1. Volume 132, Issue 3, Pages 994-1008 (March 2007)
Inhibition of TGF-β Signaling by IL-15: A New Role for IL-15 in the Loss of Immune Homeostasis in Celiac Disease 
Mélika Benahmed, Bertrand Meresse, Bertrand Arnulf, Ullah Barbe, Jean–Jacques Mention, Virginie Verkarre, Matthieu Allez, Christophe Cellier, Olivier Hermine, Nadine Cerf–Bensussan 
Gastroenterology 
Volume 132, Issue 3, Pages (March 2007)
DOI: /j.gastro
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2. Figure 1
IL-15 inhibits TGF-β signaling in human T-cell lymphocytes. (A, B) Lymphocytes were stimulated with IL-2 (300 IU/mL) or IL-15 (10 ng/mL) in the absence or presence of TGF-β1 (10 ng/mL). Proliferative responses were assessed by [3H] thymidine uptake in triplicate. (A) PBMC cultured for 24 hours to 96 hours (results representative of 5 independent experiments). (B) PBMC, IEL, and LPL from controls stimulated for 96 hours (results of 3 to 4 independent experiments). Bars indicate means ± SD. *P < .05 when compared with the condition without TGF-β. (C) PBMC cultured in the presence or not of IL-15 (10 ng/mL) for 24 hours (upper panel) or 96 hours (lower panel) were stimulated by TGF-β1 (10 ng/mL) for 45 minutes. Nuclear extracts were analyzed by electromobility shift assay using a 32P-labeled probe derived from PAI-1 promoter. Fifty molar excess of nonradiolabeled PAI-1 promoter was added as a specific competitor (cold probe). (D) Peripheral CD3+ and CD8+ lymphocytes from controls were cultured in medium added or not with IL-15 (10 ng/mL) for 1, 2, 6, and 24 hours and then stimulated with 10 ng/mL TGF-β1 for 2 hours. TGIF mRNA levels were quantified by real-time PCR. Results are expressed in arbitrary units and are representative of 3 independent experiments. (Asterisks indicate that inhibition is significant at 1,2,6, and 24 hours). (E) Peripheral CD3+, CD4+, and CD8+ lymphocytes, IEL, and LPL from controls were cultured in medium added or not with IL-15 (10 ng/mL) for 24 hours and/or 96 hours and then stimulated with 10 ng/mL TGF-β1 for 2 hours. TGIF mRNA levels were quantified as in D. Results are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
IL-15 acts downstream from Smad3 nuclear translocation. (A) PBMC were cultured for 24 hours to 96 hours in medium added or not with IL-15 (10 ng/mL). Membrane expression of TGF-β RII was monitored by flow cytometry. Results are representative of 3 independent experiments. (B) Messenger RNA expression of Smad7 was monitored by real-time PCR in PBMC cultured for 2 hours to 96 hours in the presence or not of IL-15 (10 ng/mL) or TGF-β1 (10 ng/mL). Results are representative of 2 independent experiments. (C) Peripheral CD3+, CD4+, and CD8+ lymphocytes, IEL, and LPL from controls were cultured in medium added or not with IL-15 (10 ng/mL) for 24 hours and/or 96 hours and then stimulated with 10 ng/mL TGF-β1 for 2 hours. Smad7 mRNA levels were quantified by real-time PCR. Results expressed are representative of 3 independent experiments. (D) Immunocytochemical staining of Smad3 in peripheral CD3+ T cells stimulated overnight with TGF-β1 (10 ng/mL) after a 5-day culture in the presence or not of IL-15 (10 ng/mL). Percentage of cells with nuclear staining was determined among 500 cells. In 3 independent experiments, TGF-β-induced nuclear translocation of Smad3 was comparable in lymphocytes cultured in medium alone or in the presence of IL-15 (85%–99%). Nuclear translocation was observed in <2% of lymphocytes cultured in medium alone but in 25%–35% of lymphocytes cultured in IL-15, likely due to the induction of an endogenous production of TGF-β in the latter cells (data not shown). Note that IL-15-activated T cells become enlarged blast cells, while T cells cultured in control medium remain small and round.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
IL-15 induces a long-lasting expression of phospho-c-jun in peripheral and intestinal lymphocytes. (A) Peripheral lymphocytes were cultured with IL-15 (10 ng/mL) or IL-2 (300 IU/mL) for 30 minutes to 96 hours. JNK kinase assay was performed on cell extracts precipitated with GST-c-jun as described in Materials and Methods, and proteins were separated by 10% SDS-PAGE (upper panel). Loading control was obtained by running an identically loaded SDS-polyacrylamide gel that was stained by Coomassie blue (lower panel). (B–D) Peripheral and intestinal lymphocytes were cultured with IL-15 (10 ng/mL) or IL-2 (300 IU/mL) for 10 days. Intracellular staining of phospho-c-jun (p-c-jun) was monitored by flow cytometry. (B) Histogram analysis of intracellular staining of phospho-c-jun in the lymphocyte gate at the indicated time points. Isotype control was identical to unstimulated PBL (data not shown). (C) Representation of the increase of phospho-c-jun median fluorescence in PBMC stimulated for 10 days. Results were pooled from 3 independent experiments. Bars indicate mean ±SD. (D) Increase of phospho-c-jun median fluorescence after a 96-hour culture is compared in different subsets of peripheral and intestinal lymphocytes. Results are the mean value (±SD) of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Antisense oligonucleotides for c-jun restore TGF-β effects in lymphocytes cultured with IL-15. Lymphocytes were cultured in the presence of cytokines for 96 hours. Sense or antisense c-jun oligonucleotides (25 μg/mL) were added every 24 hours to the cultures. (A) Histogram analysis of intracellular phospho-c-jun (p-c-jun) expression in PBMC at 96 hours. (B) Proliferative responses of PBMC after a 96-hour culture in the presence of the indicated reagents. Oligonucleotides were added at 24 hours. Results are expressed as mean cpm of 3H thymidine incorporation (±SD) and are representative of 3 independent experiments. (C) TGIF and Smad7 mRNA induction was quantified by real-time PCR in peripheral CD8+ lymphocytes stimulated or not with TGF-β1 (10 ng/mL) for 2 hours after a 5-day culture in the indicated conditions. Results, expressed in arbitrary units, are from 2 independent experiments. *P < .05 when compared with the condition without IL-15.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Intestinal lymphocytes from active CD have defective Smad-dependent response to TGF-β. (A) Expression of TGF-β1 was analyzed by immunohistochemistry (left panel) and immunoblotting (right panel) in duodenal biopsies of 5 patients with active CD (ACD) and of 5 controls. Results of the immunoblots are shown in the upper right panel for 2 active CD patients and 2 controls. The ratios of TGF-β1 to β-actin assessed by densitometry were comparable between patients and controls (bars indicate the median value). (B) Messenger RNA expression of TTP was quantified by real-time PCR in duodenal samples of 13 active CD and 8 controls. (C) IEL and LPL were isolated from active CD and controls and stimulated for 2 hours with TGF-β1 (10 ng/mL). TGIF and Smad7 mRNA levels were quantified by real-time PCR. Results are expressed in arbitrary units. Bars indicate the median values. *P < .05 compared with controls.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
The early steps of TGF-β-mediated signaling are not impaired in situ in active CD. (A) Messenger RNA expression of Smad7 was quantified by real-time PCR in duodenal samples of 13 active CD (ACD) and 8 controls and expressed as arbitrary units (AU). *P < .05 when compared with controls. (B) Expression of Smad7 was analyzed by immunoblotting in total extracts from 5 active CD patients and 4 controls. The ratios of Smad7 to β-actin assessed by densitometry (right panel) were comparable between patients and controls (bars indicate the median value of arbitrary units). Results of the immunoblot are shown in the left panel for 4 active CD patients and 3 controls. (C) Expression of phospho-Smad2/3 was analyzed by immunoblotting in total extracts from 4 active CD patients and 4 controls. The ratios of phospho-Smad2/3 to β-actin assessed by densitometry (right panel) were comparable between patients and controls. (D) Phospho-Smad2/3 distribution was studied by immunohistochemistry in intestinal biopsies of 4 patients with active CD. Staining is shown at 100× (left panel) and 200× (right panel) magnifications. Arrows show positive nuclei in lamina propria and epithelium.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Phospho-c-jun is up-regulated in intestinal lymphocytes in active celiac disease. (A) Small intestinal sections from 4 active CD (ACD) and 4 controls were stained with antiphospho-c-jun antibody (10 μg/mL). Immunohistochemical staining is shown at 100× (left panel) and at 400× magnification (right panel). (B) IEL and LPL were isolated from active CD and controls. Intracellular staining of phospho-c-jun (p-c-jun) was studied by flow cytometry (results representative of 3–4 experiments).
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Effect of c-jun antisense and anti-IL-15 antibody ex vivo in duodenal biopsies from patients with active CD. Duodenal biopsies were cultured for 24–36 hours with medium containing sense or antisense c-jun oligonucleotides (50 μg/mL), monoclonal anti-IL-15 antibody or control mouse IgG1 (20 μg/mL) in the absence or presence of 10 ng/mL exogenous TGF-β added 3 hours before the end of the culture. (A) Immunohistochemical staining of organ cultures (OC) with antiphospho-c-jun antibody. (B) Quantification by real-time PCR of TTP, Smad7, or IFN-γ mRNAs expression in duplicate biopsies after incubation in the presence of the indicated reagents. Results, expressed as relative induction, are means (±SD) of 3 to 4 independent experiments. *P < .05 when compared with the control condition.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions