Aminoacyl tRNA Synthetase−Interacting Multifunctional 1 Acts as a Novel B Cell−Activating Factor In Vitro and In Vivo

This information is current as Myun Soo Kim and Tae Sung Kim of September 28, 2021. J Immunol 2015; 194:4729-4736; Prepublished online 13 April 2015; doi: 10.4049/jimmunol.1401352 http://www.jimmunol.org/content/194/10/4729 Downloaded from

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Aminoacyl tRNA Synthetase–Interacting Multifunctional Protein 1 Acts as a Novel B Cell–Activating Factor In Vitro and In Vivo

Myun Soo Kim and Tae Sung Kim

Endogenous B cell–activating factors play pivotal roles in defense mechanisms by regulating B cell responses. We previously reported that aminoacyl tRNA synthetase–interacting multifunctional protein 1 (AIMP1) functions as a novel proinflammatory that activates macrophages and dendritic cells. However, roles of AIMP1 in B cell responses have not been studied. In this study, we investigated the effects of AIMP1 on B cell responses and their underlying mechanisms. AIMP1 induced the expression of surface activation markers on murine B cells and the proliferation of B cells. Additionally, AIMP1 increased the expression of activation-induced deaminase and class switch recombination in B cells. AIMP1 also had synergistic effects on B cell activation Downloaded from when combined with CD40 stimulus. Intracellular signaling experiments showed that AIMP1 activated B cells through a protein kinase C/NF-kB signaling pathway. Importantly, i.v. injection of AIMP1 into mice increased the expression of CD69 on splenic B cells and significantly enhanced Ag-specific Ab production. Taken together, our results show that AIMP1 acts as a novel B cell– activating factor. AIMP1-mediated B cell activation and the involvement of AIMP1 in diseases will provide additional information for therapeutic strategies. The Journal of Immunology, 2015, 194: 4729–4736. http://www.jimmunol.org/ ntibody production requires B cell activation, and B cells these factors, thereby activating B cells and inducing the matu- can be activated through both –dependent and ration and survival of B cells (6). Importantly, BAFF-deficient A T cell–independent manners. Although somatic hyper- mice showed abnormal B cell development and reduced serum mutation and clonal expansion are thought to occur with the aid of levels of IgM and IgG (7), suggesting that endogenous B cell– T cells within germinal centers (1), T cell–independent activation activating factors affect total B cell responses. APRIL-deficient also plays pivotal roles in proper immune responses. For example, mice also have revealed impaired Ab production (8). Therefore, important roles have been suggested for IgM Abs generated by endogenous B cell–activating factors are regarded as important T-independent B cell responses to microbial infections (2). Addi- modulators of total B cell responses. However, the mechanisms tionally, intestinal levels of IgA are normal in CD40-deficient underlying B cell activation via endogenous factors are still not by guest on September 28, 2021 mice (3), suggesting that T cell–independent class switching well understood, and there are novel B cell–activating factors yet occurs spontaneously in the intestine. As a defense mechanism, to be identified. B cells can be activated by numerous exogenous molecules, in- Aminoacyl tRNA synthetase–interacting multifunctional protein cluding TLR ligands and specific Ags for BCR through a T- 1 (AIMP1) has been identified as an aminoacyl tRNA synthetase independent manner (4). In addition to these exogenous factors, complex component and acts to stabilize the complex (9). AIMP1 various endogenous factors have been shown to induce T- is also secreted and has been shown to have various activities, independent B cell responses. For example, BAFF and a prolifer- including effects on wound healing (10) and glucagon-like func- ation-inducing ligand (APRIL) have been reported to induce IgA tions that control blood glucose levels (11). Furthermore, we production in the intestine in T cell–independent manners (5). demonstrated that AIMP1 functions as a proinflammatory cyto- Various non–T cells, including macrophages, monocytes, dendritic kine that induces macrophage activation, DC maturation, and IL- cells (DCs), and epithelial cells, have been reported to secrete 12 production, indicating that AIMP1 is a novel immune modu- lator (12, 13). However, it is unclear whether AIMP1 affects other APCs, such as B cells. Division of Life Sciences, College of Life Sciences and Biotechnology, Korea To our knowledge, this study is the first to show that AIMP1 is University, Seoul 136-701, Republic of Korea a novel B cell–activating factor. AIMP1 induced the expression Received for publication May 27, 2014. Accepted for publication March 14, 2015. of surface activation markers on B cells, such as CD19, CD40, This work was supported by National Research Foundation of Korea Grant NRF- CD69, CD80, CD86, and MHC class II (MHC II). The prolifer- 2014R1A2A2A01005031 funded by the Korea government (Ministry of Science, ICT and Future Planning). ation and Ag uptake ability of B cells were also dramatically in- Address correspondence and reprint requests to Prof. Tae Sung Kim, Division of Life creased after AIMP1 treatment. AIMP1 and CD40 signaling Sciences, College of Life Sciences and Biotechnology, Korea University, 5-ga, synergistically enhanced B cell activation, whereas AIMP1 showed Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea. E-mail address: only moderate effects on B cell activation via BCR signaling. [email protected] AIMP1 also increased the expression of activation-induced de- The online version of this article contains supplemental material. aminase (AID) and class switch recombination (CSR) in mesen- Abbreviations used in this article: AID, activation-induced deaminase; AIMP1, ami- noacyl tRNA synthetase–interacting multifunctional protein 1; APRIL, a prolifera- teric lymph node (MLN) B cells. Experiments using signaling tion-inducing ligand; CSR, class switch recombination; DC, dendritic cell; MHC II, inhibitors revealed that AIMP1 induced the protein kinase C MHC class II; MLN, mesenteric lymph node; PKC, protein kinase C; PMB, poly- (PKC)–mediated NF-kB signaling pathway. Additionally, i.v. in- myxin B; RT, room temperature. jection of AIMP1 into mice increased CD69 expression on splenic Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 B cells and enhanced OVA-specific IgG production. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401352 4730 AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR

Materials and Methods 2-ME (50 mM, Sigma-Aldrich, St. Louis, MO), HEPES (10 mM, Welgene), Mice and cells sodium pyruvate (1 mM, Invitrogen, Carlsbad, CA), penicillin (100 U/ml, Invitrogen), and streptomycin (0.1 mg/ml, Invitrogen). Recombinant AIMP1 Seven- to 10-wk-old C57BL/6 mice (OrientBio, Seongnam, Korea) were was prepared as described previously (13). His-tagged AIMP1 was con- used for this study. The animals were housed in a specific pathogen-free structed. AIMP1 protein was produced and purified by Young In Frontier facility and the experiments were performed according to the guidelines (Seoul, Korea). The level of endotoxin in each lot was determined using of Korea University Institutional Animal Care and Use Committee. Ramos a Limulus amebocyte lysate QCL-1000 (Lonza, Walkersville, MD). Lots B cell line (KCLB 21596) was obtained from Korean Cell Line Bank (Seoul, containing ,0.1 endotoxin unit/1 mg protein were used for this study. Korea). CD19+ primary B cells were isolated using CD19 microbeads and a MACS system (Miltenyi Biotec, Auburn, CA). Antibodies Media and PE-conjugated anti-B220 (RA3-6B2), PE-conjugated anti-CD40 (3/23), PE-conjugated anti-CD80 (16-10A1), and PE-conjugated anti-CD86 Animal cells and Ramos cells were cultured in RPMI 1640 medium (Thermo (GL1) were from BD Biosciences (San Diego, CA). Anti-mouse CD40 Scientific, Rockford, IL) supported with FBS (10%, Welgene, Daegu, Korea), (1C10), allophycocyanin-conjugated anti-CD19 (MB19-1), PE-conjugated Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. AIMP1 increased the expression of surface activation markers on MLN B cells. CD19+ B cells were isolated from MLNs as described in Materials and Methods.(A) MLN B cells were cultured in the presence of AIMP1 at the indicated concentrations for 3 d, and then the surface MHC IIhi CD86hi population was evaluated by flow cytometric analysis. Results from (A) are summarized in (B) as mean 6 SD (n = 3). *p , 0.05. (C) MLN B cells were treated with AIMP1 (100 nM) for 3 d, and then the surface expression of CD19, CD23, CD40, and CD80 was compared with that on cells treated with medium alone (negative control). (D) MLN B cells were treated for 3 d with AIMP1 (100 nM) or LPS (500 ng/ml) in the presence or absence of PMB (10 mg/ml). Afterward, the levels of MHC II and CD86 on B cells were evaluated and summarized in (E) as mean 6 SD (n =3).**p , 0.01. (F)MLNBcells were treated with AIMP1 in the presence or absence of PMB (5 mg/ml) for 15 h. Surface levels of CD69 were analyzed by flow cytometric analysis, and the results are summarized in (G)asmean6 SD (n =3).*p , 0.05. AIMP1 alone and LPS alone groups in (E)and(G) showed significant induction of MHC IIhiCD86hi and CD69+ cells, respectively, compared with media control (p , 0.05). The numbers in (A), (D), and (F) are the percentages of cells in the gates or the quadrants. The numbers in (C) are the geometric means of the histograms. Results shown in (A), (C), (D), and (F) are representative of data from three independent experiments. The Journal of Immunology 4731 anti-CD23 (B3B4), FITC-conjugated anti-CD69 (H1.2F3), and FITC- Ag uptake, proliferation assay, and Ca2+ assay conjugated anti-MHC II (NIMR-4) were purchased from eBioscience 3 6 (San Diego, CA). Alexa Fluor 488–conjugated anti-rabbit IgG Ab was from MLN B cells (1 10 cells/well) were treated with AIMP1 (100 nM) or LPS (1 mg/ml) for 48 h in 24-well plates and then preincubated for 30 min Molecular Probes (Eugene, OR). F(ab9)2 fragment anti-mouse IgM was from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti–NF- at 4˚C or 37˚C. Afterward, the cells were incubated for 1 h with 50 mg/ml kB p65, anti-IkBa, anti–p-IkBa, and anti-GAPDH Abs were purchased FITC-conjugated dextran (Sigma-Aldrich) at 4˚C or 37˚C and harvested from Santa Cruz Biotechnology (Dallas, TX). for flow cytometric analysis. For B cell proliferation assay, CFSE-labeled CD19+ B cells (1 3 105 In vitro and in vivo B cell activation by AIMP1 cells/well, 96-well plate) were cultured for 3 d with AIMP1 (100 nM) in the presence or absence of PMB. Harvested B cells were then stained with MLN B cells or splenic B cells were seeded into each well of 48-well plates allophycocyanin-conjugated CD19 Ab and flow cytometric analysis was 3 5 (5 10 cells/well) and then treated with various concentrations of performed. AIMP1 for up to 48 h to analyze CD69 expression and for 72 h to study the For detection of cytosolic Ca2+ levels, MLN B cells were loaded with other expression markers. For inhibitor studies, MLN B cells were pre- Fluo-4 (3 mM in RPMI 1640, TEF Labs, Austin, TX) for 45 min at 37˚C treated for 1 h with the inhibitors for NF-kB (Bay 11-7082, 5 mM, Biomol, and rested for 20 min at room temperature (RT). The Ca2+ flux was de- Hamburg, Germany), JNK (SP600125, 1 mM, Sigma-Aldrich), PKC termined after AIMP1 (100 nM) or anti-IgM Ab (10 mg/ml) by flow (chelerythrine, 10 mM, Tocris Cookson, Bristol, U.K.), MEK1/2 (PD98059, cytometry. 5 mM, Tocris Cookson), or p38 (SB203580, 5 mM, Sigma-Aldrich), and then the cells were treated with AIMP1 (100 nM). CD69 expression was de- Flow cytometry and FACS sorting termined at 15 h and MHC IIhiCD19hi B cell populations were evalua- ted at 72 h of AIMP1 treatment. The analysis for the B cell activation For cell surface or intracellular staining, single-cell suspensions were markers was done by flow cytometry. For in vivo experiments, AIMP1 washed and stained in 13 FACS buffer. Fixation and intracellular staining (150 mg/mouse) or PBS was injected i.v. into the tail vein of the mice, and were performed in Cytofix/Cytoperm and Perm/Wash solutions (BD Bio- Downloaded from spleen cells and PBMCs were isolated 20 h postinjection. The expression sciences), according to the manufacturer’s instructions. Abs were used at of CD69 on B cells was evaluated by flow cytometric analysis. For 1:250 dilution for both surface and intracellular staining. Nonspecific quantitation of Ab levels in sera, OVA (150 mg/mouse) was injected two staining for every Ab used in the study was monitored using fluorescent- times i.v. in the presence or absence of AIMP1 (150 mg/mouse) with conjugated isotype Abs to each Ab. All of the flow cytometric analyses, a 1-wk interval. At day 5 after the last injection, sera were collected and except the viability assay, were performed with gating of live cells using OVA-specific IgG was detected by ELISA. FACSCalibur with CellQuest software (BD Biosciences). http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. AIMP1 activates both IgM+IgD+ naive B cells and IgM2 B cells. (A) IgM+IgD+ B cells were isolated from MLN cells by FACS (top) and cultured for 15 h with AIMP1 (100 or 500 nM) or LPS (100 ng/ml). CD69 expression was evaluated by flow cytometry and summarized as mean 6 SD. (B) IgM2 B cells were sorted by FACS (top). B cells were cultivated and analyzed as described in (A). n =3.*p , 0.05, **p , 0.01, ***p , 0.005. 4732 AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR

IgD+IgM+ naive B cells and CD19+IgM2 B cells were isolated from inhibitors were pretreated for 1 h before addition of AIMP1. The whole- MLN cells using a FACSAria II (BD Biosciences). In brief, total MLN cell lysates were prepared in lysis buffer and centrifuged for 15 min at cells were stained with fluorescence-conjugated IgD, IgM, and CD19 Abs, 20,000 3 g. and then the IgD+IgM+ cells and CD19+IgM2 cells were sorted. The For cytoplasmic/nuclear protein extraction, MLN B cells (4 3 106 cells/ sorting efficacy was determined by FACSCalibur. well, 12-well plates) were cultured for 90 min with AIMP1 (100 nM). Cytoplasmic were prepared by incubation in cytosol extract buffer Evaluation of AID expression (10 mM HEPES, 15 mM MgCl2, 10 mN KCl, and protease inhibitors), MLN B cells were plated in 12-well plates (2 3 106 cells/well), followed by followed by centrifugation (3000 rpm, 10 min). Nuclear proteins were addition of AIMP1 (100 nM) or LPS (200 ng/ml). After 2-d cultivation, RNA prepared by incubation in nuclear extract buffer (20 mM HEPES, 15 mM MgCl , 420 mM NaCl, and protease inhibitors), followed by centrifugation was isolated (TRI Reagent, Molecular Research Center, Cincinnati, OH) and 2 (14,000 rpm, 20 min). reverse transcription was performed to synthesize cDNA. AID expression was determined from the cDNA by PCR (516 bp; sense, 59-AAATGTCC- SDS-PAGE was performed for the separation and proteins were GCTGGGCCAA-39, antisense, 59- CATCGACTTCGTACAAGGG -39). transferred to nitrocellulose membranes. The membranes were blocked for 1 h with 5% skim milk. Primary Abs were diluted to 1/5000 in washing Western blot analysis buffer and applied for overnight at 4˚C. The HRP-conjugated secondary Abs (diluted to 1/5000 in washing buffer) were treated for 1 h at RT. The Ramos B cells (1 3 106 cells/well) or MLN B cells (4 3 106 cells/well) bands were visualized with the chemiluminescent HRP substrate (Mil- were seeded in 12-well plates, and then AIMP1 (100 or 200 nM) was lipore, Billerica, MA) and the Fuji LAS-3000 (Fuji Photo Film, Tokyo, treated for the indicated times. For the inhibitor study, indicated signal Japan). Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. AIMP1 induces proliferation, AID expression, and CSR in B cells. (A) CFSE-labeled MLN B cells were cultured for 3 d with AIMP1 (100 nM) or LPS (100 ng/ml) in the presence or absence of PMB (10 mg/ml). The proliferation of B cells was determined by flow cytometric analysis and summarized as mean 6 SD (n = 3). ***p , 0.005. AIMP1 alone and LPS alone groups showed significant induction of proliferation when compared with media control (p , 0.005). (B) MLN B cells were cultured for 4 d with AIMP1 (100 nM) or LPS (100 ng/ml). CD138 expression was determined by FACS and summarized as mean 6 SD (n = 3). *p , 0.05, **p , 0.01. (C) MLN B cells were treated with LPS (200 ng/ml) or AIMP1 (100 nM) for 2 d, and then AID expression was determined by RT-PCR. (D) MLN B cells were cultured for 72 h with AIMP1 (200 nM) or LPS (100 ng/ml). AID expression was evaluated by Western blot analysis from total cell lysates and is summarized as mean 6 SD (n = 3). *p , 0.05. Numbers in (C)and(D) represent relative intensity of AID to a loading control. (E) MLN B cells were prepared as described in Materials and Methods. Ig classes were analyzed by flow cytometry and summarized in (F) as mean 6 SD (n = 3). The numbers in (A), (B), and (E) are the percentages of cells in the quadrants or the regions. *p , 0.05, **p , 0.01, ***p , 0.005. The Journal of Immunology 4733

Fluorescent microscopy B cells was also significantly induced after 15 h of culture in the MLN B cells were cultivated in the absence or presence of AIMP1 (100 nM) presence of AIMP1 in a dose-dependent manner (Fig. 1F, 1G), for 90 min. Cells were then fixed in 4% paraformaldehyde solution for 30 suggesting that AIMP1 has direct B cell–activating activities. min at RT and blocked with staining solution (0.3% Triton X-100 and 5% Furthermore, the results showed that AIMP1 activated both MLN FBS in PBS) for 1 h at RT. Fixed cells were incubated with primary anti–NF- and splenic B cells (Supplemental Fig. 3A, 3B). kB Ab (rabbit IgG, 1:200 dilution) overnight at 4˚C, followed by staining with Alexa Fluor 488–conjugated anti-rabbit IgG Ab (1:300 dilution) for AIMP1 has subset-independent B cell–stimulatory activity 1 h at RT. Cells were stained with rhodamine phalloidin (Molecular Probes, 1:40 dilution, 20 min, RT) and DAPI (Molecular Probes, 3 mM, 3 To identify subsets of MLN B cells activated by AIMP1, we min, RT). Cells were observed with a fluorescence microscope (Olympus isolated IgM+IgD+ naive B cells or IgM2 B cells containing IgM2 IX71, Olympus, Tokyo, Japan) equipped with fluorescence attachment memory B cells. As shown in Fig. 2A, both 100 and 500 nM (IX-FLA, Olympus) and CoolSNAP-Pro (Media Cybernetics, Silver + + Spring, MD). AIMP1 significantly induced CD69 expression in IgM IgD naive B cells. Because ∼80% of MLN B cells were IgM+IgD+ B cells, Statistical analyses most of AIMP1-activated B cells from the culture of total B cells A paired Student t test for flow cytometric analysis and a two-tailed Stu- were thought to be naive B cells. Interestingly, low concentrations of dent t test for Western blot analysis were used to compare experimental AIMP1 (100 nM) failed to activate IgM B2 cells, whereas high groups and control groups, respectively. A p value , 0.05 was considered concentrations of AIMP1 (500 nM) significantly activated IgM B2 to be statistically significant. cells (Fig. 2B), suggesting that naive B cells are more sensitive to AIMP1 stimulation. Additionally, AIMP1 also tended to activate Results GL7+ germinal center B cells (data not shown), although the pop- Downloaded from AIMP1 induces surface activation markers in B cells ulation of GL7+ B cells were ,5% of total MLN B cells. Taken To investigate the effects of AIMP1 on B cells, MLN B cells were together, our results demonstrated that AIMP1 activated B cells in used because AIMP1 is known to be highly expressed in intestinal a subset-independent manner, although the sensitivities for AIMP1- tissues (11). CD19+ MLN B cells were isolated and then cultured mediated activation were various according to the subsets. in the presence or absence of AIMP1 for 72 h. Surprisingly, hi hi AIMP1 induces the proliferation and Ig CSR AIMP1 increased the population of CD86 MHC II activated http://www.jimmunol.org/ B cells in a dose-dependent manner (Fig. 1A), and this increase in Because AIMP1 significantly increased the expression of B cell the number of activated B cells was significant (Fig. 1B). Next, we activation markers, and B cell activation usually results in in- determined the surface expression of various activation markers. creased proliferation, we next evaluated the proliferation of MLN The results showed that surface levels of CD19, CD23, CD40, and B cells after AIMP1 treatment. As expected, the proliferation of CD80 were increased by AIMP1 treatment 72 h after cultivation MLN B cells was significantly induced by AIMP1 and the in- (Fig. 1C). Because we used a high purity of B cells and TACI-Fc duction was not due to endotoxin contamination (Fig. 3A). Ad- treatment had no effect on AIMP1-induced B cell activation ditionally, AIMP1 enhanced the uptake of FITC-conjugated (Supplemental Fig. 1), we concluded that the B cell activation was dextran by MLN B cells (Supplemental Fig. 3C), suggesting that not mediated by BAFF or APRIL secreted from contaminated DCs AIMP1 increased the activation-induced endocytosis of B cells by guest on September 28, 2021 or monocytes. Because we used live cell gatings (Supplemental (14). Therefore, our results clearly indicate that AIMP1 activates Fig. 2A) and AIMP1 did not affect the cell viability (Supplemental B cells, resulting in the increased proliferation and the activation- Fig. 2B), the increased B cell activation by AIMP1 was not due to induced endocytosis. changes of the cell viability. AIMP1-induced B cell activation was Because AIMP1 activated B cells significantly, we next inves- also not due to endotoxin contamination. As shown in Fig. 1D and tigated whether AIMP1 promoted plasma cell differentiation. 1E, AIMP1-induced B cell activation was not affected by the When we cultivated MLN B cells with AIMP1 for 4 d, CD138- presence of polymyxin B (PMB), a LPS inhibitor, whereas LPS- expressing B cells were significantly increased by AIMP1 induced activation was significantly inhibited by PMB. Addi- (Fig. 3B), indicating that AIMP1 efficiently generates plasma cells tionally, the expression of CD69, an early activation marker, on from B cells.

FIGURE 4. Combination effects of AIMP1 and CD40 or BCR stimulation on MLN B cell activa- tion. Isolated MLN B cells were cultured in the presence of AIMP1 alone (100 nM) or in combi- nation with the indicated stimuli for 3 d. (A) The effects of AIMP1 and an anti-CD40 Ab on in- duction of MHC IIhiCD86hi activated B cells were determined by flow cytometric analysis and the results (mean 6 SD) from four independent ex- periments are summarized in (B). *p , 0.05, **p , 0.01 (paired Student t test). (C)MLNB cells were cultivated with AIMP1 and an anti-IgM Ab, and the population of activated B cells was examined. The numbers in the plots are the per- centages of cells in each gate. The values shown in (C) are representative of data from two independent experiments. 4734 AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR

As activation and proliferation of B cells are required for AID combined treatment with AIMP1 and a CD40 Ab showed syner- expression and CSR (15), we examined whether AIMP1 induced gistic effects on MLN B cell activation (Fig. 4A, 4B). In contrast, CSR in MLN B cells. As shown in Fig. 3C, the mRNA expression the effect of combined treatment with AIMP1 and an anti-IgM Ab of AID increased dramatically in MLN B cells following AIMP1 was mild, and decreased activation was observed with low-level treatment. The level of AID protein was also significantly in- BCR stimulation (0.25 mg/ml) (Fig. 4C), suggesting that crosstalk creased by AIMP1 treatment (Fig. 3D). We next investigated between AIMP1 signaling and CD40 or BCR signaling differs. AIMP1-induced CSR in Ig levels. Interestingly, AIMP1 alone led AIMP1 activates B cells via the PKC/NF-kB signaling CSR into IgG1, IgG2a/b, IgG3, and IgE, but not into IgA, in MLN pathway B cells (Fig. 3E, 3F), suggesting that AIMP1 is involved in broad classes of Ab CSR. Taken together, our results revealed that Various signaling pathways, including the NF-kB, JNK, MAPK, 2+ AIMP1 was also involved in CSR as well as B cell activation. ERK, and Ca pathways, are involved in B cell activation (16, 17). Therefore, we examined their involvement in AIMP1-mediated AIMP1 and CD40 signaling has synergistic effects on B cell B cell activation via signaling studies using inhibitors of signaling activation molecules. We pretreated MLN B cells with various inhibitors and Because AIMP1 has B cell–activating effects, we investigated then activated the cells with AIMP1 for 72 h. The results indicated whether AIMP1 had synergistic effects with CD40 or BCR stimu- that NF-kB, JNK, and PKC are involved in AIMP1-induced B cell lation on B cell activation. We treated MLN B cells with AIMP1 in activation. In contrast, treatment with MEK and p38 inhibitors combination with an anti-CD40 or anti-IgM Ab. Interestingly, the showed no effect (Supplemental Fig. 4A). We also evaluated Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. AIMP1 activates B cells via the PKC/NF-kB signaling pathway. (A) MLN B cells were pretreated with the indicated inhibitors for 1 h, and then AIMP1 (100 nM) was added. After 15 h of cultivation, CD69 expression was determined by flow cytometry and the results are summarized as mean 6 SD (n = 3). *p , 0.05, ***p , 0.005 (paired Student t test). Numbers in (A) are the percentages of activated B cells. (B) Ramos B cells were treated with AIMP1 (100 nM) for the indicated time periods. The levels of p-IkBa in the lysates were measured by Western blotting. Numbers indicate densitometry of p-IkBa, presented relative to loading control. The density of 0 min was compared with that of 60 min as mean 6 SD (n = 3). *p , 0.05 (Student t test). (C) Ramos B cells were pretreated with various concentrations of PKC inhibitor, and the cells were treated with AIMP1 (100 nM) for 90 min. The levels of p-IkBa in the lysates were measured by Western blotting. Data represent relative intensity of p-IkBa to a loading control. (D) MLN B cells were cultured in the presence or absence of AIMP1 (100 nM) and nuclear translocation of NF-kB p65 was determined by Western blot analysis. Numbers represent relative intensity of nuclear p65 to a loading control. The results were summarized as mean 6 SD (n = 4). *p , 0.05 (Student t test). (E) MLN B cells were cultured for 90 min in the presence or absence of AIMP1 (100 nM) and then the cells were incubated with anti-NF-kB (rabbit IgG) Ab, followed by stain with anti- rabbit IgG (Alexa Fluor 488), rhodamine phalloidin, and DAPI. The location of NF-kB p65 was analyzed by fluorescent microscopy. The Journal of Immunology 4735 whether AIMP1 initiated intracellular calcium signaling using Fluo- expression, an early activation marker, to determine the direct 4 staining. However, there was no change in cytosolic Ca2+ levels in B cell–activating effects of AIMP1 and to exclude DC-mediated AIMP1-treated B cells until 300 s after treatment, whereas BCR B cell activation, because AIMP1 is known to induce the activa- stimulation immediately increased Ca2+ flux into the cytosol tion and maturation of DCs (13). Spleens and blood samples were (Supplemental Fig. 4B), suggesting that Ca2+ signaling is not in- collected 20 h postinjection, and CD69 expression on CD19+ volved in AIMP1-induced B cell activation. We also confirmed the B cells was evaluated. As shown in Fig. 6A and 6B, CD69 ex- effects of these screened inhibitors on the expression of an early pression on splenic B cells was significantly increased in AIMP1- activation marker, CD69. The results showed that only the JNK injected mice, whereas CD69 expression on blood B cells was not inhibitor failed to reduce AIMP1-induced CD69 expression (Fig. changed, strongly suggesting that AIMP1 is a direct B cell–acti- 5A), whereas NF-kB and PCK inhibitors significantly reduced CD69 vating factor. To determine whether AIMP1 also enhanced pro- expression, suggesting that PKC and NF-kB are directly involved duction of Ag-specific Ab, we injected mice with OVA alone or in AIMP1-mediated B cell activation. Additionally, Western blot together with AIMP1. As expected, the levels of OVA-specific IgG analysis showed that AIMP1 increased the phosphorylation of IkBa in sera were significantly higher in the AIMP1-treated group than significantly in Ramos B cells (Fig. 5B). Because treatment with those in the OVA alone group. Therefore, based on our in vitro and a PKC inhibitor decreased AIMP1-induced B cell activation, we in vivo results, we conclude that AIMP1 directly activates B cells treated Ramos cells with a PKC inhibitor and investigated the effects and enhances Ab production. on AIMP1-induced phosphorylation of IkBa. As shown in Fig. 5C, inhibition of PKC decreased IkBa phosphorylation. Next, we in- Discussion vestigated whether AIMP1 induced NF-kBtranslocationintothe The present study reports AIMP1 as a novel B cell–activating Downloaded from nuclei in MLN B cells. As shown in Fig. 5D, the results indicated factor. AIMP1 was shown to induce the expression of B cell that AIMP1 significantly increased the translocation of NF-kB p65. activation markers and proliferation of B cells. Additionally, Furthermore, NF-kB translocation following AIMP1 treatment was AIMP1 also induced AID expression and CSR in B cells. The also observed by fluorescent microscopy (Fig. 5E), suggesting that in vivo administration of AIMP1 induced CD69 expression on AIMP1 activates B cells via PKC-mediated NF-kB activation. splenic B cells, indicating that AIMP1 is a critical B cell acti-

vator. We previously reported that AIMP1 activates macrophages http://www.jimmunol.org/ AIMP1 induces B cell activation and Ab production in vivo and DCs (12, 13). Because stimulated macrophages and DCs are To investigate the effects of AIMP1 on B cell activation in vivo, we known to express BAFF and APRIL (18), the in vivo data might injected AIMP1 into the tail vein of the mice. We measured CD69 be, at least in part, mediated by these cells. However, it is con- by guest on September 28, 2021

FIGURE 6. In vivo B cell activation and OVA-specific Ab production by AIMP1. (A) Mice were injected i.v. with AIMP1 (150 mg/mouse) or PBS, after which PBMCs and splenocytes were collected after 20 h. CD69 expression on B cells was compared by flow cytometric analysis, and the results are summarized in (B). (C) Mice were injected as described in (A) together with OVA (150 mg/mouse) twice with a 1-wk interval. At day 5 after the last injection, sera were isolated and the levels of OVA-specific IgG were evaluated by ELISA. Data shown in (B) and (C) are mean 6 SD (n = 3) (three mice per group). The numbers in (A) are the percentages of cells in the quadrants. *p , 0.05, **p , 0.01, ***p , 0.005. 4736 AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR troversial that BAFF and APRIL induce CD69 expression on References B cells. In particular, it has been reported that BAFF does not 1. Victora, G. D., and M. C. Nussenzweig. 2012. Germinal centers. Annu. 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Immunol. 7: 355–359. 23. Jackson, L., C. T. Cady, and J. C. Cambier. 2009. TLR4-mediated signaling induces Acknowledgments MMP9-dependent cleavage of B cell surface CD23. J. Immunol. 183: 2585–2592. We thank J.H. Song and H.J. Hong for helpful discussion and technical 24. Lemieux, G. A., F. Blumenkron, N. Yeung, P. Zhou, J. Williams, A. C. Grammer, R. Petrovich, P. E. Lipsky, M. L. Moss, and Z. Werb. 2007. The low affinity IgE assistance. receptor (CD23) is cleaved by the metalloproteinase ADAM10. J. Biol. Chem. 282: 14836–14844. 25. Croft, M., W. Duan, H. Choi, S. Y. Eun, S. Madireddi, and A. Mehta. 2012. TNF Disclosures superfamily in inflammatory disease: translating basic insights. Trends Immunol. The authors have no financial conflicts of interest. 33: 144–152.