Role of TLR in B Cell Development: Signaling through TLR4 Promotes B Cell Maturation and Is Inhibited by TLR2 This information is current as Elize A. Hayashi, Shizuo Akira and Alberto Nobrega of September 28, 2021. J Immunol 2005; 174:6639-6647; ; doi: 10.4049/jimmunol.174.11.6639 http://www.jimmunol.org/content/174/11/6639 Downloaded from References This article cites 45 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/174/11/6639.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 28, 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Role of TLR in B Cell Development: Signaling through TLR4 Promotes B Cell Maturation and Is Inhibited by TLR21 Elize A. Hayashi,* Shizuo Akira,† and Alberto Nobrega2* The role of TLR4 in mature B cell activation is well characterized. However, little is known about TLR4 role in B cell development. Here, we analyzed the effects of TLR4 and TLR2 agonists on B cell development using an in vitro model of B cell maturation. Highly purified B220؉IgM؊ B cell precursors from normal C57BL/6 mouse were cultured for 72 h, and B cell maturation in the presence of the TLR agonists was evaluated by expression of IgM, IgD, CD23, and AA4. The addition of LPS or lipid A resulted in a marked increase in the percentage of CD23؉ B cells, while Pam3Cys had no effect alone, but inhibited the increase of CD23؉ B cell population induced by lipid A or LPS. The TLR4-induced expression of CD23 is not accompanied by full activation of the lymphocyte, as suggested by the absence of activation Ag CD69. Experiments with TLR2-knockout mice confirmed that the inhibitory effects of Pam3Cys depend on the expression of TLR2. We studied the effects of TLR-agonists on early steps of B cell Downloaded from differentiation by analyzing IL-7 responsiveness and phenotype of early B cell precursors: we found that both lipid A and Pam3Cys impaired IL-7-dependent proliferation; however, while lipid A up-regulates B220 surface marker, consistent with a more mature phenotype of the IgM؊ precursors, Pam3Cys keeps the precursors on a more immature stage. Taken together, our results suggest that TLR4 signaling favors B lymphocyte maturation, whereas TLR2 arrests/retards that process, ascribing new roles for TLRs in B cell physiology. The Journal of Immunology, 2005, 174: 6639–6647. http://www.jimmunol.org/ n the adult mouse, B lymphopoiesis occurs in the bone mar- cules, the TLR family, which includes at least 10 members in the row through the commitment and differentiation of hemopoi- mouse species (15). TLRs are present in the cell types involved in I etic stem cells (1, 2). B lineage cells are characterized by cell innate immunity and are capable of recognizing several pathogen- surface expression of CD19 and B220, and the earliest B cell pre- associated molecular patterns, playing a key role in the activation cursors express several well-described surface markers such as of monocytes and granulocytes. TLR4 recognizes the LPS present AA4, c-kit, CD43, BP-1, and heat stable antigen (3–6). At a later in cell walls of Gram-negative bacteria (16, 17), while TLR2 rec- stage characterized as pre-B-II stage (6), or fraction D (4), c-kit ognizes bacterial lipoproteins and is important for triggering innate and CD43 are down-modulated, while IL-2R is expressed on cell immune response against Gram-positive bacteria (18). The activa- surface and maintained until the immature B lymphocyte stage (6). tion of mature B lymphocytes to proliferation and Ig secretion by by guest on September 28, 2021 Along B cell development, V-D-J rearrangements lead to forma- LPS and bacterial CpG-DNA has been confirmed to be mediated tion of the clonotypic BCR and surface expression of IgM mole- ϩ ϩ by TLR4 and TLR9, respectively (17, 19). Triggering of murine B cules (7). Newly formed surface IgM (sIgM ) B lymphocytes lymphocytes by bacterial cell wall lipoproteins is very likely me- are still immature cells bearing AA4, which is progressively lost as diated by TLR2 (12, 18). B lymphocytes mature (3), and must pass through transitional The final differentiation of the maturing B cell is thought to be stages before the full maturation (8–10). During the late differen- dependent on the positive selection of the lymphocyte upon en- tiation steps, transitional B lymphocytes acquire CD23 and IgD, gagement of the BCR with self-Ags, suggesting a role for cellular which are considered as markers of B cell maturity (8–11). activation in B cell maturation (20–22). Although TLRs are well Mature B cells can be activated to proliferation and Ig secretion, characterized as functionally triggering receptors for mature B in a BCR-independent manner, by microbial products such as LPS, lipoproteins, and CpG-enriched DNA (12–14). Recently, receptors lymphocytes, little is known about their expression and function in for such microbial ligands and their molecular signaling pathways B cell development. TLR and BCR signaling can exhibit synergy have been characterized. These receptors belong to a recently de- in activation of mature B cell (23) and it is not known whether scribed family of germline-encoded and highly conserved mole- triggering through TLRs could act in B cell development as well. Here, using an in vitro model of B cell differentiation, we have studied the role of TLR4 and TLR2 agonists, lipid A/LPS, and *Department of Immunology, Institute of Microbiology, Federal University of Rio de Pam3Cys respectively, in B cell maturation. Our results show that Janeiro, Rio de Janeiro, Brazil; †Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, and Exploratory Research for Advanced Tech- signaling through TLR4 and TLR2 modulates B cell differentiation nology, Japan Science and Technology Agency, Osaka, Japan from early precursor stage. We found that, while TLR4 promotes Received for publication September 7, 2004. Accepted for publication March maturation, TLR2 engagement arrests/retards B cell maturation. 14, 2005. Simultaneous addition of TLR2 and TLR4 ligands revealed a pre- The costs of publication of this article were defrayed in part by the payment of page viously unknown antagonism between those stimuli, suggesting an charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibitory cross-talk between the respective signaling pathways in 1 This work was supported by grants from Conselho Nacional de Pesquisas, Fundac¸a˜o maturing B lymphocytes. Experiments with TLR2-knockout (KO) de Amparo á Pesquisa do Estado do Rio de Janeiro, and Fundac¸a˜o Universitária José mice showed that Pam3Cys effects depend on the expression of Bonifácio. TLR2. This study reveals a new role for TLRs in B cell maturation, 2 Address correspondence and reprint requests to Dr. Alberto Nobrega, Department of Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de raising questions about their physiological role in B lymphopoiesis Janeiro 21941-590, RJ, Brazil. E-mail address: [email protected] and repertoire selection in vivo. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6640 ROLE OF TLR IN B CELL DEVELOPMENT Materials and Methods of 10%. Harvested cells were submitted to MACS for the depletion of IgMϩ cells. The recovered B220ϩIgMϪ B cell precursors at the end of the Mice and cells ϩ fifth day represented ϳ85% of the total cells after elimination of IgM C57BL/6 and C57BL/10ScCr mice were from Fluminense Federal Uni- cells. Cells were recultured in 96-well flat-bottom plates at 2 ϫ 105 cells versity and from the Oswaldo Cruz Foundation. TLR2-KO mice were ob- per well with rIL-7 for 48 h, without or with either Pam3Cys (1 ␮g/ml), tained from Centro de Pesquisas Rene´Rachou from breeding stock orig- lipid A (1 ␮g/ml), or both at 1 ␮g/ml. Living cells were counted excluding inally provided by Dr. Akira (Osaka University). Bone marrow (BM)3 cells dead cells with trypan blue dye and processed for FACS analyses. from 2- to 3-mo-old mice were flushed out of femurs with ice-cooled OptiMEM plus 10% FCS (Invitrogen Life Technologies) by using a 10-ml Proliferation assay syringe with a 21-gauge needle. Spleen cell suspensions were obtained by gently teasing the spleen. Erythrocytes were lysed with NH4Cl lysis buffer Spleen cells from C57BL/6 or TLR2-KO mice were cultured with different and BM and spleen cells were washed with OptiMEM plus 10% FCS. concentrations of Pam3Cys or lipid A at 2 ϫ 105 cells per well in 96-well flat-bottom plates in 200 ␮l/well of OptiMEM supplemented with 10% MACS FCS in a humidified atmosphere of 7% CO2 at 37°C. After 48 h, cultures 3 ϩ were pulsed with 1 ␮Ci of [ H]thymidine and incubated for further 24 h.