T Cell-mediated Pathology in Two Models of Experimental
Colitis Depends Predominantly on the Interleukin 12/Signal
Transducer and Activator of Transcription (Stat)-4 Pathway,
but Is Not Conditional on Interferon
Expression by T Cells
By
Stephen J.
Simpson,*
Samir
Shah,*
Martina
Comiskey,*
Ype P.
de Jong,*
Baoping
Wang,*
Emiko
Mizoguchi,
Atul K.
Bhan,
and
Cox
Terhorst*
From the * Division of Immunology, Beth Israel Deaconess Medical Center and the
Department of
Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115
 |
Abstract |
The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4
signaling and induction of T cell-specific interferon (IFN)-
expression in the development of
T helper cell (Th)1-type pathology were examined in two different models of experimental
colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient
mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-
and tumor necrosis factor (TNF)-
-producing T cells. We show here that
treatment with anti-IL-12 antibodies in one of the models, or reconstitution with T cells from
Stat-4-deficient (Stat-4null) mice in both models resulted in a milder disease in the majority of
recipient animals, compared with those that were left untreated or that had been reconstituted
with wt cells. Protected mice in each group also harbored lower frequencies of IFN-
-producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from
the attenuation of IFN-
expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell-specific IFN-
expression, IFN-
null donors were used for T cell reconstitution in each system. Surprisingly, large
numbers of IFN-
null-reconstituted mice developed wasting and colitis, which in many cases
was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T
cells from these animals expressed TNF-
, demonstrating that they had retained the ability to
produce another proinflammatory cytokine. Taken together, these results demonstrate that in
some forms of chronic experimental colitis the development of pathogenic T cells is influenced
predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or
other Th1 promoting pathways are not limited to the induction of IFN-
gene expression in T lymphocytes.
 |
Introduction |
Several rodent models of chronic intestinal inflammation
share features of immunopathology with human inflammatory bowel disease (IBD),1 which exists in the two distinct forms of Crohn's disease and ulcerative colitis (1).
In particular, the finding that human and many animal
forms of IBD show evidence of aberrant Th1 responses has
come under close scrutiny (5, 7). Most recently, it has
been proposed that mucosal inflammation, such as that
found in IBD, emerges from an alteration in the normal
balance between the effects of proinflammatory cytokines
such as IFN-
and regulatory cytokines such as transforming growth factor
(TGF-
; reference 13). The observation that TGF-
-deficient mice develop inflammation of
various tissues, including the intestine, provides strong evidence in support for this (14). IL-12, a cytokine produced
by activated macrophages and dendritic cells, plays a central
role in the generation of Th1-type responses, characterized
predominantly by the induction of IFN-
production in T
cells (15). In light of this, elucidation of the mechanisms
by which IL-12 might promote mucosal inflammation is of
clear importance for understanding the pathogenesis of IBD.
Evidence that IL-12 is important in intestinal inflammation has recently been provided in an experimental model
of acute granulomatous colitis, induced by administration
of trinitrobenzene sulphonic acid (TNBS), which could be
prevented by treatment with an anti-IL-12 antibody (21).
The absence of IFN-
expression in protected animals in
these experiments was consistent with other data that has
shown that IL-12 activity correlates strongly with IFN-
expression by T cells (19, 22, 23). Recent studies have extended these observations by demonstrating that induction
of IFN-
expression in Th1 cells depends largely upon the
activity of intracellular signal transducer and activator of
transcription 4 (Stat-4) proteins, which mediate signals via
the IL-12 receptor (24). Although IL-12 activates other
members of the Stat protein family (including Stat-3; reference 27), the effects of this cytokine on Th1 cell development have been shown to depend specifically on expression of Stat-4. Consistent with this, T cells from Stat-4null
mice are almost completely unable to produce IFN-
in
response to IL-12 and show reduced Th1 responses equivalent to those in IL-12null mice (28).
Although the IL-12/Stat-4 pathway clearly predominates in the development of Th1 type T cells, Stat-4-deficient T cells are nevertheless capable of limited IFN-
production in response to IL-12-independent stimuli, such as
CD3-IL-2 costimulation (28, 29). Additionally, IFN-
has
been shown to efficiently augment IL-12-mediated differentiation of Th1 cells (31). These data leave open the possibility that pathways other than that provided by IL-12 and
Stat-4 contribute to the expression of IFN-
and development of Th1-mediated inflammation. Furthermore, such
experiments beg the question as to whether induction of
IFN-
gene expression in T cells does in fact represent the
critical step in the development and action of pathogenic
Th1 type cells. For example, it is possible that T cells could
be induced into a Th1-like state in the absence of autocrine
IFN-
expression, and induce pathology through expression of other proinflammatory mediators. In this study we
sought to examine these questions by testing the extent to
which Stat-4 signaling and the resulting IFN-
production
by T cells contribute to pathology in two distinct Th1
models of colitis.
In each colitis model, wasting and intestinal pathology
has been shown to be generated by the abnormal reconstitution of the T cell compartment in nonallogeneic,
immunodeficient mice. In the first system, we have previously shown that disease develops after bone marrow cell
(BMC) reconstitution of T cell and NK cell-deficient Tg
26
(C57BL/6 × CBA/J) mice, using (C57BL/6 × CBA/J)F1
donor animals (BM
Tg
26) (32). In the second system, IBD was originally shown to be induced upon reconstitution of CB.17 Scid mice with CD45RBhi CD4+ T cells
from wt Balb/c or CB.17 animals (35). In both models, strong evidence exists to suggest that pathology develops
through a lack of normal T cell regulation. In the case of
the CD45RBhi transfer model, this occurs due to the absence of CD45RBlo CD4+ T cells, which exert their regulatory effects through the expression of IL-10 and TGF-
(38).
Similarly, we have provided evidence in the BM
Tg
26
model that abnormal T cell regulation, resulting from aberrant development of thymus-derived T cells, is causal in the
development of pathology in these mice (34). Central to
our hypothesis is the observation that the thymi of Tg
26
mice lack a normal stromal architecture and thus are unable
to support normal ontogeny of wt donor-derived thymocytes
supplied by bone marrow inoculation. Consequently, we
have suggested that this defect in thymocyte development
prevents the establishment of a regulated T cell repertoire
in BM
Tg
26 mice.
In both the BM
Tg
26 and CD45RBhi transfer models, the notion that T cell dysregulation leads to aberrant
Th1 responses is underscored by the presence of very large
numbers of activated IFN-
- and TNF-
-secreting T cells
in the peripheral lymphoid tissue and the colon (38, 40).
However, despite the similarity of the T cell phenotype in
each model, the resulting pathology in each case is clearly
distinct. Thus, in the BM
Tg
26 mice, inflammation is
limited to the mucosa of the colon and shares some features
of UC, including crypt abscess formation, crypt cell proliferation, and extensive mononuclear cell infiltration. Although these are also characteristic of the CD45RBhi transfer model, colitis in these mice displays additional features resembling some of those observed in Crohn's disease, including transmural inflammation, formation of granulomas,
and occasional involvement of the distal small intestine.
These distinctions provide a useful means by which to
compare mechanisms important in alternative forms of
chronic intestinal inflammation. Furthermore, since each
system uses adoptive transfer of cells into T-cell deficient
host animals, both are amenable to a novel experimental design whereby alternative chimeras can be generated using
donors genetically deficient in specific proteins of interest.
Using this as one of our approaches, we investigated the requirements for IL-12/Stat-4 signaling and induction of T
cell-specific IFN-
gene expression in the development of
pathology in each form of colitis.
 |
Materials and Methods |
Mice.
Donor (C57BL/6 × CBA/J)F1, C57BL/6, C57BL/6/
Scid, and C57BL/6/IFN-
null mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). The Stat-4null mice were a
gift from Dr. J.N. Ihle (St. Jude's Children's Hospital, Memphis,
TN). 129/SvEv RAG-2null and wt mice were purchased from
Taconic Farms (Germantown, NY). The Tg
26 recipient mice
were generated as previously described by transgenic overexpression of a full-length human CD3
gene (32) and bred on the
original C57BL/6 × CBAJ background in the Beth Israel Deaconess Medical Center animal facility. All mice were kept under
standard conditions in microisolator cages with autoclaved food,
water, and bedding. In RAGnull and Scid reconstitution experiments, recipients were between 4 and 6 wk of age. Tg
26 recipient mice were between 5 and 10 wk of age.
Cell and Bone Marrow Purification and Cell Transfer.
The CD45RBhi
model was generated as described by Powrie et al. with minor
modifications (37). CD4+ T cells obtained from the spleens of
donor animals and were enriched by magnetic sorting. For cell
purification, the following biotinylated anti-mouse antibodies
were used to label non-CD4+ T cells isolated from the spleens of
donor mice: B220 (RA3-6B2), Mac-1 (M1/70), Gr-1(RB6-8C5), and CD8
(53-6.7). Magnetically labeled streptavidin
beads (Miltenyi Biotec Inc., Sunnyvale, CA) were used to bind
the biotinylated antibodies. All antibodies were obtained from
PharMingen (San Diego, CA). Negative selection was accomplished using a MACS magnetic cell sorter (Miltenyi Biotec Inc.).
The enriched CD4+ cells were then labeled for cell sorting with
FITC-conjugated CD45RB (16A) and PE-conjugated CD4
(Becton Dickinson, Mountain View, CA). Subsequently, cells
were sorted under sterile conditions by flow cytometry for CD4+
CD45RBhi cells, using criteria similar to that described in previous studies (37). This resulted in a >98% pure population of T
cells. Harvested cells were resuspended at 106/400 ml PBS (no
FCS) and injected into the tail veins of recipient mice.
For the BM
Tg
26 model, bone marrow was prepared as
previously described (34). In brief, BMCs were depleted of T
cells using two rounds of treatment with anti-Thy1.2 antibody
(30-H12) followed by rabbit complement lysis (Cedarlane Labs,
Westbury, NY). This procedure resulted in <0.1% CD4+ and
CD8
+ cells within the inoculum, as determined by flow cytometry. Bone marrow recipients were total body irradiated with a
sublethal dose (400 rads) 6-8 h before transplant or were treated
with 5-Fluorouracil (75 mg/kg) 48 h before transplantation. 5 × 106-107 cells were resuspended in 400 ml PBS (no FCS) and
transferred by tail vein injection.
To reduce the possibility of graft versus host disease (GVHD)
in the CD45RBhi model, cell transfers were made, where possible,
between mice of the same backgrounds. Stat-4null mice were of the
mixed background C57BL/6 × 129/SvEv, making responses against
segregated minor antigens from the 129/SvEv donor background
a possibility. However, no evidence of GVHD, such as skin inflammation, was observed in any of the animals studied. In the
Tg
26 bone marrow transfer model, significant differences were
present between donor and recipient mice in some cases. However, preconditioning of bone marrow was effective in depleting
the BMCs of T cells (see above). Inflammation of the skin and
small bowel were not apparent in the mice included in this study.
Antibody Treatment.
The anti-IL-12 antibody (clone C 17.8;
reference 16) was a gift from Dr. T. Veldman (Genetics Institute,
Cambridge, MA). 260 µg of purified anti-IL-12 antibody (or vehicle control [PBS]) was injected intraperitoneally into mice on a
weekly basis starting at the time of BMC transfer.
Disease Monitoring and Scoring.
Mice were weighed twice a
week and monitored for appearance and signs of loose stool and
diarrhea. In the BM
Tg
26 model, the level of wasting was determined by percentage of loss of weight from the starting
weight, measured at the time of transplant. Weights were subsequently ranked 1 point for every 10% of body weight (or part
thereof) lost: 0 = no weight loss; 1 = 1-10% weight loss; 2 = 11-20% weight loss; and 3 =
21% weight loss. At necropsy animals were assessed for the level of colitis, which they displayed using two parameters: colon thickening and stool consistency, which were subsequently ranked as follows: for colon enlargement, 0 = no colon thickening; 1 = mild thickening; 2 = moderate thickening; 3 = extensive thickening; and for stool consistency, 0 = normal beaded stool; 1 = soft stool; 2 = diarrhea; and
an additional point was added if gross blood was noted. The total
score given for disease was the combined scores for colon thickening, stool consistency, and weight loss, divided by three.
Histological Analysis.
Colon tissue samples were fixed in 10%
buffered formalin and embedded in paraffin. Sections (4 µm)
were cut and placed on gelatin-coated microscope slides for staining with hematoxylin and eosin using standard techniques as previously described (34). The severity of colitis was determined
based on histological examination of the distal colon, whereby
the extent of colonic inflammation was graded on a scale of 0-3
in each of three criteria: cell infiltration, thickening of the bowel
wall, and the number of crypt abscesses. Histological grades were
assigned in a blinded fashion by the same pathologist (A.K. Bhan).
Cell Preparations and Cytokine Analysis.
Mesenteric lymphocytes
were harvested and single cell suspensions prepared in PBS with
2% FCS (Intergen, Purchase, NY). Viable cells were counted and
determined by trypan blue exclusion. Surface and cytoplasmic
staining and FACS® (Becton Dickinson, Indianapolis, IN) analysis
were performed as previously described (40). All antibodies were
purchased from PharMingen and were conjugated to PE, FITC,
or biotin. In the case of biotin-conjugated antibodies, labeling
was accomplished by secondary staining with streptavidin-RED670
(GIBCO BRL). Cytoplasmic staining for IFN-
(clone XMG1.2)
and TNF-
(clone MP6-XT22) was done as previously described
(40). In brief, freshly isolated cells were stimulated overnight with
plate-bound anti-CD3 (clone 145-2C11). Brefeldin A was added
for the final 2 h of incubation at a final concentration of 10 mg/
ml. Cells were stained with anti-CD4 (clone RM4-5) and anti-
CD3-biotin (145-2C11), washed, and fixed with 2% paraformaldehyde (Sigma Chemical Co., St. Louis, MO). Cells were permeabilized with Saponin (0.2% in PBS) to facilitate cytoplasmic
cytokine staining. Cells were analyzed by flow cytometry using a
FACscan® flow cytometer in conjunction with FACScan® software
(Becton Dickinson) and CellQuest Software (Becton Dickinson).
 |
Results |
The IL-12/Stat-4 Pathway Predominates in Development of
Wasting and Intestinal Pathology in Two Models of Colitis.
To examine the role of IL-12 in the BM
Tg
26 colitis
model, Tg
26 mice were transplanted with BMCs from
(C57BL/6 × CBA)F1 donors as previously described (34)
and given a weekly dose of anti-IL-12 antibody, or vehicle
control (PBS). The requirement for Stat-4 expression in T
cells was tested by comparing disease in Tg
26 mice transplanted with BMCs from Stat-4null mice or wt control animals of the same genetic background (129/SvEv). Using a
similar approach in the CD45RBhi model, we used 129/
SvEv RAGnull mice, rather than CB.17/SCID mice, as recipients, allowing us to maintain an equivalent genetic
background to the Stat-4null donor animals (see Materials
and Methods). RAGnull mice were thus reconstituted with
FACS®-sorted splenic CD4+ CD45RBhi T cells from Stat-4null mice (Stat-4null
RAGnull). Control RAGnull mice in
these experiments were transplanted with wt 129/SvEv CD4+ CD45RBhi T cells (129/wt
RAGnull).
Treatment of BM
Tg
26 mice with anti-IL-12 antibody resulted in a significant reduction in disease compared
with control (untreated) mice. A gross clinical disease activity score, which measures stool consistency, gross colon enlargement, and weight loss, is shown in Fig. 1. The difference in the extent of disease was most apparent between 4 and 5 wk after BMC transfer, where control animals
showed signs of severe cachexia, and in many cases diarrhea
which was occasionally accompanied by visible blood. By
comparison, anti-IL-12-treated animals showed reduced
wasting and generally did not develop diarrhea. At
necropsy, the colons of treated animals were also visibly less
thickened than those of control mice. Similarly, the majority of Tg
26 reconstituted with Stat-4null BMCs showed
milder disease compared with control animals that had received wt BMC (Fig. 1). 129/wt
RAGnull mice developed
wasting and colitis 7-10 wk after cell transfer. In contrast,
Statnull
RAGnull retained a healthy appearance over the
same period of time and showed mild colitis at necroscopy,
similar to that seen in RAGnull mice that had received
sorted CD4+ CD45RBlo T cells (36, 37).

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Fig. 1.
Disease is reduced in the absence of the IL-12/Stat-4 pathway. Disease activity scores of Tg 26 mice that were transplanted with
(C57BL/6 × CBA/J)F1 BMCs (F1 Tg 26) and received a weekly dose
of anti-IL-12 antibody are compared with PBS-treated F1 Tg 26 mice
(untreated). Tg 26 and RAGnull mice were reconstituted with Stat-4null
BMCs and CD45RBhi CD4+ T cells, respectively, and compared with
animals that received 129/SvEv/wt cells. For comparison, results are also
presented from RAGnull mice that received CD45RBlo CD4+ T cells
from wt and Stat-4null donors. Clinical disease activity scores were determined according to parameters of weight loss, colon thickening, and stool
consistency (described in Materials and Methods). The means of collective individual scores, obtained from three separate experiments, are plotted ± SD (BM Tg 26 and CD45RBhi transfer groups, n = 10-15/
group; CD45RBlo, n = 3-4/group).
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The reduced severity of disease found in each experimental group of mice was reflected both in the extent of
loss of body mass (Fig. 2 A) and for the most part in the level
of mucosal inflammation in the large bowel, determined by
examination of histological tissue sections (Fig. 2 B). Thus,
the extent of mucosal inflammation in all anti-IL-12-treated
F1
Tg
26 mice, and the majority of Stat-4null reconstituted mice, was lower than in control animals. Nevertheless, significant histological colitis could be detected in a
proportion of Stat-4null recipient animals, even in cases
where severe gross colitis (extensive colon thickening and
diarrhea) was not apparent. In Stat-4null
RAGnull experiments, a large majority of animals showed considerably lower grades of colitis than did control mice. Representative histological sections from colons of CD45RBhi
RAGnull
and BM
Tg
26 mice from different groups are shown in
Fig. 3, illustrating the principal differences between mild
and severe forms of colitis in each model. The limited extent of mucosal inflammation in most of the Stat-4null-recipients and in those subjected to anti-IL-12 antibody
treatment was evident from the nominal level of cellular
infiltration, crypt cell hyperplasia, and the paucity of crypt
cell abscesses formation. However, it was noted, in the colons of some Stat-4null
RAGnull mice, that even in the absence of continuous mucosal inflammation there was a
moderate frequency of focal inflammatory involvement. In
some cases this was accompanied by the appearance of
granulomas, a characteristic feature of the CD45RBhi transfer model (Fig. 3 D). Collectively, the use of anti-IL-12 antibodies and Stat-4null mutant mice as donors revealed a
predominant role for IL-12 and Stat-4 proteins in the development of both the wasting syndrome and colon inflammation in two distinct models of IBD.

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Fig. 2.
Wasting and colitis
are both affected by the absence
of IL-12/Stat-4 signaling. The
divisible components of disease,
weight loss and histological colitis, are represented. (A) Weight
loss. The body weights of animals measured at the end of each
experiment was divided by the
starting body weight (on the day
of cell transfer), to calculate the
percentage of starting body weight.
Each point represents data from
an individual animal. (B) The
extent of mucosal inflammation
was examined histologically and
graded (see Materials and Methods). Represented are the combined scores for cell infiltration
(0-3) and colon thickening (0-3)
on a scale of 1-6. Each point
represents the score assigned to
an individual animal.
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Fig. 3.
Histology of colitis. Sections taken from the distal region of colon are compared after hematoxylin and eosin staining. Objectives are all ×10
and scoring is as described in Materials and Methods and Fig. 2. (A) Normal colon taken from an untransplanted Scid mouse. (B) Severe disease (6+) in a
RAGnull mouse transplanted with CD45RBhi T cells from a 129/SvEv/wt donor showing extensive cellular infiltration and crypt destruction. (C) Mild
disease (2+) in a RAGnull mouse transplanted with CD45RBhi T cells from a Stat-4null donor. The basic structure of the mucosa is intact, but reveals some
infiltration and crypt distortion. (D) Mild disease (2+) and presence of a granuloma in a RAGnull mouse transplanted with CD45RBhi T cells from a Stat-4null donor. (E) Severe disease (5+) in a Tg 26 mouse transplanted with wt (129/SvEv) bone marrow. Evident is extensive crypt elongation, and a pronounced cellular infiltration. (F) Mild disease (2+) in a BM Tg 26 mouse treated with a weekly dose of anti-IL-12 antibody. This section reveals a low
level of cellular infiltrate, moderate crypt elongation, and reduced thickening of the bowel wall.
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IL-12 and Stat-4 Cooperate in the Expression of IFN-
by
Th1 Cells.
To examine whether disease correlated directly with the level of Th1 involvement in the two colitis
models, we examined the development of Th1-type T cells
in each system. Since previous studies have shown a direct
effect of IL-12 and Stat-4 on the production of IFN-
by
T cells, we used cytoplasmic staining of IFN-
and FACS®
analysis to determine the frequencies of T cells capable of
IFN-
expression. As shown in Fig. 4, the frequency of
IFN-
-producing CD4+ T cells in the mesenteric lymph
nodes (MLNs) in anti-IL-12-treated F1
Tg
26 mice and
in Stat-4null
Tg
26 and Stat-4null
RAGnull mice was 60-
80% lower than in control animals. Additionally, numbers
of colon lamina propria CD4+ T cells capable of IFN-
production were also markedly reduced in protected animals (data not shown). Previous reports have suggested that
in the absence of Stat-4 signaling T cells develop a Th2-like phenotype (29). Examination of IL-4 and IL-10 expression by MLN T cells in Stat-4null recipients revealed no
detectable expression of either cytokine (data not shown).

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Fig. 4.
The effect of the IL-12/Stat-4 pathway on IFN- production by T cells. T cells from the MLNs from each group of BM Tg 26
and CD45RBhi RAGnull mice were compared for production of IFN- .
T cells were stimulated overnight using anti-CD3 antibody and stained
for cytoplasmic expression of IFN- , and the frequency of cytokine positive cells was assessed by flow cytometry. (A) Dot plots comparing the
frequency IFN- production by CD4+ MLN T cells from BM Tg 26
mice treated with anti-IL-12 antibody or PBS, Stat-4null Tg 26 and
wt Tg 26 controls, and CD45RBhi RAGnull mice that had received
Stat-4null and 129/SvEv/wt (129/wt) cells. Plots show staining of cytoplasmic IFN- after analytical gating on CD4+ T cells. Gates are drawn after
determination of background staining after preblocking with anti-IFN-
antibodies and staining with isotype control antibodies of irrelevant specificity. The percentages of positive cells falling in the designated gates are
shown. Frequencies of T cells from healthy wt mice expressing IFN- after identical CD3 stimulation or T cells from each of the represented
groups without CD3 staining fall below 1% (data not shown) (B) The
percentages of IFN- -positive T cells from the MLNs of different groups
of mice were determined. Shown are the mean values ± SD calculated
for each group (n = 5-9/group).
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IFN-
-deficient T Cells Mediate Disease and Produce TNF-
.
Together, the above data implicate a causal relationship between the numbers of T cells capable of IFN-
production
and the extent of wasting and colitis. We reasoned that if
induction of IFN-
gene expression did in fact represent a
critical step in the development of pathogenic Th1 cells, then
the ablation of this, specifically in T cells, should be sufficient to prevent disease. To test this, we reconstituted the
T cell compartment of Tg
26 and Scid or RAGnull mice using IFN-
-deficient donors (IFN-
null mice; reference 41).
In the BM
Tg
26 model, animals injected with BMCs
from IFN-
null donors backcrossed to the C57BL/6 background were compared with controls injected with C57BL/
6/wt BMCs. In the CD45RBhi transfer model, CD45RBhi
CD4+ T cells from IFN-
null mice were transferred into
C57BL/6/Scid or RAGnull mice. Scid and RAGnull mice transplanted with C57BL/6/wt CD4+ CD45RBhi T cells served
as controls in these experiments. Fig. 5 shows the cumulative
results from these experiments, which compare the mean
disease activity scores from each group. In both BM
Tg
26 and CD45RBhi
Scid RAGnull animals little difference was
apparent between the overall gross disease in each group of
mice (Fig. 5). However, in some cases, weight loss and/or
histological colitis were milder in IFN-
null-recipient mice
(Fig. 6, A and B). A large number of IFN-
null
Tg
26
mice showed a range of histological scores and, in some cases, despite showing clinical evidence of significant colitis, revealed comparatively mild inflammation of the mucosa by
histological examination (Fig. 6 B). In CD45RBhi
Scid/
RAGnull experiments, most animals showed visible signs of
wasting. However, due to their young age at the time of
cell transfer, many of these mice increased in body mass
due to compensatory growth. The extent of colon involvement observed in the large majority of CD45RBhi
Scid/
RAGnull mice was equivalent whether or not animals had
received IFN-
null or C57BL/6/wt T cells. Representative
histological sections from mice that developed colitis in the
presence of IFN-
null T cells are shown in Fig. 7. In each
case the salient features of pathology were similar to those
in animals reconstituted with wt T cells.

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Fig. 5.
T cells deficient in IFN- production can cause disease in
each of the colitis models. Tg 26 mice and C57BL/6/Scid or RAGnull
mice received CD45RBhi CD4+ T cells and BMCs, respectively, from
C57BL/6/IFN- null donors, and were monitored for disease. These animals were compared with mice that received cells from C57BL/6/wt
(B6/wt) donors. Plotted are the mean clinical disease activity scores ± 1 SD for each group of animals (n = 4-12 mice/group).
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Fig. 6.
Wasting and colitis
in the absence of IFN- expression in T cells. (A) Change in
body weights and (B) histological
colitis are represented as described in Fig. 2 in animals reconstituted with IFN- null T
cells. In the CD45RBhi transfer
model, closed symbols represent
RAGnull, rather than Scid recipients mice.
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Fig. 7.
Histological colitis in IFN- null T cell-reconstituted mice.
Sections taken from distal colon are shown from (A) an IFN- null Tg 26
mouse with mild colitis (2+ histological score), (B) IFN- null Tg 26
mouse severe colitis (4+ histological score), and (C) IFN- null Scid mouse
with severe colitis (5+ histological score).
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Finally, we observed that large numbers of T cells from
IFN-
-reconstituted animals expressed a pattern of cell
surface markers consistent with an activation or memory
status, including low CD45RB and L-selectin expression
and elevated CD69 expression (data not shown). To examine whether, in the absence of IFN-
expression, these T
cells retained other characteristics consistent with a pathogenic phenotype, their ability to produce TNF-
was tested. As shown in Fig. 8, equivalent frequencies of CD4+
T cells from IFN-
null and wt recipients in each model expressed TNF-
. Taken together, these data demonstrate
that without producing IFN-
themselves, T cells in each
of the IBD models were nevertheless capable of mediating
significant levels of wasting and colitis that correlated with
the retained capacity of these cells to produce a proinflammatory cytokine.

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Fig. 8.
T cells from IFN- null-recipient mice produce TNF- .
MLN T cells were examined for expression of IFN- (hatched bars) and
TNF- (closed bars) using flow cytometry after overnight in vitro stimulation with an anti-CD3 antibody. The mean percentages of cytokine-positive cells in each group are shown ± SD (n = 5-10/group).
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Discussion |
In our study we used a novel experimental approach to
examine the mechanisms leading to development and action of pathogenic Th1-type T cells in colitis. The significant effect of Stat-4 deficiency in limiting progression of
disease in both the BM
Tg
26 and the CD45RBhi transfer systems establishes a principal role for this pathway in
the development of pathogenic Th1-type T cells in colitis. Furthermore, the similar attenuation of both disease and T
cell-specific IFN-
expression seen in anti-IL-12-treated
BM
Tg
26 mice and Stat-4null
Tg
26 mice underscores
the link between the actions of IL-12 and Stat-4. In the
two colitis models studied here, inhibition of IL-12 and/or
Stat-4 signaling affected two divisible aspects of disease,
colitis and wasting, implicating involvement of Th1 cells in
both components of pathology.
To an extent, our experimental results appear consistent
with those obtained in the more acute TNBS-induced
model of colitis (21). However, we observed that although
disease was for the most part attenuated in the absence of
IL-12/Stat-4 signaling, many animals nevertheless revealed
evidence of pathology. Furthermore, this correlated with
the continued (although strongly reduced) presence of T
cells capable of IFN-
expression. These data differ from
those in the TNBS colitis model, where an almost complete inhibition of both disease and IFN-
production
were observed after anti-IL-12 treatment. These differences make apparent that the mechanisms of pathology in
colitis models studied here and that induced by TNBS are
not equivalent, and they suggest that factors other than IL-12 contribute to the development of more chronic forms
of intestinal inflammation. In this capacity, the recently described IFN-
-inducing factor (IL-18) is a potential candidate, since this cytokine has been shown to have a significant effect on the long-term ability of T cells to produce
IFN-
(42). By inference it is likely that this could significantly affect the development of pathogenic Th1-like T cells.
Notwithstanding the above arguments, the results from
our study suggest a degree of independence between the
actions of IL-12 and/or other Th1-promoting cytokines
and the specific expression of IFN-
by T cells. Thus, although the absence of IFN-
expression by T cells altered
the severity of colitis in many cases, the overall effects on
disease were less pronounced than might have been anticipated. On first assessment this is surprising, principally for
three reasons. First, the dependence of IFN-
expression
on IL-12 signaling suggests that the effects of IL-12 (via
Stat-4) result from its ability to induce IFN-
transcription
in T cells (22). Second, IFN-
has been shown to be required for efficient IL-12 priming of Th1 cells, presumably
through autocrine expression (45). Principally, this appears to be achieved through the upregulation of the IL-12
receptor (48). Third, a general consensus exists that the
well-documented proinflammatory effects of IFN-
are central to pathology in IBD (4, 7, 13). The validity of this
last assumption in experimental colitis has been strengthened by a previous study by Powrie et al., who demonstrated that anti-IFN-
treatment was effective in reducing
disease in the CD4+ CD45RBhi transfer model (38). It is
possible that in this latter case anti-IFN-
antibody treatment might block all available IFN-
. By contrast, in our
experiments non-T cells, resident in the host animals,
might remain a sufficient source of IFN-
to promote effects on both T cells and macrophages. However, the development of colitis in the absence of IFN-
expression is
not unprecedented, since IL-2null mice, which succumb to
a Th1-type colitis similar to that seen in BM
Tg
26 mice,
also develop disease after import of the IFN-
null mutation
(Zand, M., C. Stevens, and T. Strom, personal communication). In addition, a recent study by Berg et al. revealed
that antibody neutralization of IFN-
in IL-10null mice was
sufficient to prevent disease only when administered to animals at 3 wk of age and not in animals aged 3 mo or older (50). Thus, although IFN-
was required to establish colitis in IL-10null mice, other inflammatory mediators were clearly
sufficient to mediate disease once a pathogenic T cell phenotype had been established.
In two autoimmune models classically associated with
Th1-type T cell responses, it has been observed that Th2
cells are also capable of mediating disease (51, 52). These
studies support the contention that, in some cases, immunopathology normally associated with Th1-type responses
can also be attributable to cells that have undergone immune deviation, leading to the expression of Th2-associated cytokines. However, in our studies we were unable to
detect any IL-4 or IL-10 expression in the absence of either Stat-4null or IFN-
expression by T cells. Although these
findings do not unequivocally demonstrate lack of immune
deviation, they do argue that this is not predominant in the
colitis models studied here.
Collectively, the results offered here lend credence to
the argument that Th1-type cells develop pathogenicity via
a complex range of mechanisms, not all of which fall under
the influence of IL-12 or their ability to produce IFN-
.
Ultimately, this is most likely achieved by the concerted
expression of a range of cytokines and cytotoxic molecules
which could act directly or indirectly to promote inflammation. TNF-
, which is produced by T cells as well as
macrophages, is a cytokine that encompasses these characteristics and is known to induce potent inflammatory effects. Consistent with this, we have shown that in colitic
mice T cells expressed TNF-
at a similar frequency
whether IFN-
was expressed by the same cells or not.
Furthermore, we have found that anti-TNF-
antibody
treatment is highly effective in reducing disease in the
BM
Tg
26 model.2 Similarly, Powrie et al. demonstrated
an effect of anti-TNF-
treatment in the CD45RBhi
Scid model (38). These data are consolidated by the finding that anti-TNF-
antibody therapy has been shown to be
efficacious in treating Crohn's disease (53).
In summary, our study offers insights into the requirements for both the development and pathogenic activity of
Th1-type cells in two distinct models of inflammatory colitis. The observations that IL-12 is upregulated in human
IBD (7) and that interruption of the TNF-
pathway inhibits Crohn's disease correlate well with our observations
in the two models studied here (38).2 Further dissection of
the pathways which lead to aberrant Th1 pathology in animal models of IBD will undoubtedly provide a useful source of information for future therapy in IBD.
 |
Footnotes |
Address correspondence to Cox Terhorst, Division of Immunology, Beth Israel Hospital, 330 Brookline
Ave., Harvard Medical School, Boston, MA 02115. Phone: 617-667-7147; Fax: 617-667-7140.
Received for publication 22 September 1997 and in revised form 22 December 1997.
1Abbreviations used in this paper: BM
Tg
26, (C57BL/6 × CBA/J)F1;
BMC; bone marrow cells; IBD; inflammatory bowel disease; Stat, signal
transducer and activator of transcription; TGF, transforming growth factor.
Stephen J. Simpson's present address is Education and Research Centre, St. Vincent's Hospital, Dublin 4, Ireland.
Simpson and Shah contributed equally to this work.
2
Mackay, F., J. Browning, P. Lawton, S. Shah, M. Comiskey, A.K. Bhan,
E. Mizoguchi, C. Terhorst, and S. Simpson, manuscript submitted for
publication.
We thank Dr. James Ihle for providing us with the Stat-4null mice and Dr. T. Veldman for providing anti-
IL-12 antibody.
This work was supported by grants from the Crohn's and Colitis Foundation of America (C. Terhorst) and
from the National Institutes of Health (P30 DK-43551 and RO1 DK-47677 to A.K. Bhan). Samir A. Shah
is a Howard Hughes Medical Institute Physician Postdoctoral Fellow. Stephen J. Simpson was supported by
a Research Fellowship Award from the Crohn's and Colitis Foundation of America.
 |
References |
1.
|
Podolsky, D.K..
1991.
Inflammatory bowel disease.
N. Engl.
J. Med.
325:
928-937
[Medline].
|
2.
|
MacDonald, T.T..
1994.
Gastrointestinal inflammation. Inflammatory bowel disease in knockout mice.
Curr. Biol
3:
261-263
.
|
3.
|
Sartor, R.B..
1995.
Current concepts of the etiology and
pathogenesis of ulcerative colitis and Crohn's disease.
Gastroenterol. Clin. North Am.
24:
475-507
[Medline].
|
4.
|
Sartor, R.B..
1994.
Cytokines in intestinal inflammation:
pathophysiological and clinical considerations.
Gastroenterology.
106:
533-539
[Medline].
|
5.
| Simpson, S.J., G.A. Hollander, E. Mizoguchi, A.K. Bhan, B. Wang, and C. Terhorst. 1996. Defects in T-cell regulation:
lessons for inflammatory bowel disease. In Essentials of Mucosal Immunology. M.F. Kagnoff and H. Kiyono, editors.
Academic Press Inc., San Diego, CA. 291-304.
|
6.
| Stenson, W.F. 1994. Animal models of inflammatory bowel
disease. In Inflammatory Bowel Disease: From Bench to Bedside. S.R. Targan, editor. Williams and Wilkins, Baltimore,
MD. 180-192.
|
7.
|
Parronchi, P.,
P. Romagnani,
F. Annunziato,
S. Sampognaro,
A. Becchio,
L. Giannarini,
E. Maggi,
C. Pupilli,
F. Tonelli, and
S. Romagnani.
1997.
Type 1 T-helper cell predominance and interleukin-12 expression in the gut of patients
with Crohn's disease.
Am. J. Pathol.
150:
823-832
[Abstract].
|
8.
|
Fiocchi, C.,
D.G. Binion, and
J.A. Katz.
1994.
Cytokine
production in the human gastrointestinal tract during inflammation.
Current Opinion in Gastroenterology.
2:
639-644
.
|
9.
|
Murata, Y.,
Y. Ishiguro,
J. Itoh,
A. Munakata, and
Y. Yoshida.
1995.
The role of proinflammatory and immunoregulatory cytokines in the pathogenesis of ulcerative colitis.
J. Gastroenterol.
30(Suppl.):
56-60
[Medline].
|
10.
|
Niessner, M., and
B.A. Volk.
1995.
Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed
transcribed polymerase chain reaction (RT-PCR).
Clin. Exp.
Immunol.
101:
428-435
[Medline].
|
11.
|
Radford-Smith, G., and
D.P. Jewell.
1996.
Cytokines and inflammatory bowel disease.
Bailliere's Clin. Gastroenterol.
10:
151-164
[Medline].
|
12.
|
Powrie, F..
1995b.
T cells in inflammatory bowel disease: protective and pathogenic roles.
Immunity.
3:
171-174
[Medline].
|
13.
|
Strober, W.,
B. Kelsall,
T. Marth,
B. Ludviksson,
R. Erhardt, and
M. Neurath.
1997.
Reciprocal IFN- and TGF- responses regulate the occurrence of mucosa inflammation.
Immunol. Today.
18:
61-64
[Medline].
|
14.
|
Shull, M.M.,
I. Ormsby,
A.B. Kier,
S. Pawlowski,
R.J. Diebold,
M. Yin,
R. Allen,
C. Sidman,
G. Proetzel,
D. Calvin, et al
.
1992.
Targeted disruption of the mouse transforming
growth factor-beta 1 gene results in multifocal inflammatory
disease.
Nature.
359:
693-699
[Medline].
|
15.
|
Trinchieri, G..
1995.
Interleukin-12: a pro-inflammatory cytokine with immunoregulatory functions that bridge innate
resistance and antigen-specific adaptive immunity.
Annu.
Rev. Immunol.
13:
251-276
[Medline].
|
16.
|
Ozmen, L.,
M. Pericin,
J. Hakimi,
R.A. Chizzonite,
M. Wysocka,
G. Trinchieri,
M. Gately, and
G. Garotta.
1994.
Interleukin 12, interferon , and tumor necrosis factor are
the key cytokines of the generalized Shwartzman reaction.
J.
Exp. Med.
180:
907-915
[Abstract].
|
17.
|
Gerosa, F.,
C. Paganin,
D. Peritt,
F. Paiola,
M.T. Scupoli,
M. Aste-Amezaga,
I. Frank, and
G. Trinchieri.
1996.
Interleukin-12 primes human CD4 and CD8 T cell clones for high
production of both interferon and interleukin 10.
J. Exp.
Med
183:
2559-2569
[Abstract].
|
18.
|
Heufler, C.,
F. Koch,
U. Stanzl,
G. Topar,
M. Wysocka,
G. Trinchieri,
A. Enk,
R.M. Steinman,
N. Romani, and
G. Schuler.
1996.
Interleukin-12 is produced by dendritic cells
and mediates T helper 1 development as well as interferon-
production by T helper 1 cells.
Eur. J. Immunol
26:
659-668
[Medline].
|
19.
|
Wysocka, M.,
M. Kubin,
L.Q. Vieira,
L. Ozmen,
G. Garotta,
P. Scott, and
G. Trinchieri.
1995.
Interleukin-12 is required
for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice.
Eur. J. Immunol.
25:
672-676
[Medline].
|
20.
|
Gracie, J.A., and
J.A. Bradley.
1996.
Interleukin-12 induces
interferon- -dependent switching of IgG alloantibody subclass.
Eur. J. Immunol.
26:
1217-1221
[Medline].
|
21.
|
Neurath, M.F.,
I. Fuss,
B.L. Kelsall,
E. Stuber, and
W. Strober.
1995.
Antibodies to interleukin 12 abrogate established experimental colitis in mice.
J. Exp. Med.
182:
1281-1290
[Abstract].
|
22.
|
Ryffel, B..
1997.
Interleukin-12: role of interferon- in IL-12
adverse effects.
Clin. Immunol. Immunopathol
83:
18-20
[Medline].
|
23.
|
Schmitt, E.,
P. Hoehn,
C. Huels,
S. Goedert,
N. Palm,
E. Rude, and
T. Germann.
1994.
T helper type 1 development
of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon- and is inhibited by transforming growth factor- .
Eur. J. Immunol.
24:
793-798
[Medline].
|
24.
|
Cho, S.S.,
C.M. Bacon,
C. Sudarshan,
R.C. Rees,
D. Finbloom,
R. Pine, and
J.J. O'Shea.
1996.
Activation of STAT4
by IL-12 and IFN- : evidence for the involvement of ligand-induced tyrosine and serine phosphorylation.
J. Immunol.
157:
4781-4789
[Abstract].
|
25.
|
Bacon, C.M.,
E.F.R. Petricoin,
J.R. Ortaldo,
R.C. Rees,
A.C. Larner,
J.A. Johnston, and
J.J. O'Shea.
1995.
Interleukin 12 induces tyrosine phosphorylation and activation of
STAT4 in human lymphocytes.
Proc. Natl. Acad. Sci. USA
92:
7307-7311
[Abstract].
|
26.
|
Ihle, J.N..
1996.
STATs: signal transducers and activators of
transcription.
Cell.
84:
331-334
[Medline].
|
27.
|
Jacobson, N.G.,
S.J. Szabo,
R.M. Weber-Nordt,
Z. Zhong,
R.D. Schreiber,
J.E. Darnell Jr., and
K.M. Murphy.
1995.
Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.
J. Exp. Med
181:
1755-1762
[Abstract].
|
28.
|
Thierfelder, W.E.,
J.M. van Deursen,
K. Yamamoto,
R.A. Tripp,
S.R. Sarawar,
R.T. Carson,
M.Y. Sangster,
D.A. Vignali,
P.C. Doherty,
G.C. Grosveld, and
J.N. Ihle.
1996.
Requirement for Stat-4 in interleukin-12-mediated responses of
natural killer and T cells.
Nature.
382:
171-174
[Medline].
|
29.
|
Kaplan, M.H.,
Y.L. Sun,
T. Hoey, and
M.J. Grusby.
1996.
Impaired IL-12 responses and enhanced development of Th2
cells in Stat4-deficient mice.
Nature.
382:
174-177
[Medline].
|
30.
|
Magram, J.,
S.E. Connaughton,
R.R. Warrier,
D.M. Carvajal,
C.Y. Wu,
J. Ferrante,
C. Stewart,
U. Sarmiento,
D.A. Faherty, and
M.K. Gately.
1996.
IL-12-deficient mice are
defective in IFN production and type 1 cytokine responses.
Immunity.
4:
471-481
[Medline].
|
31.
|
Wenner, C.A.,
M.L. Guler,
S.E. Macatonia,
A. O'Garra, and
K.M. Murphy.
1996.
Roles of IFN- and IFN- in IL-12-
induced T helper cell-1 development.
J. Immunol
156:
1442-1447
[Abstract].
|
32.
|
Wang, B.,
C. Biron,
J. She,
K. Higgins,
M.J. Sunshine,
E. Lacy,
N. Lonberg, and
C. Terhorst.
1994.
A block in both
early T lymphocyte and natural killer cell development in
transgenic mice with high-copy numbers of the human
CD3 gene.
Proc. Natl. Acad. Sci. USA
91:
9402-9406
[Abstract/Free Full Text].
|
33.
|
Hollander, G.A.,
B. Wang,
A. Nichogiannopoulou,
P.P. Platenberg,
W. Van Ewijk,
S.J. Burakoff,
J.C. Gutierrez-Ramos, and
C. Terhorst.
1995.
Developmental control point in induction of thymic cortex regulated by a subpopulation of
prothymocytes.
Nature.
373:
350-353
[Medline].
|
34.
|
Hollander, G.A.,
S.J. Simpson,
E. Mizoguchi,
A. Nichogiannopoulou,
J. She,
J.C. Gutierrez-Ramos,
A.K. Bhan,
S.J. Burakoff,
B. Wang, and
C. Terhorst.
1995.
Severe colitis in
mice with aberrant thymic selection.
Immunity.
3:
27-38
[Medline].
|
35.
|
Powrie, F.,
M.W. Leach,
S. Mauze,
L.B. Caddle, and
R.L. Coffman.
1993.
Phenotypically distinct subsets of CD4+ T
cells induce or protect from chronic intestinal inflammation
in CB-17 Scid mice.
Int. Immunol.
5:
1461-1471
[Abstract].
|
36.
|
Morrissey, P.J.,
K. Charrier,
S. Braddy,
D. Liggitt, and
J.D. Watson.
1993.
CD4+ T cells that express high levels of
CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T
cells.
J. Exp. Med.
178:
237-244
[Abstract].
|
37.
|
Powrie, F.,
M.W. Leach, and
S. Mauze.
1994.
Inhibition of
TH1 responses prevents IBD in SCID mice reconstituted
with CD45RBhigh CD4+ T cells.
Immunity.
1:
553-562
[Medline].
|
38.
|
Powrie, F.,
J. Carlino,
M.W. Leach,
S. Mauze, and
R.L. Coffman.
1996.
A critical role for transforming growth factor- but not interleukin 4 in the suppression of T helper
type 1-mediated colitis by CD45RBlow CD4+ T cells.
J. Exp.
Med.
183:
2669-2674
[Abstract].
|
39.
|
Powrie, F.,
R. Correa-Oliveira,
S. Mauze, and
R.L. Coffman.
1994.
Regulatory interactions between CD45RBhigh
and CD45RBlow CD4+ T cells are important for the balance
between protective and pathogenic cell-mediated immunity.
J. Exp. Med.
179:
589-600
[Abstract].
|
40.
|
Simpson, S.J.,
G.A. Hollander,
E. Mizoguchi,
D. Allen,
B.P. Wang, and
C. Terhorst.
1997.
Expression of pro-inflammatory cytokines by TCR + and TCR + T cells in an
experimental model of colitis.
Eur. J. Immunol.
27:
17-25
[Medline].
|
41.
|
Dalton, D.K.,
S. Pitts-Meek,
S. Keshav,
I.S. Figari,
A. Bradley, and
T.A. Stewart.
1993.
Multiple defects of immune cell
function in mice with disrupted interferon- genes.
Science.
259:
1739-1742
[Medline].
|
42.
|
Kohno, K.,
J. Kataoka,
T. Ohtsuki,
Y. Suemoto,
I. Okamoto,
M. Usui,
M. Ikeda, and
M. Kurimoto.
1997.
IFN- -inducing factor (IGIF) is a costimulatory factor on the activation of
Th1 but not Th2 cells and exerts its effect independently of
IL-12.
J. Immunol.
158:
1541-1550
[Abstract].
|
43.
|
Micallef, M.J.,
T. Ohtsuki,
K. Kohno,
F. Tanabe,
S. Ushio,
M. Namba,
T. Tanimoto,
K. Torigoe,
M. Fujii,
M. Ikeda, et al
.
1996.
Interferon- -inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism
with interleukin-12 for interferon- production.
Eur. J. Immunol
26:
1647-1651
[Medline].
|
44.
|
Okamura, H.,
H. Tsutsi,
T. Komatsu,
M. Yutsudo,
A. Hakura,
T. Tanimoto,
K. Torigoe,
T. Okura,
Y. Nukada,
K. Hattori, et al
.
1995.
Cloning of a new cytokine that induces
IFN- production by T cells.
Nature.
378:
88-91
[Medline].
|
45.
|
Gollob, J.A.,
H. Kawasaki, and
J. Ritz.
1997.
Interferon-
and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression.
Eur. J. Immunol.
27:
647-652
[Medline].
|
46.
|
Bradley, L.M.,
D.K. Dalton, and
M. Croft.
1996.
A direct
role for IFN- in regulation of Th1 cell development.
J. Immunol.
157:
1350-1358
[Abstract].
|
47.
|
Nakamura, T.,
R.K. Lee,
S.Y. Nam,
E.R. Podack,
K. Bottomly, and
R.A. Flavell.
1997.
Roles of IL-4 and IFN- in
stabilizing the T helper cell type 1 and 2 phenotype.
J. Immunol.
158:
2648-2653
[Abstract].
|
48.
|
Szabo, S.J.,
A.S. Dighe,
U. Gubler, and
K.M. Murphy.
1997.
Regulation of the interleukin (IL)-12R 2 subunit expression in developing T helper 1 (Th1) and Th2 cells.
J. Exp.
Med.
185:
817-824
[Abstract/Free Full Text].
|
49.
|
Rogge, L.,
L. Barberis-Maino,
M. Biffi,
N. Passini,
D.H. Presky,
U. Gubler, and
F. Sinigaglia.
1997.
Selective expression of an interleukin 12 receptor component by human T
helper 1 cells.
J. Exp. Med.
185:
825-831
[Abstract/Free Full Text].
|
50.
|
Berg, D.J,
N. Davidson,
R. Kuhn,
W. Muller,
S. Menon,
G. Holland,
L. Thompson-Snipes,
M.W. Leach, and
D. Rennick.
1996.
Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production
and CD4+ Th-1-like responses.
J. Clin. Invest.
98:
1010-1020
[Abstract/Free Full Text].
|
51.
|
Lafaille, J.J.,
F. van de Keere,
A.L. Hsu,
J.L. Baron,
W. Haas,
C.S. Raine, and
S. Tonegawa.
1997.
Myelin basic protein-
specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather
than protect them from disease.
J. Exp. Med.
186:
307-312
[Abstract/Free Full Text].
|
52.
|
Pakela, S.,
M.O. Kurrer, and
J.D. Katz.
1997.
T helper cells
(Th2) T cells induce acute pancreatitis and diabetes in immune-compromised nonobese diabetic (NOD) mice.
J. Exp.
Med.
186:
299-306
[Abstract/Free Full Text].
|
53.
|
van Dullemen, H.M.,
S.J. van Deventer,
D.W. Hommes,
H.A. Bijl,
J. Jansen,
G.N. Tytgat, and
J. Woody.
1995.
Treatment of Crohn's disease with anti-tumor necrosis factor
chimeric monoclonal antibody (cA2).
Gastroenterology.
109:
129-135
[Medline].
|