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Am J Physiol Gastrointest Liver Physiol 290: G827-G838, 2006; doi:10.1152/ajpgi.00513.2005
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INFLAMMATION/IMMUNITY/MEDIATORS

IL-22 is increased in active Crohn’s disease and promotes proinflammatory gene expression and intestinal epithelial cell migration

Stephan Brand,1,* Florian Beigel,1,* Torsten Olszak,1 Kathrin Zitzmann,1 Sören T. Eichhorst,1 Jan-Michel Otte,2 Helmut Diepolder,1 Andreas Marquardt,3 Wolfgang Jagla,3 Andreas Popp,3 Stéphane Leclair,3 Karin Herrmann,4 Julia Seiderer,1 Thomas Ochsenkühn,1 Burkhard Göke,1 Christoph J. Auernhammer,1 and Julia Dambacher1

1Department of Medicine II, University-Hospital Munich-Grosshadern, University of Munich, Munich, Germany; 2Department of Medicine I, St. Josef-Hospital, Ruhr-University, Bochum, Germany; 3Ingenium Pharmaceuticals, Martinsried, Germany; and 4Institute of Radiology, University-Hospital Munich-Grosshadern, University of Munich, Munich, Germany

Submitted 1 November 2005 ; accepted in final form 18 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-{alpha}, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-{alpha}, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn’s disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn’s disease and promotes proinflammatory gene expression and IEC migration.

interleukin-10-like cytokines; interleukin-22; defensin

IL-22 WAS ORIGINALLY DESCRIBED as an IL-9-induced gene and was named for IL-10-related T cell-derived inducible factor (IL-TIF) (22). This cytokine shows 22% amino acid identity with IL-10 and belongs to a family of cytokines with limited homology to IL-10, namely IL-10, IL-19, IL-20, IL-24, IL-26, IL-28A, IL-28B, and IL-29 (the latter 3 also known as IFN-{lambda}s). IL-22 binds at the cell surface to a receptor complex composed of two chains belonging to the class II cytokine receptor family (CRF2): IL-22R1 and IL-10R2 (23, 32, 56). The ligand-binding chains for IL-22, IL-26, IL-28A/B, and IL-29 are distinct from that used by IL-10. However, all of these cytokines use a common second chain, IL-10 receptor-2 (IL-10R2; CRF2–4) to assemble their active receptor complexes. The binding of IL-22 to its respective R1 chain induces a conformational change that enables IL-10R2 to interact with the newly formed ligand-receptor complexes. This, in turn, activates a signal-transduction cascade that results in rapid activation of several transcription factors, including STAT proteins in several cell lines such as mesangial cells, lung epithelial cells, melanomas, hepatomas, and keratinocytes (6, 22, 23, 35, 53, 56).

Major sources of IL-22 are activated T and natural killer cells (54). As discovered thus far, IL-22 activities include upregulation of acute-phase reactants in the liver and hepatoma cells (22, 23) as well as induction of pancreatitis-associated protein in pancreatic acinar cells (2), suggesting a role for this cytokine in inflammatory processes. To date, no comprehensive analysis of IL-22-inducible genes has been published; however, several IL-22-inducible genes have been identified including chemokine genes in hepatocytes such as IFN-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), and IL-8 (19).

IL-22Rs are expressed on a number of tissues, including kidney, pancreas, and liver (32). The biological activities induced by IL-22 are only beginning to be defined. For example, a recent study (42) supports a potential therapeutic role for IL-22 as a protective factor in hepatocellular injury.

Although expression of the IL-22 receptor complex has been demonstrated in colonic epithelial cells (38), the regulation of the receptor expression, its detailed signal transduction including its specific biological functions in intestinal epithelial cells (IEC) and role in human gastrointestinal disease still have to be established, which was therefore the aim of this study.

Here, we demonstrate that the expression of IL-22 is upregulated in intestinal inflammation as seen in patients with Crohn’s disease. IL-22 signaling in IEC resulted in increased proinflammatory gene transcription. Importantly, IL-22 promotes the intestinal barrier integrity in vitro through stimulation of IEC migration and defensin expression. Overall, our data indicate a role for this cytokine system in protecting the intestinal barrier by enhancing IEC migration, suggesting an important function in intestinal inflammation and wound healing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Polyclonal antibodies to phosphorylated extracellular signal-regulated kinase (ERK)1/2 (Thr183/Tyr185), phosphorylated stress-activated protein kinase (c-Jun NH2-terminal kinase), SAPK/JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), and phospho-Akt (Ser473) were purchased from Cell Signaling (Beverly, MA). Anti-ERK1/2, anti-SAPK/JNK, anti-p38, and anti-Akt antibodies were also from Cell Signaling. Horseradish peroxidase-linked anti-rabbit secondary antibody was purchased from Amersham (Arlington Heights, IL). Recombinant human IL-22, TNF-{alpha}, and IL-1beta were obtained from R&D Systems (Minneapolis, MN). LPS from Escherichia coli (O26:B6) prepared by phenol extraction was purchased from Sigma (St. Louis, MO) and prepared as dispersed sonicate in endotoxin-free water (Life Technologies, Rockville, MD) before diluting to final concentration in supplemented media. MEK-1 inhibitor PD98059, SAPK/JNK inhibitor SP600125, and phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin were from Tocris Cookson (Bristol, UK).

Cell culture. The human colorectal cancer-derived IEC lines SW480, Caco-2, HT-29, HCT116, and DLD-1 were obtained from American Type Culture Collection (Rockville, MD). Cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO-BRL/Life Technologies, Gaithersburg, MD) with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (PAA, Pasching, Austria) in a humidified 5% CO2 atmosphere at 37°C. For signal-transduction experiments with IL-22, cells were starved overnight in serum-free medium.

RT-PCR. RT-PCR was performed as previously described (13). Briefly, total RNA was isolated using TRIzol reagent (GIBCO-BRL/Life Technologies). For RT-PCR, RNA was treated with ribonuclease (RNase)-free deoxyribonuclease (DNA-free Kit, Ambion) to remove potential genomic DNA contaminants. Three micrograms of total RNA were reverse transcribed using Roche first-strand cDNA synthesis kit. To control for genomic contamination, an identical parallel PCR reaction was performed containing starting material that had not been reverse transcribed. The following conditions were used for semiquantitative PCRs: 25–36 cycles (depending on the specific PCR) of denaturing at 95°C for 45 s, annealing at 61°C for 45 s, extension at 72°C for 45 s. The primers for the PCR reactions are shown in Table 1. The PCR products were subcloned into pCR 2.1 vector (Invitrogen, Carlsbad, CA) and sequenced. Densitometric analysis was performed using software TINA (version 2.10g, Raytest Isotopenmessgeräte, Straubenhardt, Germany).


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  		Table 1. PCR primers used for RT-PCR analysis

 
Quantitative PCR. Real-time PCR was performed with a Rotorgene RG-3000 cycler (Corbett Research, Sydney, Australia) using the Quantitect SYBR Green PCR kit from Qiagen (Hilden, Germany) following the manufacturer’s guidelines. Oligonucleotide primers were designed not to amplify genomic DNA, according to the published sequences, and the following primer pairs were used: human IL-22 forward 5'-gcaggcttgacaagtccaact-3', reverse 5'-gcctccttagccagcatgaa-3'; beta-actin: forward 5'-gccaaccgcgagaagatga-3', reverse 5'-catcacgatgccagtggta-3'; IL-8 forward 5'-ccaggaagaaaccaccgga-3', reverse 5'-gaaatcaggaaggctgccaag-3'; TNF-{alpha} forward 5'-ccaggcagtcagatcatcttctc-3', reverse 5'-agctggttatctctcagctccac-3' (MWG-Biotech, Ebersberg, Germany). IL-22 mRNA expression was normalized to beta-actin expression in the respective cDNA preparation. To compare IL-8 and IL-22 expression levels between inflamed and noninflamed colonic lesions in patients with inflammatory bowel disease (IBD), expression in noninflammatory tissue was arbitrarily set to 1.0.

Signal-transduction experiments, gel electrophoresis and immunoblotting. The signal-transduction experiments were performed in overnight serum-starved intestinal epithelial cell lines as indicated. Cells were stimulated with 100 ng/ml IL-22, unless indicated differently. This concentration was used based on pilot experiments demonstrating a significantly higher effect of 100 ng/ml for the activation of certain kinases and cell migration than lower concentrations. Cells were solubilized in lysis buffer containing 1% Nonidet P-40, 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and phosphatase inhibitors (400 mM sodium orthovanadate and 4 mM NaF) and were passed six times through a 21-gauge needle. After 30 min on ice, lysates were cleared by centrifugation at 10,000 g for 20 min. The protein concentration of each sample was quantified by the Bradford method. Immunoblotting was performed as previously described (37).

ELISA. For the quantification of IL-8 release, BD OptEIA Human IL-8 Elisa kit II (BD Biosciences, Bedford, MA) was used according to the manufacturer’s instructions.

Colonic biopsies. Biopsies were taken from patients with Crohn’s disease and ulcerative colitis undergoing diagnostic colonoscopy. The study was approved by the Ethics Committee of the Medical Faculty of the University of Munich. All participating subjects gave written, informed consent before biopsy sampling. From each patient four biopsies were collected: two from macroscopically noninflamed sites and two from macroscopically inflamed mucosa. IL-22 and IL-8 mRNA levels were measured in each individual biopsy. For quantification, the average IL-22 and IL-8 mRNA expression of the two noninflamed biopsies was compared with the average expression in the two inflamed biopsies. For calculation of the correlation coefficient, for each patient, mRNA expression of IL-22 was correlated to expression of IL-8 mRNA in the four individual biopsies.

Wounding assay. Wounding assays were performed as previously described (18). Briefly, SW480 cells, which were the most suitable human IEC line in pilot experiments, were grown in six-well plates to complete confluence. With the use of a sterile razorblade, eight standardized wounds were created in each plate. Detached cells were removed by three washes with PBS, and the cell medium was changed from 10% FCS-containing medium to 1% FCS-containing medium. The cells were stimulated with IL-22 (10 and 100 ng/ml) or 1% FCS. The cells were washed with PBS after 24 h, and the number of migrated cells (over the wounding edge) was counted under a microscope (Olympus IX50, x10 magnification). For each group (IL-22 stimulated and medium stimulated), three dishes were analyzed, whereas for each dish, eight separate fields were counted containing more than 300 migrated cells per group.

Cell proliferation assay. HT-29 cells were seeded onto 96-well plates at a density of 10,000 cells per well and were allowed to attach overnight. Cells were then stimulated with 10, 100, or 1,000 ng/ml IL-22 or with cytokine-free medium (negative control) for 48 h. The cell proliferation rate was determined by MTS assay on day 2 using the CellTiter 96 aqueous non-radioactive cell-proliferation assay (Promega, Madison, WI) according to the manufacturer’s instructions.

Apoptosis assays. Apoptosis assays were performed as described previously (24). For induction of CD95-mediated cell death, ligand-specific anti-APO-1 mAb at concentrations of 100 and 500 ng/ml was used. Cells were trypsinized and lysed in a hypotonic lysis buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 µg/ml of propidium iodide. After incubation at 4°C overnight, the nuclei were then analyzed for DNA content by flow cytometry.

Dextran sulfate sodium colitis model. C57BL/6 and C3H/HeJ mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Experimental colitis was induced by adding 3% and 4.5% dextran sulfate sodium (DSS; molecular weight 36,100–45,500; TdB Consultancy, Uppsala, Sweden) to the drinking water of C57BL/6 and C3H/HeJ mice, respectively, for 5 days. At day 6, mice were euthanized by CO2 asphyxiation and the large intestine was removed for further analysis. Total RNA of the colon was isolated using Qiagen RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. This study was approved by the Animal Care and Use Committee of the State of Bavaria (Regierung von Oberbayern) following the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Statistical analysis. Statistical analysis was performed using two-tailed Student’s t-test. P values <0.05 were considered as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IEC express the IL-22 receptor complex and IL-22 binding protein. To determine whether the IL-22 receptor complex consisting of IL-10R2 and IL-22R1 is expressed in IEC and to use an IEC model to study this ligand-receptor system, we analyzed IL-10R2 and IL-22R1 mRNA expression in several human IEC lines (Caco-2, DLD-1, SW480, HCT116, and HT-29). RT-PCR analysis demonstrated IL-10R2 and IL-22R1 mRNA expression in all cell lines tested (Fig. 1). Moreover, all cell lines with the exception of SW480 cells expressed mRNA for IL-22 binding protein (IL-22BP). Interestingly, a previously described alternative splicing variant of IL-22BP (21) was found in Caco-2 cells (Fig. 1). However, none of the cell lines analyzed expressed IL-22 (Fig. 1).


Figure 1
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  		Fig. 1. IL-10 receptor 2 (R2), IL-22R1, and IL-22 binding protein (IL-22BP) but not IL-22 are expressed in intestinal epithelial cells (IEC). mRNA expression of IL-10R2, IL-22R1, IL-22BP, and IL-22 as analyzed by RT-PCR analysis of mRNA derived from IEC lines as indicated.

 
IL-22 induces STAT1/3-, ERK1/2-, SAPK/JNK-1/2, and Akt phosphorylation. Having demonstrated that IEC lines express the IL-22 receptor complex, we next analyzed whether this complex is functional in IEC investigating various signaling pathways activated by IL-22. Previous studies (6, 22, 23, 35, 53, 56) in other cell lines reported activation of STAT signaling by IL-22. Therefore, we investigated the influence of IL-22 on phosphorylation levels of STAT1 and STAT3 in IEC. Compared with basal levels of tyrosine phosphorylation of STAT1 in unstimulated controls, tyrosine phosphorylation of STAT1 was clearly induced by 100 ng/ml IL-22 (Fig. 2A). Similarly, IL-22 strongly induced tyrosine phosphorylation of STAT3 with a maximal phosphorylation level after 15–30 min (Fig. 2B).


Figure 2
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  		Fig. 2. IL-22 induces STAT-1 and STAT-3 phosphorylation in IEC. On stimulation of HT-29 cells with IL-22 (100 ng/ml), STAT1 (A) and STAT3 (B) proteins are strongly phosphorylated. One representative experiment (n = 3) is shown.

 
Moreover, IL-22 (100 ng/ml) induced a transient activation of ERK1/2 (Fig. 3A). During the observed time interval, total ERK1 and ERK2 levels remained unchanged (Fig. 3A). To identify upstream signaling events, we investigated the effect of the MEK-1 inhibitor PD98059 on the IL-22-mediated ERK regulation. PD98059 downregulated ERK1/2 phosphorylation after IL-22 stimulation significantly (Fig. 3B), suggesting MEK-1 as an upstream signal transducer of the IL-22-induced ERK activation. Cross talk between the PI3-kinase and the MEK-ERK pathway has been proposed (43). However, ERK activation after IL-22 stimulation was not significantly affected by pretreatment with wortmannin (Fig. 3C), suggesting a PI3-kinase-independent activation of ERK-MAP kinases by IL-22. Similarly, the JNK kinase inhibitor SP600125 did not influence the IL-22-induced ERK activation (Fig. 3C).


Figure 3
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  		Fig. 3. IL-22 activates ERK-MAP kinases in IEC. Activation and expression of phospho-ERK1/2 in HT-29 cells was assessed by immunoblotting. A: phospho-ERK1/2 activation after IL-22 stimulation (100 ng/ml). B: the IL-22-induced ERK activation is MEK-1 dependent. Pretreatment with the MEK-1 inhibitor PD98059 (10 µM 1 h before IL-22 stimulation) resulted in a decreased ERK activation. C: The IL-22-induced ERK activation is SAPK/JNK and phosphatidylinositol 3 (PI3)-kinase independent. Pretreatment with the SAPK/JNK inhibitor SP600125 (20 µM) or the PI3-kinase inhibitor wortmannin (25 µM) did not influence ERK1/2 activation. One representative experiment (n = 3) is shown.

 
Activation of the IL-22 receptor in hepatocytes results in activation of p38 and SAPK/JNK kinases (35). Similarly, stimulation of IEC with IL-22 resulted in the phosphorylation of SAPK/JNK kinases (Fig. 4A), which was significantly suppressed by pretreatment with the JNK inhibitor SP600125 (Fig. 4B). However, stimulation of IEC with IL-22 did not result in a significant phosphorylation of p38 (data not shown). Furthermore, IL-22 binding to its receptor complex also resulted in increased phosphorylation of Akt (Fig. 4C). Pretreatment with the PI3-kinase inhibitor wortmannin caused a complete dephosphorylation of Akt (Fig. 4D).


Figure 4
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  		Fig. 4. IL-22 activates SAPK/JNK MAP kinases and Akt in IEC. A: stimulation of HT-29 cells with IL-22 (100 ng/ml) resulted in increased phosphorylation of SAPK/JNK kinases. B: pretreatment with the SAPK/JNK inhibitor SP600125 (20 µM) decreased SAPK/JNK activation. C: IL-22 induces PI3-kinase-dependent Akt phosphorylation. D: pretreatment with the PI3-kinase inhibitor wortmannin (25 µM) resulted in a complete dephosphorylation of Akt. One representative experiment (n = 3) is shown.

 
IL-22 increases expression of proinflammatory cytokines in IEC. After having established that the IL-22 receptor complex is functional in IEC, we next examined transcriptional targets of this cytokine. First, we analyzed mRNA levels for SOCS-3, which we previously identified as an immediate-early STAT1/3-dependent gene (4). As shown in Fig. 5A, IL-22 treatment induced SOCS-3 mRNA expression with maximal levels detected after 12 h of stimulation.


Figure 5
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  		Fig. 5. IL-22 increases expression of proinflammatory cytokines and SOCS-3 in IEC. A: SOCS-3 mRNA expression after stimulation of HT-29 cells with IL-22 (100 ng/ml). The mRNA expression levels were normalized to GAPDH mRNA levels using TINA software. B: upregulation of IL-8 and TNF-{alpha} mRNA expression after stimulation of HT-29 cells with 100 ng/ml IL-22. TNF-{alpha} and IL-8 mRNA expression was measured by quantitative PCR and normalized with respect to beta-actin expression levels in the respective cDNA preparation. mRNA expression at the start of the experiment (0 h) was arbitrarily set to 1.0. C: IL-8 protein levels increase and reach a maximum after 12 h of IL-22 stimulation. IL-8 protein concentration was determined by ELISA. Rel., relative.

 
Furthermore, previous studies in other cell lines demonstrated that IL-22 increases gene expression of acute phase proteins (19, 38). Therefore, we focused on the proinflammatory cytokines TNF-{alpha} and IL-8, two major mediators of inflammatory responses in IECs, as a downstream readout of IL-22-mediated gene expression. The mRNA expression of both cytokines was significantly increased (TNF-{alpha}: 5.2-fold; IL-8: 27.7-fold) as measured by quantitative PCR (Fig. 5B). Accordingly, IL-8 protein expression measured in ELISA assays increased 4.2-fold after IL-22 stimulation (Fig. 5C).

IL-22R1 mRNA expression is upregulated after stimulation with proinflammatory cytokines. Having established that IL-22 upregulates the expression of proinflammatory cytokines, we next analyzed whether mRNA expression of the IL-22 receptor subunits IL-22R1 and IL-10R2 is regulated by proinflammatory cytokines. Although IL-10R2 is a promiscuous receptor subunit, which is not only used by IL-22 but also by IL-26, IFN-{lambda}s, and the anti-inflammatory cytokine IL-10, signaling through IL-22R1 is restricted to IL-22. As shown in Fig. 6, LPS, IL-1beta, and TNF-{alpha} upregulated IL-22R1 mRNA expression but had no effect on IL-10R2 mRNA expression levels. Maximal induction of IL22R1 was detected after 2–4 h of stimulation.


Figure 6
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  		Fig. 6. IL-22R1 mRNA expression is upregulated after stimulation with various proinflammatory stimuli. Regulation of IL-10R2 and IL-22R1 mRNA expression in HT-29 cells after stimulation with LPS (1 µg/ml; A) with IL-1beta (10 ng/ml; B) and TNF-{alpha} (50 ng/ml; C). The expression levels of IL-10R2 and IL-22R1 mRNA were normalized to GAPDH mRNA levels using TINA software.

 
IL-22 mRNA expression is increased in inflamed colonic mucosa of patients with IBD. Because we have shown that IL-22 upregulates the expression of proinflammatory cytokines and that the expression of the specific receptor subunit IL-22R1 is increased by proinflammatory stimuli, we analyzed its expression in intestinal inflammation in vivo using real-time PCR. In these experiments, we compared IL-22 mRNA expression levels in 80 biopsy samples from 20 patients with IBD (Crohn’s disease: n = 9; ulcerative colitis: n = 11) taken from sites with endoscopically (macroscopic) inflamed colonic mucosa with those of endoscopically noninflamed colonic mucosa taken from the same 20 patients. The IL-8 mRNA expression, which was used as a control marker for inflammation, was significantly increased (P < 0.05) in the inflamed biopsy samples in Crohn’s disease (Table 2, Fig. 7A). The increase in IL-8 mRNA expression ranged from 1.4- to 31-fold compared with the noninflamed tissues. Similarly, IL-22 mRNA expression levels were significantly higher in inflamed colonic biopsy samples than in noninflamed colonic lesions (P < 0.05; increase between 1.4- and 13.6-fold; Table 2, Fig. 7A). Interestingly, the highest IL-22 mRNA levels were found in a patient with severe Crohn’s disease (Fig. 7A, red mark) affecting the ileocecal valve and terminal ileum (Fig. 7C), which also had the highest IL-8 mRNA expression levels (Fig. 7A). Histopathological analysis of the biopsies demonstrated severe mucosal ulceration with massive infiltration of neutrophils and other inflammatory cells (Fig. 7, D and E). Magnetic resonance imaging enteroclysis demonstrated wall thickening of the terminal ileum resulting in ileal stenosis (Fig. 7, F and G).


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  		Table 2. Comparison of IL-22 and IL-8 expression in inflamed and noninflamed colonic lesions of patients with CD and UC

 

Figure 7
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  		Fig. 7. IL-22 is upregulated in inflammatory bowel disease. IL-22 mRNA expression in colonic and ileal biopsies correlated with IL-8 mRNA expression in Crohn’s disease (A) and ulcerative colitis (B). The highest IL-22 mRNA levels of all patients examined in this study (A; Table 2) were found in a patient with severe Crohn’s disease (A; see arrow) particularly affecting the ileocecal valve and terminal ileum (C), which had also the highest IL-8 mRNA expression levels (A). Histopathological analysis of the biopsies demonstrated severe inflammation with extensive mucosal ulcerations and massive infiltration of neutrophils (D and E). Magnetic resonance imaging enteroclysis demonstrated wall thickening of the terminal ileum and ileal stenosis (F and G).

 
In patients with ulcerative colitis, the average IL-8 and IL-22 expression levels in inflamed tissues were lower compared with expression levels in patients with Crohn’s disease (Table 2, Fig. 7, A and B). Although IL-8 expression was significantly upregulated to eightfold in the inflamed lesions of patients with ulcerative colitis (P = 0.02), the difference of IL-22 mRNA expression in inflamed and noninflamed biopsies did not reach statistical significance (P = 0.09). Interestingly, IL-22 was downregulated in 5 of 11 patients with ulcerative colitis, with 3 of these patients also having low IL-8 expression levels (Table 2, Fig. 7B).

Overall, the upregulation of IL-22 expression was more pronounced in inflamed lesions of patients with Crohn’s disease compared with patients with ulcerative colitis (3.65- vs. 1.84-fold), confirming its role in TH1-mediated inflammation (54). Moreover, the IL-22 mRNA levels correlated highly with the IL-8 mRNA expression levels in patients with Crohn’s disease (r = 0.840, Table 2). In eight of nine samples, the correlation coefficient was even higher than 0.90. In patients with ulcerative colitis, the observed correlation between IL-22 and IL-8 mRNA expression was lower (0.598; Table 2), and only 4 of 11 patients showed correlation coefficients >0.90.

IL-22 mRNA expression is increased in murine DSS colitis. Next, we studied the IL-22 mRNA expression levels in intestinal inflammation in vivo in the acute phase of colitis in the murine DSS colitis model. As shown in Fig. 8, 6 days after DSS treatment, IL-22 was among the genes most strongly upregulated, whereas it was almost undetectable in untreated mice. This effect was more pronounced in C3H/HeJ mice than in C57BL/6 mice. The IL-22 mRNA expression correlated with the expression of other proinflammatory cytokines and chemokines such as IL-6 and MIP-2{alpha} (Fig. 8).


Figure 8
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  		Fig. 8. IL-22 mRNA expression is upregulated in murine dextran sulfate sodium (DSS) colitis. C57BL/6 and C3H/FeJ mice were stimulated with 3% and 4.5% DSS, respectively, added to the drinking water for 5 days. Six days (d6) after DSS treatment, mice were euthanized and colonic gene expression was determined by semiquantitative RT-PCR as described in MATERIALS AND METHODS using the primers listed in Table 1. The PCR for actin was performed with 28 PCR cycles, whereas for all other genes, 36 cycles were used. IL-22 mRNA expression was calculated relative to actin mRNA expression in the respective cDNA preparation using TINA software. Control mice received no treatment ("healthy").

 
IL-22 induces intestinal epithelial restitution and promotes intestinal barrier integrity in vitro. Having established that IL-22 plays a role in mucosal injury in vivo, we next analyzed whether IL-22 itself promotes IEC injury or is a counterregulatory cytokine released to promote wound healing. This was analyzed in previously established IEC restitution assays (11). In these "wounding assays," standardized, sterile wounds were created in SW480 cells, which were shown to be the most suitable human IEC line for migration experiments, forming evenly distributed monolayers in pilot experiments (11). Twenty-four hours after wounding, the number of migrated cells was counted under the microscope. This experiment demonstrated a highly significant, dose-dependent 290% increase of the cell migration rate in the IL-22-stimulated cells (P = 0.00000002 for 10 ng/ml and P = 0.00000006 for 100 ng/ml IL-22 vs. unstimulated controls, Fig. 9A).


Figure 9
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  		Fig. 9. IL-22 promotes intestinal barrier integrity in vitro by enhancing IEC restitution, cell proliferation, and human beta-defensin-2 expression. A: "wounding" assays were used to analyze the influence of IL-22 on IEC migration. IL-22 (10 and 100 ng/ml) induced a significant increase of the cell migration rate (*P < 0.00001 vs. unstimulated control). B: preincubation with the PI3-kinase inhibitor wortmannin (25 µM) inhibited IL-22-mediated IEC migration, whereas the MEK-1 inhibitor PD98059 (10 µM) did not affect the IL-22-induced cell migration (*P < 0.00001; **P = 0.002 vs. unstimulated control). C: IL-22 does not influence Fas ligand-mediated apoptosis in SW480 cells. Cells were treated with 100 and 500 ng/ml anti-APO-1 antibody and either IL-22 stimulated or unstimulated for 24 h. Apoptosis was measured by FACS analysis. D: low doses of IL-22 increased cell proliferation in HT-29 cells. HT-29 cells were seeded onto 96-well plates at a density of 10,000 cells per well and were grown for 1 day. After starvation in serum-free medium overnight, the cells were stimulated with IL-22 as indicated or with cytokine-free serum-free medium (negative control). The cell proliferation rate was determined by MTS assay after 48 h. With lower doses of IL-22 (10 and 100 ng/ml), there was a statistical significant increase in cell proliferation whereas on stimulation with high doses of IL-22 (1,000 ng/ml) a decrease of cell proliferation was observed (*P < 0.005; **P = 0.07 vs. unstimulated control). E: after stimulation with IL-22 (100 ng/ml), human beta-defensin-2 (hBD-2) mRNA is upregulated 6-fold in HT-29 cells as determined by semiquantitative PCR. F: densitometric analysis of the results shown in E.

 
IL-22 promotes intestinal barrier integrity by PI3-kinase-dependent mechanisms and through increased IEC proliferation and beta-defensin-2 production. Next, we analyzed the mechanisms by which IL-22 promotes IEC restitution and intestinal barrier integrity. The activation of MAP kinases such as ERK1/2 and the activation of Akt have been linked to cell migration (5, 48). Therefore, we repeated the restitution assays using a specific MEK-1 inhibitor (PD98059) and the PI3-kinase inhibitor wortmannin. The inhibition of IL-22-induced Akt activation through pretreatment with wortmannin significantly decreased IL-22-mediated cell migration (P < 0.00001 compared with cells stimulated with IL-22 only; Fig. 9B). In contrast, the inhibition of ERK activation using the MEK-1 inhibitor PD98059 did not block IL-22-mediated cell migration (Fig. 9B; P < 0.00001 compared with unstimulated cells, P = 0.19 compared with cells stimulated with IL-22 only), suggesting that IL-22-induced cell migration is PI3-kinase but not MEK-1 dependent. Furthermore, we also analyzed whether the IL-22-dependent IEC restitution is caused by increased cell proliferation or decreased apoptosis, particularly because ERK1/2 and Akt activation have also been shown to mediate antiapoptotic pathways and to increase cell proliferation (20, 25). Therefore, we investigated the IL-22-mediated effect on apoptosis using previously established experimental conditions (24). In these experiments, SW480 cells were used, which are less resistant to Fas-induced apoptosis than HT-29 cells (1). However, no significant difference between the number of apoptotic cells in the IL-22-stimulated group and the unstimulated group was found (Fig. 9C). In contrast, IL-22 at concentrations of 10 and 100 ng/ml significantly increased cell proliferation (P = 0.002 and P = 0.001, respectively), whereas there was a trend for an antiproliferative effect using higher IL-22 concentrations (P = 0.07, Fig. 9D).

Finally, intestinal barrier integrity is also mediated by the expression of "barrier protective" proteins such as defensins. Therefore, we analyzed whether IL-22 regulates human beta-defensin-2 (hBD-2) expression in IEC. As demonstrated in Fig. 9E, IL-22 upregulated hBD-2 mRNA expression in the IEC line HT-29 up to sixfold.


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IBD, such as Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract. Although the etiology is incompletely understood, initiation and aggravation of the inflammatory process seem to be due to a massive local mucosal immune response. IL-10 is a regulatory cytokine that inhibits both antigen presentation and subsequent proinflammatory cytokine release. IL-10-deficient mice spontaneously develop severe intestinal inflammation (34). Therefore, the use of IL-10 has been proposed as anti-inflammatory biological therapy in chronic IBD (36).

In contrast, we showed in this study that the novel IL-10-related cytokine IL-22, which shares with IL-10 the IL-10R2 subunit for signaling, has proinflammatory functions in IEC and is upregulated in Crohn’s disease. Moreover, we demonstrated that the IL-22 receptor complex consisting of IL-22R1 and IL-10R2 is expressed in IEC and is functional in these cells. After stimulation with IL-22, MAP kinases, Akt, and STAT proteins are activated resulting in an increased expression of proinflammatory cytokines and hBD-2. An increased hBD-2 expression after IL-22 stimulation has also been demonstrated in keratinocytes (55), suggesting that IL-22 increases the innate immunity of epithelial tissues such as skin and intestine. Interestingly, an increased mRNA expression of hBD-2 has been recently described in inflamed colonic lesions of patients with Crohn’s disease (51). This is consistent with the increased IL-22 mRNA expression in inflamed colonic lesions in Crohn’s disease found in this study. The IL-22 mRNA expression correlated highly with the IL-8 mRNA expression (r = 0.840). It has been shown that activated T cells are major sources of IL-22 (54). Polarization of T cells toward the type 1 (TH1) phenotype further increases the activation-induced IL-22 expression, whereas polarization toward type 2 (TH2) reduces it (54). Interestingly, it has been proposed that Crohn’s disease represents a TH1-mediated intestinal inflammation, whereas ulcerative colitis resembles more a TH2-mediated colitis (7). Consistent with this observation, IL-22 expression was significantly increased in inflamed lesions in Crohn’s disease but overall only slightly elevated in active ulcerative colitis, further illustrating the potential importance of IL-22 in the pathogenesis of Crohn’s disease. These results are supported by a very recent study that also demonstrated increased IL-22 expression in IBDs, particularly in Crohn’s disease (3). Furthermore, this study characterized IL-22R1-expressing subepithelial myofibroblasts as additional targets of IL-22 in the intestine (3).

PCR analysis revealed that the mRNA expression of the IL-22 specific subunit of the receptor complex (IL-22R1), but not the promiscuous IL-10R2 subunit, which is also part of the anti-inflammatory IL-10 receptor complex, is under transcriptional control of proinflammatory cytokines. Moreover, IL-22 increased IL-8 mRNA and protein expression in IEC. Similarly, IL-22 upregulated expression of several chemokine genes in hepatocytes, including IL-8, IP-10, and MCP-1 (19). In addition to liver (19) and intestine, proinflammatory properties of IL-22 were also reported in the skin (6, 55), lung (53), and pancreas (2, 28). Hence, consistent with the IL-22R1 receptor expression, epithelial tissues seem to be a preferred target of IL-22. For example, IL-22 overexpression in mice causes neonatal lethality with skin abnormalities reminiscent of psoriatic lesions in humans (17).

Furthermore, we demonstrated that IEC also express mRNA for IL-22BP, which specifically binds IL-22 and does not bind other IL-10-related cytokines (21, 33). This suggests that IEC may regulate the intensity of IL-22 signaling by differential expression of IL-22BP. This is further supported by their ability to secrete anti-inflammatory IL-10 on IL-22 stimulation as demonstrated in a recent study (38).

In this study, IL-22 activated STAT1 and STAT3, which resulted in increased SOCS-3 mRNA expression in IEC, confirming our previous results that SOCS-3 is a transcriptional target of STAT1/3 (4). This is also in agreement with increased SOCS-3 mRNA in a hepatoma cell line following IL-22 stimulation (33) and a very recent study demonstrating STAT1/3 activation after stimulation with IL-22 in the colonic epithelial cell line Colo205 (38). In this study, IL-22 signaling could be inhibited by IL-22BP and a neutralizing antibody against IL-10R2 (38). Interestingly, colonic tissue samples of patients with Crohn’s disease demonstrated increased STAT1 phosphorylation levels and, compared with samples taken from ulcerative colitis patients, increased SOCS-3 levels (44) further underlining the proinflammatory properties of IL-22 in Crohn’s disease. Interestingly, in a murine colitis model SOCS-3 has been demonstrated to play a negative regulatory role in STAT3 activation and intestinal inflammation (46). Moreover, we recently demonstrated that signaling of other IL-10-like cytokines such as IL-28A and IL-29 is abrogated by increased expression of SOCS proteins (15), suggesting that signaling of IL-10-like cytokines is regulated by SOCS proteins. Interestingly, we found a similar mechanism for the signaling mediated by IFN-{alpha} (49).

In addition, IL-22 activates ERK and SAPK/JNK MAP kinases in IEC, which is similar to the signaling described for IL-22 in hepatic cells (35). However, in contrast to IL-22 signaling in hepatocytes (35), IL-22 did not significantly alter the phosphorylation levels of p38 MAP kinases in IEC. Particularly, signaling via SAPK/JNK in IEC has gained interest because two recent studies demonstrated that SAPK/JNK is activated in Crohn’s disease (30, 50) and that inhibition of SAPK/JNK resulted in significant clinical benefit and rapid mucosal healing (30). In this study, the IL-22-mediated ERK activation was MEK-1 dependent, whereas the activation of Akt was entirely dependent on PI3-kinase.

Importantly, the activation of ERK-MAP kinases and Akt has been implicated in cell migration (5, 11, 27, 45, 57). Similarly, our experiments demonstrated that IL-22-receptor activation results in increased IEC migration and epithelial wound healing, which could be blocked using a PI3-kinase inhibitor. Generally, the integrity of the intestinal mucosal surface barrier is rapidly reestablished even after extensive destruction because of an enormous regenerative capability of the mucosal surface epithelium. As demonstrated in wounding assays in this study, IL-22 stimulation may facilitate this epithelial restitution.

The IL-22-mediated barrier integrity in the IEC wounding assays was partly due to an increased cell proliferation rate. Interestingly, only low IL-22 concentrations (10 and 100 ng/ml) increased the IEC proliferation rate, whereas high doses (1,000 ng/ml) decreased cell proliferation. This inverted U-shaped dose-response curve is similar to the biological response observed for other cell migration-mediating cytokines such as the chemokine CXCL12 (41). We recently demonstrated similar antiproliferative properties for high doses of IFN-{lambda}s in IEC and hepatic cells, which also belong to the IL-10-like cytokine family (8–10). However, similar to IFN-{lambda}s (9, 10), no effect on cell apoptosis could be demonstrated for IL-22 in IEC. This is in contrast to studies in the hepatic cell line HepG2, where stable overexpression of IL-22 induced the expression of several antiapoptotic genes including Bcl-2, Bcl-x, and Mcl-1 (42).

There is also increasing evidence that Crohn’s disease is a polygenic disease with several genes being involved in its pathogenesis. Interestingly, CARD15/NOD2, the first susceptibility gene of Crohn’s disease (29, 31, 40), has been linked to increased intestinal permeability (16) and diminished defensin production (52). Although our in vitro data demonstrated that IL-22 increases defensin expression and promotes the integrity of the intestinal barrier, additional in vivo experiments are necessary to clarify the role of IL-22 in intestinal inflammation.

Recently, we demonstrated that in addition to CARD15/NOD2, there are several other genes [e.g., Toll-like receptor 4 (14), organic cation transporter cluster (47), and fractalkine receptor (CX3CR1) polymorphisms (12)] involved in the pathogenesis of Crohn’s disease. The high expression of some of these receptor proteins in monocytes and dendritic cells (DC) suggests a central role of these cell populations in the pathogenesis of Crohn’s disease. As recently demonstrated by us and others, DCs are able to sample bacteria from the intestinal lumen particularly in the ileum (39). Although IL-22 is not expressed by DCs, a very recent study demonstrated that a particular DC subset, which expresses Nectinlike protein-2, regulates IL-22 expression in activated CD8(+) T cells (26).

In summary, we demonstrated that IEC express the IL-22-receptor complex. Binding of IL-22 to its surface receptor in IEC leads to phosphorylation of STAT1/3, Akt, ERK-, and SAPK/JNK MAP kinases. In addition, IL-22 upregulated the mRNA expression of proinflammatory cytokines and of hBD-2. IL-22 also increased IEC migration but had no effect on apoptosis. Moreover, the mRNA expression of IL-22 is upregulated in inflamed colonic lesions in patients with Crohn’s disease. Taken together, our data indicate a role for this cytokine in promoting proinflammatory gene transcription and IEC migration, suggesting an important function in intestinal inflammation and wound healing.


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This study was supported by grants of the Deutsche Forschungsgemeinschaft (BR 1912/3–1 and 5–1), Else-Kröner-Fresenius-Stiftung, and Friedrich-Baur-Stiftung.


   ACKNOWLEDGMENTS
 
We thank Z. Sisic, G. Spöttl, and J. Meinecke (all University of Munich) for excellent technical support.

This work contains parts of the unpublished doctoral theses of F. Beigel and J. Dambacher at Ludwig-Maximilians-University Munich, Germany.

Parts of this paper were presented as an oral presentation at the Annual Meeting of the American Gastroenterological Association and Digestive Disease Week (Chicago, May 14–19, 2005) and have been published in abstract form. Additional parts were presented at the United European Gastroenterology Week (Copenhagen, October 15–19, 2005) as an oral presentation and have been published in abstract form.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Brand, Univ.-Hospital Munich-Grosshadern, Dept. of Medicine II, Univ. of Munich, Marchioninistr. 15, 81377 Munich, Germany (e-mail: stephan.brand{at}med.uni-muenchen.de)

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.

* S. Brand and F. Beigel contributed equally to this work. Back


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