From the Institute of Medical Microbiology and
Immunology, the Panum Institute, and the Institute for Molecular
Biology, University of Copenhagen, 2200 Copenhagen, Denmark, the
¶ Department of Medicine V, Aarhus University Hospital and the
Institute of Human Genetics, the Bartholin Building, University
of Aarhus, 8000 Aarhus C, Denmark, and the ** Tissue
Typing Laboratory, Department of Clinical Immunology, National
University Hospital, 2200 Copenhagen, Denmark
Received for publication, August 6, 2002, and in revised form, February 20, 2003
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ABSTRACT |
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Via cytoplasmic signal transduction pathways,
cytokines induce a variety of biological responses and modulate the
outcome of inflammatory diseases and malignancies. Crohn's disease is a chronic inflammatory bowel disease of unknown etiology. Perturbation of the intestinal cytokine homeostasis is believed to play a pivotal role, but the pathogenesis of Crohn's disease is not fully understood. Here, we study intestinal T cells from Crohn's disease and healthy volunteers. We show that STAT3 and STAT4 are constitutively activated in Crohn's patients but not in healthy volunteers. The activation is
specific, because other STAT proteins are not constitutively activated.
Furthermore, the STAT3 regulated protein, SOCS3, is also constitutively
expressed in Crohn's patients but not in healthy volunteers. Taken
together, these data provide evidence of abnormal STAT/SOCS signaling
in Crohn's disease. This aberrant activation, so far noted only in
malignant cells, establish a new critical approach for better
understanding the immunopathogenesis of Crohn's disease.
Cytokines play an important role in activation, effector function,
and homeostasis of the immune system. Dysregulation, imbalanced production, or aberrant expression of cytokines is believed to be
involved in the immunopathogenesis of infectious, allergic, and
autoimmune diseases. To transmit their messages, cytokines bind to
specific cell surface receptors and activate cytoplasmic signal
transduction pathways, often involving the Janus kinase (Jak)1/signal transducers and
activators of transcription (STAT) signaling pathway (1, 2). The Jak
kinase family consists of non-receptor protein kinases that are
constitutively associated with the cytoplasmic membrane-proximal
regions of various receptors. Upon ligand binding, Jak kinases become
catalytically activated by phosphorylation. STAT proteins are
subsequently recruited via highly specific recognition of
phosphotyrosine docking sites on the receptor complex by the STAT SH2
domains (3). Upon tyrosine phosphorylation by Jaks, STAT proteins homo-
or heterodimerize and translocate to the nucleus, where the interaction
with specific DNA sequences triggers transcription of target genes (4).
In healthy individuals, Jak/STAT activation is rapid, transient, and
strictly regulated at multiple levels. Down-regulation of STAT
activation involves inhibitors such as suppressors
of cytokine signaling (SOCS),
protein inhibitor of activated
Stat (PIAS), protein tyrosine phosphatases (5), and proteolytic degradation (6-9). The importance of controlling STAT
activation has been underscored by the findings that the constitutively
activated form of STAT3 induces an uncontrolled cellular growth
in vitro and functions as an oncogene in vivo (10-12). Furthermore, constitutive activation of STAT3 is observed in
breast cancer and head and neck cancer as well as leukemia and
lymphomas (13-16). Crohn's disease and other human chronic inflammatory bowel diseases are characterized by disturbances in the
immune homeostasis involving CD4+ T cells, regulatory T
cells, and a number of cytokines and cytokine receptors (17).
Experimental and clinical data point toward a dysregulation of the
cytokine network with a dominance of T helper cell type 1 (Th1)
cytokines (IFN- Reagents--
Antibodies against human SOCS3 (M20), STAT3 (C20),
STAT6 (S20), and ERK-1/2 (K23) were from Santa Cruz Biotechnology
(Santa Cruz, CA). Antibodies against human STAT5 phospho-tyrosine 694 and human STAT6 phospho-tyrosine 641 were from New England Biolabs (Beverly, MA). The antibody against human STAT3 phospho-tyrosine 704 was purchased from Nanotools (Denzlingen, Germany). The antibody against human STAT4 phospho-tyrosine 693 was from Zymed
Laboratories Inc. Antibodies against human STAT4 and human
STAT5 were from BIOSOURCE. Horseradish
peroxidase-conjugated secondary antibodies against mouse-IgG,
rabbit-IgG, and goat-IgG were from DAKO (Glostrup, Denmark). RhIL-2
Proleukin was purchased from Chiron (Emeryville, CA). RhIL-4, rhIL-10,
rhIL-12, and rhIFN- Patients and Healthy Donors--
The intestinal CD4+
T cells were obtained from colon mucosa biopsies from seven healthy
volunteers (median age 38, range 20-50 years) and from seven patients
(median age 40, range 22-50 years) with an established diagnosis of
Crohn's disease. All patients had active disease treated with 5-ASA
(2-4 g/day). Two patients received additional treatment with
azathioprine (150-200 mg/day) and/or prednisone (5-20 mg/day). None
of the patients received anti-TNF treatment.
Cell Purification and Culture--
Intestinal T cells were
cultured in RPMI 1640 (Sigma) supplemented with 10% pooled human serum
(Blood Bank, State University Hospital, Copenhagen, Denmark), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin (all from Sigma), and 103 units/ml
rhIL-2 and 25 ng/ml rhIL-4. Neither antigen nor feeder cells were added
to the intestinal T cells cultures, which consisted of more than 97%
CD3+ and CD4+ T cells. Alloantigen-specific
peripheral human CD4+ T cell lines were obtained from
healthy donors as described previously (20). Peripheral T cell cultures
were IL-2-dependent and grown in RPMI 1640 supplemented
with 10% pooled human serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin and 100 units/ml rhIL-2. Cutaneous T cells from the affected skin of patients with atopic dermatitis and psoriasis were IL-2- and
IL-4-dependent and grown under the same conditions
described above for cultures of intestinal T cells. The cutaneous T
cell lymphoma cell line was established from skin biopsies from a
patient with mycosis fungoides (MF) as described (21). In this study we
used a long-term culture of an MF tumor cell line (MyLa 2000), which
grows independently of cytokines. MyLa 2000 was cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Cytokine Stimulations--
Prior to all experiments, both
intestinal and peripheral T cells were washed twice and "rested"
in the absence of exogenous cytokines by culturing the cells
overnight (>10 h) in normal culture media depleted of cytokines.
Subsequently, cells were stimulated with 500 units/ml rhIL-2 or 25 ng/ml rhIL-4 for 15 min at 37 °C. Longer IL-2-stimulations of 45 min
or 90 min were also performed in peripheral T cells. Stimulations by
1-50 ng/ml rhIL-10 or rhIL-12 and stimulations by 104
units/ml of rhIFN- Protein Extraction and Western Blotting--
After stimulation
with or without cytokines, cells were lysed in ice-cold 1% Nonidet
P-40 lysis buffer with the following protease/phosphatase inhibitors: 1 mM phenylmethylsulfonyl fluoride (in
Me2SO), 5 mM EDTA, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µl/ml indoleacetic acid, and 10 mM NaF. The cytoplasmic extracts
were analyzed by SDS-PAGE, and gels were transferred to nitrocellulose membrane. Blots were incubated with primary antibodies overnight at
4 °C, washed, and then incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h at room
temperature. The bands were detected using enhanced chemiluminiscence
(Amersham Biosciences) according to the manufacturer's manual. For
repeated immunoblotting, membranes were stripped and reprobed using the
procedure described above.
Statistical Analysis--
MEDSTAT was used for statistical
analyses. Data were analyzed using a two-tailed Fisher's exact test
with assumption of no variance. A p value of less than 0.05 was considered statistically significant.
First, we studied STAT activation in intestinal CD4+ T
cells obtained from a patient with Crohn's disease (CD) and
peripheral, allospecific CD4+ T cell lines from healthy
donors. As expected, STAT proteins were not tyrosine phosphorylated in
rested peripheral T cells from healthy donors (Fig.
1, A and B,
lanes 1), whereas cytokine stimulation induced profound
tyrosine phosphorylation of STAT3/STAT5 (IL-2), STAT6 (IL-4), or STAT3
(IL-10) (Fig. 1, A, lanes 2 and 3, and
B, lane 3). Surprisingly, high levels of tyrosine
phosphorylated STAT3 was observed in rested intestinal T cell lines
obtained from CD patients (Fig. 1, A and B,
lanes 4). The constitutive tyrosine phosphorylation of STAT3
was highly specific, because STAT1/5/6 were not constitutively tyrosine
phosphorylated in intestinal T cells from Crohn's patients (Fig.
1A, lane 4). Moreover, IL-2 and IL-4 did not
trigger tyrosine phosphorylation of STAT5 and STAT6 in intestinal T
cells from Crohn's patients (Fig. 1A, lanes 5 and 6), whereas high concentrations of IL-10 induced an
enhanced tyrosine phosphorylation of STAT3 (Fig. 1B,
lane 6).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, TNF-
, IL-12) and a deficiency in Th2 cytokines
(IL-4 and IL-10) (18, 19). Because most of these cytokines/cytokine
receptors transduce signals through the Jak/STAT signaling pathways, we
hypothesized that disturbances in STAT signaling might be involved in
the pathogenesis of Crohn's disease. Accordingly, the present study
was undertaken to investigate STAT signaling in intestinal T cells from
Crohn's patients and healthy volunteers.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
were purchased from PeproTech (Rocky Hill, NJ).
Tyrphostin AG 1478 was from Alexis, Laufelfingen, Switzerland), and
Jak3-Inhibitor I was purchased from Calbiochem.
were carried out for 15 min at 37 °C. In all
experiments, controls of cytokine-unstimulated cells were always run in parallel.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of STAT3, STAT5, and STAT6 in
cytokine-stimulated human T cells. Total cell extracts were
analyzed by immunoblotting with specific anti-phospho-STAT3, -5, -6 (p-Y-stat3/5/6) and anti-STAT3, -5, and -6 (Total-stat3/5/6)
antibodies. A, peripheral CD4+ T cells
(left panel) and intestinal CD4+ T
cells from Crohn's disease patients (right panel) were
unstimulated (lanes 1 and 4) or stimulated with
500 units/ml IL-2 (lanes 2 and 5) and 25 ng/ml
IL-4 (lanes 3 and 6) for 15 min. B,
peripheral CD4+ T cells (left panel)
and intestinal CD4+ T cells from patients with Crohn's
disease (right panel) were unstimulated
(lanes 1 and 4) or stimulated with IL-10 at 1 ng/ml (lanes 2 and 5) or 50 ng/ml (lanes
3 and 6)
Because Th1 cytokines are believed to play a crucial role in chronic
inflammatory bowel diseases (18) and STAT4 is a key regulator of Th1
responses, we studied IL-12-induced STAT4 activation in CD patients and
healthy individuals. As expected, in rested peripheral T cells from
healthy donors, IL-12 induced a profound tyrosine phosphorylation of
STAT4 (Fig. 2, lane 3),
whereas STAT4 was not tyrosine phosphorylated in the absence of IL-12
(Fig. 2, lane 1). In contrast, STAT4 was constitutively
tyrosine phosphorylated in rested intestinal T cells from CD patients
(Fig. 2, lane 4), and the phosphorylation was further
up-regulated following IL-12 stimulation (Fig. 2, lane
6).
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Next, we compared STAT signaling in intestinal T cells from the mucosa
of healthy volunteers and Crohn's disease patients. As shown in Fig.
3, STAT3 and STAT4 were constitutively
tyrosine phosphorylated in rested T cells from five of seven different CD patients (Fig. 3, A and B, lanes 4 and 7, and data not shown). In contrast, STAT3 and STAT4
were not constitutively tyrosine phosphorylated in rested intestinal T
cells from any of seven healthy volunteers (Fig. 3, A and
B, lanes 1, and C, p = 0.02). In T cells from healthy donors, IL-2 and IL-4 induced tyrosine phosphorylation of STAT3 (Fig. 3A, lanes 2 and
3), whereas high concentrations of IL-12 induced STAT4
tyrosine phosphorylation (Fig. 3B, lane 3). In
intestinal T cells from CD patients, the constitutive tyrosine
phosphorylation of STAT3 and STAT4 was enhanced following stimulation
with IL-4 (Fig. 3A, lanes 5 and 8) and
IL-12 (Fig. 3B, lanes 6 and 9),
respectively, whereas IL-2 had little effect on STAT3 phosphorylation
and did not induce tyrosine phosphorylation of STAT5 (Fig.
3A, lanes 6 and 9, and data not
shown). Moreover, IL-4 did not induce tyrosine phosphorylation of STAT6
(data not shown), confirming the results shown previously in Fig.
1A. The difference in STAT activation between Crohn's
disease patients and healthy donors was observed throughout a 3-month
period, indicating that the phenotype was stable over time and not
caused by the culture conditions (data not shown).
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Although the intestinal tract is the primary target organ, Crohn's
disease is often a systemic disease with extra intestinal manifestations such as arthritis, conjunctivitis, and erythema nodosum.
Therefore, we examined whether an aberrant STAT activation was observed
only in intestinal T cells or also in peripheral T cells. As shown Fig.
4A, STAT3 was constitutively
activated in peripheral T cells from all of the five patients tested.
In contrast, we were unable to detect significant levels of tyrosine phosphorylated STAT3 in peripheral T cells from healthy controls (Fig.
4B), indicating that an aberrant STAT activation was a
systemic feature in Crohn's disease.
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In an attempt to address whether the constitutive STAT activation was caused by an autocrine production of cytokines, co-culture experiments were performed using a culture system where cells are separated by diffusible microporous membranes. Different combinations of T cells from Crohn's patients and healthy volunteers had no influence on the constitutive STAT3 activation, indicating that cytokines (and other soluble factors) might not be involved in the constitutive activation of STAT proteins (data not shown). Likewise, repeated washing and starvation for up to 72 h did not inhibit the constitutive activation of STAT3 (data not shown), indicating that exogenous factors are not involved in constitutive STAT activation.
To address whether STAT3 and STAT4 were specifically activated in
Crohn's disease or were a secondary event to chronic inflammation, we
tested the activation of the STAT protein in T cells from the affected
skin of patients with atopic dermatitis and psoriasis. As shown in Fig.
5, STAT proteins were not constitutively
activated in rested cutaneous T cells from patients with atopic
dermatitis and psoriasis (Fig. 5, A, lane 4, and
B, lane 1). However cytokine stimulation induced
profound tyrosine phosphorylation of STAT3 (IL-2 and IL-4) in T cells
from atopic dermatitis (Fig. 5A, lanes 5 and
6). Likewise, in T cells from psoriasis, IL-2 induced
tyrosine phosphorylation of STAT5 (Fig. 5B, lane
2), and IFN- induced tyrosine phosphorylation of STAT1/3/4
(Fig. 5B, lane 4).
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Several tyrosine kinases and oncogenes have been implicated in
the constitutive tyrosine phosphorylation of STAT3 in cancer cells
(11). Because the Janus kinase 3 is involved in the constitutive activation of STAT3 in T cell lymphomas, we addressed whether Jak3
might also be involved in the constitutive tyrosine phosphorylation of
STAT3 in T cells from CD patients. As shown in Fig.
6, STAT3 tyrosine phosphorylation is
almost completely blocked by a selective inhibitor of Jak3 (Fig. 6,
lane 3). In contrast, an inhibitor of the epidermal growth
factor (EGF) receptor tyrosine kinase (which is involved in STAT3
tyrosine phosphorylation in several cancer cells) did not affect the
constitutive STAT3 tyrosine phosphorylation in intestinal T cells from
CD patients (Fig. 6, lane 4) even at concentrations that
block epidermal growth factor-induced STAT3 activation in the IEC cell
line (Ref. 22, and data not shown).
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To address whether the constitutive STAT activation was caused by a deficient negative regulation by tyrosine phosphatases, we performed pulse-chase experiments comparing the kinetics of STAT3 dephosphorylation following treatment with the Jak3 inhibitor. The decrease in phosphotyrosine STAT3 followed the same kinetics in intestinal T cells from Crohn's patients and IL-2 treated intestinal T cells from healthy donors, indicating that the constitutive STAT activation was not caused by a deficiency in the expression or function of tyrosine phosphatases (data not shown). In keeping with these observations, Crohn's T cells did not have a deficient expression of tyrosine phosphatases such as Src homology domain 2 containing inositol 5-phosphatase 1 (SHIP1) (data not shown)
Because STAT3 is a transcription factor regulating the expression of
SOCS3 (9), we addressed whether SOCS3 was expressed in Crohn's
disease. Indeed, SOCS3 was constitutively expressed in intestinal T
cells from CD patients (Fig. 7,
A and B) and in T cell lymphomas (MyLa 2000) with
a constitutively activated STAT3 (Fig. 7A, right
panel). Moreover, the constitutive expression of SOCS3 found
in intestinal CD4+ T cells of CD patients was up-regulated
by IL-2-stimulation for 45 min (Fig. 7B, right
panel). In contrast, SOCS3 was not constitutively expressed in
both rested intestinal T cells from healthy individuals (Fig.
7B, left panel) and rested peripheral
T cells from healthy blood donors (Fig. 7A, left
panel). In rested T cells from healthy individuals, SOCS3
was expressed only upon IL-2-stimulation (Fig. 7, A and
B, left panel), supporting the
hypothesis that STAT3 might trigger a constitutive SOCS3 expression in
intestinal T cells from CD patients.
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DISCUSSION |
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The present data provide the first evidence of a constitutive activation of STAT3 and STAT4 and expression of SOCS3 in T cells from colon mucosa of patients with Crohn's disease. Thus, a selective tyrosine phosphorylation of STAT3 and STAT4 was seen in intestinal T cells from CD patients. In contrast, STAT3 and STAT4 were not constitutively (but inducibly) tyrosine phosphorylated in intestinal T cells from healthy volunteers, suggesting that a constitutive activation of STAT3 and STAT4 might be a specific feature of intestinal T cells from Crohn's disease. In support of this hypothesis, Suzuki et al. have recently shown that STAT3 is tyrosine phosphorylated and SOCS3 expressed in a murine colitis model and in whole colon tissue extracts from ulcerative colitis and Crohn's disease patients (23). In accordance with the present study, Suzuki et al. also reported that STAT1, STAT5, and STAT6 were not constitutively activated in human whole colon tissue extracts, whereas the possible activation of STAT4 was not commented (23). Using experimental animal models, it has been proposed that the STAT4 pathway plays a key a role in chronic intestinal inflammation (24, 25). This notion is supported by the present findings of an aberrant activation of STAT4.
We also investigated whether the aberrant STAT activation was restricted to T cells from the gastrointestinal area. We found a clear constitutive STAT3-activation in peripheral mononuclear cells from patients with Crohn's disease compared with healthy controls, suggesting that disturbances in STAT-activation are a generalized feature of T cells from Crohn's disease patients. Studies are in progress to elucidate whether aberrant STAT activation can be utilized as an early diagnostic or prognostic marker.
To our knowledge, the present study comprises the first example of a constitutive activation of STAT3 and STAT4 in non-transformed human T cells. Indeed, we looked for but were unable to find constitutive STAT activation in rested T cells from the affected skin of patients with atopic dermatitis, chronic eczema (data not shown), and psoriasis. The regulation of tyrosine phosphorylation and dephosphorylation is orchestrated by the coordinated action of phosphatase and protein-tyrosine kinases. Modulation of the function of either phosphatases or protein-tyrosine kinases results in imbalanced activity, leading to altered signal transduction and response. We did not observe any modification of the phosphatase activity in the intestinal T cells from Crohn's disease patients, indicating that the constitutive STAT activation is not caused by a deficiency in negative regulation by tyrosine phosphatases. As mentioned earlier, several human cancers, including T cell lymphomas, have a constitutive activation of STAT3 mediated by known and as yet unidentified tyrosine kinases and oncogenes. Our observation that a selective inhibitor of Jak3 almost completely blocked the constitutive STAT activation suggests that Jak3 is involved in the aberrant STAT activation in CD patients. Interestingly, the Jak3 inhibitor also blocks the constitutive activation of STAT3 in T cell lymphomas (14). Studies are in progress to identify whether Jak3 is mutated or associated with cytokine receptors, tyrosine kinases, and/or oncogenes known to activate STAT3 in tumor cells.
It was a consistent finding that intestinal T cells from CD patients responded weakly to stimulation with exogenous cytokines such as IL-2 and IL-4. Recently, it has become clear that intracellular inhibitors such as SOCS and PIAS proteins negatively regulate cytokine signaling. STAT3 is a transcription factor regulating SOCS3 expression (9), and SOCS3 was constitutively expressed in intestinal T cells from CD patients but not in T cells from healthy volunteers. It is therefore possible that the partial resistance to exogenous cytokines is caused by the constitutive expression of SOCS3 in intestinal T cells from CD patients.
The constitutive expression of SOCS3 in cells that have a constitutive activation of STAT3 indicates that SOCS3 is not able to sufficiently block the tyrosine kinase responsible for tyrosine phosphorylation of STAT3. One possible explanation might be that the expression level is not sufficient to block the kinase. Alternatively, mutations in the kinase, docking proteins/receptors, and/or SOCS3 could explain this apparent paradox. Because deficient expression of another inhibitor of STAT3, PIAS3, has also been linked to the constitutive expression of STAT3 in T lymphomas (26), we also looked for but were unable to find a deficient expression of PIAS3 in CD patients (data not shown). Studies are in progress to address whether PIAS3 and/or SOCS3 are mutated in CD patients.
In conclusion, we provide the first evidence of an aberrant activation
of STAT3, STAT4, and SOCS3 in intestinal T cells from patients with
Crohn's disease. These findings suggest a possible pathogenetic link
between cancer and autoimmunity. Moreover, they open a new battlefield
in the fight of chronic inflammatory bowel disease and possibly other
autoimmune diseases.
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FOOTNOTES |
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* This work was supported by the Danish Colitis Crohn Foundation, the Danish Research Councils, the Danish Biotechnological Center for Cellular Communication, the Danish Biotechnology Program, the Danish Allergy Research Center, the Novo Nordic Foundation, Becketts Fond (founded December 17, 1981), the Danish Medical Associations Research Foundation, the Danish Cancer Research Foundation (Dansk Kræftsforsknings Fond), the Danish Cancer Society (Kræftens Bekæmpelse), Gerda and Aage Haensch's Foundation, and the Danish Rheumatism Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Alfred Benzon Foundation.
To whom correspondence should be addressed: Inst. of Medical
Microbiology and Immunology, Panum 22.5, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen, Denmark. Tel.: 0045-35327879; Fax:
0045-35327876; E-mail: n.odum@immi.ku.dk.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M207999200
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ABBREVIATIONS |
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The abbreviations used are: Jak, Janus kinase; STAT, signal transducers and activators of transcription; SOCS, suppressors of cytokine signaling; PIAS, protein inhibitor of activated STAT; IFN, interferon; TNF, tumor necrosis factor; IL, interleukin; ERK, extracellular signal-regulated kinase; CD, Crohn's disease; EGF, epidermal growth factor.
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