Immune Netw.  2013 Feb;13(1):10-15. 10.4110/in.2013.13.1.10.

Alum Directly Modulates Murine B Lymphocytes to Produce IgG1 Isotype

• Jin BR
• Kim SJ
• Lee JM
• Kang SH
• Han HJ
• Jang YS
• Seo GY
• Kim PH

• KMID: 2150765
• DOI: http://doi.org/10.4110/in.2013.13.1.10

Abstract

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTgamma1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.

Keyword

Alum; IgG1; B lymphocyte; Isotype switch

MeSH Terms

Alum Compounds
Aluminum Hydroxide
Animals
B-Lymphocytes
Cell Count
Enzyme-Linked Immunosorbent Assay
Enzyme-Linked Immunospot Assay
Humans
Hydroxides
Immunoglobulin Class Switching
Immunoglobulin G
Mice
Spleen
Vaccines
Alum Compounds
Aluminum Hydroxide
Hydroxides
Immunoglobulin G
Vaccines

Figure

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  Figure 1 Effect of alum on Igs secretion in mouse spleen B cells. Mouse spleen B cells were cultured with LPS (12.5µg/ml) and alum (30µg/ml, 60µg/ml, 120µg/ml). After 7 days of culture, supernatants were collected and Igs productions were determined by isotype-specific ELISA. Data are means of triplicate samples± SEM.

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  Figure 2 Effect of alum on the viability of mouse spleen B cells. Mouse spleen B cells were cultured with LPS (12.5µg/ml) and alum (30µg/ml, 120µg/ml). Cell viability was assessed using trypan blue staining. Data are means of triplicate samples±SEM.

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  Figure 3 Effect of alum on Ig secretion in the presence of IL-4 and TGF-β1. Mouse spleen B cells were cultured with LPS (12.5µg/ml), alum (120µg/ml), IL-4 (10 ng/ml), and TGF-β1 (0.5 ng/ml) for 7 days. Culture supernatants were collected and Igs productions were determined by isotype-specific ELISA. Data are means of triplicate samples±SEM. *p<0.05.

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  Figure 4 Effect of alum on numbers of IgG1 secreting cells. Mouse spleen B cells were cultured with LPS (12.5µg/ml) and alum (120µg/ml). After 4 days, IgG1 spot-forming cells were enumerated by ELISPOT assay (left panel). The bar graph represents the average of IgG1 spot numbers±SEM.

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  Figure 5 Effect of alum on the expression of Ig GLTs and AID. Mouse spleen B cells were cultured with LPS (12.5µg/ml) and alum as indicated for 2 days. (A) Diagram of DNA recombination occurring during switching to IgG1. Rectangles and ovals represent exons and S regions, respectively. RNA transcripts are indicated beneath the DNA diagrams. (B) Levels of GLTs, CTγ1, and β-actin were measured by RT-PCR. (C-1) Effect of alum on endogenous AID transcriptional level was measured after 2 days of culture by RT-PCR. (C-2) CH12F3-2A cells were transfected with 5µg of pAID1. Luciferase activity was determined following alum treatment as indicated for 48 h. Transfection efficiency was normalized to β-gal activities. Data represent average of two independent samples.

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  Figure 6 Effect of alum on T cell differentiation. Mouse spleen whole cells were stimulated with plate bound anti-CD3 mAb, soluble anti-CD28 mAb and IL-2. Spleen whole cells were incubated with alum (120µg/ml) for 2 days. Levels of IFN-γ, T-bet, IL-4, GATA-3, CXCR5, and Foxp3 transcripts were determined by RT-PCR. Fold increases represent relative DNA level normalized with the expression of β-actin cDNA.

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