Immune Netw.  2012 Jun;12(3):89-95. 10.4110/in.2012.12.3.89.

Kinetic Analysis of CpG-Induced Mouse B Cell Growth and Ig Production

• Kim YH
• Lee SH
• Yoo YC
• Lee J
• Park JH
• Park SR

• KMID: 2168006
• DOI: http://doi.org/10.4110/in.2012.12.3.89

Abstract

Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-beta1-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-beta1-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-beta1-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-beta1-nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.

Keyword

CpG; B cell; TLR9; Immunoglobulin

MeSH Terms

Animals
Cell Survival
DNA, Bacterial
Immune System
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Immunoglobulins
Kinetics
Mice
Toll-Like Receptors
DNA, Bacterial
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Immunoglobulins
Toll-Like Receptors

Figure

• 

  Figure 1 Effect of CpG on mouse B cell growth and Igs production. (A) Purified mouse spleen resting B cell population (CD43-B220+) was measured by flow cytometric analysis. Data shown are representative of all the experiments carried out in this study. (B) Resting B cells were stimulated with the indicated doses of CpG (0 nM, open triangle; 20 nM, closed triangle; 40 nM, open circle; 60 nM, closed circle; 80 nM, open square; 100 nM, closed square). After 2 and 3 days of culture, cell viability was measured by EZ-Cytox cell viability assay. Data are averages of duplicate samples with ranges (bars). (C) Culture conditions were the same as in (B). After 2 and 3 days of culture, cell proliferation was measured by the dilution of CFSE. The percentage indicates cells (%) with low level of CFSE intensity. (D) Culture conditions were the same as in (B). After 7 days of culture, supernatants were harvested and the levels of Igs production were determined by isotype-specific ELISA. Data represent means±SEM of triplicate samples.

• 

  Figure 2 Comparison of effects of CpG and LPS on B cell growth and Igs production. Mouse spleen resting B cells were stimulated with CpG (100 nM), LPS (12.5µg/ml), and TGF-β1 (0.2 ng/ml). (A) After 2 and 3 days of culture, cell viability was measured by EZ-Cytox cell viability assay. Data are averages of duplicate samples with ranges (bars). (B) After 2 and 3 days of culture, cell proliferation was measured by the dilution of CFSE. (C) After 7 days of culture, supernatants were harvested and the levels of Igs production were determined by isotype-specific ELISA. Data represent means±SEM of triplicate samples. *p<0.05.

• 

  Figure 3 Effects of lower doses of CpG on B cell viability and proliferation. Mouse spleen resting B cells were stimulated with the indicated doses of CpG (0 nM, open circles; 2.5 nM, closed triangles; 5 nM, open squares; 10 nM, closed circles; 20 nM, open triangles; 40 nM, closed squares). After 1, 2, 3, and 4 days of culture, cell viability was measured by EZ-Cytox cell viability assay (A), and cell proliferation was measured by the dilution of CFSE (B). CFSE intensity (%) of panel B represents the percentage of cells with a low level of CFSE intensity. Data are averages of duplicate samples with ranges (bars).

• 

  Figure 4 Effect of low dose CpG on TGF-β1- and RA-induced Igs production. Mouse spleen resting B cells were stimulated with CpG (10 nM) and LPS (12.5µg/ml). TGF-β1 (0.2 ng/ml) and retinoic acid (RA, 20 nM) were added on day 0 (D0) or day 1 (D1). After 7 days of culture, supernatants were harvested and the levels of IgM and IgA production were determined by isotype-specific ELISA. Data represent means±SEM of triplicate samples. *p<0.05.

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