The Role of Physiological Vitamin C Concentrations on Key Functions of Neutrophils Isolated from Healthy Individuals

Abstract

Vitamin C (ascorbate) is important for neutrophil function and immune health. Studies showing improved immune function have primarily used cells from scorbutic animals or from individuals with infectious conditions or immune cell disorders. Few studies have focused on the requirements of neutrophils from healthy adults. Therefore, we have investigated the role of vitamin C, at concentrations equivalent to those obtained in plasma from oral intakes (i.e., 50-200 µmol/L), on key functions of neutrophils isolated from healthy individuals. Cells were either pre-loaded with dehydroascorbic acid, which is rapidly reduced intracellularly to ascorbate, or the cells were activated in the presence of extracellular ascorbate. We measured the effects of enhanced ascorbate uptake on the essential functions of chemotaxis, oxidant production, programmed cell death and neutrophil extracellular trap (NET) formation. We found that neutrophils isolated from healthy individuals already had replete ascorbate status (0.35 nmol/10⁶ cells), therefore they did not uptake additional ascorbate. However, they readily took up dehydroascorbic acid, thus significantly increasing their intracellular ascorbate concentrations, although this was found to have no additional effect on superoxide production or chemotaxis. Interestingly, extracellular ascorbate appeared to enhance directional mobilityin the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Stimulation of the cells in the presence of ascorbate significantly increased intracellular ascorbate concentrations and, although this exhibited a non-significant increase in phosphatidylserine exposure, NET formation was significantly attenuated. Our findings demonstrate the ability of neutrophils to regulate their uptake of ascorbate from the plasma of healthy humans to maintain an optimal level within the cell for proper functioning. Higher oral intakes, however, may help reduce tissue damage and inflammatory pathologies associated with NET formation.

Keywords: ascorbate; chemotaxis; immunity; neutrophil extracellular traps; neutrophils; vitamin C.

Figure 1

Uptake of ascorbate by neutrophils isolated from healthy individuals. (A) Cells were incubated with ascorbate (○) or DHA (■), 200 µmol/L, in HBSS for up to 90 min. Intracellular ascorbate was measured by HPLC and expressed as the increase above that of cells processed at 0 min (0.38 ± 0.05 nmol/10⁶ cells), n = 3. (B) Cells were incubated with increasing concentrations of DHA for 15 min and the intracellular ascorbate concentration expressed as the increase above that of cells in HBSS (0.32 ± 0.01 nmol/10⁶ cells), n = 3. Data represent mean ± SEM, * p < 0.05.

Figure 2

The effect of ascorbate uptake on neutrophil chemotaxis. (A) The directional motility of preloaded cells towards a microbial chemoattractant (fMLP). Graphical representation of the fluorescence detected in the lower assay compartment after 30 min. (B) The intracellular ascorbate concentration of the preloaded cells was measured using HPLC. Data represent mean ± SEM, n = 3, * p < 0.05.

Figure 3

The effect of extracellular ascorbate on neutrophil chemotaxis. The directional motility of cells towards fMLP alone, or with extracellular ascorbate (100 µmol/L), was measured. Graphical representation of the fluorescence detected in the lower assay compartment after 30 min. Data represent mean ± SEM, n = 3.

Figure 4

The effect of ascorbate uptake on superoxide generation by stimulated neutrophils. (A) Cells pre-loaded with DHA were stimulated with PMA (100 ng/mL) and the rate of superoxide generation measured over 5 min. (B) The intracellular ascorbate concentration of preloaded cells was measured by HPLC. Data represent mean ± SEM, n = 3. * p < 0.05.

Figure 5

Ascorbate uptake by stimulated neutrophils. (A) Cells were incubated with extracellular ascorbate (100 µmol/L) for the indicated times, in the absence (○) or presence (■) of PMA (100 ng/mL). The time zero ascorbate concentration was 0.34 ± 0.134 nmol/10⁶ cells, n = 4. (B) Cells were incubated with increasing concentrations of ascorbate for 45 min in the presence of PMA (100 ng/mL). The starting ascorbate concentration was 0.29 ± 0.11 nmol/10⁶ cells, n = 3. The intracellular ascorbate concentration was measured by HPLC. Data represent mean ± SEM, * p < 0.05.

Figure 6

Effect of ascorbate on phosphatidylserine exposure of activated neutrophils. Cells were incubated with extracellular ascorbate (0, 100 or 200 µmol/L) and stimulated with PMA (100 ng/mL) for 2 h (white bars) or 4 h (grey bars). Phosphatidylserine exposure was measured by flow cytometry with Annexin-V FITC. Data represent mean ± SEM, n = 5.

Figure 7

Effect of ascorbate on NET production by activated neutrophils. Cells were incubated with extracellular ascorbate (0, 100 or 200 µmol/L) and stimulated with PMA (2 μg/mL) for 4 h. Extracellular DNA was stained with SytoxGreen™ and fluorescence measured as a marker of NET production. Data represent mean ± SEM, n = 5.