Volume 128, Issue 5, Pages 1243-1257 (May 2005) Different Sensitivity of Lamina Propria T-Cell Subsets to Nitric Oxide-Induced Apoptosis Explains Immunomodulatory Activity of a Nitric Oxide--Releasing Derivative of Mesalamine in Rodent Colitis  Luca Santucci, John Wallace, Andrea Mencarelli, Silvana Farneti, Antonio Morelli, Stefano Fiorucci  Gastroenterology  Volume 128, Issue 5, Pages 1243-1257 (May 2005) DOI: 10.1053/j.gastro.2005.01.051 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Early administration of NCX-456 protects against the development of TNBS-induced colitis in mice. Colitis was induced by intrarectal instillation of 2 mg of TNBS per mouse, and animals were killed 7 days after TNBS administration. NCX-456 and mesalamine were administered per os (po) daily for 7 days, starting at the same time of intrarectal instillation of TNBS. (A and B) NCX-456 reduces mortality and prevents wasting disease induced by intrarectal instillation of TNBS. Each point represents the mean ± SE of 8–12 mice. (C and D) The severity of TNBS-induced inflammation (microscopic score and MPO activity) is reduced by NCX-456 administration in a dose-dependent manner. Bars represent the mean ± SE of 8–12 mice per group. *P < .05 vs control; **P < .05 vs TNBS alone. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Early administration of NCX-456 protects against the development of TNBS-induced colitis. Histological analysis (H&E staining) of the colon is shown from mice killed 7 days after intrarectal instillation of TNBS, with or without treatment with NCX-456 or mesalamine. (A) H&E-stained paraffin section of a control (ethanol-treated) mouse (original magnification, 100×). (B) H&E-stained paraffin section from a mouse killed 7 days after TNBS administration alone, showing thickening of the colon wall and massive inflammatory infiltrate in the lamina propria (original magnification, 100×). (C) Administration of 100 mg/kg mesalamine had no effect on the histological damage induced by TNBS (original magnification, 200×). (D) The effect of daily administration of 100 mg/kg NCX-456 per os on histological colitis induced by TNBS. Subepithelial edema with no inflammatory infiltrate in the mucosa and submucosa is shown (original magnification, 100×). Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 NCX-456 administered intracolonically protects against the development of TNBS-induced colon inflammation. *P < .05 vs control; **P < .05 vs TNBS alone. I.R., intrarectally. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Early administration of NCX-456 suppresses Th1-type and increases regulatory-type cytokine production. Colon specimens were taken from mice killed 7 days after intrarectal instillation of TNBS in 50% ethanol or ethanol alone. NCX-456 or mesalamine was administered per os or intrarectally daily for 7 days. (A) Reverse-transcription polymerase chain reaction (RT-PCR) analysis of expression of inflammatory mediators in colons obtained 7 days after TNBS administration. M, molecular weight; lane 1, control (ethanol treated); lane 2, colon from a mouse treated with TNBS alone; lane 3, colon of a mouse treated with TNBS plus 100 mg/kg NCX-456 given intrarectally; lane 4, colon of a mouse treated with TNBS plus 100 mg/kg mesalamine given intrarectally. Each RT-PCR is representative of 3 separate experiments. (B-D) Concentrations of Th1 and regulatory-type cytokines in colon homogenates. Cytokines were measured by using specific enzyme-linked immunosorbent assays as described in Materials and Methods. Bars represent the mean ± SE of 5 mice per group. *P < .05 vs all groups. I.R., intrarectally. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of early administration of NCX-456 on LP T-cell apoptosis in vivo. (A) Percentages of LP T-cell populations and PMN cells isolated 7 days after TNBS administration. NCX-456 or mesalamine was administered intracolonically at 100 mg/kg for 7 days. Treatment with NCX-456 resulted in a marked decrease in the percentage of LP CD4+ T cells and in the percentage of PMN cells (Gr1+Mac1+ cells). Isolated cells were analyzed by using a flow cytometer (Coulter Epics XL). *P < .05 vs TNBS alone and mesalamine. (B and C) representative flow cytometry analysis of apoptotic CD4+ LP T cells freshly isolated from mice killed 7 days after TNBS administration. NCX-456 or mesalamine was administered intracolonically at 100 mg/kg for 7 days. After purification with magnetic beads, freshly isolated LP CD4+ T cells were stained with propidium iodide, and the percentage of apoptotic nuclei was analyzed by a flow cytometer (Coulter Epics XL). (D-F) NCX-456 causes apoptosis of LP CD4+ T cells. Apoptotic cells in a cryosection of colon tissue were determined with a double immunofluorescence assay (anti-CD4/terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling). TNBS-injected mice were treated for 4 days with 100 mg/kg NCX-456 or mesalamine administered intracolonically. Infiltrating CD4+ T cells (red) were seen at the bottom and between the colonic glands of a mouse killed 4 days after TNBS administration and treated with 100 mg/kg NCX-456 given intracolonically. As shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (green) and merged (yellow) studies, most of these T cells were apoptotic (original magnification, 200×). Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 NCX-456 abrogates IFN-γ production and stimulates IL-10 and TGF-β secretion by isolated LP CD4+ T cells. LP T cells were isolated from mice killed 7 days after intrarectal administration of TNBS. NCX-456 100 mg/kg or mesalamine was administered intracolonically for 7 days. Bars represent the mean ± SE of 3 experiments (4–6 mice per group). *P < .05 vs control (ethanol-treated) mice; **P < 0.05 vs mice treated with TNBS alone. , medium alone; , anti-CD3/CD28. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 NCX-456 inhibits proliferation and causes selective apoptosis of activated CD4+CD25− LP T cells. CD4+ LP T cells were purified from untreated mice by negative selection with magnetic beads (MACS; Miltenyi Biotech, Auburn, CA). CD4+ LP T cells were then further separated into CD25+ (regulatory T cells) and CD25− populations by MACS with phycoerythrin-labeled anti-CD25 MAbs. Anti-CD3/CD28 activated LP T cells were then incubated with increasing doses of NCX-456 or mesalamine for 24 hours. NCX-456 inhibits the proliferative response (A) and causes selective apoptosis (B) only in activated CD4+CD25− LP T cells and is ineffective in regulatory CD25+ LP T cells. A total of 10 μmol/L carboxy-PTIO, an NO scavenger, reverses the effects exerted by NCX-456 on T-cell function and survival. *P < .05 vs mesalamine and NCX-456 plus carboxy-PTIO. (C) Representative scatter plots of the 2 LP T-cell populations after incubation with 100 μmol/L NCX-456 or mesalamine. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 (A) Changes in ΔΨm as measured by JC-1 relative red fluorescence intensity. The mitochondrial potential was assessed at the time points indicated. Data are mean ± SE of 4 independent measures. *P < .05 vs medium alone, mesalamine, and NCX-456 plus carboxy-PTIO. (B) Caspase 3 activity measured after 2 and 18 hours of incubation with or without 100 μmol/L mesalamine or NCX-456. Caspase 3 activity was measured by using a specific ELISA kit. (C) ROS generation was measured by flow cytometry with the fluorogenic probe DCFH-DA. *P < .05 vs mesalamine and NCX-456 plus carboxy-PTIO. (D) GSH content and the GSH/GSSG ratio in activated CD4+CD25− LP T cells and in CD25+ regulatory T cells. LP T-cell subsets were activated for 24 hours by using anti-CD3/CD28 monoclonal antibodies. GSH and GSSG content was measured by using a specific ELISA kit. *P < .05 vs activated CD4+CD25− LP T cells. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Therapeutic administration of NCX-456 abrogates fully established colitis induced by TNBS administration. NCX-456 or mesalamine was administered intracolonically at 100 mg/kg daily starting 14 days after intrarectal instillation of TNBS. Mice were killed 21 days after TNBS administration. NCX-456, but not mesalamine, significantly reduced the histological damage score and colon MPO activity. Each point represents average weight data pooled from 10 mice. *P < .05 vs control; **P < .05 vs TNBS alone and mesalamine. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Therapeutic administration of NCX-456 exerts therapeutic activity in fully established colitis in 20-week-old IL-10–deficient mice. NCX-456 or mesalamine was administered intracolonically at 100 mg/kg daily for 14 days. (A) Starting body weight of 20-week-old IL-10−/− and IL-10+/+ mice. *P < .01 vs IL-10+/+ mice. Effect of NCX-456 administration on body weight (B), histological score of colitis (C), and colonic MPO activity (D). *P < .05 vs control and mesalamine. Gastroenterology 2005 128, 1243-1257DOI: (10.1053/j.gastro.2005.01.051) Copyright © 2005 American Gastroenterological Association Terms and Conditions