Aminoacyl tRNA Synthetase−Interacting
Multifunctional Protein 1 Acts as a Novel B
Cell−Activating Factor In Vitro and In Vivo
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of June 14, 2017.
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J Immunol 2015; 194:4729-4736; Prepublished online 13
April 2015;
doi: 10.4049/jimmunol.1401352
http://www.jimmunol.org/content/194/10/4729
http://www.jimmunol.org/content/suppl/2015/04/11/jimmunol.140135
2.DCSupplemental
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Copyright © 2015 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Supplementary
Material
Myun Soo Kim and Tae Sung Kim
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
A
ntibody production requires B cell activation, and B cells
can be activated through both T cell–dependent and
T cell–independent manners. Although somatic hypermutation and clonal expansion are thought to occur with the aid of
T cells within germinal centers (1), T cell–independent activation
also plays pivotal roles in proper immune responses. For example,
important roles have been suggested for IgM Abs generated by
T-independent B cell responses to microbial infections (2). Additionally, intestinal levels of IgA are normal in CD40-deficient
mice (3), suggesting that T cell–independent class switching
occurs spontaneously in the intestine. As a defense mechanism,
B cells can be activated by numerous exogenous molecules, including TLR ligands and specific Ags for BCR through a Tindependent manner (4). In addition to these exogenous factors,
various endogenous factors have been shown to induce Tindependent B cell responses. For example, BAFF and a proliferation-inducing ligand (APRIL) have been reported to induce IgA
production in the intestine in T cell–independent manners (5).
Various non–T cells, including macrophages, monocytes, dendritic
cells (DCs), and epithelial cells, have been reported to secrete
Division of Life Sciences, College of Life Sciences and Biotechnology, Korea
University, Seoul 136-701, Republic of Korea
Received for publication May 27, 2014. Accepted for publication March 14, 2015.
This work was supported by National Research Foundation of Korea Grant NRF2014R1A2A2A01005031 funded by the Korea government (Ministry of Science, ICT
and Future Planning).
Address correspondence and reprint requests to Prof. Tae Sung Kim, Division of Life
Sciences, College of Life Sciences and Biotechnology, Korea University, 5-ga,
Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AID, activation-induced deaminase; AIMP1, aminoacyl tRNA synthetase–interacting multifunctional protein 1; APRIL, a proliferation-inducing ligand; CSR, class switch recombination; DC, dendritic cell; MHC II,
MHC class II; MLN, mesenteric lymph node; PKC, protein kinase C; PMB, polymyxin B; RT, room temperature.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401352
these factors, thereby activating B cells and inducing the maturation and survival of B cells (6). Importantly, BAFF-deficient
mice showed abnormal B cell development and reduced serum
levels of IgM and IgG (7), suggesting that endogenous B cell–
activating factors affect total B cell responses. APRIL-deficient
mice also have revealed impaired Ab production (8). Therefore,
endogenous B cell–activating factors are regarded as important
modulators of total B cell responses. However, the mechanisms
underlying B cell activation via endogenous factors are still not
well understood, and there are novel B cell–activating factors yet
to be identified.
Aminoacyl tRNA synthetase–interacting multifunctional protein
1 (AIMP1) has been identified as an aminoacyl tRNA synthetase
complex component and acts to stabilize the complex (9). AIMP1
is also secreted and has been shown to have various activities,
including effects on wound healing (10) and glucagon-like functions that control blood glucose levels (11). Furthermore, we
demonstrated that AIMP1 functions as a proinflammatory cytokine that induces macrophage activation, DC maturation, and IL12 production, indicating that AIMP1 is a novel immune modulator (12, 13). However, it is unclear whether AIMP1 affects other
APCs, such as B cells.
To our knowledge, this study is the first to show that AIMP1 is
a novel B cell–activating factor. AIMP1 induced the expression
of surface activation markers on B cells, such as CD19, CD40,
CD69, CD80, CD86, and MHC class II (MHC II). The proliferation and Ag uptake ability of B cells were also dramatically increased after AIMP1 treatment. AIMP1 and CD40 signaling
synergistically enhanced B cell activation, whereas AIMP1 showed
only moderate effects on B cell activation via BCR signaling.
AIMP1 also increased the expression of activation-induced deaminase (AID) and class switch recombination (CSR) in mesenteric lymph node (MLN) B cells. Experiments using signaling
inhibitors revealed that AIMP1 induced the protein kinase C
(PKC)–mediated NF-kB signaling pathway. Additionally, i.v. injection of AIMP1 into mice increased CD69 expression on splenic
B cells and enhanced OVA-specific IgG production.
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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
cytokine 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
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.
4730
Materials and Methods
Mice and cells
Seven- to 10-wk-old C57BL/6 mice (OrientBio, Seongnam, Korea) were
used for this study. The animals were housed in a specific pathogen-free
facility and the experiments were performed according to the guidelines
of Korea University Institutional Animal Care and Use Committee. Ramos
B cell line (KCLB 21596) was obtained from Korean Cell Line Bank (Seoul,
Korea). CD19+ primary B cells were isolated using CD19 microbeads and
a MACS system (Miltenyi Biotec, Auburn, CA).
Media and cytokines
Animal cells and Ramos cells were cultured in RPMI 1640 medium (Thermo
Scientific, Rockford, IL) supported with FBS (10%, Welgene, Daegu, Korea),
AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR
2-ME (50 mM, Sigma-Aldrich, St. Louis, MO), HEPES (10 mM, Welgene),
sodium pyruvate (1 mM, Invitrogen, Carlsbad, CA), penicillin (100 U/ml,
Invitrogen), and streptomycin (0.1 mg/ml, Invitrogen). Recombinant AIMP1
was prepared as described previously (13). His-tagged AIMP1 was constructed. AIMP1 protein was produced and purified by Young In Frontier
(Seoul, Korea). The level of endotoxin in each lot was determined using
a Limulus amebocyte lysate QCL-1000 kit (Lonza, Walkersville, MD). Lots
containing ,0.1 endotoxin unit/1 mg protein were used for this study.
Antibodies
PE-conjugated anti-B220 (RA3-6B2), PE-conjugated anti-CD40 (3/23),
PE-conjugated anti-CD80 (16-10A1), and PE-conjugated anti-CD86
(GL1) were from BD Biosciences (San Diego, CA). Anti-mouse CD40
(1C10), allophycocyanin-conjugated anti-CD19 (MB19-1), PE-conjugated
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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) MLN B cells
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) as mean 6 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
anti-CD23 (B3B4), FITC-conjugated anti-CD69 (H1.2F3), and FITCconjugated anti-MHC II (NIMR-4) were purchased from eBioscience
(San Diego, CA). Alexa Fluor 488–conjugated anti-rabbit IgG Ab was from
Molecular Probes (Eugene, OR). F(ab9)2 fragment anti-mouse IgM was
from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti–NFkB p65, anti-IkBa, anti–p-IkBa, and anti-GAPDH Abs were purchased
from Santa Cruz Biotechnology (Dallas, TX).
In vitro and in vivo B cell activation by AIMP1
Ag uptake, proliferation assay, and Ca2+ assay
MLN B cells (1 3 106 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
at 4˚C or 37˚C. Afterward, the cells were incubated for 1 h with 50 mg/ml
FITC-conjugated dextran (Sigma-Aldrich) at 4˚C or 37˚C and harvested
for flow cytometric analysis.
For B cell proliferation assay, CFSE-labeled CD19+ B cells (1 3 105
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
allophycocyanin-conjugated CD19 Ab and flow cytometric analysis was
performed.
For detection of cytosolic Ca2+ levels, MLN B cells were loaded with
Fluo-4 (3 mM in RPMI 1640, TEF Labs, Austin, TX) for 45 min at 37˚C
and rested for 20 min at room temperature (RT). The Ca2+ flux was determined after AIMP1 (100 nM) or anti-IgM Ab (10 mg/ml) by flow
cytometry.
Flow cytometry and FACS sorting
For cell surface or intracellular staining, single-cell suspensions were
washed and stained in 13 FACS buffer. Fixation and intracellular staining
were performed in Cytofix/Cytoperm and Perm/Wash solutions (BD Biosciences), according to the manufacturer’s instructions. Abs were used at
1:250 dilution for both surface and intracellular staining. Nonspecific
staining for every Ab used in the study was monitored using fluorescentconjugated isotype Abs to each Ab. All of the flow cytometric analyses,
except the viability assay, were performed with gating of live cells using
FACSCalibur with CellQuest software (BD Biosciences).
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.
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MLN B cells or splenic B cells were seeded into each well of 48-well plates
(5 3 105 cells/well) and then treated with various concentrations of
AIMP1 for up to 48 h to analyze CD69 expression and for 72 h to study the
other expression markers. For inhibitor studies, MLN B cells were pretreated for 1 h with the inhibitors for NF-kB (Bay 11-7082, 5 mM, Biomol,
Hamburg, Germany), JNK (SP600125, 1 mM, Sigma-Aldrich), PKC
(chelerythrine, 10 mM, Tocris Cookson, Bristol, U.K.), MEK1/2 (PD98059,
5 mM, Tocris Cookson), or p38 (SB203580, 5 mM, Sigma-Aldrich), and then
the cells were treated with AIMP1 (100 nM). CD69 expression was determined at 15 h and MHC IIhiCD19hi B cell populations were evaluated at 72 h of AIMP1 treatment. The analysis for the B cell activation
markers was done by flow cytometry. For in vivo experiments, AIMP1
(150 mg/mouse) or PBS was injected i.v. into the tail vein of the mice, and
spleen cells and PBMCs were isolated 20 h postinjection. The expression
of CD69 on B cells was evaluated by flow cytometric analysis. For
quantitation of Ab levels in sera, OVA (150 mg/mouse) was injected two
times i.v. in the presence or absence of AIMP1 (150 mg/mouse) with
a 1-wk interval. At day 5 after the last injection, sera were collected and
OVA-specific IgG was detected by ELISA.
4731
4732
IgD+IgM+ naive B cells and CD19+IgM2 B cells were isolated from
MLN cells using a FACSAria II (BD Biosciences). In brief, total MLN
cells were stained with fluorescence-conjugated IgD, IgM, and CD19 Abs,
and then the IgD+IgM+ cells and CD19+IgM2 cells were sorted. The
sorting efficacy was determined by FACSCalibur.
Evaluation of AID expression
MLN B cells were plated in 12-well plates (2 3 106 cells/well), followed by
addition of AIMP1 (100 nM) or LPS (200 ng/ml). After 2-d cultivation, RNA
was isolated (TRI Reagent, Molecular Research Center, Cincinnati, OH) and
reverse transcription was performed to synthesize cDNA. AID expression
was determined from the cDNA by PCR (516 bp; sense, 59-AAATGTCCGCTGGGCCAA-39, antisense, 59- CATCGACTTCGTACAAGGG -39).
Western blot analysis
Ramos B cells (1 3 106 cells/well) or MLN B cells (4 3 106 cells/well)
were seeded in 12-well plates, and then AIMP1 (100 or 200 nM) was
treated for the indicated times. For the inhibitor study, indicated signal
AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR
inhibitors were pretreated for 1 h before addition of AIMP1. The wholecell lysates were prepared in lysis buffer and centrifuged for 15 min at
20,000 3 g.
For cytoplasmic/nuclear protein extraction, MLN B cells (4 3 106 cells/
well, 12-well plates) were cultured for 90 min with AIMP1 (100 nM).
Cytoplasmic proteins were prepared by incubation in cytosol extract buffer
(10 mM HEPES, 15 mM MgCl2, 10 mN KCl, and protease inhibitors),
followed by centrifugation (3000 rpm, 10 min). Nuclear proteins were
prepared by incubation in nuclear extract buffer (20 mM HEPES, 15 mM
MgCl2, 420 mM NaCl, and protease inhibitors), followed by centrifugation
(14,000 rpm, 20 min).
SDS-PAGE was performed for the separation and proteins were
transferred to nitrocellulose membranes. The membranes were blocked
for 1 h with 5% skim milk. Primary Abs were diluted to 1/5000 in washing
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
bands were visualized with the chemiluminescent HRP substrate (Millipore, Billerica, MA) and the Fuji LAS-3000 (Fuji Photo Film, Tokyo,
Japan).
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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
Fluorescent microscopy
MLN B cells were cultivated in the absence or presence of AIMP1 (100 nM)
for 90 min. Cells were then fixed in 4% paraformaldehyde solution for 30
min at RT and blocked with staining solution (0.3% Triton X-100 and 5%
FBS in PBS) for 1 h at RT. Fixed cells were incubated with primary anti–NFkB 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
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
min, RT). Cells were observed with a fluorescence microscope (Olympus
IX71, Olympus, Tokyo, Japan) equipped with fluorescence attachment
(IX-FLA, Olympus) and CoolSNAP-Pro (Media Cybernetics, Silver
Spring, MD).
Statistical analyses
A paired Student t test for flow cytometric analysis and a two-tailed Student t test for Western blot analysis were used to compare experimental
groups and control groups, respectively. A p value , 0.05 was considered
to be statistically significant.
Results
To investigate the effects of AIMP1 on B cells, MLN B cells were
used because AIMP1 is known to be highly expressed in intestinal
tissues (11). CD19+ MLN B cells were isolated and then cultured
in the presence or absence of AIMP1 for 72 h. Surprisingly,
AIMP1 increased the population of CD86hiMHC IIhi activated
B cells in a dose-dependent manner (Fig. 1A), and this increase in
the number of activated B cells was significant (Fig. 1B). Next, we
determined the surface expression of various activation markers.
The results showed that surface levels of CD19, CD23, CD40, and
CD80 were increased by AIMP1 treatment 72 h after cultivation
(Fig. 1C). Because we used a high purity of B cells and TACI-Fc
treatment had no effect on AIMP1-induced B cell activation
(Supplemental Fig. 1), we concluded that the B cell activation was
not mediated by BAFF or APRIL secreted from contaminated DCs
or monocytes. Because we used live cell gatings (Supplemental
Fig. 2A) and AIMP1 did not affect the cell viability (Supplemental
Fig. 2B), the increased B cell activation by AIMP1 was not due to
changes of the cell viability. AIMP1-induced B cell activation was
also not due to endotoxin contamination. As shown in Fig. 1D and
1E, AIMP1-induced B cell activation was not affected by the
presence of polymyxin B (PMB), a LPS inhibitor, whereas LPSinduced activation was significantly inhibited by PMB. Additionally, the expression of CD69, an early activation marker, on
FIGURE 4. Combination effects of AIMP1 and
CD40 or BCR stimulation on MLN B cell activation. Isolated MLN B cells were cultured in the
presence of AIMP1 alone (100 nM) or in combination with the indicated stimuli for 3 d. (A) The
effects of AIMP1 and an anti-CD40 Ab on induction of MHC IIhiCD86hi activated B cells were
determined by flow cytometric analysis and the
results (mean 6 SD) from four independent experiments are summarized in (B). *p , 0.05,
**p , 0.01 (paired Student t test). (C) MLN B
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 percentages of cells in each gate. The values shown in
(C) are representative of data from two independent
experiments.
B cells was also significantly induced after 15 h of culture in the
presence of AIMP1 in a dose-dependent manner (Fig. 1F, 1G),
suggesting that AIMP1 has direct B cell–activating activities.
Furthermore, the results showed that AIMP1 activated both MLN
and splenic B cells (Supplemental Fig. 3A, 3B).
AIMP1 has subset-independent B cell–stimulatory activity
To identify subsets of MLN B cells activated by AIMP1, we
isolated IgM+IgD+ naive B cells or IgM2 B cells containing IgM2
memory B cells. As shown in Fig. 2A, both 100 and 500 nM
AIMP1 significantly induced CD69 expression in IgM+IgD+ naive
B cells. Because ∼80% of MLN B cells were IgM+IgD+ B cells,
most of AIMP1-activated B cells from the culture of total B cells
were thought to be naive B cells. Interestingly, low concentrations of
AIMP1 (100 nM) failed to activate IgM B2 cells, whereas high
concentrations of AIMP1 (500 nM) significantly activated IgM B2
cells (Fig. 2B), suggesting that naive B cells are more sensitive to
AIMP1 stimulation. Additionally, AIMP1 also tended to activate
GL7+ germinal center B cells (data not shown), although the population of GL7+ B cells were ,5% of total MLN B cells. Taken
together, our results demonstrated that AIMP1 activated B cells in
a subset-independent manner, although the sensitivities for AIMP1mediated activation were various according to the subsets.
AIMP1 induces the proliferation and Ig CSR
Because AIMP1 significantly increased the expression of B cell
activation markers, and B cell activation usually results in increased proliferation, we next evaluated the proliferation of MLN
B cells after AIMP1 treatment. As expected, the proliferation of
MLN B cells was significantly induced by AIMP1 and the induction was not due to endotoxin contamination (Fig. 3A). Additionally, AIMP1 enhanced the uptake of FITC-conjugated
dextran by MLN B cells (Supplemental Fig. 3C), suggesting that
AIMP1 increased the activation-induced endocytosis of B cells
(14). Therefore, our results clearly indicate that AIMP1 activates
B cells, resulting in the increased proliferation and the activationinduced endocytosis.
Because AIMP1 activated B cells significantly, we next investigated whether AIMP1 promoted plasma cell differentiation.
When we cultivated MLN B cells with AIMP1 for 4 d, CD138expressing B cells were significantly increased by AIMP1
(Fig. 3B), indicating that AIMP1 efficiently generates plasma cells
from B cells.
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AIMP1 induces surface activation markers in B cells
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4734
As activation and proliferation of B cells are required for AID
expression and CSR (15), we examined whether AIMP1 induced
CSR in MLN B cells. As shown in Fig. 3C, the mRNA expression
of AID increased dramatically in MLN B cells following AIMP1
treatment. The level of AID protein was also significantly increased by AIMP1 treatment (Fig. 3D). We next investigated
AIMP1-induced CSR in Ig levels. Interestingly, AIMP1 alone led
CSR into IgG1, IgG2a/b, IgG3, and IgE, but not into IgA, in MLN
B cells (Fig. 3E, 3F), suggesting that AIMP1 is involved in broad
classes of Ab CSR. Taken together, our results revealed that
AIMP1 was also involved in CSR as well as B cell activation.
AIMP1 and CD40 signaling has synergistic effects on B cell
activation
Because AIMP1 has B cell–activating effects, we investigated
whether AIMP1 had synergistic effects with CD40 or BCR stimulation on B cell activation. We treated MLN B cells with AIMP1 in
combination with an anti-CD40 or anti-IgM Ab. Interestingly, the
AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR
combined treatment with AIMP1 and a CD40 Ab showed synergistic effects on MLN B cell activation (Fig. 4A, 4B). In contrast,
the effect of combined treatment with AIMP1 and an anti-IgM Ab
was mild, and decreased activation was observed with low-level
BCR stimulation (0.25 mg/ml) (Fig. 4C), suggesting that crosstalk
between AIMP1 signaling and CD40 or BCR signaling differs.
AIMP1 activates B cells via the PKC/NF-kB signaling
pathway
Various signaling pathways, including the NF-kB, JNK, MAPK,
ERK, and Ca2+ pathways, are involved in B cell activation (16, 17).
Therefore, we examined their involvement in AIMP1-mediated
B cell activation via signaling studies using inhibitors of signaling
molecules. We pretreated MLN B cells with various inhibitors and
then activated the cells with AIMP1 for 72 h. The results indicated
that NF-kB, JNK, and PKC are involved in AIMP1-induced B cell
activation. In contrast, treatment with MEK and p38 inhibitors
showed no effect (Supplemental Fig. 4A). We also evaluated
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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 antirabbit IgG (Alexa Fluor 488), rhodamine phalloidin, and DAPI. The location of NF-kB p65 was analyzed by fluorescent microscopy.
The Journal of Immunology
AIMP1 induces B cell activation and Ab production in vivo
To investigate the effects of AIMP1 on B cell activation in vivo, we
injected AIMP1 into the tail vein of the mice. We measured CD69
expression, an early activation marker, to determine the direct
B cell–activating effects of AIMP1 and to exclude DC-mediated
B cell activation, because AIMP1 is known to induce the activation and maturation of DCs (13). Spleens and blood samples were
collected 20 h postinjection, and CD69 expression on CD19+
B cells was evaluated. As shown in Fig. 6A and 6B, CD69 expression on splenic B cells was significantly increased in AIMP1injected mice, whereas CD69 expression on blood B cells was not
changed, strongly suggesting that AIMP1 is a direct B cell–activating factor. To determine whether AIMP1 also enhanced production of Ag-specific Ab, we injected mice with OVA alone or
together with AIMP1. As expected, the levels of OVA-specific IgG
in sera were significantly higher in the AIMP1-treated group than
those in the OVA alone group. Therefore, based on our in vitro and
in vivo results, we conclude that AIMP1 directly activates B cells
and enhances Ab production.
Discussion
The present study reports AIMP1 as a novel B cell–activating
factor. AIMP1 was shown to induce the expression of B cell
activation markers and proliferation of B cells. Additionally,
AIMP1 also induced AID expression and CSR in B cells. The
in vivo administration of AIMP1 induced CD69 expression on
splenic B cells, indicating that AIMP1 is a critical B cell activator. We previously reported that AIMP1 activates macrophages
and DCs (12, 13). Because stimulated macrophages and DCs are
known to express BAFF and APRIL (18), the in vivo data might
be, at least in part, mediated by these cells. However, it is con-
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.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
whether AIMP1 initiated intracellular calcium signaling using Fluo4 staining. However, there was no change in cytosolic Ca2+ levels in
AIMP1-treated B cells until 300 s after treatment, whereas BCR
stimulation immediately increased Ca2+ flux into the cytosol
(Supplemental Fig. 4B), suggesting that Ca2+ signaling is not involved in AIMP1-induced B cell activation. We also confirmed the
effects of these screened inhibitors on the expression of an early
activation marker, CD69. The results showed that only the JNK
inhibitor failed to reduce AIMP1-induced CD69 expression (Fig.
5A), whereas NF-kB and PCK inhibitors significantly reduced CD69
expression, suggesting that PKC and NF-kB are directly involved
in AIMP1-mediated B cell activation. Additionally, Western blot
analysis showed that AIMP1 increased the phosphorylation of IkBa
significantly in Ramos B cells (Fig. 5B). Because treatment with
a PKC inhibitor decreased AIMP1-induced B cell activation, we
treated Ramos cells with a PKC inhibitor and investigated the effects
on AIMP1-induced phosphorylation of IkBa. As shown in Fig. 5C,
inhibition of PKC decreased IkBa phosphorylation. Next, we investigated whether AIMP1 induced NF-kB translocation into the
nuclei in MLN B cells. As shown in Fig. 5D, the results indicated
that AIMP1 significantly increased the translocation of NF-kB p65.
Furthermore, NF-kB translocation following AIMP1 treatment was
also observed by fluorescent microscopy (Fig. 5E), suggesting that
AIMP1 activates B cells via PKC-mediated NF-kB activation.
4735
4736
Acknowledgments
We thank J.H. Song and H.J. Hong for helpful discussion and technical
assistance.
Disclosures
The authors have no financial conflicts of interest.
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troversial that BAFF and APRIL induce CD69 expression on
B cells. In particular, it has been reported that BAFF does not
induce CD69 expression on primary B cells (19). Therefore, i.v.
injection of AIMP1 may induce CD69 expression on B cells
directly.
AIMP1 is generally secreted in stressful conditions. It has been
shown that AIMP1 is produced upon exposure to apoptosisinducing agents and hypoxia from epithelial cells (20). TNF-a
also induces the secretion of AIMP1 from macrophages at sites of
skin injury (10). Furthermore, as AIMP1 is highly expressed in
intestinal tissues (11), it is likely to be involved in intestinal immune responses, especially in B cell activation and subsequent
generation of plasma cells in the intestines. AIMP1 alone failed to
lead CSR into IgA (Fig. 3F). However, AIMP1 is still thought to
be involved in IgA production in the gut as a B cell–activating
factor. Because of the importance of B cell responses in the intestine, further studies to identify sources and conditions of
AIMP1 secretion in intestinal tissues will provide us better understanding about the gut immunity.
Recently CD23, also known as Fcε receptor II, was reported to
be a functional receptor of AIMP1 in monocytes (21). Because
CD23 is also a marker of mature B cells, we hypothesized that
AIMP1 activates B cells via CD23. However, it was difficult to
find evidence to support our hypothesis because treatment with
anti-CD23 blocking Abs activated B cells (22), which made it
impossible to demonstrate inhibition of AIMP1-induced B cell
activation. It is also known that CD23 is cleaved into a soluble
form during cultivation (23). ADAM10 and MMP9 mediate the
cleavage of surface CD23, and the loss of CD23 on the cell surface is observed during B cell culture (23, 24). Therefore,
knockdown of CD23 via RNA interference is also not suitable
because the expression of surface CD23 is also decreased in the
control groups.
To our knowledge, this is the first time that AIMP1 has been shown
to induce B cell activation in vitro and in vivo, which suggests that
AIMP1 is a novel endogenous B cell–activating factor. AIMP1 is
a good candidate for vaccination because it directly activates
macrophages (12), DCs (13), and B cells. Our results also showed
that AIMP1 enhanced Ab production (Fig. 6C). Additionally,
AIMP1 may be involved in various inflammatory and autoimmune
diseases, as many B cell–activating factors are involved in asthma,
rheumatoid arthritis, lupus, encephalomyelitis, and colitis (25).
Therefore, further studies of AIMP1-mediated B cell activation and
the involvement of AIMP1 in diseases will provide additional information for therapeutic strategies.
AIMP1 AS A NOVEL B CELL–ACTIVATING FACTOR