A crucial role for T-bet in selectin ligand expression in T helper 1 (Th1) cells - PubMed (original) (raw)

A crucial role for T-bet in selectin ligand expression in T helper 1 (Th1) cells

Greg H Underhill et al. Blood. 2005.

Abstract

Proinflammatory T helper 1 (Th1) cells express high levels of carbohydrate ligands for the endothelial selectins, but the molecular basis for this phenotype is incompletely understood. We document here a significant role in selectin ligand formation for the recently described Th1 transcription factor T-bet. Th1 cells generated from T-bet-/- mice showed significantly lower levels of ligands for both E-selectin and P-selectin, compared with wild-type (WT) Th1 cells. Enforced expression of T-bet in WT Th0 cells only modestly up-regulated P-selectin ligands and had no effect on E-selectin ligands. To define a mechanism for the defects observed in T-bet-/- mice, we examined expression of glycosyltransferases involved in selectin ligand biosynthesis. T-bet-/- Th1 cells expressed significantly lower levels of core 2 beta1,6 N-acetylglucosaminyltransferase I (C2GlcNAcT-I), but no differences in levels of alpha 2,3-sialyltransferase IV (ST3Gal-IV). Further, we show that T-bet is responsible for the signal transducer and activator of transcription 4 (Stat4)-independent increase in Th1 cells of fucosyltransferase VII (FucT-VII). We also identify ST3Gal-VI, which is thought to play an important role in E- and P-selectin ligand formation, as an interleukin 12 (IL-12)-regulated, T-bet-dependent gene. These data show that T-bet controls selectin ligand formation in Th1 cells via control of expression of multiple key enzymes in response to IL-12 signaling and establishes an independent transcriptional pathway for control of Th1 cell traffic.

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Figures

Figure 1.

Figure 1.

Defective induction of selectin ligands in T-bet-/- Th1 cells. Purified CD4+ T cells were cultured under either Th1 (IL-2 plus IL-12 plus anti–IL-4) or Th0 (IL-2 only) conditions and analyzed on day 8 for rolling on (A) E-selectin and (B) P-selectin. One representative of at least 2 experiments. **Significantly different (P < .05) from all other groups; *significantly different (P < .05).

Figure 2.

Figure 2.

Kinetics of expression of selectin ligands is not altered by the absence of T-bet. Cells from T-cell activation cultures as in Figure 1 were also analyzed over time by FACS with selectin chimeras to determine the fraction which expressed ligands for (A) E-selectin or (B) P-selectin. Filled symbols, Th1; open symbols, Th0; squares, WT; circles, T-bet-/-. One of at least 5 experiments.

Figure 3.

Figure 3.

Direct and indirect effects of T-bet on selectin ligand induction. (A) Enforced overexpression of T-bet in WT Th0 cells fails to induce maximal levels of selectin ligands. WT CD4+ T cells were activated under Th0 conditions, infected with retroviruses (RVs) expressing no cDNA (empty) or T-bet, and cultured under Th0 conditions. Cells were then stained with P- or E-selectin chimeras or 1B11. Uninfected WT Th1 cells were included side-by-side for comparison. Numbers given for retrovirally transduced cells are the percentage of the GFP+ cells which stain for either 1B11 (first column), P-selectin ligands (second column), or E-selectin ligands (third column). One representative of 3 similar experiments. (B) T-bet deficiency inhibits functional expression of the IL12R. WT and T-bet-/- CD4 cells were cultured under Th1 conditions for 6 days, washed, and restimulated with IL-12. At the indicated time points, cells were harvested, lysed, and subjected to Western blotting for tyrosine phosphorylated, and total Stat4. Results are representative of 2 experiments. KO indicates knock-out.

Figure 4.

Figure 4.

T-bet is required for up-regulation of C2GlcNAcT-I and FucT-VII in Th1 cells. (A) qRT-PCR of FucT-VII mRNA levels of WT and T-bet-/- Th0 and Th1 cells. Levels are normalized to pgk mRNA levels and expressed as fold induction over WT Th0 cells. Error bars indicate SD. (B) Expression of the C2GlcNAcT-I reporter epitope defined by the 1B11 mAb on WT and T-bet-/- Th0 and Th1 cells over time. Results are representative of at least 3 experiments. Filled symbols, Th1; open symbols, Th0; squares, WT; circles, T-bet-/-, as in Figure 2. **Significantly different from all other groups.

Figure 5.

Figure 5.

Role of ST3Gal-IV in selectin ligand formation in Th1 cells. Rolling of WT and ST3Gal-IV-/- Th0 and Th1 cells on (A) E-selectin and (B) P-selectin reveals significantly decreased rolling of ST3Gal-IV-/- Th1 cells, compared with WT Th1 cells. (C) qRT-PCR shows no significant difference in ST3Gal-IV-/- mRNA levels between WT or T-bet-/- Th0 or Th1 cells. (D) qRT-PCR also shows no significant difference in ST3Gal-IV mRNA levels between WT and Stat4-/- Th0 or Th1 cells. Data are depicted as in Figure 4A. Results are representative of at least 3 experiments. **Significantly different from all other groups.

Figure 6.

Figure 6.

Possible role of ST3Gal-VI in selectin ligand formation. WT and ST3Gal-IV-/- Th1 cells were either untreated or treated with neuraminidase, and rolling on (A) E-selectin or (B) P-selectin was measured. Neuraminidase blocked rolling of cells of both genotypes on both selectins. (A-B) **Significantly different from the corresponding group without neuraminidase treatment. (C) qRT-PCR reveals significant induction of ST3Gal-VI mRNA by IL-12 in WT cells but virtually no effect of IL-12 in T-bet-/- cells. (D) A similar effect on ST3Gal-VI induction is observed with CD4 cells from Stat4-/- mice, although the magnitude of the effect of Stat4 deficiency is smaller than that for T-bet (note different scales). (C-D) **Significantly different from all other groups.

Figure 7.

Figure 7.

Pathways controlling expression of glycosyltransferases in Th1 cells. Naive T cells express little or no FucT-VII, C2GlcNAcT-I, or ST3Gal-VI. T-cell activation through the TCR leads to activation of H-Ras and induction of FucT-VII, as well as induction or maintenance of constitutive levels of ST3Gal-IV. Whether ST3Gal-IV expression requires H-Ras is unknown. In addition, T-cell activation is associated with induction of the IL12R, particularly the IL12Rβ2 chain, and the IL12R is essential for activation of Stat4, which plays a major role in induction of C2GlcNAcT-I and a minor role in induction of ST3Gal-VI. T-bet plays a role through induction/maintenance of the IL12Rβ2, allowing up-regulation of C2GlcNAcT-I by Stat4; through direct up-regulation of C2GlcNAcT-I and ST3Gal-VI; and through up-regulation of FucT-VII by an as yet undetermined mechanism. Bold lines represent strong, presumably or known to be direct, effects; thin lines represent weak effects.

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