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The Journal of Experimental Medicine
ARTICLE
The Rockefeller University Press $30.00
J. Exp. Med. Vol. 205 No. 9 2099-2110
www.jem.org/cgi/doi/10.1084/jem.20072484
2099
Crohn ’ s disease (CD) and ulcerative colitis (UC)
are the two key forms of in ammatory bowel
diseases (IBDs) in humans ( 1 – 3 ). Growing evi-
dence indicates a central role of a genetically de-
termined dysregulation of the mucosal immune
response toward the resident bacterial ora in
the pathogenesis of human IBD ( 4 ). This patho-
logical immune response is characterized by an
accumulation of antigen-presenting cells and T
cells that represent the vast majority of activated
mononuclear cells in ltrating the gut ( 5 – 8 ).
The di erentiation and activation of CD4
+
T cells in the lamina propria play a major role in
the pathogenesis of IBD ( 9 ). Whereas CD is as-
sociated with increased production of Th1-like
cytokines such as IFN- ␥ and TNF, the cytokine
pro le in chronic UC is characterized by the
increased production of several Th2 cytokines,
such as IL-5, -6, and -13 ( 7, 8, 10, 11 ). Interest-
ingly, both Th1- and Th2-type cytokines have
been shown to play an important pathogenic
role in various animal models of IBD, suggesting
that both T helper subsets can induce chronic
intestinal in ammation in vivo ( 11, 12 ). This
pathogenic function of Th1 and Th2 cells can be
counteracted by immunosuppressive cytokines
such as IL-10 and TGF-  , which are produced
by regulatory T cells and Th1 cells ( 11 – 15 ).
T lymphocytes transit through sequential
stages of cytokine activation, commitment, silenc-
ing, and physical stabilization during polarization
CORRESPONDENCE
Markus F. Neurath:
neurath@
1-med.klinik.uni-mainz.de
Abbreviations used: AAD,
amino-actinomycin D; CD,
Crohn ’ s disease; IBD, in am-
matory bowel disease; LPMC,
lamina propria mononuclear
cell; NFAT, nuclear factor
of activated T cells; UC, ulcer-
ative colitis.
The online version of this article contains supplemental material.
The transcription factor NFATc2 controls
IL-6 – dependent T cell activation
in experimental colitis
Benno Weigmann ,
1
Hans A. Lehr ,
2
George Yancopoulos ,
3
David Valenzuela ,
3
Andrew Murphy ,
3
Sean Stevens ,
3
Jan Schmidt ,
5
Peter R. Galle ,
6
Stefan Rose-John ,
4
and Markus F. Neurath
1,6
1
Institute of Molecular Medicine, Johanes Gutenberg Univeristy, 55131 Mainz, Germany
2
Institut Universitaire de Pathologie, Universite de Lausanne, CH-1011 Lausanne, Switzerland
3
Regeneron Pharmaceuticals, Tarrytown, NY 10591
4
Institute of Biochemistry, University of Kiel, 24098 Kiel, Germany
5
Department of Surgery, University of Heidelberg, 69120 Heidelberg, Germany
6
Laboratory of Immunology, I. Medical Clinic, University of Mainz, 55131 Mainz, Germany
The nuclear factor of activated T cells (NFAT) family of transcription factors controls
calcium signaling in T lymphocytes. In this study, we have identi ed a crucial regulatory
role of the transcription factor NFATc2 in T cell – dependent experimental colitis. Similar to
ulcerative colitis in humans, the expression of NFATc2 was up-regulated in oxazolone-
induced chronic intestinal in ammation. Furthermore, NFATc2 de ciency suppressed colitis
induced by oxazolone administration. This nding was associated with enhanced T cell
apoptosis in the lamina propria and strikingly reduced production of IL-6, -13, and -17 by
mucosal T lymphocytes. Further studies using knockout mice showed that IL-6, rather than
IL-23 and -17, are essential for oxazolone colitis induction. Administration of hyper-IL-6
blocked the protective effects of NFATc2 de ciency in experimental colitis, suggesting
that IL-6 signal transduction plays a major pathogenic role in vivo. Finally, adoptive trans-
fer of IL-6 and wild-type T cells demonstrated that oxazolone colitis is critically dependent
on IL-6 production by T cells. Collectively, these results de ne a unique regulatory role for
NFATc2 in colitis by controlling mucosal T cell activation in an IL-6 – dependent manner.
NFATc2 in T cells thus emerges as a potentially new therapeutic target for in ammatory
bowel diseases.
© 2008 Weigmann et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the rst six months
after the publication date (see http://www.jem.org/misc/terms.shtml). After six
months it is available under a Creative Commons License (Attribution–Noncom-
mercial–Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).
2100 NFACTC2 IN COLITIS | Weigmann et al.
been shown to control Th2 cytokine production, STAT-1,
STAT-4, and T-bet are associated with signaling events in Th1
cells and play a key role in Th1-speci c cytokine production
in peripheral T cells ( 16, 17 ). In the mucosal immune system,
several studies have suggested important roles for STAT-4 and
T-bet in Th1 cell e ector functions in the gut in experimental
colitis and CD ( 9, 18, 19 ). However, the functional role of
other T cell transcription factors such as nuclear factor of acti-
vated T cells (NFAT) in IBD is poorly understood.
The NFAT family of transcription factors consists of
ve members: NFATc1 (also known as NFATc or NFAT2),
NFATc2 (NFATp or NFAT1), NFATc3 (NFATx or NFAT4),
NFATc4 (NFAT3), and NFAT5 (TonEBP or OREBP) ( 20 – 23 ).
All NFAT proteins have a highly conserved DNA-binding
domain that is structurally related to the DNA-binding
domain of the REL family of transcription factors ( 23, 24 ).
This REL-homology region (RHR) is the unifying char-
acteristic of NFAT proteins and confers a common DNA-
binding speci city.
NFAT proteins seem to play a pivotal role in the activa-
tion and di erentiation of T lymphocytes and are activated by
calcium signaling ( 22 – 25 ). In fact, calcium-bound calmodulin
activates the calcineurin phosphatase complex, which dephos-
phorylates NFATc1 and causes its import into the nucleus,
where NFATc1 and its nuclear partner, NFATn, cooperatively
bind to DNA. NFATc1 complexes act as “ coincidence de-
tectors ” and allow the integration of multiple signaling path-
ways at the level of DNA binding. In addition to NFATc1,
NFATc2 is constitutively expressed in T cells and controls T
cell activation and survival ( 22, 24-26 ). Consistently, NFATc2
KO mice developed a hyperproliferative syndrome with in-
creased numbers of peripheral lymphocytes caused by defects
in activation-induced cell death and reduced expression of sev-
eral proapoptotic genes, such as CD95L and TNF ( 27 – 29 ).
NFATc2-de cient mice also showed alterations in T cell cyto-
kine production. Whereas NFATc2-de cient splenic T cells
displayed reduced early IL-4 production, increased levels of
IL-4 were found at later time points during Th2 development,
suggesting that NFATc2 is important for cytokine production
by peripheral T cells ( 27, 28 ).
In contrast to peripheral T cells, the function of NFATc2
in mucosal T cells remains largely unknown. Therefore, the
aim of this study was to analyze the role of NFATc2 signal
transduction in intestinal in ammation. A signi cantly higher
expression of NFATc2 was found in UC tissues compared
with control samples. Furthermore, NFATc2 was found to
play a pivotal regulatory role in T cell – dependent experimen-
tal colitis by controlling IL-6 – dependent T cell activation.
RESULTS
Enhanced expression of NFATc2 in patients with IBDs
The NFAT plays a fundamental role in controlling calcium-
dependent T cell activation ( 23, 30-32 ). As T cells have
been suggested to play a major role in patients with IBDs, we
analyzed expression of NFATc2 by immunohistochemistry.
Accordingly, colonic cryosections from IBD patients were
into e ector subsets, a process that is tightly controlled by reg-
ulatory transcriptional events ( 3, 16, 17 ). Although transcrip-
tion factors such as STAT-6, GATA-3, c-Maf and JunB have
Figure 1. Enhanced expression of NFATc2 transcription factors in
patients with IBDs compared with control patients. (A) Immunohisto-
chemistry for NFATc2 expression. Colon cross sections were incubated with
NFATc2-speci c antibodies and analyzed by microscopy. An increased ex-
pression of NFATc2 was observed in sections from UC and CD patients
compared with control patients. Representative stainings from 5 to 10
patients per group are shown. (B) Quantitative analysis of positive cells
revealed a signi cantly increased number of NFATc2-positive cells in IBD
patients compared with control patients. Data represent mean values ± the
SD per high power eld. *, P < 0.05. (C) Detection of NFATc2-expressing
T cells in the lamina propria of patients with IBDs. Samples from CD, UC, and
control patients were stained with anti-NFATc2 and anti-CD3 antibodies,
followed by confocal laser microscopy. Nuclei were stained with DAPI. Rep-
resentative pictures are shown. Staining with an isotype control antibody
served as negative control. CD3/NFATc2 double-positive, yellowish cells
were seen in the lamina propria in CD and UC. Bars: (A) 80 μ m; (C) 30 μ m.
JEM VOL. 205, September 1, 2008
ARTICLE
2101
T lymphocytes are key e ector cells in the pathogenesis
of IBD and produce several proin ammatory cytokines that
contribute to tissue destruction in IBD ( 4, 12 ). To deter-
mine whether T lymphocytes in IBD express NFAT proteins,
stained with NFATc2-speci c antibodies. As shown in Fig. 1
(A and B) , NFATc2-positive cells were found in the lamina
propria of control patients, and the number of these cells was
signi cantly increased in patients with IBD.
Figure 2. A regulatory role of NFATc2 in oxazolone-induced experimental colitis. (A) Enhanced expression of the NFATc2 transcription factor in
oxazolone-induced colitis. Colonic cryosections from WT and NFATc2 KO mice were incubated with anti-NFATc2 antibodies followed by tyramide signal
ampli cation. An increased expression of NFATc2 was observed in colonic tissue from WT mice with oxazolone colitis (WT oxa) as compared with WT un-
challenged mice. Colonic tissue from NFATc2 KO mice (NFATc2 oxa) served as negative control and did not reveal any speci c staining as expected.
(B) Oxazolone colitis was induced by sensitizing mice with oxazolone, followed by intrarectal administration of the hapten reagent after 1 wk. The body
weight of the mice was monitored after oxazolone rechallenge at the indicated time points. Mean values ± the SEM from two representative experiments
out of six are shown. The average weight of the mice at the beginning of the experiments was 22.9 g (WT group) and 22.5 g (NFATc2 KO group), respec-
tively. For this experiment, 6 WT and 7 NFATc2 KO mice were used. (C) Histological sections (left) of colonic in ammation in WT or NFATc2-de cient mice
upon oxazolone administration. Signs of in ammation such as goblet cell depletion, ulcers, and accumulation of mononuclear cells were noted in WT
mice, whereas NFATc2 KO mice showed little or no evidence of colitis. Quantitative histopathologic assessment of colitis activity (right) showed a signi -
cant (*P < 0.05) protection from in ammation and tissue injury in NFATc2 KO mice compared with WT mice. Data represent mean values ± the SEM from
one representative experiment out of six. (D) High-resolution miniendoscopic analysis (left) of the colon of NFATc2 KO and WT mice in oxazolone colitis.
Marked erosions and ulcers were seen in the WT group, whereas an almost normal colon architecture was noted in NFATc2-de cient mice. Quantitative
endoscopic analysis (right) of in ammation (MEICS score) in WT and NFATc2 KO mice in oxazolone colitis was done at day 2 after administration of oxa-
zolone. A signi cantly (P < 0.05) lower endoscopic score was observed in NFATc2-de cient mice compared with WT mice. (E) Adoptive T cell transfer from
WT and NFATc2-de cient mice in SCID mice. CD4
+
T cells were transferred into CB-17/SCID mice, followed by oxazolone sensitization and intrarectal oxa-
zolone administration. The body weight of reconstituted mice was analyzed at indicated time points. Mean values ± SEM from one representative experi-
ment out of three are shown. Whereas mice reconstituted with WT T cells showed a marked weight loss, mice given NFATc2-de cient T cells were
protected from colitis and gained weight. For this experiment ve SCID mice in each group were used. Bars: (A) 80 μ m; (C) 100 μ m.
2102 NFACTC2 IN COLITIS | Weigmann et al.
Figure 3. Augmented T cell apoptosis in the colon of NFATc2-de cient mice in oxazolone-induced colitis. (A) Cryosections of colonic tissue
from WT and NFATc2-de cient mice in oxazolone-induced colitis were made, and apoptotic cells were stained using the TUNEL reaction. Staining of the
nuclei was done with DAPI. Representative stainings from WT and NFATc2 KO mice, as well as negative and positive control stainings are shown (left).
(right) Quantitative analysis of apoptotic cells in 10 randomly selected high power elds per sample. Data represent mean values ± the SD. A signi cantly
(P < 0.05) higher number of apoptotic cells was observed in colonic tissue from NFATc2-de cient mice in oxazolone-induced colitis compared with WT
control mice. Bar, 100 μ m. (B) Double staining analysis of colonic tissue from WT and NFATc2-de cient mice in oxazolone-induced colitis using TUNEL
assays and anti-CD3 antibodies (left). Cryosections were analyzed by confocal laser microscopy for apoptotic T cells, and representative pictures are
shown (right). A higher number of apoptotic/CD3 double-positive cells (FITC TUNEL staining; Cy3 stained CD3) was observed in NFATc2 mice compared
with WT mice. (C) An increased number of apoptotic LPMCs in NFATc2-de cient mice upon administration of oxazolone. LPMCs from WT and NFATc2-
de cient mice were isolated and stained with 7-AAD and anti – Annexin V antibodies for FACS analysis. In comparison to WT cells, LPMCs from NFATc2-
de cient mice showed an increased number of apoptotic cells in oxazolone-induced colitis. Representative FACS analyses of different groups of mice for
annexin V are shown. Quantitative analysis of apoptotic LPMCs was performed in three independent experiments (bottom). Data represent mean values ±
the SD. There was a signi cant (*, P < 0.05) increase of apoptotic LPMCs in NFATc2-de cient mice as compared with WT control mice. (D) Increased num-
ber of apoptotic lamina propria CD4
+
T cells in NFATc2-de cient mice in oxazolone-induced colitis. LPMCs from WT and NFATc2-de cient mice were
JEM VOL. 205, September 1, 2008
ARTICLE
2103
prepared, and CD4
+
T cells were isolated by using the MACS-System. Cells were stained with 7-AAD and anti – Annexin V antibodies for subsequent analy-
sis (top). Lamina propria T cells from NFATc2 KO mice showed an increased percentage of apoptotic cells in colitis compared with WT mice. Quantitative
analysis of apoptotic CD4 T cells was performed in two experiments (bottom). Data represent mean values ± the SD. There was a signi cant (*, P < 0.05)
increase of apoptotic lamina propria T cells in NFATc2-de cient mice compared with WT control mice.
double staining for CD3 and NFATc2 was performed ( Fig.
1 C ). Interestingly, many T lymphocytes in the gut of IBD
patients were positive for NFATc2, which is consistent with
a potential regulatory role of this transcription factor in mu-
cosal T cells.
A key regulatory role of NFATc2 in oxazolone-
induced colitis
In subsequent studies, we found an induction of NFATc2 ex-
pression in WT mice with oxazolone-induced colitis as com-
pared with unchallenged mice, which is consistent with a
potential regulatory role of NFATc2 in T cell – mediated coli-
tis ( Fig. 2 A ). To analyze the functional role of NFATc2 in
experimental colitis, we then took advantage of genetically
engineered mice in which the NFATc2 gene was inactivated
by homologous recombination ( 27 ). In these studies, we de-
termined whether NFATc2-de cient mice exhibit an altered
susceptibility to oxazolone colitis that (similarly to UC) is
characterized by IL-5 and -13 production by T cells ( 33, 34 ).
It was found that NFATc2-de cient mice were almost com-
pletely protected from oxazolone colitis. Although WT mice
challenged with oxazolone showed a marked weight loss, no
diarrhea and weight loss were noted in mice lacking NFATc2
( Fig. 2 B ). Consistently, histological analysis showed signi -
cant suppression of colitis activity in NFATc2-de cient mice
compared with WT mice after administration of oxazolone
( Fig. 2 C ). Finally, endoscopic analysis revealed signi cant
suppression of oxazolone-induced colitis in the former as
compared with the latter group of mice ( Fig. 2 D ).
To prove that the observed protective e ect in NFATc2-
de cient mice was caused by T lymphocytes, we next per-
formed adoptive transfer studies. Accordingly, splenic CD4
+
T lymphocytes from WT and NFATc2-de cient mice were
adoptively transferred into immunode cient mice, followed
by sensitization and challenge of the reconstituted mice with
oxazolone. As shown in Fig. 2 E , mice given WT T cells de-
veloped severe colitis with weight loss, whereas mice recon-
stituted with NFATc2-de cient T cells were protected from
such colitis, suggesting that NFATc2 mediates its pathogenic
role in oxazolone colitis via its e ects on T lymphocytes.
Increased apoptotic rate of NFATc2-de cient lamina
propria T cells in oxazolone-induced colitis
Because it is known that NFATc2-de cient mice have a de-
fect in lymphocyte apoptosis ( 27, 29 ), we next assessed the
apoptotic rate of lamina propria mononuclear cells (LPMCs)
in oxazolone-induced colitis. Accordingly, cryosections of
oxazolone-treated WT and NFATc2 KO mice were stained
by TUNEL assays ( Fig. 3 A ). Surprisingly, a signi cantly higher
number of apoptotic cells was observed in NFATc2-de cient
mice as compared with WT control mice ( Fig. 3 A ). Further-
more, double staining analysis for CD3 and TUNEL ( Fig. 3 B )
revealed a higher number of apoptotic T cells in the former
as compared with the latter mice, suggesting that NFATc2-
de cient lamina propria T cells in oxazolone colitis are more
susceptible to undergo programmed cells death than WT
T cells. To further analyze this possibility, we analyzed freshly
isolated T cell enriched lamina propria cells from WT and
NFAT-de cient mice for apoptosis by FACS analysis using
7-amino-actinomycin D (7AAD) and anti – annexin V antibodies.
In oxazolone-induced colitis, a signi cantly higher rate of
apoptotic lamina propria cells was seen in NFATc2 KO mice
compared with WT mice ( Fig. 3 C ). Furthermore, there was a
signi cantly higher percentage of apoptotic T lymphocytes in
the lamina propria of NFATc2 KO mice compared with WT
mice in oxazolone-induced colitis ( Fig. 3 D ). Interestingly,
NFATc2-de cient T cells expressed lower amounts of anti-
apoptotic proteins, such as bcl-xl and bcl-2, compared with
WT T cells in colitis (Fig. S1, available at http://www.jem
.org/cgi/content/full/jem.20072484/DC1). As NFATc2-
de cient T cells are normally resistant to undergo apoptosis
( 27, 29 ), our data thus suggested the possibility that external
factors such as the cytokine milieu may cause increased muco-
sal NFATc2
⫺ / ⫺
T cell apoptosis in experimental colitis.
Reduced production of IL-6, -13, and -17 by NFATc2-
de cient lamina propria T cells
To further test the aforementioned hypothesis, we next as-
sessed the e ects of NFATc2 on T cell cytokine production in
oxazolone-induced colitis. Splenic CD4
+
T cells isolated from
NFATc2-de cient mice produced signi cantly lower amounts
of IFN- ␥ , IL-6, and IL-17 but higher amounts of IL-4 than
T cells from WT mice ( Fig. 4 A ). Furthermore, colonic LPMCs
from NFATc2-de cient mice produced signi cantly lower
amounts of IL-6, -13, and -17 in oxazolone-induced colitis
compared with control WT cells ( Fig. 4 B ). Whereas T cell
survival at the end of the cell cultures di ered by only ⵑ 10%
between the groups (79% in WT vs. 70% in NFAT KO mice),
the production of the above cytokines was reduced by > 70%
in the absence of NFATc2, suggesting that NFATc2 controls
cytokine production by mucosal T cells. In contrast to IL-6,
-13, and -17, no signi cant changes in IL-4 production were
noted. Furthermore, a signi cant induction of IL-5 produc-
tion by NFATc2-de cient cells was observed, suggesting that
only distinct cytokines produced by T cells are reduced by
the absence of the transcription factor NFATc2. The reduced
IL-17 production by T cells lacking NFATc2 was remarkable,
as both splenic and lamina propria cells from NFATc2-de -
cient mice expressed normal or even increased amounts of the
Th17 master transcription factor ROR- ␥ t (unpublished data).
2104 NFACTC2 IN COLITIS | Weigmann et al.
Figure 4. Cytokine production in oxazolone-induced colitis.
(A) Cytokine production by splenic CD4
+
T cells from WT and NFATc2-
de cient mice in oxazolone-induced colitis. CD4
+
T cells were stimulated
with antibodies to CD3 and CD28 for 48 h, followed by analysis of culture
supernatants using cytomix (see Materials and methods). Data represent
mean values of four to eight mice per group. CD4
+
T cells from WT mice
produced signi cantly (P < 0.05) higher amounts of IFN- ␥ , but lower
amounts of IL-4 than NFATc2-de cient T cells. Furthermore, a signi -
cantly lower production of IL-6 and -17 was observed in the supernatant
of NFATc2-de cient CD4
+
T cells in comparison to WT T cells. (B) Cytokine
production by T cell – enriched lamina propria cells from WT and NFATc2-
de cient mice in oxazolone-induced colitis. Lamina propria cells were
stimulated with PMA/ionomycin, and the cell supernatant was analyzed
using cytomix. Data represent mean values of three to six mice per group.
T cells from WT mice produced higher amounts of IFN- ␥ , IL-6, IL-13, and
IL-17 when compared with cells from NFATc2-de cient mice. At the end
of the culture, cell survival rates differed by ⵑ 10% between WT and KO
cells only, suggesting that the marked differences in cytokine production
under our experimental conditions are not caused by primary effects on
T cell apoptosis. (C) IL-6 production by puri ed CD4
+
lamina propria T cells
from WT and NFATc2-de cient mice in oxazolone-induced colitis. LPMCs
from WT and NFATc2-de cient mice were prepared, and CD4
+
T cells were
isolated by using the MACS System. T cells were stimulated with PMA/
ionomycin, and supernatants were analyzed using an IL-6 – speci c ELISA.
Data represent mean value ± the SD from two experiments with four
mice per group. CD4
+
lamina propria T cells from WT mice produced sig-
ni cantly (P < 0.05) higher amounts of IL-6 compared with cells from
NFATc2-de cient mice.
Figure 5. Reduced capacity of IL-6 and IL-13 – de cient mice to
develop oxazolone-induced colitis. (A) Oxazolone colitis was induced in
IL-13 – de cient mice and WT mice by sensitizing mice with oxazolone,
followed by intrarectal administration of the hapten reagent after 1 wk.
The body weight of the mice was monitored after oxazolone rechallenge
at indicated time points (top left). Mean values ± SEM from one represen-
tative experiment out of two are shown. The average weight of mice at
the beginning of the experiment was 25.8 g (WT group) and 25.8 g (IL-13
KO group), respectively. For this experiment, 5 WT and 5 IL-13 KO mice
were used. Endoscopic assessment of colonic in ammation in WT (A, top
right) or IL-13 – de cient (A, bottom right) mice upon oxazolone adminis-
tration. In contrast to WT mice, IL-13 – de cient mice showed little evi-
dence of colitis. Mean endoscopic scores ± the SEM from one
representative experiment out of two are shown (bottom left). (B) IL-6 –
de cient mice and WT mice were sensitized with oxazolone, followed by
intrarectal administration of the hapten reagent after 1 wk. The body
weight of the mice was monitored after oxazolone rechallenge at the
indicated time points. Mean values ± the SEM from one representative
experiment out of three are shown (top left). The average weight of mice
at the beginning of the experiment was 25.9 g (WT group) and 24.5 g (IL-
6 KO group), respectively. For this experiment, four WT and four IL-6 KO
mice were used. One representative experiment out of three is shown.
Endoscopic assessment of colonic in ammation in WT (B, top right) or
JEM VOL. 205, September 1, 2008
ARTICLE
2105
IL-6 showed normal susceptibility to oxazolone-induced colitis
comparable to WT control mice, as shown by weight curves
( Fig. 6 A ), histopathologic criteria ( Fig. 6, B and C ), and
miniendoscopic criteria ( Figs. 6, D and E ). In fact, hyper-
IL-6 abrogated the protection of NFATc2 KO mice in oxa-
zolone-treated mice and induced mucosal and systemic IL-6
and -13 production as well as an increased local T cell num-
ber ( Fig. 6, F and G ) strongly suggesting that the pathogenic
role of NFATc2 in T cell-mediated colitis is caused by regu-
lation of IL-6 production.
IL-6 de ciency in T cells controls mucosal in ammation
in oxazolone-induced colitis
As the above ndings indicated a key role of IL-6 signaling in
oxazolone colitis, we determined in a nal series of studies
the e ect of IL-6 de ciency in T cells on mucosal in amma-
tion in oxazolone-induced colitis. Accordingly, splenic CD4
+
T cells were puri ed from both WT and IL-6 – de cient mice
and adoptively transferred into immunode cient mice, followed
by induction of oxazolone-induced colitis. As shown in Fig. 7 A ,
mice given WT T cells developed severe colitis with weight
loss, whereas mice reconstituted with IL-6 – de cient T cells
were protected from such colitis. Furthermore, endoscopic
analysis showed signi cant reduction of colitis activity in the
latter compared with the former mice ( Figs. 7, B and C ), sug-
gesting that T cell – derived IL-6 controls mucosal in amma-
tion in oxazolone-induced colitis.
DISCUSSION
UC is an IBD characterized by chronic relapsing, immuno-
logically mediated in ammation of the intestine ( 1, 35 ). Here,
we have identi ed a central pathogenic role for the transcrip-
tion factor NFATc2 in chronic intestinal in ammation. In an
initial series of studies, we demonstrated that NFATc2 ex-
pression is increased in lamina propria T cells in UC compared
with control patients. We then took advantage of genetically
altered mice that lack NFATc2 to uncover a critical role
for this factor in chronic intestinal in ammation. We found
that NFATc2 – and IL-6 – de cient T cells fail to induce T cell –
mediated oxazolone colitis. In further mechanistic studies, we
found that NFATc2 de ciency blocks IL-6 production by mu-
cosal T lymphocytes thereby inducing T cell apoptosis and
preventing development of IL-6/-13 – producing T cells. Fi-
nally, we showed that activation of IL-6 signaling in vivo
via hyper-IL-6 abrogates the protective e ects of NFATc2
de ciency in T cell – mediated colitis and induces IL-6 and -13
production. These data identify NFATc2 as a master regu-
lator for IL-6 production and subsequent T cell activation in
experimental colitis in vivo.
Consistent with a recent proteomic study in UC ( 36 ), we
observed that expression of NFATc2 proteins is increased in
IBD patients. The nding that lamina propria T lymphocytes
in patients with IBD expressed increased amounts of NFATc2
led us to further investigate the functional role of this tran-
scription factor in a T cell – mediated animal model of IBD
( 33 ). These results demonstrated a potent regulatory role of
IL-6 – de cient (B, bottom right) mice upon oxazolone administration. IL-
6 – de cient mice were signi cantly (*, P < 0.05) protected from oxazo-
lone-induced colitis.
However, IL-17F production was also reduced in the absence
of NFATc2 as compared with WT mice in oxazolone colitis
(Fig. S2, available at http://www.jem.org/cgi/content/full/
jem.20072484/DC1), suggesting that NFATc2 controls Th17
cytokine production in experimental colitis.
In subsequent experiments, we puri ed lamina propria CD4
+
T cells from WT and NFATc2-de cient mice in oxazolone-
induced colitis using immunomagnetic beads, and thereby
determined IL-6 production. As shown in Fig. 4 C , NFATc2-
de cient lamina propria T cells produced signi cantly less
IL-6 than WT T cells, suggesting that NFATc2 controls IL-6
production by mucosal T cells in experimental colitis. Con-
sistently, a lower amount of IL-6 – expressing cells was noted
in the lamina propria of NFATc2 KO mice compared with
WT mice in oxazolone colitis (Fig. S3, available at http://
www.jem.org/cgi/content/full/jem.20072484/DC1).
IL-6 and -13, but not the IL-23/IL-17A axis, is required
for oxazolone-induced colitis
To determine the functional role of the aforementioned cy-
tokines in vivo, we next performed studies in oxazolone-
induced colitis using speci c KO mice. As shown in Fig. S4
(A and B, available at http://www.jem.org/cgi/content/full/
jem.20072484/DC1), both IL-23p19 – and IL-17A – de cient
mice showed no signi cant reduction of colitis activity, as
determined by weight curves, endoscopic scores, and histo-
logical assessment (unpublished data), suggesting that other
cytokines produced by T cells could play a pathogenic role.
Interestingly, IL-6 levels produced by IL-17A – and IL-
23p19 – de cient lamina propria cells were comparable to WT
cells (Fig. S2). However, mice de cient for IL-13 ( Fig. 5 A )
and IL-6 ( Fig. 5 B ) showed signi cant protection from oxa-
zolone-induced colitis. These mice showed a reduction of
weight loss and endoscopic colitis activity compared with
WT mice. Collectively, these data suggested that IL-6 and -13,
rather than IL-23 and -17A, play a major pathogenic role in
oxazolone colitis.
IL-6 signaling reverses the protective effect of NFATc2
de ciency in experimental colitis
The above ndings were consistent with the hypothesis that
the reduced susceptibility of NFATc2-de cent mice for oxa-
zolone-induced colitis is caused by suppression of IL-6 pro-
duction by mucosal T cells with consecutive T cell apoptosis.
To test whether activation of IL-6 signaling in vivo could
overcome the reduced susceptibility of NFATc2-de cient
mice to oxazolone-induced colitis, we next treated NFATc2-
de cient mice upon oxazolone administration with hyper-
IL-6. In these studies, NFATc2-de cient mice were injected
i.p. with hyper-IL-6 1 d before intrarectal challenge with oxa-
zolone. Interestingly, NFATc2-de cient mice given hyper-
2106 NFACTC2 IN COLITIS | Weigmann et al.
Figure 6. Administration of hyper-IL-6 restores the susceptibility of NFATc2-de cient mice to oxazolone-induced colitis. (A) Oxazolone coli-
tis was induced by sensitizing mice with oxazolone, followed by intrarectal administration after 7 d and monitoring of the body weight at indicated time
points. Hyper IL-6 (hIL-6; 1 μ g per mouse) was given i.p. before oxazolone administration. Weight curves from one representative experiment out of three
are shown. Data represent mean values ± the SEM. Hyper-IL-6 administration led to colitis development and weight loss in oxazolone-treated NFATc2 KO
mice, but had little effect on colitis activity in WT mice. This experiment was performed three times with groups of four to ve mice. (B) Histological anal-
ysis of colonic in ammation in WT and NFATc2-de cient mice given PBS or hyper-IL-6. Signs of in ammation such as goblet cell depletion, erosions, and
accumulation of mononuclear cells were noted in NFATc2-de cient mice treated with hyper-IL-6, but not control-treated NFATc2 KO mice. (C) Quantita-
tive histopathologic assessment of colitis activity showed a signi cant (P < 0.05) protection from in ammation and tissue injury in NFATc2-de cient mice
compared with WT mice, whereas no signi cant difference was noted between WT mice and NFATc2 KO mice given hyper-IL-6. Data represent mean val-
ues ± the SEM. (D) Endoscopic analysis of the colon of WT and NFATc2-de cient mice at day 2 after application of oxazolone. Oxazolone-induced erosions
and ulcers were seen in the WT and NFATc2/hyper-IL-6 groups, whereas a normal colon architecture was noted in NFATc2-de cient mice. (E) Quantitative
endoscopic score of in ammation (MEICS score). A signi cantly lower endoscopic score was observed in NFATc2-de cient mice compared with WT mice
and NFATc2-de cient mice treated with hyper-IL-6. (F) IL-6 and -13 cytokine production by isolated CD4
+
CD25
⫺
splenic T cells and LPMCs in oxazolone
colitis. Whereas NFATc2-de cient cells produced lower amounts of IL-6 and -13 than WT cells, hyper-IL-6 administration led to a strong induction of IL-6
and -13 production by cells lacking NFATc2. No changes in IFN- ␥ production were noted between NFATc2 KO mice and NFATc2 KO mice given hIL-6 (not
depicted). (G) Increased numbers of CD4
+
T cells in NFATc2-de cient mice after administration of hIL-6 in oxazolone colitis. Cryosections of colonic tissue
were incubated with anti-CD4 antibodies and stained with conjugated Cy3 antibodies. An increased number of CD4
+
T cells was observed in colonic tissue
from WT mice during oxazolone-induced colitis compared with NFATc2 KO mice. After administration of hIL-6, NFATc2-de cient mice showed increased
numbers of CD4
+
T cells compared with NFATc2 mice without hIL-6 administration. Negative control staining is shown. Bars: (B) 100 μ m; (G) 80 μ m.
JEM VOL. 205, September 1, 2008
ARTICLE
2107
Figure 7. Adoptive T cell transfer from WT and IL-6 – de cient
mice in SCID mice. (A) CD4
+
T cells from WT and IL-6 donor mice were
transferred into CB-17/SCID mice, followed by oxazolone sensitization
and intrarectal oxazolone administration. The body weight of reconsti-
tuted mice was analyzed at the indicated time points. Mean values ± the
SEM from one representative experiment are shown. Whereas mice re-
constituted with WT T cells showed a marked weight loss, mice given
IL-6 – de cient T cells were protected from colitis. For this experiment,
four SCID mice in each group were used. (B) Endoscopic assessment of
colonic in ammation of SCID mice reconstituted with WT (left) or IL-6 –
de cient (right) T cells upon oxazolone administration. Endoscopy was
performed 2 d after intrarectal oxazolone challenge. (C) Quantitative
endoscopic analysis of in ammation (MEICS score) of SCID mice in oxa-
zolone colitis was done 2 d after administration of oxazolone. A signi -
cantly (P < 0.05) lower endoscopic score was observed in SCID mice
reconstituted with T cells from IL-6 – de cient mice as compared with the
WT group.
NFATc2 in experimental colitis. In fact, NFATc2 KO mice
were protected from oxazolone colitis, and this protective
e ect could be adoptively transferred by T lymphocytes,
strongly suggesting that NFATc2 regulates mucosal T cell
activity in vivo.
Previous studies have shown that inactivation of the
NFATc2 gene leads to increased numbers of peripheral lym-
phocytes ( 23, 27, 29 ). This nding is at least partially caused
by a defect of NFATc2-de cient splenic T cells to undergo
activation-induced cell death in a Fas/FasL-dependent path-
way. In contrast to these ndings in peripheral T cells, we
observed a signi cantly increased number of apoptotic mu-
cosal CD4
+
T cells in NFATc2-de cient mice in experi-
mental colitis, suggesting that augmented T cell death in the
absence of NFATc2 might contribute to protection from
experimental colitis. To analyze the potential mechanism for
these di erences in mucosal T cell apoptosis, we next assessed
the production of proin ammatory cytokines in experimen-
tal colitis. Consistent with previous studies on augmented
Th2 T cell development by NFATc2-de cient T cells ( 27 ),
we observed that splenic T cells from NFATc2 KO mice
in oxazolone colitis produced signi cantly more IL-4 than
T cells from WT mice. However, no signi cant di erences
in the production of this cytokine were observed between
WT and NFATc2-de cient mucosal T cells, whereas IL-5
production was induced in the absence of NFATc2. In con-
trast, the production of IL-6, -13, and -17 was signi cantly
reduced by NFATc2-de cient mucosal T cells compared
with control WT T cells. Interestingly, IL-17 production was
reduced in spite of normal or even increased levels of the
Th17 master transcription factor ROR ␥ t ( 37 – 39 ), suggesting
that both ROR ␥ t and NFATc2 are required to induce opti-
mal IL-17 production by a large number of mucosal T cells.
However, both IL-23 – and IL-17A – de cient mice showed
normal susceptibility for oxazolone colitis, suggesting that
other cytokines might be crucial for the decreased suscepti-
bility of NFATc2-de cient mice to oxazolone colitis.
IL-6 is a multifunctional cytokine with pro- and antiin-
ammatory properties ( 40 ), and it stimulates target cells via a
membrane receptor complex consisting of the IL-6 receptor
(IL-6R) and the signaling receptor subunit gp130 ( 41 ).
Although gp130 is ubiquitously expressed, the IL-6R is only
found on hepatocytes and some hematopoietic cells. Interest-
ingly, a soluble form of the IL-6R (sIL-6R) has been shown
to bind IL-6 and to stimulate gp130 on cells that do not express
the IL-6R. This process has been named IL-6 trans-signaling
( 42 – 44 ) and activates T cells lacking the IL-6R such as most
mucosal T cells. As IL-6 signaling has been identi ed as a key
regulator of T cell apoptosis in experimental colitis ( 45 – 48 ),
we have focused on the e ects of IL-6 trans-signaling in
NFATc2-de cient mice. It was found that the administration
of hyper-IL-6, a potent activator of IL-6/sIL-6R signal trans-
duction ( 44, 49 ), completely restores IL-13 production by
NFATc2-de cient T cells and abrogates the protective e ect
of NFATc2 de ciency in oxazolone colitis in vivo. These
data suggest that NFATc2 regulates experimental colitis in vivo
in an IL-6 – dependent fashion and that IL-6 signaling is
important to augment mucosal IL-6 and -13 production.
Finally, adoptive transfer studies showed that IL-6 produc-
tion by T lymphocytes is important for activity of oxazolone-
induced colitis.
NFATc2 de ciency in RAG KO mice results in the develop-
ment of severe colitis ( 50 ), suggesting that this transcription
2108 NFACTC2 IN COLITIS | Weigmann et al.
in 100 μ l 1X PBS 1 d before sensitization and challenge with oxazolone.
Control mice were injected with PBS at the same time point. In some ex-
periments, splenic CD4
+
T cells (5 × 10
6
) from WT, NFATc2-de cient, or
IL-6 – de cient mice were isolated and injected into syngenic SCID mice,
followed by sensitization with oxazolone. At day 7 after T cell transfer,
SCID mice were challenge intrarectally with oxazolone and the body weight
was monitored at indicated time points.
In vivo high resolution miniendoscopic analysis of the colon. For
monitoring of colitis activity, a high resolution video endoscopic system
(Karl Storz) for mice was used ( 53 ). Prominent endoscopic signs of in am-
mation in CB-17/SCID mice were abrogation of the normal vascular pat-
tern, the presence of mucosal granularity, and the appearance of ulcers. To
determine colitis activity in reconstituted CB-17/SCID-de cient mice or
hapten-treated mice the mice were monitored by miniendoscopy at indi-
cated time points, and endoscopic scoring of ve parameters (translucent,
granularity, brin, vascularity, and stool) was performed.
Isolation of LPMCs. LPMCs were isolated from freshly obtained colonic
specimens using a modi cation of previously described techniques ( 54 – 56 ).
LPMCs were collected at the interphase of the Percoll gradient, washed
once, and resuspended in FACS bu er or cell culture medium. For some
experiments, CD4
+
T cells were isolated from LPMC preparations by using
anti-CD4 antibodies conjugated with microbeads (Miltenyi Biotec) accord-
ing to the manufacturer ’ s instructions.
Preparation of cytospins. Lamina propria cells were isolated and resuspended
in PBS/1% FCS at a density of 10
6
cells/ml in PBS. Slides were prepared in a
Cytospin2-centrifuge (Shandon) and loaded with 200 μ l cell suspension. After
centrifugation at 500 upm for 5 min, slides were removed and dried for 2 h.
Slides were subsequently stained using speci c anti-bcl-xl and anti-bcl-2 anti-
bodies, as speci ed above. Samples were analyzed by microscopy.
T cell culture and cytokine assays. To measure cytokine production, 10
6
splenic T cells per ml were activated with 10 μ g/ml puri ed hamster anti –
mouse CD3 (clone 145-2C11) and 5 μ g/ml soluble hamster anti – mouse
CD28 (clone 37.51) and cultured in complete medium or serum-free me-
dium. LPMCs were stimulated with PMA/ionomycin or anti-CD3/CD28
antibodies. Cytokine concentrations were determined by using commer-
cially available mouse FlowCytomix systems (Bender MedSystems). For
measurement of IL-6, -13, and -17F levels, commercially available ELISA
kits (R & D Systems and Bender MedSystems) were used.
Histological analysis of colon cross sections. Colon samples were re-
moved from colitic mice at indicated points of time. 4 μ m para n sections
were made and stained with hematoxylin and eosin. For colitis induced by
oxazolone, the degree of in ammation and epithelial injury on microscopic
cross sections of the colon was graded semiquantitatively from 0 to 6 ( 18 ).
Grading of colitis activity was done in a blinded fashion by the same patholo-
gist (H.A. Lehr). Small bowel sections were taken from the same animals as
an additional control and showed no evidence of in ammation.
FACS analysis. For FACS analysis, apoptotic cells were detected by stain-
ing with Annexin V antibodies, and necrotic cells were stained with 7-AAD
and the cells were analyzed using a FACSCalibur (BD Biosciences).
Analysis of cell apoptosis in colonic specimens. To visualize apoptotic
cells, cryosections of colonic tissue were analyzed by TUNEL assay using a
commercially available kit (Oncor-Appligene) according to the manufactur-
er ’ s instructions. Nuclear staining was done with DAPI. For detection of
CD3
+
TUNEL
+
(double-positive) cells, immunohistochemical studies were
done after TUNEL staining. Blocking was done by treatment of samples
with immunoblock kit (Roth) before incubation with a monoclonal anti-
body against CD3 (BD Biosciences). Detection was achieved using a Cy3-
conjugated secondary antibody against mouse IgG (Dianova).
factor plays a protective role in innate mucosal immune re-
sponses. In contrast, we observed here that NFATc2 plays an
important pathogenic role in controlling T cell e ector re-
sponses in experimental colitis. In fact, this study identi es
NFATc2 as a master regulator for T cell-mediated oxazolone
colitis and the regulation of IL-6 production by mucosal
T cells with subsequent e ects on T cell cytokine production
and apoptosis. Thus, modulation of NFATc2 function in
T cells appears to be an attractive target for therapeutic inter-
vention in T cell – mediated chronic intestinal in ammation
such as is observed in IBDs.
MATERIALS AND METHODS
Patients. Gut specimens obtained from patients with CD or UC or from
control patients were studied. Collection of surgical samples was approved
by the ethical commitee and the institutional review board of the University
of Mainz.
Immunohistochemistry. Immunohistochemistry was performed on 7- μ m
cryosections from gut specimens of control and IBD patients, as previously
described ( 18 ). For staining of NFATc2 transcription factor, the mouse anti-
bodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used. Immuno-
uorescence was performed using the tyramide signal ampli cation Cy3
system (PerkinElmer). Accordingly, tissues were xed in 4% paraformalde-
hyde/PBS, followed by sequential incubation with avidin/biotin- (Vector
Laboratories), peroxidase-, and protein-blocking reagent (Dako) to elimi-
nate unspeci c background staining. Sections were incubated with primary
antibodies speci c for human CD3 (Dianova) or NFATc2 proteins (Santa
Cruz Biotechnology), dissolved in PBS/0.5% BSA/0.2% saponin. Sections
incubated with isotype matched control antibodies served as negative
control. Next, samples were incubated with biotinylated goat anti – rabbit
secondary IgG antibody or uorescence-conjugated antibody (Dianova) fol-
lowed by incubation with streptavidin-conjugated Cy3 (Dianova) or with
streptavidin-HRP and stained with tyramide-Cy3, according to the manu-
facturer ’ s instructions. For confocal microscopy (Leitz Microscope) before
examination, the nuclei were counterstained with DAPI (Vector Labora-
tories), mounted, and analyzed. NFATc2-positive cells in 6 – 10 high power
elds were subsequently counted in all patients per condition.
For immunostaining of CD4
+
T cells in the lamina propria, a rat anti –
mouse CD4 (BD Biosciences) antibody was used. Biotinylated goat anti –
rat antibodies (Dianova) and Streptavidin-Cy3 were used for subsequent
analysis, as well as counterstaining with DAPI. For immunostaining of anti-
apoptotic proteins, rabbit-anti-bcl-xl (Cell Signaling Technology) and
Armenian hamster anti-bcl-2 (BD Biosciences) antibodies were chosen.
Anti – rabbit or anti – Armenian hamster biotinylated antibodies were used for
subsequent staining in combination with streptavidin-HRP and tyramide-
Cy3 signal ampli cation.
Animals. 2-4-mo-old BALB/c mice were obtained from the central breed-
ing facility at the University of Mainz or from Charles River Laboratories;
NFATc2-de cient mice have been described elsewhere ( 27 ). NFATc2-de-
cient mice used in the experimental studies were between 4 and 12 wk of
age. CB-17/SCID mice were obtained from M & B. IL-6 – and IL-13 – de -
cient mice were previously described ( 51, 52 ). IL-23p19 – and IL-17A – de -
cient mice were obtained from Regeneron Pharmaceuticals. All animal
studies were kept in speci c pathogen – free conditions and approved by the
Institutional Animal Care and Use Committee of the University of Mainz.
Oxazolone-induced colitis. Oxazolone-induced colitis in BALB/c mice
was induced using a previously described method ( 18 ). After 2 d, the mice
were analyzed by miniendoscopy to monitor the manifestation of colitis.
For the analysis of hyper-IL-6 (IL-6/sIL-6R fusion protein [ 49 ]) func-
tions, mice were injected i.p. with 1 μ g hyper-IL-6 (per 20 g mice weight)
JEM VOL. 205, September 1, 2008
ARTICLE
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Statistical analysis. Statistical analysis was made using the Student ’ s
t test. P values < 0.05 were considered as statistically signi cant and identi ed
with asterisks.
Online supplemental materials. The supplemental gures provide infor-
mation on the expression of the antiapoptotic proteins bcl-2 and bcl-xl in
lamina propria T cells from NFATc2-de cient mice in oxazolone-induced
colitis (Fig. S1), and the IL-6, -13, and -17F production in oxazolone-in-
duced colitis (Fig. S2). Fig. S3 demonstrates IL-6 expression during oxazo-
lone-induced colitis in NFATc2-de cient mice, and the capacity of
IL-23p19 –
and IL-17A – de cient mice to develop oxazolone-induced colitis
(Fig. S4). The online version of this article is available at http://www.jem
.org/cgi/content/full/jem.20072484/DC1.
The authors would like to thank Professor Laurie Glimcher for generously providing
NFATc2 KO mice and Alexei Nikolaev for excellent work in immunohistochemistry.
The research of M.F. Neurath was supported by the MAIFOR program of the
University of Mainz and the Sonderforschungsbereich SFB548 of the German
Research Council (DFG). S. Rose-John was funded by the DFG within the
Sonderforschungsbereich SFB415.
The authors have no con icting nancial interests.
Submitted: 21 November 2007
Accepted: 7 July 2008
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