Differential impact of L-arginine deprivation on the activation and effector functions of T cells and macrophages

Abstract

The metabolism of the amino acid L-arginine is emerging as a crucial mechanism for the regulation of immune responses. Here, we characterized the impact of L-arginine deprivation on T cell and macrophage (MPhi) effector functions: We show that whereas L-arginine is required unconditionally for T cell activation, MPhi can up-regulate activation markers and produce cytokines and chemokines in the absence of L-arginine. Furthermore, we show that L-arginine deprivation does not affect the capacity of activated MPhi to up-regulate L-arginine-metabolizing enzymes such as inducible NO synthase and arginase 1. Thus, our results show that to exert their effector functions, T cells and MPhi have different requirements for L-arginine.

Fig. 1.

l-arginine deprivation impairs T cells proliferation and cytokine production. Spleen cells or purified T cells were stimulated with anti-CD3 and anti-CD28 mAb in grading concentration of l-arginine (+l-arginine=400 μM; -l-arginine=0 μM). The capacity of T cells from total spleen (A–C) or purified T cells (D) to proliferate and the capacity of splenic T cells (E) and purified T cells (G) to produce cytokines were determined by flow cytometry. (F) The level of cytokine production was determined by ELISA. The error bars represent sd, and data show the results of one representative experiment out of four independent experiments.

Fig. 2.

l-arginine deprivation impairs expression of activation markers by T cells. Spleen cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence (400 μM, black line) or absence (0 μM, gray line) of l-arginine, and the expression of CD28, CD62L, and CD25 on TcRβ⁺ cells was determined by flow cytometry. Data show the results of one representative experiment out of three independent experiments.

Fig. 3.

l-arginine is essential for efficient effector functions following restimulation of activated T cells. Spleen cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence (400 μM) of l-arginine for 4 days, rested for 3 day, and restimulated for 24 h in the presence (400 μM) or absence (0 μM) of l-arginine. The capacity of T cells to proliferate (A) and produce cytokines (B) was determined by flow cytometry. The capacity of purified T cells from splenocytes to produce cytokines (C) was also determined. The error bars represent sd, and data show the results of one representative experiment out of three independent experiments.

Fig. 4.

l-arginine depletion by arginase-expressing MΦ impairs T cells functions. Mature BMMΦs were stimulated with IL-4/IL-10 in the presence of 100 μM l-arginine. Two days later, MΦ were washed, and purified T cells (preactivated with anti-CD3 and anti-CD28 for 24 h) were added to the MΦ. l-arginine (400 μM) was added twice/day to some of the wells (+l-arginine). The capacity of T cells to proliferate (A) and produce cytokines (B) was determined by flow cytometry. The error bars represent sd, and data show the results of one representative experiment out of three independent experiments.

Fig. 5.

l-arginine is not required for the expression of MΦ activation markers. Mature BMMΦ were differentiated in CAMΦ or AAMΦ or left unstimulated (nil) in the presence (400 μM, shaded bars) or absence (0 μM, open bars) of l-arginine, and the capacity to express cell-surface markers was analyzed by flow cytometry. The error bars represent sd, and data show the results of one representative experiment out of three independent experiments.

Fig. 6.

l-arginine is not required for the expression of Ym1 and Fizz1, two hallmarks of AAMΦ and does not impair iNOS expression by CAMΦ. (A) Mature BMMΦ were differentiated in CAMΦ or AAMΦ or left unstimulated in the presence (400 μM) or absence (0 μM) of l-arginine, and the expression of Ym1, Fizz1, and iNOS was determined by Western blot. The same concentration of protein (14 μg) was loaded for each group. Data show the results of one representative experiment out of three independent experiments. (B) Mature BMMΦ were differentiated in CAMΦ or AAMΦ or left unstimulated in the presence (400 μM) or absence (0 μM) of l-arginine, and the production of NO was measured by the Griess reaction (B). The horizontal line represents the detection limit (5 μM). The error bars represent sd, and data show the results of one representative experiment out of five independent experiments. N.D., Not detected.

Fig. 7.

l-arginine is not required for the expression and activity of arginase. Mature BMMΦ were activated with IFN-γ/TNF-α (CAMΦ), IL-4 (AAMΦ), or IL-4/IL-10 or left unstimulated in the presence (400 μM) or absence (0 μM) of l-arginine, and the expression of arginase was determined by Western blot (upper panel), and the activity of arginase was determined by enzymatic assay (lower panel). The same concentration of protein (14 μg) was loaded for each group. The error bars represent sd, and data show the results of one representative experiment out of five independent experiments.