BALB/c mice were purchased from Damool Science (Daejeon, Korea) and maintained on an 8:16 h light:dark cycle in an animal environmental control chamber. Eight- to twelve-week-old mice were used, and animal care was in accordance with the institutional guidelines of Institutional Animal Care and Use Committee of Konyang University.

Untouched mouse spleen resting B cells were obtained by depletion of CD43⁺ cells using anti-CD43 microbeads and high-gradient magnetic cell separation (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer's instructions. Briefly, BALB/c mouse spleen cell suspensions were washed with HBSS (WelGENE, Daegu, Korea) and treated with 0.83% ammonium chloride to lyse red blood cells. Spleen cells were treated with anti-mouse CD43 microbeads and depleted with LS column and MACS Separator (Miltenyi Biotec). The purity of resting B cells (CD43^-B220⁺) assessed by FACSCalibur (BD Biosciences, San Jose, CA, USA) following staining of the cells with anti-CD43 FITC (eBioscience, San Diego, CA, USA) and anti-B220 PE (BD Biosciences). Cells were cultured at 37℃ in a humidified CO₂ incubator (Forma Scientific, Marietta, OH, USA) in RPMI-1640 medium (WelGENE) supplemented with 10% fetal bovine serum (PAA Laboratories, Etobicoke, ON, Canada). Cells were stimulated with CpG ODN1826 (Invivogen, San Diego, CA, USA), LPS (12.5µg/ml, E. coli 0127:B8, Sigma, St. Louis, MO, USA), TGF-β1 (0.2 ng/ml, R&D Systems, Minneapolis, MN, USA), and retinoic acid (20 nM, Sigma).

Cell viability was determined by EZ-Cytox cell viability assay kit (Daeil Lab Service Co, Seoul, Korea) according to the manufacturer's instructions. Briefly, 20µl of EZ-Cytox kit reagent was added to each cell cultured well of a 96-well microplate and then incubated at 37℃ in a humidified CO₂ incubator for 3 h. After incubation, optical density (OD) was measured at a wavelength of 450 nm using an Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). For cell proliferation assay, purified mouse resting B cells were labeled with CFSE (eBioscience) and then added with stimuli (CpG, LPS, TGF-β1, and retinoic acid). Dilution of CFSE was measured by counting 10,000 cells with a FACSCalibur.

Antibodies produced in B cell cultures were detected by using isotype-specific ELISA. Affinity purified anti-isotype specific antibodies were added at 1.2µg/ml in 0.05 M sodium bicarbonate buffer (pH 9.3) to 96-well U bottomed polyvinyl microplates (Falcon, Becton Dickinson & Co., Oxnard, CA, USA). Plates were washed with PBS containing 0.05% Tween-20 (PBST) followed by overnight incubation at 4℃, and blocked for 1 h with 0.5% BSA solution. After washing, 50µl of standard myeloma proteins and culture supernatants were added to each well and incubated for 1 h at 37℃. After washing, horseradish-peroxidase (HRPO) conjugated anti-isotype specific antibodies (Southern Biotechnology, Birmingham, AL, USA) were added to each well and incubated for 1 h. Plates were then washed and TMB substrate (BD Biosciences) was added. After incubation, 0.05 M sulfuric acid was added to each well and colorimetric reaction was measured at 450 nm with an Absorbance Microplate Reader.

Statistical differences between experimental groups were determined by analysis of variances. Values with p<0.05 by an unpaired two tailed Student's t test were considered significant.

To elucidate the direct effect of CpG on B cell activation and differentiation, we used untouched resting B cells (CD43^-B220⁺) from mouse spleen in this study. As shown in Fig. 1A, purity of the resting B cells assessed by flow cytometric analysis was higher than 97%. To determine the basic property of CpG in mouse B cell activation, we first examined the effect of CpG on mouse B cell growth. As shown in Fig. 1B, CpG constantly increased cell growth up to 2 days and dose-dependently sustained cell viability on day 3 of culture. Overall cell viability on day 3 was lower than day 2. This seems due to the exhaustion of nutrients in the culture media. In addition, we observed that CpG induces cell proliferation in a dose-dependent manner (Fig. 1C). Thus, CpG can act as a potent B cell mitogen and this is consistent with an earlier report that CpG induces B cell cycle entry and cell survival (19). Since CpG strongly increased B cell proliferation, it was important to explore whether it affects Igs production. As shown in Fig. 1D, CpG dose-dependently increased all Ig isotypes production and this function was consistent with results from cell growth.

LPS is a well-known mitogen for mouse B cells and a stimulant for TLR4 (20,21). Therefore, we next compared the effects of CpG and LPS on B cell growth and Igs production. Along with LPS, we adopted TGF-β1 to determine whether CpG can support TGF-β1-induced IgA expression. TGF-β1 is a well-known IgA class switching factor in mouse B cells (22,23). Here, we used 100 nM CpG because this dosage showed great B cell viability and proliferation as shown in Fig. 1B and C. CpG enhanced cell viability and proliferation and this effect was greater than that of LPS on day 2 of culture (Fig. 2A and B). In addition, we found that these two stimuli have neither additive nor synergetic effect on cell viability. As previously shown by other (24), TGF-β1 markedly inhibited the increase of cell viability and proliferation caused by LPS on day 2 of culture (Fig. 2A and B). However, TGF-β1 did not inhibit CpG-induced cell viability and had a little effect on inhibiting CpG-induced cell proliferation. This different effect of TGF-β1 between LPS and CpG is likely to arise because mitogenic potency of CpG is greater than that of LPS. As for Ig production, CpG was less stimulatory than LPS, especially IgM and IgG2b isotypes (Fig. 2C). Interestingly, CpG decreased LPS-induced IgM production (Fig. 2C) although the cell viability and proliferation were not decreased under the same conditions (Fig. 2A and B). On the other hand, CpG was unable to support TGF-β1-induced IgA and IgG2b production (Fig. 2C). This result is sharply contrasted to that of LPS. In addition, CpG also down-regulated TGF-β1-induced IgA/IgG2b production in the presence of LPS. We assumed that this effect of CpG is attributed to its excessive mitogenic property. To resolve these unexpected results, we decided to test low doses of CpG in cell viability and cell proliferation. As shown in Fig. 3, we observed that 10 nM CpG looks sufficient for supporting cell viability and proliferation. Therefore, 10 nM CpG was used for further experiments.

Next, we tested the effect of 10 nM CpG on Igs production in parallel with LPS. We used TGF-β1 and retinoic acid (RA) as anti-proliferative agents to alleviate the excessive proliferative activity of CpG. It was reported that RA specifically induces IgA expression (25,26). As shown in Fig. 4, 10 nM CpG was still less stimulatory than LPS in IgM and IgA production as seen in high concentration of CpG. LPS supported TGF-β1-induced IgA secretion but not CpG (Fig. 4). Interestingly, CpG supported RA-induced IgA production. This different action of CpG may be caused by different anti-proliferative effects of TGF-β1 and RA to properly regulate B cell growth and differentiation into cells producing Abs. In addition, unlike 100 nM CpG, 10 nM CpG did not reduce LPS/TGF-β1-induced IgA production. Moreover, when RA was added on day 0, CpG substantially increased LPS/RA-induced IgA production (Fig. 4). These results suggest that CpG can regulate the mouse B cell proliferation and differentiation and that optimum dosage of CpG can cooperate with LPS to produce Igs.

In the present study, we found that CpG, a TLR9 stimulant, shows a powerful mitogenic potency for mouse spleen B cells, which was greater than LPS, a well-known mouse B cell polyclonal activator. Nevertheless, CpG was less stimulatory than LPS in all Ig isotypes production. Surprisingly, CpG down-regulated the LPS-stimulated Igs production. It is assumed that this function of CpG is attributed to the excessive mitogenic property. When low concentration of CpG was used, it supported RA-induced IgA production and did not down-regulate LPS/TGF-β1-induced IgA production. Moreover, CpG further augmented LPS/RA-induced IgA production. In conclusion, our results indicate that the optimal dose of TLR9 agonist CpG and TLR4 agonist LPS cooperate to direct stimulate B cells to proliferate and differentiate into cells producing Igs.