Role of TL1A and its receptor DR3 in two models of chronic murine ileitis

Abstract

TL1A is a TNF-like cytokine that binds to the death-domain receptor (DR)3 and provides costimulatory signals to activated lymphocytes. Through this interaction, TL1A induces secretion of IFN-gamma and may, therefore, participate in the development of T helper-1-type effector responses. In this study, we investigated whether interactions between TL1A and DR3 are involved in the pathogenesis of chronic murine ileitis. We demonstrate that alternative splicing of DR3 mRNA takes place during the activation of lymphocytes, which results in up-regulation of the complete/transmembrane (tm) form of DR3. Using two immunogenetically distinct animal models of Crohn's disease, we demonstrate that induction of intestinal inflammation is associated with significant up-regulation of TL1A and tm DR3 in the inflamed mucosa. In addition, within isolated lamina propria mononuclear cells from mice with inflammation, TL1A is primarily expressed on CD11c(high) dendritic cells. We also report that TL1A acts preferentially on memory CD4(+)/CD45RB(lo) murine lymphocytes by significantly inducing their proliferation, whereas it does not affect the proliferation of the naïve CD4(+)/CD45RB(hi) T helper cell subpopulation. Finally, we demonstrate that TL1A synergizes with both the cytokine-dependent IL-12/IL-18 pathway and with low-dose stimulation of the T cell receptor to significantly induce the secretion of IFN-gamma via an IL-18-independent pathway. Our results raise the possibility that interaction(s) between TL1A expressed on antigen-presenting cells and tm DR3 on lymphocytes may be of particular importance for the pathogenesis of chronic inflammatory conditions that depend on IFN-gamma secretion, including inflammatory bowel disease. Blockade of the TL1A/DR3 pathway may, therefore, offer therapeutic opportunities in Crohn's disease.

Fig. 1.

Effects of TL1A stimulation on TCR-mediated IFN-γ secretion. CD4⁺ splenocytes were purified and cultured as described in Materials and Methods and indicated on the axes of the graphs. (A) Cells were stimulated with anti-CD3 (0.5 μg/ml) and anti-CD28 (1 μg/ml) Abs, then rmTL1A (100 ng/ml) was added to the cultures. The concentration of IFN-γ was measured at 72 h. A representative of four experiments is shown. (B) Cells were stimulated with anti-CD3/anti-CD28, then rmTL1A was added to the cultures. The concentration of IFN-γ was measured at 24–96 h. A representative of three experiments is shown. Asterisks indicate values of P < 0.05 for comparison between IFN-γ secretion with or without rmTL1A for each time point. (C) Cells were stimulated with different doses of anti-CD3 (0.5–10 μg/ml). The concentration of IFN-γ was measured at 72 h. Pooled data from four independent experiments are shown. All data are presented as mean ± SEM.

Fig. 2.

TL1A synergizes with IL-12. (A) CD4⁺ splenocytes were stimulated with the combination of IL-12/IL-18, then rmTL1A was added to the cultures. A representative of four experiments is shown. (B) CD4⁺ splenocytes were stimulated with anti-CD3 (0.5 μg/ml), anti-CD28 (1 μg/ml), rmIL-12 (10 ng/ml), rmIL-18 (100 ng/ml), and rmTL1A (100 ng/ml), as indicated. The concentration of IFN-γ in the supernatant was measured after 72 h. Pooled data from six experiments are shown. All data are presented as mean ± SEM. (C) CD4⁺ splenocytes were stimulated with rmIL-12, with or without the addition of TL1A (200 ng/ml). The concentration of IFN-γ in the supernatant was measured after 72 h. Six individual experiments are shown. Horizontal lines indicate the average for each group.

Fig. 3.

TL1A acts on memory lymphocytes. Naïve (CD4⁺/CD45RB^hi) and memory (CD4⁺/CD45RB^hi) splenocytes were purified and cultured as described in Materials and Methods. Stimulatory conditions are indicated on the axes of the graphs. Proliferation of naïve (A) and memory (B) lymphocytes was estimated by measuring thymidine incorporation. Representatives of two experiments are shown. (C) Average proliferative responses for naïve and memory lymphocytes from four independent experiments are shown. All data are presented as mean ± SEM.

Fig. 4.

Expression of tm DR3 mRNA during activation of lymphocytes and in chronic ileitis. Specific amplification of tm and total DR3 mRNA was performed as described in Materials and Methods. (A) Lymphocytes (CD4⁺ and CD8⁺) were isolated from mouse spleens, and the ratio of the relative expression of tm vs. total DR3 mRNA was measured in freshly isolated, overnight resting or overnight stimulated (aCD3 + aCD28) cells (n = 4 per group). (B) The relative expression of tmDR3 mRNA was measured by quantitative real-time RT-PCR in total tissue RNA extracted from the terminal ileum of SAMP1/YitFc mice with ileitis (>20-wk-old) or before the development of ileitis (4-wk-old) and age-matched normal AKR control mice (n = 6–7 mice per group). (C) Relative expression of tmDR3 mRNA was measured in total tissue RNA extracted from the terminal ilea of TNF^ΔARE mice with ileitis (24-wk-old) or before the development of ileitis (4-wk-old) and age-matched wild-type mice (n = 5–6 mice per group). All data are presented as mean ± SEM.

Fig. 5.

TL1A is up-regulated during chronic ileitis and is expressed on mucosal dendritic cells. (A) Relative expression of TL1A mRNA was measured in total tissue RNA extracted from the terminal ilea of TNF^ΔARE mice (>8-wk-old, n = 13) and age-matched wild-type mice (n = 14). All data are presented as mean ± SEM. (B) Flow-cytometric analysis of LPMCs from inflamed SAMP1/YitFc mice after staining with a specific anti-TL1A Ab demonstrated that expression of TL1A is confined to the CD11c-positive fraction. (C) Confocal microscopy of LPMCs incubated with antibodies against mouse TL1A (red) and CD11c (green) demonstrated that the two markers were colocalized. (D) LPMCs were incubated with mAbs against mouse TL1A, CD11c, and MHC-II. Gates were set as indicated in the figure. Histogram plots indicate the expression of TL1A (green) or negative control (red) in each subpopulation. Negative control indicates staining with secondary Ab alone.