. 1999 Sep;117(3):482–488. doi: 10.1046/j.1365-2249.1999.01009.x

Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction

PMCID: PMC1905365 PMID: 10469051

Abstract

Decreased glutamine concentrations are found during catabolic stress and are related to susceptibility to infections. However, little is known about the mechanism of glutamine modulation of lymphocyte functions. Glutamine is not only an important energy source in mitochondria, but is also a precursor of glutamate, which is used for cellular glutathione (GSH) biosynthesis in lymphocytes. In this study, we investigated the effects of glutamine on the redox reaction during lymphocyte proliferation. Peripheral blood mononuclear cells, obtained from healthy adult volunteers, were cultured and stimulated by phytohaemagglutinin (PHA) in the presence of different glutamine concentrations. Cells were harvested and prepared for analysis of lymphocyte proliferation, cell cycle propagation, intracellular glutathione levels and reactive oxygen species (ROS) production. We found that glutamine supplementation significantly enhanced PHA-stimulated lymphocyte proliferation and propagation of the cell cycle from the G₁ to S and G₂/M phases. Glutamine also enhanced production of both intracellular ROS and GSH levels in PHA-stimulated lymphocytes. Flow cytometric analysis by the mercury orange staining method showed that glutamine significantly enhanced intracellular non-protein thiols in PHA-stimulated CD4⁺, but not CD8⁺ lymphocyte subsets. Furthermore, intracellular GSH detected by monochlorobimane dye probe showed that glutamine enhanced GSH both in PHA-stimulated CD4⁺ and CD8⁺ lymphocyte subsets. Inadequate glutamine supplementation resulted in decreased lymphocyte proliferation in association with decreased levels of intracellular GSH. Addition of exogenous GSH significantly enhanced lymphocyte proliferation, whereas blockade of GSH synthesis enhanced ROS production and suppressed lymphocyte proliferation. These results suggest that the modulation of PHA-stimulated lymphocyte proliferation by glutamine is closely related to the maintenance of appropriate intracellular redox status.

Keywords: lymphocyte proliferation, cell cycle, glutamine, glutathione, oxidative stress

INTRODUCTION

Glutamine is the most abundant amino acid in the body, accounting for approximately 50–60% of the intracellular free amino acid pool in skeletal muscle, and it has the highest plasma concentration of all the amino acids [1–3]. Glutamine can be produced in sufficient quantities under stable conditions, but becomes a limiting substrate during such metabolic stresses as burns, trauma, sepsis, major surgery, and exhaustive exercise [4–6], which are associated with an increased susceptibility to infection and impairment of the immune response [3, 6]. Low levels of glutamine have been shown to compromise lymphocyte proliferation [7–10], production of IL-2 and interferon-gamma (IFN-γ) [7–9], and expression of certain surface activation markers (CD25, CD45RO, CD71) [8]. Optimal lymphocyte proliferation has been demonstrated when the glutamine concentration is set around 0.6–2.0 mm [8, 9]. Although glutamine is known to be important for lymphocyte proliferation, the mechanism by which glutamine modulates lymphocyte functions is not known.

Glutamine acts not only as a precursor in nucleotide synthesis but also as a primary fuel in lymphocytes [4, 11, 12]. The rate of glutamine utilization by lymphocytes is either similar to or greater than that of glucose [11, 12]. Glutamine is metabolized in mitochondria through the citric acid cycle and therefore by aerobic oxidative phosphorylation, whereas glucose is metabolized by anaerobic glycolysis in lymphocytes [12, 13]. Mitochondria, consuming about 90% of the inhaled oxygen, are a particularly rich source of endogenous reactive oxygen species (ROS) [14]. It seems likely that glutamine is crucial for endogenous ROS production during lymphocyte activation. Moreover, a high rate of glutamine utilization can provide glutamate, which acts as a precursor for intracellular glutathione (GSH) synthesis [15, 16]. In this study we attempted to explore whether glutamine influences lymphocyte proliferation via modulation of the redox status.

MATERIALS AND METHODS

Cell preparation

Whole blood was obtained from healthy adult volunteers after informed consent was obtained. All subjects agreed to provide blood samples for this study. Peripheral blood mononuclear cells (PBMC) were separated from whole blood by density gradient centrifugation [17]. PBMC (2 × 10⁶ cells/ml) were suspended in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Gibco). For the experiments, various concentrations of glutamine (Sigma, St Louis, MO), with or without 5 μg/ml phytohaemagglutinin (PHA; Sigma), were added and the cultures were incubated at 37°C in a humidified incubator containing 5% CO₂ [18]. Aliquots of the PBMC were harvested for experiments as indicated. To evaluate the effect of GSH depletion on lymphocyte proliferation and ROS production, PBMC were incubated with 100 μm l-buthionine-[S,R]-sulfoximine (BSO; Sigma) (an inhibitor of GSH synthesis) in some experiments.

Lymphocyte proliferation assay

PBMC (2 × 10⁵ cells/well) were incubated in 96-well culture plates for 72 h in a humidified incubator containing 5% CO₂at 37°C. PBMC were then pulsed with 1 μCi/well of ³H-thymidine for 6 h before being harvested with a harvester (Cambridge Technology, Watertown, MA). Lymphocyte proliferation, as determined by ³H-thymidine uptake, was assessed with a β-counter (Beckman Instruments, Fullerton, CA) [19].

Cell cycle assay

PBMC (1 × 10⁶ cells) harvested from different treatment conditions were gently suspended in a 1.5-ml hypotonic propidium iodide (PI) solution (50 μg/ml PI in 0.1% sodium citrate and 0.1% Triton X-100; Sigma). Cells were kept at 4°C in the dark overnight before analysis with flow cytometry (Becton Dickinson, Mountain View, CA) [20, 21]. Cell cycle propagation, as determined by DNA ploidy, was assessed on the basis of PI incorporation. Gating upon FL-2 area versus width was used to exclude cell debris from cell cycle analysis [22].

Measurement of intracellular ROS

ROS generation was evaluated by means of the ROS indicator 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). PBMC (1 × 10⁶ cells) were loaded with 5 μm DCFH-DA (Kodak, Rochester, NY) and the cultures were incubated at 37°C for 15 min. DCF peroxidation was determined using flow cytometry [23, 24]. To measure the endogenous ROS production at the single-cell level, we gated the PHA-stimulated lymphocyte and lymphoblast populations on the basis of their light scattering profile by flow cytometry analysis [25].

Measurement of intracellular GSH levels by Tietze assay

The total intracellular GSH was determined by the method of Tietze [26, 27]. PBMC, harvested from different incubation conditions, were washed and precipitated with 50 μl of 5% trichroloacetic acid containing 0.1 n HCl and 10 mm EDTA. The reaction tubes were vortexed vigorously and then centrifuged. The supernatants were then harvested and transferred to 96-well plates. Following this step, 140 μl of 0.3 mm NADPH in stock buffer solution (125 mm sodium phosphate, 6.3 mm EDTA, pH 7.5) and 100 μl GSH reductase (Sigma; 1 U/ml) were added to each well. Finally, the substrate (20 μl of 6 mm 5,5′-dithio-bis-2-nitrobenzoic acid; Sigma) was added to the reaction. The absorbance of each well at 405 nm was quantified by used of a microplate reader (MR 700; Dynatech, UK).

Flow cytometry measurement of intracellular non-protein thiols in T cell subsets

Intracellular non-protein thiols in different lymphocyte subsets were measured by flow cytometry according to a previously described method [28, 29] with some modifications. Briefly, mercury orange [1-(4-chloromercuriphenylazo)-2-naphthol] (Sigma) was dissolved in acetone to a final concentration of 100 μm. T cells were further purified from PBMC by removing adherent monocytes on a 24-well plastic plate at 37°C for 30 min [19]. Purified T cells (2 × 10⁶ cells) were suspended in ice-cold PBS and centrifuged for 5 min at 500 g. The cell pellet was suspended in the mercury orange/acetone solution and incubated on ice for 5 min. After this, cells were washed twice with cold PBS and resuspended in cold PBS. Samples were then stained for MoAbs (CD4–PerCP, CD8–PerCP) (Becton Dickinson, San Jose, CA) in cold PBS for 30 min, washed twice with cold PBS, and subsequently analysed by flow cytometry.

Flow cytometry measurement of intracellular GSH levels in T cell subsets

PBMC (2 × 10⁶ cells/ml) were washed twice with PBS and then incubated with 40 μm monochlorobimane (MCB) (Molecular Probe, Eugene, OR) at room temperature for 20 min. To measure cellular GSH levels in specific lymphocyte subsets, cells were washed twice with cold PBS and then stained with CD4–PerCP and CD8–PerCP MoAbs at 4°C. These cells were then washed with cold PBS and kept on ice until analysed by FACS vantage flow cytometry (Becton Dickinson). The CD4⁺ or CD8⁺ lymphocyte subsets were identified by binding of PerCP-conjugated MoAbs. The bimane–GSH complex fluorescence was detected by an argon ion laser, which excited at 351–361 nm (50 mW) and emitted at 515–545 nm. MCB fluorescence measurement was displayed as histogram of log fluorescence intensity (abscissa) versus cell number (ordinate) [29, 30].

Statistical analysis

Results are presented as the mean ± s.e.m. Student's t-test was applied to assess the differences in results after incubation in the presence or absence of glutamine. P < 0.05 was considered statistically significant.

RESULTS

Glutamine enhanced PHA-stimulated lymphocyte proliferation and cell cycle propagation

Lymphocyte proliferation was highly dependent on the glutamine concentration, as shown in Fig. 1. Glutamine exerted its maximal effect on lymphocyte proliferation at a concentration of 0.6 mm; lymphocyte proliferation with 0.6 mm glutamine was three-fold higher than that without the addition of glutamine. Lymphocytes that were incubated with PHA alone for 24 h did not substantially propagate from the G₁to S phase (Table 1). Glutamine, at a concentration range of 0.6–2.0 mm, significantly promoted the transition of lymphocytes through the S and G₂/M phases of the cell cycle at 48 h and 72 h.

Table 1.

Percentage of cell cycle propagation of peripheral blood mononuclear cells (PBMC) stimulated with phytohaemagglutinin (PHA) at various concentrations of glutamine

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Results presented are means of percentages of total cell numbers (n = 6).

Glutamine modulated ROS production in PHA-stimulated lymphocytes

Intracellular ROS production, as determined by DCF peroxidation, increased markedly in the PHA-stimulated lymphocytes (Fig. 2). The peak ROS levels in PHA-stimulated lymphocytes occurred at 48 h, and at that time cells supplemented with 2 mm glutamine had ROS values two-fold higher than cells not supplemented with glutamine.

Glutamine enhanced PHA-stimulated lymphocyte intracellular GSH levels

Intracellular GSH levels in PHA-stimulated lymphocytes were highly dependent on the glutamine concentrations used (Fig. 3). The greatest increase in GSH levels was noted after 24 h incubation with 2 mm glutamine. At 24 h, the GSH levels in cells treated with 2 mm glutamine were 1.5-fold higher than in the absence of glutamine. Concerning the more specific mechanism of GSH modulation by glutamine in selected T cells, we found, by flow cytometric analysis using the mercury orange staining method, that addition of glutamine significantly enhanced intracellular non-protein thiol levels in PHA-stimulated CD4⁺ lymphocytes (1007 ± 166 versus 555 ± 116; P < 0.05), but not PHA-stimulated CD8⁺ or non-stimulated T cells (Fig. 4). Furthermore, experiments using MCB as a dye probe to detect intracellular GSH clearly demonstrated that glutamine did not affect the MCB fluorescence in non-stimulated CD4⁺ and CD8⁺ T lymphocytes (Fig. 5a,b). However, glutamine did enhance the MCB fluorescence in PHA-stimulated CD4⁺ and CD8⁺ lymphocyte subsets (Fig. 5c,d). These results indicate that glutamine affects intracellular GSH levels in activated CD4⁺ and CD8⁺ T cells, but not those in resting (non-stimulated) T cells.

Fig. 4 — Glutamine modification of the non-protein thiols in CD4⁺ and CD8⁺ lymphocytes. Peripheral blood mononuclear cells (PBMC; 2 × 10⁶ cells/ml) were cultured in the absence (□) or presence (▪) of glutamine (2 mm) and stimulated with phytohaemagglutinin (PHA; 5 μg/ml) for 24 h. (a) Glutamine significantly enhanced mean fluorescence in PHA-stimulated CD4⁺ lymphocytes. (b) Glutamine did not significantly change the mean fluorescence in PHA-stimulated CD8⁺ lymphocytes. Results presented are mean ±s.e.m. (n = 3). *Significant difference between presence and absence of glutamine (P < 0.05).

Fig. 5 — Effect of glutamine on the intracellular glutathione (GSH) levels in CD4⁺ and CD8⁺ lymphocytes. Peripheral blood mononuclear cells (PBMC; 2 × 10⁶ cells/ml) were cultured in the absence or presence of glutamine (2 mm) and stimulated with phytohaemagglutinin (PHA; 5 μg/ml) for 24 h. Histograms of the GSH in cells cultured with glutamine (solid line) or without glutamine (dotted line) of (a) non-stimulated CD4⁺ lymphocytes; (b) non-stimulated CD8⁺ lymphocytes; (c) PHA-stimulated CD4⁺ lymphocytes; (d) PHA-stimulated CD8⁺ lymphocytes. Monochlorobimane (MCB) fluorescence shift to the right in PHA-stimulated CD4⁺ and CD8⁺ lymphocyte subsets. Results shown were a representative study from three reproducible experiments.

Exogenous GSH promoted, but blockade of GSH formation suppressed, PHA-stimulated lymphocyte proliferation

Since glutamine was found to augment intracellular GSH levels, as well as proliferation of PHA-stimulated lymphocytes, the next set of experiments was directed at evaluating the influence of exogenous GSH supplementation on lymphocyte proliferation. Addition of exogenous GSH, in the range 2–10 mm, appeared to enhance PHA-stimulated lymphocyte proliferation, with the optimal response occurring at 5 mm GSH (Fig. 6). When both PHA and BSO were added and co-cultivated for 48 h, we found that BSO, an inhibitor of GSH synthesis, augmented ROS production in the presence or absence of glutamine (2 mm) (Fig. 7a). Addition of BSO significantly suppressed glutamine-enhanced PHA-stimulated lymphocyte proliferation (Fig. 7b).

Fig. 6 — Effect of exogenous glutathione (GSH) supplementation on lymphocyte proliferation. Peripheral blood mononuclear cells (PBMC; 2 × 10⁶ cells/ml) were cultured in the presence GSH at various concentrations and stimulated with phytohaemagglutinin (PHA; 5 μg/ml) for 72 h. Results presented are mean ±s.e.m. (n = 6). *Significant difference between presence and absence of GSH (P < 0.05).

Fig. 7 — Blockade of glutathione (GSH) synthesis by l-buthionine-[S,R]-sulfoximine (BSO)-enhanced reactive oxygen species (ROS) production and suppressed lymphocyte proliferation. (a) BSO enhanced ROS production in phytohaemagglutinin (PHA)-stimulated lymphocytes in the presence or absence of glutamine after 48 h cultivation. (b) BSO suppressed PHA-stimulated lymphocyte proliferation in the presence or absence of glutamine. Results presented are mean ±s.e.m. (n = 6). *Significant difference between treatment with and without BSO (P < 0.05). □, PHA; ▪, PHA + BSO.

DISCUSSION

This study has demonstrated that glutamine modulates PHA-stimulated lymphocyte proliferation via a redox-based mechanism. There are several lines of evidence that support the idea of glutamine modulation of redox reactions: (i) glutamine significantly enhanced PHA-stimulated lymphocyte proliferation; (ii) glutamine increased both intracellular ROS production and GSH levels in PHA-stimulated lymphocytes; (iii) addition of exogenous GSH significantly augmented lymphocyte proliferation; and (iv) blockade of GSH synthesis elevated ROS production and suppressed lymphocyte proliferation.

Glutamine provides precursors for purine, pyrimidine, and nucleotide synthesis, which are required for the biosynthetic processes of lymphocyte proliferation and cell cycle propagation [4, 31, 32]. Accordingly, glutamine is used at high rates during lymphocyte proliferation [11, 12]. Glutamine that is metabolized in lymphocytes via aerobic oxidation is converted to glutamate, aspartate, lactate, and ammonia [12, 13, 33]. Presumably, the oxidation of glutamine could create a metabolic condition that facilitates the mitochondrial production of ROS in PHA-stimulated lymphocytes. We did indeed find that glutamine supplementation enhanced endogenous ROS production in PHA-stimulated lymphocytes. It is possible that the long-lasting substantial ROS production after 24-h cultivation of PHA-stimulated lymphocytes is related to the obligatory requirement for glutamine during lymphocyte proliferation.

In contrast to the finding of increased ROS levels, we also demonstrated that supplementation with glutamine increased intracellular GSH levels in PHA-stimulated lymphocytes, in a dose-dependent fashion. Similar results were found in concanavalin A (Con A)- and pokeweed mitogen (PWM)-stimulated lymphocyte proliferation (data not shown).

It has been demonstrated that inadequate concentrations of glutamine compromise the expression of lymphocyte surface activation markers (CD25, CD45RO) [8], and decrease the proportion of CD4⁺ lymphocytes and the CD4⁺/CD8⁺ ratio [10, 34]. Therefore, it is important to investigate the GSH modulating effect in specific lymphocyte subsets. Because MCB is more specific and reliable than mercury orange in the detection of GSH by flow cytometry [29], using MCB as a dye probe, we found that glutamine enhanced GSH levels in both PHA-stimulated CD4⁺ and CD8⁺ T cells. Based on these results, we speculate that glutamine is crucial for GSH synthesis or regeneration, especially in T lymphocyte activation and proliferation.

Since the ratio of reduced GSH to oxidized glutathione (GSSG) is a direct indication of the extent of the oxidation reaction required for proliferation, it is important to know the glutamine effect on the GSH/GSSG ratio in cells. It has been shown that the ratio of GSH to GSSG under physiological conditions is 30–100:1 [35, 36]. The ways by which glutamine affects the GSH/GSSG ratio may be by elevating the GSH levels, and subsequently the GSH/GSSG ratio, in cells. For instance, (i) glutamine converts to glutamate which acts as a precursor for GSH synthesis [15, 16]; (ii) glutamine increases NADPH production via the malate:pyruvate reaction and therefore serves as hydrogen donor to promote GSH regeneration [37, 38]; and (iii) glutamine enhances adenosine triphosphate (ATP) production, which catalyses the ATP-dependent reactions by γ-glutamylcysteine synthetase and GSH synthetase for GSH synthesis [11, 35].

The fact that glutamine supplementation enhanced both cellular ROS and GSH levels in PHA-stimulated lymphocytes indicates a causal link between redox reactions and lymphocyte proliferation. Oxidative stress in lymphocytes causes a reduced response to mitogens or antigens [39]. GSH, the most abundant non-protein thiol in mammalian cells, is important in protecting cells from ROS-mediated damage [38, 40]. Addition of exogenous GSH mimicked glutamine supplementation, enhancing PHA-stimulated lymphocyte proliferation. Reciprocally, BSO, which has been reported to decrease cellular GSH concentrations [41, 42], enhanced ROS production and inhibited glutamine enhancement of PHA-stimulated lymphocyte proliferation (Fig. 7). These findings suggest that glutamine not only provides biosynthetic precursors for lymphocyte proliferation and cell cycle propagation, but also protects lymphocytes from ROS-mediated damage by increasing GSH production. Further studies are needed to clarify how glutamine provides certain precursors for lymphocyte proliferation and modulates redox reactions for lymphocyte proliferation.

It has been shown that GSH therapy has widespread applications in ageing, oncology, liver disease, acute respiratory distress syndrome, and AIDS [38]. For example, patients with HIV were found to have abnormally low plasma glutamine and cellular GSH levels, which may contribute to oxidative damage and consequent immunological dysfunction [38, 43]. Replenishing GSH in deficient states effectively blocked HIV expression in models of acute and chronic infection [38]. The fact that human lymphocyte proliferation can be modulated by glutamine through the redox reaction suggests that supplementation with glutamine may be beneficial for patients with certain infections or compromised immune status.

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PERMALINK

Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction

W K Chang

K D Yang

M F Shaio

Abstract

INTRODUCTION