Eosinophils Regulate Peripheral B Cell Numbers in Both Mice and Humans Tina W. Wong, Alfred D. Doyle, James J. Lee and Diane F. Jelinek This information is current as of September 26, 2021. J Immunol published online 10 March 2014 http://www.jimmunol.org/content/early/2014/03/07/jimmun ol.1302241 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/03/07/jimmunol.130224 Material 1.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 10, 2014, doi:10.4049/jimmunol.1302241 The Journal of Immunology Eosinophils Regulate Peripheral B Cell Numbers in Both Mice and Humans Tina W. Wong,* Alfred D. Doyle,† James J. Lee,† and Diane F. Jelinek*,‡ The view of eosinophils (Eos) as solely effector cells involved in host parasite defense and in the pathophysiology of allergic diseases has been challenged in recent years. In fact, there is a growing realization that these cells interact with other components of innate and adaptive immunity. For example, mouse Eos were recently demonstrated to promote plasma cell retention in the bone marrow. However, it remains unknown whether Eos influence the biology of normal B lymphocytes. In this study, we specifically assessed the effect of Eos on B cell survival, proliferation, and Ig secretion. Our data first revealed that the genetic deletion of Eos from NJ1638 IL-5 transgenic hypereosinophilic mice (previously shown to display profound B cell expansion) resulted in the near abolishment of the B cell lymphocytosis. In vitro studies using human tissues demonstrated Eos’ proximity to B cell follicles and their ability to promote B cell survival, proliferation, and Ig secretion via a contact-independent mechanism. Additionally, this ability of Eos to Downloaded from enhance B cell responsiveness was observed in both T-independent and T-dependent B cell activation and appears to be inde- pendent of the activation state of Eos. Finally, a retrospective clinical study of hypereosinophilic patients revealed a direct correlation between peripheral blood eosinophil levels and B cell numbers. Taken together, our study identifies a novel role for Eos in the regulation of humoral immunity via their impact on B cell homeostasis and proliferation upon activation. The Journal of Immunology, 2014, 192: 000–000. http://www.jimmunol.org/ osinophils (Eos) are innate immune cells that originate Traditionally, Eos have been best known for their anti-helminthic from pluripotent progenitor cells in the bone marrow effector functions in host defense against infections as well as their E (BM). Developmentally, their survival, expansion, and involvement in the pathophysiology of airway dysfunction and terminal differentiation are driven by the cytokines IL-3, IL-5, and tissue remodeling in asthma (10). However, in recent years, these GM-CSF (1). Upon maturation, these cells exit the BM, circulate cells were demonstrated to be much more multifunctional than briefly in the peripheral blood (PB), and then home to and reside originally understood. With respect to immunoregulation, for ex- in tissues that include the gut, uterus, thymus, BM, and mammary ample, Eos mediate aluminum hydroxide–induced B cell priming, gland (2). serve as APCs for T cells, and release cytokines that influence by guest on September 26, 2021 Of the three aforementioned cytokines, IL-5 is the most specific T cell differentiation (i.e., Th1 versus Th2) (11–13). Eos also se- for eosinophilopoiesis (3, 4). To this extent, the IL-5 gene has been crete chemoattractants for the recruitment of T cells, macrophages, used as a genetic tool to create mouse models that have altered and dendritic cells to tissue sites (10, 14–17). numbers of Eos for the study of these cells. IL-5 overexpression Recently, a role for Eos in the homeostasis of long-lived plasma from various promoters uniformly results in Eos expansion (5–7). cells (PCs) within mouse BM was described (18, 19). Specifically, Conversely, genetic deletion of IL-5 or its receptor, IL-5Ra, causes PC retention in the marrow was significantly diminished in the reduced Eos numbers (8, 9). absence of Eos. We subsequently demonstrated that in the human PC malignancy, multiple myeloma, Eos within the tumor mi- croenvironment can induce proliferation of the malignant cells, *Department of Immunology, Mayo Clinic, Rochester, MN 55905; †Division of thereby contributing to disease pathology (20). Based on these Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo findings, we questioned whether the proliferation-inducing effect Clinic Arizona, Scottsdale, AZ 85259; and ‡Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905 of Eos on myeloma cells is a phenomenon that is restricted to this Received for publication August 22, 2013. Accepted for publication February 7, malignancy, or perhaps it is applicable to normal B-lineage cells 2014. as well. Indeed, via both transgenic mouse models and in vitro This work was supported by the Mayo Foundation, National Institutes of Health study of human Eos, we provide strong evidence that eosinophils Grants HL058723 and HL065228, and National Institutes of Health Pre-doctoral do in fact impact the biology of normal B cells. Significantly, this Immunology Training Grant T32 AI07425. This work was also supported by grants from the Mayo Clinic Medical Scientist Training Program, the Mayo Graduate conclusion was supported by our retrospective evaluation of clinical School, and the Mayo Medical School (to T.W.W.), as well as by a grant from the records from patients with idiopathic hypereosinophilic syndrome Mayo Graduate School and by a Sidney Luckman Family Pre-doctoral Fellowship (HES), which demonstrated a direct correlation between Eos levels (to A.D.D.). and circulating B cell numbers. Address correspondence and reprint requests to Dr. Diane F. Jelinek, Department of Immunology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905. E-mail address: [email protected] Materials and Methods The online version of this article contains supplemental material. Mice Abbreviations used in this article: BM, bone marrow; CBC, complete blood count; Mouse strains employed in these studies include C57BL/6J wild-type (WT) Eos, eosinophil; EPX, eosinophil peroxidase; HES, hypereosinophilic syndrome; IRB, Institutional Review Board; MBP, major basic protein; PB, peripheral blood; controls (The Jackson Laboratory, Bar Harbor, ME), eosinophil-deficient PHIL mice (21), NJ1638 IL-5 transgenic mice (6), and NJ1638.PHIL PC, plasma cell; RPMIEos, RPMI1640 1 20% FCS 1 1 ng/ml IL-5; TBNK, T cell/B cell/NK cell; WT, wild-type. mice generated by crossing NJ1638 and PHIL. All mice were analyzed between 3 and 5 mo of age. All mice used in these studies have been Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 backcrossed to C57BL/6J for .20 generations and were maintained in the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302241 2 REGULATION OF B CELL HOMEOSTASIS BY EOSINOPHILS Mayo Clinic Arizona Small Animal Facility (a specific pathogen-free fa- mean cell counts of each cohort 6 SEM. A Spearman correlation coeffi- cility). Studies involving animals were performed in accordance with cient between PB Eos and B cell counts was calculated for data pooled National Institutes of Health and Mayo Clinic Institutional Animal Care from all cohorts. and Use Committee guidelines. Cell isolation and culture Flow cytometry analysis of mouse PB, marrow, and spleen B cells were isolated from human PB, tonsils, and spleens via B cell en- Single-cell suspensions from PB, BM (flushed from a single femur), and richment kits (StemCell Technologies, Vancouver, BC, Canada) and an homogenized spleen were treated with Pharm Lyse (BD Biosciences, San automated RoboSep cell separator (StemCell Technologies). B cell subsets Jose, CA) to deplete erythrocytes. Cell suspensions were then stained with were isolated using naive and memory B cell enrichment kits (StemCell various Abs following blockade of FcR with 5 mg/ml Fc blocker (CD16/32; Technologies) or by sorting on a FACSAria cell sorter (BD Biosciences) BD Biosciences). Abs used to identify Eos include: anti–CCR3-allophycocyanin after pan–B cell enrichment and staining with anti-CD27 mAb (BD (83101; R&D Systems, Minneapolis, MN) and anti–Siglec-F-PE (E50- Biosciences). Eos were isolated from human PB and BM as previously 2440; BD Biosciences). Abs for identifying B cells include: anti–B220- described (24). Unless otherwise indicated, all cells were cultured in allophycocyanin (RA3-6B2; eBioscience, San Diego, CA), anti–CD19- RPMIEos (RPMI1640 1 20% FCS 1 1 ng/ml IL-5) at 37˚C with 5% FITC (1D3; BD Biosciences), anti–CD19-PE-Cy7 (eBio1D3; eBioscience), CO2 (24). anti–IgM-FITC (RMM-1; BioLegend, San Diego, CA), anti–CD5-PE (53- 7.3; eBioscience), anti–CD11b-allophycocyanin (M1/70; eBioscience), Histology and immunofluorescence microscopy anti–CD273-PE (TY25; BioLegend), anti–CD80-allophycocyanin (16-10A1; Human tonsil and spleen samples were processed for histological analysis as eBioscience), and anti–CD73-V450 (TY/23; BD Horizon). B1a cells previously described (20). Slides were stained either with H&E or with are defined as CD19+IgM+CD11b+CD5+ cells.