Protein arginine methylation plays crucial roles, including signal transduction, transcriptional control, cell proliferation and/or differentiation. B cells undergo clonal division, isotype switching and differentiate into antibody forming cells following stimulation with Toll-like receptor-ligand, lipopolysaccharide (LPS) and T cell-derived signals, including CD40-ligand (CD40-L) and interleukin 4 (IL-4). Whether protein arginine methylation affects B cell division and/or isotype switching to IgG1 in response to LPS, IL-4, and CD40-L was examined using the arginine methyl transferase inhibitor adenosine-2′,3′-dialdehyde (AdOx). Addition of AdOx substantially reduced the number of division cycles of stimulated B cells, whereas cell viability remained intact. Upon stimulation with LPS/IL-4/CD40-L, the proportion of surface IgG1 positive cells in each division cycle was slightly diminished by AdOx. However, the degree of expression of γ1 germ line transcript and activation-induced cytidine deaminase (AID) in response to LPS/IL-4/CD40-L were unaffected by addition of AdOx, suggesting that AdOx influences class switch recombination independent of AID expression through transcriptional control. Taken together, arginine methylation appears to be involved in B cell isotype switching, as well as in clonal expansion of B cells in response to LPS/IL-4/CD40-L.
activation-induced cytidine deaminase
cluster of differentiation
carboxyfluorescein diacetate succinimidyl ester
germ line transcript
protein arginine methyl transferase
Modification of proteins, including phosphorylation, ubiquitination and methylation, reportedly influences cell fate; activation, division and differentiation of cells, including immune cells [1, 2]. For example, arginine methylation plays an important role in lymphocyte signaling . Modification of arginine residues in protein is catalyzed by PRMTs through transfer of a methyl group to a nitrogen atom of arginine. PRMTs are classified as type I (PRMT1, 3, 4) and type II (PRMT5 and 7) according to the production of asymmetric and symmetric dimethylarginine, respectively . Arginine methylation inhibits cytokine production by type I and type II T helper cells . PRMT1-deficient mouse embryonic fibroblasts established through the Cre/lox system show diminished proliferation . Moreover, we have recently shown that membrane immunoglobulin-mediated growth arrests at the G1 phase in WEHI-231 B lymphoma cells, which represent immature B cells, is partially abrogated by knockdown of PRMT1 .
B lymphocytes undergo cell division, differentiation into antibody forming cells and/or isotype switching under the influence of T cell-derived signals including cytokines [7, 8]. In addition to T cell help, signals through TLRs contribute to activation and differentiation of B cells . Thus, together with CD40-L and IL-4, LPSs, agonists for TLR-4, induce isotype switching to IgG1 and IgE , whereas transforming growth factor-β plus LPS favor isotype switching to IgG2b . It has been proposed that the isotype-switching event accompanies cell division and that both are tightly linked [10, 11]. Isotype switching is preceded by germ line transcription, followed by removal of S-region-mediated intervening sequences, forming a circular DNA . Class switch recombination involves several components including AID .
In the present study, we used the arginine methyltransferase inhibitor AdOx to determine whether arginine methylation is involved in IgG1 production by LPS, IL-4 and CD40-L. AdOx inhibited IgG1 antibody production resulting from this combined stimulation; the proportion of IgG1-generating cells from each division was also slightly decreased. The number of cell division cycles was markedly decreased in the inhibitor-treated cells, suggesting that AdOx inhibits IgG1 production through isotype switching as well as cell cycle progression. The role of arginine methylation in the regulation of antibody responses is discussed.
MATERIALS AND METHODS
Purification of B cells and in vitro cell culture
C57BL/6 mice were obtained from Japan SLC (Hamamatsu, Japan) and maintained under pathogen-free conditions at the Tokyo Medical University. They were handled according to the guidelines of the Ethical Committee for Animal Experiments. B cells were purified as described previously . Briefly, red cell-depleted spleen cells were incubated with biotin-conjugated anti-CD43, anti-CD4, and anti-Ter-119 Abs, followed by incubation with magnetic microbeads conjugated with anti-biotin Abs, using a B cell isolation kit (Military Biotech, GmbH, Bergisch Gladbach, Germany). The labeled cells were isolated using autoMACS Pro (Military Biotech). Purified B cells were stimulated with LPS O55:B5 (Sigma, St Louis, MO, USA; 20 µg/mL), recombinant IL-4 (10 ng/mL), and CD40-L-CD8 fusion protein (1:10 dilution of culture supernatant) in the presence or absence of AdOx (Sigma) for the indicated periods.
Western blotting analysis was performed as previously described (6). Briefly, samples (50 µg/lane) were separated by SDS–PAGE, and then transferred to polyvinylidene difluoride membranes Millipore, Billerico, MA, USA). The blots were incubated with primary Abs, anti-asymmetric dimethylarginine (ASYM24, 1:100; Upstate, Lake Placid, NY, USA). The bound primary Abs were then detected with HRP-conjugated with HRP-conjugated goat anti-rabbit IgG (Fc; 1:10,000; ICN Pharmaceuticals, Aurora, OH, USA). Membrane-bound HRP-conjugated Abs were visualized with ECL (GE Healthcare, Little Chalfont, UK).
B cells stimulated or unstimulated with LPS, IL-4, and CD40-L in the presence or absence of AdOx were stained with anti-IgG1-APC (BD Biosciences, San Jose, CA, USA). For determination of number of cell division cycles, resting B cells were labeled with CFSE (Invitrogen, Carlsbad, CA, USA) according to a modified procedure of Lyons and Parish . Briefly, B cells in PBS containing 0.1% BSA were incubated with 1 µM CFSE for 5 min at room temperature. The labeling reaction was quenched by addition of a tenfold volume of PBS/0.1% BSA, and the cells washed twice. Labeled cells were analyzed using flow cytometer (FACSCalibur, Nippon Becton Dickinson, Tokyo, Japan).
Cell viability was determined by a standard dye exclusion test using trypan blue.
Quantification of Ig G1 by enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay was carried out as previously described . Briefly, ELISA plates were coated with 1 µg/mL anti-mouse IgG1 (Southern Biotech, Birmingham, AL, USA). After incubation at 4°C overnight, the supernatants were discarded, followed by washing. Plates pre-incubated with a blocking buffer were washed three times and then samples were added into the wells, followed by incubation at 4°C overnight. After washing three times, alkaline phosphatase-labeled goat IgG Abs specific for IgG1 were added and the plates incubated at 37°C for 1 hr. Color development reaction was performed by incubation with phosphate substrate p-nitorophenyl phosphate (Sigma) for 30 min. The concentrations of IgG1 were determined from a standard curve generated using standard antibody preparations.
Reverse transcription-polymerase chain-reaction
Total RNA was isolated from B cells using TRI REAGENT (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's recommendations. RT-PCR was carried out as previously described . PCR reactions utilized Taq polymerase (Takara, Kyoto, Japan) in combination with primers specific for γ1 germ line transcript (GLT) (forward, 5′-TACAGCCTGGTGTCAACTAG-3′; reverse, 5′-CTGTACATATGCAAGGCTTAC-3′), Aicda (forward, 5′-CGCGGATCCATGGACAGCCTTCTGATGAAGC-3′; reverse, 5′-TTCAAAATCCCAACATACGAAATG-3′), and Igβ (forward, 5′-CAGCAATGACAAGCAGTAGTGAC-3′; reverse, 5′-CCGAAGAGTCACTATGTCTTC-3′). The PCR products were resolved on 2% agarose gels.
Data are expressed as mean ± SD of several independent experiments. Statistical significance was determined by Student's t-test; a difference of P < 0.05 was considered significant.
Protein arginine methylation in B cells stimulated with lipopolysaccharide, interleukin-4, and CD40-ligand
B cells were stimulated with or without LPS, IL-4, and CD40-L (LPS/IL-4/CD40-L) in the presence or absence of AdOx for 24–48 hr. Lysates from the stimulated or control cells were assayed for arginine-methylated protein by western blotting using Abs specific for asymmetric dimethylarginine. Protein arginine methylation of 52 kDa protein (p52), p21, and p19 detected by anti-asymmetric dimethylarginine Abs was enhanced following stimulation with LPS/CD40-L/IL-4, which was reduced by AdOx to unstimulated levels (Fig. 1). Amounts of arginine-methylated proteins in the control unstimulated cultures were also attenuated by AdOx. These results indicate that signaling through TLR, IL-4-receptor and CD40 modulates protein arginine methylation in B cells.
Adenosine-2′,3′-dialdehyde mediates inhibition number of division cycles, but not viability, of B cells
To examine whether protein arginine methylation affects B cell growth in the presence of the PRMT inhibitor AdOx, B cells were cultured with or without various concentrations of AdOx in the presence of LPS/IL-4/CD40-L for 4 days, followed by assay of total viable cells. As shown in Figure 2a, AdOx at 12 µM prevented B cell growth in the presence of LPS/IL-4/CD40-L. However, cell viability remained intact even in the presence of 12 µM AdOx (Fig. 2b). To assess the role of AdOx in cell proliferation, B cells stimulated with LPS/IL-4/CD40-L in the presence or absence of various concentrations of AdOx for the indicated days were assayed for the number of cell division cycles. B cells stimulated with the combination progressively underwent more than seven cycles of division, whereas AdOx markedly reduced the number of cycles in a dose-dependent manner up to 12 µM (Fig. 2c, d). These results suggest that AdOx reduces the number of B cell division cycles induced by LPS/IL-4/CD40-L, while retaining cell viability.
Adenosine-2′,3′-dialdehyde mediates partial inhibition of IgG1 antibody generation following stimulation with lipopolysaccharide, interleukin-4 and CD40-ligand
To examine whether protein arginine methylation affects IgG1 production, B cells were stimulated with or without LPS/IL-4/CD40-L in the presence or absence of 8 µM AdOx for 4 days. In the absence of AdOx, the B cells differentiated into IgG1 positive cells (30.7%) upon combined stimulation, as assessed by flow cytometry (Fig. 3a). Addition of AdOx to the B cell cultures inhibited differentiation of B cells into IgG1 positive cells (15.5%). AdOx inhibited IgG1 generation between 3 µM and 12 µM, as assessed by % IgG1 positive cells (Fig. 3b). A similar tendency was observed when assessed by total IgG1 positive cells, although this was not significant at 3 µM AdOx (Fig. 3c). Interestingly, AdOx-mediated inhibition of LPS/IL-4/CD40-L-induced IgG1 secretion was more pronounced even at 1.5 µM than that of IgG1 positive cells (Fig. 3d). These results suggest that arginine methylation is involved in the generation of IgG1 positive cells, and also in secretion of IgG1.
Adenosine-2′,3′-dialdehyde mediates inhibition of IgG1 positive cell generation in each division cycle
To further examine whether cellular differentiation into IgG1 is influenced by treatment with AdOx, surface IgG1 positive cells in each division cycle were enumerated in the presence of LPS/IL-4/CD40-L with or without 8 µM AdOx (Fig. 4a). Upon combined stimulation, IgG1 positive cells in each division cycle increased in a division cycle number-dependent manner up to seven (Fig. 4b), suggesting that isotype switching is linked to cell division, as proposed by Hodgkin et al. . Addition of AdOx severely reduced the number of IgG1 positive and total cells in late division cycles (numbers 6 and 7), whereas it enhanced them in early division cycles (numbers 3 and 4; Fig. 4b, c). The percentage of surface IgG1 positive cells was slightly reduced by AdOx in the cell divisions from cycles four to six (Fig. 4d). These findings suggest that AdOx inhibits differentiation into IgG1 positive cells as well as cell division.
To examine whether AdOx-mediated inhibition of differentiation into IgG1 positive cells is accounted for by class switch recombination, B cells were stimulated with or without AdOx in the presence of LPS/IL-4/CD40-L for 4 days. Degrees of expression of γGLT and AID were very similar in the AdOx and control groups after stimulation with the LPS/IL-4/CD40-L (Fig. 4e). These results suggest that AdOx inhibits IgG1 generation independent of transcriptional control of AID.
Arginine methylation plays a crucial role in transcription, RNA processing, protein–protein interaction, cell proliferation and differentiation in a variety of cell types, including immune cells [1-3]. For example, arginine methylation regulates cytokine gene expression in T cells [3, 4]. However, the effects of arginine methylation in B cells remain largely unclear. We have shown that arginine methyl inhibitor AdOx inhibits IgG1 antibody generation following stimulation with LPS/IL-4/CD40-L. B cells undergo cell division and differentiate into antibody forming cells and/or undergo isotype switching [7, 8]. In the present study, we used AdOx to determine whether arginine methylation is involved in cell division and/or isotype switching in B cells.
B cell growth is regulated by proliferation and/or cell death. Following stimulation with LPS/IL-4/CD40-L, AdOx reduces the total number of viable cell cycles without altering cell viability, suggesting that arginine methylation plays a crucial role in B cell proliferation. AdOx-mediated diminished proliferation could be explained by a delay in cell cycle progression. This notion is supported by the findings that AdOx mediates delay in the kinetics of B cell division upon stimulation with LPS/IL-4/CD40-L. Consistent with our findings, arginine methylation plays an important role in proliferation of fibroblasts . Regulation of cell cycle progression could be associated with checkpoint activation including G1/S. Indeed, activation of PRMT1 is reportedly involved in G1 checkpoint induced by engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells . Thus, arginine methylation is required for cell cycle progression and checkpoint activation of B cells.
Cell division is reportedly closely associated with isotype switching of B cells . Numbers of IgG1 positive cell cycles in B cells stimulated with LPS/IL-4/CD40-L increased in a cell division-dependent manner, whereas progression of IgG1 positive cells halted around cell division number 4–5 in the AdOx-treated group, probably reflecting an accumulation of cells in division numbers 4–5. Moreover, arginine methylation is required for differentiation to IgG1 positive cells, as evidenced by the ratio of surface IgG1 positive cells to total cell number in each cell division cycle being moderately lower in the AdOx-treated group. Thus, arginine methylation appears to be required for switching from IgM to IgG1 positive cells as well as for cell cycle progression.
Class switch recombination is regulated by several factors including germ line transcription and AID [13, 17]. AID expression is regulated at transcription; AID activity is also modulated at a post-translation level [17, 18]. Because degrees of expression of γ1GLT and AID in the AdOx-treated group were almost identical with those of controls, it is possible that arginine methylation plays a crucial role in the post-translational control of AID activity. Moreover, IgG1 secretion from B cell cultures stimulated with LPS/IL-4/CD40-L is substantially inhibited even at 1.5 µM AdOx, which did not affect generation of total and IgG1 positive cells. Thus, arginine methylation also regulates differentiation from IgG1-switched cells to IgG1-producing plasma cells.
Although the arginine-methylated-molecule(s) critical for cell cycle progression after combined stimulation remain unclear, it would be interesting to check whether arginine-methylated p21 and/or p19 are involved in cell cycle progression and/or differentiation of B cells. In terms of molecular weight, one possible candidate is p19ARF, which is reported to be a negative regulator of p53-destabilizing oncogene murine double minute 2 . Moreover, because AdOx is a general inhibitor of PRMTs, which PRMT is involved in the cell cycle progression and/or differentiation of B cells remains unresolved. Because targeted disruption of PRMT1 results in embryonic lethality , we started to explore the role of PRMT1, a major enzyme among PRMT family members in mammalian cells , in B cell function and established B cell-specific PRMT1-deficient mice via a Cre-lox approach. PRMT1-deficient B cells show decreased IgG1 production in response to LPS/IL-4/CD40-L compared with controls (Hata and Mizuguchi, unpublished observation, 2012). Such an approach in combination with chemical inhibitor AdOx would be valuable for understanding the role of arginine methylation in B cell development and function.
This work was supported by a grant from the Intractable Immune System Disease Research Center of Tokyo Medical University, which is supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The authors declare no competing financial interest.