Developmental Checkpoints of the Basophil Mast Cell Lineages in Adult Murine Hematopoiesis
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Developmental checkpoints of the basophil͞mast cell lineages in adult murine hematopoiesis Yojiro Arinobu*†, Hiromi Iwasaki*†‡, Michael F. Gurish§, Shin-ichi Mizuno*, Hirokazu Shigematsu*, Hidetoshi Ozawa*, Daniel G. Tenen¶, K. Frank Austen§ʈ, and Koichi Akashi*‡** *Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, and ¶Harvard Institute of Medicine, Harvard Medical School, Boston, MA 02115; ‡Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka 812-8582, Japan; and §Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115 Contributed by K. Frank Austen, October 21, 2005 Basophils and mast cells, which are selectively endowed with the populations that have committed to the basophil and͞or mast high-affinity IgE receptor and mediate a range of adaptive and cell lineages. Our data show that basophils and mast cells share innate immune responses, have an unknown developmental rela- the common progenitor stage, where CCAAT͞enhancer- tionship. Here, by evaluating the expression of the 7 integrin, a binding protein ␣ (C͞EBP␣), the transcription factor essential molecule that is required for selective homing of mast cell pro- for granulocyte development (10), plays a critical role in their genitors (MCPs) to the periphery, we identified bipotent progen- fate decision. itors that are capable of differentiating into either cell type in the mouse spleen. These basophil͞mast cell progenitors (BMCPs) gave Materials and Methods rise to basophils and mast cells at the single-cell level and recon- Mice. C57BL͞6J, B6.SJL-Ptprca Pep3b͞BoyJ, and WBB6F1͞J- stituted both mucosal and connective tissue mast cells. We also KitW͞KitWϪv mice were purchased from The Jackson Labora- identified the basophil progenitor (BaP) and the MCP in the bone tory. C͞EBP␣F/F mice were developed as described in ref. 11. marrow and the gastrointestinal mucosa, respectively. We further They were bred and maintained in the Research Animal Facility show that the granulocyte-related transcription factor CCAAT͞ at Dana–Farber Cancer Institute in accordance with institutional enhancer-binding protein ␣ (C͞EBP␣) plays a primary role in the guidelines. fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common Antibodies, Cell Staining, and Sorting. Myeloid and lymphoid developmental stage at which their fate decision might be con- progenitors were purified as described in refs. 12 and 13. For trolled principally by C͞EBP␣. the staining of lineage antigens, phycoerythrin (PE)-Cy5- conjugated rat antibodies specific for CD3 (CT-CD3), CD4 integrin ͉ progenitor ͉ CCAAT͞enhancer-binding protein (RM4–5), CD8 (5H10), B220 (6B2), Gr-1 (8C5), and CD19 (6D5) (Caltag, Burlingame, CA) were used. For basophil͞mast asophils and mast cells exhibit distinct differentiation pat- cell progenitor (BMCP) sorting, spleen cells were stained with Bterns despite their multiple shared phenotypic characteris- FITC-conjugated anti-T1͞ST2, PE-conjugated anti-7 inte- tics. Both cell types express the ␣␥2 form of the high-affinity grin, allophycocyanin (APC)-conjugated anti-c-Kit (2B8), and receptor for IgE (FcRI). Crosslinking of the high-affinity biotinylated anti-Fc␥RII͞III (2.4G2) (BD Pharmingen), fol- receptor for IgE (FcRI) on tissue mast cells triggers immediate lowed by avidin-APC͞Cy7 (Caltag). For basophil progenitor hypersensitivity with local symptoms, and sequential recruit- (BaP) or mast cell progenitor (MCP) sorting, bone marrow or ment of basophils to these tissue sites expands the spectrum of intestinal cells were stained with FITC-conjugated anti-CD34 this inflammatory process (1). By secreting diverse inflamma- (RAM34) (BD Pharmingen), PE-conjugated anti-FcRI␣ tory protein mediators, they provide innate defense against (MAR-1), APC-conjugated anti-c-Kit (2B8), and͞or APC͞ parasitic or bacterial infections (2–4) and also play a critical role Cy7-conjugated anti-CD45.2 (104). Cells were double-sorted in the development of a variety of autoimmune disorders (5). A by using a highly modified double-laser (488-nm͞350-nm marked increase in activated intraepithelial and bronchial Enterprise II and 647-nm Spectrum) FACS (Moflo-MLS, smooth muscle mast cells characterizes bronchial asthma, and a Cytomation, Fort Collins, CO). Cells were doubly sorted and prominent basophil infiltration is noted with fatal diseases. In were deposited into 60-well Terasaki plates by using an cutaneous allergic diseases, mast cell activation is the basis of automatic cell deposition unit system (14). itching hives, whereas basophils are abundant in contact hyper- sensitivity eruptions (6). However, basophils exist as mature cells Infection with Trichinella spiralis and Ovalbumin (OVA) Sensitization. in the circulation, whereas mast cells circulate as progenitors and The method for T. spiralis infection has been reported previously complete their maturation after migration into peripheral tissues (14). For the OVA sensitization, mice were immunized i.p. twice such as the skin, heart, lung, and the gastrointestinal mucosa. with OVA (10 g, Sigma-Aldrich) adsorbed to 1 mg of alum Their origin and developmental relationships remain as one of (Pierce) 7 days apart. Ten days after the second immunization, the major issues in the biology of hematopoiesis (7) and in the pathobiology of allergic diseases. Conflict of interest statement: No conflicts declared. In adult mice, the intestine is the main peripheral tissue IMMUNOLOGY harboring mast cell colony-forming activity (8). We have shown Abbreviations: MCP, mast cell progenitor; BMCP, basophil͞mast cell progenitor; BaP,   basophil progenitor; C͞EBP␣, CCAAT͞enhancer-binding protein ␣; OVA, ovalbumin; CMP, that the 7-integrin ( 7) is an essential molecule for tissue- common myeloid progenitor; MEP, megakaryocyte͞erythrocyte progenitor; GMP, granu- specific homing of putative precursors for intestinal mast cells (8) locyte͞monocyte progenitor; EoP, eosinophil lineage-committed progenitor. ϩ and that mast cell potential resides mostly in the c-Kit fraction, †Y.A. and H.I. contributed equally to this work. at least in the murine bone marrow (9). To identify a candidate ʈTo whom correspondence may be addressed. E-mail: [email protected]. population that seeds putative intestinal progenitors for mast **To whom correspondence may be addressed at: Department of Cancer Immunology and cells, we evaluated the expression level of 7 in hematopoietic AIDS, Dana–Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail: progenitor populations in the bone marrow, the spleen, and the koichi[email protected]. intestine. This approach enabled us to isolate a set of progenitor © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0509148102 PNAS ͉ December 13, 2005 ͉ vol. 102 ͉ no. 50 ͉ 18105–18110 Downloaded by guest on September 27, 2021 Fig. 1. Identification of BMCPs in C57BL͞6 murine hematopoiesis. (A) The expression of 7 and T1͞ST2 in the bone marrow myeloerythroid progenitors and peritoneal mast cells (MC). A fraction of myeloerythroid progenitors expressed a low level of 7. (B) The surface phenotype of 7hi BMCPs in the spleen. (C)(Left) Morphology of purified LinϪc-KitϩFc␥RII͞IIIhi7hi BMCPs. These cells gave rise exclusively to mast cells and basophils (Right) in the presence of a panel of myeloerythroid cytokines (see Materials and Methods). May–Giemsa staining was used. (Magnification: ϫ1,000.) (D) A basophil͞mast cell colony developed from a single BMCP in vitro. May–Giemsa staining was used. (Magnification: ϫ1,000.) (E) Clonogenic analysis of 7lo bone marrow myeloerythroid progenitors and 7hi BMCP. Note that a fraction of the spleen BMCP and the bone marrow 7lo GMP gave rise to both basophils and mast cells at the single-cell level. (F) FACS analysis of day-7 progeny of the bone marrow 7Ϫ and 7lo GMP. Both populations gave rise to FcRI␣ϩCD11cϩ basophils and FcRI␣ϩCD11cϪ mast cells expressing c-Kit. the mice were exposed to an aerosol of 1% OVA in PBS for 30 C͞EBP␣F/F mice and infected with MIG-Cre retroviruses, as min by using a PARI nebulizer (PARI Respiratory Equipment, reported in ref. 11. Midlothian, VA). Mice were analyzed 1 day after the fifth daily exposure. Results A Population Expressing a High Level of 7 Resides in the Spleen but Cell Cultures. Cells were cultured in Iscove’s modified Dulbecco’s Not in the Bone Marrow. We evaluated the expression of 7 within medium supplemented with 20% FCS. Cultures were performed stem͞progenitor populations that did not express a panel of in the presence of cytokine cocktails containing murine stem lineage antigens (Lin) but expressed c-Kit. In the bone marrow, cell factor (20 ng͞ml), IL-3 (20 ng͞ml), IL-5 (50 ng͞ml), IL-6 LinϪSca-1ϩc-Kitϩ hematopoietic stem cells (HSCs) did not (20 ng͞ml), IL-7 (20 ng͞ml), IL-9 (50 ng͞ml), IL-11 (10 express 7, but a low level of 7 expression was found in a ng͞ml), granulocyte–macrophage colony-stimulating factor fraction of progenitor populations, such as common myeloid (10 ng͞ml), erythropoietin (2 units͞ml), and thrombopoietin progenitors (CMPs), megakaryocyte͞erythrocyte progenitors (10 ng͞ml) (R & D Systems). Cells were cultured at 37°C in a (MEPs), and granulocyte͞monocyte progenitors (GMPs) (13)  humidified chamber under 5% CO2. (Fig. 1A). In contrast, cells expressing 7 at high levels were found in the LinϪc-Kitϩ fraction of the spleen. These 7hi cells In Vivo Reconstitution Assay. Four hundred splenic BMCPs or existed only in a fraction expressing Fc␥RII͞III (Fig. 1B), at a intestinal MCPs were purified from C57BL͞6 (Ly5.1) mice and level comparable to that in GMPs (13), neutrophils, and mono- injected i.p. into nonirradiated W͞Wv mice. Eight weeks after cytes (not shown). 7hi spleen cells expressed CD34, a marker for the injection, peritoneal lavage cells were analyzed. Three hematopoietic progenitors that can generate mast cells and thousand purified BMCPs (Ly5.1) were also injected intrave- basophils (15) and for mature mast cells (16). They also ex- nously into W͞Wv mice after 2.5-Gy irradiation.