In Vitro and In Vivo Activation Induces BAFF and APRIL Expression in B Cells Van Trung Chu, Philipp Enghard, Gabriela Riemekasten and Claudia Berek This information is current as of September 27, 2021. J Immunol 2007; 179:5947-5957; ; doi: 10.4049/jimmunol.179.9.5947 http://www.jimmunol.org/content/179/9/5947 Downloaded from References This article cites 40 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/179/9/5947.full#ref-list-1 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 27, 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology In Vitro and In Vivo Activation Induces BAFF and APRIL Expression in B Cells1 Van Trung Chu,* Philipp Enghard,† Gabriela Riemekasten,† and Claudia Berek2* B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88- dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up- regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmem- brane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture Downloaded from supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo ؋ activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB NZW)F1 mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of ؋ BAFF. In (NZB NZW)F1 mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease. The Journal of Immunology, 2007, 179: 5947–5957. http://www.jimmunol.org/ wo closely related cytokines of the TNF superfamily, B BAFF is produced by a number of different cell types. Expres- cell-activating factor (BAFF,3 also termed BLyS, sion of BAFF by follicular dendritic cells (FDC) may be essential T TALL-1, zTNF-4, THANK, and TNF13B) and a prolif- for B cell homeostasis (12–14). However, the main sources of eration-inducing ligand (APRIL), are central players in B cell de- BAFF are monocytes, macrophages, dendritic cells, and neutro- velopment and homeostasis. BAFF-deficient mice have an almost phils, although subpopulations of B and T cells have also been complete loss of follicular (FO) and MZ B cells, although there is shown to express it (15–21). BAFF is found to be membrane as- normal development of early B cells in the bone marrow (1–4). sociated and its expression is enhanced by cytokines, such as Normal numbers of newly formed immature B cells leave the bone IFN-␣, IFN-␥, and IL-10 or growth factors (17, 19, 20, 22). marrow and develop into the transitional T1 stage. In the absence Much less is known about APRIL which has no essential func- by guest on September 27, 2021 of BAFF. B cells do not progress past the transitional T2 stage, tion in normal B cell development and plays only a minor role in resulting in impaired humoral immune responses (5). However, B cell homeostasis (23). In immune responses APRIL acts as a when BAFF is overexpressed, for example in BAFF- transgenic costimulator for B and T cell proliferation and supports class mice, self-reactive B cells may be rescued from peripheral deletion switch (2, 4, 22). (6, 7). As a consequence, BAFF-transgenic mice develop a lupus- BAFF binds to three separate receptors, the BAFF receptor like autoimmune disease (8–11). From these data, it is apparent (BAFF-R, BR3), the transmembrane activator and calcium mod- that the level of BAFF has to be tightly regulated to ensure B cell ulator ligand interactor (TACI), and the B cell maturation Ag (24– survival on the one hand and to prevent autoimmunity on the other. 27). APRIL binds only to TACI and B cell maturation Ag. All three receptors are expressed on B cells, although their expression level changes with B cell maturation. The analysis of human B cell tumor lines, such as B cell chronic *Deutsches Rheuma ForschungsZentrum; and †Department of Rheumatology and Clinical Immunology, Charite University Hospital, Berlin, Germany lymphatic leukemia (B-CLL), multiple myeloma, and Hodgkin’s lymphoma cells, suggested that BAFF and APRIL support tumor Received for publication April 6, 2007. Accepted for publication August 21, 2007. survival by an autocrine pathway (21, 28–30). There is also evi- 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 dence that normal nonmalignant human B cells up-regulate BAFF with 18 U.S.C. Section 1734 solely to indicate this fact. and APRIL upon activation (21, 28). However, it is thought that 1 This work was supported by a grant from the Government of Vietnam and the murine B cells do not express BAFF or APRIL (3), although an DAAD (to V.T.C.), the Bundesministerium fu¨r Bildung und Forschung Grant NGFN2 analysis of early murine B cell development suggested that B cell and the SFB 650. The DRFZ is supported by the Berlin Senate of Research and Education. survival in the bone marrow may be supported by an autocrine 2 Address correspondence and reprint requests to Dr. Claudia Berek, Deutsches pathway (31). Rheuma ForschungsZentrum, Chariteplatz 1, Berlin, Germany. E-mail address: In this study, we show a detailed analysis of BAFF and APRIL [email protected] expression in different B cell subsets isolated from the bone mar- 3 Abbreviations used in this paper: BAFF, B cell-activating factor; APRIL, a prolif- row, spleen, and peritoneal cavity. In vitro cultures demonstrate eration-inducing ligand; MZ, marginal zone; FO, follicular; GC, germinal center; FDC, follicular dendritic cells; CMK, chloromethylketone; phOx, 2-phenyl-ox- that upon stimulation with LPS or CpG-oligodeoxynucleotides azolone; PNA, peanut agglutinin; MFI, mean fluorescence intensity; TACI, trans- (CpG-ODN), splenic B2 cells as well as peritoneal B1 cells up- membrane activator and calcium modulator ligand interactor; ODN, oligodeoxynucle- regulate BAFF and APRIL expression. MyD88-deficient mice otide; BAFF-R, BAFF receptor. demonstrated that BAFF expression in B cells is regulated by the Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 TLR signaling pathway. Immunization with the T-dependent Ag www.jimmunol.org 5948 ACTIVATED B CELLS EXPRESS BAFF AND APRIL FIGURE 1. Expression of BAFF and APRIL in B cells. A, To determine the level of BAFF and APRIL expression, B cell sub- sets were sorted by FACS. Gating for the isolation of pro-, pre-, immature (Imm) bone marrow; T1, T2, MZ, and FO (spleen); B1 and B2 (peritoneal cavity) B cell subsets is indicated. Representative dot blots show the purity of pro-, pre-, and immature B cells (Post-sort). B, B cell subsets isolated from the bone marrow and the peritoneal cavity (PC) express APRIL cDNA. Amplification was done for 40 cycles. C, The relative level of BAFF mRNA was determined by real- time PCR. Three mice per group were ana- lyzed. Results of three independent experi- ments are shown as mean values plus SD. D, Expression of BAFF, APRIL, and CD11c mRNA in defined numbers of MZϪ,FOϪ, ϩ peritoneal B1 B, and CD11c cells. Number Downloaded from of sorted cells is indicated. For each cell population, one-fifth of the cDNA was used for PCR amplification of BAFF, APRIL, CD11c, and ␤-actin. E, Splenocytes and peritoneal cells from BALB/c mice were tri- ple stained for CD5, B220, and BAFF (Buffy-2). Dot blots show the gating for http://www.jimmunol.org/ splenic newly formed B220low (NF-B), ma- ture (M-B), and B1 B cells and for peritoneal (PC) B1a, B1b and B2 subsets. Histograms in the upper row show staining of splenic B cells with anti-BAFF-R (left panel) and with BAFF-specific Abs (middle and right pan- els). Surface expression of BAFF before (nor- mal line) and after incubation of B cells with soluble BAFF (heavy line) is shown. Isotype by guest on September 27, 2021 control (shaded area) is included. The different B cell subsets (spleen and peritoneal cavity) were stained with the mAb Buffy-2 only. Data are representative of three (spleen) to five (peritoneum) experiments. 2-phenyl-oxazolone (phOx) induced BAFF expression in germinal with polyclonal rabbit anti-BAFF (Sigma-Aldrich) or anti-APRIL (Stress- ϫ gen) Ab, respectively.