1. Volume 141, Issue 6, Pages 2026-2038 (December 2011)
Antibodies Against Tumor Necrosis Factor (TNF) Induce T-Cell Apoptosis in Patients With Inflammatory Bowel Diseases via TNF Receptor 2 and Intestinal CD14+ Macrophages 
Raja Atreya, Michael Zimmer, Brigitte Bartsch, Maximilian J. Waldner, Imke Atreya, Helmut Neumann, Kai Hildner, Arthur Hoffman, Ralf Kiesslich, Andreas D. Rink, Tilman T. Rau, Stefan Rose–John, Hermann Kessler, Jan Schmidt, Markus F. Neurath 
Gastroenterology 
Volume 141, Issue 6, Pages (December 2011)
DOI: /j.gastro
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2. Figure 1
Effects of anti-TNF agents on apoptosis in CD14+ blood monocytes. CD14+ and CD4+ cells were isolated from PBMCs, cultured for 24 hours, and untreated or treated with anti-TNF antibodies (10 μg/mL). Induction of apoptosis was analyzed by FACS after annexin and PI staining. (A) Induction of annexin-positive/PI-negative (lower right quadrant) and annexin-positive/PI-positive (upper right quadrant) cells after anti-TNF treatment is shown for one representative FACS plot. Statistical analysis of the induction of apoptosis in (B) CD14+, (C) CD4+, and (D) cocultured CD4/CD14+ PBMCs on treatment with the anti-TNF antibodies in the different patient groups (n = 8–15). Baseline apoptosis was 15%–30%. Data represent mean ± SEM. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Significant up-regulation of mTNF in intestinal CD14+ macrophages and TNFR2 in intestinal CD4+ T cells in patients with IBD. (A) Representative immunofluorescence staining for mTNF with FITC-labeled adalimumab (green) and CD11b, CD14, or CD4 (all red) in colonic cryosections from patients with CD. Double-positive cells in yellow (arrows). Confocal microscopy with original magnification 200× to 400×. (B) Significantly more CD4+ and CD11b+ than CD14+ cells in all patient groups. (C) Higher percentage of mTNF-positive cells among intestinal APCs compared with CD4+ T cells in all patients (n = 7–11). (D) Marked up-regulation of mTNF expression in mucosal CD14+ compared with CD4+ cells of patients with IBD was found by FACS analysis. One representative experiment out of 4 is shown. (E) Significantly higher TNFR2 expression in mucosal CD4+ T cells of patients with IBD compared with controls, as analyzed by FACS (n = 7). (F) Significant up-regulation of TNFR2 mRNA, in relation to CD4, in inflamed versus noninflamed mucosa in the same patient with IBD, quantified by qRT-PCR (n = 14). Data represent mean values ± SEM. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Significant up-regulation of TRAF2 and NF-κB in mucosal T cells from patients with IBD. CD4+ T cells from gut specimens of patients with IBD and controls were stimulated for 4 hours with phytohemagglutinin/ionomycin. (A) Cell lysates were analyzed by immunoblot analysis. One representative experiment is shown. Densitometric quantification revealed significant up-regulation of TRAF2 expression in CD4+ T cells from patients with IBD (n = 15–18). (B) Significant up-regulation of TRAF2 mRNA in inflamed versus noninflamed mucosa taken from the same patient with IBD as quantified by qRT-PCR. TRAF2 expression levels are shown in relation to CD4 mRNA expression (n = 15). (C) Nuclear extracts of stimulated gut CD4+ T cells were analyzed by electrophoretic mobility shift assay using a radiolabeled NF-κB consensus DNA binding site. Densitometric quantification of NF-κB activity revealed heightened expression in CD4+ T cells from patients with IBD compared with controls (n = 9). (D) Significant NF-κB up-regulation in nuclear proteins of unstimulated mucosal CD4+ T cells from patients with IBD (n = 18) compared with controls (n = 9), as analyzed by enzyme-linked immunosorbent assay. (E) Marked up-regulation of NF-κBp65 mRNA in relation to CD4 in inflamed versus noninflamed mucosa in the same patient with IBD, quantified by qRT-PCR (n = 13). Data represent mean values ± SEM. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Significant induction of apoptosis in coincubated CD4+ T cells/CD14+ macrophages from patients with IBD but not controls on treatment with clinically effective anti-TNF antibodies in vitro. Analysis of apoptosis induction on anti-TNF antibody treatment in mucosal CD4+ and CD14+ cells from control patients and patients with IBD. Cells were cultured together or separately from each other (24 hours), stimulated with lipopolysaccharide, and treated with the anti-TNF antibodies or IgG control. Induction of apoptosis was analyzed by FACS after annexin/PI staining. (A) There was no significant induction of annexin-positive CD4+/CD14+ cells from control patients after anti-TNF antibody treatment (n = 3–7). (B) Statistical analysis of the induction of annexin-positive CD4+/CD14+ cells from patients with IBD after anti-TNF treatment compared with baseline levels (n = 3–12). With the exception of adalimumab in CD4+ T cells, there was no significant induction of apoptosis in mono-cultured CD14+ or CD4+ cells. However, there was a significant induction of apoptosis on treatment with all anti-TNF antibodies except etanercept in cocultured CD4+ and CD14+ cells. Baseline apoptosis was 20%–45%. Data represent mean values ± SEM. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Anti-TNF antibody treatment with infliximab and adalimumab leads to the induction of apoptosis in lamina propria CD4+ T cells in patients with IBD: in vivo and ex vivo evidence. (A) LPMCs of patients with IBD were isolated from intestinal biopsy specimens, cocultured for 24 hours, stimulated with lipopolysaccharide, and treated with an anti-TNFR1 or anti-TNFR2 antibody (10 μg/mL). Induction of apoptosis was analyzed after annexin/PI staining. There was a significant induction of apoptotic cells from patients with IBD on treatment with the anti-TNFR2 but not with the anti-TNFR1 antibody compared with baseline levels (n = 15). (B) Mucosal CD4+ and CD14+ cells from patients with IBD were stimulated with lipopolysaccharide and treated with 10 μg/mL infliximab or IgG control for 24 hours. Induction of apoptosis in CD4+ T cells was determined via FACS by double staining with annexin. Data represent one representative experiment. (C) CD4+TNFR2+ and CD4+TNFR2− T cells were cocultured with CD14+ LPMCs from patients with IBD, stimulated with lipopolysaccharide, and treated with an anti-TNF antibody for 24 hours. There was a significant induction of apoptosis on anti-TNF treatment in coincubated CD4+TNFR2+ T cells versus CD4+TNFR2− T cells when cocultured with CD14+ macrophages. Baseline apoptosis was 35%–53%. (D) Immunofluorescence staining for CD4 (red) and caspase (green) in sections from intestinal biopsy specimens obtained from the same area before and 4 weeks after treatment with infliximab or adalimumab in the same patient with CD. Double-positive cells appear yellow (original magnification 200× to 600×). (E and F) There is significant induction of apoptosis in LPMCs and CD4+ T cells in CD after anti-TNF treatment (n = 6). Data represent mean values ± SEM. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Inhibition of IL-6 or STAT3 synergizes with anti-TNF induced apoptosis in cocultured CD4+/CD14+ cells. (A) LPMCs were isolated from patients with IBD and control patients and stimulated with anti-CD2/CD28 for 48 hours. IL-6 concentration in the supernatant was significantly higher in patients with IBD (n = 10–14). (B) Intestinal CD4+ and CD14+ cells from patients with IBD were cultured together for 24 hours and treated with IgG or infliximab (10 μg/mL) in the presence or absence of 100 ng/mL hyper-IL-6. Induction of apoptosis was analyzed by FACS after annexin/PI staining. Data show one representative experiment. (C) Induction of apoptosis was significantly inhibited by hyper-IL-6 treatment (n = 7). (D) LPMCs of patients with IBD were cocultured for 24 hours, stimulated with lipopolysaccharide, and treated with an anti-TNF antibody or additionally with an anti–IL-6R antibody. There was a significant induction of apoptosis on additional treatment with the anti–IL-6R antibody compared with anti-TNF treatment alone. (E) Induction of apoptosis in gated CD4+ T cells was analyzed by annexin/TNFR2 staining using FACS. There was a significant induction of apoptosis selectively in CD4+TNFR2+ but not in CD4+TNFR2− T cells compared with baseline levels (n = 11). (F) Addition of the STAT3 inhibitors Stattic (10 μg/mL) or WP1066 (10 μg/mL) significantly increased anti-TNF–induced apoptosis in cocultured LPMCs from patients with IBD (n = 5), as analyzed by FACS. Baseline apoptosis was 35%–45%. Data represent mean values ± SEM. *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Model of action of clinically effective anti-TNF antibodies in IBD based on indirect induction of apoptosis in CD4+ T cells. Apoptosis resistance of mucosal CD4+ T cells in IBD appears to be mediated by mTNF-expressing CD14+ macrophages, which activate CD4+ T cells via TNFR2, leading to NF-κB induction, IL-6 production, and increased T-cell resistance against apoptosis. IL-6 signaling exerts synergistic effects to TNF signaling in IBD by inducing STAT3 activation, thereby augmenting T-cell resistance against apoptosis.
Treatment with clinically effective anti-TNF antibodies leads to the binding of these agents to mTNF-expressing CD14+ macrophages, thereby causing inhibition of TNFR2 activation on CD4+ T cells. Blockade of the TNFR2-dependent TRAF2/NF-κB signaling pathway suppresses IL-6 gene transcription. This mechanism finally results in the induction of apoptosis in CD4+ T cells in IBD. Blockade of IL-6R signaling synergizes with anti-TNF antibody treatment in inducing T-cell apoptosis in IBD.
Gastroenterology  , DOI: ( /j.gastro )
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