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Downloaded From Rap1b Regulates B Cell Development, Homing, and T Cell-Dependent Humoral Immunity This information is current as Haiyan Chu, Aradhana Awasthi, Gilbert C. White II, of September 27, 2021. Magdalena Chrzanowska-Wodnicka and Subramaniam Malarkannan J Immunol 2008; 181:3373-3383; ; doi: 10.4049/jimmunol.181.5.3373 http://www.jimmunol.org/content/181/5/3373 Downloaded from References This article cites 63 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/181/5/3373.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 27, 2021 *average 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Rap1b Regulates B Cell Development, Homing, and T Cell-Dependent Humoral Immunity1 Haiyan Chu,* Aradhana Awasthi,* Gilbert C. White II,† Magdalena Chrzanowska-Wodnicka,‡ and Subramaniam Malarkannan2*§ Rap1 is a small GTPase that belongs to Ras superfamily. This ubiquitously expressed GTPase is a key regulator of integrin functions. Rap1 exists in two isoforms: Rap1a and Rap1b. Although Rap1 has been extensively studied, its isoform-specific functions in B cells have not been elucidated. In this study, using gene knockout mice, we show that Rap1b is the dominant isoform in B cells. Lack of Rap1b significantly reduced the absolute number of B220؉IgM؊ pro/pre-B cells and B220؉IgM؉ immature B cells in bone marrow. In vitro culture of bone marrow-derived Rap1b؊/؊ pro/pre-B cells with IL-7 showed similar proliferation levels but reduced adhesion to stromal cell line compared with wild type. Rap1b؊/؊ mice displayed reduced splenic marginal zone ؊ ؊ (MZ) B cells, and increased newly forming B cells, whereas the number of follicular B cells was normal. Functionally, Rap1b / Downloaded from mice showed reduced T-dependent but normal T-independent humoral responses. B cells from Rap1b؊/؊ mice showed reduced migration to SDF-1, CXCL13 and in vivo homing to lymph nodes. MZ B cells showed reduced sphingosine-1-phosphate-induced migration and adhesion to ICAM-1. However, absence of Rap1b did not affect splenic B cell proliferation, BCR-mediated acti- vation of Erk1/2, p38 MAPKs, and AKT. Thus, Rap1b is crucial for early B cell development, MZ B cell homeostasis and T-dependent humoral immunity. The Journal of Immunology, 2008, 181: 3373–3383. http://www.jimmunol.org/ ␣ ␤ he small GTPase, Rap1, belongs to the Ras superfamily effector of Rap1-GTPase in immune cells and associates with L 2 and cycles between an inactive GDP-bound and an active integrin (9). RIAM contains Ras-associated and pleckstrin homol- T GTP-bound conformation (1, 2). This ubiquitously ex- ogy domains and interacts with profilin and Ena/VASP proteins pressed molecular switch regulates cell proliferation, differentia- that regulate actin dynamics (10). Rap1 signaling is terminated by tion, and adhesion by distinct mechanisms through integrin acti- GTPase-activating proteins (GAPs), such as SPA-1 (signal-in- vation and MAPK cascades (3, 4). Multiple extracellular stimuli duced proliferation-associated protein 1) and RapGAPs, which can activate Rap1. This activation is mediated by various guanine convert active GTP-bound to inactive GDP-bound form (11, 12). 3 nucleotide exchange factors (GEFs), such as C3G, CalDAG-GEF, Integrins mediate cellular contacts to extracellular matrix or to their by guest on September 27, 2021 and EPAC (5–7). C3G is activated by receptor-associated protein counter receptors, thereby regulating cell motility, polarity, growth, tyrosine kinases (5). CalDAG-GEF responds to calcium and diac- and survival (13). Rap1 is expressed in multiple hematopoietic cells ylglycerol, downstream of phospholipase C signaling (8). EPAC is and one of its key functions is to regulate integrin-mediated cell ad- a cAMP-dependent GEF (7). Active Rap1-GTPase recruits multi- hesion (4). This amplifies a series of hematopoietic cellular processes ple effectors (e.g., RapL/Nore1B and RIAM) to execute their cel- including aggregation, migration, extravasation, and homing (4, 14, lular functions (9, 10). RapL, a Rap1-binding molecule, is a major 15). Mice lacking CalDAG-GEFI show impaired platelet adhesion and aggregation leading to impaired thrombus formation (16). Defi- ciency of C3G, one of the major Rap-specific GEFs, resulted in severe *Laboratory of Molecular Immunology, †Laboratory of Platelet, ‡Vascular Signaling, Blood Research Institute, and §Division of Neoplastic Diseases and Related Disor- embryonic lethality, partly due to aberrant cell adhesion and spreading ders, Department of Medicine, and Department of Microbiology and Molecular Ge- (17). Overexpression of Rap1, RapGEFs, GAPs, or their mutant pro- netics, Medical College of Wisconsin, Milwaukee, WI 53226 teins altered the activation of Rap1 and helped to uncover its signaling Received for publication November 15, 2007. Accepted for publication June 26, 2008. functions in different cell types (18–21). Rap1 can be activated through Ag or chemokine receptors (18, 22, 23). Active Rap1 plays an The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance important role in immunological synapse formation between T cells with 18 U.S.C. Section 1734 solely to indicate this fact. and APCs (22). 1 This work was supported in part by an American Cancer Society Scholar Grant Recent studies have well documented that interference in Rap1 RSG-02-172-LIB (to S.M.), by Roche Organ Transplantation Research Foundation Grant 111662730 (to S.M.), by National Institutes of Health Grants R01 functions can result in severe pathological conditions. In human, A1064826-01 (to S.M.), U19 AI062627-01 (to S.M.), NO1-HHSN26600500032C (to leukocyte adhesion deficiency syndrome III is associated with de- S.M.), and HL-45100 (to G.C.W.), and by American Heart Association Grant fective Rap1 activation and impaired stabilization of integrin SDG0235127N (to M.C.W.). H.C. is a recipient of Wisconsin Breast Cancer Show House Postdoctoral Fellowship. S.M. is a recipient of The American Society for bonds (24). Additionally, knockout mice for SPA-1 or RapL have Blood and Marrow Transplantation Young Investigator Award. provided critical insights into in vivo functions of Rap1-GTPase 2 Address correspondence and reprint requests to Dr. Subramaniam Malarkannan, (25, 26). SPA-1 is the major Rap1GAP in the hematopoietic pro- Laboratory of Molecular Immunology, Medical College of Wisconsin, 8701 Water- Ϫ/Ϫ town Plank Road, Milwaukee, WI 53226. E-mail address: [email protected] genitors in bone marrow (26). SPA-1 mice show an increase in the hematopoietic stem cells and eventually develop leukemia 3 Abbreviations used in this paper: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; MZ, marginal zone; FO, follicular; TNP, trinitrophenol; (26). SPA-1 deficiency also results in aberrant BCR repertoire KLH, keyhole limpet hemocyanin; S1P, sphingosine-1-phosphate; SDF, stromal cell- and autoimmune diseases (27). RapL deficiency causes defec- derived factor; LN, lymph node; WT, wild type. tive chemokine-triggered lymphocyte adhesion and migration Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 (25). However, many of these RapGEFs, GAPs, and effectors www.jimmunol.org 3374 Rap1b IN B CELL DEVELOPMENT AND FUNCTION are promiscuous and can associate with multiple Rap1 GTPases; Immunohistochemistry staining therefore the direct evaluation of Rap1 functions is limited in the Spleens were embedded in Tissue-Tek (Sakura Finetek), frozen with dry described knockout mouse models. ice-isopropanol mixture, and kept at Ϫ80°C until sectioning. The 10-␮m Rap1 is closely related to the Rap2 family (Rap2a/2b/2c) and cryostat sections were generated, air-dried for1hatroom temperature, and shares 60% homology (1, 2, 28). Rap1 exists in two isoforms, fixed in acetone for 10 min. The sections were blocked with 0.1% BSA/ Rap1a and Rap1b, which are encoded by different genes. Rap1 PBS containing anti-CD16/CD32 mAb for 45 min, incubated for1hat room temperature in dark with the rat anti-mouse CD3␧-PE, anti-B220- isoforms share more than 95% amino acid homology and differ Cy5, MOMA-1-FITC, anti-mouse IgM-PE, or anti-mouse IgD-FITC only in eight amino acids with five located at their C termini. Due mAbs. Sections were mounted in aqueous mounting medium and visual- to the high homology of Rap1a and Rap1b, their functions have ized on inverted Nikon Eclipse TE200 fluorescence microscope. Images been studied undistinguished in some studies (22, 23, 29). How- are at a magnification of ϫ40. ever, the different subcellular localization and tissue expression Bone marrow transplantation patterns of these two isoforms have been described and indicate Bone marrow cells from tibias and femurs of wild-type (WT) and Rap1Ϫ/Ϫ their distinct functions (30–35). Studies have indicated the role of mice were prepared and resuspended in 2% FBS/RPMI 1640 (Invitrogen Rap1 isoforms in neuron, T cells, and platelets (19, 34, 35). Rap1a Life Technologies). A total of 5 ϫ 106 bone marrow cells (200 ␮l) were has been shown to positively regulate T cell functions in both injected i.v. to reconstitute 6- to 8-wk-old, sublethally irradiated (300 rad) Ϫ Ϫ Ϫ Ϫ transgenic and knockout mouse models (19, 36).
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