Thiol-mediated redox regulation of intestinal lamina propria T lymphocytes

Abstract

Intestinal lamina propria T lymphocytes (LP-Ts) have a markedly low proliferative potential both in vivo and in vitro. Here, we have identified that the capacity of antigen-presenting cells to release cysteine upon receptor-ligand interactions represents a critical parameter for proliferation of LP-Ts. The availability of cysteine is limiting for the intracellular production of glutathione, which in turn is essential for cell cycle progression. When cysteine is provided either directly or by addition of the reducing agent 2-mercaptoethanol to cystine-containing culture medium, proliferation of LP-T is fully restored. Importantly, coculture with peripheral blood monocytes that easily take up cystine, reduce cystine, and secrete cysteine also restores reactivity of LP-Ts to T cell receptor/CD3 stimulation. In marked contrast, lamina propria macrophages lack this capacity to elaborate cysteine, and thereby secure physiological unresponsiveness to antigen exposure in the intestinal microenvironment. The well-documented local recruitment of blood monocytes in inflammatory bowel disease (IBD) may thus represent an important parameter underlying hyperresponsiveness of T cells, an essential component of the pathogenesis of IBD.

Figure 1

Redoxregulation of DNA synthesis in LP-Ts. (A) Influence of antioxidative (2-ME) and prooxidant (hydrogen peroxide, BSO, BCNU) culture conditions on the proliferation of LP-Ts after CD3 (10 U/ml IL-2 added) or CD2 stimulation. (B) Protective effect of 2-ME on the hydrogen peroxide or BSO-induced suppression of proliferation.

Figure 2

Costimulation of LP-Ts by PB-MOs or 2-ME, but not by LP-MOs. (A) Irradiated LP-MOs or autologous PB-MOs were added to LP-Ts (5 × 10⁴/well) in graded amounts up to 30% of total cell number. (B) Costimulatory potential of 2-ME in comparison with irradiated PB-MOs and LP-MOs (30% of total cell number). unstim., unstimulated.

Figure 4

Involvement of CD2–CD58 interactions in PB-MO–mediated costimulation of LP-Ts. CD58 mAb (1A3) was used at saturating concentrations (10 μg/ml). Irradiated PB-MOs were added at 30% of total cell number. The dotted line indicates the baseline as defined by the proliferation induced by the CD2 mAb M1+M2.

Figure 3

Protective effect of PB-MOs versus LP-MOs on the suppression of proliferation of LP-Ts by hydrogen peroxide, which was added at the beginning of the culture. unstim., unstimulated.

Figure 6

Thiol-dependent proliferation of LP-T. (A) Cystine-deficient RPMI 1640/10% FCS was supplemented with graded amounts of cystine at the indicated final concentrations. Concentration of 2-ME was 10 μM. Irradiated PB-MOs were added at 30% of total cell number. (B) Stimulatory potential of cysteine versus equimolar amounts of cystine on the proliferation of LP-Ts after CD3 (OKT3) or CD2 (M1+M2) stimulation. LP-Ts were plated at 8 × 10⁵/well in an initial volume of 1 ml/well in 24-well plates. Cultures were primarily set up in cystine-deficient medium, and were supplemented with cysteine in 15-μl volumes every 6 h at a final concentration of 30 μM each time. Cysteine had to be added repeatedly because of rapid oxidation to cystine under culture conditions. Alternatively, cultures received equimolar amounts of cystine. After 84 h of culture, wells were pulsed with [³H]-TdR at 5 μCi/ml, and cells were harvested 18 h later.

Figure 5

(A) Release of cysteine (acid-soluble thiol) by PB-MOs versus LP-MOs after 40 h of culture. Cells were plated at 5 × 10⁵/ml in a total volume of 1 ml/well in 48-well culture plates, and were either left untreated or activated with LPS or IFN-γ. For cross-linking of CD58 and CD54, wells were precoated with 1A3 and GP89 mAb, respectively, at 10 μg/ml in PBS for 2 h at 37°C followed by two washes with PBS/5% FCS. Data represent one of three independent experiments performed with identical results. (B) Effect of 2-ME, hydrogen peroxide, BSO, and BCNU, respectively, on release of cysteine (acid-soluble thiol) by PB-MOs.

Figure 5

(A) Release of cysteine (acid-soluble thiol) by PB-MOs versus LP-MOs after 40 h of culture. Cells were plated at 5 × 10⁵/ml in a total volume of 1 ml/well in 48-well culture plates, and were either left untreated or activated with LPS or IFN-γ. For cross-linking of CD58 and CD54, wells were precoated with 1A3 and GP89 mAb, respectively, at 10 μg/ml in PBS for 2 h at 37°C followed by two washes with PBS/5% FCS. Data represent one of three independent experiments performed with identical results. (B) Effect of 2-ME, hydrogen peroxide, BSO, and BCNU, respectively, on release of cysteine (acid-soluble thiol) by PB-MOs.