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.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: TNF-like ligand 1A (TL1A), which binds its cognate receptor DR3 and the decoy receptor DcR3, is an identified member of the TNF superfamily. TL1A exerts pleiotropic effects on cell proliferation, activation, and differentiation of immune cells, including helper T cells and regulatory T cells. TL1A and its two receptors expression is increased in both serum and inflamed tissues in autoimmune diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), and ankylosing spondylitis (AS). Polymorphisms of the TNFSF15 gene that encodes TL1A are associated with the pathogenesis of irritable bowel syndrome, leprosy, and autoimmune diseases, including IBD, AS, and primary biliary cirrhosis (PBC). In mice, blocking of TL1A-DR3 interaction by either antagonistic antibodies or deletion of the DR3 gene attenuates the severity of multiple autoimmune diseases, whereas sustained TL1A expression on T cells or dendritic cells induces IL-13-dependent small intestinal inflammation. This suggests that modulation of TL1A-DR3 interaction may be a potential therapeutic target in several autoimmune diseases, including IBD, RA, AS, and PBC.Mediators of Inflammation 01/2013; 2013:258164. · 3.88 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4+ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A's role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.PLoS ONE 01/2014; 9(8):e105627. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Objectives: To investigate the role of Death Receptor 3 (DR3) and its ligand TNF-like protein 1A (TL1A) in early stages of inflammatory arthritis.Methods: C57BL/6 mice genetically deficient in the DR3 gene (DR3KO) and their DR3WT littermates were subjected to antigen-induced arthritis (AIA) by priming and intra-articular injection of methylated BSA. Joints were sectioned and analyzed histochemically for damage to cartilage and expression of DR3, TL1A, Ly6G (a marker for neutrophils), the gelatinase matrix metallopeptidase 9 (MMP-9), the aggrecanase a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) and the neutrophil chemoattractant CXCL1. In vitro production of MMP-9 was measured from cultures of fibroblasts, macrophages and neutrophils following addition of TL1A and other pro-inflammatory stimuli.Results: DR3 expression was upregulated in the joint following induction of AIA in DR3WT mice. DR3KO mice were protected from cartilage damage compared to DR3WT mice, even at early timepoints prior to the main accumulation of effector T cells in the joint. Early protection from AIA in vivo correlated with reduced levels of MMP-9. In vitro, neutrophils were found to be major producers of MMP-9, while in vivo neutrophil numbers were reduced in DR3KO joints. However, TL1A neither induced MMP-9 release, nor affected survival of, neutrophils. Instead, reduced levels of CXCL1 were recorded in DR3KO joints.Conclusions: DR3 drives early cartilage destruction in the AIA model of inflammatory arthritis through the release of CXCL1, maximizing neutrophil recruitment to the joint leading to enhanced local production of cartilage destroying enzymes. © 2014 American College of Rheumatology.Arthritis & Rheumatology. 06/2014;
Role of TL1A and its receptor DR3 in two models
of chronic murine ileitis
Giorgos Bamias*, Margarita Mishina*, Mark Nyce*, William G. Ross*, Giorgos Kollias†, Jesus Rivera-Nieves*,
Theresa T. Pizarro*, and Fabio Cominelli*‡
*Digestive Health Center of Excellence, University of Virginia Health Sciences Center, Charlottesville, VA 22908; and†Institute of Immunology,
Alexander Fleming Biomedical Sciences Research Center, 16672 Vari, Greece
Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved March 31, 2006 (received for review
December 16, 2005)
Through this interaction, TL1A induces secretion of IFN-? and may,
therefore, participate in the development of T helper-1-type ef-
fector responses. In this study, we investigated whether interac-
tions between TL1A and DR3 are involved in the pathogenesis of
chronic murine ileitis. We demonstrate that alternative splicing of
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 in-
flammation 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 CD11chighdendritic cells. We also
report that TL1A acts preferentially on memory CD4??CD45RBlo
murine lymphocytes by significantly inducing their proliferation,
whereas it does not affect the proliferation of the naı ¨ve CD4??
CD45RBhiT 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-? via an IL-18-independent
pathway. Our results raise the possibility that interaction(s) be-
tween TL1A expressed on antigen-presenting cells and tm DR3 on
lymphocytes may be of particular importance for the pathogenesis
including inflammatory bowel disease. Blockade of the TL1A?DR3
pathway may, therefore, offer therapeutic opportunities in
Crohn’s disease ? cytokines ? mucosal inflammation
involves several cell types, costimulatory molecules, transcrip-
tion factors, and secreted cytokines (1). Antigen-presenting cell
(APC)-derived IL-12 is essential for the induction of IFN-?, 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-?, 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 abun-
dantly expressed in the immune system, and are critically in-
volved in the differentiation, proliferation, and apoptosis of
immune cells (5). Several members of these families induce
secretion of IFN-? upon ligand?receptor binding, thereby en-
hancing Th1-type responses (6–8). TL1A (TNFSPF15) is a
recently identified, TNF-like factor that is currently the only
he differentiation of naı ¨ve CD4?lymphocytes into IFN-?-
secreting Th1 ‘‘effector’’ cells is a multistep process that
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-?B-mediated path-
ways (9, 12). TL1A specifically induces secretion of IFN-? 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-? 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 patho-
genesis 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 CD11chighdendritic cells, raising the possibility
that there is an interaction between lymphocytic tmDR3 and
APC-derived TL1A. We also show that TL1A acts as a costimu-
lator for lymphocytes, enhancing IFN-? 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 naı ¨ve CD4?lym-
phocytes, a finding that supports a role for TL1A during the
effector phase of Th1 responses.
TL1A Enhances TCR-Mediated Secretion of IFN-? by Murine Lympho-
cytes. We initially investigated whether TL1A affects TCR-
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: APC, antigen-presenting cell; CD, Crohn’s disease; DR, death-domain re-
ceptor; 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.
See Commentary on page 8303.
‡To whom correspondence should be addressed at: Digestive Health Center of Excellence,
University of Virginia Health Sciences Center, P.O. Box 800708, Charlottesville, VA 22908.
© 2006 by The National Academy of Sciences of the USA
May 30, 2006 ?
vol. 103 ?
no. 22 ?
recombinant murine (rm)TL1A to cultured CD4?cells, in the
presence of immobilized anti-CD3 and soluble anti-CD28 Abs,
significantly increased IFN-? secretion in a dose-dependent
fashion (Fig. 1 A and B). In addition, we found that low-dose (0.2
?g?ml) anti-CD3 stimulation provided optimal conditions for
TL1A function (average increase in IFN-? secretion, 223%;
range, 143–349%) (Fig. 1C). In contrast, the effect was greatly
diminished when 1 ?g?ml anti-CD3 was used (122%; range,
stimulation (100%; range, 78–117%).
TL1A Acts in Synergy with IL-12 and Independently of IL-18 for the
Induction of IFN-?. It has been demonstrated that a combination
of IL-12 and IL-18 can up-regulate IFN-? in the absence of
Ag?TCR stimulation (19). We investigated whether TL1A could
also synergize with this cytokine pathway for IFN-? 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-? secreted, in the
complete absence of TCR-mediated signals (Fig. 2A).
To further explore the role played by TL1A in the pathways that
CD4?cells, TL1A synergized with the combination of TCR stim-
ulation and IL-12; however, when used in combination with IL-18
and TCR stimulation, TL1A failed to induce higher levels of
secretion of IFN-? 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
and indicated on the axes of the graphs. (A) Cells were stimulated with
anti-CD3 (0.5 ?g?ml) and anti-CD28 (1 ?g?ml) Abs, then rmTL1A (100 ng?ml)
was added to the cultures. The concentration of IFN-? 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 concentra-
tion of IFN-? was measured at 24–96 h. A representative of three experiments
is shown. Asterisks indicate values of P ? 0.05 for comparison between IFN-?
secretion with or without rmTL1A for each time point. (C) Cells were stimu-
lated with different doses of anti-CD3 (0.5–10 ?g?ml). The concentration of
IFN-? was measured at 72 h. Pooled data from four independent experiments
are shown. All data are presented as mean ? SEM.
Effects of TL1A stimulation on TCR-mediated IFN-? secretion. CD4?
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 ?g?ml), anti-CD28 (1 ?g?ml), rmIL-12 (10 ng?
ml), rmIL-18 (100 ng?ml), and rmTL1A (100 ng?ml), as indicated. The concen-
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-? in the supernatant was mea-
sured after 72 h. Six individual experiments are shown. Horizontal lines
indicate the average for each group.
TL1A synergizes with IL-12. (A) CD4?splenocytes were stimulated
www.pnas.org?cgi?doi?10.1073?pnas.0510903103 Bamias et al.
a significant costimulatory effect, leading to a 3- to 17-fold increase
in IFN-? secretion (Fig. 2C).
TL1A Acts on Memory Th Cells. Next, we tested whether TL1A
stimulates the naı ¨ve or memory lymphocytic subpopulations. We
therefore studied the proliferative responses of highly purified
CD4??CD45RBhi(naı ¨ve) and CD4??CD45RBlo(memory) cells
isolated from the spleens of C57?Bl6 mice. As expected, naı ¨ve
splenocytes were highly responsive to stimulation with IL-12 (Fig.
3A). However, no increase in proliferation was observed after
addition of TL1A to cultured naı ¨ve lymphocytes, nor was there any
synergistic effect when IL-12 and TL1A were combined (Fig. 3A).
By contrast, CD4??CD45RBlocells 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 en-
hanced (Fig. 3B). Indeed, whereas TL1A induced only a modest
proliferative response from naı ¨ve lymphocytes (148% ? 88% over
(380% ? 141%) (Fig. 3C).
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
second lacks the tm region and is, therefore, predicted to encode a
soluble protein. Finally, the third variant lacks one of the cysteine-
is limited to activated lymphocytes (10, 11), we hypothesized that
lymphocytic activation might be associated with up-regulation of
in the expression of a fully functional tm receptor and increased
To test our hypothesis, we designed a dual amplification
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.
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
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?AREmice with ileitis (24-wk-old) or before the devel-
group). All data are presented as mean ? SEM.
Expression of tm DR3 mRNA during activation of lymphocytes and in
memory (CD4??CD45RBhi) splenocytes were purified and cultured as de-
estimated by measuring thymidine incorporation. Representatives of two
experiments are shown. (C) Average proliferative responses for naı ¨ve and
are presented as mean ? SEM.
TL1A acts on memory lymphocytes. Naı ¨ve (CD4??CD45RBhi) and
Bamias et al.
May 30, 2006 ?
vol. 103 ?
no. 22 ?
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.
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 immunoge-
netically diverse models of chronic ileitis, namely SAMP1?YitFc
and TNF?AREmice. 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 expres-
sion 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?AREmice, with older mice again
demonstrating significantly higher expression of tmDR3 (270%
increase vs. age-matched wild-type and 230% vs. young, prein-
flamed TNF?AREmice) (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?AREand 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?AREmice.
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 colo-
calize by using confocal microscopy of stained LPMCs (Fig. 5C).
To determine whether the major source of TL1A is true den-
dritic 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 CD11chigh?
MHC-IIposcells. Secondly, TL1A was also expressed on a
RNA extracted from the terminal ilea of TNF?AREmice (?8-wk-old, n ? 13) and age-matched wild-type mice (n ? 14). All data are presented as mean ? SEM. (B)
to the CD11c-positive fraction. (C) Confocal microscopy of LPMCs incubated with antibodies against mouse TL1A (red) and CD11c (green) demonstrated that the
plots indicate the expression of TL1A (green) or negative control (red) in each subpopulation. Negative control indicates staining with secondary Ab alone.
www.pnas.org?cgi?doi?10.1073?pnas.0510903103 Bamias et al.
population of CD11clow?MHC-IInegcells. On the other hand,
only very low expression of TL1A was observed on CD11clow?
MHC-IIposcells, 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.
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-? by murine lymphocytes.
Similar effects were recently reported in studies of human lympho-
TL1A plays in the pathways that lead to production of IFN-?. 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 ampli-
fication of IFN-? 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 alter-
native route for the cytokine-mediated induction of IFN?, 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
The majority of the TNFRSFPs are produced as tm proteins
under normal circumstances, with the soluble forms being gener-
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 phenom-
functional ligation by TL1A under physiological conditions. The
tight regulation of TL1A is further strengthened by the existence of
only as a soluble protein (9). DcR3 competes with DR3 for the
as was recently shown in studies with DcR3-deficient mice. TL1A
signaling appears to be compromised in these mice, resulting in
defective IFN-? production and a bias toward Th2 polarization of
the immune response, resulting in increased susceptibility to infec-
tion (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 mech-
of tmDR3 expression reported in our study. This up-regulation
could then lead to unrestricted TL1A signaling and deleterious
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 overexpres-
sion 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 prolif-
eration of effector lymphocytes in the inflamed mucosa. Expan-
sion of lamina propria T cells is a central characteristic of both
experimental ileitis (including SAMP1?YitFc and TNF?ARE
mice) and the human condition (16, 17, 21). Binding of TL1A to
DR3 on mucosal lymphocytes would lead to increased secretion
of IFN-? from the lymphocytes. This increased secretion may be
of particular importance, because CD is considered a prototypic
Th1-mediated condition in which IFN-? plays a central patho-
genetic 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 Euro-
pean cohorts (28). In addition to our findings, recent studies by
other research groups have implicated TL1A?DR3 in the patho-
genesis of other inflammatory conditions. An association with
rheumatoid arthritis has recently been reported for a destabi-
lizing 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
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 costimula-
tory signals that significantly amplify production of IFN-?. 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 con-
clude that interactions between TL1A and DR3 participate in the
pathogenesis of Th1-mediated inflammation, including the inflam-
mation 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 en-
coded 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?ARE/?,
and wild-type TNF?/?mice were maintained under specific
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). Lym-
phocytes were cultured in 96-well round-bottom plates at 106
Bamias et al.
May 30, 2006 ?
vol. 103 ?
no. 22 ?
cells per ml of complete medium (RPMI medium 1640, 10%
FBS, 2 mM L-glutamine, 1 ? 10?5mol?l ?-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
available ELISA kit (BD Biosciences Pharmingen). For mea-
surement of DR3 mRNA content, cells were cultured overnight
in 24-well plates at 2 ? 106cells per ml and then recovered from
the wells and stored as pellets at ?80°C.
Proliferation Assay for Naı ¨ve and Memory Lymphocytes. Naı ¨ve and
memory lymphocytes were purified from the magnetically sorted
CD4?splenocytes by incubation with fluorochrome-conjugated
antibodies against CD4?and CD45RB. Separation of CD4??
CD45RBhi(naı ¨ve) and CD4??CD45RBlo(memory) cells was
performed on a FACScalibur flow cytometer (Becton Dickin-
son). To determine the levels of proliferation, cells (105per
condition) were cultured in triplicate for 96 h and then pulsed
with [3H]thymidine [1 ?Ci (1 Ci ? 37 GBq) per well] (MP
Biomedicals, Irvine, CA) overnight. Proliferation was estimated
by measuring incorporation of the thymidine.
cell pellets by using the RNeasy Mini kit (Qiagen, Valencia, CA)
and converted to cDNA with the GeneAmp RNA PCR kit (Ap-
in a final reaction volume of 20 ?l). 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
used: forward 5?-TGGCTTCTATATACGTGGCAATGA-3?; re-
verse 5?-GCACCTGGACCCAAAACATCT-3?; probe 5?-AGC-
CACAGACAGCAGTGCAAGCCT-3?. For total DR3 (all splice
variants), the primers were: forward 5?-AAGAGGCCCT-
TCAAGTGACC-3?; reverse 5?-AGTCAACACACCAGCCT-
GAC-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 reac-
tion mix consisting of 3 mM MgCl2, 200 ?M 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 ?l. TL1A mRNA
detection was quantified by real-time RT-PCR using the iQTM
SYBR green Supermix (Bio-Rad). The following primers were
used: forward, GCTGCCTGTTGTCATTTCC; reverse, TCTGG-
GAGGTGAGTAAACTTG. A separate reaction mix was set up
with primers for ?-actin mRNA as the reference standard: forward
5?-CAGGGTGTGATGGTGGGAATG-3?, reverse 5?-GTA-
GAAGGTGTGGTGCCAGATC-3?. For detection of ?-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 ?l that included iQ SYBR green Supermix (Bio-Rad) according
to manufacturer’s directions.
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 sec-
ondary 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 pur-
chased from BD Pharmingen.
Statistical Analysis. The Student t test was used for statistical
analysis, with an ? level of 0.05 considered to be significant
(P ? 0.05).
We thank the Histology?Imaging Core and the Immunology?Cell Iso-
of Diabetes and Digestive and Kidney Diseases, University of Virginia
(UVA) Digestive Health Research Center; Paul Moore of Human
Genomic Sciences for the kind gift of the human anti-mouse TL1A
antibody; and Dr. Sarah A. De La Rue for critically reviewing the
Grants DK-42191, DK-44540, and DK-55812 (to F.C.); the UVA
Digestive Health Research Center (1P30DK67629); and a Research
Fellowship Award from the Crohn’s and Colitis Foundation of America
1. Murphy, K. M. & Reiner, S. L. (2002) Nat. Rev. Immunol. 2, 933–944.
2. Trinchieri, G. (2003) Nat. Rev. Immunol. 3, 133–146.
3. Szabo, S. J., Kim, S. T., Costa, G. L., Zhang, X., Fathman, C. G. & Glimcher,
L. H. (2000) Cell 100, 655–669.
4. Trinchieri, G., Pflanz, S. & Kastelein, R. A. (2003) Immunity 19, 641–644.
5. Locksley, R. M., Killeen, N. & Lenardo, M. J. (2001) Cell 104, 487–501.
7. Huard, B., Schneider, P., Mauri, D., Tschopp, J. & French, L. E. (2001)
J. Immunol. 167, 6225–6231.
8. Wen, T., Bukczynski, J. & Watts, T. H. (2002) J. Immunol. 168, 4897–4906.
9. Migone, T. S., Zhang, J., Luo, X., Zhuang, L., Chen, C., Hu, B., Hong, J. S.,
Perry, J. W., Chen, S. F., Zhou, J. X., et al. (2002) Immunity 16, 479–492.
10. Screaton, G. R., Xu, X. N., Olsen, A. L., Cowper, A. E., Tan, R., McMichael,
A. J. & Bell, J. I. (1997) Proc. Natl. Acad. Sci. USA 94, 4615–4619.
11. Tan, K. B., Harrop, J., Reddy, M., Young, P., Terrett, J., Emery, J., Moore, G.
& Truneh, A. (1997) Gene 204, 35–46.
12. Wen, L., Zhuang, L., Luo, X. & Wei, P. (2003) J. Biol. Chem. 278,
13. Bamias, G., Martin, C., III, Marini, M., Hoang, S., Mishina, M., Ross, W. G.,
Sachedina, M. A., Friel, C. M., Mize, J., Bickston, S. J., et al. (2003) J. Immunol.
14. Papadakis, K. A., Prehn, J. L., Landers, C., Han, Q., Luo, X., Cha, S. C., Wei,
P. & Targan, S. R. (2004) J. Immunol. 172, 7002–7007.
15. Prehn, J. L., Mehdizadeh, S., Landers, C. J., Luo, X., Cha, S. C., Wei, P. &
Targan, S. R. (2004) Clin. Immunol. 112, 66–77.
16. Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F. & Kollias, G.
(1999) Immunity 10, 387–398.
M. J., Moskaluk, C. A., Cohn, S. M. & Cominelli, F. (2003) Gastroenterology
18. Bamias, G., Martin, C., Mishina, M., Ross, W. G., Rivera-Nieves, J., Marini, M.
& Cominelli, F. (2005) Gastroenterology 128, 654–666.
19. Yang, J., Murphy, T. L., Ouyang, W. & Murphy, K. M. (1999) Eur. J. Immunol.
20. Wang, E. C., Kitson, J., Thern, A., Williamson, J., Farrow, S. N. & Owen, M. J.
(2001) Immunogenetics 53, 59–63.
21. Bouma, G. & Strober, W. (2003) Nat. Rev. Immunol. 3, 521–533.
22. Watts, T. H. (2005) Annu. Rev. Immunol. 23, 23–68.
23. Papadakis, K. A., Zhu, D., Prehn, J. L., Landers, C., Avanesyan, A., Lafkas, G.
& Targan, S. R. (2005) J. Immunol. 174, 4985–4990.
24. Levine, S. J. (2004) J. Immunol. 173, 5343–5348.
25. Hsu, T. L., Wu, Y. Y., Chang, Y. C., Yang, C. Y., Lai, M. Z., Su, W. B. & Hsieh,
S. L. (2005) J. Immunol. 175, 5135–5145.
26. Ashkenazi, A. (2002) Nat. Rev. Cancer 2, 420–430.
27. Hughes, D. P. & Crispe, I. N. (1995) J. Exp. Med. 182, 1395–1401.
Cardon, L., Takazoe, M., Tanaka, T., Ichimori, T., et al. (2005) Hum. Mol.
Genet. 14, 3499–3506.
29. Borysenko, C. W., Furey, W. F. & Blair, H. C. (2005) Biochem. Biophys. Res.
Commun. 328, 794–799.
30. Kang, Y. J., Kim, W. J., Bae, H. U., Kim, D. I., Park, Y. B., Park, J. E., Kwon,
B. S. & Lee, W. H. (2005) Cytokine 29, 229–235.
www.pnas.org?cgi?doi?10.1073?pnas.0510903103Bamias et al.