LIGHT Is Constitutively Expressed on T and
NK Cells in the Human Gut and Can Be
Induced by CD2-Mediated Signaling
This information is current as
of July 31, 2017.
Offer Cohavy, Jaclyn Zhou, Carl F. Ware and Stephan R.
Targan
J Immunol 2005; 174:646-653; ;
doi: 10.4049/jimmunol.174.2.646
http://www.jimmunol.org/content/174/2/646
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References
The Journal of Immunology
LIGHT Is Constitutively Expressed on T and NK Cells in the
Human Gut and Can Be Induced by CD2-Mediated Signaling1
Offer Cohavy,* Jaclyn Zhou,* Carl F. Ware,† and Stephan R. Targan2*
B
oth Crohn’s disease (CD)3 and ulcerative colitis (UC),
collectively defined as inflammatory bowel diseases
(IBD) (1), are the result of a dysregulated mucosal inflammatory response (2). The intestinal immune compartment is
differentially regulated, and its antigenic repertoire is independently shaped to accommodate the heavy antigenic load characteristic of the gut environment (3). In IBD, tolerance to intestinal
Ags is perturbed (4, 5), and strong evidence implicates a skewed T
cell-mediated Th1 response in CD (6), as well as mouse models of
IBD (7, 8). TNF is implicated as an important contributor to intestinal inflammation with a subset of patients with CD responding
to anti-TNF therapy (9). However, the partial success of blocking
TNF emphasizes the complexity of mucosal immune regulatory
mechanisms, prompting an investigation of other TNF-related ligands, such as lymphotoxin (LT) ␣␤ and lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT), in human IBD
pathology (2, 10, 11).
TNF, LT␣␤, and LIGHT form a network of signaling systems
involved in many aspects of immune function and inflammation
*Cedars-Sinai Inflammatory Bowel Disease Center, Los Angeles, CA 90048; and
†
Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology,
San Diego, CA 92121
Received for publication September 2, 2004. Accepted for publication October
20, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants
F32DK10139 (to O.C.), R01DK57328/R01DK43211 (to S.R.T.), and R37AI33068
(to C.F.W.).
2
Address correspondence and reprint requests to Dr. Stephan R. Targan, Cedars-Sinai
Inflammatory Bowel Disease Center, 8700 Beverly Boulevard, Suite D4063, Los
Angeles, CA 90048. E-mail address: [email protected]
3
Abbreviations used in this paper: CD, Crohn’s disease; CHX, cycloheximide;
HVEM, herpesvirus entry mediator; IBD, inflammatory bowel disease; LIGHT, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells; LP, lamina propria; LPMC, mononuclear cells
from LP; LT, lymphotoxin; PB, peripheral blood; P/I, PMA and ionomycin; UC,
ulcerative colitis.
Copyright © 2005 by The American Association of Immunologists, Inc.
(reviewed in Refs. 12 and 13). LIGHT signaling is transduced via
two members of the TNFR family, herpesvirus entry mediator
(HVEM, TNFRSF14) and LT␤R (TNFRSF3) (14, 15), which also
binds the LT␣␤ heterotrimer involved in the development and organization of peripheral lymphoid tissue (16). In addition, LIGHT
is bound and putatively regulated by DcR3 (TNFRSF6B), a soluble decoy receptor (17). LT␤R is broadly expressed, including
stromal and myeloid cells, but absent on lymphocytes, whereas
HVEM is expressed prominently on lymphocytes. LIGHT is primarily expressed in the lymphoid compartment by activated T
cells, but also by monocytes (14). LIGHT-HVEM engagement is
likely to play an important immunomodulatory role mediating
stimulatory T-T interactions, because HVEM can deliver a costimulatory signal augmenting proinflammatory cytokine production and T cell proliferation (18). Disruption of the LIGHT gene in
the mouse (19 –21) revealed no abnormalities in lymphoid organ
development, but CD8⫹ T cell differentiation in response to Ag
was compromised (20).
Aberrant regulation of some TNF-related cytokines, including
TNF (22), Fas ligand (23), or B cell-activating factor of the TNF
family (24), can precipitate autoimmune diseases (25). Transgenic
expression of LIGHT (TNFSF14) by T cells induced severe intestinal inflammation with autoimmune-like pathology in mice, specifically linking LIGHT-mediated signaling to the intestinal immune compartment (26, 27). In the gut, inhibition of the LT␤R
signaling pathway with a LT␤R-Fc chimera decoy receptor alleviated inflammatory symptoms in the CD4⫹CD45RBhigh T cell
transfer model of colitis, suggesting a role for LIGHT or LT␣␤ in
this CD4⫹ T cell-mediated pathology (10, 28). Moreover, the human LIGHT locus is closely linked to the TNF family members,
CD27 ligand (CD70, TNFSF7) and 4-1BB ligand (TNFSF9),
within the MHC paralogous region on chromosome 19p13.3 (29),
which contains a candidate susceptibility locus for CD (30). These
reports suggest that regulation of LIGHT expression or signaling
may play a key role modulating mucosal immunity, possibly contributing to the pathological inflammation of human IBD. We recently reported an enhanced potential for LIGHT expression by
intestinal T cells in vitro, suggesting that mucosal location of T
0022-1767/05/$02.00
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The TNF superfamily cytokine, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus
entry mediator on T cells (LIGHT; TNFSF14), can augment T cell responses inducing IFN-␥ production and can drive pathological gut inflammation when expressed as a transgene in mouse T cells. LIGHT expression by human intestinal T cells suggests
the possibility that LIGHT may play a key role in regulation of the mucosal immune system. A nonenzymatic method was
developed for the isolation of T cells from the human lamina propria, permitting analysis of native cell surface protein expression.
Cell surface LIGHT was constitutively expressed on mucosal T and NK cells and a subpopulation of gut-homing CD4ⴙ T cells in
the periphery. In addition, CD2-mediated stimulation induced efficient LIGHT expression on intestinal CD4ⴙ T cells, but not on
peripheral blood T cells, suggesting a gut-specific, Ag-independent mechanism for LIGHT induction. By contrast, herpesvirus
entry mediator expression on gut T cells was unperturbed, implicating the transcriptional regulation of LIGHT as a mechanism
modulating signaling activity in the gut. Quantitative analysis of LIGHT mRNA in a cohort of inflammatory bowel disease patients
indicated elevated expression in biopsies from small bowel and from inflamed sites, implicating LIGHT as a mediator of mucosal
inflammation. The Journal of Immunology, 2005, 174: 646 – 653.
The Journal of Immunology
cells might promote LIGHT induction. In the present study, we
provide direct evidence that LIGHT is constitutively expressed on
human intestinal T and NK cells, but can also be activated through
a gut-specific CD2-dependent mechanism. Expression of LIGHT
mRNA was elevated in small bowel and inflamed intestinal tissue
biopsy specimens from a cohort of IBD patients, further implicating LIGHT as a mediator of mucosal inflammation.
Materials and Methods
Cohort and specimen procurement
Cell isolation and culture
PBMC were isolated from uncoagulated blood by standard Ficoll-Hypaque
density gradient centrifugation. Mononuclear cells from lamina propria
(LPMC) were isolated, as described previously (32). Briefly, epithelial
cells were removed by washing in EDTA, followed by enzymatic disruption of the lamina propria (LP) matrix, mincing, and density gradient purification of LPMC. For analysis of native surface protein expression, mucosal specimens were cut perpendicularly to the mucosal surface (⬍2
mm2) following the EDTA wash step, and lightly smashed to mechanically
disrupt the tissue and facilitate lymphocyte leakage. Mechanically disrupted mucosa was incubated in RPMI 1640 for 2– 6 h, and lymphocyte
accumulation in the medium was monitored periodically for optimal yield,
followed with exclusion of tissue debris through a macroporous (105-␮m
opening) polypropylene mash (Spectrum Laboratories) and centrifugation
at 28 ⫻ g to exclude remaining epithelial cells. Intraepithelial lymphocytes
were excluded from these preparations by means of the initial EDTA wash
similar to the standard enzymatic isolation protocol. Lymphocytes were
cultured at 0.25–1 ⫻ 106 cells/ml in RPMI 1640 containing 2 mM
L-glutamine and 25 mM HEPES buffer (Mediatech), supplemented with
10% heat-inactivated FBS (Atlanta Biologicals), 50 ␮g/ml gentamicin
(Omega Scientific), and LPMCs with additional 0.25 ␮g/ml amphotericin
B (Gemini Bio-Products). Where indicated, lymphocytes were stimulated
by 40 ng/ml PMA and 1 ␮g/ml ionomycin (Sigma-Aldrich); or by Ab
cross-linking of cell surface CD2 and CD28 used at 0.4 and 1 ␮g/ml,
respectively.
T cell subset purification
Adherent cells were partially depleted from LPMC and PBMC preparations by adherence to plastic for 16 –20 h before further purification or
experimentation. CD4⫹, CD8⫹, CD4⫹/CCR9⫹, and CD4⫹/CCR9⫺ cells
were purified from PBMC by flow cytometry (FACStar; BD Biosciences)
gating on CD3⫹ staining for the CD4/8⫹ subsets or on CD4⫹ staining for
the CD45RA/RO subsets. Purity was consistently greater than 99% for the
gated markers when reanalyzed by flow cytometry (FACScan; BD
Biosciences).
Ab reagents
Gem1A.1 is an anti-human LIGHT combinatorial Ab containing VH and
V␬ chains generated from a BALB/c mouse immunized with soluble
rLIGHT (29). Gem1A.1 is recognized by anti-mouse Ig ␬. Mouse antimethamphetamine was used as an isotype control and was provided by G.
Valkirs (Biosite Diagnostics). An anti-LIGHT polyclonal antiserum was
produced in rats immunized with soluble human rLIGHT protein (33). A
polyclonal goat anti-HVEM was generated by immunization with purified
human HVEM-Fc. The serum was absorbed with immobilized human IgG,
and purified IgG was prepared by protein G affinity chromatography. AntiLIGHT Ab was used at 20 ␮g/ml in blocking experiments, and anti-HVEM
was used at 0.2 ␮g/ml for T cell stimulation. The anti-CD2 mAb pair,
clones GD10 and CB6, was a gift of C. Benjamin (Biogen Idec). AntiCD28 mAb ascites, clone 9.3, was obtained from Bristol-Meyers Squibb
Pharmaceutical Research Institute. The ascites was purified over a protein
G column and quantified by ELISA. Anti-CCR9 was the 3C3 clone (34)
from R&D Systems, anti-human integrin ␤7 from BD Pharmingen, and
PC5-conjugated anti-CD56 from Beckman-Coulter. Additional chromophore-conjugated Abs specific for human CD3, CD4, and CD8 were
from Caltag Laboratories.
Cell staining for flow cytometry
Cells were blocked with goat IgG for 20 min on ice, then indirectly stained
for membrane-associated LIGHT using a mouse anti-human rLIGHT Fab,
or isotype control Fab (Jackson ImmunoResearch Laboratories), and detected after washing by a FITC-conjugated goat anti-mouse (H and L) Fab
(Jackson ImmunoResearch Laboratories) for 30 min per step. To avoid
secondary Ab detection of mouse anti-CD2 Abs used for T cell stimulation,
a rat anti-LIGHT serum was used and detected with a mouse-adsorbed,
anti-rat secondary (Jackson ImmunoResearch Laboratories). After washing
and blocking with mouse IgG or normal rat serum for 20 min, cells were
stained for additional surface markers, for 20 min on ice. Flow cytometric
analysis included at least 2 ⫻ 104 events on a FACScan (BD Biosciences)
and analyzed with CellQuest software. Nonspecific staining by control isotypes or staining of unstimulated cells was subtracted from percentage
staining for each cell subset to determine specific mean fluorescence.
Real-time PCR
Isolated cells or whole biopsy specimens were lysed in guanidium thiocyanate
buffer, and total RNA was isolated using the RNeasy kit (Qiagen). For
micropurification of RNA from ⬍105 cells, lysates were supplemented with 12
␮g of rRNA as a carrier (Sigma-Aldrich). LIGHT mRNA levels were quantified by real-time RT-PCR (iCycler; Bio-Rad) using One-Step RT-PCR
mixture (Qiagen) with a LIGHT-specific dual-labeled probe and intronspanning primers, normalized to a primer limiting 18S ribosomal RNA
amplification measured in duplex. The following primer (Integrated DNA
Technologies) and probe (Qiagen) sets were designed using Primer 3 software
(35): LIGHT, forward primer, 5⬘-TGGCGTCTAGGAGAGATGGT-3⬘ and
reverse primer, 5⬘-GGTTGACCTCGTGAGACCTT-3⬘; Hyb probe, 5⬘-6FAM-AGCTGCTCCCAGGAGCCTGC-BHQ1–3⬘; 18S, forward primer, 5⬘AAACGGCTACCACATCCAAG-3⬘ and reverse primer, 5⬘-CCTCCAATG
GATCCTCGTTA-3⬘; Hyb probe, 5⬘-TxRed-AGCAGGCGCGCAAATTA
CCC-BHQ3–3⬘.
Results
LIGHT is constitutively expressed on mucosal T and NK cells
LIGHT expression has only been detected on in vitro activated
intestinal or peripheral blood (PB) T cells (11), but no direct
evidence exists for LIGHT expression by gut lymphoid cells in
vivo. We developed a mechanical disruption technique for the
isolation of lymphocytes from the LP, because traditional enzymatic release of lymphocytes from intestinal tissue may remove
cell surface proteins. Cells isolated using the mechanical disruption approach showed significant constitutive LIGHT expression
primarily on CD8⫹ and CD4⫹ T cells (CD3⫹, CD4/8⫹) and NK
cells (CD56⫹, CD3⫺) (Fig. 1A), contrary to cells isolated from
enzymatically disrupted tissue, which do not stain for surface
LIGHT protein. Cells from the inflamed or uninvolved colon
(shown), small bowel, or rectum consistently expressed membrane
LIGHT, although expression was not detected in cells from the PB
or draining mesenteric lymph nodes whether inflamed or not (11).
LIGHT expression diminished over 24 – 48 h in culture, but could
be rapidly induced to maximal levels by PMA and ionomycin (P/I)
activation, in accordance with the induction profile characteristic
of LP T cells (Fig. 1A) (11).
LIGHT expression was further validated by quantitative realtime RT-PCR analysis of LIGHT mRNA levels in LP lymphocyte
preparations isolated by mechanical disruption of the mucosa and
staining positive for membrane LIGHT protein in comparison with
enzymatically isolated LP lymphocytes or nonactivated PBLs that
do not stain for surface LIGHT (Fig. 1B). LIGHT mRNA was
detected in freshly isolated, some rested, and activated LPMC,
although only activated lymphocytes from PB expressed LIGHT
mRNA transcript. Although this approach does not permit further
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Blood leukocytes were obtained by venipuncture from healthy adult volunteers. Intestinal specimens were obtained from patients undergoing intestinal resection for clinical reasons, and biopsy specimens from patients
undergoing diagnostic endoscopy to evaluate the location and severity of
intestinal inflammation, or to characterize an intestinal tumor (non-IBD
patients). Patient diagnosis was defined as CD, UC, or non-IBD, using
standard clinical, radiographic, and endoscopic criteria (31), and gross tissue involvement was validated microscopically. Patients treated with cyclosporin A and patients with indeterminate colitis were omitted from the
study. Procedures for subject recruitment, informed consent, and specimen
procurement were in accordance with protocols approved by the Institutional Review Board for Human Subject Protection of the Cedars-Sinai
Medical Center.
647
648
separation of highly pure cell subsets, it is likely that detected
LIGHT mRNA is produced by T and NK cells, which are the
predominant cell type in these preparations and the exclusive cell
type expressing membrane LIGHT protein by flow cytometry.
LIGHT is expressed on circulating mucosa-associated T cell
subsets
The constitutive expression of LIGHT on T cells directly isolated
from the intestinal mucosa suggested that gut-homing T cells in PB
may express LIGHT. Gut-homing T cells were identified by surface expression of ␤7 integrin and CCR9 (36). In support of our
hypothesis, LIGHT was identified on a small subset of integrin
␤7⫹ T cells (Fig. 2A) and on a major subset (⬎40%) of unstimulated CD4⫹/CCR9⫹ T cells (Fig. 2B). In addition, LIGHT was
preferentially induced on the integrin ␤7⫹ subset of T cells (␤7⫹ ⬎
6% vs ␤7⫺ ⬍ 0.5%) following P/I stimulation (Fig. 2A), but expression was diminished on the CD4⫹/CCR9⫹ subset when compared with the CD4⫹CCR9⫺ population (Fig. 2B), suggesting
maximal expression levels in the unstimulated state. Interestingly,
while nonspecific stimulation induces a broad range of LIGHT
expression levels on T cells, constitutive LIGHT expression, or
lack of, clearly defines two major unique subsets of CCR9⫹ T cells
(Fig. 2B). In support of LIGHT protein expression, constitutive
LIGHT transcript expression was detected by quantitative real-
FIGURE 2. LIGHT is primarily expressed on PB T cells expressing
gut-homing markers. PBMC were surface stained with anti-LIGHT Ab
(shaded), or isotype control Ab (unshaded), anti-CD3, and anti-integrin ␤7
or anti-CCR9 following 16-h incubation in the absence (top panels) or
presence (lower panels) of P/I. A, Lymphocyte-gated CD3⫹ histograms are
shown resolving LIGHT expression based on ␤7 integrin expression; B,
resolving LIGHT expression based on CCR9 expression. C, LIGHT
mRNA levels were quantified by real-time RT-PCR in duplex with primer
limiting 18S rRNA amplification in flow-sorted CD3⫹/CCR9⫹ (⬎99% purity) cells, vs control unseparated PBMC activated for 16 h, where indicated. The 18S-adjusted threshold cycle for LIGHT signal is presented.
Data are representative of a minimum of three experiments. ND ⫽ not
detected.
time RT-PCR in highly purified (⬎99% by flow cytometry)
CD4⫹/CCR9⫹ cells, but not in unstimulated CD4⫹/CCR9⫺ or unseparated PBLs (Fig. 2C).
HVEM is expressed and regulated normally on mucosal T cells
HVEM is the primary receptor for LIGHT constitutively expressed
on PB T cells (18, 37). T cells from PB constitutively express
HVEM, but can down-regulate expression following activation
(38). Interestingly, while fresh mechanically isolated mucosal
CD4⫹ and CD8⫹ T cells uniformly express HVEM (Fig. 3A),
expression levels are relatively low, similar to levels expressed
by activated PB T cells (Fig. 3B) (38). Surface expression of
HVEM was regained by resting cells in culture, but following
cell activation, HVEM was partially down-regulated, as occurs
on peripheral T cells. Therefore, regulatory mechanisms governing HVEM expression in intestinal T cells do not differ from
PB cells (Fig. 3).
The relatively reduced levels of HVEM in fresh, mechanically
isolated gut T cells suggest in vivo activation before isolation from
the mucosa. This finding agrees with the reported activated state of
LP T cells (39) and the expression of LIGHT (Fig. 1), which is
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FIGURE 1. LIGHT is constitutively expressed on mucosal T and NK
cells. A, LPMC were isolated from the mucosa using a modified nonenzymatic method and surface stained with anti-LIGHT Ab (shaded), or isotype control Ab (unshaded), anti-CD3, and anti-CD4/8 or anti-CD56.
Where indicated, cells were rested in culture for 24 h and then stimulated
for an additional 6 h with P/I. Gated CD3⫹/CD4⫹ or CD3⫹/CD8⫹ T cell
and CD3⫺/CD56⫹ NK cell histograms are shown. B, LIGHT mRNA levels
were quantified by real-time RT-PCR in duplex with primer limiting 18S
rRNA amplification in fresh mechanically isolated LPMC, or rested/activated LPMC in comparison with a PBL control. The 18S-adjusted threshold cycle for LIGHT signal is presented. Data are representative of three
experiments, and a discontinuous rested LPMC error bar indicates variable
mRNA detection (mean representing LIGHT mRNA levels where detected,
in three of eight experiments). ND ⫽ not detected.
LIGHT EXPRESSION IN HUMAN GUT
The Journal of Immunology
649
regulated reciprocally to HVEM (38). HVEM expression is unlikely to be compromised considering the rapid nonenzymatic cell
isolation technique and unperturbed expression of other cell surface markers such as CD3, CD4, CD8, or CD56 (data not shown).
LIGHT can be induced on mucosal T cells by the CD2 signaling
pathway
FIGURE 4. LIGHT is induced on mucosal T cells via
CD2 signaling. Lymphocytes were isolated from the LP or
mesenteric lymph nodes, as described in Materials and
Methods, and activated with P/I CD2 or CD2 and CD28.
Cells were then surface stained with anti-LIGHT Ab and
anti-CD3/8. A, Lymphocyte-gated, CD4/8⫹ T cells following activation are plotted (shaded), in reference to the
unstimulated profile (unshaded). B, Mean fluorescence
changes from the isotype control are plotted for CD4⫹ and
CD8⫹ T cells for the activation time indicated. C, LIGHT
mRNA levels were quantified by real-time RT-PCR in
duplex with primer limiting 18S rRNA amplification. The
18S-adjusted threshold cycle for LIGHT signal is presented for the activation time indicated. Data are representative of a minimum of three experiments.
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FIGURE 3. The LIGHT receptor, HVEM, is expressed and normally
regulated by gut T cells. A, LPMC were isolated from the mucosa using a
modified nonenzymatic method and surface stained with anti-HVEM Ab
(shaded), or isotype control Ab (unshaded), anti-CD3, and anti-CD4/8.
Where indicated, cells were rested in culture for 24 h and then stimulated
for an additional 6 h with P/I. Gated CD3⫹/CD4⫹ or CD3⫹/CD8⫹ T cell
and CD3⫺/CD56⫹ NK cell histograms are shown. B, Mean MFI changes
vs isotype controls in three separate experiments are shown for CD8⫹ (f)
or CD4⫹ (䡺) T cells.
CD2 can act as an Ag-independent activation pathway for intestinal T cells (40), which could account for constitutive expression of
LIGHT. Cell surface staining and flow cytometry indicated efficient LIGHT induction by CD2 cross-linking, which was further
augmented by costimulation with CD28 on LP and mesenteric T
cell populations (Fig. 4A). Interestingly, CD2 cross-linking induced higher levels of LIGHT expression in the CD4⫹ T cell subset, when compared with P/I stimulation (Fig. 4A, top and middle
panels). Hence, although peripheral CD8⫹ T cells constitute the
predominant LIGHT-expressing T cell subset (11, 41), CD2-mediated activation provides a novel mechanism for efficient LIGHT
induction on CD4⫹ T cells in the intestinal compartment.
In addition, LIGHT induction via CD2 cross-linking followed
similar kinetics to P/I stimulation (Fig. 4B), in accordance with the
rapid LIGHT induction profile characteristic of LP T cells (11).
LIGHT transcription was induced to similar peak levels and kinetics following CD2, CD2⫹CD28, or P/I stimulation, indicating
LIGHT up-regulation at the transcriptional level (Fig. 4C).
Conversely, PB T cells rendered responsive to CD2 by treatment
with PHA (42) expressed LIGHT primarily on the CD8⫹ T cell
650
FIGURE 5. CD2 mediates induction
of LIGHT on PHA-activated PB-T cells,
but not following LP-T kinetics of induction. PBMC were treated with PHA for
72 h, and cells were then surface stained
with anti-LIGHT Ab and anti-CD3/8. A,
Lymphocyte-gated, CD4/8⫹ T cells following activation are plotted (shaded), in
reference to the unstimulated profile (unshaded), comparing LIGHT induction on
PHA-treated vs untreated cells. B, Mean
fluorescence changes from the isotype
control are plotted for CD8⫹ (top) and
CD4⫹ (bottom) T cells for the activation
time indicated for PHA treated (shaded) or
untreated (unshaded). C, LIGHT mRNA
levels were quantified by real-time RTPCR in duplex with primer limiting 18S
rRNA amplification. The 18S-adjusted
threshold cycle for LIGHT signal is presented for the activation time indicated
comparing PHA treated (shaded top) vs
untreated (unshaded bottom). Data are representative of a minimum of three
experiments.
mRNA levels revealed unperturbed induction following stimulation in the presence of CHX, indicating that the LIGHT transcriptional machinery is preformed in the resting cell (Fig. 6C). Taken
together, these data suggest transcriptional regulation as a primary
mechanism governing LIGHT expression in T cells.
LIGHT mRNA expression is elevated in the inflamed and small
bowel mucosa
Gut-related LIGHT expression on T and NK cells (Figs. 1 and 2)
(11) and the capacity of T cell-expressed LIGHT to induce a
pathological gut-specific inflammation (26, 27) suggest that intestinal T cell activation induces LIGHT expression and a potential
self-propagating proinflammatory signal driven by this elevated
LIGHT expression. Accordingly, we examined whether LIGHT
expression levels correlated with inflammation in the human gut
mucosa. RNA samples were extracted from intestinal biopsy specimens representing a cohort of IBD patients, and LIGHT mRNA
levels were measured by real-time RT-PCR. When LIGHT mRNA
levels were compared between biopsies from inflamed and uninvolved intestinal regions from each patient, a significant trend for
higher LIGHT mRNA levels was detected ( p ⬍ 0.019, n ⫽ 11
pairs) in biopsies from inflamed tissue, regardless of diagnosis or
intestinal location (Fig. 7A). However, no significant difference
was detected (less than one cycle) in 3 of the 11 biopsy pairs
analyzed (Fig. 7A, dotted lines). Interestingly, significantly higher
levels of LIGHT mRNA ( p ⬍ 0.036) were detected in small bowel
biopsy specimens when compared with colonic biopsy specimens
(Fig. 7B), in agreement with a previous report indicating a higher
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population, with only minimal levels on the CD4⫹ PB T cell subset (Fig. 5A). Furthermore, up-regulation on both CD8⫹ and CD4⫹
PB T cells was consistent with the slower induction kinetics profile
typical of PB T cells regardless of PHA pretreatment, with LIGHT
protein levels increasing through a 24-h time point (Fig. 5B).
LIGHT mRNA was effectively induced by CD2 in PHA-treated
cells, peaking later than in LP T cells (by 2 h vs 1 h in LP T cells),
and detected through 24 h (Fig. 5C). Interestingly, induction was
transient in untreated cells, falling bellow detection by 6 h following P/I or CD2 stimulation, yet stabilized by CD2 and CD28
through 24 h (Fig. 5C). These data indicate that although PHA
treatment can potentiate PB T cell response to CD2 signaling, gutspecific regulatory mechanisms in addition to basic CD2 responsiveness must account for the rapid induction kinetics and enhanced CD4⫹ T cell responsiveness unique to the LP T cell
population (11).
LIGHT synthesis can possibly be regulated at the level of transcription, as LIGHT mRNA levels were significantly increased
following T cell stimulation (Figs. 5C and 6C). Alternatively, intracellular stores of LIGHT protein have been reported (43) that
could facilitate up-regulation of membrane LIGHT. Both CD2and PMA-ionomycin-mediated up-regulation of LIGHT protein
was effectively abrogated by cycloheximide (CHX) (⬎90% inhibition) (Fig. 6, A and B), thus demonstrating that de novo protein
synthesis is essential to membrane LIGHT up-regulation via CD2
or P/I stimulation. In addition, effective inhibition of membrane
LIGHT up-regulation was attained by transcription blocker, actinomycin D (Fig. 6, A and B, lower panels), indicating a requirement for transcriptional activation. Finally, analysis of LIGHT
LIGHT EXPRESSION IN HUMAN GUT
The Journal of Immunology
651
FIGURE 6. LIGHT is induced de novo by CD2 via a pre-existing transcriptional mechanism. PHA-treated PBMC were activated with P/I, CD2,
or CD2 following 15-min incubation with CHX. Cells were then surface
stained with anti-LIGHT Ab and anti-CD3/8. Lymphocyte-gated, CD4/8⫹
T cells following activation are plotted (shaded), in reference to the unstimulated profile (unshaded) for: A, P/I-activated cells, or B, CD2-activated cells. C, LIGHT mRNA levels were quantified by real-time RT-PCR
in duplex with primer limiting 18S rRNA amplification. The 18S-adjusted
threshold cycle for LIGHT signal is presented for the activation time indicated comparing mRNA levels in the presence of CHX treated (shaded)
vs its absence (unshaded). Data are representative of a minimum of three
experiments.
potential for LIGHT expression on small bowel T cells (11). However, variable levels of LIGHT transcript were detected in the majority of mucosal specimens analyzed not significantly correlating
with IBD pathology in this 16-patient cohort (3 non-IBD, 5 UC, 6
CD, and 2 indeterminant colitis).
Discussion
CD4⫹ T cells are key to maintaining immune homeostasis in the
gut and can be pathogenic when reactive to bacterial Ags (44).
More specifically, CD4⫹ CD45RBhigh, Th1, and a deficiency in T
regulatory type 1 cells have been implicated in IBD pathology (6,
7, 28, 45). However, although excessive immune reactivity to bacterial Ags has been associated with human IBD (4), the mechanism
by which breakdown of tolerance occurs remains largely unknown.
The present study provides evidence for LIGHT expression in
the human intestinal mucosa, thus proposing a role for this TNF
family cytokine in IBD pathology. A nonenzymtic protocol for
gentle extraction of lymphocytes from the intestinal mucosa permitted the analysis of cell surface proteins that may more closely
reflect in vivo expression. Using this approach, we detected constitutive LIGHT expression on both CD8⫹ and CD4⫹ T cells, as
well as NK cells (Fig. 1). These observations correlate with the
semiactivated state of LP T cell population (39), and denote the
applicability of previous in vitro analysis indicating enhanced potential for LIGHT expression on LP T cells (11). From studies of
mice, constitutive expression of LT␣␤ and TNF is a crucial mechanism serving as a homeostatic regulatory pathway between lymphocytes and the stroma to control tissue microenvironments
within lymphoid organs. The work in this study demonstrating
constitutive LIGHT expression in T cells resident in mucosal lymphoid tissue and a unique subset of mucosal trafficking T cells in
blood suggests the involvement of LIGHT in homeostatic immune
regulation.
Intensive immune interaction with intestinal Ags at the mucosal
interface plays a key role in IBD pathology (5, 44, 46). However,
T cell maturation and repertoire shaping via Ag exposure are not
sufficient in themselves for the induction of an inflammatory response, given that Ag-specific T cells can be regulatory as well as
effector cells (47). Constitutive LIGHT expression on NK cells
that are effective IFN-␥ producers (Fig. 1), and an almost exclusive
LIGHT expression by IFN-␥-producing CD4⫹ T cells (11), further
link LIGHT to a skewed Th1 response associated with CD pathology (6, 7). Moreover, LIGHT expression on NK cells could potentially play an additional role mediating NK regulation of DC
cells, thus modulating Ag-presenting functions in the gut mucosa
and perturbing the mucosal immune repertoire (4, 48).
We recently reported an enhanced potential for LIGHT expression on LP and mature peripheral CD4⫹ T cells (CD45RO), presumed to have encountered Ag (11). Because the gut is the primary
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FIGURE 7. LIGHT mRNA expression is elevated in the inflamed and
small bowel mucosa. RNA was extracted from biopsy specimens of an IBD
patient cohort, and LIGHT mRNA levels were quantified by real-time RTPCR in duplex with primer limiting 18S rRNA amplification. The 18Sadjusted threshold cycle for LIGHT signal is presented. A, Paired analysis
comparing LIGHT mRNA levels from inflamed vs uninvolved sites for
each patient. Solid lines indicate biopsy pairs with significantly elevated
mRNA levels in the inflamed sample; dotted lines indicate pairs not varying significantly. B, Comparing LIGHT mRNA levels in small bowel vs
colon (two-tailed p ⬍ 0.019).
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complex gut-specific regulatory mechanisms must be in place, because CD2 responsiveness by itself could not confer the rapid
LIGHT induction profile to PB T cells (Fig. 5).
LIGHT is transcribed following activation (Figs. 4C and 5C),
and LIGHT mRNA was detected in gut cells (Figs. 1C, 2C, and 7),
suggesting that LIGHT expression is regulated at the transcriptional level. Alternatively, intracellular stores of LIGHT have been
reported (43), proposing a presynthesized protein pool that can be
rapidly transported to the cell surface. Our data indicate that
LIGHT induction is actinomycin D and CHX sensitive (Fig. 6),
suggesting a requirement for transcription and de novo protein
synthesis, and supporting LIGHT regulation primarily at the transcriptional level. Rapid LIGHT mRNA induction resistant to protein synthesis inhibitor, CHX, indicates the presence of preformed
LIGHT transcriptional machinery in resting T cells (Fig. 6C), providing a rapid mechanism for LIGHT up-regulation. Furthermore,
full-length LIGHT mRNA was not detected in unstimulated T cells
(Figs. 5C and 6C), but a variant form of LIGHT has been described
that lacks the transmembrane domain and cannot be displayed on
the cell surface (29), most likely accounting for intracellular
LIGHT detected in resting PB T cells (43). However, a more precise real-time protein-trafficking analysis will be required to exclude intracellular stores of LIGHT as a mechanism for rapid induction and to establish whether signaling activity may be
terminated by shedding LIGHT (29).
In summary, our findings demonstrate for the first time that
LIGHT is constitutively expressed on human mucosal T and NK
cells. Furthermore, constitutive LIGHT expression on a subset of
circulating small bowel-homing T cells and elevated LIGHT transcript levels in small bowel biopsies localize LIGHT expression to
the small bowel compartment, suggesting a link to Th1-driven CD
pathogenesis. In addition, activation of the CD2 pathway is proposed as a novel gut-specific, yet Ag-independent mechanism for
LIGHT induction on CD4⫹ T cells. Finally, LIGHT regulation on
T cells is proposed to occur primarily at the transcriptional level
via a preformed transcriptional apparatus. In conclusion, gut-specific regulation of LIGHT could have significant pathological implications in human IBD, and thus, further investigation of
LIGHT-associated immune regulatory mechanisms unique to the
intestinal immune compartment is merited.
Acknowledgments
We thank Kostas Papadakis for advice and help with peripheral CCR9⫹ T
cell analysis, Phillip Fleshner for facilitating intestinal specimen procurement, Patricia Lin for advice and help with flow cytometry, Walker R.
Force for helpful discussions, and John L. Prehn for helpful discussions
and critical reading of this manuscript.
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