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-γ 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-γ 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-γ secretion, including inflammatory bowel disease. Blockade of the TL1A/DR3 pathway may, therefore, offer therapeutic opportunities in Crohn's disease.

The differentiation of naïve CD4⁺ lymphocytes into IFN-γ-secreting Th1 “effector” cells is a multistep process that involves several cell types, costimulatory molecules, transcription factors, and secreted cytokines (1). Antigen-presenting cell (APC)-derived IL-12 is essential for the induction of IFN-γ, an effect that is greatly enhanced by IL-18 (2). IL-12 up-regulates T-bet, a transcription factor that is critical for the stabilization of a T helper (Th)1-polarized phenotype (3). Recently, additional cytokines that play prominent roles during Th1 responses have been described, such as IL-27 and IL-23 (4). Engagement of the T cell receptor (TCR) provides further signals for the induction of IFN-γ, both in parallel to and independently of cytokine-mediated pathways (1).

Members of the TNF and TNF-receptor superfamilies of proteins (TNFSFPs and TNFRSFPs, respectively) are abundantly expressed in the immune system, and are critically involved in the differentiation, proliferation, and apoptosis of immune cells (5). Several members of these families induce secretion of IFN-γ upon ligand/receptor binding, thereby enhancing Th1-type responses (6–8). TL1A (TNFSPF15) is a recently identified, TNF-like factor that is currently the only known ligand for death-domain receptor (DR)3 (9), which is primarily expressed on activated lymphocytes (10, 11).

Binding of TL1A to DR3 triggers proliferative/activation signals, most likely through activation of NF-κB-mediated pathways (9, 12). TL1A specifically induces secretion of IFN-γ by human T cells (9), raising the possibility that TL1A/DR3 may participate in Th1-mediated responses. Indeed, we and others have recently reported up-regulation of both TL1A and DR3 in inflammatory bowel disease (IBD), particularly Crohn's disease (CD) (13–15). Whereas original reports indicated that TL1A expression was confined to endothelial cells (9), subsequent studies demonstrated that in involved intestinal tissue from patients with IBD, TL1A was also expressed on lymphocytes, plasma cells, and monocytes. TL1A may participate in the pathogenesis of IBD, likely by inducing secretion of IFN-γ from lamina propria mononuclear cells (LPMCs) and the subsequent generation of proinflammatory responses (13–15).

In contrast to the aforementioned human studies, limited data have been published describing the function of TL1A and DR3 in mice. In this study, we investigated the hypothesis that interaction(s) between TL1A and DR3 participate in the pathogenesis of murine chronic small intestinal inflammation. Using two mouse models of CD (16–18), we have demonstrated that expression of DR3 is significantly up-regulated during chronic ileitis in an inflammation-specific manner. This up-regulation involves alternative splicing of the mRNA that encodes DR3, which results in predominant expression of the transmembrane (tm) form of the receptor in preference to the soluble form. In addition, we show that there is abundant expression of TL1A on the surface of CD11c^high dendritic cells, raising the possibility that there is an interaction between lymphocytic tmDR3 and APC-derived TL1A. We also show that TL1A acts as a costimulator for lymphocytes, enhancing IFN-γ production in synergy with low-level stimulation of the TCR or with IL-12, whereas its actions are independent of IL-18. Finally, we report that TL1A induces the proliferation of memory but not naïve CD4⁺ lymphocytes, a finding that supports a role for TL1A during the effector phase of Th1 responses.

Results

TL1A Enhances TCR-Mediated Secretion of IFN-γ by Murine Lymphocytes.

We initially investigated whether TL1A affects TCR-mediated secretion of IFN-γ by murine lymphocytes. Addition of recombinant murine (rm)TL1A to cultured CD4⁺ cells, in the presence of immobilized anti-CD3 and soluble anti-CD28 Abs, significantly increased IFN-γ secretion in a dose-dependent fashion (Fig. 1A and B). In addition, we found that low-dose (0.2 μg/ml) anti-CD3 stimulation provided optimal conditions for TL1A function (average increase in IFN-γ secretion, 223%; range, 143–349%) (Fig. 1C). In contrast, the effect was greatly diminished when 1 μg/ml anti-CD3 was used (122%; range, 112–136%), and there was no effect with high-dose (5–10 μg/ml) stimulation (100%; range, 78–117%).

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.

TL1A Acts in Synergy with IL-12 and Independently of IL-18 for the Induction of IFN-γ.

It has been demonstrated that a combination of IL-12 and IL-18 can up-regulate IFN-γ in the absence of Ag/TCR stimulation (19). We investigated whether TL1A could also synergize with this cytokine pathway for IFN-γ secretion. Our data demonstrate that synergy between TL1A and the IL-12/IL-18 pathway does indeed occur. Addition of TL1A to cultures of IL-12/IL-18-stimulated CD4⁺ cells caused a dose-dependent increase in the levels of IFN-γ secreted, in the complete absence of TCR-mediated signals (Fig. 2A).

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.

To further explore the role played by TL1A in the pathways that lead to secretion of IFN-γ, we studied the effect of combining TL1A with IL-12 or IL-18, in the presence of TCR stimulation. In cultured CD4⁺ cells, TL1A synergized with the combination of TCR stimulation and IL-12; however, when used in combination with IL-18 and TCR stimulation, TL1A failed to induce higher levels of secretion of IFN-γ than were induced by TCR stimulation and IL-18 alone (Fig. 2B). These data indicate that there is a potent synergy between TL1A and IL-12 that is independent of IL-18 signaling. This finding was further confirmed in experiments that tested the synergy between TL1A and IL-12 in the absence of TCR engagement. Addition of TL1A to IL-12-stimulated CD4⁺ cells had a significant costimulatory effect, leading to a 3- to 17-fold increase in IFN-γ secretion (Fig. 2C).

TL1A Acts on Memory Th Cells.

Next, we tested whether TL1A stimulates the naïve or memory lymphocytic subpopulations. We therefore studied the proliferative responses of highly purified CD4⁺/CD45RB^hi (naïve) and CD4⁺/CD45RB^lo (memory) cells isolated from the spleens of C57/Bl6 mice. As expected, naïve splenocytes were highly responsive to stimulation with IL-12 (Fig. 3A). However, no increase in proliferation was observed after addition of TL1A to cultured naïve lymphocytes, nor was there any synergistic effect when IL-12 and TL1A were combined (Fig. 3A). By contrast, CD4⁺/CD45RB^lo cells did not respond to stimulation with IL-12 (Fig. 3B). However, when TL1A was added to cultures of memory lymphocytes, the proliferation rate was greatly enhanced (Fig. 3B). Indeed, whereas TL1A induced only a modest proliferative response from naïve lymphocytes (148% ± 88% over baseline), it exerted a far greater stimulatory effect on memory cells (380% ± 141%) (Fig. 3C).

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.

Activation of Lymphocytes Is Associated with Alternative Splicing of DR3 and Up-Regulation of the tm Form of DR3.

Three splice variants of DR3 have been described in the mouse (20). The first encodes the full protein, including both the tm and extracellular regions. The second lacks the tm region and is, therefore, predicted to encode a soluble protein. Finally, the third variant lacks one of the cysteine-rich regions in the extracellular domain. Because expression of DR3 is limited to activated lymphocytes (10, 11), we hypothesized that lymphocytic activation might be associated with up-regulation of the splice variant that encodes the full-length DR3 protein, resulting in the expression of a fully functional tm receptor and increased TL1A signaling.

To test our hypothesis, we designed a dual amplification system for the detection of DR3 mRNA by real-time PCR. A first set of primers and probe, referred to as tm, was designed to amplify a region that is present only in the full/tm form of DR3 mRNA. The second set, referred to as total, amplifies a region that is present in all three variants. We then used this system to compare the relative ratio of the tm to the total mDR3 in lymphocytes at different stages of activation. As shown in Fig. 4A, the ratio of tm to total mDR3 was relatively low in freshly isolated or resting T cells, whereas activation for 24 h resulted in a definitive increase in the relative amount of tmDR3 (>5-fold increase over baseline). In fact, rather than altering the total levels of mRNA for DR3, activation of the cells increased the level of tmDR3 (data not shown). This result indicates that the increased ratio of tm to total mDR3 after cell activation is a direct result of an increased proportion of the tm splice variants.

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.

Chronic Small Intestinal Inflammation Is Associated with Mucosal Up-Regulation of tmDR3.

To explore the importance of the TL1A/DR3 pair under inflammatory conditions in vivo, we investigated the hypothesis that the expression of tmDR3 should be increased in IBD, because this immunological condition is characterized by heavy infiltration of the intestinal lamina propria by activated lymphocytes (21). We tested our hypothesis in two immunogenetically diverse models of chronic ileitis, namely SAMP1/YitFc and TNF^ΔARE mice. Both models share clinical, pathological, and immunological characteristics with CD, and are considered to be representative of the human condition (16, 17). We measured the relative expression of the mRNA for tmDR3 in the terminal ileum of inflamed SAMP1/YitFc mice and compared it to the levels in both uninflamed control AKR and young SAMP1/YitFc mice before the development of ileitis, with the latter serving as the internal, strain-specific control.

As shown in Fig. 4B, there was significantly increased expression of tmDR3 in inflamed SAMP1/YitFc mice compared with age-matched AKR controls (230% increase). More importantly, the up-regulated expression of tmDR3 was clearly related to inflammation, because it was absent in the young, noninflamed SAMP1/YitFc mice (350% increase after the development of ileitis). On the other hand, no difference was observed between young and old AKR mice. A similar, ileitis-specific up-regulation of tmDR3 was observed in TNF^ΔARE mice, with older mice again demonstrating significantly higher expression of tmDR3 (270% increase vs. age-matched wild-type and 230% vs. young, preinflamed TNF^ΔARE mice) (Fig. 4C).

TL1A Is Up-Regulated in Chronic Ileitis and Is Primarily Expressed on APCs.

Next, we measured the expression of TL1A in the terminal ileum of TNF^ΔARE and wild-type mice. As shown in Fig. 5A, there was significantly increased expression of TL1A in mice with ileitis as compared with uninflamed control mice (3.4-fold increase, P < 0.001). Similar to our studies in CD patients (13), immunostaining for TL1A protein showed increased TL1A expression in the lamina propria of mice with ileitis compared with wild type controls (data not shown). The significant up-regulation of both TL1A and the active/tm form of its receptor DR3 during chronic ileitis indicates that binding of TL1A and downstream signaling takes place within the inflamed mucosa. It is, therefore, critically important to identify the cellular source(s) of TL1A. To achieve this end, we used flow cytometry to study the expression of TL1A in small intestinal LPMCs isolated from inflamed SAMP1/YitFc and TNF^ΔARE mice. Using a monoclonal antibody against TL1A, we demonstrated that expression of TL1A was confined to CD11c-positive cells (Fig. 5B). We further confirmed that CD11c and TL1A colocalize by using confocal microscopy of stained LPMCs (Fig. 5C). To determine whether the major source of TL1A is true dendritic cells, or nondendritic CD11c-positive cells, we further stained for expression of MHC-II. By applying sequential gating, we clearly identified two major populations of cells that express TL1A (Fig. 5D). Firstly, TL1A is highly expressed on CD11c^high/MHC-II^pos cells. Secondly, TL1A was also expressed on a population of CD11c^low/MHC-II^neg cells. On the other hand, only very low expression of TL1A was observed on CD11c^low/MHC-II^pos cells, and no expression was detected on CD11c-negative cells. Taken together these results indicate that lamina propria dendritic cells are the major source of TL1A in the intestinal mucosa of mice with spontaneous ileitis.

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.

Discussion

Members of the TNF/TNFR superfamilies of proteins are abundantly expressed throughout the immune system and exert a wide array of effects on immune cells (5). An important property of many of these proteins is their ability to provide costimulatory signals for the enhancement of immune responses (6–8, 22). In the present study, we provide evidence that the TNF-like cytokine TL1A and its functional receptor DR3 also act as costimulators for T cells. In addition we show that interactions between lymphocytic tmDR3 and APC-derived TL1A occur and may be of pathophysiological importance during chronic CD-like ileitis in mice.

Our studies clearly demonstrate that one of the main functions of TL1A is to induce production of IFN-γ by murine lymphocytes. Similar effects were recently reported in studies of human lymphocytes (9, 13, 15, 23). This study further explored the specific role that TL1A plays in the pathways that lead to production of IFN-γ. First, the effects of TL1A are more pronounced in the presence of suboptimal (i.e., low-dose) stimulation through the TCR, which may indicate that TL1A is involved in the preservation or amplification of IFN-γ responses when the antigenic load is low. Second, the in vitro experiments we carried out demonstrated that TL1A synergizes with IL-12 and that this synergy occurs independently of IL-18. Combining IL-12 and TL1A, therefore, provides an alternative route for the cytokine-mediated induction of IFNγ, which may be of particular importance when IL-18 signaling is absent or defective. Finally, TL1A preferentially acts on memory cells, which indicates that TL1A may be particularly important during the late/effector phase of Th1 immunity, increasing the amount of IFN-γ that is available and, thereby, increasing the magnitude of the immune response.

The majority of the TNFRSFPs are produced as tm proteins under normal circumstances, with the soluble forms being generated by proteolytic cleavage (24). It is therefore of particular interest that a soluble form of DR3 can be directly generated by means of alternative splicing (20). Our study clearly demonstrates that tmDR3 predominates only after T cell activation and that this predominance is a result of mRNA rearrangement. This phenomenon may be indicative of a mechanism that prevents inappropriate functional ligation by TL1A under physiological conditions. The tight regulation of TL1A is further strengthened by the existence of an inhibitory receptor, decoy receptor 3 (DcR3)/TR6, which occurs only as a soluble protein (9). DcR3 competes with DR3 for the binding of TL1A and abrogates its stimulatory effects on T cells (9), as was recently shown in studies with DcR3-deficient mice. TL1A signaling appears to be compromised in these mice, resulting in defective IFN-γ production and a bias toward Th2 polarization of the immune response, resulting in increased susceptibility to infection (25). Similar control mechanisms also exist for other members of the TNF/TNFRSFPs, such as Fas (26, 27). It is possible that, under conditions of chronic inflammation, these regulatory mechanisms are compromised, as indicated for IBD by the up-regulation of tmDR3 expression reported in our study. This up-regulation could then lead to unrestricted TL1A signaling and deleterious proinflammatory effects mediated by the increased levels of IFN-γ.

CD results from a dysregulated activation of the gut-associated mucosal immune system in genetically predisposed individuals (21). Many of the findings presented herein indicate that TL1A/DR3 may participate in the pathogenesis of CD. Our data show that chronic mucosal inflammation is associated with alternative splicing of DR3 and up-regulated expression of the tm form of the receptor. At the same time, there is overexpression of TL1A primarily on mucosal APCs. A central pathogenic mechanism in CD involves aberrant presentation of lumenal bacterial antigens by APCs to lamina propria T cells (21). The localization of TL1A and DR3 in APCs and T cells, respectively, therefore raises the possibility that a functional association takes place between these proteins during chronic ileitis. Our in vitro findings indicate that such an association would lead to proliferation of effector lymphocytes in the inflamed mucosa. Expansion of lamina propria T cells is a central characteristic of both experimental ileitis (including SAMP1/YitFc and TNF^ΔARE mice) and the human condition (16, 17, 21). Binding of TL1A to DR3 on mucosal lymphocytes would lead to increased secretion of IFN-γ from the lymphocytes. This increased secretion may be of particular importance, because CD is considered a prototypic Th1-mediated condition in which IFN-γ plays a central pathogenetic role. In fact, as we and others have shown, the TL1A/DR3 system is up-regulated during CD (13–15). Interestingly, it was recently reported that single nucleotide polymorphisms conferred susceptibility to CD in one Japanese and two European cohorts (28). In addition to our findings, recent studies by other research groups have implicated TL1A/DR3 in the pathogenesis of other inflammatory conditions. An association with rheumatoid arthritis has recently been reported for a destabilizing mutation in the dr3 gene (29). Furthermore, TL1A and DR3 may be involved in the development of atherogenesis through the induction of proinflammatory cytokines and matrix metalloproteinases (30).

The data presented herein lead us to propose that, under physiological conditions, expression of tmDR3 on lymphocytes is minimal, and functional signaling is inhibited. However, when chronic inflammation develops, T cells up-regulate the expression of the active, tm form of DR3. In addition, TL1A expressed on APCs binds to tmDR3 on lymphocytes, which triggers costimulatory signals that significantly amplify production of IFN-γ. This interaction and the subsequent functional outcomes may be of particular importance during disease phases when there is only low-level stimulation with antigens, such as during the early/induction or low-activity/maintenance stages. We therefore conclude that interactions between TL1A and DR3 participate in the pathogenesis of Th1-mediated inflammation, including the inflammation observed in patients with CD. Manipulation of these interactions may have therapeutic potential for these conditions.

Materials and Methods

Recombinant Proteins and Antibodies.

A DNA sequence that encoded the C-terminal extracellular domain of mouse TL1A (Ser-81 to Leu-252) was cloned into the pET 28a(+) expression vector (pET System; Novagen) and expressed in Escherichia coli. The resulting rmTL1A was purified via its His-tag under native conditions by metal chelation chromatography (Ni-NTA His Bind Resins; Novagen). Lipopolysaccharide was removed by using Detoxi-Gel Endotoxin Removing Gel (Pierce). A human anti-mouse anti-TL1A mAb (clone MT101) was kindly provided by Human Genome Sciences, Rockville, MD. Anti-CD3e (145-2C11) and anti-CD28 (37.51) mAbs were purchased from BD Biosciences, and rm IL-12 and IL-18 from R & D Systems.

Mice.

SAMP1/YitFc, control AKR, heterozygous TNF^ΔARE/+, and wild-type TNF^+/+ mice were maintained under specific pathogen-free conditions in the Animal Facility at the University of Virginia. All protocols were approved by the Animal Care and Use Committee of the University of Virginia.

Cell Isolation and Culture.

Highly enriched suspensions of CD4⁺ lymphocytes (>90%) were obtained by positive selection using an immunomagnetic cell-sorting system (Miltenyi Biotec). Lymphocytes were cultured in 96-well round-bottom plates at 10⁶ cells per ml of complete medium (RPMI medium 1640, 10% FBS, 2 mM l-glutamine, 1 × 10⁻⁵ mol/l β-mercaptoethanol, and 1% penicillin/streptomycin) under the conditions indicated in the figure legends. After 24–72 h, supernatants were collected and stored at −80°C until further use. The concentration of IFN-γ in the supernatants was measured by using a commercially available ELISA kit (BD Biosciences Pharmingen). For measurement of DR3 mRNA content, cells were cultured overnight in 24-well plates at 2 × 10⁶ cells per ml and then recovered from the wells and stored as pellets at −80°C.

Proliferation Assay for Naïve and Memory Lymphocytes.

Naïve and memory lymphocytes were purified from the magnetically sorted CD4⁺ splenocytes by incubation with fluorochrome-conjugated antibodies against CD4⁺ and CD45RB. Separation of CD4⁺/CD45RB^hi (naïve) and CD4⁺/CD45RB^lo (memory) cells was performed on a FACScalibur flow cytometer (Becton Dickinson). To determine the levels of proliferation, cells (10⁵ per condition) were cultured in triplicate for 96 h and then pulsed with [³H]thymidine [1 μCi (1 Ci = 37 GBq) per well] (MP Biomedicals, Irvine, CA) overnight. Proliferation was estimated by measuring incorporation of the thymidine.

Real-Time PCR.

Total RNA was isolated from homogenized tissue or cell pellets by using the RNeasy Mini kit (Qiagen, Valencia, CA) and converted to cDNA with the GeneAmp RNA PCR kit (Applied Biosystems) by using random hexamers (0.75 μg of total RNA in a final reaction volume of 20 μl). cDNA was quantified by real-time PCR using an iCycler detection system (Bio-Rad). The primers and TaqMan probes were designed with the assistance of Beacon Designer software (premier; Biosoft). To specifically quantify the full/tm DR3 splice variants, the following primers were used: forward 5′-TGGCTTCTATATACGTGGCAATGA-3′; reverse 5′-GCACCTGGACCCAAAACATCT-3′; probe 5′-AGCCACAGACAGCAGTGCAAGCCT-3′. For total DR3 (all splice variants), the primers were: forward 5′-AAGAGGCCCTTCAAGTGACC-3′; reverse 5′-AGTCAACACACCAGCCTGAC-3′; probe 5′-CTCGGCAAAGTCGGACACCCACTG-3′. The real-time PCR was performed with iTaq DNA polymerase (Bio-Rad) as per the manufacturer's recommendations, in a reaction mix consisting of 3 mM MgCl₂, 200 μM solutions of each dNTP, 400 nM solutions of each primer, and a 200 nM solution of the probe. Five percent of the volume of the first-strand synthesis was added and the total volume adjusted to 25 μl. TL1A mRNA detection was quantified by real-time RT-PCR using the iQ^TM SYBR green Supermix (Bio-Rad). The following primers were used: forward, GCTGCCTGTTGTCATTTCC; reverse, TCTGGGAGGTGAGTAAACTTG. A separate reaction mix was set up with primers for β-actin mRNA as the reference standard: forward 5′-CAGGGTGTGATGGTGGGAATG-3′, reverse 5′-GTAGAAGGTGTGGTGCCAGATC-3′. For detection of β-actin by real-time PCR, a 400 nM solution of each primer and 5% of the volume of the first-strand synthesis were used in a total volume of 25 μl that included iQ SYBR green Supermix (Bio-Rad) according to manufacturer's directions.

Thermocycling conditions for all targets were as follows: 95°C for 3 min (to activate the iTaq DNA polymerase), then 40 cycles of 95°C for 15 sec, 60°C for 15 sec, and 72°C for 15 sec. The standard curve method was used to quantify the relative mRNA level of mDR3, as described in Essentials of Real-Time PCR (Applied Biosystems). Results were expressed as a relative ratio to the lowest control sample. All samples were assayed in duplicate.

Flow Cytometry.

LPMCs were isolated as described in ref. 18. The expression of TL1A on the surface of LPMCs was then analyzed by FACS, using a mAb specific for murine TL1A (MT101). APC-conjugated mouse anti-human IgG was used as the secondary Ab (BD Pharmingen). Antibodies against murine CD11c (HL3), MHC-II (IA/IE, clone 114.15.2), and CD16/CD32 (clone 2.4G2, to block nonspecific FcR binding) were all purchased from BD Pharmingen.

Statistical Analysis.

The Student t test was used for statistical analysis, with an α level of 0.05 considered to be significant (P < 0.05).

Abbreviations

APC

antigen-presenting cell

CD

Crohn's disease

DR

death-domain receptor

IBD

inflammatory bowel disease

LPMC

lamina propria mononuclear cell

rm

recombinant murine

tm

transmembrane

TCR

T cell antigen receptor

Th

T helper

TNFSFPs

TNF superfamily of proteins

TNFRSFPs

TNF receptor superfamily of proteins.