Ascorbate recycling in human neutrophils: induction by bacteria

Abstract

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen Candida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor in C. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.

Figure 1

Induction of ascorbate recycling by different microorganisms. Neutrophils were incubated with 100 μM ascorbate for 45 min with the indicated microorganism/neutrophils (effector/target) ratios for the following microorganisms: E. coli CP9 (○) or CP922 (•), E. faecalis (▪), M. catarrhlis (□), K. oxytoca (▾), A. baumanii (▵), and C. albicans (▴). Neutrophils incubated with ascorbate and no microorganisms are indicated by (). Intracellular ascorbate was measured by scintillation spectrometry and is shown as mM (left axis) and nmol/mg protein (right axis).

Figure 2

Dependence of ascorbate recycling on time and extracellular ascorbate concentrations. Intracellular ascorbate was measured by scintillation spectrometry and is shown as mM (left axis) and nmol/mg protein (right axis). Neutrophils were incubated with E. coli CP9 (effector/target ratio 28:1) for the indicated times with extracellular ascorbate concentrations of 10 μM (▪), 25 μM (□), 50 μM (▴), 75 μM (▵), 100 μM (•), and 200 μM (▿). Control neutrophils (○) were incubated without bacteria and 100 μM ascorbate for the times indicated. (Inset) Dependence of recycling on extracellular ascorbate concentrations at one fixed time. Neutrophils were incubated with (•) or without (○) E. coli CP9 (effector/target ratio, 28:1) and the indicated extracellular ascorbate concentrations for 30 min. The y axis is the same as in Fig. 2.

Figure 3

Ascorbate recycling in chronic granulomatous disease neutrophils. Chronic granulomatous disease neutrophils were incubated with 100 μM ascorbate (• and ○) for 45 min or 300 μM dehydroascorbic acid (▪ and □) for 5 min and the indicated target/effector ratios for E. coli CP9 (• and ▪) or E. coli CP922 (○ and □). Intracellular ascorbate was measured by HPLC and is shown as mM (left axis) and nmol/mg protein (right axis).

Figure 4

(A) Uptake of glucose, ascorbate, and dehydroascorbic acid by E. coli CP9. Bacteria were incubated with 400 μM [¹⁴C]ascorbate (▪), [¹⁴C]dehydroascorbic acid (•), [¹⁴C]glucose (▴), or [¹⁴C]galactose (▾) (Inset) for the indicated times. Uptake was measured by scintillation spectrometry. (B) Ascorbate recycling in either neutrophils or microorganisms. Different microorganisms or neutrophils were incubated with the indicated concentrations of extracellular dehydroascorbic acid for 5 min. Intracellular ascorbate was measured by HPLC. Symbols represent neutrophils (⧫), E. coli CP9 (○), E. faecalis (▪), M. catarrhlis (□), K. oxytoca (▾), A. baumanii (▿), and C. albicans (▾).