Anat Cell Biol.  2010 Mar;43(1):25-35. 10.5115/acb.2010.43.1.25.

Vitamin C acts indirectly to modulate isotype switching in mouse B cells

• Woo A
• Kim JH
• Jeong YJ
• Maeng HG
• Lee YT
• Kang JS
• Lee WJ
• Hwang YI

• KMID: 2168894
• DOI: http://doi.org/10.5115/acb.2010.43.1.25

Abstract

Vitamin C, one of essential micronutrients, has been reported to modulate the humoral immune responses in some mammals. We investigated whether vitamin C might modulate this response in mice by directly affecting B cells. Splenic B cells were isolated and activated by CD40- and B cell receptor-ligation in vitro. The cells were cultured with a pretreatment of vitamin C from 0 to 1 mM of concentrations. Vitamin C slightly increased apoptosis of B cells dose-dependently and behaved as an antioxidant. We found that in vivo administration of vitamin C by intraperitoneal injection affected isotype switching as previously reported: the titer of antigen-specific IgG1 antibody was decreased, while that of IgG2a was unaffected. Somewhat different from those observed in vivo, in vitro exposure to vitamin C slightly decreased isotype switching to IgG1 and increased isotype switching to IgG2a. Pretreatment with vitamin C in the safe range did not affect either proliferation of cultured B cells or the expression of CD80 and CD86 in those cells. Taken together, in vivo results suggest that vitamin C acts to modulate isotype switching in the mouse. However, because of our in vitro results, we suggest that the modulation exerted by vitamin C in vivo is by indirectly affecting B cells, perhaps by directly influencing other immune cells such as dendritic cells.

Keyword

Vitamin C; antioxidant; reactive oxygen species; mouse B cell; isotype switching

MeSH Terms

Animals
Apoptosis
Ascorbic Acid
B-Lymphocytes
Dendritic Cells
Immunity, Humoral
Immunoglobulin Class Switching
Immunoglobulin G
Injections, Intraperitoneal
Mammals
Mice
Micronutrients
Reactive Oxygen Species
Vitamins
Ascorbic Acid
Immunoglobulin G
Micronutrients
Reactive Oxygen Species
Vitamins

Figure

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  Fig. 1 Administration of vitamin C in vivo markedly reduced serum IgG1 titer. Mice were injected with nothing (control group), PBS, 0.625 mg, or 5 mg of vitamin C during primary immunization with 100 µg Keyhole limpet hemmaglutinin (KLH). Seven days after second immunization on day 21, sera were obtained and ELISA was performed for titration of total, IgG1, and IgG2a KLH-specific antibodies. Titers are expressed as relative units to standard serum, which was regarded as 2.5. IgG1 titers of two vitamin C-injected groups were significantly lower (*) that that of PBS-injected group (P=0.018 and 0.012, respectively). Values are mean±SD (n=8 in each group).

• 

  Fig. 2 Vitamin C slightly induced apoptosis of activated B cells dose-dependently. B cells were seeded at a density of 1×106/well in 24-well plates and pre-incubated for 1 hour with varying concentrations of vitamin C as indicated. Cells were then activated with anti-IgM and anti-CD40 antibodies for 24 hours, were harvested and stained with Annexin V-FITC and propidium iodide, and flowcytometric analysis was performed. The concentrations of vitamin C are marked at the top of each panel. The data are representative of three independent experiments.

• 

  Fig. 3 Vitamin C lowered ROS levels in activated B cells. B cells were loaded with 50 µM DCFH-DA for 30 minutes, and then washed and plated to 96-well plates at a density of 4×105 cells/well. After that, cells were pre-treated with vitamin C at concentrations indicated (mM) for 1 hour before activation with anti-IgM and anti-CD40 antibodies. The fluorescence intensities were measured with 485 nm and 538 nm of excitation and emission frequencies, respectively. At 60 minutes, cultures treated with 0.0625 to 0.5 mM vitamin C lowered intracytoplasmic level of ROS compared to that of the 0 mM group (P<0.05). For 1 mM group, the ROS level was still lower than that of 0 mM group (P<0.05), but higher than those of other vitamin C-treated groups (P<0.05). Values represent mean±SD of triplicate wells, from a typical one out of three independent experiments.

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  Fig. 4 Vitamin C did not affect the proliferation of activated B cells. B cells in 96-well plates at a density of 4×105 cells/200 µL/well were activated with anti-IgM and anti-CD40 antibodies in the presence or absence of vitamin C. After 48 hours, cells were pulsed with 1 µCi of 3H-thymidine for an additional 18 hours. Vitamin C did not induce significant change of proliferating ability of activated B cells (P>0.05). Values represent mean cpm±SD of triplicate cultures.

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  Fig. 5 Vitamin C did not affect the expression of CD80 and CD86 molecules on activated B cells. B cells were pre-treated with vitamin C of various concentrations as indicated for 1 hour and then activated with anti-IgM and anti-CD40 antibodies. Cells were harvested and stained for surface expression of co-stimulatory molecules, CD80 and CD86, and were analyzed by flow cytometry. The numbers in each panel indicate the ratio of B220 and either CD80 or CD86 double positive cells. The concentrations of vitamin C are marked at the top of each panel. The data are representative of three independent experiments.

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  Fig. 6 The in vitro effects of vitamin C on immunoglobulin production by activated B cells. B cells in 24-well plates at a density of 1×106 cells/1 mL/well were pre-treated with vitamin C, activated with anti-IgM and anti-CD40 antibodies, and cultured for 6 days. Vitamin C was added repeatedly every second day during culture periods. Cell supernatants were collected and the titers were determined for total Ig, IgG1, and IgG2a by ELISA. Results for total immunoglobulin are expressed as relative values to standard serum, which was regarded as 1,000 as described in MATERIALS AND METHODS. Values are mean±SD of triplicate culture wells. *P<0.05.

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