The cystine/glutamate antiporter regulates dendritic cell differentiation and antigen presentation

Abstract

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.

FIGURE 1

Human DC maturation is associated with increased transport of cystine and glutamate. A and B, Immature (IMDC) and mature (LPS-DC) DCs were incubated for 5 min at 37°C or 4°C in medium containing [³H] glutamate or [¹⁴C] cystine. The quantity of glutamate or cystine transported into the cell is expressed in picomoles per milligram of cellular protein. Data are mean values ± SEM of triplicate samples from three independent experiments (i.e. three different donors). C, Immature DCs were treated for 30 min with 1mM dimethyl amiloride (DMA), washed and then incubated for 5 min at 37°C in uptake medium containing [³H] glutamate. Data are mean values ± SEM of triplicate samples from two independent experiments. D, IMDC and LPS-DC were incubated for 5 min at 37°C in medium containing [³H] glutamate. The quantity of glutamate transported into the cell is expressed in picomoles per milligram of cellular protein. Data are mean values ± SEM of triplicate samples using DCs prepared from 23 different donors. The means are indicated by open diamonds. Data are displayed as paired data points.

FIGURE 2

The functional subunit of the cystine/glutamate antiporter is transcriptionally regulated during human DC maturation. Immature DCs were incubated with or without LPS for 4 h and then cultured in complete medium or in cystine/cysteine-free medium for 6 or 16 h. RNA transcripts encoding xCT were quantified by digital mRNA profiling. Data are mean values ± SEM of duplicate samples from three independent experiments.

FIGURE 3

Functional characterization of the cystine/glutamate antiporter in human DCs. Immature (IMDC) and mature (LPS-DC) DCs were incubated in medium for 30 min at 37°C with or without LHC (10mM) or in medium containing excess glutamate (2.5mM) or cystine (1mM) and then examined for transport of [³H] glutamate (200μM) (A) or [¹⁴C] cystine (200μM) (B). Data are mean values ± SEM of triplicate samples from two independent experiments.

FIGURE 4

The cystine/glutamate antiporter regulates human DC glutathione homeostasis. A-C, Immature (IMDC) and mature (LPS-DC) DCs were cultured for 24 h in complete medium, LHC-containing medium (2.5mM), or cystine/cysteine-free medium. Cells were then lysed and GSH and GSSG were measured in lysates by HPLC. Data were normalized to total cellular protein. Reduced glutathione (A), oxidized glutathione (B) and the GSH-to-GSSG ratio (C) were plotted (note differences in the y-axis scales between A and B). Data represent single measurements from four independent experiments. D and E, Glutathione efflux was measured by culturing immature (IMDC) and mature (LPS-DC) DCs for 24 h in complete medium, medium containing LHC (2.5mM) or cystine/cysteine-free medium. Cells were then removed by centrifugation and GSH and GSSG in the medium was measured by HPLC. Data were normalized to total cellular protein. Data are plotted as GSH or GSSG in nmol per mg of protein (D) and as GSH content relative to the non-treated immature and mature DCs (=100%) (E). Data represent single measurements from four independent experiments.

FIGURE 5

The cystine/glutamate antiporter regulates human DC development from monocyte precursors. Monocytes were stained for markers following culture for five days with IL-4 and GM-CSF in the presence or absence of LHC (10mM). The MFI (log scale) is plotted on the y-axis. Data are mean values ± SEM of triplicate samples from four independent experiments.

FIGURE 6

The activity of the cystine/glutamate antiporter does not regulate the phenotypic maturation of human DCs. Immature DCs were incubated with or without LPS for 24 h and then cultured with or without LHC (10mM) an additional 24 h. The cell surface expression of MHC class I, MHC class II, CD80, CD83 and CD86 was monitored by flow cytometry. The MFI (log scale) is plotted on the y-axis. Data are mean values ± SEM of triplicate samples from three independent experiments.

FIGURE 7

Blocking the activity of the cystine/glutamate antiporter in human DCs does not interfere with antigen uptake. Immature DCs were treated with or without LHC (10mM) for 2 or 24 h, washed and incubated with FITC-dextran in the presence or absence of LHC for 30, 60 and 90 mins. The data were normalized to the MFI of non-treated DC incubated with FITC-dextran for 90 mins (= 100%). Means ± SEM of triplicate measurements for each time point from two independent experiments are shown.

FIGURE 8

The cystine/glutamate antiporter regulates murine splenic DC antigen presentation to T cells. Murine splenic DCs were incubated for 2 or 16 h in complete medium or in complete medium containing LHC (10mM) and then pulsed with OVA (0–1,000μg). DCs were cultured with OT-I or OT-II cells for 3 days after which co-cultures were spiked with 3H-thymidine and incubated overnight. T cell proliferation was measured by liquid scintillation counting (A and C) and granzyme B and IFN-γ were quantified in culture supernatants by ELISA (B and D). Data are from one experiment in which samples were analyzed in triplicate (nd= not detectable).

FIGURE 9

The cystine/glutamate antiporter regulates DC cross-presentation. Splenic DCs were incubated in complete medium or in complete medium containing LHC (10mM) for 2 or 16 h and then pulsed with OVA (500μg). DCs were then cultured with CFSE-labeled OT-I cells at a ratio of 1:1 (A) or 1:3 (B) for 3 days after which T cell proliferation was measured by CFSE dilution by flow cytometry. Data are from one experiment in which samples were analyzed in triplicate.