Anat Cell Biol.  2016 Jun;49(2):88-98. 10.5115/acb.2016.49.2.88.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro

Affiliations
  • 1Department of Anatomy and Cell Biology, Seoul National University College of Medicine, Seoul, Korea. hyi830@snu.ac.kr

Abstract

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Keyword

Vitamin C; Human T cell; T cell activation; Sodium-dependent vitamin C transporter 2; Anti-oxidant

MeSH Terms

Adaptive Immunity
Antioxidants
Apoptosis
Ascorbic Acid*
Culture Media
Humans*
In Vitro Techniques*
Interleukin-2
Micronutrients
Sodium-Coupled Vitamin C Transporters*
T-Lymphocytes*
Vitamins*
Antioxidants
Ascorbic Acid
Culture Media
Interleukin-2
Micronutrients
Sodium-Coupled Vitamin C Transporters
Vitamins

Figure

  • Fig. 1 SVCT-2 expression in human T cells. (A) Total RNA was extracted from human T cells and cDNA was synthesized. The expression of SVCT-1 and -2 was assessed using reverse transcription polymerase chain reaction. (B) T cells were lysed and subjected to western blotting for the expression of SVCT-2. HepG2 (hepatoma cell line) cells were used as positive control of SVCT-1 and -2. (C) Human T cells were collected 24 hours or 48 hours after activation with phorbol-12-myristate-13-acetate/ionomycin, attached on a slide, and briefly fixed with formalin. The expression of SVCT-2 was assessed by immunofluorescence staining. The staining was observed under a confocal microscope.

  • Fig. 2 Vitamin C uptake by human T cells. Cells were activated with phorbol-12-myristate-13-acetate/ionomycin for 48 hours, re-allocated in a various culture condition (4×106 cells/group), and added with vitamin C at a concentration of 0.5 mM. After 2 hours, cells were harvested and vitamin C concentration was measured using DNPH method. The experiment was repeated 3 times with triplicated samples per each experiment, and a representative one is presented. No VC, not treated with vitamin C; VC, treated with vitamin C; DTT+VC, treated with vitamin C in the presence of dithiothreitol (DTT); Na+ free buffer+VC, cells stayed in Na+ free buffer with vitamin C; Na+ free buffer+ DTT+VC, cells stayed in Na+ free buffer with vitamin C and DTT in it. **P<0.01.

  • Fig. 3 The effects of vitamin C on proliferation of human T cells. Cells (1×105/well) were plated in flat bottom 96-well and activated with phorbol-12-myristate-13-acetate (PMA)/ionomycin or anti-CD3 and anti-CD28 antibodies. Vitamin C was treated 2 hours before (A) or 24 hours after activation (B) with various concentrations of vitamin C as indicated. [3H]-thymidine was added at 48 hours, cultured additional 16 hours, and counter per minute (CPM) values were measured. The experiment was repeated three times and a representative one is shown. All the samples were quadriplicated. **P<0.01.

  • Fig. 4 Human T cells (1×105 cells/well) were cultured in a 96-well plate and activated with phorbol-12-myristate-13-acetate (PMA)/ionomycin. Cells were treated with 0.5 mM vitamin C 3, 6, 12, or 24 hours after activation, added with [3H]-thymidine at 48 hours, cultured for additional 16 hours, and counter per minute (CPM) values were measured. All the samples were quadriplicated. The experiment was repeated three times and a representative one is shown. Control group was cultured without vitamin C treatment. **P<0.01.

  • Fig. 5 The effects of vitamin C on human T cell apoptosis. Cells were activated with phorbol-12-myristate-13-acetate/ionomycin, added with 0.5 mM vitamin C 2 hours before or 24 hours after activation, stained with Annexin V and propidium iodide (PI), and subjected to flow cytometric analysis. The experiment was repeated three times and a representative one is shown. Control group was cultured without vitamin C treatment.

  • Fig. 6 Expression of CD69 and CD25 under vitamin C treatment at various times before and after activation. Human T cells (5×105 cells/well) were cultured for 2 hours in a 24-well plate with 1 mM vitamin C before phorbol-12-myristate-13-acetate/ionomycin activation and 3, 6, and 12 hours after activation Twenty-four hours after activation, cells were harvested, stained with appropriate antibodies, and were subjected to flow cytometric analysis. The experiment was repeated three times and a representative one is shown. Numbers are in %. SSC, side-scattered light; FSC, forward-scattered light.

  • Fig. 7 Effects of high concentration vitamin C on interleukin 2 (IL-2) secretion in human T cells. Cells from 4 individuals were cultured in a 24-well plate and activated with phorbol-12-myristate-13-acetate/ionomycin. Vitamin C was added to the culture media 2 hours before or 24 hours after activation, or not added (control). Culture supernatants were obtained at 36 hours and IL-2 amount were analysed by enzymelinked immunosorbent analysis. Data are presented as absolute concentrations (A) or normalized values (B) to the control data. Samples were quadriplicated. *P<0.05, **P<0.01.


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