs were defined as LinϪSca-1ϪCD34ϩc-kitlowIL- 5R␣ϩ cells. The percentages indicate the percent of cells with defined phenotypes within the gated cell population. D, WT or STAT5Ϫ/Ϫ FL cell reconstituted mice were or were not injected with the IL-3 complex. Three days after injection, BaPs in the bone marrow were analyzed by FACS. The numbers within the FACS plots indicate the percentage of BaPs within the gated cell population. The absolute number of donor-derived BaPs was calculated by multiplying the total number of donor-derived bone marrow cells in the reconstituted mice by the percentage of donor-derived BaPs. weeks later, cells were isolated from the bone marrow of the reconstituted tween two groups with and without the IL-3 complex injection. mice and were subjected to flow cytometric analysis. Interestingly, we observed an increase in the number of ␧ ␣ϩ Ϫ Statistical analysis Fc RI c-kit basophils by about 8-fold in the bone marrow (Fig. 1, A, C, and D), 8-fold in the peripheral blood, and 5-fold in the All of the error bars in this report represent the SD. The difference between two samples was analyzed with Student’s t test. spleen (Fig. 1, C and D). The increase caused by a single injection lasted for 6 days (Fig. 1B). By contrast, injection with IL-3 or with Results the sham control (anti-IL-3 Ab alone) did not increase the number IL-3 specifically and dramatically increases the number of of Fc␧RI␣ϩc-kitϪ basophils (Fig. 1A). Binding IL-3 with anti-IL-3 basophils in vivo Ab has been shown to increase the half-life of IL-3 significantly ϩ ϩ Treatments with the IL-3 complex (IL-3 plus anti-IL-3 Ab) for 8 (28). No significant changes in the number of Fc␧RI␣ c-kit mast days induced the expansion of basophils, mast cells, and eosino- cells were detected in the peritoneal cavity (Fig. 1, C and D)(p Ͼ phils (6). Because basophils might have expanded more rapidly 0.05). Injection of the IL-3 complex only caused a Ͻ2-fold in- ϩ than mast cells, we tested a short-term expansion protocol. Three crease in the number of Siglec-F eosinophils in the bone marrow, days after a single injection of the IL-3 complex, the total number peripheral blood, and the spleen (Fig. 1, C and D). Further of bone marrow, spleen, and peritoneal cells was comparable be- phenotypical and morphological analyses of the isolated The Journal of Immunology 2839 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. BaPs but not EoPs up-regulate IL-3 receptor expression independent of IL-3 or STAT5 signaling. A, Bone marrow or spleen cells were prepared from C57BL/6J mice that were or were not injected with the IL-3 complex 3 days before the assay. IL-3 receptor expression on CMPs, GMPs, BMCPs, BaPs, basophils, EoPs, and eosinophils was analyzed by FACS. Mean fluorescence intensity (MFI). The numbers indicate the percentage of the defined cells within the gated cell population. Data are representative of two independent experiments. Bone marrow cells were prepared from C57BL/6J, IL-3Ϫ/Ϫ (B), or WT or STAT5Ϫ/Ϫ chimera mice (C). IL-3 receptor expression on CMPs, GMPs, BaPs, and/or basophils was analyzed by FACS. The numbers indicate the percentage of the defined cells within the gated cell population. Data are representative of two independent experiments.

Fc␧R1␣ϩc-kit Ϫ cells revealed that expanded Fc␧R1␣ϩc-kitϪ cells complete absence of STAT5 results in perinatal death (20). We resembled those basophils reported previously (Fig. 2 and Ref. 29). generated STAT5Ϫ/Ϫ chimera mice by injecting embryonic day These results indicate that short-term IL-3 treatment specifically 14.5 fetal liver (E14.5 FL) cells into sublethally irradiated (600 induces expansion of basophils in vivo. rad) recipient mice. Mice reconstituted with STAT5Ϫ/Ϫ FL cells contained 10% of donor-derived cells, while mice reconstituted STAT5 signaling is essential in basophil development and with WT littermate control FL cells contained 80% of donor-de- IL-3-driven basophil expansion rived cells. Total number of basophils was greatly reduced in It has been shown that IL-3 activates STAT5 (30) and that STAT5 STAT5Ϫ/Ϫ chimeric mice (Fig. 3, a 93% reduction), demonstrat- signaling is required for the mast cell development, survival, and ing that basophil development was greatly impaired in the absence functions (31, 32). Although basophils and mast cells share many of STAT5 signaling. Although the percentage of STAT5Ϫ/Ϫ FL similarities, basophils do differ significantly from mast cells. Thus, cell-derived basophils increased in response to IL-3, the total it merits an investigation of the role of STAT5 in basophil devel- number of STAT5Ϫ/Ϫ FL cell-derived basophils failed to increase opment and IL-3-driven basophil expansion. In this study, we significantly (Fig. 3). These results demonstrate that STAT5 is chose to evaluate the role of STAT5 in basophils in an experimen- required for basophil development and IL-3-driven expansion of tal system where the STAT5 was completely absent. The basophils. 2840 BASOPHIL DEVELOPMENT AND EXPANSION

IL-3 greatly facilitates developmental transition from GMPs to which IL-3 dramatically increases the number of basophils. We used BaPs in the bone marrow a short-term in vivo expansion system to examine the effects of IL-3 To analyze the mechanisms by which IL-3 induces the dramatic ex- on the differentiation of myeloid progenitors into basophil lineage. In pansion of basophils in vivo, we first established the methods for our short-term expansion experimental system, we found that IL-3 analyzing CMPs, GMPs, and BaPs in the bone marrow according to induced basophil-specific expansion. This result differs from that ob- published protocols (Fig. 4A and Ref. 24–26). Using the defined sort- served by Finkleman and colleagues (6). They used an 8-day IL-3 ing gates, we were able to achieve a 52-fold enrichment for CMPs, a treatment protocol and observed that IL-3 induced dramatic expansion 36-fold enrichment for GMPs, and a 5-fold enrichment for BaPs (Fig. of mast cells and modest expansion of basophils and eosinophils in 4B). The BaPs-derived colonies were highly enriched with basophils, the spleen (6). The discrepancy might be explained by the different consisting of 87% basophils (Fig. 4B). Mature basophils failed to time points at which we chose to analyze basophil numbers. Three- generate any colonies (data not shown). Thus, the relative low colony- day treatment with the IL-3 complex resulted in a 4-fold increase in forming efficiency of the enriched basophil progenitors may reflect a the number of basophils in the spleen but did not increase the number lower proliferation capacity for such committed basophil progenitors. of mast cells or eosinophils. Consistent with these observations, we Upon treatment with the IL-3 complex, we observed that the num- found that the 3-day IL-3 treatment regimen caused a 4-fold increase ber of BaPs increased dramatically by ϳ8-fold (Fig. 5, A and B). CMP in the number of BMCPs. Compared with the degree of increase in numbers did not change significantly by the IL-3 treatment, while the the number of BaPs, the fold of increase in the number of BMCPs number of GMPs increased moderately (Fig. 5, A and B). The IL-3 was lower. We believe that this lower increase is due to a lower treatment did not significantly enhance the development of GMPs into percentage of BMCPs that express the IL-3 receptor. Mast cells are EoPs (Fig. 5C). Total number of BaPs was greatly reduced in known to live longer and undergo a slower maturation process (19, Downloaded from STAT5Ϫ/Ϫ chimeric mice (Fig. 5D, a 93% reduction). Although the 34). Eight days of treatment with the IL-3 complex might prefer- percentage of STAT5Ϫ/Ϫ FL cell-derived BaPs increased in response entially induce mast cell expansion. Our time course experiment, to IL-3, the total number of STAT5Ϫ/Ϫ FL cell-derived BaPs failed to showing that IL-3-induced basophil expansion that lasted up to increase significantly (Fig. 5D). Together, these results demonstrate seven days, was consistent with this notion. Taken together, our that IL-3 expands basophils by directing GMPs to differentiate into data demonstrate that a short-term IL-3 treatment leads to baso- BaPs in the bone marrow. phil-specific expansion in the bone marrow and in the spleen. http://www.jimmunol.org/ An additional pathway for basophil differentiation has also been Based on in vitro data, IL-3 is believed to be a hematopoietic factor reported. Akashi and colleagues (24) have described a subset of BM- that has broad effects; it has been shown to promote , CPs in the spleen, which expresses high levels of ␤7 integrin. BMCPs eosinophil, basophil, and mast cell differentiation (35). In vitro, we have been proposed as the immediate progeny of GMP in the bone have consistently observed that bone marrow progenitors gave rise to marrow and are thought to give rise to mature basophils without going 10–20% of basophils in the presence of IL-3. Yet, the in vivo effect through the intermediate BaP developmental stage. We found that the of IL-3 stimulation is remarkably specific in expanding the number of IL-3 complex treatment resulted in ϳ4-fold increase in the number of basophils. Our analysis of IL-3 receptor expression patterns on pro- BMCPs in the spleen (Fig. 5, A and B). This result demonstrates that genitors may provide part of the explanation. Acquisition of IL-3 re- by guest on September 29, 2021 the IL-3 complex treatment also expands basophils in the spleen by ceptor expression by myeloid progenitors indicates the readiness of directing BMCPs to differentiate into basophils. particular myeloid progenitors to respond to IL-3 robustly. We found that at the GMP developmental stage, 7% of GMPs began to express GMPs begin to express IL-3 receptor expression independent of low levels of IL-3 receptor. At the BaP stage, all BaPs expressed high IL-3 and STAT5 signaling levels of the IL-3 receptor. Up-regulation of the IL-3 receptor did not occur on EoPs. Acquisition of IL-3 receptor expression appears to be To understand why a short-term IL-3 treatment resulted in the expan- controlled stochastically. Mice deficient in IL-3 have normal IL-3 sion of basophils in remarkable fashion, we examined the develop- receptor expression throughout basophil development. Thus, IL-3 mental stage at which IL-3 receptor begins to be expressed. The IL- does not appear to regulate its own receptor expression. Furthermore, ␣ 3R -chain expression on CMPs, GMPs, BMCPs, BaPs, basophils, expression of IL-3 receptor on basophils did not require STAT5 EoPs, and eosinophils was measured using six-color FACS analysis. signaling. ␣ Ϫ Ϫ We found that CMPs did not express a detectable IL-3R -chain while The present study revealed that the development of STAT5 / ϳ ␣ 7% of GMPs expressed the IL-3R -chain (Fig. 6A), consistent with FL-derived basophils was greatly impaired in the chimera model previous report (33). Approximately 38% of BMCPs expressed the when sublethally irradiated recipient mice were used. We also ob- ␣ ␣ Ϫ Ϫ IL-3R -chain. Nearly all BaPs and basophils expressed the IL-3R - served that the mice reconstituted with STAT5 / FL cells contained chain at high levels. The up-regulation of IL-3 receptor expression did only 10% of donor-derived cells, while those reconstituted with WT not occur on EoPs or eosinophils (Fig. 6A). FL cells contained 80% of donor-derived cells. STAT5 is activated by Although IL-3 treatment increased the percentage of IL-3 receptor- multiple including IL-2, IL-3, IL-5, IL-7, SCF, Flt-3 ligand, expressing GMPs by 2-fold (presumably by inducing the division of GM-CSF, and erythropoietin (36) and was shown to play a critical the IL-3 receptor-expressing GMPs), IL-3 treatment did not increase role in functions (37–39); thus, we cannot the mean fluorescence intensity of IL-3 receptor expression on pro- rule out the possibility that STAT5 deficiency might have affected the Ͼ genitors at any developmental stages examined (Fig. 6A; p 0.05). earlier hematopoietic stem cell development. Indeed, STAT5 has The IL-3 receptor expression on myeloid progenitors was not affected been demonstrated to play a critical role in hematopoietic stem cell in the absence of IL-3 (Fig. 6B) or in the absence of STAT5 signaling functions (40). One of the caveats of interpreting the STAT5 data (Fig. 6C). Together, our data indicate that selective up-regulation of obtained in this study is that we cannot identify the exact develop- the IL-3 receptor on BaPs and BMCPs might explain the remarkable mental stage at which STAT5 is required. STAT5 could be required expansion of baosphils as a result of the IL-3 treatment. at the GMP to BaP stage; alternatively, it could act at a much earlier stage. Ideally, if basophil-specific Cre mice were available, we would Discussion cross the basophil-specific Cre mice with STAT5 flox/flox mice. Be- Expansion of basophils is imperative to the development of type-2 cause basophil-specific Cre mice were not available, a more definitive . In this study, we sought to examine the mechanisms by answer to which developmental stage STAT5 is required awaits the The Journal of Immunology 2841 generation of basophil-specific Cre mice. Although our results do not non-B, non-T cells in response to cross-linkage. J. 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