Volume 114, Issue 1, Pages 93-102 (January 1998) Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells  Andrew L. Salzman, Tonyia Eaves–Pyles, Stephen C. Linn, Alvin G. Denenberg, Csaba Szabó  Gastroenterology  Volume 114, Issue 1, Pages 93-102 (January 1998) DOI: 10.1016/S0016-5085(98)70637-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 IFN-γ primes DLD-1 cells for NO production in response to S. dublin. DLD-1 cells were pretreated with 0.5 ng/mL IL-1β, 100 U/mL IFN-γ, or IL-1β/IFN-γ (simultaneous addition) for 3 hours, followed by the addition of S. dublin for 3 hours. After treatment, cells were washed, resuspended in medium containing antibiotics, and incubated for 18 hours, and NO2−/NO3− production was measured (n = 4 wells per condition). Controls (C) were normal, untreated cells. Cells were also pretreated for 1 hour with NG-methyl-L-arginine (L-NMA), followed by the addition of IFN-γ and S. dublin using the same protocol as above. Data are expressed as mean ± SEM. S. dublin (102-107 CFU/mL) induced NO synthesis in IFN-γ–treated cells (P < 0.005). SD, Salmonella dublin. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 IFN-γ primes DLD-1 cells for NO production in response to LPS. Cells were treated for 3 hours with 0.5 ng/mL IL-1β or 100 U/mL IFN-γ, followed by the addition of 0.1-10.0 μg/mL LPS for 18 hours (n = 8 wells per condition), and NO2−/NO3− production was measured. Controls (C) were normal, untreated cells. Data are expressed as mean ± SEM. LPS (1-10 μg/mL) induced NO synthesis in IFN-γ–treated cells (P < 0.005). Cells treated with IL-1β and IFN-γ, in the presence or absence of LPS, induced significant NO production (P < 0.001). Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 IFN-γ primes Caco-2BBe cells for NO production in response to S. dublin but not LPS. Cells were treated for 3 hours with 0.5 ng/mL IL-1β or 100 U/mL IFN-γ, followed by the addition of 1 μg/mL LPS or 107 CFU/mL S. dublin for 3 hours. Cells were incubated for 18 hours (n = 8 wells per condition), and NO2−/NO3− production was measured. Controls (C) were normal, untreated cells. Data are expressed as mean ± SEM. *Significant difference vs. control (P < 0.05). **Significant difference vs. IFN-γ–treated cells (P < 0.05). S. dublin, but not LPS, induced significant NO synthesis in IFN-γ–treated cells (P < 0.005). SD, Salmonella dublin. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Characterization of S. dublin–mediated induction of NO production in IFN-γ–primed DLD-1 cells. Cells were pretreated with 100 U/mL IFN-γ for 3 hours, followed by the addition of 106 CFU/mL S. dublin, S. dublin invA (an invasion deficient S. dublin mutant; 106 CFU/mL), or 1 μg/mL LPS. Heat-inactivated (h.i.) S. dublin and LPS were treated at 95°C for 10 minutes before addition. NO2−/NO3− content was measured after incubations (n = 6 wells per condition). Data are expressed as mean ± SEM. **Significant difference between unstimulated cells and the groups indicated (P < 0.01). ##Significant difference between cells treated with IFN and S. dublin vs. the groups indicated (P < 0.01). Heat treatment of S. dublin abolished its ability to induce NO production (P < 0.001). Heat treatment of LPS had only a minor effect on the ability of LPS to induce NO production (P < 0.001). S. dublin invA was a less potent stimulus of NO synthesis than the wild-type strain (P < 0.001). SD, S. dublin. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of priming with IFN-γ, LPS, and S. dublin on NO production in DLD-1 cells. Cells were pretreated with 100 U/mL IFN-γ for 3 hours, followed by its removal and the subsequent addition for 3 hours of 1 μg/mL LPS or S. dublin (106 CFU/mL; n = 8 wells per condition). The reverse order was also performed using the same conditions and incubation times. NO2−/NO3− content was measured after incubations. Data are expressed as mean ± SEM. **Significant difference between unstimulated cells and the groups indicated (P < 0.01). ##Significant difference between the groups in which the order of treatments has been reversed (P < 0.01). Pretreatment with S. dublin, compared with LPS, had a greater effect on NO2−/NO3− production in DLD-1 cells subsequently exposed to IFN-γ. SD, S. dublin. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 LPS and S. dublin induce iNOS mRNA expression in IFN-γ–primed DLD-1 cells. Cells were pretreated with IFN-γ, IL-1β, or a combination of IFN-γ and IL-1β for 3 hours before the addition of 1 μg/mL LPS or 106 CFU/mL S. dublin for 3 additional hours. Some cells were pretreated for 1 hour with 1 μg/mL actinomycin D or 10 μg/mL cycloheximide, followed by the addition of IFN-γ and LPS or S. dublin as stated above. Northern hybridization analysis was performed after cell incubations. Representative blots (n = 3), which were hybridized with an iNOS complementary DNA probe, are shown. iNOS mRNA expression was clearly visible in cells treated with IL-1β/IFN-γ and IFN-γ followed by the addition of S. dublin or LPS. IL-1β/IFN-γ–treated cells also showed iNOS mRNA expression. Actinomycin D and cycloheximide treatment eliminated iNOS mRNA expression in IFN-γ–primed DLD-1 cells treated with S. dublin or LPS. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 NO formation is blocked by inhibitors of NF-κB and tyrosine kinase activity in DLD-1 cells. Cells were pretreated for 1 hour with PDTC (100 μmol/L; ▨), TLCK (300 μmol/L; 2), genistein (100 μmol/L; ▩), and dexamethasone (100 μmol/L; ▩), followed by stimulation with IFN-γ (100 U/mL) for 3 hours and the subsequent addition of LPS (1 μg/mL) for 18 hours or S. dublin (106 CFU/well) for 3 hours (n = 6 wells per condition). After treatment with S. dublin, cells were washed, resuspended in medium containing antibiotics, and incubated for an additional 18 hours. PDTC, TLCK, and genistein inhibited NO production in IFN-γ–primed cells treated with S. dublin or LPS (P < 0.01). Dexamethasone pretreatment showed no inhibitory effects on NO production in treated cells. Data are expressed as mean ± SEM. **Significant reduction (P < 0.01) in NO production vs. immunostimulated cells in the absence of inhibitors. SD, S. dublin. ■, Vehicle. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Response of iNOS promoter reporter constructs to (A) IFN-γ priming and LPS and (B) S. dublin. End points of the promoter fragments, in kilobases, are indicated. Luciferase activities were measured from cells cultured under the following conditions: C (control), IFN-γ/LPS (2-hour pretreatment with 100 U/mL of IFN-γ, followed by a 22-hour exposure to 1 μg/mL of LPS), and IFN-γ/S. dublin (2-hour pretreatment with 100 U/mL of IFN, followed by a 3-hour exposure to S. dublin [106 CFU/well]. Values are presented relative to those obtained in unstimulated cells with the −7.2-kb construct (included in all experiments) and are expressed as the mean ± SEM (n = 8 wells per condition). Data for each construct were obtained from a minimum of 6 independently transfected wells, with and without cytokine treatment. Values marked with asterisks differed significantly from control in an analysis of variance test. **P < 0.01. ***P < 0.001. (A) ■, LPS + IFN; ▨, controls; (B) ■ S. dublin + IFN; ▨, controls. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Response of iNOS promoter reporter constructs to (A) IFN-γ priming and LPS and (B) S. dublin. End points of the promoter fragments, in kilobases, are indicated. Luciferase activities were measured from cells cultured under the following conditions: C (control), IFN-γ/LPS (2-hour pretreatment with 100 U/mL of IFN-γ, followed by a 22-hour exposure to 1 μg/mL of LPS), and IFN-γ/S. dublin (2-hour pretreatment with 100 U/mL of IFN, followed by a 3-hour exposure to S. dublin [106 CFU/well]. Values are presented relative to those obtained in unstimulated cells with the −7.2-kb construct (included in all experiments) and are expressed as the mean ± SEM (n = 8 wells per condition). Data for each construct were obtained from a minimum of 6 independently transfected wells, with and without cytokine treatment. Values marked with asterisks differed significantly from control in an analysis of variance test. **P < 0.01. ***P < 0.001. (A) ■, LPS + IFN; ▨, controls; (B) ■ S. dublin + IFN; ▨, controls. Gastroenterology 1998 114, 93-102DOI: (10.1016/S0016-5085(98)70637-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions